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BMC Biology
Lactoferrin deciency duringlactation
increases therisk ofdepressive-like behavior
inadult mice
Wenli Wang1, Zhimei Cheng1, Xiong Wang1, Qin An1, Kunlun Huang1, Yunping Dai2, Qingyong Meng2 and
Yali Zhang1*
Abstract
Background Lactoferrin is an active protein in breast milk that plays an important role in the growth and develop-
ment of infants and is implicated as a neuroprotective agent. The incidence of depression is currently increasing,
and it is unclear whether the lack of lactoferrin during lactation affects the incidence of depressive-like behavior
in adulthood.
Results Lack of lactoferrin feeding during lactation affected the barrier and innate immune functions of the intestine,
disrupted the intestinal microflora, and led to neuroimmune dysfunction and neurodevelopmental delay in the hip-
pocampus. When exposed to external stimulation, adult lactoferrin feeding-deficient mice presented with worse
depression-like symptoms; the mechanisms involved were activation of the LPS–TLR4 signalling pathway in the intes-
tine and hippocampus, reduced BDNF-CREB signaling pathway in hippocampus, increased abundance of depression-
related bacteria, and decreased abundance of beneficial bacteria.
Conclusions Overall, our findings reveal that lactoferrin feeding deficient during lactation can increase the risk
of depressive-like behavior in adults. The mechanism is related to the regulatory effect of lactoferrin on the develop-
ment of the "microbial–intestinal–brain" axis.
Keywords Lactation, Lactoferrin, Depression, Microbial–intestinal–brain, Innate immunity, Inflammation
Background
Depressive disorders are a global public health concern
and a leading cause of disease burden, substantially
impairing patients’ quality of life and increasing mortal-
ity risk. According to the World Health Organization
(WHO), the number of people affected by depression
increased by 18% worldwide in the decade from 2005 to
2015. In the first year of the COVID-19 pandemic, the
global prevalence of anxiety and depression increased
by a massive 25%, according to a scientific brief released
by the WHO. Depression is caused by complex interac-
tions of multiple factors. e pathogenesis is not fully
understood, although many hypotheses have been pro-
posed, such as the inflammatory hypothesis [1], “leaky
gut” hypothesis [2], gut microbiota hypothesis [3],
and monoamine hypothesis [4]. Biochemical signals
that occur between the gastrointestinal tract and cen-
tral nervous system are known as the gut brain axis.
e bidirectional communication between the brain
and gut occurs through the autonomic, enteric, neu-
roendocrine, and immune systems [3]. Studies of the
*Correspondence:
Yali Zhang
zhangyali@cau.edu.cn
1 College of Food Science and Nutritional Engineering, China Agricultural
University, Beijing, China
2 College of Biological Sciences, China Agricultural University, Beijing,
China
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Page 2 of 26
Wangetal. BMC Biology (2023) 21:242
microbiota–gut–brain axis may lead to novel approaches
for preventing and treating mental illnesses, including
anxiety and depression.
Immune system disorders caused by impaired gastro-
intestinal barrier integrity are considered as important
mechanisms of depression [5]. e intestinal epithelial
barrier serves as the first boundary of defence between
the organism and luminal environment [6]. Increased
intestinal permeability caused by intestinal barrier injury
leads to systemic chronic inflammation, whereby nerv-
ous system inflammation is an important pathological
feature of depression. Lipopolysaccharide (LPS) from
bacteria in the intestinal lumen, mainly gram-negative
bacteria, enters the systemic circulation through the
increased intercellular space and cross-cellular path-
ways [7] and, LPS binds to TLR-4 in the central nervous
system, thus triggering neuroinflammation [8]. Depres-
sion is a common comorbidity associated with gastro-
intestinal diseases, such as irritable bowel syndrome
and inflammatory bowel diseases (IBDs) [9]. A previous
study showed that chronic gastrointestinal inflamma-
tion induces anxiety-like behaviour and alters the central
nervous system biochemistry [10]. A population- based
study revealed that individuals with IBD had a higher
prevalence of depression than matched controls [11].
Gampierakis etal. showed that acute and chronic experi-
mental colitis affects adult hippocampal neurogenesis
and innate immune cell responses, which may be mecha-
nisms of cognitive and mood dysfunction in patients with
IBD [12].
e gut microbiota is a key regulator of the gut–brain
axis and influences brain function and behaviour through
the microbiota–gut–brain axis. Many antibodies against
intestinal symbiotic bacteria LPS have been detected in
the serum of patients with depression, supporting the
notion that inflammation is related to intestinal symbi-
otic bacteria [2]. Animal experiments add further support
to this idea; interestingly, faecal microbiota transplanta-
tion from depressed patients to microbiota-depleted rats
induced anxiety and depressive behaviours and altered
tryptophan metabolism in recipient animals [13]. Fur-
thermore, bacterial species regulate the production of
neurotransmitters and their precursors, such as seroto-
nin, γ-aminobutyric acid type A (GABA), and tryptophan
[14]. Gut microbes also promote the release of short-
chain fatty acids (SCFAs) and brain-derived nutritional
factors (BDNFs), which have been shown to ameliorate
depression [10].
Lactation is a critical period for the development of the
intestinal tract and nervous system and the establishment
of the gut microbiota; breast milk plays important roles
in these processes. However, many newborns cannot be
breastfed due to the nutritional and health status of their
mothers, social factors, and uncertain circumstances. In
low- and middle-income countries, only 37% of children
younger than six months of age are exclusively breast-
fed; this rate is even lower in high-income countries [15].
Lactoferrin (LF) is an important active whey protein in
breast milk present at concentrations of 1–7 and 0.2–
1.5g/L in humans and bovines, respectively [16]. LF plays
an important role in neonatal development by promoting
maturation of the neonatal small intestine and increas-
ing the integrity of the intestinal barrier [17]. Several
studies have shown that LF can improve neural develop-
ment, neuroprotection, and cognitive abilities [18]. e
early gut microbiota has been predicted to contribute to
disease progression later in life, and the foundation for a
stable adult gut microbiota has already been established
in infancy [19]. According to a previous study, early life
LF intervention can modulate the microbial community
of the cecum [20]; however, whether the effects of LF on
the microbiota of suckling mice affect adult depression
remains unclear. Formula is an important alternative
to breast milk, but the addition of LF to formula is not
widespread. us, considering that exclusive breastfeed-
ing rates are < 50% in most countries, LF deficiency dur-
ing lactation may be prevalent worldwide. e effect of
LF feeding deficiency during lactation on adult health,
particularly depression, remains unknown.
In this study, we generated an Ltf-knockout (KO)
mouse model as a mother mouse that provided LF-free
milk. Two groups of mice were evaluated: wild-type
(WT) neonates breast-fed by WT or KO female mice
(WT-WT or KO-WT). e chronic unpredictable mild
stress (CUMS) depression model was established when
the mice reached 9 weeks of age, and differences in
depressive phenotypes were compared among groups.
To investigate the mechanism by which LF feeding defi-
ciency during lactation increases the risk of depressive-
like behavior in adult mice, the intestinal and brain
development and intestinal microbe composition at dif-
ferent growth stages of mice were investigated.
Results
LF feeding deciency duringsuckling period increased
depressive‑like behavior risk inadult mice
Male and female mice that consumed normal milk and
LF-free milk during the suckling period were fed a nor-
mal diet until 9weeks of age, then SPT was tested before
CUMS, after which, the CUMS model was established
for 4weeks, then SPT was tested again after CUMS, OFT
was carried out on the second day after CUMS, serum
was collected on the fourth day after CUMS (Fig. 1A).
As shown in Fig. 1B (F = 1.820, df = 6, p = 0.919) and
1C (F = 0.298, df = 8, p = 0.857), wt-wt and ko-wt mice
did not differ before CUMS; however, after 4weeks of
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Page 3 of 26
Wangetal. BMC Biology (2023) 21:242
CUMS treatment, mice without LF intake during the
suckling period (ko-wtM and ko-wtF) showed a lower
sucrose preference in the SPT (Mann–Whitney U tests,
Fig.1B: p = 0.009 < 0.05; Fig. 1C: p = 0.031 < 0.05). In LF-
feeding mice the sucrose preference before CUMS has
no difference with after in both male and female mice
(Fig. 1B: F = 2.146, df = 8, p = 0.429; Fig. 1C: F = 1.916,
df = 8, p = 0.575), but the difference was significant in
LF-feeding deficient mice (Mann–Whitney U tests,
Fig.1B: p = 0.019 < 0.05; t test, Fig. 1C: F = 0.259, df = 5,
p = 0.000 < 0.05).
In the TST (Fig.1D), there was no significant differ-
ence between the wt-wtM and ko-wtM groups during
the first week of CUMS (F = 2.39, df = 14, p = 0.696). At
week 4, the immobility time of the wt-wtM (F = 1.128,
df = 14, p = 0.099 > 0.05) and ko-wtM (Mann–Whit-
ney U tests, p = 0.017 < 0.05) groups was higher than
that of week 1 (wt-wtM). Although a similar trend was
observed in the female group (Fig.1E), no significant dif-
ference was observed between the wt-wtF and ko-wtF
groups at weeks 1 (F = 0.382, df = 14, p = 0.297) and week
4 (F = 1.75, df = 14, p = 0.798). In the FST (Fig.1F), there
was no difference between wt-wtM and ko-wtM mice at
week 1 (F = 0.796, df = 14, p = 0.362); at week 4, the immo-
bility time of the wt-wtM (F = 0.445, df = 14, p = 0.22) and
ko-wtM (F = 6.571, df = 9.342, p = 0.011 < 0.05) groups
increased compared with that of week 1, and the immo-
bility time of the ko-wtM mice was longer than that of the
wt-wtM mice (F = 4.69, df = 9.337, p = 0.02 < 0.05). Fig-
ure1G shows that the immobility time in the FST of the
ko-wt female mice (ko-wtF) was significantly longer than
that of the wt-wtF group at week 1 (F = 0.207, df = 14,
p = 0.034 < 0.05), and increased compared with that of the
wt-wtF mice at week 4, but the difference was not signifi-
cant (F = 0.028, df = 14, p = 0.14). What’s more, there were
no interaction effects between time and group (Fig.1D-
G). In the OFT, the distance in the central area for the
ko-wtM group was significantly smaller than that for the
wt-wtM group (F = 2.942, df = 14, p = 0.022 < 0.05), with
no difference between the ko-wtF and wt-wtF groups
(F = 5.474, df = 9.393, p = 0.501 > 0.05) (Fig. 1H). e
influence of lactoferrin intake during lactation on OFT is
significant (F (1,28) = 5.801, p = 0.023 < 0.05). Male mice
with LF feeding deficiency during lactation showed more
severe anxiety-like behaviours in the OFT compared
to male mice that drank normal milk. e influence of
gender on open field is also significant (F (1,28) = 4.586,
p = 0.041 < 0.05). Figure1H shows that the distance in the
central area for the wt-wtF group was lower than that for
the wt-wtM group (F = 0.062, df = 14, p = 0.062 > 0.05),
indicating that female mice may exhibited more severe
anxiety-like behaviours compared to male mice.
Serum indicators associated with depression were
measured. BDNF is a well-known growth factor in the
brain [21], and the level of BDNF in the ko-wt group was
significantly lower than that in the wt-wt group, for both
male and female mice (ko-wtM vs. wt-wtM (F = 9.376,
df = 10.191, p = 0.004); ko-wtF vs. wt-wtF (F = 0.626,
df = 14, p = 0.000 < 0.05)). e levels of BDNF in female
mice were lower than those in male mice (wt-wtF vs. wt-
wtM (F = 19.838, df = 8.369, p = 0.059 > 0.05); ko- wtF vs.
ko-wtM (F = 0.215, df = 14, p = 0.004 < 0.05)) (Fig.1I). Cor-
ticosterone (CORT) and adrenocorticotropic hormone
(ACTH) are hormones related to the hypothalamic–pitu-
itary–adrenal axis and are associated with depression.
Our results revealed a significantly increased production
of CORT and ACTH in the ko-wt mice compared to that
in the wt-wt mice (ko-wtM vs. wt-wtM (F = 1.945, df = 14,
p = 0.00 < 0.05), (F = 3.67, df = 14, p = 0.002 < 0.05); ko-
wtF vs. wt-wtF(Mann–Whitney U tests, p = 0.009 < 0.05,
p = 0.009 < 0.05) (Fig . 1J, K); the wt-wtF group showed
significantly higher CORT levels than the wt-wtM group
(Mann–Whitney Test, p = 0.009 < 0.05). e levels of pro-
inflammatory cytokines, such as tumour necrosis factor
(TNF)-α (Fig. 1L) and interleukin (IL)-1β (Fig.1M), in
the ko-wt group were significantly higher than those in
the wt-wt group (ko-wtM vs. wt-wtM (F = 0.07, df = 14,
p = 0.027 < 0.05), (F = 0.266, df = 14, p = 0.000 < 0.05);
ko-wtF vs. wt-wtF(F = 0.086, df = 14, p = 0.005 < 0.05),
(F = 0.91, df = 14, p = 0.006 < 0.05)), and IL-1β levels in
the wt-wtF group were significantly higher than those in
the wt-wtM group (F = 3.173, df = 14, p = 0.009 < 0.05).
Additionally, LPS content was significantly increased in
the ko-wt mice serum (ko-wtM vs. wt-wtM (F = 0.446,
df = 14, p = 0.026); ko-wtF vs. wt-wtF (F = 0.425,
df = 14, p = 0.002 < 0.05) (Fig. 1N), which can induce
(See figure on next page.)
Fig. 1 LF feeding deficiency during suckling period increased depressive-like behaviors of CUMS mice in adult. A Study design of mice
experiment. B Percent of sucrose consumption in SPT of male mice. n = 4–6. C Percent of sucrose consumption in SPT of female mice. n = 3–6. D
Immobility time in the TST of male mice. E Immobility time in the TST of female mice. F Immobility time in the FST of male mice. G Immobility
time in the FST of female mice. H Trajectory map and distance in central area in OFT of male and female mice. I Serum levels of brain-derived
neurotrophic factor (BDNF), J corticosterone (CORT), K adrenocorticotropic hormone (ACTH), L tumor necrosis factor-α (TNF-α). M interleukin-1β
(IL-1β), N lipopolysaccharide (LPS). D-N n = 8. Data are presented as mean ± SEM. Two-way ANOVA with multiple comparisons and two-tailed t test
for normally distributed data, two-tailed Mann–Whitney test for non-normally distributed data, *P < 0.05; **P < 0.01; ***P < 0.001
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Wangetal. BMC Biology (2023) 21:242
Fig. 1 (See legend on previous page.)
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Page 5 of 26
Wangetal. BMC Biology (2023) 21:242
inflammation. Furthermore, the LPS levels in the ko-wtF
group were significantly higher than those in the ko-wtM
group (F = 0.111, df = 14, p = 0.04 < 0.05). What’s more,
there were no interaction effects between gender and
group (Fig.1H-N).
CUMS mice withlactation LF feeding deciency showed
intestinal, brain andmicrobial ora disorders
Dysfunction of the microbiota–gut–brain axis is thought
to be the main pathological basis of depression. We com-
pared the histological changes in the colon, inflammation
of the colon and hippocampus and composition of intes-
tinal microorganisms in depressed mice with LF feeding
deficiency during lactation with those in depressed mice
that consumed normal mouse milk during lactation. As
shown in Fig.2A, the crypt depth of ko-wtM mice was
significantly lower than that of wt-wtM mice (F = 0.202,
df = 13, p = 0.046 < 0.05), there was no significant differ-
ence in colonic crypt depth between the wt-wtF and ko-
wtF groups (F = 1.881, df = 9, p = 0.612 > 0.05). Damage
to the intestinal epithelial barrier increased the translo-
cation of this luminal LPS to systemic circulation, and
expression of the zonula occludens gene (Zo-1) in ko-wt
mice has no significant difference with wt-wt mice (ko-
wtM vs wt-wtM, F = 7.672, df = 7.323, p = 0.084 > 0.05;
ko-wtF vs wt-wtF, F = 6.858, df = 5.12, p = 0.067 > 0.05).
However, the results of two-way ANOVA analysis
showed that the expression level of Zo-1 was signifi-
cantly affected by lactoferrin intake during lactation (F
(1,28) = 6.76, p = 0.015 < 0.05), Zo-1 expression in ko-
wtF group was significantly lower than that in ko-wtM
(F = 2.265, df = 14, p = 0.003 < 0.05). ere was no signifi-
cant difference in Occludin expression between ko-wtM
mice and wt-wtM mice (F = 0.03, df = 14, p = 0.188 > 0.05)
(Fig. 2B). Occludin expression in wt-wtF was signifi-
cantly lower than that in wt-wtM (F = 1.12, df = 14,
p = 0.035 < 0.05). erefore, the intestinal barrier func-
tion was more severely damaged in female mice than in
male mice, which may be the reason for the higher LPS
concentration in ko-wtF mice compared with ko-wtM
(Fig.1N). However, our results showed that the expres-
sion of ZO-1 and Occludin in LF lacking mice has no
difference with LF feeding mice (ko-wtF vs wt-wtF and
ko-wtM vs wt-wtM). It is well known that the increase in
serum LPS level is associated with intestinal microflora
disorder and intestinal mucosal permeability [22]. ere-
fore, intestinal microflora disorder may be primarily
responsible for the increase of LPS.
As shown in Fig. 2C, the infiltration score of colons
showed that no significant difference was found between
LF-lacking mice and LF-feeding mice (ko-wtM vs wt-
wtM, ko-wtF vs wt-wtF). We further examined the
activation of the LPS-TLR4 signaling pathway in the
colon and hippocampus. In the colon (Fig.2D) of male
mice, the expression of Myd88 (p = 0.01 < 0.05), CD14
(F = 0.506, df = 14, p = 0.045 < 0.05), nuclear factor
(NF)-κB (p = 0.001 < 0.05), P38MAPK (p = 0.027 < 0.05)
and IL-1β (F = 0.921, df = 14, p = 0.01 < 0.05) was sig-
nificantly higher in ko-wtM group than in the wt-wtM
group and expression of TLR4 was higher in the ko-
wtM group than in wt-wtM group, but the difference
was not significant (P = 0.059 > 0.05). JNK expression
in the wt-wtM group was significantly higher than that
in the ko-wtM group (F = 3.4, df = 14, p = 0.038 < 0.05);
other genes showed no significant differences between
these two groups. In female mice, the expression
of TLR-4 (F = 3.834, df = 14, p = 0.000 < 0.05), CD14
(F = 5.039, df = 8.566, p = 0.016 < 0.05), p38MAPK
(p = 0.027 < 0.05), JNK (F = 0.053, df = 14, p = 0.001 < 0.05)
and IL-1β (F = 3.119, df = 14, p = 0.005 < 0.05) was sig-
nificantly lower in ko-wtF group than in the wt-wtF
group, TNF-α (F = 14.463, df = 5.102, p = 0.054 > 0.05)
also show a lower trend in ko-wtF group than in the
wt-wtF group. In LF-feeding mice, the expression of
NF-κB (p = 0.001 < 0.05) and IL-1β (F = 0.103, df = 14,
p = 0.045 < 0.05) in female mice was siginificantly higher
than male mice. However, in LF-free mice, the expres-
sion of TLR4 (F = 6.524, df = 9.16, p = 0.000 < 0.05), CD14
(F = 3.006, df = 14, p = 0.006 < 0.05), NF-κB (F = 24.853,
df = 8.439, p = 0.037 < 0.05), p38MAPK (p = 0.006 < 0.05),
Fig. 2 The damage of colon, hippocampus and microorganism composition in lactation LF feeding deficient mice after CUMS model. A Crypt
depth in the colon of mice and representative images of H.E-stained colonic sections, 200 × , scale bar = 100 μm. n = 5–8. B Zo-1, Occludin mRNA
expression in the colon. n = 6–8. C Inflammation infiltration score and representative images in the colon, 200 × , scale bar = 100 μm. n = 6–8. D
Activation of LPS-TLR4 signaling pathway in the colon. n = 6–8. E Activation of LPS-TLR4 signaling pathway and microglia in the hippocampus.
n = 6–8. F The mRNA expression of BDNF signaling pathway in the hippocampus. n = 4–8. G The alpha diversity analysis, including Shannon diversity
and Chao diversity, Wilcoxon rank-sum test. n = 8. H PCoA analysis results of male and female CUMS mice and evaluated using Analysis of Similarities
(ANOSIM). n = 8. I Analysis on the composition of microbial community at phylum level. n = 8. J Student’s t-test on Phylum level of male and female
depressive-like behavior mice. n = 8. K Linear discriminant analysis (LDA > 3) scores derived from LEfSe analysis at genus level of male and female
depressive-like behavior mice. n = 8. L Heatmap of Spearman’s correlation between gut microbiota (at the genus level) and depressive-like
behavior related indices. n = 8. Data are presented as mean ± standard error. A-FTwo-way ANOVA with multiple comparisons and two-tailed t test
for normally distributed data, two-tailed Mann–Whitney test for non-normally distributed data, *P < 0.05; **P < 0.01; ***P < 0.001
(See figure on next page.)
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Wangetal. BMC Biology (2023) 21:242
Fig. 2 (See legend on previous page.)
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Page 7 of 26
Wangetal. BMC Biology (2023) 21:242
JNK (F = 0.017, df = 14, p = 0.001 < 0.05), IL-1β (F = 1.656,
df = 14, p = 0.000 < 0.05) and TNF-α (p = 0.013 < 0.05) in
female mice was siginificantly lower than male mice. In
the hippocampus (Fig. 2E), the expression of JNK was
significantly higher in the ko-wtM group than in the
wt-wtM group (F = 1.738, df = 11, p = 0.017 < 0.05). e
expression of TLR-4 (F = 1.93, df = 13, p = 0.031 < 0.05),
LBP (F = 1.601, df = 13, p = 0.011 < 0.05), Myd88
(F = 14.221, df = 6.098, p = 0.048 < 0.05), NFκB (F = 3.946,
df = 13, p = 0.019 < 0.05) and IL-1β (p = 0.019 < 0.05)
was significantly higher in ko-wtF mice than in wt-wtF
mice. e expression of Iba-1 (a marker of activation for
microglia) was significantly higher in ko-wtF mice than
in wt-wtF mice (F = 9.926, df = 6.586, p = 0.018 < 0.05).
In LF-feeding mice, the expression of Myd88 (F = 0.115,
df = 11, p = 0.017 < 0.05) in female mice was siginificantly
higher than male mice. In LF-free mice, the expression
of JNK (p = 0.008) in female mice was siginificantly lower
than male mice.
us, after 4weeks of CUMS, the colon of LF-free male
mice showed more severe inflammation which occurred
through activation of the TLR4-Myd88-NF-κB signaling
pathway. However, female mice that consumed normal
mouse milk showed more severe intestinal inflamma-
tion via activation of the MAPK and JNK signals in
the TLR4 signaling pathway. e difference in colonic
inflammation between female and male mice may be
related to different levels of oestrogen [23]. Based on the
significantly different levels of BDNF in the serum, we
measured the expression of genes related to the BDNF
signaling pathway in the hippocampus of depressed mice.
None of the genes differed between the ko-wtM and wt-
wtM group (Fig.2F); however, in the female groups, the
expression of Grb2 (F = 0.915, df = 13, p = 0.000 < 0.05),
CaMK1 (F = 1.652, df = 9, p = 0.012 < 0.05), and Creb
(F = 2.283, df = 9, p = 0.048 < 0.05) in the ko-wtF group
was significantly lower than that in the wt-wtF group,
and the expression of BDNF in the ko-wtF mice was
lower than that in the wt-wtF group but not signifi-
cantly (F = 0.346, df = 9, p = 0.094 > 0.05). What’s more,
gender and group (LF intake or not during lactation)
had an significant interactive effect on the expression
of BDNF (F (1,17) = 6.267, p = 0.023 < 0.05), GRB2 (F
(1,24) = 9.39, P = 0.005 < 0.05) and CREB ((F(1,17) = 5.394,
p = 0.033 < 0.05).
Next, we examined the composition of the gut flora.
Alpha diversity represents the richness and diversity of
the microbial community. e Shannon formula was used
to estimate diversity, and the richness of the microbial
community was estimated using the Chao index. Mice
without LF intake during the suckling period (ko-wtM
and ko-wtF) showed a lower Shannon index of the intes-
tinal microbiota but the difference was not significant
(Fig.2G). e Chao index was significantly lower in the
ko-wtF than in the wt-wtF mice, but there was no signifi-
cant difference between the ko-wtM and wt-wtM groups
(Fig.2G). Principal coordinate analysis (PCoA) revealed
major differences between the ko-wtM and wt-wtM
mice (p = 0.002 < 0.05) as well as differences in the micro-
bial composition between the ko-wtF and wt-wtF mice
(Fig.2H, P = 0.097 > 0.05). ere was a significant differ-
ence in the pattern of intestinal flora between the ko-wt-
depressed and wt-wt-depressed mice. e predominant
phyla (Fig. 2I) were Firmicutes, Bacteroidetes, Desul-
fobacterota, Patescibacteria, and Actinobacteria in the
faecal samples of the four groups, and there was no dif-
ference between ko-wtM and wt-wtM mice. In contrast,
the abundances of Desulfobacterota, Actinobacteria, and
Chloroflexi significantly increased in the ko-wtF group
(Fig.2J). At the genus level, we used linear discriminant
analysis effect size (LEfSe) to identify bacteria whose
relative abundance significantly differed between ko-wt
and wt-wt mice. Figure2K shows that there was a greater
abundance of Eubacterium_xylanophilum_group, and a
lower abundance of Lactobacillus and SCFA-producing
bacteria (Alistipes and Dubosiella) in the ko-wtM group
than in the wt-wtM group. SCFAs exert anti-inflamma-
tory functions and are beneficial to the composition of
the gut microbiota and intestinal barrier [24]. Figure2K
shows that Lactobacillus, Desulfovibrio, Arenimonas,
Enterorhabdus, and Bifidobacterium were significantly
enriched in the ko-wtF group, and Ileibacterium was sig-
nificantly enriched in the wt-wtF group.
To further explore the correlation between the gut
microbiota (significantly different microbiota at the
genus level) and depression-related behavioural indices
(OFT, SPT, TST, and FST), depression-related hormones
(ACTH and CORT), serum inflammatory factors (IL-1β
and TNF-α), and BDNF, a heatmap of Spearman’s corre-
lation analysis was generated (R and P value was showed
in Additional file: Table. S1, S2). As shown in Fig. 2L,
Desulfovibrio was significantly negatively correlated with
SPT and positively correlated with FST (P = 0.06); Bifido-
bacterium was negatively correlated with TST (P = 0.05);
and Alistipes was significantly positively correlated with
OFT. Furthermore, Lactobacillus and Enterorhabdus
were positively correlated with TST, ACTH, CORT,
IL-1β, TNF-α, and LPS, and negatively correlated with
OFT, SPT, and BDNF. Blautia showed a positive correla-
tion with ACTH, FST, TST, CORT, IL-1β, TNF-α, LPS,
and depressive-like behavior.
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Page 8 of 26
Wangetal. BMC Biology (2023) 21:242
Intestinal growth retardation andintestinal
microbiological disorder in18‑day‑old suckling mice
withLF feeding deciency duringlactation
Our experiments showed that LF intake during lactation
significantly affected depressive-like behavior in adult
mice, which was induced by differences in body develop-
ment caused by LF intake during lactation. erefore, we
used 18-day-old mice with almost no feed intake for anal-
ysis. e body weight of 18-day-old suckling mice with-
out LF feeding during lactation did not differ from that of
control mice (Fig.3A) but the intestinal index and density
of the small intestine (F = 0.145, df = 16, p = 0.000 < 0.05;
F = 1.282, df = 15, p = 0.000 < 0.05) and colon (F = 1.39,
df = 16, p = 0.031 < 0.05; F = 0.025, df = 15, p = 0.002 < 0.05)
decreased significantly (Fig.3B, C). Crypt depth did not
differ significantly between the two groups (Fig.3D). e
number of colonic goblet cells in the ko-wt group was sig-
nificantly lower than that in the control group (F = 0.012,
df = 13, p = 0.000 < 0.05) (Fig. 3E). ere was no signifi-
cant difference in the number of Paneth cells per crypt in
colon between two group (Additional file: Fig. S1).
To further explore the effect of LF feeding defi-
ciency on intestinal development during lactation, we
performed genome-wide transcriptional profiling of
the small intestine of the two groups of mice for RNA
sequencing. PCoA showed that the transcriptomes of
ko-wt and wt-wt mice were clearly separated (Fig.3F).
Transcriptome sequencing showed that 143 genes were
upregulated and 72 genes downregulated in the small
intestine of the ko-wt mice compared to those of the
wt-wt mice (fold-change > 1, P adjust < 0.05, Fig. 3G).
ese DEGs were subjected to pathway analysis using
the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database, which showed that upregulated mRNAs were
mainly involved in infectious, immune, and neurode-
generative diseases, and were enriched in the nervous,
immune, and endocrine systems (Fig.3H). We explored
immune-related genes in the small intestine using an
RNA-seq transcriptomic profiling approach, as shown
in Fig.3I; genes encoding α-defensin, such as Defa23,
Defa36, Defa3, Defa17, Defa38, and Defa39, were sig-
nificantly decreased in the ko-wt group. e RNA-
Seq results showed no difference in the expression of
the tight junction proteins zo-1 and occludin between
the two groups. However, the expression of the gene
encoding the actin protein (Gm12715) was significantly
decreased (Fig.3J); this gene is related to the assembly
of tight junctions.
To determine the effect of LF deficiency during lac-
tation on intestinal microorganisms, we analysed the
gut microbiota composition by 16S rRNA gene ampli-
con sequencing of the caecal contents of 18-day-old
mice. Alpha diversity measures (Shannon and Chao
diversity indices), calculated for each group, showed
that the Chao diversity index of the ko-wt group did
not differ from that of the wt-wt group; however, the
Shannon index was significantly higher than that
of the control group (Fig. 3K). These results indi-
cate that the intestinal microbial alpha diversity was
increased in 18-day-old mice not fed LF. PCoA of the
beta-diversity comparison revealed significant sepa-
ration between the two groups of microbial com-
munities (Fig. 3L). At the phylum level, Firmicutes,
Bacteroidetes, and Desulfobacterota accounted for
the majority of the bacteria (Fig.3M), with no differ-
ences between the two groups (Fig.3N). At the genus
level, the LEfSe results revealed a greater abundance
of Muribaculum, Oscillibacter, Bilophila, Colidextri-
bacter, Harryflintia, Bifidobacterium, Odoribacter,
UCG-009, Lachnospiraceae_UCG-006, Eubacterium_
xylanophilum_group, Ruminococcus, and some unclas-
sified microbiotas; there was a lower abundance of
Blautia, Desulfovibrio, Parabacteroides, Romboutsia,
Eubacterium_brachy_group, and Turicibacter in the
ko-wt group (Fig.3O).
(See figure on next page.)
Fig. 3 Effects of LF feeding deficiency during lactation on intestinal development and intestinal microorganisms in 18-day-old suckling mice.
A Body weight of 18-day-old mice. n = 8. B Small intestine and colon index of 18-day-old mice. n = 8–9. C Intestinal density of small intestinal
and colon of 18-day-old mice. n = 8–9. D Crypt depth in the colon of 18-day-old mice and representative images of H.E-stained colonic sections,
200 × , scale bar = 100 μm. n = 6–8. E Counts of the goblet cells in the colon of 18-day-old mice and representative images of AB-stained colonic
sections, 200 × , scale bar = 100 μm. n = 6–9. (A-E, data are presented as mean ± standard error. Student’s t-test was employed for comparisons
between two groups; *P < 0.05; **P < 0.01; ***P < 0.001). F Principal components analysis (PCA) of small intestinal RNAseq. G Differential expression
genes (DEGs) between wt-wt group and ko-wt group mice. (P adjust < 0.05, fold change ≥ 2). H KEGG functional annotation analysis of differential
genes of wt-wt and ko-wt group mice. I The clustering heatmap of immune related DEGs between wt-wt group and ko-wt group. J Tight junction
genes expression in RNAseq. F-J, n = 3. K Alpha diversity index difference analysis of the wt-wt and ko-wt group: Shannon index, Chao index.
Wilcoxon rank-sum test. L Principal coordinates analysis (PCoA) of bacterial beta-diversity based on the Bray–Curtis dissimilarity index and evaluated
using ANOSIM. Each symbol represents a single sample of feces. M Composition of microbial community at phylum level of 18-day-old sucking
mice. N Student’s t-test on Phylum level of 18-day-old sucking mice. O Linear discriminant analysis (LDA > 3) scores derived from LEfSe analysis
at genus level of 18-day-old sucking mice, K–O, n = 8
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Wangetal. BMC Biology (2023) 21:242
Fig. 3 (See legend on previous page.)
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Page 10 of 26
Wangetal. BMC Biology (2023) 21:242
LF feeding deciency duringlactation induced
hippocampal growth retardation
Considering that depression is a mental illness, it is nec-
essary to explore the effects of LF deficiency during lac-
tation on the hippocampus. Figure4A shows that there
was no significant difference in the brain index between
ko-wt and wt-wt 18-day-old mice. e CA1 region of the
hippocampus is involved in cognitive processes, learning,
and memory [25]. e hippocampal dentate gyrus (DG)
is a key target region for both antidepressant effects and
stress-related processes [26]. In the wt-wt group, pyrami-
dal cells in the CA1 region were arranged neatly in dense
layers and had regular and clear structures. No significant
difference was observed between the ko-wt and wt-wt
groups in cell morphology in the DG region (Fig.4B).
RNA-seq of the hippocampi of 18-day-old mice was
performed. PCA showed that the transcriptomes of the
two groups were clearly distinct (Fig.4C). Transcriptome
sequencing results showed that 199 genes were upregu-
lated and 297 genes downregulated in the hippocampi
of the ko-wt mice compared to those of the wt-wt mice
(fold-change > 1, P < 0.05, Fig. 4D). e KEGG path-
way annotation results showed that the downregulated
mRNAs mainly participated in signalling molecules and
interactions, and signal transduction, and were enriched
in the immune system (Fig. 4E). Figure4F shows that
the expression of genes involved in innate immunity,
such as Lrg1, Gbp4 [27], Gbp10 [27], and Stx11 [28], was
decreased. Genes involved in adaptive immunity, such
as Ccl21a [29], Cd247 [30], Cd28 [31], and IL25 [32],
showed increased expression. e gene expression of Pigr
[33], Ltf, Lcn2 [34], S100a9 [35], and Ccl28 [36], which are
involved in both innate and adaptive immune responses,
decreased. Some genes involved in the development of
the nervous system in the ko-wt group were significantly
different from those of the wt-wt group. e gene expres-
sion of BDNF, Cdh3, Ccn5, and Folr1 decreased, and that
of Prdm12 and Pcdha8 increased (Fig.4G). ese differ-
ences may lead to the delayed development of the nerv-
ous system. Figure4H shows that the expression of genes
related to neuronal signal transduction (Trpv4, Gabra6,
Kcne2, and Kcnj13) decreased in the ko-wt group.
To explore whether the influence of LF-deficient feed-
ing during lactation on the hippocampus extends into
adulthood, we assayed differential gene expression in
the hippocampus of adult mice. Figure 4I shows that
there was no significant difference between KO-WT
mice (mKO-WT, fKO-WT) and WT-WT mice (mWT-
WT, fWT-WT) in immune-related differential expres-
sion genes (DEGs), but gene expression in female mice
was significantly higher than that in male mice. Fig-
ure4J shows that the expression of BDNF, Ccn5, Cdn3,
Prdm12 and Pcdha8 in KO-WT mice (mKO-WT, fKO-
WT) did not differ from that in WT-WT mice (mWT-
WT, fWT-WT). However, the effect of sex on these
genes is significant. e expression of BDNF (F = 0.188,
df = 14, p = 0.021 < 0.05), Cdn3 (F = 3.116, df = 14,
p = 0.000 < 0.05), Prdm12 (p = 0.004 < 0.05) and Pcdha8
(p = 0.004 < 0.05) in fWT-WT group was significantly
higher than mWT-WT group. e expression of BDNF
(F = 0.84, df = 14, p = 0.013 < 0.05), Ccn5 (F = 7.919,
df = 5.673, p = 0.012 < 0.05), Cdn3 (F = 56.901, df = 7.29,
p = 0.018 < 0.05), Prdm12 (F = 61.522, df = 5.006,
p = 0.02 < 0.05) and Pcdha8 (F = 34.899, df = 5.574,
p = 0.006) in fWT-WT group was significantly higher
than mWT-WT group. Figure4K shows that the expres-
sion of Trpv4, Gabra6, Kcne2, and Kcnj13 in KO-WT
mice (mKO-WT, fKO-WT) did not differ from that in
WT-WT mice (mWT-WT, fWT-WT), and the expres-
sion of Gabra6, Kcne2, and Kcnj13 was significantly
higher in female mice than in male mice. When the mice
reached adulthood, the difference induced by LF feeding
deficiency in the hippocampus was disappeared.
Lactation LF feeding‑decient adult mice still
showed some dierences inintestinal andintestinal
microorganisms compared withthecontrol group
To explore whether the effects of LF feeding defi-
ciency during lactation on intestinal and intestinal
Fig. 4 LF feeding deficiency during lactation affects the development of hippocampus in 18-day old and adult mice. A The brain index
of 18-day-old mice in wt-wt and ko-wt group. B The representative H.E staining of hippocampus (50 × , scale bar = 500 μm), hippocampus CA1
and DG region (200 × , scale bar = 100 μm) in 18-day-old mice. A-B, n = 8–9. (C)Principal components analysis (PCA) of hippocampus RNAseq
in 18-day-old mice. (D)Differential gene expression between wt-wt group and ko-wt group mice (18-day-old), (P value < 0.05, fold change ≥ 2).
E KEGG functional annotation analysis of differential genes in 18-day-old mice. F, G The clustering heatmap of immune, nervous development
response-related DEGs between wt-wt group and ko-wt group (18-day-old). H Expression of nervous signal transduction related genes in 18-day
old mice RNAseq. I Expression of immune related genes in the hippocampus of adult mice. J Expression of nervous development related genes
in the hippocampus of adult mice. K Expression of nervous signal transduction related genes in the hippocampus of adult mice. C-H, n = 3.
(I)-(K), n = 7–8, Two-way ANOVA with multiple comparisons and two-tailed t test for normally distributed data, two-tailed Mann–Whitney test
for non-normally distributed data, *P < 0.05; **P < 0.01; ***P < 0.001
(See figure on next page.)
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Wangetal. BMC Biology (2023) 21:242
Fig. 4 (See legend on previous page.)
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Page 12 of 26
Wangetal. BMC Biology (2023) 21:242
microorganisms continue into adulthood, mice were fed
normally until adulthood after weaning.
Intestinal development was evaluated using four groups
(male: mWT-WT, mKO-WT; female: fWT-WT, fKO-
WT) of 9-week-old mice which was different from that of
18-day-old mice. ere were no significant differences in
the body weight (Fig.5A), small intestinal index (Fig.5B),
small intestinal density (Fig. 5C) and colon length
(Fig. 5D) between WT-WT and KO-WT mice either
male or female. However, the body weight (fWT-WT vs
mWT-WT: F = 0.012, df = 14, p = 0.002 < 0.05; fKO-WT
vs mKO-WT: Mann–Whitney test, p = 0.005 < 0.05) and
colon length (fWT-WT vs mWT-WT: F = 2.543, df = 14,
p = 0.027 < 0.05; fKO-WT vs mKO-WT: F = 0.206, df = 15,
p = 0.021 < 0.05) of female mice were significantly lighter
and shorter than male mice (Fig.5A, D), the small intes-
tinal index of female mice were significantly higher than
male mice (fWT-WT vs mWT-WT: F = 0.012, df = 14,
p = 0.002 < 0.05; fKO-WT vs mKO-WT: F = 1.521, df = 13,
p = 0.002 < 0.05) (Fig.5B). e results of two-way ANOVA
in Fig.5C showed that LF intake situation had a certain
effect on small intestine density, but the difference was
not significant enough (F (1,27) = 3.563, p = 0.07 > 0.05).
The maltase/lactase values of the ileum in the mKO-
WT and fKO-WT mice were significantly lower than
those in the mWT-WT and fWT-WT mice, respec-
tively (Fig. 5E (F = 0.02, df = 10, p = 0.049 < 0.05), 5F
(Mann–Whitney test, p = 0.028 < 0.05)). As shown in
Fig.5G, there was no difference between KO-WT and
WT-WT mice in the crypt depth (mKO-WT vs. mWT-
WT; fKO-WT vs. fWT-WT). Figure5H shows that the
expression of Zo-1 and Occludin in KO-WT mice has
no significant difference with WT-WT mice (mKO-
WT vs. mWT-WT; fKO-WT vs. fWT-WT). Zo-1 and
Occludin expression was significantly lower in fKO-
WT mice than in mKO-WT mice (F = 0.029, df = 14,
p = 0.015 < 0.05; F = 7.404, df = 5.234, p = 0.006 < 0.05).
Thus, the influence of LF feeding deficiency on the
intestines of normal adult mice was weakened; how-
ever, female mice that consumed LF-free milk during
lactation may have a greater risk of the intestinal bar-
rier dysfunction compared with male mice in adult-
hood. In addition, the ileal maturity of LF-deficient
mice was lower than that of LF-drinking mice, and we
have previously shown that the maturity of the three
segments of the small intestine of 18-day old mice
drinking LF-free milk was lower than that of normal
mice [17].
To examine the effects of different feeding methods
during lactation on the composition of intestinal micro-
flora in healthy adult mice (male: mWT-WT, mKO-WT;
female: fWT-WT, fKO-WT), the cecal content was inves-
tigated using 16S rRNA gene sequencing. Alpha-diversity
analysis revealed no difference in the Shannon and Chao
indices between WT-WT and KO-WT mice (mKO-WT
vs mWT-WT; fKO-WT vs fWT-WT, Fig.5I). PCoA of the
beta-diversity comparison revealed separation between
the mKO-WT and mWT-WT groups, but the difference
was not significant (P = 0.053). PCoA showed no separa-
tion among fKO-WT and fWT-WT groups on OUT level
(Fig.5J). ese results suggest that there was no difference
in alpha and beta diversity between lactating LF-deficient
adult mice and normal mice. e phylum level was domi-
nated by Firmicutes, Bacteroidetes, Desulfobacterota,
Actinobacteria, and Patescibacteria (Fig.5K). Patescibac-
teria was significantly higher in the mWT-WT group, and
there was no difference between the fKO-WT and fWT-
WT groups at the phylum level (Fig. 5L). At the genus
level, the LEfSe results demonstrated a greater abundance
of Lactobacillus, Lysinibacillus, Ileibacterium, Bifidobac-
terium, and Lachnospiraceae_UCG-001, whereas there
was a lower abundance of Kurthia, Enterorhabdus, Can-
didatus, Saccharimonas, Eubacteriumxylanophilum_
group, Monoglobus, and ASF356 in the mKO-WT group.
Dubosiella, Lactococcus, Sporosarcina, Pseudograciliba-
cillus, and Eubacteriumbrachygroup were significantly
higher in the fKO-WT group; Staphylococcus and Eubac-
terium_nodatum_group were significantly higher in the
fWT-WT group (Fig.5M).
(See figure on next page.)
Fig. 5 Effects of LF feeding deficiency during lactation on intestinal development and intestinal microorganisms in adult mice. A Body weight
of four groups of adult mice. n = 8. B Small intestinal index of four groups of adult mice. n = 8. C Small intestinal density of four groups of adult mice.
n = 8. D Colon length of four groups of adult mice. n = 8. E The ratio of maltase/ lactase in the duodenum, jejunum and ileum of male mice. n = 6–8.
F The ratio of maltase/ lactase in the duodenum, jejunum and ileum of female mice. n = 5–8. G H.E stain colonic tissue sections and crypt depth
of four groups of adult mice, 200 × , scale bar = 100 μm. n = 6–8. H Zo-1, Occludin mRNA expression in the colon of four groups of adult mice. n = 6–8.
A-H Two-way ANOVA with multiple comparisons and two-tailed t test for normally distributed data, two-tailed Mann–Whitney test for non-normally
distributed data, *P < 0.05; **P < 0.01; ***P < 0.001. I The alpha diversity analysis of four groups of adult mice, including Shannon diversity and Chao
diversity, Wilcoxon rank-sum test. J PCoA analysis results of male and female adult mice and evaluated using ANOSIM. K The composition
of microbial community at phylum level of four groups of adult mice. L Student’s t-test on Phylum level between WT-WT and KO-WT of male
and female mice. M Linear discriminant analysis (LDA > 3) scores derived from LEfSe analysis at genus level of male and female mice. I-M n = 7–8
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Fig. 5 (See legend on previous page.)
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Page 14 of 26
Wangetal. BMC Biology (2023) 21:242
Lactation LF feeding‑decient adult mice showed severe
intestinal injury andintestinal microbial disorder based
onDSS‑induced colitis
Following LF deficiency during lactation, the intestinal
development and composition of intestinal microorgan-
isms in 18-day-old mice significantly differed from those
of normal mice; however, these differences were covered
and became not obvious after 9 weeks of normal feed-
ing under physiological conditions. To examine whether
the two types of mice exhibited the same pathological
reaction, a 1-week acute DSS model was established in
8-week-old mice.
In the DSS colitis model mice, the body weight of
mDKO-WT mice was significantly lower than mDWT-
WT mice (F (1,15) = 7.526, P = 0.015 < 0.05) (Fig. 6A).
In female mice, the body weights of fDKO-WT mice
were consistently lower than those of fDWT-WT mice,
but the difference was not significant (F (1,15) = 1.695,
P = 0.213 > 0.05)), and the body weights of fDWT-WT
and fDKO-WT mice decreased to 77.3% and 74.6%,
respectively (Fig.6B). The disease activity index (DAI)
score is an effective indicator of colon inflammation
and damage. The DAI score of the mDKO-WT group
was significantly higher than that of the mDWT-WT
group (F (1,15) = 5.873, p = 0.028 < 0.05)) (Fig. 6C).
The DAI score of the fDKO-WT group was also sig-
nificantly higher than that of the fDWT-WT group
(F (1,15) = 5.668, p = 0.031 < 0.05)), and the difference
was significant at d 6 and 7 (t test, F = 1.582, df = 15,
p = 0.034; F = 3.737, df = 15, p = 0.05) (Fig. 6D). The
survival rate of the mDKO-WT group was lower than
that of the mDWT-WT group, and there were no
deaths in the two female mouse groups (Fig.6E). There
was no difference in colon length between the two
groups of male mice but the colon length of fDKO-
WT mice was significantly shorter than that of fDWT-
WT mice (F = 0.975, df = 14, p = 0.046 < 0.05) (Fig.6F).
Hematoxylin and eosin (H.E) staining of the colonic
tissue sections showed that DSS-treated colons had
great histological damage (cellular infiltration, goblet
cell depletion, and damage to the crypt architecture),
but the difference between KO-WT and WT-WT mice
was unclear. The crypt depth of fDKO-WT mice was
lower than that of fDWT-WT mice, but the difference
was not significant (Fig.6G). Figures6H–K shows that
the expression of IL-1β (p = 0.028 < 0.05) and IL-10
(F = 0.029, df = 14, p = 0.041 < 0.05) in the colon in the
fDKO-WT group was significantly higher than that in
the fDWT-WT group. Thus, LF-deficient feeding of
mice during lactation led to more severe DSS-induced
weight loss (female), an increased DAI score (male and
female), decreased survival (male mice), and colon
shortening (female mice).
Ulcerative colitis is typically associated with dysbiosis
of the gut microbiota. Alpha-diversity analysis showed
that the Shannon index of the fDKO-WT group was sig-
nificantly lower than that of the fDWT-WT group. e
Chao index at the OTU level of fDKO-WT mice was sig-
nificantly lower than that of fDWT-WT mice (Fig.6L).
ese results suggest that alpha diversity was lower in
LF-deficient female mice compared to those with nor-
mal milk; no difference was observed in male mice after
acute DSS injury. PCoA revealed significant separation
between the mDKO-WT and mDWT-WT groups at
the OTU level, and the microflora of female LF-deficient
DSS mice (fDKO-WT) differed significantly from that
of the fDWT-WT group (Fig.6M). At the phylum level,
Bacteroidetes, Firmicutes, Proteobacteria, Campilobac-
terota, Desulfobacterota, Actinobacteriota, and Defer-
ribacterota accounted for the majority of the bacteria
(Fig.6N). e relative abundance of Firmicutes was sig-
nificantly higher in the mDKO-WT group, and of Pro-
teobacteria was significantly higher in the mDWT-WT
group. In the fDKO-WT group, the relative abundance
of Campilobacterota was significantly higher, whereas
that of Desulfobacterota and Cyanobacteria was sig-
nificantly lower (Fig.6O). At the genus level, the rela-
tive abundance of Lachnospiraceae_NK4A136 group,
Ruminococcus_torques_group, Odoribacter, Romboutsia,
Marvinbryantia, and Family_XIII_AD3011_group was
Fig. 6 Lactation LF feeding deficiency mice show more severe intestinal damage and gut microbial disorders in DSS model. A Changes in body
weight of male mice in DSS model within 7 days. B Changes in body weight of female mice in DSS model within 7 days. C DAI scores of male mice
during 7 days of DSS-induced colitis experiments. D DAI scores of female mice during 7 days of DSS-induced colitis experiments. A-D Repeated
measures two-way ANOVA test. n = 8. E Survival rate of mice in DSS model. n = 8. F Colon length of mice in DSS model. n = 7–8. G H.E stain colonic
tissue sections and crypt depth of DSS model mice, 200 × , scale bar = 100 μm. n = 5–8. H–K The level of TNF-α, IL-1β, IL-6, IL-10 in colon tissue
of DSS model mice. n = 5–8. Data are presented as mean ± standard error. F-K Two-way ANOVA with multiple comparisons and two-tailed t test
for normally distributed data, two-tailed Mann–Whitney test for non-normally distributed data, *P < 0.05; **P < 0.01; ***P < 0.001. L Alpha diversity
index difference analysis in DSS model: Shannon index, Chao index. Wilcoxon rank-sum test. M Principal coordinates analysis (PCoA) of bacterial
β-diversity based on the Bray–Curtis dissimilarity index in DSS model and evaluated using ANOSIM. N Composition of microbial community
at phylum level in DSS model. O Student’s t-test on Phylum level in DSS model. P Linear discriminant analysis (LDA > 3) scores derived from LEfSe
analysis at genus level. L-P n = 6–8, *P < 0.05; **P < 0.01; ***P < 0.001
(See figure on next page.)
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Fig. 6 (See legend on previous page.)
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Page 16 of 26
Wangetal. BMC Biology (2023) 21:242
higher in the mDKO-WT group than in the mDWT-WT
group (Fig.6P). As shown in Additional file: Fig. S2A, the
abundance of Ruminococcus_torques _group, Odoribac-
ter, Romboutsia, and Family_XIII_AD3011_group was
higher in the mDKO-WT group than in the mKO-WT
group, indicating that mDKO-WT mice experienced a
more substantial increase in these genera. e relative
abundances of Leucobacter and Parvibacter were lower
in the mDKO-WT group than in the mDWT-WT group
(Fig. 6P). In female mice, the relative abundances of
pathogenic bacteria such as Escherichia-Shigella, Entero-
coccus, Clostridium_sensu_stricto_1, Erysipelatoclostrid-
ium, Colidextribacter, and Akkermansia were higher
in the fDKO-WT group than in the fDWT-WT group
(Fig.6P). e relative abundances of beneficial genera,
such as Alloprevotella, Desulfovibrio, and many unclassi-
fied bacteria on genus level were lower in the fDKO-WT
group than in the fDWT-WT group (Fig.6P). Similarly,
the abundance of Escherichia-Shigella, Enterococcus,
Erysipelatoclostridium, Colidextribacter and Akkerman-
sia were increased and Desulfovibrio was decreased in
the DSS model (Additional file: Fig. S2B). fDKO-WT
mice showed a more significant increase in Escherichia-
Shigella, Enterococcus, Erysipelatoclostridium, Coli-
dextribacter and Akkermansia and a serious decline in
Desulfovibrio. LF feeding-deficient mice clearly showed
more severe microbial disorders after DSS processing. In
addition, the effects of DSS enteritis on intestinal micro-
biome of WT mice at genus level are shown in Addi-
tional file: Fig. S2C, D.
Discussion
LF is an important functional protein in breast milk
that has profound effects on the growth and develop-
ment of infants and young children. Formulas are the
main breast milk substitute for infants who cannot
breastfeed. However, LF has not been widely added
to formulas, commonly leading to LF feeding defi-
ciency during lactation. LF has a protective effect on
newborns, particularly premature infants. However,
the effects of lactation LF feeding deficiency on adult
health remain unclear. Depression is the most common
mental health problem in adults and a leading risk fac-
tor for physical disability. Here, we explored the influ-
ence of LF feeding deficiency during lactation on the
incidence of depressive-like behavior in adult mice and
related mechanisms. Unexpectedly, we found that lac-
tating LF feeding-deficient mice showed more severe
depressive behaviour after induction of CUMS depres-
sion in adulthood (Fig.1).
LF feeding deciency duringlactation impairs intestinal
barrier function andimmunomodulatory function
Dysfunction of the microbiota–gut–brain axis is the main
pathological basis of depression and may directly influ-
ence and cause psychiatric disorders [37]. Our results
showed that the expression level of tight junction Zo-1
was significantly affected by lactoferrin intake during
lactation after depression in adulthood, and LF feeding
deficiency male mice showed more severe inflammation
in the colon after adult CUMS. Based on these observa-
tions in the intestines, we further explored the effects of
LF deficiency during lactation on intestinal development
in mice in different growth periods. e small intestine
of 18-day-old LF-deficient mice showed decreased intes-
tinal barrier function and innate immunity. ZO fam-
ily proteins bind directly to F-actin and barrier-forming
claudins are necessary for the assembly of tight junc-
tions [38]. Actin cytoskeletal can also regulate epithelial
permeability, and knockdown of myosin IIA decreases
epithelial permeability in several intestinal epithelial cell
lines [39]. erefore, the low expression of actin protein
in the ko-wt group (18-day-old) may have weakened
intestinal barrier function. e DEGs related to immunity
were mainly enriched in the NOD-like receptor signaling
pathway, which plays a key role in pathogen identification
and innate immune response (Additional file: Fig. S3).
Paneth cells in the crypts of the small intestine secrete
microbicidal α-defensins as components of the enteric
innate immunity. Moreover, dysregulation of α-defensins
has been observed under pathogenic conditions, such as
in IBD [40]. e lack of LF during lactation reduces the
secretion of α-defensin in the small intestine of suckling
mice, which may reduce innate immunity and increase
the incidence of inflammatory intestinal disease. When
the mice reached adulthood, the difference between the
KO-WT and WT-WT groups (mKO-WT vs. mWT-WT,
fKO-WT vs. fWT-WT) in the intestinal physiological
structure of healthy mice was decreased. However, after
DSS enteritis treatment, KO-WT mice showed more
serious damage and inflammation, particularly female
mice. us, loss of LF feeding during lactation hinders
the development of intestinal physiological structure,
barrier function, and innate immune function in suck-
ing mice, and the gap in intestinal physiological structure
gradually narrows with increasing age but a functional
gap remains, particularly during negative stress. ere-
fore, LF feeding deficiency during lactation impairs intes-
tinal barrier and immunomodulatory functions, causing
serious damage to the intestinal barrier and inducing sys-
temic inflammation, under the stimulation of depression
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 17 of 26
Wangetal. BMC Biology (2023) 21:242
modelling, which in turn leads to neuroinflammation and
depressive-like behavior.
Eect ofLF feeding deciency duringlactation ongut
microbiota frominfancy toadulthood
e gut microbiota plays a prominent role in gut-brain
interactions. e results of gut microbiota are summa-
rized in Additional file: Table. S3. LF feeding deficiency
during lactation affects the composition of intesti-
nal microorganisms in mice, and this effect continues
into adulthood. e overall microbial composition of
18-day-old ko-wt mice deviated from that of the con-
trol group. is separation disappeared in 9-week-old
healthy mice, possibly because they had the same die-
tary conditions. However, this separation reappeared
under stimulation of depression and DSS. e effects
of LF deficiency during lactation on gut microbes are
persistent. Alpha diversity is often used as a proxy
for community stability and functioning. e effect
of depression on intestinal microbial alpha-diversity
is inconclusive [14]. In our study, the microbiota of
LF feeding-deficient mice showed lower alpha diver-
sity after CUMS. Similarly, LF feeding-deficient mice
showed lower alpha diversity under the influence of
DSS enteritis. Alpha diversity was increased in 18-day-
old ko-wt mice, possibly because of the antibacterial
activity of LF. In contrast, another study showed that
LF intervention in early life increased the diversity of
the caecal microbiota [20].
LF had no significant influence on the abundance of
phylum Firmicutes and Bacteroidota in 18-day-old mice
and CUMS model mice. Similar to the results of existing
research [22], DSS treatment reduces the level of Firmi-
cutes and increases the level of Bacteroidetes (Additional
file: Fig. S4). It is worth noting that Desulfobacterota is
enriched in female ko-wt mice in the depression model,
but enriched in female wt-wt mice in the DSS model
(Additional file: Table. S3). Study found that Desulfobac-
terota was increased in the depression group [41], the
LF feeding deficiency during lactation aggravated the
increase of Desulfobacterota caused by depression. Other
study showed that Desulfobacterota was increased in
the DSS group [42]. However, our study found that Des-
ulfobacterota was decreased in the DSS group, and this
phenomenon occurs only in KO-WT mice (mKO-WT
vs mDKO-WT, fKO-WT vs fDKO-WT) (Additional file:
Fig. S4). e effect of LF deficiency during lactation at
the genus level of intestinal microorganisms was com-
plex and diverse. In the intestine of 18-day-old suckling
mice, many microorganisms enriched in the ko-wt group
were related to inflammation. For example, many studies
have reported that Oscillibacter is increased in depres-
sion [43] and is positively correlated with obesity and
increased permeability of the mouse colon [44]. Similarly,
the increasing relative abundance of clusters in the ko-wt
group, including Bilophila, Colidextribacter, Harryflintia
[44], Odoribacter [45], Lachnospiraceae_UCG-006 [46],
and Ruminococcus [47], has also been reported as posi-
tively associated with inflammation due to progressive
obesity or type 2 diabetes mellitus. In our study, Blautia,
Desulfovibrio, Parabacteroides, Romboutsia, Eubacte-
rium_brachy_group, and Turicibacter were enriched in
wt-wt group. Blautia may function as a probiotic in the
host and has gained attention because of its ability to alle-
viate inflammatory diseases and metabolic diseases, as
well as because of its antibacterial activity against specific
microorganisms [48]. However, we found that Blautia
was positively related to depression. Parabacteroides and
Turicibacter resulted in increased formation of the fae-
cal SCFAs propionate and butyrate [49]. In our RNA-seq
results, LF feeding deficiency during lactation decreased
the expression of α-defensin in the small intestine of
18-day-old sucking mice (Fig.3I), which may contribute
to host-microbe dysbiosis and enhance inflammatory
responsiveness associated with the pathogenesis of IBD
[50]. erefore, the disorder of intestinal microorganisms
in 18-day-old ko-wt mice may be directly related to the
antibacterial effect of LF. Additionally, it may be indi-
rectly related to the effect of LF on the development and
gene expression of the intestine.
When the mice reached adulthood, the abundance
of beneficial bacteria in LF-feeding deficient mice was
lower than in LF-fed mice. Among the intestinal micro-
organisms of 9-week-old healthy mice, the abundance
of Kurthia, Enterorhabdus, Candidatus_Sacchari-
monas, Monoglobus, ASF356, and Eubacterium_xylano-
philum_group was lower in the mKO-WT group and
decreased in the DSS model compared to in healthy
mice (Additional file: Fig. S2A). A previous study showed
that the abundance of Enterorhabdus spp. decreased
after chronic DSS induction [51]. According to previ-
ous reports, ASF356 exerts probiotic effects [52]. Can-
didatus_Saccharimonas can suppress the production
of TNF-α in macrophages, suggesting its potential for
immune suppression [53]. Eubacterium_nodatum_group
enriched in fWT-WT is SCFAs-producing bacteria [54].
ese results suggest that the adverse effect of LF feed-
ing deficiency during lactation on the composition of
intestinal microorganisms continued into adulthood.
is effect was also observed in the DSS model. Among
intestinal microorganisms in the DSS model, more
harmful bacteria were enriched in LF-deficient mice
(Fig. 6P). Ruminococcus_torques_group and Family_
XIII_AD3011_group were enriched in the mDKO-WT
group; these genera are significantly positively core-
lated with inflammation [24, 55]. Escherichia-Shigella,
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Page 18 of 26
Wangetal. BMC Biology (2023) 21:242
Enterococcus, Clostridium_sensu_stricto_1, Erysipela-
toclostridium, Colidextribacter and Akkermansia were
increased in the fDKO-WT group compared to in the
fDWT-WT group. Escherichia-Shigella and Enterococ-
cus are potential pathogens [56, 57], and studies found
that Clostridium_sensu_stricto_1, Erysipelatoclostridium
and Colidextribacter were increased in high-fat diet-fed
mice [44, 45, 58]. Similar to our results, Xu etal. found
that the relative abundance of Akkermansia was mark-
edly increased after chronic DSS induction [51]. Con-
sidering the close relationship between the intestinal
microbial composition and depression, LF deficiency
during lactation is likely to affect intestinal microor-
ganisms in the CUMS model. In the CUMS model,
Eubacterium_xylanophilum_group was enriched in the
ko-wtM group and was positively correlated with FST
(Fig.2L), which was positively correlated with the sever-
ity of depression. Study found that Eubacterium_xylano-
philum_group was the characteristic genera of valproic
acid-induced rat autism model [59]. To the best of our
knowledge, our results are the firsttoreportthatEubac-
terium_xylanophilum_group might be associated with
depression. We also found that Alistipes was positively
correlated with the OFT, indicating that Alistipes is
negatively correlated with the severity of depressive
symptoms. e abundance of Alistipes is decreased in
patients with IBD, which is characterised by overexpres-
sion of indoleamine 2,3-dioxygenase (IDO) in the colon
[60]. IDO can reduce 5-hydroxytryptamine (5-HT) lev-
els by promoting the metabolism of tryptophan, a pre-
cursor of 5-HT synthesis, leading to depression. In our
study, the abundance of Lactobacillus was decreased in
the ko-wtM group but increased in the ko-wtF group
(Fig.2K). Studies of the role of Lactobacillus in depres-
sion have reported conflicting results. Mice with chronic
restraint stress-induced depressive-like behaviorshowed
a significant increase in Lactobacillus [61], whereas a low
abundance of Lactobacillus was found in people with
depression [62]. In addition, this difference may be sex-
dependent, and Lactobacillus was shown to be inversely
associated with depression scores among males but was
positively associated with anxiety scores among females
[63], which is similar to our results. Bifidobacterium was
enriched in the ko-wtF group, and is present at high
levels in major depressive disorder/depression groups
in many studies [14]. Interestingly, specific strains of
Bifidobacterium are commonly considered as anti-
inflammatory and used as probiotics. However, a higher
abundance of Bifidobacterium has been associated with
IBD, indicating that specific strains have inflammatory
potential [64]. However, we found that Bifidobacterium
was negatively corelated with depressive-like behav-
ior (Fig.2L), based on the negative correlation between
Bifidobacterium and depressive-like behavior in male
mice (Additional file: Fig. S5), and the correlation was
greater than that in female mice, thus affecting the over-
all correlation. Bifidobacterium was also enriched in the
18-day-old ko-wt (Fig. 3O) group and 9-week mKO-
WT (Fig.5M) group, indicating that colonization of the
neonatal intestine byBifidobacterium can be reduced if
milk is concurrently supplemented with bovine LF [65].
erefore, a lack of LF during lactation may be benefi-
cial for colonization of Bifidobacterium. Many studies
revealed higher levels of Desulfovibrio in patients with
major depressive disorder than in controls [14, 62].
Similarly, Desulfovibrio was enriched in ko-wtF group
(Fig. 2K) and was positively related to the severity of
depressive symptoms in our study (Fig.2L).
e lack of LF during lactation increased the pro-
inflammatory microflora in the intestinal microflora of
18-day-old mice and decreases the number of benefi-
cial bacteria in adult mice, which is disadvantageous for
establishing a healthy intestinal ecosystem. LF feeding
deficiency during lactation may decreased the stability of
intestinal microorganisms, rendering them more vulner-
able to dysregulation during adverse stimuli. However,
further investigation is needed to understand how the
impact of lactoferrin on microbial composition translates
into host health. Future studies should consider con-
ducting metabolomic analysis to shed more light on this
aspect.
LF feeding deciency duringlactation inhibits
hippocampal development frominfancy toadulthood
e hippocampus is a key brain region involved in
cognitive processes, mood regulation, and the patho-
physiology of depression [12]. In our study, the ko-wtF
group exhibited more severe hippocampal inflamma-
tion (Fig.2E) and lower BDNF levels (Fig.1I, 2F). In
the hippocampus of 18-day-old mice, genes involved
in innate immunity were downregulated in the ko-wt
group (Fig. 4F). Family-wide loss-of-function analysis
showed that Gbp10 conferred cell-autonomous immu-
nity to listerial or mycobacterial infection within mac-
rophages and gene-deficient animals [27]. Another
study revealed thatstx11knockdown reduced phago-
cytosis of Escherichia coli in interferon-γ-activated
macrophages [28]. e innate immune response can
eliminate most invading pathogenic microorgan-
isms and participate in immune self-stabilization of
the body. Low expression of innate immune related
genes is unfavorable for pathogen clearance in time,
thus aggravating infection. Furthermore, activation of
innate immunity is indispensable for inducing adaptive
immune responses. Downregulation of innate immune
function may affect the intensity and type of adaptive
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 19 of 26
Wangetal. BMC Biology (2023) 21:242
immunity. In our study, the genes involved in adap-
tive immunity were upregulated in the hippocampus
of LF-deficient mice (Fig. 4F). Ccl21a promotes the
establishment of self-tolerance in T cells in the thymic
medulla [29]. CD28 is the second signal required for
T-cell activation during the immune response [31].
CD247 is part of the T-cell antigen receptor com-
plex, which plays a key role in receptor expression and
signaling leading to optimal effector T-cell functions
[30]. IL-25 can induce and enhance 2 type immune
response [32]. Some genes participate not only in the
innate immunity of the mucous membrane but also in
acquired immunity. Pigr mainly functions to cooperate
with IgA transport, which plays an important role in
mucosal immunity [33]. CCL28 is an anchoring point
that bridge innate and adaptive immunities [36]. e
Ltf gene encodes LF, which has a defensive role in the
body as its levels were found to be profoundly elevated
under pathological conditions such as neurodegenera-
tion and inflammatory disease [66]. Lcn2 can control
neuroinflammation by modulating cytokine produc-
tion and exerts neuroprotective functions in the brain
by suppressing the secretion of pro-inflammatory
cytokines [34]. S100a9 encodes a pro-inflammatory
protein (calgranulin) that has been implicated in mul-
tiple diseases. A previous study has shown that S100a9
prevents hyperinflammatory responses without impair-
ing pathogen defence [35]. e Ltf, Lcn2, and S100a9
genes play important roles in controlling the bal-
ance of the immune response, and low expression of
these genes may induce immune response disorders.
Although our results showed that the disorder of hip-
pocampal immunomodulatory function caused by LF
feeding deficiency in 18-day-old mice disappeared in
adult mice (Fig.4I), another study showed that early
life inflammation causes depressive symptoms in ado-
lescence [67], suggesting that disorder of hippocampal
immunomodulatory function likely led to neuroinflam-
mation in 18-day-old ko-wt mice, which may have been
related to depressive-like behavior in adult mice.
Mice fed an LF-deficient diet during lactation may
be at risk of neurodevelopmental delay. Prdm12 regu-
lates the proliferation and differentiation of neural stem
cells. Overproduction of prdm12 impaired cell prolif-
eration and increased the G1 population [68], and high
expression of Prdm12 in the ko-wt group (18-day-old
mice (Fig.4G) may inhibit neurogenesis. Pcdha8 is a
member of the protocadherin cluster, which regulates
the ability of neurons to migrate and of dendrites to
avoid each other. Many brain diseases, such as autism
spectrum disorders and depression, are caused by
abnormal neuronal migration and connections [69].
Low Pcdha8 expression increases the risk of depression
[69], but the influence of high Pcdha8 expression on
depression is unclear. In our study, the expression
of Pcdha8 was higher in the ko-wt group (18-day-old
mice (Fig. 4G)). e Cdh3 gene encodes cadherin, a
cell adhesion molecule that is important in a variety
of morphogenetic events during neural development
including cell migration, segmentation of the neural
tube, neurite outgrowth, axon targeting, and synapse
formation [70]. A previous study showed that the over-
expression of Wisp2/Ccn5 potently enhanced neurite
outgrowth. e low expression of Cdh3 and Ccn5 in
18-day-old ko-wt mice (Fig.4G) may negatively affect
neural development. We found that BDNF expres-
sion was significantly lower in the 18-day-old ko-wt
mice (Fig.4G); similarly, the ko-wtM and ko-wtF mice
showed lower BDNF serum levels after CUMS (Fig.1I).
LF has been suggested to promote early neurodevelop-
ment and cognition in postnatal piglets by upregulat-
ing the BDNF signalling pathway [71]. erefore, a lack
of LF during lactation reduces the expression of BDNF,
which may increase the risk of depressive-like behav-
ior in adult mice. In the hippocampi of 18-day old mice,
the expression of Folr1 was lower in the ko-wt group
(Fig.4G). Folr1 is a membrane protein that mediates
folic acid uptake by cells. Recent studies have shown
that mouse Folr1 is essential for neural tube closure
and can induce central nervous system axon regenera-
tion mediated by folate [72].
In our study, the expression of genes related to neu-
ronal signal transduction in the 18-day-old ko-wt mice
significantly decreased (Fig. 4H), but did not differ
from that in adult mice (Fig.4K). Gabra6 is a GABA
receptor. GABA is a neurotransmitter involved in the
mechanism of anxiety and depression. A rat experi-
ment suggested that depressive behaviour caused by
maternal-foetal separation is related to decreased
expression of Gabra6 [73], and low GABA trans-
mission has been reported in victims of suicide [74].
TRPV4 is a Ca2+ nonselective cation channel involved
in modulating membrane potentials in neurons. A pre-
vious study showed that downregulation of hippocam-
pal TRPV4 affects depression-like behavior in mice by
regulating neurogenesis [75]. Kcne2 and Kcnj13 regu-
late the return of the membrane potential to resting
potential, and loss of Kcne2 can increase excitability
in neurones [76]. Decreased expression of Kcne2 and
Kcnj13 may lead to continuous excitation of neurons,
resulting in neurotoxicity. In summary, LF feeding
deficiency during lactation decreased the expression
of Gabra6, TRPV4, Kcne2, and Kcnj13 in 18-day-old
mice, which may increase the depressive-like behav-
ior rate of in mice; however, as the mice aged, this
difference disappeared. The choroid plexus is the site
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Page 20 of 26
Wangetal. BMC Biology (2023) 21:242
of cerebrospinal fluid (CSF) production and of the
blood–CSF barrier, and claudin2 is present in the epi-
thelial tight junctions of the choroid plexus forming
the blood–CSF barrier [77]. Our results showed that
Cldn2 expression was decreased in 18-day old ko-wt
mice (Additional file: Fig. S6), which may have led to
increase in permeability of the blood–CSF barrier and
thus more severe neuroinflammation and an increased
risk of depressive-like behavior.
Sex dierences
We evaluated mice of both sexes and observed differ-
ences in some results between male and female mice.
Epidemiological studies have shown that women have
much higher rates of major depression and higher lev-
els of inflammatory markers than men [78]. Similarly,
female mice showed a more severely depressed pheno-
type in our study (Fig. 1I-J). Furthermore, female mice
had lower intestinal tight junction expression and higher
level of inflammatory cytokines (Fig. 2B, Fig. 1M-N),
which induced more severe hippocampal inflammation
compared to in male mice in our CUMS model (Fig.2E).
LF-feeding deficient male mice showed more severe
intestinal inflammatory damage after CUMS (Fig.2). In
contrast, the LF-feeding deficient female mice showed
mild intestinal inflammatory damage after CUMS. is
difference may be related to the anti-inflammatory effect
of oestrogen, study found that oestrogen decreased the
production of macrophage migration inhibitory factor,
which affected the susceptibility to inflammation in the
colon by reducing the TNF-α and IL-1β production in
colitis [23, 79]. However, why did the anti-inflammatory
effects of oestrogen manifest only in the ko-wtF and not
in wt-wtF mice? We speculated that the LF feeding defi-
ciency during lactation may influenced the signal trans-
duction of estrogen. In 18-day-old suckling mice, the
genes encoding Fkbp5 and Hsp70 in the oestrogen sig-
nalling pathway were up regulated in the ko-wt group
(Additional file: Fig. S7). As receptor associated proteins,
Fkbp5 and Hsp70 affect oestrogen receptor nucleocy-
toplasmic shuttling [80]. Oestrogen receptors are abun-
dantly expressed in the intestine and cells of the immune
system [81], and binding of these receptors to oestrogen
induces gene regulation. is phenomenon did not occur
in the DSS enteritis model, possibly because of the differ-
ent pathogeneses of chronic enteritis caused by depres-
sion and acute enteritis established by DSS. Further
studies are needed to investigate the underlying reasons
for this result. In our DSS model, in agreement with the
results of previous studies [79], male mice showed more
severe weight loss and higher mortality than female mice,
which was also related to the anti-inflammatory func-
tion of oestrogen. Sex-specific relationships between
gastrointestinal microbiota and depressive-like behavior
were also observed in this study. e abundance of Lac-
tobacillus and Bifidobacterium was negatively related to
depressive-like behavior among male mice and positively
related to depressive-like behavior among female mice.
Currently, the relationship between Lactobacillus and
Bifidobacterium and depression in individuals of differ-
ent sex is still inconclusive, which is also an important
reason to prevent these two bacteria from being widely
used in the clinical treatment of depression [63].
Conclusions
Our study demonstrated for the first time that LF feeding
deficiency during lactation increases the risk of depres-
sive-like behavior in adults. During negative stress, LF
deficiency causes more serious inflammatory responses,
thus increasing systemic inflammation and neuroin-
flammation, leading to depressive-like behavior. Lack of
LF during lactation disrupts the homeostasis of intesti-
nal microorganisms in mice from infancy to adulthood
and causes more serious flora disorders after negative
stress stimulation. Additionally, lack of LF during lacta-
tion leads to neuroimmune dysfunction and neurode-
velopmental retardation in the hippocampus, which in
turn leads to increased susceptibility to depressive-like
behavior. In short, LF feeding deficiency during lacta-
tion increases the risk of depressive-like behavior in adult
mice by inducing microbiota–gut–brain axis disorders.
Methods
Animal
e heterozygous Ltf gene knockout mice were custom-
ized from Biocytogen Co., Ltd. (Beijing, China). Het-
erozygous breeding produced Ltf gene knockout (KO)
and wild-type littermate (WT) C57BL/6N mice, which
were used for homozygous breeding to produce a larger
number of age-matched WT and KO mice for the cur-
rent experiments. Before the experiment, KO and WT
homozygous breeding produced mice at the same time.
On the second day after birth, all litters born to KO dams
were replaced with pups of the same age born to WT
dams (KO-WT group). Control litters suckling WT dams
were also standardized to pups on the second day after
birth (WT-WT group). Offspring were weaned and sep-
erated from mothers on d21, males and females separated
into cages of 4 mice each cage on 4weeks. e mice in
KO-WT group drank LF-free milk during sucking time,
and these two group mice would be used in subsequent
experiments.
Mice were housed in a controlled environment (12:12h
light: dark cycle; temperature: 22 ± 2 °C; humidity: 60%;
15 − 20 fresh air changes per hour) with free access
to food (fed with sustaining fodder) and water. Cage
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 21 of 26
Wangetal. BMC Biology (2023) 21:242
cleaning was performed once per week. Each cage size
is 25cm × 15cm with four mice per cage. All mice were
treated in accordance with the guidelines of the Institu-
tional Animal Care and Use Committee (SYXK 2020–
0052). All experiments were approved by the Animal
Experimentation Committee of the China Agricultural
University (Beijing, China).
Procedure ofCUMS
16 male 9-week-old mice (8 from WT-WT group and 8
from KO-WT group) and 16 female 9-week-old mice (8
from WT-WT group and 8 from KO-WT group), were
divided into 4 groups: wt-wtM, ko-wtM, wt-wtF, ko-wtF
(Figs.1and2). e CUMS procedure was a variation of
methods described by Agnieszka [82]. e list of stress-
ors, their details, and the day they were applied was given
in Additional file: Table. S4.
Behavioral evaluations
Open field test (OFT).
e test consisted of one 5min trial in a white opaque
50 cm × 50 cm × 50 cm arena. e center zone was
defined as a 25cm × 25cm central square. To begin each
test, a mouse was introduced to the center of the square
and its behavior was captured on video (Samsung, South
Korea) over the course of 5min, the area was cleaned
with 50% ethanol and allowed to dry completely between
each test. e distance that each mouse walked in either
the peripheral or central regions was quantified using
KEmaze software.
Sucrose preference test (SPT)
e sucrose preference test was performed according to
a previous report [83] with minor modifications. Before
the test, mice were acclimatized to sucrose solution. At
first, two bottles of 1% sucrose solution (w/v) were placed
in each cage for 24h to avoid sucrose neophobia. After
adaptation, mice were deprived of liquid and food for
24 h and then given the sucrose preference test. Each
mouse had free access to 2 bottles for 24h: one bottle
with 1% sucrose solution (w/v) and another bottle with
tap water. To avoid position effects, the positions of the
2 bottles were reversed at intervals of 12h. Sucrose con-
sumption and water consumption were measured by
comparing the weights of the bottles before and after the
test. e sucrose preference was calculated as sucrose
preference (%) = (sucrose solution intake)/ (sucrose solu-
tion intake + tap water intake) *100%.
Forced swimming test (FST)
Each mouse was placed in an open cylindrical container
(diameter 10cm, height 30cm) with 23 ± 1℃ water to a
depth of 20cm and allowed to swim for 6 min. Immo-
bility times were measured during the last 4min of the
test. During the test, the behavior of each mouse was
recorded using a video camera (Samsung, South Korea),
and immobility time was measured using SuperFst soft-
ware (KEWBIO Co., Ltd. Nanjing, China).
Tail suspension test (TST)
Mice were habituated to the testing room for 30 min
before the experiments. Animals were attached by their
tails to a shelf with medical adhesive tape (placed approx-
imately 1cm from the distal end of the tail). e last
4min was recorded for immobility time during a 6-min
test. During the test, the behavior of each mouse was
recorded using a video camera (Samsung, South Korea),
and immobility time was measured using SuperTst soft-
ware (KEWBIO Co., Ltd. Nanjing, China).
Dextran Sodium Sulfate (DSS)‑induced colitis
Colitis was induced by the administration of 2.5% w/v
DSS (MW = 36,000–50,000kDa; MP Biomedicals) in the
drinking water for 7 days [12]. e 8-week-old female
mice (fDWT-WT, fDKO-WT) and male mice (mDWT-
WT, mDKO-WT) were weighed daily and the disease
activity index (DAI) score can be assessed (Fig. 6). e
DAI is the combined score of weight loss compared to
initial weight, stool consistency and bleeding [84].
Sample collection andmeasurement
After 4weeks of CUMS, all animals were sacrificed under
anesthesia with 10% chloral hydrate and the colon tis-
sue, hippocampus, serum and cecal content of all animals
were collected and stored at − 80 °C for the following
experiments. Blood was collected from the retro-orbital
sinus under 10% chloral hydrate general anesthesia.
Serum was obtained from the whole blood by centrifu-
gation at 5000rpm for 20min. Collected colon samples
were fixed in 4% paraformaldehyde and embedded in
paraffin. Sections of the colon were stained with H.E. e
inflammation score (0: normal cell pattern, 1: scattered
inflammatory cells in the lamina propria, 2: increased
number of inflammatory cells in the lamina propria,
3: confluence of inflammatory cells extending into the
submucosa, 4: transmural extension of the infiltrating
inflammatory cells) [85].
18-day-old mice (wt-wt, ko-wt) were sacrificed under
anesthesia with isoflurane without fasting. e body
weight, whole brain, small intestinal and colon were
weighted, length of small intestinal and colon were
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 22 of 26
Wangetal. BMC Biology (2023) 21:242
measured. e small intestinal, colon, hippocampus and
cecal content were collected and immediately snapfro-
zen in liquid nitrogen. Collected colon and hippocampus
samples were fixed in 4% paraformaldehyde and embed-
ded in paraffin. Sections of the colon were stained with
H.E and alcian blue (AB) staining by using commercial
kits (Beijing Solarbio Technology Co., Ltd. Beijing, China)
according to the manufacturer’s instructions. Paneth cells
staining by using Lendrum’s fluorescent Peach Red stain-
ing Kit (Xi’an Qiyue biology, Xian, China) Sections of the
hippocampus were stained with H.E. (Figs.3and4A-H).
9 weeks female mice (fWT-WT, fKO-WT) and male
mice (mWT-WT, mKO-WT) (Figs.4I-K and5) were sac-
rificed under anesthesia with 10% chloral hydrate. e
small intestine and colon were removed, and total length
and weight were measured. Intestinal density is indices of
intestinal mucosal growth. Intestinal density = weig ht of
small intestine or colon/length of small intestine or colon.
e small intestinal, half of colon, hippocampus and cecal
content were collected and immediately snap-frozen in
liquid nitrogen. e other half of colon was fixed in 4%
buffered formaldehyde for the histological observation.
e activities of lactase and maltase in each section of
the small intestine were measured using commercial kits
(Nanjing Jiancheng Institute of Bioengineering, Jiangsu,
China) according to manufacturer’s instructions. Sec-
tions of the intestinal were stained with H.E.
e DSS induced colitis mice (mDWT-WT, mDKO-
WT, fDWT-WT, fDKO-WT) (Fig.6) were sacrificed on
day 7 and colon length was measured. Half of colon and
cecal content of all animals were collected and stored
at − 80 °C for following experiments. e other half of
colon was fixed in 4% buffered formaldehyde for the his-
tological observation. Cytokine levels in colonic tissues
were detected by enzyme-linked immunosorbent assay
(ELISA) kit (Cloud-Clone, Wuhan, China) according to
the manufacturer’s instructions.
RNA sequencing
e small intestinal and hippocampus of 18-day-old
mice (wt-wt, ko-wt) were collected and the total RNA
was extracted by using TRIZOL reagent (Invitrogen,
USA). e concentration and purity of the RNA were
detected by Nanodrop2000, RNA integrity was detected
by agarose gel electrophoresis, the RIN value was meas-
ured by Agilent2100. e construction of a single data-
base requires that the total amount of RNA ≥ 1 μg,
concentration ≥ 35 ng/μl, OD260/280 = 1.8 ~ 2.2,
OD260/230 ≥ 2.0.RIN ≥ 6.5, 28S:18S ≥ 1.0, Libraries were
constructed using the Illumina TruseqTM RNA sam-
pleprepKit (Illumina, San Diego, CA, USA) according to
the manufacturer’s instructions.
Sequencing of the libraries was performed on an Illu-
mina HiSeq2000 instrument by Shanghai Majorbio Biop-
harm Biotechnology (Shanghai, China), and individually
assessed for quality using FastQC. To identify DEGs
between two different samples, the expression level of
each transcript was calculated according to the tran-
scripts per million reads (TPM) method. RSEM (http://
dewey lab. biost at. wisc. edu/ rsem/) was used to quantify
gene abundances. Differential expression analysis was
performed using the edgeR with Q value ≤ 0.05. Statisti-
cal significance was assessed using a negative binomial
Wald test, then corrected for multiple hypothesis testing
with the Benjamini–Hochberg method.
Quantitative real‑time PCR
Total RNA was extracted from the tissue using an RNA
extraction kit (Magen, Guangzhou, China) according
to the manufacturer’s instructions. e purity and con-
centration of RNA were determined using a NanoDrop
spectrophotometer (ermo Fisher, Waltham, MA). One
microgram of total RNA was reverse transcribed into
cDNA using a HiFiScript cDNA synthesis kit (NUOWEI-
ZAN, Nanjing, China). RT-PCR was performed using
Rotor-Gene Q (Qiagen, Hilden, Germany) with an Taq
Pro Universal SYBR qPCR Master Mix Kit (NUOWEI-
ZAN, Nanjing, China). RT-PCR amplification conditions
included denaturation at 95°C for 10s, annealing at 55°C
for 30s, and extension for 32s at 72°C. Geometric mean
glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was
used for normalization. All primer pairs were synthesized
by Synbio Technologies (Jiangsu, China), the sequence
was showed in Table1.
16S rRNA gene sequencing andanalysis
Microbial community genomic DNA was extracted from
cecal content samples using the E.Z.N.A.® soil DNA
Kit (Omega Bio-Tek, Norcross, GA, USA) according to
the manufacturer’s instructions. e DNA extract was
checked on 1% agarose gel, and DNA concentration and
purity were determined with NanoDrop 2000 UV–vis
spectrophotometer (ermo Scientific, Wilmington,
USA). e hypervariable region V3-V4 of the bacterial
16S rRNA gene were amplified. e PCR product was
extracted from 2% agarose gel and purified using the
AxyPrep DNA Gel Extraction Kit (Axygen Biosciences,
Union City, CA, USA) according to the manufacturer’s
instructions and quantified using Quantus™ Fluorometer
(Promega, USA).
Purified amplicons were pooled in equimolar and
paired-end sequenced on an Illumina MiSeq PE300 plat-
form/NovaSeq PE250 platform (Illumina, San Diego,
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 23 of 26
Wangetal. BMC Biology (2023) 21:242
USA) according to the standard protocols by Majorbio
Bio-Pharm Technology Co. Ltd. (Shanghai, China).
e raw 16S rRNA gene sequencing reads were demul-
tiplexed, quality-filtered by fast version 0.20.0 and
merged by FLASH version 1.2.7. Operational taxonomic
units (OTUs) with a 97% similarity cutoff were clustered
using UPARSE version 7.1, and chimeric sequences were
identified and removed. e taxonomy of each OTU
representative sequence was analyzed by RDP Classifier
version 2.2 against the 16S rRNA database using a confi-
dence threshold of 0.7.
Statistical analysis
e results are expressed as mean ± SEM. Statistical anal-
yses were performed using IBM SPSS Statistic 23. e
data were tested for normality using the Shapiro–Wilk
normality test. Data that passed the normality test were
analyzed using Students two-tailed t-test and ANOVA
(two-way ANOVA and repeated measures two-way
ANOVA). While non-parametric Mann–Whitney U tests
were performed on samples that did not pass the Shap-
iro–Wilk normality test (P < 0.05). Statistical sig nificance
was set at P < 0.05.
Abbreviations
5-HT Hydroxytryptamine
AB Alcian blue;
ACTH Adrenocorticotropic hormone
BDNFs Brain-derived nutritional factors
CORT Corticosterone
CSF Cerebrospinal fluid
CUMS Chronic unpredictable mild stress
DAI Disease activity index
DEGs Differentially expressed genes
DG Dentate gyrus
DSS Dextran sodium sulfate
ELISA Enzyme-linked immunosorbent assay
FST Forced swimming test
GABA γ-Aminobutyric acid type A
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
H.E Haematoxylin and eosin
IBD Inflammatory bowel disease
IDO Indoleamine 2,3-dioxygenase
IL Interleukin
KEGG Kyoto Encyclopedia of Genes and Genomes
KO Knockout
LF Lactoferrin
LPS Lipopolysaccharide
MIF Macrophage migration inhibitory factor
NF-κB Nuclear factor-κB
OFT Open field test
Table 1 Sequences of primers for real-time PCR
Gene name F‑primer (5’‑3’) R‑primer (5’‑3’)
Gapdh TCT CCT GCG ACT TCA ACA TGT AGC CGT ATT CAT TGT CA
Zo-1 GCA TCA TTC GCC TTC ATA C GAC ACA ACC TCA TCC TCA T
Occludin GGC TGC TGC TGA TGA ATA ATC CTC TTG ATG TGC GAT AA
TLR4 CAC TGT TCT TCT CC T GCC
TGA GGA ATG TCA TCA GGG ACT
TTGC
LBP GTC GTG GGC AGT ACG AGT
TT CCT TCC ATT TGC CTC GGA CA
MyD88 CGC ATG GTG GTG GTT GTT TC AGT CGC TTC TGT TGG ACA CC
CD14 ACA ATT CAC TGC GGG ATG
CT AGC TCA TCT GGG CTA GGG TT
JNK CCA CCA AAG ATC CCG GAC
AA GGC TGC CCT CT T ATG ACT CC
NFκB CCC TAC GGA ACT GGG
CAA AT GCA AAT TTT GAC CTG TGG GT
IκBα CCT GAC CTG GTT TCG CTC TT CTG TAT CCG GGT ACT TGG GC
P38MAPK TTC TAC CGG CAG GAG CTG
AA ATC AAA AGC AGC ACA CAC CG
IL-1β CTT CAG GCA GGC AGT ATC CAG CAG GTT ATC ATC ATC ATC
TNF-α ACT GAA CTT CGG GGT GAT
CG CCA CTT GGT GGT TTG TGA GTG
Bdnf TAA ACG TCC ACG GAC AAG
GC AGT GTC AGC CAG TGA TGT CG
Grb2 AAC ATC CGT GTC CAG GAA
CC AAG TCT CCT CTG CGA AAG CC
CaMK1 TGA TCC TGG CAG AGG ACA
AGA GCT ACA ATG TTG GGG TGC TTG
Creb AAC CAG CAG AGT GGA
GAT GC GAT GTT GCA TGA GCT GCT GG
Lrg1 TGT CCA GCC TCA AGG AAT
GC TTC CAC CGA CAG ATG GAC AG
Gbp4 TGA ACC AGG AAG CCA TAG
AGA GGG AAA CCT TTG GCT GGT
AGG
Stx11 ATG ACT TTG ACG CTC CTC GG GCG CCT CAC GTC TAT CAG AA
Pigr GAC TCT CGC TGG AGA ACC
AC ACG GAT AGT GGC AGG AAA CG
Ltf AGC TGA AGT CTA CGG GAC
CA CTC AGG CCT TGG AGT TGG TT
Lcn2 ACA ACC AGT TCG CCA TGG
TAT AAG CGG GTG AAA CGT TCC TT
S100a9 ACC ACC ATC ATC GAC ACC
TTC AAA GGT TGC CAA CTG TGC
TTC
Ccl28 CTC ACA CTC ATG GCT GTG
GCT AGT ACG ATT GTG CGG GCT GA
IL-25 GAC CTG TAC CAC GCT CGA TG AGA AGA CCG TCT GGT TGT GG
Ccn5 TGT GTG ACC AGG CAG TGA
TG GTG CTC CAG TTT GGA CAG GG
Cdh3 GCA TGC ACC ATG CAG ACA
AT GTG GCA TCA CCC ACT CTC TC
Prdm12 TCA AGT GTG CCC GGA ATG
AG TCC TGG TCT GGA GGG ATC AT
Pcdha8 TCA AAG GGT TTT GGG GAC
CT GAG CCT TTT GTG TCG CTG GA
Trpv4 TGG AAC CAG AAC TTG GGC
AT GGA CCA ACG ATC CCT ACG AA
Gabra6 AGG AGT CAG TCC CAG CAA
GA ATG AAC CAA TCC ATG GCG GT
Table 1 (continued)
Gene name F‑primer (5’‑3’) R‑primer (5’‑3’)
Kcne2 CCA GAG TGG ATG CCG AGA
AC CTT CGA CTT CAC CGT GCT CA
Kcnj13 TTT GTG GCG AAG ATT GCA
CG ACA CGA ACG TTG GTC AGA GG
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 24 of 26
Wangetal. BMC Biology (2023) 21:242
OTU Operational taxonomic unit
PCoA Principal coordinate analysis
RT-PCR Quantitative real-time PCR
SCFAs Short-chain fatty acids
SPT Sucrose preference test
TLR Toll-like-receptor
TNF-α Tumour necrosis factor-α
TPM Transcripts per million reads
TST Tail suspension test
WHO World Health Organization
WT Wild-type
zo-1 Zonula occludens
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12915- 023- 01748-2.
Additional le1: Fig. S1. Paneth cell numbers in the colon of 18-day-
old mice and representative images of fluorescence red tartar yellow
stained colonic sections. Fig. S2. Analysis of species difference at intestinal
microbial genus level between DSS enteritis mice and normal mice. Fig.
S3. KEGG pathway enrichment analysis of immune related differential
genes in 18-day-old mice small intestinal. Fig. S4. The abundance of
phylm Firmicutes, Bacteroidota, Desulfobacterota in 18-day-old mice,
9-week-old mice, DSS model mice and CUMS depression mice. Fig. S5.
The abundance of Bifidobacterium in CUMS mice. Fig. S6. Expression of
Cldn2 in the hippocampus RNAseq of 18-day-old mice. Fig. S7. Expression
of genes encode FKBP5 and HSP70 in 18-day-old small intestinal RNAseq.
Table. S1. The R value of Fig.2L. Table. S2. The P value of Fig.2L. Table. S3.
The summary of gut microbiota results in our study. Table. S4. Chronic
unpredictable mild stress schedule.
Acknowledgements
We would like to thank Editage (www. edita ge. cn) for English language editing.
Authors’ contributions
YZ, YD, WW and ZC conceived and designed the study. WW and ZC conducted
the experiments and performed the analyses. WW wrote the manuscript. XW,
QA helped collect and analyze the data. KH, QM checked the manuscript. All
authors read and approved the final manuscript.
Funding
We gratefully acknowledge the financial support provided by
National Key R&D Program of China (2021YFD1200903), the China Agriculture
Research System of MOF and MARA, Ministry of Agriculture and Villages Trans-
genic Major Project of China (2018ZX08007001).
Availability of data and materials
All data generated or analyzed during this study are included in this published
article, its supplementary information files and publicly available reposito-
ries. The raw reads have been deposited at the NCBI Short Read Archive with
the project accession numbers: PRJNA1030344 [86] (Effects of lactoferrin defi-
ciency during lactation on intestinal microbial composition in adult depressed
mice), PRJNA1030310 [87] ( Effects of lactoferrin deficiency during lactation
on intestinal microbial composition in 18-day mice, 9-week health mice and
DSS incuced colitis mice), PRJNA1030326 [88] (Effect of lactoferrin deficiency
during lactation on small intestinal transcriptome in mice), PRJNA1030504 [89]
(Effect of lactoferrin deficiency during lactation on small intestinal transcrip-
tome in mice).
Declarations
Ethics approval and consent to participate
All mice were treated in accordance with the guidelines of the Institutional
Animal Care and Use Committee (SYXK 2020–0052). All experiments were
approved by the Animal Experimentation Committee of the China Agricultural
University (Beijing, China).
Consent for publication
All the co-authors and participants have given their consent for publication.
in Journal of neuroinflammation.
Competing interests
The authors declare no competing financial interests or potential conflicts of
interest.
Received: 4 April 2023 Accepted: 24 October 2023
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