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Citation: Zhang, M.; Guo, C.; Li, Z.;
Cai, X.; Wen, X.; Lv, F.; Lin, C.; Ji, L.
Mulberry Twig Alkaloids Improved
the Progression of Metabolic-Associated
Fatty Liver Disease in High-Fat
Diet-Induced Obese Mice by
Regulating the PGC1α/PPARα
and KEAP1/NRF2 Pathways.
Pharmaceuticals 2024,17, 1287.
https://doi.org/10.3390/ph17101287
Academic Editors: Sabina
Lachowicz-Wi´sniewska, Marco
G. Alves and Ariane Zamoner
Received: 17 August 2024
Revised: 19 September 2024
Accepted: 25 September 2024
Published: 27 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceuticals
Article
Mulberry Twig Alkaloids Improved the Progression
of Metabolic-Associated Fatty Liver Disease in High-Fat
Diet-Induced Obese Mice by Regulating the PGC1α/PPARα
and KEAP1/NRF2 Pathways
Mengqing Zhang †, Chengcheng Guo †, Zonglin Li, Xiaoling Cai * , Xin Wen, Fang Lv, Chu Lin and Linong Ji *
Department of Endocrinology and Metabolism, Peking University People’s Hospital, Beijing 100044, China
*Correspondence: dr_junel@sina.com (X.C.); jiln@bjmu.edu.cn (L.J.)
†These authors contributed equally to this work.
Abstract: Background/Objectives: Metabolic-associated fatty liver disease (MAFLD) is one of the
most common liver disorders associated with obesity and metabolic syndrome, and poses a signifi-
cant global health burden with limited effective treatments. The aim of this study was to assess the
protective effects of mulberry twig alkaloids (SZ-A) on MAFLD and to further investigate the under-
lying mechanisms including the specific targets or pathways. Methods: Diet-induced obesity (DIO)
and normal mouse models were established by feeding C57Bl/6J mice with a high-fat diet (HFD) or
common diet for 12 weeks. SZ-A, dapagliflozin, and placebo were administered to corresponding
mouse groups for 8 weeks. Data of fasting blood glucose, glucose tolerance, insulin tolerance, and the
body weight of mice were collected at the baseline and termination of the experiment. Serum liver
enzymes and lipids were measured by ELISA. Western blotting, qPCR, and pathological section stain-
ing were implemented to evaluate the degrees of liver steatosis, fibrosis, and oxidative stress in mice.
Results: In DIO mouse models, high-dose SZ-A (800 mg/kg/d) treatment significantly inhibited
HFD-induced weight gain, improved insulin tolerance, and reduced serum alanine aminotransferase,
total cholesterol, and triglyceride levels compared with placebo. In DIO mice, SZ-A could alleviate
the pathological changes of hepatic steatosis and fibrosis compared with placebo. Lipid catabolism
and antioxidant stress-related proteins were significantly increased in the livers of the high-dose SZ-A
group (p< 0.05). Inhibition of PGC1
α
could inhibit the function of SZ-A to enhance lipid metabolism
in hepatocytes. PGC1
α
might interact with NRF2 to exert MAFLD-remedying effects. Conclusions:
By regulating the expression of PGC1
α
and its interacting KEAP1/NRF2 pathway in mouse liver
cells, SZ-A played important roles in regulating lipid metabolism, inhibiting oxidative stress, and
postponing liver fibrosis in mice with MAFLD.
Keywords: MAFLD; mulberry twig alkaloids; PGC1
α
; NRF2; lipid metabolism; anti-oxidative stress
1. Introduction
Metabolic-associated fatty liver disease (MAFLD) originates from metabolic stress-
related liver injury, which is strongly associated with insulin resistance and relevant genetic
susceptibility [
1
,
2
]. MAFLD, as a disease with similar prevalence to obesity, could po-
tentially progress to nonalcoholic steatohepatitis, liver fibrosis, and eventually cirrhosis
and hepatocellular carcinoma (HCC) [
3
,
4
]. In the last decade, a growing body of studies
has demonstrated that the disability-adjusted life years associated with MAFLD increased
greatly from countries with low to moderate sociodemographic indices [
5
]. The prevalence
of MAFLD has also risen sharply in China in recent years, in which it affects over 30%
of the total population and has exceeded twice the prevalence in Western countries [
5
].
The surge in the incidence of MAFLD poses a huge disease burden worldwide, and more
effective ways to treat MAFLD still await further refinement.
Pharmaceuticals 2024,17, 1287. https://doi.org/10.3390/ph17101287 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2024,17, 1287 2 of 16
The primary pathogenesis of MAFLD was considered to be lipid deposition and
oxidative stress in the liver, where hyperglycemia would aggravate this process [
6
–
8
].
When the lipid content surpasses the normal regulatory load of the body due to chronic
over-intake, the balance between liver lipid production and catabolism is disturbed, and
free fatty acids (FFAs) would then accumulate in the liver. These deposited lipids serve as
substrates to produce toxic lipid metabolites that induce endoplasmic reticulum (ER) stress,
mitochondrial dysfunction, oxidative stress, cellular damage, and cytokine release, which
can result in liver injuries and therefore instigate MAFLD [9].
Mulberry twig alkaloids (known as Sangzhi-alkaloid in Chinese, hereafter referred to
as SZ-A) is extracted from Mulberry Ramulus. It has demonstrated effectiveness in man-
aging type 2 diabetes (T2D) in China [
10
,
11
]. The main components are 1-deoxynojirycin
(1-DNJ), fagopycin (FA), and 1,4 dideoxy-1,4-iminod-arabinitol (DAB) [
12
]. SZ-A has a
highly selective and precise inhibitory effect on intestinal glycosidases, and
in vitro
experi-
ments have confirmed that its inhibitory effect on disaccharidase activity is stronger than
or equivalent to acarbose [
12
]. In addition to the regulatory effects on glucose metabolism,
SZ-A could also reduce the inflammatory response to a certain extent by blocking the
activation of the p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-
regulated kinase (ERK), and stress-activated protein kinase (c-Jun N-terminal kinase, JNK)
signaling pathways [
13
]. Regarding lipid metabolism, previous studies have demonstrated
that SZ-A could lower serum lipids through multiple mechanisms including reducing
body weight, improving overall energy metabolism, and enhancing lipid consumption in
hepatocytes [
10
]. Since the progression of MAFLD is closely associated with glucolipid
metabolism disorder (mostly dyslipidemia) and inflammation [
6
–
8
], SZ-A may exert po-
tential therapeutic effects for MAFLD. In previous research, it was observed that SZ-A
ameliorated the progression of MAFLD in mice with high-fat diet (HFD) [
10
]. However,
the specific regulatory mechanisms by which SZ-A relieves MAFLD have not been fully
revealed. Whether SZ-A introduces liver benefits through interacting with other potential
targets in multiple tissues and organs throughout the body still needs exploration.
Therefore, the aim of this study was to evaluate the therapeutic effects of SZ-A on
MAFLD and to investigate its mechanism of action in depth, providing further reference
for the treatment of MAFLD in the future.
2. Results
2.1. High-Dose SZ-A Decreased Body Weight and Improved Insulin Tolerance in Mice Fed
with HFD
To investigate the therapeutic effects of SZ-A on MAFLD, an experiment was com-
pleted with five groups of mice. The results showed that at week 20, mice receiving the HFD
had a significantly higher body weight and more impaired glucose tolerance and insulin
tolerance compared with the control group (CTRL). There was no significant difference in
food consumption between all experimental groups. In HFD mice, after 8 weeks of drug
intervention, mice in the SZ-A high-dose group had significantly inhibited HFD-induced
weight gain and improved insulin tolerance (Figure 1A–D).
Figure 1. Cont.
Pharmaceuticals 2024,17, 1287 3 of 16
Figure 1. The figure presents a comprehensive analysis of the impact of Sangzhi alkaloids (SZA) and
dapagliflozin on mice subjected to a high-fat diet (HFD). (A) illustrates the growth curve, detailing the
average body weight of mice across a 20-week study, with the control group (CTRL) on a standard
diet and experimental groups on a HFD with varying interventions: low-dose SZA (HFD + SZA-L),
high-dose SZA (HFD + SZA-H), and dapagliflozin (HFD + Dapa). Each data point is accompanied by
an error bar indicating the standard error of the mean (SEM), showcasing the variability within each
group. (B) illustrates the average daily food intake measured during the final week for each group,
with error bars representing SEM. (C) displays the terminal intraperitoneal glucose tolerance test
(IPGTT) results, measuring glucose levels at specific time intervals post-glucose administration, while
(D) presents the intraperitoneal insulin tolerance test (IPITT) outcomes, tracking glucose levels at
different time points after insulin injection. (E) depicts the terminal body weight of the mice with SEM
error bars representing the variability in these measurements. Additionally, (F) shows the terminal
fasting plasma glucose (FPG) levels, providing a snapshot of each group’s glucose metabolism under
fasting conditions. Asterisks (*) denote significant differences compared to the control group (p< 0.05),
and double asterisks (**) indicate highly significant differences compared to the control group (p< 0.01).
Hashes (#) are used to show significant differences compared to the HFD group (p< 0.05).
2.2. High-Dose SZ-A Significantly Reduced the Serum Liver Enzyme Concentrations and
Improved Lipid Profiles in MAFLD Induced by HFD
At week 20, compared with the CTRL group, the HFD group showed significantly
increased levels of blood glucose, TG, TC, and LDL-C, as well as serum liver injury indexes
including ALT, AST, and ALP. In HFD mice, after 8 weeks of high-dose SZ-A intervention,
the serum ALT, AST, ALP, TC, and TG levels were significantly reduced, which were more
prominent in the high-dose SZ-A group. In addition, there was a significant reduction
in serum TC levels in the HFD + dapagliflozin group compared with the HFD group
(Figure 2A–H).
2.3. SZ-A Could Improve Liver Hepatic Lipids in MAFLD Induced by HFD
In the hematoxylin and eosin (HE) staining assessment of liver sections, we com-
paratively analyzed the non-steatotic regions among the different groups. The CTRL
group exhibited a significantly higher area of non-steatosis compared to the HFD group
(p< 0.0001). The HFD group demonstrated a significantly lower non-steatotic area com-
Pharmaceuticals 2024,17, 1287 4 of 16
pared to the high-dose SZ-A treatment group (HFD + SZA-H) (p< 0.0001), suggesting that
high-dose SZ-A effectively ameliorates steatosis. Additionally, the low-dose SZ-A treatment
group (HFD + SZA-L) had a significantly smaller non-steatotic area than the HFD + SZA-H
group (p< 0.01), indicating a dose-dependent effect of SZ-A on reducing steatosis. Lastly,
the HFD + SZA-H group exhibited a significantly higher area of non-steatosis compared to
the dapagliflozin treatment group (HFD + Dapa) (p< 0.0001), suggesting that SZ-A has
a greater advantage in reducing liver steatosis compared to dapagliflozin and indicating
that the beneficial effects of SZ-A in reducing liver fat accumulation are independent of its
glucose-lowering effects (Figure 3A,B).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 4 of 17
the serum ALT, AST, ALP, TC, and TG levels were significantly reduced, which were more
prominent in the high-dose SZ-A group. In addition, there was a significant reduction in
serum TC levels in the HFD + dapagliflozin group compared with the HFD group (Figure
2A–H).
Figure 2. All comparisons of interventions were conducted at week 20. (A) The concentration of ALT
in mouse serum (n = 10). (B) The concentration of AST in mouse serum (n = 10). (C) The concentration
of ALP in mouse serum (n = 10). (D) The concentration of glucose in mouse serum (n = 10). (E) The
concentration of TC in mouse serum (n = 10). (F) The concentration of TG in mouse serum (n = 10).
(G) The concentration of HDL-C in mouse serum (n = 10). (H) The concentration of LDL-C in mouse
serum (n = 10). The results of body weight change in mice (n = 10). Abbr: CTRL, control group; HFD,
high-fat diet; SZ-A, Sangzhi-alkaloids; SZA-L, low-dose Sangzhi-alkaloids; SZA-H, high-dose Sang-
zhi-alkaloids; Dapa, dapagliflozin; ALT, alanine aminotransferase; AST, aspartate aminotransferase;
ALP, alkaline phosphatase; TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein
cholesterol; LDL-C, low-density lipoprotein cholesterol. “*” means p < 0.05 compared to CTRL
group, “#” means p < 0.05 compared to HFD group.
2.3. SZ-A Could Improve Liver Hepatic Lipids in MAFLD Induced by HFD
In the hematoxylin and eosin (HE) staining assessment of liver sections, we compar-
atively analyzed the non-steatotic regions among the different groups. The CTRL group
exhibited a significantly higher area of non-steatosis compared to the HFD group (p <
0.0001). The HFD group demonstrated a significantly lower non-steatotic area compared
to the high-dose SZ-A treatment group (HFD + SZA-H) (p < 0.0001), suggesting that high-
dose SZ-A effectively ameliorates steatosis. Additionally, the low-dose SZ-A treatment
group (HFD + SZA-L) had a significantly smaller non-steatotic area than the HFD + SZA-
H group (p < 0.01), indicating a dose-dependent effect of SZ-A on reducing steatosis.
Lastly, the HFD + SZA-H group exhibited a significantly higher area of non-steatosis com-
pared to the dapagliflozin treatment group (HFD + Dapa) (p < 0.0001), suggesting that SZ-
A has a greater advantage in reducing liver steatosis compared to dapagliflozin and indi-
cating that the beneficial effects of SZ-A in reducing liver fat accumulation are independ-
ent of its glucose-lowering effects (Figure 3A,B).
Figure 2. All comparisons of interventions were conducted at week 20. (A) The concentration of
ALT in mouse serum (n= 10). (B) The concentration of AST in mouse serum (n= 10). (C) The
concentration of ALP in mouse serum (n= 10). (D) The concentration of glucose in mouse serum
(n= 10). (E) The concentration of TC in mouse serum (n= 10). (F) The concentration of TG in mouse
serum (n= 10). (G) The concentration of HDL-C in mouse serum (n= 10). (H) The concentration of
LDL-C in mouse serum (n= 10). The results of body weight change in mice (n= 10). Abbr: CTRL,
control group; HFD, high-fat diet; SZ-A, Sangzhi-alkaloids; SZA-L, low-dose Sangzhi-alkaloids;
SZA-H, high-dose Sangzhi-alkaloids; Dapa, dapagliflozin; ALT, alanine aminotransferase; AST,
aspartate aminotransferase; ALP, alkaline phosphatase; TC, total cholesterol; TG, triglyceride; HDL-C,
high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. “*” means p< 0.05
compared to CTRL group, “#” means p< 0.05 compared to HFD group.
We assessed the mRNA expression levels of genes implicated in hepatic lipid metabolism.
In the HFD group, there was a significant upregulation of the mRNA levels for the genes
PPGR
γ
and PCSK9, which are associated with lipid synthesis, compared to the CTRL
group. Conversely, the mRNA levels of SCAD, SOD1, and LXRA, which are involved in
lipid catabolism and antioxidant defense, were significantly downregulated in the HFD
group. In comparison to the HFD group, the HFD + SZA-H group exhibited a significant
decrease in PPGR
γ
mRNA levels. Additionally, the mRNA levels of SCAD, SOD1, and
LXRA were significantly upregulated in the HFD + SZA-H group, suggesting an enhance-
ment of lipid catabolism and antioxidant mechanisms. However, in contrast, the HFD +
SZA-H group showed a significant increase in PCSK9 mRNA levels, which may suggest a
complex regulation of lipid metabolism by SZA-H. These findings indicate that the SZA-H
Pharmaceuticals 2024,17, 1287 6 of 16
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Figure 3. (A) Microscopic view of mouse liver after hematoxylin–eosin, Masson, and Sirius Red
staining. (B) The results of hematoxylin–eosin (HE) staining of liver tissue of mice. (C) The results
of Masson staining of liver tissue of mice. (D) The results of Sirius staining of liver tissue of mice.
(E) The expression of SCD1 gene compared to GAPDH. (F) The expression of ACC1 gene compared
to GAPDH. (G) The expression of SCAD gene compared to GAPDH. (H) The expression of PCSK9
gene compared to GAPDH. (I) The expression of PPARγ gene compared to GAPDH. (J) The expres-
si on of H MGCR g ene co mpare d to GA PDH. ( K) The expression of SOD1 gene compared to GAPDH.
(L) The expression of LXRα gene compared to GAPDH. (M) The result of immunobloing. (N) The
expression of MFN2 compared to β-actin. (O) The expression of FASN compared to β-actin. (P) The
expression of ACC compared to β-actin. (Q) The expression of PPARα compared to β-actin. (R) The
expression of PGC1α compared to β-actin. (S) The expression of TOM20 compared to β-actin. Abbr:
CTRL, control diet; HFD, high-fat diet; SZA-L, low-dose Sangzhi-alkaloids; SZA-H, high-dose Sang-
zhi-alkaloids; Dapa, dapagliflozin; SCD1, stearoyl-CoA desaturase 1; ACC1, acetyl-CoA carboxylase
1; SCAD, short-chain acyl-coenzyme A dehydrogenase; PCSK9, proprotein convertase subtil-
isin/kexin type 9; PPARγ, peroxisome proliferator-activated receptor γ; HMGCR, 3-hydroxy-3-
methylglutaryl-coenzyme A reductase; SOD1, superoxide dismutase-1; LXRα, liver X receptor α;
MFN2, mitochondrial fusion protein; FASN, fay acid synthase; ACC, acetyl-CoA carboxylase;
PPARα, peroxisome proliferator-activated receptor α; PGC1α, peroxisome proliferator-activated re-
ceptor-γ coactivator 1α; TOM20, translocase of outer mitochondrial membrane 20. The symbols *,
**, ***, and **** denote p-values of less than 0.05, 0.01, 0.001, and 0.0001, respectively. Note: The green
color in (B,C) is used to distinguish annotations where lines overlap and does not indicate any spe-
cific meaning.
We assessed the mRNA expression levels of genes implicated in hepatic lipid metab-
olism. In the HFD group, there was a significant upregulation of the mRNA levels for the
genes PPGRγ and PCSK9, which are associated with lipid synthesis, compared to the
Figure 3. (A) Microscopic view of mouse liver after hematoxylin–eosin, Masson, and Sirius Red
staining. (B) The results of hematoxylin–eosin (HE) staining of liver tissue of mice. (C) The results
of Masson staining of liver tissue of mice. (D) The results of Sirius staining of liver tissue of mice.
(E) The expression of SCD1 gene compared to GAPDH. (F) The expression of ACC1 gene compared to
GAPDH. (G) The expression of SCAD gene compared to GAPDH. (H) The expression of PCSK9 gene
compared to GAPDH. (I) The expression of PPAR
γ
gene compared to GAPDH. (J) The expression
of HMGCR gene compared to GAPDH. (K) The expression of SOD1 gene compared to GAPDH.
(L) The expression of LXR
α
gene compared to GAPDH. (M) The result of immunoblotting. (N)
The expression of MFN2 compared to
β
-actin. (O) The expression of FASN compared to
β
-actin.
(P) The expression of ACC compared to
β
-actin. (Q) The expression of PPAR
α
compared to
β
-actin.
(R) The expression of PGC1
α
compared to
β
-actin. (S) The expression of TOM20 compared to
β
-actin.
Abbr: CTRL, control diet; HFD, high-fat diet; SZA-L, low-dose Sangzhi-alkaloids; SZA-H, high-
dose Sangzhi-alkaloids; Dapa, dapagliflozin; SCD1, stearoyl-CoA desaturase 1; ACC1, acetyl-CoA
carboxylase 1; SCAD, short-chain acyl-coenzyme A dehydrogenase; PCSK9, proprotein convertase
subtilisin/kexin type 9; PPAR
γ
, peroxisome proliferator-activated receptor
γ
; HMGCR, 3-hydroxy-
3-methylglutaryl-coenzyme A reductase; SOD1, superoxide dismutase-1; LXR
α
, liver X receptor
α
; MFN2, mitochondrial fusion protein; FASN, fatty acid synthase; ACC, acetyl-CoA carboxylase;
PPAR
α
, peroxisome proliferator-activated receptor
α
; PGC1
α
, peroxisome proliferator-activated
receptor-
γ
coactivator 1
α
; TOM20, translocase of outer mitochondrial membrane 20. The symbols
*, **, ***, and **** denote p-values of less than 0.05, 0.01, 0.001, and 0.0001, respectively. Note: The
green color in (B,C) is used to distinguish annotations where lines overlap and does not indicate any
specific meaning.
Pharmaceuticals 2024,17, 1287 7 of 16
By Western blot analysis, the protein levels of key molecules involved in lipid synthesis,
FASN and ACC, were significantly elevated in the HFD group relative to the CTRL group
(p< 0.05), suggesting an upregulation of lipogenic pathways. In contrast, the protein levels
of PGC1
α
, PPAR
α
, MFN2, and TOM20, which are associated with lipid catabolism, were
significantly reduced in the HFD group compared to the CTRL group (p< 0.05), indicating
a downregulation of fatty acid oxidation mechanisms.
In the HFD + SZA-H group, treatment with high-dose SZ-A led to a significant
decrease in the protein levels of FASN and ACC compared to the HFD group (p< 0.05),
implying a reduction in lipid synthesis. Concurrently, there was a significant increase in the
protein levels of PGC1
α
, PPAR
α
, MFN2, and TOM20 in the HFD + SZA-H group compared
to the HFD group (p< 0.05), which points towards an enhancement of lipid catabolism.
These results (Figure 3M–S) demonstrate that high-dose SZ-A treatment can ameliorate
the dysregulation of lipid metabolism in the liver by promoting fatty acid oxidation and
inhibiting lipid synthesis.
2.4. SZ-A Improved Liver Fibrosis in MAFLD Induced by HFD and Affected the Expression of
Oxidative Stress Kinase
In this study, we evaluated the effects of SZ-A on liver fibrosis and steatosis induced
by HFD.
In the Masson staining analysis (Figure 3A,C), significant differences in collagen fiber
areas were observed among the groups. The CTRL group demonstrated a significantly
lower collagen fiber area compared to the HFD group (p< 0.0001), indicating a substantial
reduction in fibrosis. The HFD group had a significantly higher collagen fiber area than
both the HFD + SZA-L group (p< 0.05) and the HFD + SZA-H group (p< 0.01), suggesting
that SZ-A treatment at both low and high doses effectively attenuates fibrosis. Furthermore,
the HFD + SZA-H group showed a lower collagen fiber area than the HFD + Dapa group
(p< 0.05), indicating that SZ-A may have a similar or greater effect in reducing fibrosis
compared to dapagliflozin. The HFD + SZA-H group also had a significantly lower collagen
fiber area than the HFD + Dapa group (p< 0.05), which further supports the potential
therapeutic advantage of SZ-A in fibrosis reduction. These results are depicted in the figure.
In the Sirius Red staining evaluation, the CTRL group showed a significantly lower
collagen fiber area compared to the HFD group (p< 0.001). The HFD + SZA-H group
exhibited a significantly reduced collagen fiber area when compared to the HFD group
(p< 0.05), indicating that the high-dose SZ-A treatment contributed to a decrease in collagen
deposition. These results suggest that SZ-A may have a beneficial effect in reducing liver
fibrosis as evidenced by the reduction in Sirius Red staining (Figure 3A,D).
In addition to the histological assessments, the protein levels of the liver fibrosis
markers KEAP1 and
α
-SMA were significantly elevated in the HFD group compared to
the CTRL group (p< 0.05), indicating increased fibrotic activity. Concurrently, the protein
levels of NRF2 and its downstream antioxidant enzymes SCD1, GPX6, SOD1, and SOD2
were significantly decreased in the HFD group (p< 0.05), suggesting a reduction in the
antioxidant defense mechanisms.
In contrast, the HFD + SZA-H group showed a significant reduction in the protein
levels of KEAP1 and
α
-SMA compared to the HFD group (p< 0.05), indicating that high-
dose SZ-A treatment effectively attenuated the fibrotic response. Furthermore, the protein
expression levels of NRF2, SCD1, GPX6, SOD1, and SCD2 were significantly increased
in the HFD + SZA-H group compared to the HFD group (p< 0.05), demonstrating an
enhancement in the antioxidant capacity with high-dose SZ-A treatment.
These findings (Figure 4A–I) indicate that high-dose SZ-A treatment effectively re-
verses the fibrotic and oxidative stress-induced changes in the liver.
Pharmaceuticals 2024,17, 1287 8 of 16
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In contrast, the HFD + SZA-H group showed a significant reduction in the protein
levels of KEAP1 and α-SMA compared to the HFD group (p < 0.05), indicating that high-
dose SZ-A treatment effectively aenuated the fibrotic response. Furthermore, the protein
expression levels of NRF2, SCD1, GPX6, SOD1, and SCD2 were significantly increased in
the HFD + SZA-H group compared to the HFD group (p < 0.05), demonstrating an en-
hancement in the antioxidant capacity with high-dose SZ-A treatment.
These findings (Figure 4A–I) indicate that high-dose SZ-A treatment effectively re-
verses the fibrotic and oxidative stress-induced changes in the liver.
Figure 4. (A) The result of immunobloing. (B) The expression of MMP9 compared to β-actin. (C)
The expression of α-SMA compared to β-actin. (D) The expression of KEAP1 compared to β-actin.
(E) The expression of NRF2 compared to β-actin. (F) The expression of SCD1 compared to β-actin.
(G) The expression of GPX6 compared to β-actin. (H) The expression of SOD1 compared to β-actin.
(I) The expression of SOD2 compared to β-actin. Abbr: CTRL, control diet; HFD, high-fat diet; SZA-
L, low-dose Sangzhi-alkaloids; SZA-H, high-dose Sangzhi-alkaloids; Dapa, dapagliflozin; KEAP1,
kelch-like ECH associated protein 1; MMP9, matrix metalloproteinase 9; α-SMA, alpha-smooth
muscle actin; NRF2, nuclear factor erythroid-2-related factor 2; SCD1, stearyl coenzyme A dehydro-
genase-1; GPX6, glutathione peroxidase 6; SOD1, superoxide dismutase-1; SOD2, superoxide dis-
mutase-2. “*” means p < 0.05.
2.5. SZ-A Improved Liver Lipid Metabolism by Upregulating the Synthesis of PGC1α
To investigate the role of SZ-A in lipid metabolism, potentially through the regula-
tion of the PGC1α molecule, we performed a rescue experiment using primary liver cells
from wild-type mice. The groups are as follows: OA + DMSO serving as the high-lipid
control group, OA + SR representing the group treated with oleic acid and the PGC1α
inhibitor SR18292, OA + SZA indicating the group treated with oleic acid and SZ-A, and
OA + SR + SZA for the group treated with all three agents. The specific values for the TG
content are illustrated in the figure, with Figure 5A showing the Oil Red O staining results
and Figure 5B displaying the TG content measurements.
Figure 4. (A) The result of immunoblotting. (B) The expression of MMP9 compared to
β
-actin.
(C) The expression of
α
-SMA compared to
β
-actin. (D) The expression of KEAP1 compared to
β
-actin.
(E) The expression of NRF2 compared to
β
-actin. (F) The expression of SCD1 compared to
β
-actin.
(G) The expression of GPX6 compared to
β
-actin. (H) The expression of SOD1 compared to
β
-actin.
(I) The expression of SOD2 compared to
β
-actin. Abbr: CTRL, control diet; HFD, high-fat diet; SZA-L,
low-dose Sangzhi-alkaloids; SZA-H, high-dose Sangzhi-alkaloids; Dapa, dapagliflozin; KEAP1, kelch-
like ECH associated protein 1; MMP9, matrix metalloproteinase 9;
α
-SMA, alpha-smooth muscle
actin; NRF2, nuclear factor erythroid-2-related factor 2; SCD1, stearyl coenzyme A dehydrogenase-1;
GPX6, glutathione peroxidase 6; SOD1, superoxide dismutase-1; SOD2, superoxide dismutase-2.
“*” means p< 0.05.
2.5. SZ-A Improved Liver Lipid Metabolism by Upregulating the Synthesis of PGC1α
To investigate the role of SZ-A in lipid metabolism, potentially through the regulation
of the PGC1
α
molecule, we performed a rescue experiment using primary liver cells
from wild-type mice. The groups are as follows: OA + DMSO serving as the high-lipid
control group, OA + SR representing the group treated with oleic acid and the PGC1
α
inhibitor SR18292, OA + SZA indicating the group treated with oleic acid and SZ-A, and
OA + SR + SZA
for the group treated with all three agents. The specific values for the TG
content are illustrated in the figure, with Figure 5A showing the Oil Red O staining results
and Figure 5B displaying the TG content measurements.
We observed that compared to the high-lipid control group (OA + DMSO), the TG
content in the primary liver cells of the OA + SR group was significantly increased. The
addition of SZ-A to the OA + SZA group led to a significant decrease in the TG content,
suggesting that SZ-A can effectively reduce lipid accumulation. However, the TG-lowering
effect of SZ-A was less pronounced when the PGC1
α
inhibitor SR was also present, as
indicated by the TG content in the OA + SR + SZA group.
Additionally, in the rescue experiment, compared to the control group, treatment
of hepatocytes with the PGC1
α
inhibitor resulted in an increase in the intracellular ROS
content and a decrease in the number of intracellular mitochondria, while SZ-A treatment
of hepatocytes resulted in an increase in the intracellular mitochondria number (Figure 5C).
Pharmaceuticals 2024,17, 1287 9 of 16
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 9 of 17
Figure 5. (A) The rescue experiment in hepatocytes. (B) The content of TG in hepatocytes (n = 3). (C)
The content of ROS and mitochondria in hepatocytes (n = 3). Abbr: SZA, Sangzhi-alkaloids; Dapa,
dapagliflozin; OA, oleic acid; DMSO, dimethyl sulfoxide; SR, SR18292. TG, triglyceride; ROS, reac-
tive oxygen species. “*” means p < 0.05.
We observed that compared to the high-lipid control group (OA + DMSO), the TG
content in the primary liver cells of the OA + SR group was significantly increased. The
addition of SZ-A to the OA + SZA group led to a significant decrease in the TG content,
suggesting that SZ-A can effectively reduce lipid accumulation. However, the TG-lower-
ing effect of SZ-A was less pronounced when the PGC1α inhibitor SR was also present, as
indicated by the TG content in the OA + SR + SZA group.
Additionally, in the rescue experiment, compared to the control group, treatment of
hepatocytes with the PGC1α inhibitor resulted in an increase in the intracellular ROS con-
tent and a decrease in the number of intracellular mitochondria, while SZ-A treatment of
hepatocytes resulted in an increase in the intracellular mitochondria number (Figure 5C).
2.6. Adiponectin and Leptin Levels Were Not Significantly Affected by SZ-A
It was deemed that HFD might also cause disorders in adipocytokine secretion by
adipose tissue. To further address this issue, we measured the serum levels of adiponectin
(APN) and leptin. However, we did not find a significant effect of SZ-A on serum APN
and leptin levels (Figure 6A,B).
Figure 5. (A) The rescue experiment in hepatocytes. (B) The content of TG in hepatocytes (n= 3).
(C) The content of ROS and mitochondria in hepatocytes (n= 3). Abbr: SZA, Sangzhi-alkaloids; Dapa,
dapagliflozin; OA, oleic acid; DMSO, dimethyl sulfoxide; SR, SR18292. TG, triglyceride; ROS, reactive
oxygen species. “*” means p< 0.05.
2.6. Adiponectin and Leptin Levels Were Not Significantly Affected by SZ-A
It was deemed that HFD might also cause disorders in adipocytokine secretion by
adipose tissue. To further address this issue, we measured the serum levels of adiponectin
(APN) and leptin. However, we did not find a significant effect of SZ-A on serum APN and
leptin levels (Figure 6A,B).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 10 of 17
Figure 6. (A) The concentration of leptin in mouse serum (n = 10). (B) The concentration of APN in
mouse serum (n = 10). Abbr: CTRL, control group; HFD, high-fat diet; SZA-L, low-dose Sangzhi-
alkaloids; SZA-H, high-dose Sangzhi-alkaloids; Dapa, dapagliflozin; APN, adiponectin. “#” means
p < 0.05 compared to HFD group.
2.7. PGC1α Might Interact with NRF2 at the Molecular Level
In order to assess the association between PGC1α and NRF2, we used co-immuno-
precipitation as a method to investigate the potential interaction between them. PGC1α
was present in the immunoprecipitated samples, while the control experiments using non-
specific IgG antibodies did not show significant co-immunoprecipitation, which provided
reciprocal evidence for the interaction between PGC1α and NRF2 (Figure 7).
Figure 7. PGC1α and NRF2 co-immunoprecipitation experiments. We conducted co-immunopre-
cipitation (Co-IP) experiments using primary hepatocytes from mice to explore the potential inter-
action between NRF2 and PGC1α proteins. The experimental results are depicted in the figure,
which includes the input group showing the baseline expression of PGC1α protein in whole-cell
lysates from primary mouse hepatocytes without immunoprecipitation treatment; the IP group,
where protein complexes interacting with NRF2 were precipitated using a specific antibody against
NRF2; and the negative control group, which used non-specific IgG antibody precipitation to con-
firm specific binding during the precipitation process. The figure also includes molecular weight
markers ranging from 140 kDa to 25 kDa to estimate the molecular weight of the protein bands.
Western blot technology was employed, utilizing a specific antibody against PGC1α (IB: PGC1α)
and a non-specific IgG antibody (IB: IgG) to detect and verify the specificity of the precipitation. All
experimental procedures were carried out under strict conditions to ensure the accuracy and repro-
ducibility of the results. Abbr: PGC1α, peroxisome proliferator-activated receptor-γ coactivator 1α;
IgG, immunoglobulin G; NRF2, nuclear factor erythroid-2-related factor 2; IP, immunoprecipitation;
IB, immunobloing.
Figure 6. (A) The concentration of leptin in mouse serum (n= 10). (B) The concentration of APN in
mouse serum (n= 10). Abbr: CTRL, control group; HFD, high-fat diet; SZA-L, low-dose Sangzhi-
alkaloids; SZA-H, high-dose Sangzhi-alkaloids; Dapa, dapagliflozin; APN, adiponectin. “#” means
p< 0.05 compared to HFD group.
Pharmaceuticals 2024,17, 1287 10 of 16
2.7. PGC1αMight Interact with NRF2 at the Molecular Level
In order to assess the association between PGC1
α
and NRF2, we used co-immunopreci-
pitation as a method to investigate the potential interaction between them. PGC1
α
was
present in the immunoprecipitated samples, while the control experiments using non-
specific IgG antibodies did not show significant co-immunoprecipitation, which provided
reciprocal evidence for the interaction between PGC1αand NRF2 (Figure 7).
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 10 of 17
Figure 6. (A) The concentration of leptin in mouse serum (n = 10). (B) The concentration of APN in
mouse serum (n = 10). Abbr: CTRL, control group; HFD, high-fat diet; SZA-L, low-dose Sangzhi-
alkaloids; SZA-H, high-dose Sangzhi-alkaloids; Dapa, dapagliflozin; APN, adiponectin. “#” means
p < 0.05 compared to HFD group.
2.7. PGC1α Might Interact with NRF2 at the Molecular Level
In order to assess the association between PGC1α and NRF2, we used co-immuno-
precipitation as a method to investigate the potential interaction between them. PGC1α
was present in the immunoprecipitated samples, while the control experiments using non-
specific IgG antibodies did not show significant co-immunoprecipitation, which provided
reciprocal evidence for the interaction between PGC1α and NRF2 (Figure 7).
Figure 7. PGC1α and NRF2 co-immunoprecipitation experiments. We conducted co-immunopre-
cipitation (Co-IP) experiments using primary hepatocytes from mice to explore the potential inter-
action between NRF2 and PGC1α proteins. The experimental results are depicted in the figure,
which includes the input group showing the baseline expression of PGC1α protein in whole-cell
lysates from primary mouse hepatocytes without immunoprecipitation treatment; the IP group,
where protein complexes interacting with NRF2 were precipitated using a specific antibody against
NRF2; and the negative control group, which used non-specific IgG antibody precipitation to con-
firm specific binding during the precipitation process. The figure also includes molecular weight
markers ranging from 140 kDa to 25 kDa to estimate the molecular weight of the protein bands.
Western blot technology was employed, utilizing a specific antibody against PGC1α (IB: PGC1α)
and a non-specific IgG antibody (IB: IgG) to detect and verify the specificity of the precipitation. All
experimental procedures were carried out under strict conditions to ensure the accuracy and repro-
ducibility of the results. Abbr: PGC1α, peroxisome proliferator-activated receptor-γ coactivator 1α;
IgG, immunoglobulin G; NRF2, nuclear factor erythroid-2-related factor 2; IP, immunoprecipitation;
IB, immunobloing.
Figure 7. PGC1
α
and NRF2 co-immunoprecipitation experiments. We conducted co-
immunoprecipitation (Co-IP) experiments using primary hepatocytes from mice to explore the
potential interaction between NRF2 and PGC1
α
proteins. The experimental results are depicted in
the figure, which includes the input group showing the baseline expression of PGC1
α
protein in
whole-cell lysates from primary mouse hepatocytes without immunoprecipitation treatment; the IP
group, where protein complexes interacting with NRF2 were precipitated using a specific antibody
against NRF2; and the negative control group, which used non-specific IgG antibody precipitation to
confirm specific binding during the precipitation process. The figure also includes molecular weight
markers ranging from 140 kDa to 25 kDa to estimate the molecular weight of the protein bands.
Western blot technology was employed, utilizing a specific antibody against PGC1
α
(IB: PGC1
α
)
and a non-specific IgG antibody (IB: IgG) to detect and verify the specificity of the precipitation. All
experimental procedures were carried out under strict conditions to ensure the accuracy and repro-
ducibility of the results. Abbr: PGC1
α
, peroxisome proliferator-activated receptor-
γ
coactivator 1
α
;
IgG, immunoglobulin G; NRF2, nuclear factor erythroid-2-related factor 2; IP, immunoprecipitation;
IB, immunoblotting.
3. Discussion
In this study, we established an MAFLD mouse model by feeding male C57BL/6J mice
with 60% HFD for 12 weeks and observed the progression and prognosis of MAFLD after
8 weeks of drug intervention (including SZ-A at low or high doses, dapagliflozin). The
results indicated that in HFD mice, all low-dose SZ-A, high-dose SZ-A, and dapagliflozin
interventions significantly improved insulin tolerance, lowered blood lipids and amelio-
rated liver steatosis and liver fibrosis in HFD mice compared with the intervention of
normal saline, where the effect of high-dose SZ-A was better than low-dose SZ-A and
dapagliflozin. The mechanism by which SZ-A improves the lipid metabolism in MAFLD
model mice might be related to the regulation of PGC1
α
-PPAR
α
/PPAR
γ
. In addition, the
mechanism of SZ-A ameliorating oxidative stress and hepatic fibrosis in MAFLD model
mice might be associated with PGC1α-induced KEAP1/NRF2 axis activation.
3.1. SZ-A Improved Liver Lipid Metabolism in MAFLD Mouse Models
Obesity, which leads to the development of metabolic syndrome and multiple co-
morbidities, has become a global health problem [
14
]. With the rising trend in obesity,
Pharmaceuticals 2024,17, 1287 11 of 16
the prevalence and severity of MAFLD are also increasing worldwide [
4
]. At present, the
targeted management of obesity through lifestyle interventions remains the cornerstone of
the treatment of MAFLD [
15
]. In addition to that, medications to ameliorate MAFLD were
also considered to be an important method of therapy. An increasing number of studies
were conducted to find effective agents to alleviate MAFLD through multiple mechanisms.
Our results showed that high-dose SZ-A treatment could significantly reduce the body
weight, decrease the serum lipids, improve insulin tolerance, and ameliorate liver steatosis
and fibrosis in MAFLD model mice. The body-weight-lowering effects of high-dose SZ-A
were independent of food intake, blood glucose, and glucose tolerance, suggesting that
SZ-A might reduce body weight through the regulation of molecules involved in the lipid
metabolism pathway.
In previous studies, PGC1
α
was also considered an important therapeutic target for
type 2 diabetes and obesity through regulating mitochondrial biogenesis and interacting
with transcription factors such as estrogen-related receptors, liver X receptors, and hep-
atic nuclear factor 4
α
to coordinate the expression of mitochondrial genes and indirectly
promote the transport and utilization of fatty acids (FAs), thereby regulating glucolipid
metabolism [
16
–
18
]. Meanwhile, PGC1
α
could also activate the function of PPAR
α
and
PPAR
γ
[
19
,
20
]. PPAR
α
is mainly expressed in the liver and controls many intracellular
processes involved in lipid metabolism, including peroxisome reactions and mitochondrial
fatty acid oxidation (FAO) [
19
]. In contrast, PPAR
γ
is widely expressed in liver cells and adi-
pose tissues, which mainly function in regulating adipocyte differentiation. The incitation
of PPAR
γ
could also increase the proportion of brown adipose tissues and facilitate energy
consumption [
20
]. In previous studies, it was also observed that the overexpression of
PGC1
α
in the liver significantly increased hepatic FA oxidation and decreased TG storage
and secretion both
in vivo
and
in vitro
[
17
], indicating the therapeutic potential of PGC1
α
in improving MAFLD conditions through the abovementioned mechanisms.
These are consistent with our findings. We found that the HFD decreased the ex-
pression of PGC1
α
and its downstream molecule PPAR
α
in the liver, whereas high-dose
SZ-A upregulated the expression of PGC1
α
and PPAR
α
in the liver. At the same time, our
rescue experiment in primary mouse liver cells further demonstrated that PGC1
α
may
serve as a key element for the lipid metabolism regulation effects of SZ-A. In other words,
the improvement in MAFLD by SZ-A treatment was potentially achieved by upregulating
PGC1αsynthesis in the liver.
3.2. SZ-A Improved Liver Fibrosis and Oxidative Stress in MAFLD Model Mice
MAFLD caused by HFD could not only induce simple hepatic steatosis but also cause
oxidative stress in hepatocytes and liver fibrosis, which mutually promote the progression
of MAFLD itself, eventually leading to liver cirrhosis and hepatocellular carcinoma. Ac-
cording to our study, it was suggested that SZ-A could reduce body weight, alleviate lipid
metabolism profiles, decrease indices reflecting liver injury, and retard liver pathological
progression in mice with MAFLD. Furthermore, the MAFLD therapeutic effects of SZ-A
were believed to be associated with the activation of several substances as mentioned
above. Moreover, relevant studies also suggested that SZ-A could activate the NRF2 axis to
inhibit the excitation of hepatic stellate cells, thereby alleviating liver fibrosis [
21
,
22
]. It was
also observed that PGC1
α
could regulate the expression of nuclear/mitochondrial genes
associated with oxidative phosphorylation by coactivating NRF2, indicating that NRF2
may also act as an important pathway through which SZ-A exerts MAFLD-remedying
effects [23].
The KEAP1/NRF2/ARE axis plays a major role in the regulation of cellular redox
balance. NRF2 and its endogenous inhibitor KEAP1, as a universal regulator of intracellular
defense mechanisms, often fight against oxidative stress in the body [
24
]. Under normal
conditions, KEAP1 binds to NRF2 and targets it to accelerate proteasome degradation
and the regeneration of KEAP1 [
25
]. However, in the case of oxidative stress, the interac-
tion between NRF2 and KEAP1 is interrupted, the contents of NRF2 are increased, and
Pharmaceuticals 2024,17, 1287 12 of 16
the synthesis of downstream antioxidant molecules, such as heme oxygenase-1 (HO-1),
glutathione-S-transferase (GST), glutathione peroxidase (GPX), NAD(P)H quinone ox-
idoreductase 1 (NQO1), superoxide dismutase (SOD), catalase (CAT), and glutathione
reductase (GR), is reduced [
26
]. Therefore, NRF2-related pathways have the potential to
improve MAFLD through mechanisms including liver fibrosis suppression and oxidative
stress inhibition.
Our results indicated that high-dose SZ-A could activate the inhibited NRF2 (by HFD),
thereby upregulating the expression of the downstream antioxidant molecules SOD1 and
GPX6 and repressing the expression of the liver fibrosis molecule
α
-SMA. We also found
that PGC1αmight be coactivated with NRF2 to play a downstream activation role. It was
suggested that SZ-A might counteract liver oxidative stress and liver fibrosis induced by
HFD through the KEAP1/NRF2/ARE axis.
In addition, SCD1 is a key enzyme in the production of fat in the liver, which converts
saturated fatty acids into monounsaturated fatty acids. When SCD1 is inhibited, fatty
acid synthesis is restrained and beta-oxidation is increased, resulting in decreased TG
storage in the liver [
27
]. However, previously, no systemic manifestations of lipid reduction
were observed in liver-specific SCD1 gene knockout animal models [
28
]. SCD1 inhibition
was shown to directly reduce the levels of the antioxidant enzyme GPX4 and the ratio of
reduced glutathione/oxidized glutathione (GSH/GSSG), which promoted the increase
in intracellular reactive oxygen species (ROS) and led to mitochondrial redox imbalance,
intracellular lipid peroxidation, and mitochondrial dysfunction [29].
In our study, we found that HFD significantly reduced the contents of SCD1, while
high-dose SZ-A treatment significantly upregulated SCD1 synthesis, suggesting that SCD1
may have a role mainly in antioxidant stress rather than in the promotion of fatty acid
synthesis in the liver.
3.3. Summary
To summarize, based on the study by Morieri, M [
30
], it can be inferred that da-
pagliflozin may indirectly regulate hepatic steatosis by affecting blood glucose levels. To
exclude the possibility that SZ-A also indirectly affects hepatic steatosis through blood
glucose-lowering effects, we conducted a drug control experiment using dapagliflozin. Our
results showed that, compared to dapagliflozin, SZ-A more significantly reduced hepatic
steatosis in mouse liver tissue. Furthermore, when we treated primary hepatocytes from
mice with SZ-A, we observed a notable decrease in the triglyceride content in the liver cells
treated with higher doses of the alkaloids. Thus, we can reasonably infer that SZ-A can
reduce the lipid content in hepatocytes independent of its blood glucose-lowering effects.
In our study, we investigated whether the protective effect of SZ-A on MAFLD mice
is independent of its effect on body weight. We also observed the phenotype that occurs
when SZ-A targets hepatocytes and attempted to explore the mechanism by which SZ-A
acts directly on hepatocytes. Our experimental results suggested that by regulating the
expression of PGC1
α
and its interacting KEAP1/NRF2 pathway in mouse liver cells, SZ-A
played important roles in regulating lipid metabolism, inhibiting oxidative stress, and
postponing liver fibrosis in mice with MAFLD as aforementioned.
4. Materials and Methods
4.1. Establishment of Animal Models
This study was ethically approved by the Institutional Animal Care and Use Commit-
tee (IACUC) of Peking University People’s Hospital (No. 2021PHE022). All protocols were
executed in compliance with the directives outlined in the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals [31].
Beijing Wehand-Bio Pharmaceutical Co., Ltd. (Beijing, China) generously supplied
Ramulus Mori (Sangzhi) alkaloids (SZ-A) powder (lot number: J202107010). The SZ-A
powder contains 60.62% total polyhydroxy alkaloids, with specific contents of 40.75% DNJ,
8.99% FA, and 8.59% DAB [10].
Pharmaceuticals 2024,17, 1287 13 of 16
The mice were kept in an environment maintained at a steady temperature of 26
◦
C,
following a 12 h light and 12 h dark cycle. Each cage housed 3–5 mice and provided
unrestricted access to food and water. Eight-week-old male C57BL/6J mice were fed with
CTRL or 60% HFD for 12 weeks to establish normal mouse models and diet-induced obesity
mouse models. From the 13th week, the HFD mice were administered SZ-A (divided into
two dose groups: 400 mg/kg/d as HFD plus low-dose SZ-A, and 800 mg/kg/d as HFD
plus high-dose SZ-A; SZ-A was dissolved in normal saline; n= 10 for each group) or the
sodium glucose cotransporter-2 (SGLT-2) inhibitor dapagliflozin (1 mg/kg/d, namely HFD
plus dapagliflozin; dapagliflozin was dissolved in normal saline; n= 10), and an equivalent
normal saline (HFD group, n= 10) was included. In addition to this, there was a control
diet group (abbreviated as CTRL, n= 10) that was observed until the 20th week. Fasting
blood glucose and glucose and insulin tolerance were measured at baseline and before
the end of the experiment. The weight and food intake of the mice were measured every
week during the treatment process. At week 20, the mice were sacrificed, and all necessary
samples were acquired. After fasting for 12 h, blood was taken from the heart of the mice.
The liver and adipose tissue (including the visceral fat mass, perirenal adipose tissue, and
epididymal adipose tissue) were cut, rinsed, weighed, and placed in the refrigerator at
−80 ◦C for subsequent determination of relevant measurements.
4.2. Serological Tests
Commercial kits were used to determine the contents of serum glucose, total choles-
terol (TC) (BioVision Research Products, Inc., Milpitas, CA, USA), triglyceride (TG), free
fatty acids (FFAs) (Wako Chemicals, Neuss, Germany), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), alkaline phosphatase (ALP), high-density lipoprotein
cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) (Cayman Chemical Com-
pany, Ann Arbor, MI, USA), adiponectin, and leptin (Solarbio, Beijing, China).
4.3. Histological Examination
The liver tissues were stained with hematoxylin-eosin (HE), Masson trichrome, and
Sirius Red to observe the degrees of liver morphological changes, hepatic steatosis, and
hepatic fibrosis.
4.4. Gene and Protein Level Detection
We examined the expression levels of genes and proteins related to lipid metabolism
in mouse liver tissues.
The mRNA and protein levels of adipogenesis-related factors were detected, includ-
ing fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC) peroxisome proliferator-
activated receptor
γ
(PPAR
γ
), peroxisome proliferator-activated receptor
α
(PPAR
α
), perox-
isome proliferator-activated receptor-
γ
coactivator 1
α
(PGC1
α
), liver X receptor
α
(LXR
α
),
nuclear factor erythroid-2-related factor 2 (NRF2), short-chain acyl-coenzyme A dehydro-
genase (SCAD), stearyl coenzyme A dehydrogenase-1 (SCD1), superoxide dismutase-1
(SOD1), superoxide dismutase-2 (SOD2), glutathione peroxidase 6 (GPX6), matrix metallo-
proteinase 9 (MMP9), proprotein convertase subtilisin/kexin type 9 (PCSK9), 3-hydroxy-3-
methylglutaryl-coenzyme A reductase (HMGCR), mitochondrial fusion protein (MFN2),
translocase of outer mitochondrial membrane 20 (TOM20), and alpha-smooth muscle actin
(
α
-SMA). Meanwhile, we used the co-Immunoprecipitation kit (Absin, Shanghai, China)
to detect whether PGC1
α
interacts with NRF2. All antibodies were sourced from Abmart
Shanghai Co., Ltd. (Shanghai, China)
4.5. Isolation of Murine Hepatocytes
We used the protocols developed by Malar et al., 2007 [
32
] for the isolation of hepato-
cytes. We took the liver from the animal and placed it in a sterile petri dish containing cold
phosphate buffered saline (PBS). The liver was then dissected into small pieces using sterile
instruments and transferred to a suitable digestive solution in a petri dish. After the liver
Pharmaceuticals 2024,17, 1287 14 of 16
fragments were incubated at the appropriate temperature for a period, they were digested
using pancreatic enzymes. The cell suspension was then filtered with a sterile strainer or
filter to remove undigested tissue and obtain a single-cell suspension. Cell suspensions
were washed with a culture medium to neutralize enzymes and remove debris. The cell
suspension was centrifuged, and the liver cells were collected at the bottom of the tube.
The liver cells were suspended in a suitable medium. Finally, the cells were placed in a
petri dish to promote cell attachment and growth. All consumables were purchased from
Solarbio Science & Technology Co., Ltd. (Beijing, China)
4.6. Rescue Experiment of Liver Primary Cells
The liver cells of wild-type C57BL/6J mice were collected for the rescue experiment.
Oleic acid (OA) and palmitic acid (PA) were added to 1640 complete medium at a ratio
of 200:100 to simulate a high-lipid environment, where the liver primary cells in 12-well
plates were divided into 4 treatment groups: high-fat group (oleic acid [OA] + dimethyl
sulfoxide [DMSO]); high-fat + PGC1
α
inhibitor group (SR18292, dissolved with DMSO,
concentration 25
µ
M) (OA + SR); high-fat + SZA group (OA + SZA, concentration 100
µ
M);
high-fat + SR + SZA
group (OA + SR + SZA). After being processed with the above treat-
ments for 48 h, the cells were collected for Oil Red O staining and cell TG content detection.
Kits measuring TG, reactive oxygen species (ROS), and mito-tracker were purchased from
Solaibao Technology company (Beijing, China). Other reagents and consumables were
purchased from Beyotime Biotechnology (Shanghai, China).
4.7. Statistical Analysis
The analysis of protein expression and adipocyte area was conducted using Image
J software, version 1.53t (NIH, Bethesda, MD, USA). Graphs illustrating the results were
generated with Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). Statistical
analyses were implemented with IBM SPSS Version 26.0. Statistical differences between
two groups were examined by Student’s t-test. Analysis of variance (ANOVA) and multiple
comparisons were applied for the comparisons among more than two groups. The results
are presented in the format of mean
±
standard error of the mean (SEM). Statistical
significance was considered at p< 0.05.
5. Conclusions
In this study, we found that treatment with SZ-A could promote hepatic cellular
lipid metabolism, reduce hepatic lipid deposition, alleviate hepatic oxidative stress, and
attenuate hepatic fibrosis. The key lipid metabolism (PGC1a/PPAR
γ
) and antioxidant
stress (KEA2/NRF2) pathways played essential roles in the function of SZ-A in alleviating
MAFLD progression. We also preliminarily observed a potential interaction between
PGC1a and NRF2, which also suggested that lipid metabolism and oxidative stress were
mutually complementary in MAFLD and that SZ-A was likely to be an effective drug to
simultaneously improve lipid metabolism and oxidative stress.
Author Contributions: M.Z. contributed to performing the experiment and was involved in drafting
the manuscript. C.G. made substantial contributions to performing the experiment, analyzing the
data, and drafting the manuscript. Z.L. was in charge of drafting the manuscript. X.C. was in charge
of the conception, design, and analysis of data and was also involved in drafting the manuscript,
revising it critically for important intellectual content, and giving final approval of the version to
be published. F.L. contributed to performing the experiment. X.W. was involved in the design and
conduct of the experiment. C.L. was involved in drafting the manuscript. L.J. made contributions
to designing the experiment and give final approval of the version to be published. Each author
participated sufficiently in the work to take public responsibility for appropriate portions of the
content and agreed to be accountable for all aspects of the work in ensuring that questions related
to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
All authors have read and agreed to the published version of the manuscript.
Pharmaceuticals 2024,17, 1287 15 of 16
Funding: This work was supported by the Chinese Journal of Diabetes (No. 2119000446). The
funding agencies had no roles in the study design, data collection or analysis, decision to publish, or
preparation of the manuscript.
Institutional Review Board Statement: This study was ethically approved by the Institutional
Animal Care and Use Committee (IACUC) of Peking University People’s Hospital (No. 2021PHE022).
All protocols were executed in compliance with the directives outlined in the National Institutes of
Health (NIH) Guide for the Care and Use of Laboratory Animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are provided in the original text.
Conflicts of Interest: L.J. has received fees for lecture presentations and for consulting from As-
traZeneca, Merck, Metabasis, MSD, Novartis, Eli Lilly, Roche, Sanofi-Aventis, and Takeda. All authors
have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf (accessed
on 17 August 2024, available on request from the corresponding authors) and declare no other support
from any organization for the submitted work other than that described above.
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