Available via license: CC BY 4.0
Content may be subject to copyright.
Dingetal. Critical Care (2023) 27:196
https://doi.org/10.1186/s13054-023-04488-5
RESEARCH Open Access
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco
mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Critical Care
Fibroblast growth factor 21 attenuates
ventilator-induced lung injury byinhibiting
theNLRP3/caspase-1/GSDMD pyroptotic
pathway
Peng Ding1,2†, Rui Yang1†, Cheng Li1†, Hai‑Long Fu1, Guang‑Li Ren2, Pei Wang3, Dong‑Yu Zheng1, Wei Chen1,
Li‑Ye Yang1, Yan‑Fei Mao4*, Hong‑Bin Yuan1* and Yong‑Hua Li1*
Abstract
Background Ventilator‑induced lung injury (VILI) is caused by overdistension of the alveoli by the repetitive recruit‑
ment and derecruitment of alveolar units. This study aims to investigate the potential role and mechanism of fibro‑
blast growth factor 21 (FGF21), a metabolic regulator secreted by the liver, in VILI development.
Methods Serum FGF21 concentrations were determined in patients undergoing mechanical ventilation during
general anesthesia and in a mouse VILI model. Lung injury was compared between FGF21‑knockout (KO) mice and
wild‑type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect.
Results Serum FGF21 levels in patients and mice with VILI were significantly higher than in those without VILI.
Additionally, the increment of serum FGF21 in anesthesia patients was positively correlated with the duration of
ventilation. VILI was aggravated in FGF21‑KO mice compared with WT mice. Conversely, the administration of FGF21
alleviated VILI in both mouse and cell models. FGF21 reduced Caspase‑1 activity, suppressed the mRNA levels of Nlrp3,
Asc, Il-1β, Il-18, Hmgb1 and Nf-κb, and decreased the protein levels of NLRP3, ASC, IL‑1β, IL‑18, HMGB1 and the cleaved
form of GSDMD.
Conclusions Our findings reveal that endogenous FGF21 signaling is triggered in response to VILI, which protects
against VILI by inhibiting the NLRP3/Caspase‑1/GSDMD pyroptosis pathway. These results suggest that boosting
endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for the
treatment of VILI during anesthesia or critical care.
Keywords Ventilator‑induced lung injury, Fibroblast growth factor 21, Pyroptosis, NLRP3, Caspase‑1, Gasdermin D
†Peng Ding, Rui Yang, Cheng Li contributed equally.
*Correspondence:
Yan‑Fei Mao
maoyanfei@xinhuamed.com.cn
Hong‑Bin Yuan
jczyy@aliyun.com
Yong‑Hua Li
liyonghua1207@smmu.edu.cn
Full list of author information is available at the end of the article
Page 2 of 15
Dingetal. Critical Care (2023) 27:196
Background
Mechanical ventilation (MV) is an important component
of general anesthesia and an indispensable respiratory
support therapy for critically ill patients. However, MV
can cause lung injury, namely ventilator-induced lung
injury (VILI), which predisposes patients to inflamma-
tory response syndrome or multiple organ failure with a
mortality rate of nearly 50% [1], making it an urgent clini-
cal problem to be solved. e primary causes of VILI are
mechanical power and the duration of ventilator expo-
sure [2]. Current studies suggest that VILI is not only a
mechanical trauma but also a biotrauma, which activates
a complex signaling cascade in the lung [3, 4]. Small tidal
volumes and low airway pressures may reduce the mor-
bidity and mortality of VILI [5]. In addition to canoni-
cal inflammation-related molecules including innate
immune cytokines and chemokines [6], the permeability-
originating obstruction response in which alveolar leak-
age leads to surfactant dysfunction and increases local
tissue stresses also plays a critical role in VILI [7]. us,
it is of great importance to understand the molecular
mechanism of VILI and develop new preventive/thera-
peutic interventions.
Fibroblast growth factors (FGFs) are a family of struc-
turally related proteins with diverse biological functions
during embryonic development, tissue injury/repair,
tumorigenesis, and metabolic homeostasis. To date, 23
members of the FGF family have been identified, all of
which are referred to as “pluripotent” growth factors and
as “promiscuous” growth factors due to their multiple
actions on a wide range of cell types. FGF21, a member
of the FGFs, was first identified and cloned in 2000 [8].
FGF21 is highly expressed in the liver and can be secreted
into the blood [9]. Numerous clinical and basic studies
have shown that FGF21 is involved in metabolic diseases
such as diabetes, obesity, and nonalcoholic fatty liver dis-
ease [10–12]. Interestingly, FGF21 has been reported to
be involved in lipopolysaccharide-induced lung injury
[13], and the emerging roles of FGF21 in acute lung
injury/acute respiratory distress syndrome, acute myo-
cardial injury, acute kidney injury, sepsis, and other criti-
cal diseases are increasingly noteworthy [14]. Moreover,
several FGF21 analogs, such as Pegbelfermin (Bristol-
Myers Squibb), LY2405319 (Eli Lilly), and PF05231023
(Pfizer), have passed Phase I/II trials and were reported
to be generally well tolerated and effective in treating
obesity or diabetes [15–17].
Currently, there is no knowledge regarding the roles
of FGF21 in VILI. We hypothesized that FGF21, which
is able to protect the blood–brain barrier and reduce
inflammation [18, 19], may play a role in the develop-
ment and progression of VILI and, if so, further explore
the underlying molecular mechanism.
Materials andmethods
Patient enrollment andblood sample preparation
Patients undergoing general anesthesia were recruited
consecutively from November 2020 to February 2021
at Shanghai Changzheng Hospital. Informed consent
was obtained from all subjects, and the protocol was
approved by the Ethics Committee of Biomedicine of
Naval Medical University. Patients with endotracheal
intubation and mechanical ventilation under general
anesthesia aged 45–70years, and ASA status I–II were
enrolled. Exclusion criteria included: severe lung, liver
or renal dysfunction, severe infection, malignancy, type
2 diabetes, obese patients (BMI > 30kg/m2), estimated
intraoperative blood loss > 500ml, and estimated ventila-
tion duration less than 2h.
After the patient entered the operating room, electro-
cardiogram monitoring was established and radial artery
catheterization was performed. Five milliliters of blood
was drawn through the arterial catheter and injected into
a coagulation-promoting tube, which was left at room
temperature for 30min and then centrifuged at 3000g
for 10min. Serum was collected and stored at − 80 °C.
e patients were routinely subjected to endotracheal
intubation and intravenous-inhalation combined anes-
thesia. e ventilation parameters were set as follows:
volume-controlled ventilation mode, 8–10 ml/kg tidal
volume, 12 breaths/minute, 1:2 inspiration/expiration
ratio, 3–5cm H2O positive end-expiratory pressure, and
60–100% inhalation oxygen concentration. At the end
of the operation and before extubation, 5ml of arterial
blood was extracted, and serum was collected and stored
in the same way. Two tubes of serum were collected from
each patient before and after mechanical ventilation. If
hemolysis or lipid clots occurred in any tube of serum,
the pair of samples were discarded, and the patient was
excluded. Serum levels of FGF21 were measured by an
ELISA kit (ab222506, Abcam, USA) according to the
manufacturer’s instructions.
Animals andthemouse VILI model
Male C57BL/6 mice were purchased from Sippr/BK Lab
Animal Co., Ltd (Shanghai, China). FGF21 global knock-
out mice (C57BL/6N-Fgf21em1Cyagen, NCBI ID 56,636)
were obtained from Cyagen Biosciences Inc (Santa Clara,
CA, USA). Details of the breeding and identification of
gene-edited mice are provided in the Additional file 1:
Supplementary content. e male homozygote and wild-
type mice from the same litter were used in subsequent
experiments. e mice were housed in individually ven-
tilated cages under a specific pathogen-free conditions
with a controlled temperature and a 12-h light–dark
cycle. All animal experiments were approved by the
Ethics Committee of Biomedicine of Naval Medical
Page 3 of 15
Dingetal. Critical Care (2023) 27:196
University, were performed in compliance with the
National Institutes of Health Guide for Care and Use of
Laboratory Animals, and were reported in accordance
with the Animal Research: Reporting In Vivo Experi-
ments (ARRIVE) guidelines 2.0 [20].
e mice were fasted for 12h before the experiment.
After anesthetization by an intraperitoneal injection of
ketamine (100mg/kg) and xylazine (10mg/kg), the mice
were intubated orally with a 22-G catheter and then con-
nected to a small animal-specific ventilator (VentElite,
Harvard Apparatus, USA) and placed on a warm pad.
e mice were ventilated with a tidal volume of 30ml/kg
at 70 breaths/min for 4h [21], and control mice under-
went intubation but breathed spontaneously. After the
modeling, the mice were resuscitated and kept for 24h;
then, the mice were killed for subsequent experiments.
Primary cell culture andcyclic mechanical stretch
Primary lung microvascular endothelial cells (LMEVCs)
were extracted from male neonatal C57BL/6N mice
(3 days old) using a tissue block attachment method
(Additional file 1: Supplementary content). Complete
ECM medium (ScienCell, USA) containing 5% fetal
bovine serum, 1% triple antibiotics, and endothelial cell
growth factors was used. Cells were identified by immu-
nocytochemistry with the endothelial marker CD31.
ird-generation LMVECs were inoculated into a 6-well
Bioflex plate (Flexcell, USA). Cells were subjected to
cyclic mechanical stretch (MS) in the Flexcell FX-5000
system using the following parameters: 0.5Hz (30 times/
minute); 20% max elongation; 4-h duration. After mod-
eling, the cells were treated with rFGF21 or phosphate-
buffered saline (PBS), transferred to a conventional
incubator (37°C, 5% CO2), and cultured for 24h before
subsequent experiments.
Cell viability andcytotoxicity assay
Cell viability was assessed using a cell counting kit (CCK-
8, Epizyme). e level of lactate dehydrogenase (LDH)
in the cell culture supernatant was measured by an LDH
cytotoxicity assay kit (J2380, Promega).
FGF21 administration
Male C57BL/6 mice (6–8 weeks old, 20–24 g) were
randomly divided into 5 groups (Additional file1: Sup-
plementary content). Recombinant mouse FGF21 (HY-
P7173, MedChemExpress, Monmouth Junction, NJ) was
dissolved in PBS and injected intraperitoneally at the end
of VILI modeling. e dose (0.75/1.5/3.0 mg/kg) was
based on previous studies of rFGF21 in the treatment of
blood–brain barrier injury [19] and acute kidney injury
[22].
Bronchoalveolar lavage uid
Bronchoalveolar lavage fluid (BALF) was collected,
and the cells in BALF were stained with hematoxylin &
eosin (H&E) dye for sorting and counting. e protein
concentration in the supernatant was determined using
a BCA protein assay kit (23,227, ermo Fisher).
Histology
Lung injury was assessed based on the microscopic
examination of slices stained with H&E dye and a five-
point numeric scores (Additional file1: Supplementary
content) [23], which was performed by a well-trained
colleague in a single blind manner.
TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP
nick-end labeling (TUNEL) was performed to evaluate
cell death using a commercial kit (G1501, Servicebio)
according to the manufacturer’s instructions.
Evans blue index
e mice were injected with 0.1ml of 0.5% Evans blue
dye (sc-203736A, Santa Cruz) through the femoral vein.
Equal weights of tissue were taken from each of the
two lungs; one tissue block was measured to determine
its dry weight, and the other was examined for its dye
content (Additional file1: Supplementary content). e
Evans blue index is expressed as the amount of Evans
blue dye per unit weight of lung tissue (ng/mg tissue).
Oxidation stress measurement
Reactive oxygen species (ROS) were detected by a
dichlorodihydrofluorescein diacetate (DCFH-DA)
probe with a BioTek Gen 5 instrument (Ex 488 nm,
Em 525 nm) and a fluorescence microscope (Leica
DMI4000B, Germany). Myeloperoxidase (MPO) activ-
ity was measured using a commercial kit (A044-1-1,
Jiancheng Biotech) according to the manufacturer’s
instructions. e total antioxidant capacity in lung tis-
sues/cell homogenate was measured using a commer-
cial kit (S0119, Beyotime). Superoxide dismutase (SOD)
activity was measured using a kit (S0109, Beyotime)
based on the nitroblue tetrazolium reduction reaction.
Mitochondrial membrane potential andapoptosis
detection
is experiment was performed using a trichrome flu-
orescent staining kit (C1071, Beyotime) according to
the manufacturer’s instructions. Mitochondria were
labeled with Mito-tracker Red CMXRos (red), dead
cells were labeled with Annexin V-FITC (green), and
nuclei were labeled with Hoechst 33,342 (blue). e
Page 4 of 15
Dingetal. Critical Care (2023) 27:196
fluorescence intensity was measured by a BioTex Gen
5 instrument (Biotex, USA), and Hoechst 33,342 was
used as an internal reference to compare the differences
in the fluorescence intensities of mitochondrial and
dead cells between the groups.
Caspase‑1 activity
Caspase-1 activity was assessed using a commercial
kit (C1101, Beyotime) according to the manufacturer’s
instructions. Briefly, lung tissues were homogenized and
lysed. e lysates were incubated with Ac-YVAD-pNA
(2mM) at 37°C for 2h. en, the absorbance was meas-
ured at 405nm using a BioTek Gen 5 instrument (BioTek,
USA), and the activity was calculated according to the
standard curve.
Quantitative real‑time PCR
Lung tissues were homogenized in RNAiso reagent
(9108, Takara), and total RNA was extracted and reverse-
transcribed into cDNA using PrimeScript RT Master
Mix (RR036A, Takara). Primers (Additional file1: Sup-
plementary Table3) were designed using Primer Express
software (Applied Biosystem, USA). e reaction was
performed in a QuantStudio 5 system (ermo Fisher,
USA) with a QuantiNova SYBR Green PCR Kit (208,056,
Qiagen). e housekeeping gene Actb was used as an
internal control, and the relative gene expression was
analyzed using the 2−ΔΔCt method.
Western blotting
Western blotting was performed as described previ-
ously [24]. e tissues were homogenized in RIPA lysis
buffer (P0013B, Beyotime) containing a protease inhibi-
tor (GRF101, Epizyme). e samples were separated on
a 10% SDS–PAGE gel, and the proteins were transferred
onto a nitrocellulose membrane, which was blocked in
protein-free rapid blocking buffer (PS108, Epizyme). e
membranes were incubated with the primary antibody
(Additional file1: Supplementary Table4) overnight at
4°C, washed and incubated with IRDye-conjugated sec-
ondary antibodies (LI-COR, Lincoln, NE) for 1h at room
temperature. Images were obtained using an Odyssey
infrared imaging system (LI-COR). Quantitative analysis
was performed using ImageJ software (National Insti-
tutes of Health, USA).
Statistics
Data normality was assessed by the Shapiro–Wilk test.
e data are presented as mean ± standard error of mean
(mean ± SEM) or median [quartile 1, quartile 3] accord-
ing to the distribution. e intergroup difference was
analyzed by Student’s t-test, paired t-test, or one-way
ANOVA followed by LSD post hoc test according to the
grouping design (Prism 9.0, GraphPad software, CA).
P < 0.05 was considered statistically significant.
Results
FGF21 isinduced afterMV inpatients andmice
We collected blood samples from 69 patients who under-
went MV during surgery and compared the baseline
and postsurgery serum levels of FGF21 (patient charac-
teristics in Table1). e serum levels of FGF21 in these
patients were significantly induced by MV (190.6 ± 10.2
vs. 152.9 ± 7.9 pg/ml, Fig.1A). When these patients were
divided into two subgroups based on the duration of ven-
tilation (< 4h and > 4 h), we found a more pronounced
increase in the latter group: the mean serum FGF21 level
increased from 154.9 ± 11.4pg/ml to 175.5 ± 13.3 pg/ml
in patients with MV < 4 h (Fig. 1B), while it increased
from 151.1 ± 11.4pg/ml to 204.4 ± 15.0pg/ml in patients
with MV > 4 h (Fig. 1C). Additionally, Pearson correla-
tion analysis showed that the elevated serum FGF21 lev-
els were positively correlated with the duration of MV
(Fig.1D).
Next, we investigated the influence of MV on serum
and tissue FGF21 levels in a mouse VILI model. Serum
FGF21 levels in VILI mice at 24 h or 48 h after MV
were significantly higher than those in the control mice
(Fig. 1E). We also examined FGF21 protein expression
in the liver and lung tissue of mice with VILI and found
that FGF21 protein expression was barely detectable
in the lung but was expressed abundantly in the liver.
Moreover, hepatic FGF21 protein levels were upregulated
by VILI (Fig. 1F). IL-1β and IL-18, two proinflamma-
tory cytokines, were induced by MV (Fig.1G&H). ese
Table 1 Patient characteristics
Categorical variables are presented as N (%), continuous variables are presented
as mean ± SEM or median [quartile 1, quar tile 3]. ASA, American Society of
Anesthesiologists; FGF21, broblast growth factor 21
Total (N = 69)
Age (yr) 57 ± 1
Female (%) 11 (16)
Body mass index (kg/m2) 23.7 ± 0.4
ASA physical status (%)
I 21 (30)
II 48 (70)
Mechanical ventilation duration (hr) 4.5 [3.0, 6.5]
Surgery type (%)
Spine surgery 48 (70)
Brain surgery 11 (16)
Gastrointestinal surgery 10 (14)
Baseline FGF21 (pg/ml) 152.9 ± 7.9
Postoperative FGF21 (pg/ml) 190.6 ± 10.2
Page 5 of 15
Dingetal. Critical Care (2023) 27:196
results suggest that the increased serum FGF21 may be
associated with VILI.
Deletion ofFGF21 aggravates VILI inmice
We next used a mouse strain with global knockout of
FGF21 to examine the pathophysiological role of FGF21
in VILI (Fig. 2A). Using immunoblotting, we con-
firmed the deficiency of FGF21 in KO mice (Fig. 2B).
H&E staining showed that there were no abnormali-
ties in the unventilated lung tissues of KO mice and WT
mice. However, VILI pathologies, including pulmonary
edema and inflammatory cell infiltration, were evident
Fig. 1 FGF21 is induced after MV in patients and mice. A. The average serum FGF21 increased in patients after mechanical ventilation (N = 69); B.
The average serum FGF21 increased in patients after short duration (< 4 h) of mechanical ventilation (N = 33); C. The average serum FGF21 increased
in patients after long duration (> 4 h) of mechanical ventilation (N = 36); D. The elevated FGF21 level was positively correlated with duration of
mechanical ventilation; E. Serum FGF21 increased in VILI mice (N = 6, 0 h referred to the time point immediately after VILI modeling); F. FGF21
protein expression in mice lung/liver tissues after mechanical ventilation; G. Serum IL‑1β increased in VILI mice (N = 6); H. Serum IL‑18 increased in
VILI mice (N = 6); ns, no significance, *P < 0.05, **P < 0.01; MV, mechanical ventilation; VILI, ventilator‑induced lung injury
Page 6 of 15
Dingetal. Critical Care (2023) 27:196
Fig. 2 Deletion of FGF21 aggravates VILI in mice. A. Timeline of the loss‑of‑function experiment; B. FGF21 protein expression in liver of KO/WT
mice (N = 3); C. Hematoxylin–eosin staining of lung tissues, and the average lung injury scores analysis of lung slices (N = 5, scale bar = 500 μm
in low‑power images and = 50 μm in amplified images); D. TUNEL staining of lung tissues, and dead cell count of lung slices (N = 5, scale
bar = 100 μm); E. H&E staining of exfoliated cells in BALF, and the total cell count in BALF (N = 6, scale bar = 20 μm); F. Neutrophil count in BALF; G.
MPO activity in lung tissue (N = 6); H. Protein concentration in BALF (N = 6); I. Wet/dry ratio of lung tissue (N = 6); J. The content of Evans blue dye
in lung tissue (N = 6); ns, no significance, *P < 0.05, **P < 0.01; WT, wild‑type; KO, knockout; MV, mechanical ventilation; VILI, ventilator‑induced lung
injury; TUNEL, TdT‑mediated dUTP nick end labeling; DAPI, 4’,6‑diamidino‑2‑phenylindole; BALF, bronchoalveolar lavage fluid; MPO, Myeloperoxidase
Page 7 of 15
Dingetal. Critical Care (2023) 27:196
in WT mice and more pronounced in KO mice (Fig.2C).
TUNEL staining showed that the number of TUNEL+
cells was increased by VILI in WT mice and, to a great
extent, in KO mice (Fig.2D). We used H&E staining to
assess the number of cells in BALF and found that the
number of total cells in the BALF of KO mice was sig-
nificantly higher than that in WT mice in the VILI model
(Fig.2E), most of which were neutrophils (Fig.2F). We
also measured MPO activity, which is a marker of neu-
trophils, and found that FGF21 knockout further pro-
moted MPO activity in the context of VILI (Fig. 2G).
BALF protein levels were induced by VILI and were more
pronounced in KO mice (Fig.2H), suggesting that FGF21
gene knockout aggravated pulmonary microvascular bar-
rier disruption during VILI. e results of the wet/dry
ratio of lung tissue and Evans blue staining demonstrated
that vascular barrier permeability was damaged by VILI
in WT mice and, to a great extent, in FGF21-KO mice
(Fig. 2I&J). ese aggravated VILI-related pulmonary
pathologies in FGF21-KO mice indicate that endogenous
FGF21 may be a protective factor in VILI.
FGF21 treatment alleviates VILI inmice
To further validate the therapeutic effect of FGF21,
we administered mouse-origin recombinant FGF21
(rFGF21) in a mouse VILI model. H&E staining showed
that ventilation-induced damages, including mas-
sive inflammatory cell infiltration, severe interstitial
edema, intra-alveolar hemorrhage, were found allevi-
ated in groups with mid- and high doses of rFGF21. In
contrast, a low dose of rFGF21 failed to protect against
VILI (Fig. 3A). Similar dose-dependent therapeutic
effects were observed by TUNEL staining (Fig.3B). H&E
staining showed that the number of total cells as well as
neutrophils in BALF was decreased significantly after
rFGF21 administration, suggesting that FGF21 may play
a role in reducing the infiltration and exudation of proin-
flammatory cells in the lung (Fig.3C and D). Evans blue
accumulation was enhanced in VILI mice and reduced
by rFGF21 (Fig.3E). Accordingly, the protein content in
BALF increased significantly after VILI but decreased
significantly in all FGF21-treated groups (Fig. 3F). e
increased W/D ratio of the lung was also reduced in
FGF21-treated groups (Fig. 3G). ese results suggest
that FGF21 alleviates microvascular barrier damage and
pulmonary edema in a dose-dependent manner. We next
determined oxidative stress in lung tissues. MPO activity
induced by VILI was significantly decreased by rFGF21
treatment (Fig. 3H). e decrease in total antioxidant
capacity, which was evaluated by Trolox-equivalent anti-
oxidant capacity (TEAC), was reversed by rFGF21 treat-
ment (Fig. 3I). Moreover, rFGF21 treatment restored
the ATP levels in the mitochondrial fraction of lung tis-
sue from VILI mice (Fig.3J). ese results suggest that
FGF21 might be able to alleviate VILI in mice.
FGF21 treatment reduces endothelial injury
anddownregulates pulmonary interstitial pro‑brosis
factors
Next, we evaluated endothelial injury and pulmonary
interstitial changes in a VILI mouse model. Insitu immu-
nofluorescence staining of VE-cadherin, a marker of
endothelial junctions, clearly showed that the VE-cad-
herin fluorescence intensity in the microvessels of intact
blood-perfused lungs was significantly decreased after
MV. However, FGF21 treatment attenuated the decline of
VE-cadherin fluorescence intensity in a dose-dependent
manner (Fig.4A). We also detected pulmonary intersti-
tial fibrosis tendency by immunofluorescence staining
of pro-fibrosis factors α-SMA and vimentin. e result
showed that α-SMA was induced in lung tissues by MV,
and this effect was partially prevented by mid- and high
doses of rFGF21 (Fig.4B). Immunofluorescence staining
of vimentin showed that the three doses of rFGF21 effi-
ciently suppressed MV-induced vimentin expression in
lung tissue (Fig.4C). ese results indicate that FGF21
treatment protects against MV-induced endothelial
injury and inhibits pro-fibrosis factors.
FGF21 treatment ameliorates MS‑induced injury inacell
model
FGF21 can impact whole-body metabolism and bio-
logical functions through multiple mechanisms,
including endocrine, paracrine, and autocrine effects
[25]. To clarify how FGF21 protects against VLLI in
cellular level, we established a mechanical stretch
model in cultured primary LMVECs, in which FGFR1,
the major receptor of FGF21, was highly expressed
(Fig.5A). Cell viability was decreased significantly by
cyclic MS stress and was partially restored by FGF21
Fig. 3 FGF21 treatment alleviates VILI in mice. A. Hematoxylin–eosin staining of lung tissues and average lung injury scores analysis of lung slices
(N = 5, scale bar = 500 μm in low‑power images and = 50 μm in amplified images); B. TUNEL staining of lung tissues and dead cells counting of
lung slices (N = 5, scale bar = 100 μm); C. H&E staining of exfoliated cells and total cell counting in BALF (N = 6, scale bar = 20 μm); D. Neutrophil
count in BALF; E. The content of Evans blue dye in lung tissue; F. Protein concentration in BALF; G. Wet/dry ratio of lung tissue; H. MPO activity in
lung tissue. I. Trolox‑equivalent antioxidant capacity in mice lung tissue. J. ATP level in mice lung tissue; K. Timeline of the treating experiment. ns,
no significance, *P < 0.05, **P < 0.01 vs. Control; #P < 0.05, ##P < 0.01 vs. PBS; MV, mechanical ventilation; TUNEL, TdT‑mediated dUTP nick end labeling;
DAPI, 4’,6‑diamidino‑2‑phenylindole; BALF, bronchoalveolar lavage fluid; MPO, Myeloperoxidase; TEAC, Trolox‑equivalent antioxidant capacity
(See figure on next page.)
Page 8 of 15
Dingetal. Critical Care (2023) 27:196
Fig. 3 (See legend on previous page.)
Page 9 of 15
Dingetal. Critical Care (2023) 27:196
treatment (Fig.5B). The levels of LDH in the culture
medium were examined to determine cell integrity.
We observed that the LDH content in the culture
medium was increased significantly by MS, whereas
rFGF21 treatment reduced this change (Fig.5C). We
also evaluated oxidative stress in this cell model. Intra-
cellular ROS levels were determined by a DCFH-DA
probe. Cyclic MS stress significantly induced ROS
levels in LMVECs, while rFGF21 treatment inhibited
ROS production (Fig. 5D). The TEAC assay results
showed that FGF21 rescued the impaired antioxidant
capacity induced by MS stress in LMEVCs (Fig. 5E).
The activity of SOD, an important antioxidant enzyme,
was decreased significantly by MS and was restored
by FGF21 treatment (Fig. 5F). Mitochondrial dys-
function has been reported to play a key role in the
pathophysiology of VILI [26]. We also measured the
influence of rFGF21 on mitochondrial dysfunction
in the cell model. Intracellular ATP levels were dra-
matically reduced by MS stress, but were rescued by
Fig. 4 FGF21 treatment inhibits endothelial injury and downregulates pulmonary interstitial pro‑fibrosis factors. A. Immunofluorescence of
vascular endothelial marker, VE‑cadherin, in lung tissue; B. Immunofluorescence of pulmonary interstitial marker, α‑SMA, in lung tissue; C.
Immunofluorescence of pulmonary interstitial marker, vimentin, in lung tissue; N = 6 in A‑C; ns, no significance, *P < 0.05, **P < 0.01 vs. Control;
#P < 0.05, ##P < 0.01 vs. PBS; Bar scale = 100 μm; MV, mechanical ventilation; DAPI, 4’,6‑diamidino‑2‑phenylindole
Page 10 of 15
Dingetal. Critical Care (2023) 27:196
rFGF21 (Fig. 5G). Mitochondrial membrane poten-
tial was examined by immunofluorescent staining.
The cells underwent MS stress exhibited an obvious
increase in Annexin immunofluorescence (green) and
a decrease in mitochondrion immunofluorescence
(red), which was largely inhibited by rFGF21 (Fig.5H).
These results indicate that FGF21 ameliorates cellular
injury, oxidative stress, and mitochondrial dysfunction
in a cell model of mechanical stretch.
Fig. 5 FGF21 treatment ameliorates MS‑induced injury in a cell model. A. FGFR1 expression in mouse liver, lung, and primary lung microvascular
endothelial cells; B. Cell viability assessment by CCK8 assay (N = 6); C. Relative LDH level in cell culture supernatant (N = 6); D. Detection of reactive
oxygen species by dichlorodihydrofluorescein probe in situ (N = 3, scale bar = 100 μm); E. Trolox‑equivalent antioxidant capacity in cells (N = 6); F.
Superoxide dismutase activity in cells (N = 6). G. Adenosine triphosphate level in cells (N = 6); H. Mitochondrial membrane potential and cell death
staining (N = 6, scale bar = 100 μm); ns, no significance, *P < 0.05, **P < 0.01 vs. Control; #P < 0.05, ##P < 0.01 vs. PBS; FGFR1, fibroblast growth factor
receptor 1; LMVESs, lung microvascular endothelial cells; MS, mechanical stretch; LDH, lactate dehydrogenase; DCF, dichlorodihydrofluorescein;
TEAC, Trolox‑equivalent antioxidant capacity; SOD, Superoxide dismutase
Page 11 of 15
Dingetal. Critical Care (2023) 27:196
FGF21 protects againstVILI byinhibiting theNLRP3/
Caspase‑1/GSDMD pyroptotic pathway
Next, we sought to decipher the molecular mecha-
nism underlying the protective effect of FGF21 on VILI.
Pyroptosis, which is a form of programmed cell death, is
critically involved in acute lung injury and VILI [27, 28].
Previously, pyroptosis was believed to be mediated by the
NLRP3 inflammasome [29]; currently, pore formation
in the cell membrane mediated by gasdermin (GSDM)
proteins, especially caspase-1 cleaved GSDMD [30], is
thought to be the crucial characteristic of pyroptosis.
We examined the activity of caspase-1 in the lung tissues
and found that caspase-1 activity was triggered (~ four-
fold) in VILI model mice, while the administration of
rFGF21 abolished this change (Fig.6A). e mRNA lev-
els of pyroptotic factors, including Nlrp3, Asc, Casp1 and
Gsdmd, increased significantly in VILI mouse’s lung tis-
sue (Fig.6B). Accordingly, the mRNA levels of Il-1β and
Il-18, two proinflammatory factors released by pyroptotic
cells, were enhanced by VILI but suppressed by rFGF21
treatment (Fig. 6C). Similar changes in high mobility
group protein 1 (Hmgb1), a delivery protein of pyropto-
sis [31], were observed (Fig.6D). Gene expression of the
proinflammatory factors Nf-κb and Rela was also induced
by VILI and inhibited by rFGF21 (Fig.6E).
We further examined the influence of FGF21 on the
protein expression of pyroptotic factors. Similar to the
changes in mRNA levels, the protein levels of NLRP3 and
ASC were significantly upregulated in VILI mice, and this
effect was prevented by FGF21 treatment (Fig.6F). Total
caspase-1 was slightly induced by VILI, while cleaved
caspase-1, the activated form of caspase-1, was mark-
edly induced by VILI, and FGF21 treatment success-
fully suppressed this change (Fig.6F). Mature IL-18 and
IL-1βAsp117 were significantly induced in the lung tissue
of VILI mice and were attenuated by rFGF21 treatment
in a dose-dependent manner (Fig.6G). A similar change
in HMGB1 was observed (Fig.6G). Finally, we examined
the cleavage of GSDMD. Obvious cleavage of GSDMD
was noted in the lung tissue of VILI mice, which was par-
tially blocked by rFGF21 in a dose-dependent manner
(Fig.6H). ese results suggest that the protective effect
of FGF21 might be associated with the inhibition of the
NLRP3/Caspase-1/GSDMD pyroptotic pathway.
Discussion
e pathogenesis of VILI is multifactorial and complex,
resulting predominantly from interactions between ven-
tilator-related factors and patient-related factors. In the
present study, we provided evidence that circulating lev-
els of FGF21 were increased in both patients and mice
with longtime mechanical ventilation. Using a mouse
strain with FGF21 deficiency, we demonstrated that VILI
pathologies were further aggravated by FGF21 deletion,
suggesting that FGF21 may be an endogenous mecha-
nism in response to VILI stimuli. Moreover, we showed
that the administration of FGF21 successfully amelio-
rated VILI in a mouse model and rescued mechanical
stretch-induced injury in a cell model. Mechanistically,
we found that inhibiting the NLRP3/Caspase-1/GSDMD
pyroptotic pathway may contribute to the protective
effect of FGF21 against VILI.
e first interesting finding is that FGF21 is induced
after VILI, and we speculated that the elevated circulat-
ing FGF21 might be mainly synthesized and secreted
by the liver. Previous studies have shown significantly
elevated levels of FGF21 in patients with type 2 diabetes,
nonalcoholic fatty liver and obesity [32, 33]. In addition,
circulating FGF21 was increased in response to cardiac
stress [34], ischemic stroke [35], limb ischemia/reperfu-
sion injury [36], and toxic kidney injury [37]. We found
that serum FGF21 tended to increase and peaked at
approximately 24h after VILI modeling in mice, which
was reported for the first time. It is reported in some
public databases that FGF21 is not expressed in nor-
mal lung tissues or lung cells (Additional file1: Fig.S4),
and we also found that FGF21 was enriched in the lung
after systemic administration of recombinant FGF21
(Additional file1: Fig.S4). ese findings supported the
hypothesis that exogenous rFGF21 was enriched in the
lung after rFGF21 medication. Nevertheless, we cannot
exclude that endogenous FGF21 might be triggered after
mechanical ventilation. As this preliminary conclusion is
based on only one single time point after medication, fur-
ther studies on pharmacology and pharmacokinetic pro-
files of rFGF21 may be needed in the future.
We also report for the first time that VILI is more
severe in FGF21-knockout mice, suggesting that endog-
enous FGF21 may be a protective factor. In addition to
results from the loss-of-function of FGF21, we further
explored the therapeutic effect of recombinant FGF21
and the results were supportive. As some FGF21-related
candidate drugs, such as FGF21 analogs (Pegbelfermin,
LY2405319, PF05231023) have been clinically tested to
treat diabetes and other metabolic disorders, our findings
strongly suggest that testing the efficacy of these FGF21
analogs in patients at high risk of VILI may be necessary.
e FGF receptor family includes FGFR1, FGFR2,
FGFR3, FGFR4, and an FGFR-like protein-FGFR5 [38],
among which FGFR1 is abundantly expressed in fibro-
blasts, smooth muscle cells, respiratory ciliated cells, and
endothelial cells in the lung (Additional file1: Fig.S5).
We knockdown FGFR1 by small interfering RNA and
found that the protective effect of FGF21 was attenu-
ated, indicating that FGFR1 plays a key role in mediat-
ing the biological function of FGF21 (Additional file 1:
Page 12 of 15
Dingetal. Critical Care (2023) 27:196
Fig. 6 FGF21 protects against VILI via inhibiting NLRP3/Caspase‑1/GSDMD pyroptotic pathway. A. Relative caspase‑1 activity in mice lung tissue;
B‑E. Relative mRNA expression levels of key nodes in NLRP3/caspase‑1/GSDMD pathway (N = 6, *P < 0.05, **P < 0.01); F–H. Relative protein expression
levels of key nodes in NLRP3/caspase‑1/GSDMD pathway (N = 3; ns, no significance, *P < 0.05, **P < 0.01 vs. Control; #P < 0.05, ##P < 0.01 vs. PBS). Lung
tissue samples in Fig. 6 are from the same batch of mice in Fig. 3
Page 13 of 15
Dingetal. Critical Care (2023) 27:196
Fig.S6). ese findings may help to explain the molecular
mechanisms underlying the protective role of FGF21 in
the lung, and further study on the specific ligand–recep-
tor interaction and signal transduction process is still
needed.
Oxidative stress is an important link in the pathophysi-
ological development of lung injury. During MV, alveo-
lar epithelial cells and vascular endothelial cells produce
large amounts of ROS in response to cyclic stretch and
shear forces [39]. Mitochondria are the most important
sites of ROS production. Under oxidative stress, ROS
overload leads to mitochondrial dysfunction, decreased
ATP synthesis capacity, reduced mitochondrial mem-
brane potential, and reduced scavenging free radical
capacity, leading to a further increase in ROS and form-
ing a vicious cycle [40]. When there is an imbalance
between high levels of ROS and antioxidant capacity,
cells are unable to maintain normal redox homeostasis,
leading to cellular damage and inflammatory responses
[41]. Kang et al. [42] found that FGF21 could reduce
neuroinflammation and oxidative stress by regulating
the NF-κB pathway and the AMPK/AKT pathway in an
aged diabetic mouse model. Zhang etal. [43] found that
FGF21 had a therapeutic effect on pulmonary fibrosis by
activating the Nrf-2 pathway and thus inhibiting oxida-
tive stress and extracellular matrix deposition. Our find-
ings are in line with these results and indicate that FGF21
inhibits the increase in ROS production, restores the
antioxidant capacity of cells, and stabilizes the membrane
potential and function of mitochondria.
Additionally, we found an association between FGF21
and pyroptosis. Pyroptosis, which is a form of lytic cell
death, plays a vital role in innate immune; however,
aberrant pyroptosis can contribute to injury in multi-
ple organs. e activation of caspase-1 triggers pyrop-
tosis, and GSDMD leads to pore formation, resulting in
the cleavage of inflammatory cytokines [44]. Since cas-
pase-1 plays a central role in inducing pyroptosis and the
NLRP3/caspase-1 axis has been well studied, it is possi-
ble to reduce pyroptosis by regulating NLRP3. Wei etal.
[45] reported that FGF21 improved intimal hyperplasia
in diabetic mice, which was associated with inhibition of
the FGFR1/Syk/NLRP3 pathway. Chen etal. [46] showed
that FGF21 inhibited pyroptosis in human umbilical
vein endothelial cells by suppressing ROS production.
In our study, we found that the induced mRNA levels of
NLRP3 inflammasome, the protein expression of IL-18
and IL-1β, and the cleavage of GSDMD were all inhib-
ited by FGF21 treatment. erefore, our results indicate a
critical role of the NLRP3/Caspase-1/GSDMD pyroptotic
pathway in the pathophysiology of VILI, which is con-
sistent with two recently published works [47, 48], and
further point out a potential therapeutic effect of FGF21
against VILI-related pyroptosis. However, the regulatory
mechanisms of FGF21 on pyroptosis, especially in VILI,
may need further investigation.
As mentioned above, the liver is a major manufac-
turer of FGF21, and we hypothesized that elevated cir-
culating FGF21 is secreted by the liver. However, how
the liver responds to volutrauma/biotrauma in the lung
and whether this is a causal or correlation relationship
is poorly understood. It may be the effect of specific
cytokines or mediators released by the lung or through
neurohumoral regulation. We believe that screening
signaling molecules relating to the cross talk between the
lung and the liver after mechanical ventilation using sys-
temic methods (transcriptomics, proteomics, and metab-
olomics) might be a promising approach to answer this
conundrum.
ere are several limitations in this study. First, only
male mice were used in this study for concerns about
confounding contributions from the hormone cycle in
female mice since our research target is also a circulat-
ing hormone. In addition, some studies reported the sex
difference in metabolic responses and pharmacologi-
cal effects of FGF21 [49, 50], while gender differences in
lung injury are rarely reported. Whether FGF21 benefits
VILI both in male and in female is an intriguing question
meriting further investigation. Second, the experimen-
tal design lacked a group of mice ventilated with normal
tidal volume. e tidal volume of 30ml/kg is classic in
mouse model while exceeds what would be used in any
clinical setting. It will be helpful in the clinical translation
of the findings if regular ventilated mice were tested in
future study.
Conclusion
Our study indicates that the increase in serum FGF21
levels after MV might be an endogenous protective
response. Treatment with rFGF21 protects against VILI
invivo and invitro by inhibiting the NLRP3/Caspase-1/
GSDMD pathway. ese findings suggest that FGF21
might be a promising pharmacological tool in the battle
against VILI.
Abbreviations
FGF21 Fibroblast growth factor 21
VILI Ventilator‑induced lung injury
NLRP3 NOD‑, LRR‑ and pyrin domain‑containing 3
KO Knockout
WT Wild‑type
ASC Apoptosis‑associated speck‑like protein containing a CARD
IL Interleukin
HMGB1 High mobility group box 1
GSDMD Gasdermin D
MV Mechanical ventilation
ASA American Society of Anesthesiologists
Page 14 of 15
Dingetal. Critical Care (2023) 27:196
BMI Body mass index
ELISA Enzyme‑linked immunosorbent assay
LMEVCs Lung microvascular endothelial cells
MS Mechanical stretch
PBS Phosphate‑buffered saline
LDH Lactate dehydrogenase
BALF Bronchoalveolar lavage fluid
BCA Bicinchoninic acid
H&E Hematoxylin and eosin
TUNEL Terminal deoxynucleotidyl transferase‑mediated dUTP nick‑end
labeling
ROS Reactive oxygen species
DCFH‑DA Dichlorodihydrofluorescein diacetate
MPO Myeloperoxidase
SOD Superoxide dismutase
PCR Polymerase chain reaction
SDS–PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEM Standard error of mean
TEAC Trolox‑equivalent antioxidant capacity
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13054‑ 023‑ 04488‑5.
Additional le1. Detailed methods and supplementary results.
Acknowledgements
Not applicable.
Author contributions
PD, RY, and CL performed the experiment, interpreted the data and wrote
the manuscript; GLR, DYZ, WC, and LYY analyzed and interpreted the data
and revised the manuscript; YFM and YHL designed the study, provided the
resources and reviewed the manuscript. HLF, PW and HBY performed addi‑
tional experiments, analyzed the data and revised the manuscript during the
revision. All authors read and approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation of
China (81873945, 82202365, 82272227), the Shanghai Sailing Program
(20YF1448800), the Medical Innovation Research Project of Shanghai Science
and Technology Commission (21Y11906400, 22Y11904000), the Military Medi‑
cal Talent Plan of Naval Medical University (2019‑YH‑11), and the Innovative
Clinical Research Project of the Second Affiliated Hospital of Naval Medical
University (2020YLCYJ‑Y18).
Availability of data and materials
The datasets generated and/or analyzed during the current study are available
from the corresponding authors on reasonable request.
Declarations
Ethical approval and consent to participate
The study protocol was approved by the Ethics Committee of Biomedicine
of Naval Medical University. Written informed consent was obtained from all
subjects. The animal experiments were approved by the Ethics Committee of
Biomedicine of Naval Medical University, were performed in compliance with
the National Institutes of Health Guide for Care and Use of Laboratory Animals,
and were reported in accordance with the Animal Research: Reporting In Vivo
Experiments (ARRIVE) guidelines 2.0.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Department of Anesthesiology, Changzheng Hospital, The Second Affiliated
Hospital of Naval Medical University, Shanghai, China. 2 Department of Anes‑
thesiology, PLA No.983 Hospital, Tianjin, China. 3 Department of Pharmacology,
College of Pharmacy, Naval Medical University, Shanghai, China. 4 Department
of Anesthesiology and Surgical Intensive Care Unit, Xinhua Hospital Affiliated
to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Received: 4 December 2022 Accepted: 13 May 2023
References
1. Vieillard‑Baron A, Matthay M, Teboul JL, Bein T, Schultz M, Magder S, et al.
Experts’ opinion on management of hemodynamics in ARDS patients:
focus on the effects of mechanical ventilation. Intensive Care Med.
2016;42(5):739–49.
2. Mingote Á, Marrero García R, Santos González M, Castejón R, Salas Antón
C, Vargas Nuñez J, et al. Individualizing mechanical ventilation: titration
of driving pressure to pulmonary elastance through Young’s modulus
in an acute respiratory distress syndrome animal model. Crit Care.
2022;26(1):316.
3. Curley GF, Laffey JG, Zhang H, Slutsky AS. Biotrauma and ventilator‑
induced lung injury: clinical implications. Chest. 2016;150(5):1109–17.
4. Liu C, Wu K, Sun T, Chen B, Yi Y, Ren R, et al. Effect of invasive mechani‑
cal ventilation on the diversity of the pulmonary microbiota. Crit Care.
2022;26(1):252.
5. Alessandri F, Pugliese F, Ranieri VM. Mechanical ventilation: we have
come a long way but still have a long road ahead. Lancet Respir Med.
2017;5(12):922–4.
6. Monjezi M, Jamaati H, Noorbakhsh F. Attenuation of ventilator‑induced
lung injury through suppressing the pro‑inflammatory signaling path‑
ways: a review on preclinical studies. Mol Immunol. 2021;135:127–36.
7. Gaver DP 3rd, Nieman GF, Gatto LA, Cereda M, Habashi NM, Bates JHT.
The poor get poorer: a hypothesis for the pathogenesis of ventilator‑
induced lung injury. Am J Respir Crit Care Med. 2020;202(8):1081–7.
8. Nishimura T, Nak atake Y, Konishi M, Itoh N. Identification of a novel
FGF, FGF‑21, preferentially expressed in the liver. Biochim Biophys Acta.
2000;1492(1):203–6.
9. Kharitonenkov A, Shiyanova TL, Koester A, Ford AM, Micanovic R,
Galbreath EJ, et al. FGF‑21 as a novel metabolic regulator. J Clin Invest.
2005;115(6):1627–35.
10. Fisher FM, Maratos‑Flier E. Understanding the physiology of FGF21. Annu
Rev Physiol. 2016;78:223–41.
11. Byun S, Seok S, Kim YC, Zhang Y, Yau P, Iwamori N, et al. Fasting‑induced
FGF21 signaling activates hepatic autophagy and lipid degradation via
JMJD3 histone demethylase. Nat Commun. 2020;11(1):807.
12. Kliewer SA, Mangelsdorf DJ. A dozen years of discovery: insights into the
physiology and pharmacology of FGF21. Cell Metab. 2019;29(2):246–53.
13. Gao J, Liu Q, Li J, Hu C, Zhao W, Ma W, et al. Fibroblast Growth Factor 21
dependent TLR4/MYD88/NF‑kappaB signaling activation is involved in
lipopolysaccharide‑induced acute lung injury. Int Immunopharmacol.
2020;80: 106219.
14. Yan F, Yuan L, Yang F, Wu G, Jiang X. Emerging roles of fibroblast growth
factor 21 in critical disease. Front Cardiovasc Med. 2022;9:1053997.
15. Sanyal A, Charles ED, Neuschwander‑Tetri BA, Loomba R, Harrison SA,
Abdelmalek MF, et al. Pegbelfermin (BMS‑986036), a PEGylated fibroblast
growth factor 21 analogue, in patients with non‑alcoholic steatohepatitis:
a randomised, double‑blind, placebo‑controlled, phase 2a trial. Lancet.
2019;392(10165):2705–17.
16. Talukdar S, Zhou Y, Li D, Rossulek M, Dong J, Somayaji V, et al. A long‑act‑
ing FGF21 molecule, PF‑05231023, decreases body weight and improves
lipid profile in non‑human primates and type 2 diabetic subjects. Cell
Metab. 2016;23(3):427–40.
17. Gaich G, Chien JY, Fu H, Glass LC, Deeg MA, Holland WL, et al. The effects
of LY2405319, an FGF21 analog, in obese human subjects with type 2
diabetes. Cell Metab. 2013;18(3):333–40.
Page 15 of 15
Dingetal. Critical Care (2023) 27:196
•
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research
? Choose BMC and benefit from:
? Choose BMC and benefit from:
18. Yu Z, Lin L, Jiang Y, Chin I, Wang X, Li X, et al. Recombinant FGF21 protects
against blood‑brain barrier leakage through Nrf2 upregulation in type 2
diabetes mice. Mol Neurobiol. 2019;56(4):2314–27.
19. Chen J, Hu J, Liu H, Xiong Y, Zou Y, Huang W, et al. FGF21 protects the
blood‑brain barrier by upregulating PPARγ via FGFR1/β‑klotho after
traumatic brain injury. J Neurotrauma. 2018;35(17):2091–103.
20. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The
ARRIVE guidelines 2.0: updated guidelines for reporting animal research. J
Cereb Blood Flow Metab. 2020;40(9):1769–77.
21. Anwar F, Sparrow NA, Rashid MH, Guidry G, Gezalian MM, Ley EJ, et al.
Systemic interleukin‑6 inhibition ameliorates acute neuropsychiatric phe‑
notypes in a murine model of acute lung injury. Crit Care. 2022;26(1):274.
22. Chen Q, Ma J, Yang X, Li Q, Gong F. SIRT1 mediates effects of FGF21
to ameliorate cisplatin‑induced acute kidney injury. Front Pharmacol.
2020;11:241.
23. Mikawa K, Nishina K, Takao Y, Obara H. ONO‑1714, a nitric oxide synthase
inhibitor, attenuates endotoxin‑induced acute lung injury in rabbits.
Anesth Analg. 2003;97(6):1751–5.
24. Chi C, Fu H, Li YH, Zhang GY, Zeng FY, Ji QX, et al. Exerkine fibronec‑
tin type‑III domain‑containing protein 5/irisin‑enriched extracellular
vesicles delay vascular ageing by increasing SIRT6 stability. Eur Heart J.
2022;43(43):4579–95.
25. Lewis JE, Ebling FJP, Samms RJ, Tsintzas K. Going back to the biology of
FGF21: new insights. Trends Endocrinol Metab. 2019;30(8):491–504.
26. Lu Q, Zemskov EA, Sun X, Wang H, Yegambaram M, Wu X, et al. Activation
of the mechanosensitive Ca(2+) channel TRPV4 induces endothelial bar‑
rier permeability via the disruption of mitochondrial bioenergetics. Redox
Biol. 2021;38: 101785.
27. Grailer JJ, Canning BA, Kalbitz M, Haggadone MD, Dhond RM, Andjelkovic
AV, et al. Critical role for the NLRP3 inflammasome during acute lung
injury. J Immunol. 2014;192(12):5974–83.
28. Sun J, Li Y. Pyroptosis and respiratory diseases: a review of current knowl‑
edge. Front Immunol. 2022;13: 920464.
29. He Y, Hara H, Nunez G. Mechanism and regulation of NLRP3 inflamma‑
some activation. Trends Biochem Sci. 2016;41(12):1012–21.
30. Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, et al. Cleavage of GSDMD
by inflammatory caspases determines pyroptotic cell death. Nature.
2015;526(7575):660–5.
31. Deng M, Tang Y, Li W, Wang X, Zhang R, Zhang X, et al. The endotoxin
delivery protein HMGB1 mediates caspase‑11‑dependent lethality in
sepsis. Immunity. 2018;49(4):740‑53 e7.
32. Mraz M, Bartlova M, Lacinova Z, Michalsky D, Kasalicky M, Haluzikova D,
et al. Serum concentrations and tissue expression of a novel endocrine
regulator fibroblast growth factor‑21 in patients with type 2 diabetes and
obesity. Clin Endocrinol (Oxf). 2009;71(3):369–75.
33. Dushay J, Chui PC, Gopalakrishnan GS, Varela‑Rey M, Crawley M, Fisher
FM, et al. Increased fibroblast growth factor 21 in obesity and nonalco‑
holic fatty liver disease. Gastroenterology. 2010;139(2):456–63.
34. Cheng P, Zhang F, Yu L, Lin X, He L, Li X, et al. Physiological and phar‑
macological roles of FGF21 in cardiovascular diseases. J Diabetes Res.
2016;2016:1540267.
35. Jiang Y, Liu N, Wang Q, Yu Z, Lin L, Yuan J, et al. Endocrine regulator
rFGF21 (recombinant human fibroblast growth factor 21) improves
neurological outcomes following focal ischemic stroke of type 2 diabetes
mellitus male mice. Stroke. 2018;49(12):3039–49.
36. Chen F, Zhan J, Yan X, Mamun AA, Zhang Y, Xu Y, et al. FGF21 alleviates
microvascular damage following limb ischemia/reperfusion injury by
TFEB‑mediated autophagy enhancement and anti‑oxidative response.
Signal Transduct Target Ther. 2022;7(1):349.
37. Li F, Liu Z, Tang C, Cai J, Dong Z. FGF21 is induced in cisplatin
nephrotoxicity to protect against kidney tubular cell injury. FASEB J.
2018;32(6):3423–33.
38. Yang L, Zhou F, Zheng D, Wang D, Li X, Zhao C, et al. FGF/FGFR signaling:
from lung development to respiratory diseases. Cytokine Growth Factor
Rev. 2021;62:94–104.
39. Parinandi NL, Kleinberg MA, Usatyuk PV, Cummings RJ, Pennathur A,
Cardounel AJ, et al. Hyperoxia‑induced NAD(P)H oxidase activation and
regulation by MAP kinases in human lung endothelial cells. Am J Physiol
Lung Cell Mol Physiol. 2003;284(1):L26‑38.
40. Jonckheere AI, Smeitink JA, Rodenburg RJ. Mitochondrial ATP syn‑
thase: architecture, function and pathology. J Inherit Metab Dis.
2012;35(2):211–25.
41. Cheng YJ, Chan KC, Chien CT, Sun WZ, Lin CJ. Oxidative stress during
1‑lung ventilation. J Thorac Cardiovasc Surg. 2006;132(3):513–8.
42. Kang K, Xu P, Wang M, Chunyu J, Sun X, Ren G, et al. FGF21 attenu‑
ates neurodegeneration through modulating neuroinflammation and
oxidant‑stress. Biomed Pharmacother. 2020;129: 110439.
43. Zhang S, Yu D, Wang M, Huang T, Wu H, Zhang Y, et al. FGF21 attenuates
pulmonary fibrogenesis through ameliorating oxidative stress in vivo and
in vitro. Biomed Pharmacother. 2018;103:1516–25.
44. Wallach D, Kang TB, Dillon CP, Green DR. Programmed necrosis in
inflammation: toward identification of the effector molecules. Science.
2016;352(6281):aaf2154.
45. Wei W, Li XX, Xu M. Inhibition of vascular neointima hyperplasia by FGF21
associated with FGFR1/Syk/NLRP3 inflammasome pathway in diabetic
mice. Atherosclerosis. 2019;289:132–42.
46. Chen JJ, Tao J, Zhang XL, Xia LZ, Zeng JF, Zhang H, et al. Inhibition of
the ox‑LDL‑induced pyroptosis by FGF21 of human umbilical vein
endothelial cells through the TET2‑UQCRC1‑ROS pathway. DNA Cell Biol.
2020;39(4):661–70.
47. Chavez L, Meguro J, Chen S, de Paiva VN, Zambrano R, Eterno JM, et al.
Circulating extracellular vesicles activate the pyroptosis pathway in the
brain following ventilation‑induced lung injury. J Neuroinflammation.
2021;18(1):310.
48. Shao RG, Xie QW, Pan LH, Lin F, Qin K, Ming SP, et al. Necrostatin‑1
attenuates Caspase‑1‑dependent pyroptosis induced by the RIPK1/ZBP1
pathway in ventilator‑induced lung injury. Cytokine. 2022;157: 155950.
49. Makarova E, Kazantseva A, Dubinina A, Denisova E, Jakovleva T, Balybina
N, et al. Fibroblast growth factor 21 (FGF21) administration sex‑specif‑
ically affects blood insulin levels and liver steatosis in obese A(y) mice.
Cells. 2021;10(12):3440.
50. Makarova E, Kazantseva A, Dubinina A, Jakovleva T, Balybina N, Baranov K,
et al. The same metabolic response to FGF21 administration in male and
female obese mice is accompanied by sex‑specific changes in adipose
tissue gene expression. Int J Mol Sci. 2021;22(19):10561.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
