Huang et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00509-5

BMC Molecular and Cell
Biology(2024) 25:13

Open Access

RESEARCH

mTOR signaling pathway regulation HIF-1
α effects on LPS induced intestinal mucosal
epithelial model damage
Zeyong Huang1, Wenbin Teng2†, Liuxu Yao3, Kai Xie4, Suqin Hang1, Rui He4* and Yuhong Li1,5*
Abstract
Background Sepsis-induced small-intestinal injury is associated with increased morbidity and mortality. Our
previous study and other papers have shown that HIF-1α has a protective effect on intestinal mucosal injury in septic
rats. The purpose of this study is to further verify the protective effect of HIF-1α on intestinal mucosa and its molecularmechanism in vitro experiments.
Methods Caco-2 cells were selected and experiment was divided into 2 parts. Part I: HIF-1α activator and inhibitor
were used to treat lipopolysacchrides (LPS)-stimulated Caco-2 cells respectively, to explore the effect of HIF-1α on LPS
induced Caco-2 cell epithelial model; Part II: mTOR activator or inhibitor combined with or without HIF-1α activator,
inhibitor to treat LPS-stimulated Caco-2 cells respectively, and then the molecular mechanism of HIF-1α reducing LPS
induced Caco-2 cell epithelial model damage was detected.
Results The results showed that HIF-1α activator decreased the permeability and up regulated tight junction (TJ)
expression, while HIF-1α inhibitor had the opposite effect with the HIF-1α activator. mTOR activation increased,
while mTOR inhibition decreased HIF-1α protein and expression of its downstream target molecules, which can beattenuated by HIF-1α activator or inhibitor.
Conclusion This study once again confirmed that HIF-1α alleviates LPS-induced mucosal epithelial model damage
through P70S6K signalling pathway. It is of great value to explore whether HIF-2α plays crucial roles in the regulationof mucosal epithelial model functions in the future.
Keywords Caco-2 cells, LPS induced intestinal epithelial model injury, Hypoxia-inducible factor-1α, mTOR/P70S6K
signalling pathway, In vitro†

Zeyong Huang and Wenbin Teng contributed equally to this work.

*Correspondence:
Rui He15347172@qq.com
Yuhong Li
yuh_li@zju.edu.cn
1
Department of Anesthesiology, Shulan (Hangzhou) Hospital, Shulan
International Medical College, Zhejiang Shuren College,
310015 Hangzhou, China

2

Department of Anesthesiology, the First Affiliated Hospital, College ofMedicine, Zhejiang University, 310001 Hangzhou, China
3
Rehabilitation Medicine Center, Department of Anesthesiology, Zhejiang
Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou
Medical College, 310014 Hangzhou, China
4
Department of Anesthesiology, Shaoxing People’s Hospital, Zhejiang
University, 312000 Shaoxing, China
5
Department of Anesthesiology, Shulan (Hangzhou) Hospital, Shulan
International Medical College, Shuren University, 848 Dongxin Road,
Xiacheng District, 310004 Hangzhou, Zhejiang, China© The Author(s) 2024. 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) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

Background
Sepsis, a life-threatening type of organ dysfunction
caused by a maladjusted response to infection [1], is arecognized global health problem with a high mortality
rate ranging between 30 and 50%. Although comprehensive therapies, including anti-inflammatory and anticoagulant applications, have been used clinically for several
years. Due to its narrow window, each hour of delay inproviding the right treatment results in a 7.6% drop in
survival for sepsis patients. Therefore, early and systematic diagnosis is a vital task and it is urgent to strengthen
the current knowledge of sepsis pathophysiology in orderto develop new clinical treatment methods.
The intestinal tract may be the initiator of multiple
organ dysfunction (MODS), which may be due to thedestruction of intestinal barrier and subsequent translocation of intestinal bacteria [2, 3]. The integrity of thestructure and function of intestinal epithelial cells is crucial in the intestinal barrier. Abnormal intestinal epithelial cells are often observed in patients with sepsis, whichis considered to play an important role in bacterial translocation and systemic infection [4].
Studies have shown that under physiological conditions, intestinal epithelial cells are in a “physiologicalhypoxia” environment and that intestinal epithelial cells
can adapt to hypoxia [5]. Under pathological conditions, small fluctuations in blood flow can cause a large
decrease in oxygen transport, leading to intestinal mucosal ischaemia/hypoxia. In sepsis, during the inflammatory
process, tissue metabolism is high, oxygen consumption
increases, intestinal mucosal vessels constrict, mucosal permeability increases [6], oxygen supply decreases,
and the degree of hypoxia in intestinal epithelial cells
increases [7]. Hypoxia-inducible factor−1 is a key transcription factor required for the human body to adapt tohypoxic environments and plays an important role in thedevelopment of sepsis [8].
HIF−1 is composed of an oxygen-dependent α subunit and a constitutively expressed β subunit [9]. HIF−1α
plays a key role in the acute hypoxia response [10]. Our
recent study found that the expression of HIF−1α in theintestinal mucosa of rats with sepsis was upregulated andthat the administration of the HIF−1α activator dimethyloxallyl glycine (DMOG) inhibited sepsis-induced inflammatory responses and oxidative stress levels, increased
antioxidant levels, and alleviated intestinal mucosal
barrier injury. The effect of the HIF−1α inhibitor BAY
87−2243 on the intestinal mucosal barrier was the opposite. These results suggest that HIF−1α has a protective
effect against intestinal mucosal injury due to sepsis [11].
The mechanistic target of rapamycin/70-kDa ribosomal
protein S6 kinase (mTOR/P70S6K) signalling pathway is
an upstream regulator of HIF−1α and a key pathway inmetabolism and plays an important role in the processes

Page 2 of 12

of energy regulation and autophagy [12]. Studies haveshown that activating mTOR signaling pathway canupregulate the expression of HIF−1α, increase glycolysis,
lead to metabolic reprogramming and activate “training
immunity” to improve inflammation; inhibition of themTOR signaling pathway has the opposite effect [13].
Whether mTOR/P70S6K signaling pathway regulates
the expression of HIF−1α in intestinal epithelial injury insepsis remains to be further studied.
This study established an in vitro model of the intestinal mucosal epithelial model in sepsis using LPS stimulation to induce human colorectal adenocarcinoma cells(Caco−2) to investigate whether the mTOR/P70S6K signalling pathway regulates HIF−1α expression to attenuate
sepsis-induced impairment of intestinal mucosal epithelial model function. This study provides an experimental basis for the mechanism and treatment of intestinal
mucosal injury in sepsis.

Materials and methods
Materials and reagents

Caco-2 human colorectal adenocarcinoma cells werepurchased from the Cell Bank of Type Culture Collectionof the Chinese Academy of Sciences (Shanghai, China)
in 2019. Fluorescein isothiocyanate-dextran (FD-4) andlipopolysaccharides (LPS, 2630, Sigma, USA); rabbit
anti-rat ZO-1, HIF-1α and occludin polyclonal antibodies (Invitrogen, USA); claudin-1 and β-actin antibodies(Abcam, UK); mTOR, P-mTOR, P70S6K and P-P70S6K
polyclonal antibodies (Cell Signaling Technology);
horseradish peroxidase (HRP)-labeled goat anti-rabbit
antibody (Jackson ImmunoResearch, USA); dimethyloxallyl glycine (DMOG), BAY 87-2243, rapamycin, andMHY1486 (MedChemExpress, USA); and fetal bovine
serum (FBS), Minimum Essential Medium (MEM), penicillin/streptomycin, N-L-alanyl-L-glutamine, sodium
pyruvate, nonessential amino acids, dimethyl sulfoxide(DMSO) and 0.25% trypsin-EDTA (Gibco, USA).
Cell culture

After thawing, Caco-2 cells were cultured in MEM containing 10% FBS, 100 U/100 mg/ml penicillin and streptomycin, 1% sodium pyruvate, 1% glutamine, and 1%
nonessential amino acids at 37 °C in a 5% CO2 incubator.
The medium was changed every 2 days. Cells were seeded
on 24-well at a density of 2 × 105 cells/ml (400 µl per well),when they were in the logarithmic growth phase andgrowing well. Culture medium (600 µl) was added fromthe bottom side and it was changed 24 h after seeding.
The cell culture medium was changed every other day
for the following week and then every day thereafter. Cell
growth was observed regularly. Transepithelial electrical resistance (TEER) was measured daily using a Millicell ERS-2 Epithelial Volt-Ohm Meter (MerckMillipore,Huang et al. BMC Molecular and Cell Biology(2024) 25:13

USA). After the cells were cultured for approximately 21
days, the TEER significantly increased and remained stable after the cells formed an intact and dense monolayer,
indicating that the intestinal mucosal epithelial model
was basically formed.
In the experiment, LPS, DMOG, BAY 87-2243,
rapamycin and MHY1487 were all dissolved in the cell
culture medium.
Confirmation of the optimal concentrations of LPS,
activator and inhibitor

Caco-2 cells were exposed to different concentrations of
LPS, DMOG (HIF-1α activator), BAY 87-2243 (HIF-1α
inhibitor), rapamycin (mTOR inhibitor) and MHY1487(mTOR activator), and the optimal concentration was
selected based on the effect of each concentration on theviability of Caco-2 cells or the expression of HIF-1α.
Establishment of a Caco-2 cell injury model andexperimental grouping

Caco-2 cells were selected and the experiment was
divided into 2 parts. Part I: untreated control group(CON), LPS-induced epithelial cell injury model group(LPS), LPS + DMOG (HIF-1α activator) group (L + D)
and LPS + BAY 87-2243 (HIF-1α inhibitor) group (L + B);
Part II: CON group, LPS group, LPS + rapamycin (mTOR
inhibitor) group (L + R), LPS + MHY1487 (mTOR activator) group (L + M), LPS + rapamycin + DMOG (L + R + D),
and LPS + MHY1487 + BAY 87-2243(L + M + B).
In the CON group, cells were cultured in normal
medium. In the LPS group, cells were stimulated with
600 µg/ml LPS. In the L + D group, cells stimulated with
LPS were treated with 104 nM DMOG. In the L + B
group, cells stimulated with LPS were treated with 10 nM
BAY 87-2243. In the L + R group, cells stimulated with
LPS were treated with 10 nM rapamycin. In the L + M
group, cells stimulated with LPS were treated with 100
nM MHY1487. In the L + R + D group, cells stimulatedwith LPS and treated with 10 nM rapamycin were incubated with 104 nM DMOG. In the L + M + B group, cells
stimulated with LPS and treated with 100 nM MHY1487
were incubated with 10 nM BAY 87-2243.
Caco-2 cells in all groups were cultured in culture
medium (same cell culture). Except the CON group,
Caco-2 cells in other treatment groups were pretreatedwith LPS (final concentration 600 µg/ml) for 2 h. DMOG
(final concentration 104 nM), BAY 87-2243 (final concentration 10 nM), rapamycin (final concentration 10 nM)
and MHY1487 (final concentration 100 nM) were added
in one time and continued to culture for another 24 h.

Page 3 of 12

Measurement of the TEER of the monolayer epithelialmodel

The electrode was immersed in 70% alcohol for 15 min,air-dried for 15 s, and then placed in Hanks’ balanced salt
solution (HBSS) at 37 °C for 15 min. The culture medium
was removed from the culture plate. Then, 400 µl of
prewarmed HBSS was added to each well of the upper
chamber, and 600 µl of prewarmed HBSS was added toeach well of the lower chamber. Solutions were equilibrated at 37 °C for 30 min in the incubator, debris on thecell surface was washed away, and HBSS was removed.
Preheated HBSS was added again, and the transmembrane resistance value was measured by Millicell ERS − 2epithelial volt-ohmmeter to evaluate the cell permeability. The above steps were repeated with a blank carrier toobtain the blank value.
TEER Ω · cm2 = (measuredresistancevalue − blank value)(Ω) × monolayer surface area cm2 .

Detection of fluorescein isothiocyanate–dextran (FD-4)
permeability

A Caco-2 monolayer model was established. After thecellswere treated with the experimental factors, the culture medium was removed, and 400 µl of HBSS solution
containing 1 mg/ml FD-4 was added to the upper chamber, avoiding light exposure. After incubation at 37 °C
with 5% CO2 for 2 h, 100 µl of the solution from the lower
chamber was collected and measured using a fluorescence microplate reader (SPARK®, USA). The excitation
wavelength was 490 nm, and the emission wavelength
was 530 nm. A standard curve for the concentration wasplotted, and the concentration of each sample was calculated based on the standard curve.
Detection of TJs and HIF-1α protein expression by western
blotting (WB)

After the cells in each experimental group were treatedbased on the predetermined experimental protocol, thecells were washed twice with cold phosphate bufferedsaline (PBS) and lysed with radioimmunoprecipitation
assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF). The cells were lysed on ice for 30 min. The
supernatant was collected after centrifugation at 12,000 g
for 15 min. After quantification using the bicinchoninicacid (BCA) assay, 40 mg of total protein was mixed withan equal volume of 2× loading buffer, boiled at 100 °C
for 3–5 min to denature, and loaded onto a 6-10% SDS
polyacrylamide gel for electrophoresis. The protein
bands were transferred to a membrane and blockedwith Tris buffered saline with Tween 20 (TBST) containing 5% bovine serum albumin (BSA) for 2 h at room
temperature. TBST was used to wash the membrane 3

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

times (5 min per wash). Membranes were incubated with
anti-HIF-1α (1:1000), anti-ZO-1 (1:1000), anti-occludin(1:1000), anti-claudin-1 (1:1000), anti-β-actin (1:2000),
anti-p-mTOR (1:1000), anti-mTOR (1:1000), anti-pP70S6K (1:1000), anti-P70S6K (1:1000), and anti-GAPDH(1:1000) at 4 °C overnight. After thorough washing with
TBST, HRP-labeled goat anti-rabbit antibody was added
to the membrane, and the membrane was incubated atroom temperature for 2 h and washed 3 times with TBST.
Protein signals were visualized using an enhanced chemiluminescence (ECL) kit (Beyotime Biotechnology,
China). The membrane was photographed in a gel imaging system (ChemiDoc XRS + System, Bio-Rad Company,
USA), and the greyscale value was analysed using Image J
software (Bio-Rad Company, USA).
Cell counting Kit-8 (CCK-8) assay

Caco-2 cells (70-80% confluent) were digested with trypsin and collected, followed by centrifugation to preparea cell suspension. Then, 100 µl of cell suspension wasadded to a 96-well plate at a cell density of 2 × 104 cells/
ml. After incubation for 24 h (37 °C, 5% CO2), different
groups were established, with 6 replicate wells for eachgroup. After 24 h of incubation (37 °C, 5% CO2), 10 µl of
CCK-8 solution was added to each well, followed by incubation (37 °C, 5% CO2) for 2 h. The absorbance was measured at 450 nm using a microplate reader (SpectraMax
Plus 384, Meigu Molecular Instruments (Shanghai) Co.,
LTD, China). Cell viability is calculated according to thefollowing formula:

Cell viability = [(As − Ab) / (Ac − Ab)] × 100%
As: OD of the experimental hole (including cells, culture
medium, CCK-8 solution and drugs); Ac: OD of the control hole (including cells, culture media, CCK-8 solution,
without drugs); Ab: OD of blank hole (including culture
medium, CCK-8 solution, excluding cells and drugs).
Statistical analysis

Statistical analysis was performed using GraphPad Prism(Version 7.0, California, USA) statistical software. Measurement data conforming to normal distribution were
presented as mean ± standard deviation ( −x ±SD). Oneway analysis of variance was used to analyze the differences among groups. For data with homogeneous
variance, the least significant difference (LSD) test wasused for pairwise comparisons. For data heterogeneous
variance, Dunnett’s T3 test was used. While measurement data of skewed distribution were expressed as M
(P25, P75), and the data among groups were tested via
the nonparametric test. P < 0.05 was considered statistically significant.

Page 4 of 12

Results
Confirmation of the optimal concentrations of LPS, DMOG
and BAY 87-2243

Different concentrations of LPS (0, 1, 10, 102, 103, 104,
105, 106 ng/ml) were added to the Caco-2 cell culture
medium. Compared with LPS free group (LPS 0 ng/ml),
10 ng/ml, 102 ng/ml, 103 ng/ml, 104 ng/ml and 105 ng/
ml of LPS significantly increased cell viability, while 106
ng/ml LPS significantly inhibited cell viability (Fig. 1A).
Then LPS at 0, 100, 200, 300, 400, 500, 600, 700, 800, and900 µg/ml were tested again, compared with LPS free,
more than 600 µg/ml of LPS showed significant inhibitory effect on Caco-2 cell viability (Fig. 1B). Therefore,
in this study, the optimal stimulating concentration was
600 µg/ml LPS. Caco-2 cells were exposed to different
concentrations of DMOG and BAY 87-2243. DMOG (106nM) inhibited the viability of Caco-2 cells, and 103, 104,
and 105 nM DMOG significantly increased cell viability(Fig. 1C). BAY 87-2243 (102 nM) had a significant inhibitory effect on Caco-2 cell viability, and BAY 87-2243 concentrations < 10 nM had no significant inhibitory effecton cell viability (Fig. 1E).
Different DMOG concentrations were evaluated within
the range of concentrations that did not inhibit the viability of Caco-2 cells. WB detection results indicated that104 nM DMOG most significantly up regulated HIF-1α
expression (Fig. 1D) and that 10 nM BAY22-8742 effectively inhibited HIF-1α expression (Fig. 1F). Based on the
results of CCK experiment and WB detection, the optimal concentrations of DMOG and BAY87-2243 were 104
nM and 10 nM, respectively.
Confirmation of the optimal concentrations of mTOR
activators and inhibitors

The results of the CCK-8 assay indicated that 105 nM
rapamycin significantly inhibited the viability of Caco-2
cells, while concentrations of 104 nM and lower had noeffect on the viability of Caco-2 cells (Fig. 2A). When0 ∼ 104 nM rapamycin was added, WB indicated that 1
nM rapamycin had the most significant inhibitory effecton p-mTOR/mTOR expression (Fig. 2B). MHY1487 (105nM) significantly inhibited the viability of Caco-2 cells,
while MHY1487 at concentrations ≤ 104 nM had no significant effect on the viability of Caco-2 cells (Fig. 2C).
WB detection indicated that 104 nM MHY1487 promoted the most significant upregulation of p-mTOR/
mTOR expression (Fig. 2D, P < 0.05). Based on the resultsof CCK experiment and WB detection, the optimal concentrations of rapamycin and MHY1487 were 10 nM and
100 nM, respectively.

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

Page 5 of 12

Fig. 1 Confirmation of the optimal concentrations of LPS, DMOG and BAY 87-2243 for Caco-2 cells. (A-B) Effect of different concentrations of LPS on theviability of Caco-2 cells and the optimal concentration of LPS for this study was 600ug/ml. (C) Effect of different concentrations of DMOG on the viabilityof Caco-2 cells. (D) Effect of different concentrations of DMOG on the expression of HIF-1α in Caco-2 cells. (E) Effect of different concentrations of BAY
87-2243 on the viability of Caco-2 cells. (F) Effect of different concentrations of BAY 87-2243 on the expression of HIF-1α in Caco-2 cells. Optimal concentrations for DMOG and BAY 87-2243 were 104 nM and 10 nM respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs. 0 nM

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

Page 6 of 12

Fig. 2 Confirmation of the optimal concentrations of rapamycin and MHY1487 for Caco-2 cells. (A) Effect of different concentrations of rapamycin on theviability of Caco-2 cells. (B) Effect of different concentrations of rapamycin on the expression of p-mTOR in Caco-2 cells. (C) Effect of different concentrations of MHY1487 on the viability of Caco-2 cells. (D) Effect of different concentrations of MHY1487 on the expression of p-mTOR in Caco-2 cells. Optimal
concentrations for rapamycin and MHY1487 were 10 nM and 100 nM, respectively. *P < 0.05; **P < 0.01; ****P < 0.0001. vs. 0 nM

Effect of HIF-1α activator and inhibitor on the TEER andFD-4 concentration of the Caco-2 cell epithelial model after
LPS stimulation

observed for BAY 87-2243 (P < 0.05), as shown in Fig. 3C,
E-G.

After LPS stimulation, the TEER of Caco-2 cells was significantly lower than that of the control group (P < 0.05).
Compared with LPS alone, DMOG significantly
increased the TEER (P < 0.05) (Fig. 3A) and decreased
the FD-4 concentration (P < 0.05) (Fig. 3B); the opposite
effect was observed for BAY 87-2243 (Fig. 3A, B).

The attenuation of LPS-induced epithelial model injury in
Caco-2 cells by HIF-1α is regulated by the mTOR/P70S6K
pathway

Effects of HIF-1α activators and inhibitors on TJ protein
expression in Caco-2 cells after LPS stimulation

WB indicated that LPS increased the protein expression level of HIF-1α in Caco-2 cells (P < 0.05; Fig. 3C and
D). Compared with LPS alone, DMOG upregulated theexpression of HIF-1α and the TJ-related proteins ZO-1,
occludin and claudin-1 (P < 0.05); the opposite effect was

Compared with those in the LPS group, the TEER of
the Caco-2 cell epithelial model in the L + R group significantly decreased, the FD-4 concentration significantly
increased (Fig. 4A-B), the expression levels of p-mTOR/
mTOR, p-P70S6K/P70S6K and HIF-1α significantly
decreased, and the expression levels of TJ-related proteins decreased (Fig. 4C. In the L + M group, the TEER of
the Caco-2 cell epithelial model significantly increased,
and the FD-4 concentration significantly decreased(Fig. 4A-B, P < 0.05). The expression levels of p-mTOR/
mTOR and p-P70S6K/P70S6K significantly increased

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

Page 7 of 12

Fig. 3 Effects of HIF-1α activators and inhibitors on the TEER (A), FD-4 concentration (B), and the expression of HIF-1α (C, D) and TJ-related proteins (C,E-G) in the Caco-2 cell epithelial model after LPS stimulation. LPS-induced damage to Caco-2 cell epithelial model showed the decrease of TEER value,
the increase of FD4 concentration and down-regulation of TJ-related proteins. DMOG, the HIF-1α activator, alleviated the LPS-induced damage to Caco-2cell epithelial model, increased TEER value, decreased FD4 concentration and up-regulated the expression of TJ-related proteins. BAY 87-2243, a HIF-1α
inhibitor, had the opposite effect. LPS up-regulated HIF-1α expression, DMOG further up-regulated HIF-1α expression, while BAY 87-2243 had the opposite effectCon: control group, LPS: LPS group, L + D: LPS + DMOG group, L + B: LPS + BAY 87-2243 group. *P < 0. 05, **P < 0. 01 vs. Con group; #P < 0.05, #P < 0.01 vs.
LPS groupHuang et al. BMC Molecular and Cell Biology

Fig. 4 (See legend on next page.)(2024) 25:13

Page 8 of 12

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

Page 9 of 12(See figure on previous page.)
Fig. 4 The effect of HIF-1α on LPS-induced epithelial model damage to Caco-2 cells is regulated by the mTOR/P70S6K pathway. (A) Rapamycin (mTOR
inhibitor) decreased TEER of Caco-2 cell monolayer, and MHY1487 (mTOR activator) increased TEER of Caco-2 cell monolayer. DMOG (HIF-1α activator) can
rescue the decrease of TEER caused by rapamycin, and BAY87-2243 (HIF-1α inhibitor) can rescue the increase of TEER caused by MHY1487; (B) Rapamycin
increased the FD4 concentration in Caco-2 cell monolayer, while MHY1487 had opposite effect. DMOG rescued the increase in FD4 concentration causedby rapamycin, and BAY 87-2243 rescued the decrease in FD4 concentration caused by MHY1487; (C) Western blot was used to detect the expression of
p-mTOR, mTOR, p-P70S6K, P70S6K, HIF-1α and TJs (ZO-1, occludin and claudin-1)
Con: control group, LPS: LPS group, L + R: LPS + rapamycin group, L + M: LPS + MHY1487 group, L + R + D: LPS + rapamycin + DMOG group. L + M + B:
LPS + MHY1487 + BAY 87-2243 group. *P < 0. 05, **P < 0. 01 vs. LPS group; ##P < 0. 01 vs. L + R group; △P < 0. 05, △△P < 0. 01 vs. L + M group(P < 0.05), while the expression levels of HIF-1α and TJrelated proteins did not significantly increase (P > 0.05,
Fig. 4C). The results indicated that compared with thosein the L + R group, the TEER in the L + R + D group significantly increased, the FD-4 concentration significantly
decreased (P < 0.05, Fig. 4A-B), and the expression levels of p-mTOR/mTOR and p-P70S6K/P70S6K did notchange significantly (Fig. 4C). The expression levelsof HIF-1α and TJ-related proteins increased (P < 0.05,
Fig. 4C). Compared with those in the L + M group, the
TEER of Caco-2 cells in the L + M + B group significantly
decreased (P < 0.05), the FD-4 concentration significantly increased (P < 0.05, Fig. 4A-B), and the expression
levels of p-mTOR/mTOR and p-P70S6K/P70S6K did
not change significantly (P > 0.05, Fig. 4C). The expression levels of HIF-1α and TJ-related proteins decreased(P < 0.05, Fig. 4C).

Discussion
Using Caco−2 human colorectal adenocarcinoma cells,
we showed that HIF1-alpha may alleviate LPS-induced
intestinal epithelial model in vitro.HIF−1α attenuated
LPS-induced Caco−2 cell injury by upregulating structural protein expression and decreasing permeability;
additionally, HIF−1α alleviated LPS-induced Caco−2
injury, a process that might be regulated by mTOR/
P70S6K signalling.
LPS is a major component of the outer membrane ofgram-negative bacteria, which can activate cells suchas macrophages, endothelial and epithelial cells, andenable host cells to produce cytokines and inflammatory
mediators [11]. The inflammatory response is a defense
mechanism against infection. In fact, systemic inflammatory responses, such as sepsis, can lead to multiple
organ failure or death [1]. Sepsis is a clinical syndrome
characterized by systemic inflammation and circulatory
system damage caused by infection [1, 14]. LPS plays acrucial role in causing intestinal and systemic inflammatory responses [1]. Previous studies have shown thatLPS increases intestinal TJ permeability and destroys theintestinal barrier [15, 16].
Previous studies have shown that LPS regulate inflammation and the immune system by activating signaling
pathways mediated by relevant receptors [17]. A highdose of LPS induces a strong inflammatory response,
leading to sepsis or septic shock. On the contrary, a low

dose of LPS induces a protective cross-tolerance state[18]. Calvello et al.demonstrated that 1 µg/ml of LPS cantrigger an inflammatory response in Caco−2 cells, butdoes not induce cell death [19]. Endotoxin tolerance has
been shown to prevent lethality after ischemia/reperfusion injuries, sepsis, and endotoxic shock. One study
showed that lethal propionibacterium acnes (PA)-primed
LPS-induced hepatic injury can be prevented by administering a tolerizing dose of LPS prior to PA-priming [20].
Another study [21] demonstrated that LPS reprocessing
may achieve neuroprotective effects by regulating inflammatory mediators, inducing neuroprotective factors andinhibiting the release of proinflammatory cytokines in rat
models of cerebral ischemic injury. In our present study,
we found that LPS below 105 ng/ml increased Caco−2
activity, and this concentration was many times higher
than that previously reported ones, which may be related
to different reagent manufacturers and models. The exact
reasons need further study. As a classical model for invitro simulation of the intestinal epithelial model anddrug transport, the Caco−2 monolayer epithelial model
has been widely used. Another study, which [22] successfully established an organoid intestinal injury modelsuitable for sepsis, examined the effects of LPS-induced
intestinal damage similar to sepsis. The biological characteristics of this model were systematically evaluated, andthe results revealed that this organoid-based model provides a platform for revealing the potential mechanism ofsepsis associated intestinal epithelial injury and screening
therapeutic agents.
FD−4 is a marker of changes in intestinal permeability and the intestinal barrier [23]. TJ-related proteins,
including ZO−1, occludin, and claudin−1, play important roles in maintaining intercellular connections andcell barriers [24, 25]. Occludin plays an important role inmaintaining the intestinal TEER, has adhesion functions,and serves as a fence. Claudin−1 affects the permeabilityof intercellular substances, especially cations, forming a
selective paracellular ion channel. ZO−1 binds to a variety of cytoskeletal proteins and plays a supporting role in
TJs [24, 25].
Destruction of the intestinal barrier was exacerbatedin intestinal HIF−1α-deficient mice [26]. Colgan et al.
[27] showed that compared with wild-type animals,
mice with intestinal epithelial HIF−1α mutations (inhibition of HIF−1α expression) had more severe colitis,

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

weight loss, reduced colon length, and increased intestinal permeability. By contrast, intestinal epithelium withthe von Hippel-Lindau mutation (which causes the sustained expression of HIF−1α) has a protective effect on
the intestine. Claudin−1 has been shown to regulate
dysfunction in various human diseases [28]. Singer et al.
[1] confirmed that HIF−1α has a crucial regulatory rolewith respect to claudin−1 in the intestinal epithelium,
and through knock-out (KO) and overexpression experiments, it was shown that HIF−1α plays a fundamental
regulatory role in the expression of claudin−1 at the genepromoter level. Reintroduction of the claudin−1 gene
into HIF−1β KO cells can lead to abnormal cell barrier
function and morphology. In TJ target screening, claudin−1 is the main cause of morphological abnormalities
in HIF−1α-deficient intestinal epithelial cell lines [29, 30].
In this study, HIF−1α simulator agonists or inhibitors was used with LPS in vitro. HIF−1α activation andpermeability of Caco−2 cells decreased subsequently by
up-regulating the expression of TJS; on the contrary, thepermeability of Caco−2 cells increased after HIF−1α inhibition by down-regulating the expression of TJs. These
results further confirmed that HIF−1α has protective
effects against LPS-induced intestinal mucosal epithelial
model injury through regulating the expression of TJs
and our conclusion was consistent with our previous animal research [9].
Study also demonstrated that the mTOR signalling
pathway is the main sensor that responds to the energy
status and autophagy in energy consumption [31] andhas a cascade amplification effect on HIF−1α [32, 33].
The mTOR signalling pathway may play an importantrole in intestinal dysfunction in sepsis. mTOR, a part of
mammalian target of rapamycin complex 1 (mTORC1),is activated in the form of phosphorylation, p-mTOR.
Protein kinase B (AKT) is activated downstream byphosphatidylinositol−3-kinase (PI3K) and subsequently
activates mTOR [31]. p-mTOR can phosphorylate
ribosomal protein S6 kinase (p70S6K) [32, 33]; subsequently, p70S6K activates rpS6 to promote HIF−1α protein synthesis [34, 35]. On the contrary, p-mTOR canalso phosphorylate eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) [36], which disrupts
4E-BP1 binding to eukaryotic translation inhibitory factor 4E (eIF−4E) and then inhibits eIF−4E [37], thereby
blocking HIF−1α protein synthesis [38]. mTOR inhibitors
can inhibit HIF−1α protein synthesis and reduce HIF−1α
activity [39]. In septic rats, rapamycin can significantly
reduce the levels of p-mTOR, p-P70S6K and HIF−1α inthe myocardium and exert a protective effect by inhibiting autophagy in cardiomyocytes [40].
To further ascertain the crosstalk between themTOR/70S6K signaling pathway and HIF−1α protein
synthesis. Firstly, mTOR activator/inhibitor was used

Page 10 of 12with LPS treatment on Caco−2. Consequently, mTOR
inhibition, p-mTOR/mTOR and p-P70S6K/P70S6K
ratio decreased and the downstream molecule HIF−1α
expression was down-regulated, which counteracted
the protective effect of HIF−1α on LPS-induced intestinal mucosal cell injury, while the mTOR activator partially alleviated LPS-induced intestinal mucosal injury, aprocess that may not act through HIF−1α. The specificmechanism needs to be further clarified. Secondly, when
HIF−1α activator and the mTOR jointly acted on LPSinduced intestinal mucosal injury, The results showedthat DMOG could partially alleviate the negative effectof rapamycin on intestinal mucosal injury, while the
HIF−1α inhibitor and the mTOR activator jointly acted
on LPS-induced intestinal mucosal injury. These results
further confirmed HIF−1α has a protective effect on LPSinduced intestinal mucosal cell injury, a process that isregulated by the mTOR/P70S6K signalling pathway.
There are some limitations in this study. LPS is theendotoxin on the cell membrane of Gram-negative bacteria, the main pathogen of intestinal sepsis, and is considered the prototype of pathogen-associated molecularpatterns (PAMPS) [41]. Toll-like receptors (TLRs) are
the pattern recognition receptors (PRRs) that recognize
PAMPs in microbial species, including bacterial flagellin recognized by TLR5. Of all PRRs in innate immunity,Toll-like receptor 2 (TLR2) recognizes the structurally
broadest range of different bacterial PAMPs, becauseof its biomedical importance and because its genetics
and biochemistry are presently most completely known
among all Gram-positive bacteria. Initial reports indicated that TLR2 binds peptidoglycan (PG), but the effects
were later found to be due to contaminating highly active
natural lipoproteins and/or lipopeptides of PAMPs)[42]. In contrast, peptidoglycan is recognized by nucleotide-binding oligomerization domains 1 and 2 (NOD1and NOD2) [43]. Thus, it would be helpful to use other
PAMPs for example for gram-positive bacteria to compare the effect in future studies.
In this study, we investigated the protective effect of themTOR/HIF−1α pathway on intestinal mucosal epithelial
model injury in sepsis by detecting the pathological structure of the intestinal mucosa, tight junction proteins, andresistance and permeability of the intestinal mucosal epithelial model. The effect of the mTOR/HIF−1α pathway
on the ultrastructural changes in the intestinal mucosal epithelial model induced by sepsis needs to be further explored. Previous studies have shown that HIF−1α
directly induces the expression of many metabolic genes
in cancer cells to enhance aerobic glycolysis, such as
glucose transporters (GLUTs), pyruvate dehydrogenasekinase 1 (PDHK1), lactate dehydrogenase A (LDHA), tomeet the energy needs of tumor growth [44]. Intestinal
epithelial cell GLUT2 knockout mice or by inhibiting

Huang et al. BMC Molecular and Cell Biology(2024) 25:13

glucose metabolism can improve the damage to intestinal
epithelial model function induced by hyperglycemia andinhibit intestinal bacterial ectopic [45]. Whether HIF−1ɑ
induced by hypoxia in septic intestinal epithelial cells canprovide energy for intestinal epithelial cells, and reduce
mucosal damage by recruiting GLUT1 remains to besolved. This is also the shortcoming of the study.
Additionally, both DMOG and BAY 87−2243 are indirect regulators of HIF−1α expression. Further studies
using HIF−1α gene knockout or overexpression technology could clarify the protective effect of the mTOR/
HIF−1ɑ pathway on sepsis-induced intestinal mucosa
injury. Previous studies have shown that the choice ofexperiments in normoxic [46] or hypoxic [47] environments should be based on the purpose of the experiment. In this study, the essence of sepsis-induced
intestinal mucosal injury is intestinal mucosal ischemia
and hypoxia; thus, the experiment was conducted undernormal oxygen. In the future, we will challenge the low
oxygen environment to explore the regulatory effect of
HIF−1α on sepsis intestinal mucosal injury.

Conclusions
In summary, the results from this study further verified that HIF-1α attenuated LPS-induced epithelial
model dysfunction in intestinal epithelial cells (Caco2) through a mechanism that may be regulated by themTOR/P70S6K signalling pathway to improve changes
in TJ structure and the expression of TJ-related proteins
and to reduce intestinal mucosa permeability. This study
provides the premise and basis for further research in the
future.
Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12860-024-00509-5.
Supplementary Material 1
Acknowledgements
Not applicable.
Author contributions
Wenbin Teng and Yuhong Li: conception and design of the research andwriting of the manuscript. Liuxu Yao acquisition of data. Zeyong Huang and
Kai Xie: analysis and interpretation of the data. Wenbin Teng and Liuxu Yao:
statistical analysis. Yuhong Li: obtaining financing. Yuhong Li, Rui He, andSuqin Huang: critical revision of the manuscript for intellectual content. All
authors contributed to the article and approved the submitted version.
Funding
This study was supported by the Zhejiang Provincial Department of Scienceand Technology Fund (Grant No. LY21H150001); Zhejiang Provincial HealthCommittee Fund (Grant No. 2020KY329); Shaoxing Science and Technology
Bureau Fund (Grant No. 2020A13014); and Hangzhou Medical and Health
Science and Technology Project (Grant No. B20210683).

Page 11 of 12

Data availability
Supplementary material associated with this article can be found, in the
online version.The email address of the corresponding author is yuh_li@zju.
edu.cn.

Declarations
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 14 April 2023 / Accepted: 5 April 2024

References
1. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer
M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM, Hotchkiss RS, Levy
MM, Marshall JC, Martin GS, Opal SM, Rubenfeld GD, van der Poll T, Vincent JL,
Angus DC. The third international consensus definitions for sepsis and septic
shock (Sepsis-3). JAMA. 2016;315(8):801–10.
2. Nie J, Kang F, Zhang C, Chai C, He M. [Research on the signal pathway ofhydrogen sulfide regulating autophagy to protect intestinal injury in sepsis].
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2020;32(10):1277–80. Chinese.
3. Lou X, Xue J, Shao R, Mo C, Wang F, Chen G. Postbiotics as potential new
therapeutic agents for sepsis. Burns Trauma. 2023;11:tkad022.
4. Sprague JL, Kasper L, Hube B. From intestinal colonization to systemic
infections: Candida albicans translocation and dissemination. Gut Microbes.
2022;14(1):2154548.
5. Walaas GA, Gopalakrishnan S, Bakke I, Skovdahl HK, Flatberg A, Østvik AE,
Sandvik AK, Bruland T. Physiological hypoxia improves growth and functional
differentiation of human intestinal epithelial organoids. Front Immunol.
2023;14:1095812.
6. Zhu Y, Wang Y, Teng W, Shan Y, Yi S, Zhu S, Li Y. Role of Aquaporin-3 in Intestinal Injury Induced by Sepsis. Biol Pharm Bull. 2019;42(10):1641–50.
7. Yu W, Wang Z, Yu X, Zhao Y, Xie Z, Zhang K, Chi Z, Chen S, Xu T, Jiang D, Guo
X, Li M, Zhang J, Fang H, Yang D, Guo Y, Yang X, Zhang X, Wu Y, Yang W, Wang
D. Kir2.1-mediated membrane potential promotes nutrient acquisition andinflammation through regulation of nutrient transporters. Nat Commun.
2022;13(1):3544.
8. Ben-Cheikh A, Bali A, Guerfali FZ, Atr C, Attia H, Laouini D. Hypoxia-inducible
factor-1 alpha stabilization in human macrophages during leishmania major infection is impaired by parasite virulence. Korean j Parasitol.
2022;60(5):317–25.
9. Xiao X, Ye M, Xu Y, Jiang C. Role of hypoxia-inducible factor in immunometabolism of sepsis. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2020;32(3):382–4.
Chinese.
10. Lai X, Zhong Z, Lin B, Wu Y, Ma Y, Zhang C, Yang Y, Zhang M, Qin W, Fu X,
Shu H. RNA-seq and qRT-PCR analyses reveal the physiological response toacute hypoxia and reoxygenation in Epinephelus coioides. Front Physiol.
2022;13:1049776.
11. Lei X, Teng W, Fan Y, Zhu Y, Yao L, Li Y, Zhu S. The protective effects of HIF-1α
activation on sepsis induced intestinal mucosal barrier injury in rats model ofsepsis. PLoS ONE. 2022;17(5):e0268445.
12. Chen F, Yi WM, Wang SY, Yuan MH, Wen J, Li HY, Zou Q, Liu S, Cai ZY. A longterm high-fat diet influences brain damage and is linked to the activation of
HIF-1α/AMPK/mTOR/p70S6K signalling. Front Neurosci. 2022;16:978431.
13. Cheng SC, Quintin J, Cramer RA, Shepardson KM, Saeed S, Kumar V, Giamarellos-Bourboulis EJ, Martens JH, Rao NA, Aghajanirefah A, Manjeri GR, Li Y, Ifrim
DC, Arts RJ, van der Veer BM, Deen PM, Logie C, O’Neill LA, Willems P, van de
Veerdonk FL, van der Meer JW, Ng A, Joosten LA, Wijmenga C, Stunnenberg
HG, Xavier RJ, Netea MG. mTOR- and HIF-1α-mediated aerobic glycolysis asmetabolic basis for trained immunity. Science. 2014;345(6204):1250684.
14. Abe T, Ogura H, Kushimoto S, Shiraishi A, Sugiyama T, Deshpande GA, et al.
Variations in infection sites and mortality rates among patients in intensive

Huang et al. BMC Molecular and Cell Biology

15.
16.
17.
18.
19.
20.

21.
22.
23.

24.
25.
26.

27.
28.
29.
30.
31.
32.
33.

(2024) 25:13

care units with severe sepsis and septic shock in Japan. J Intensive Care.
2019;7:28.
Wu XX, Huang XL, Chen RR, Li T, Ye HJ, Xie W, et al. Paeoniflorin prevents
intestinal barrier disruption and inhibits lipopolysaccharide (LPS)-induced
inflammation in Caco-2 cell monolayers. Inflammation. 2019;42(6):2215–25.
Siliciano JD, Goodenough DA. Localization of the tight junction protein, ZO-1,
is modulated by extracellular calcium and cell-cell contact in Madin-Darby
canine kidney epithelial cells. J Cell Biol. 1988;107(6 Pt 1):2389–99.
Zhou B, Weng G, Huang Z, Liu T, Dai F. Arctiin prevents LPS-Induced Acute
Lung Injury via Inhibition of PI3K/AKT signaling pathway in mice. Inflammation. 2018;41(6):2129–35.
Delayre-Orthez C, de Blay F, Frossard N, Pons F. Dose-dependent effects ofendotoxins on allergen sensitization and challenge in the mouse. Clin Exp
Allergy. 2004;34(11):1789–95.
Calvello R, Aresta A, Trapani A, Zambonin C, Cianciulli A, Salvatore R, et al.
Bovine and soybean milk bioactive compounds: effects on inflammatory
response of human intestinal Caco–2 cells. Food Chem. 2016;210:276–85.
Margenthaler JA, Landeros K, Kataoka M, Eilers M, Ku G, Flye MW. Effects ofendotoxin tolerance on Propionibacterium acnes-primed lipopolysaccharide
hepatic injury. J Surg Res. 2003;112(1):102–10. https://doi.org/10.1016/s00224804. PMID: 12873441.
Gong G, Huang Y, Yuan LB, Hu L, Cai L. Requirement of LRG in endotoxin-mediated brain protection. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.
2011;27(8):865–7. Chinese.
Huang S, Zhang S, Chen L, Pan X, Wen Z, Chen Y, et al. Lipopolysaccharide
induced intestinal epithelial injury: a novel organoids-based model for sepsis
in vitro. Chin Med J (Engl). 2022;135(18):2232–9.
Kim KY, Son JD, Hwang SJ, Lee JK, Park JY, Park KI, Oh TW. Fermented
glutinous rice extract mitigates dss-induced ulcerative colitis by alleviating
intestinal barrier function and improving gut microbiota and inflammation.
Antioxid (Basel). 2023;12(2):336.
Park HY, Yu JH. Hesperidin enhances intestinal barrier function in Caco-2 cellmonolayers via AMPK-mediated tight junction-related proteins. FEBS Open
Bio. 2023;13(3):532–44.
Fujikawa M, Sugimoto H, Tamura R, Fujikawa K, Yamagishi A, Ueda Y. Effects
of mucopolysaccharide polysulphate on tight junction barrier in humanepidermal keratinocytes. Exp Dermatol. 2022;31(11):1676–84.
Shao T, Zhao C, Li F, Gu Z, Liu L, Zhang L, Wang Y, He L, Liu Y, Liu Q, Chen
Y, Donde H, Wang R, Jala VR, Barve S, Chen SY, Zhang X, Chen Y, McClainCJ, Feng W. Intestinal HIF-1α deletion exacerbates alcoholic liver disease by inducing intestinal dysbiosis and barrier dysfunction. J Hepatol.
2018;69(4):886–95.
Colgan SP, Curtis VF, Lanis JM, Glover LE. Metabolic regulation of intestinal
epithelial barrier during inflammation. Tissue Barriers. 2015;3(1–2):e970936.
Capaldo CT, Nusrat A. Claudin switching: physiological plasticity of the tightjunction. Semin Cell Dev Biol. 2015;42:22–9.
Liu H, Zhang H, Zhang X, Chen Q, Xia L. Role of succinic acid in the regulationof sepsis. Int Immunopharmacol. 2022;110:109065.
Zeitouni NE, Chotikatum S, von Köckritz-Blickwede M, Naim HY. The impact ofhypoxia on intestinal epithelial cell functions: consequences for invasion bybacterial pathogens. Mol Cell Pediatr. 2016;3(1):14.
Tan VP, Miyamoto S. Nutrient-sensing mTORC1: integration of metabolic andautophagic signals. J Mol Cell Cardiol. 2016;95:31–41.
Xiang T, Lin YX, Ma W, Zhang HJ, Chen KM, He GP, Zhang X, Xu M, Feng QS,
Chen MY, Zeng MS, Zeng YX, Feng L. Vasculogenic mimicry formation in EBVassociated epithelial malignancies. Nat Commun. 2018;9(1):5009.
Wei J, Wu J, Xu W, Nie H, Zhou R, Wang R, Liu Y, Tang G, Wu J. Salvianolic acid
B inhibits glycolysis in oral squamous cell carcinoma via targeting PI3K/AKT/HIF-1α signaling pathway. Cell Death Dis. 2018;9(6):599.

Page 12 of 12

34. Xu Q, Liu LZ, Qian X, Chen Q, Jiang Y, Li D, Lai L, Jiang BH. MiR-145 directly
targets p70S6K1 in cancer cells to inhibit tumor growth and angiogenesis.
Nucleic Acids Res. 2012;40(2):761–74.
35. Occhiuzzi MA, Lico G, Ioele G, De Luca M, Garofalo A, Grande F. Recent
advances in PI3K/PKB/mTOR inhibitors as new anticancer agents. Eur J Med
Chem. 2023;246:114971.
36. Song G, Shang C, Zhu Y, Xiu Z, Li Y, Yang X, Ge C, Han J, Jin N, Li Y, Li X, Fang J.
Apoptin inhibits glycolysis and regulates autophagy by targeting pyruvatekinase M2 (PKM2) in lung cancer A549 cells. Curr Cancer Drug Targets. 2022.
37. Takahashi S, Shibutani S, Iwata H. Nuclear-targeted 4E-BP1 is dephosphorylated, induces nuclear translocation of eIF4E, and alters mRNA translation.
Exp Cell Res. 2022;418(1):113246.
38. Sui A, Yao C, Chen Y, Li Y, Yu S, Qu J, Wei H, Tang J, Chen G. Polystyrene nanoplastics inhibit StAR expression by activating HIF-1α via ERK1/2 MAPK andAKT pathways in TM3 leydig cells and testicular tissues of mice. Food Chem
Toxicol. 2023;173:113634.
39. Wang S, Shi JT, Wang XR, Mu HX, Wang XT, Xu KY, Wang QS, Chen SW.
1H-Indazoles derivatives targeting PI3K/AKT/mTOR pathway: Synthesis, antitumor effect and molecular mechanism. Bioorg Chem. 2023;133:106412.
40. Han W, Wang H, Su L, Long Y, Cui N, Liu D. Inhibition of the mTOR pathway
exerts cardioprotective effects partly through Autophagy in CLP rats. Mediators Inflamm. 2018;2018:4798209.
41. Somensi N, Rabelo TK, Guimarães AG, Quintans-Junior LJ, de Souza Araújo
AA, Moreira JCF, Gelain DP. Carvacrol suppresses LPS-induced pro-inflammatory activation in RAW 264.7 macrophages through ERK1/2 and NF-kB
pathway. Int Immunopharmacol. 2019;75:105743.
42. Zähringer U, Lindner B, Inamura S, Heine H, Alexander C. TLR2 - promiscuousor specific? A critical re-evaluation of a receptor expressing apparent broad
specificity. Immunobiology. 2008;213(3–4):205–24.
43. Keestra-Gounder AM, Tsolis RM. NOD1 and NOD2: beyond Peptidoglycan
Sensing. Trends Immunol. 2017;38(10):758–67.
44. Zhou L, Wang Y, Zhou M, Zhang Y, Wang P, Li X, Yang J, Wang H, Ding Z.
HOXA9 inhibits HIF-1α-mediated glycolysis through interacting with CRIP2
to repress cutaneous squamous cell carcinoma development. Nat Commun.
2018;9(1):1480.
45. Thaiss CA, Levy M, Grosheva I, Zheng D, Soffer E, Blacher E, Braverman S,
Tengeler AC, Barak O, Elazar M, Ben-Zeev R, Lehavi-Regev D, Katz MN, Pevsner-Fischer M, Gertler A, Halpern Z, Harmelin A, Aamar S, Serradas P, Grosfeld
A, Shapiro H, Geiger B, Elinav E. Hyperglycemia drives intestinal barrier
dysfunction and risk for enteric infection. Science. 2018;359(6382):1376–83.
46. Weng ML, Chen WK, Chen XY, Lu H, Sun ZR, Yu Q, Sun PF, Xu YJ, Zhu MM,
Jiang N, Zhang J, Zhang JP, Song YL, Ma D, Zhang XP, Miao CH. Fasting
inhibits aerobic glycolysis and proliferation in colorectal cancer via theFdft1-mediated AKT/mTOR/HIF1α pathway suppression. Nat Commun.
2020;11(1):1869.
47. Wang P, Zhao L, Gong S, Xiong S, Wang J, Zou D, Pan J, Deng Y, Yan Q, Wu N,
Liao B. HIF1α/HIF2α-Sox2/Klf4 promotes the malignant progression of glioblastoma via the EGFR-PI3K/AKT signalling pathway with positive feedback
under hypoxia. Cell Death Dis. 2021;12(4):312.

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