Metabolomics reveals that chronic restraint stress alleviates carbon tetrachloride-induced hepatic fibrosis through the INSR/PI3K/AKT/AMPK pathway

doi:10.21203/rs.3.rs-2536417/v1

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Research Article

Metabolomics reveals that chronic restraint stress alleviates carbon tetrachloride-induced hepatic fibrosis through the INSR/PI3K/AKT/AMPK pathway

https://doi.org/10.21203/rs.3.rs-2536417/v1

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published 23 Nov, 2023

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Hepatic fibrosis (HF) could be developed into liver cirrhosis or even hepatocellular carcinoma. Stress has an important role in the occurrence and development of various considerable diseases. However, the effect of a certain degree stress on HF is still controversial. In our study, stress was simulated with regular chronic restraint stress (CRS) and HF model was induced with CCl₄ in mice. We found that CRS was able to attenuate CCl₄-induced liver injury and fibrosis in mice. Surprisingly, behavioral analysis showed that the mice in the HF group exhibited depression-like behavior. Further, the metabolomic analysis revealed that 119 metabolites and 20 metabolic pathways were altered in mice liver, especially the betaine metabolism pathway. Combined with the results of ingenuity Pathway Analysis (IPA) analysis, the key proteins INSR, PI3K, AKT, and p-AMPK were identified and verified, and the results showed that CRS could upregulate the protein levels and mRNA expression of INSR, PI3K, AKT, and p-AMPK in liver tissues of HF mice. It suggested that CRS alleviated CCl₄-induced liver fibrosis in mice through upregulation of the INSR/PI3K/AKT/AMPK pathway. Proper stress might be a potential therapeutic strategy for the treatment of chronic liver disease, which provided new insights into the treatment of HF.

hepatic fibrosis

chronic restraint stress

metabolomics

INSR pathway

carbon tetrachloride

Hepatic fibrosis (HF) caused by various chronic stimuli, such as viruses, alcohol, and autoimmune diseases, which is characterized by excessive deposition of extracellular matrix (ECM), is a reversible pathological process^[1–2]. Hepatic stellate cells (HSCs) play a significant role in the development of HF, activated HSCs are the key effectors of fibrogenesis. They are activated and transformed into myofibroblasts when damaged by pathogenic factors and participate in the injury repair process^[3–4]. The nature of HF is a wound healing response, but without appropriate intervention, it can progress to cirrhosis, portal hypertension, and hepatocellular carcinoma, resulting in increased morbidity and mortality^[5]. However, there are still no approved antifibrotic therapies for liver disease. Thus, there is an exigent need to clarify the cellular and molecular mechanisms of fibrosis regression in order for treating liver disease^[6].

Stress is used to refer to a condition where various or perceived stimuli alter the homeostatic state of an organism^[7], which regular accompanies with patients and exists in the process of disease therapy. Stress has an important role in the occurrence and development of various consider able diseases among cardiovascular^[8], digestive^[9], immune^[10], and nervous system^[11].A number of early reports have suggested that stress serves a major role in the initiation, course and outcome of liver diseases. Study demonstrated that chronic restraint stress (CRS) decreased the repair potential from mesenchymal stem cells on liver injury by inhibiting TGF-β1 generation, thereby reducing HF^[12]. Another work showed that CRS reduces the development of liver fibrosis by inhibiting the activation of hepatic stellate cells via 5-HT2B receptor^[13]. In addition, it is reported that stress can attenuate hepatic lipid accumulation via elevation of hepatic β-muricholic acid levels in mice with nonalcoholic steatohepatitis^[14]. On the contrary, a few studies demonstrated that stress promoted the progression of liver fibrosis in rats or led to steatosis and non-alcoholic steatohepatitis^{[15, 16]}. Therefore, the effects of stress on liver disease, especially HF, are widely controversial and uncertain, and the mechanisms remain to be elucidated.

The term "restraint stress" refers to a specific type of stress resulting from the restriction of movement. In our study, we constructed an animal model by giving restraint stress while inducing liver fibrosis in mice with CCl₄. We attempted to elucidate the effect of CRS on liver fibrosis and the potential signaling pathways. The workflow of this study was summarized in Fig. 1.

2.1 Chemicals and reagents

The standards of reserpine and cholic acid-d4 were purchased from Sigma Aldrich (St. Louis, MO, USA), betaine (97%) was purchased from TCI (Shanghai, China). HPLC grade acetonitrile was purchased from Merck & Co (Billerica, MA, USA). Ultrapure water was produced from a Milli-Q system (Millipore, MA, USA). The alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and hydroxyproline detection kits were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.2 Animals experiments

Male C57BL/6 mice aged 6–8 weeks were obtained from the Guangzhou Baiyun District Suibei Experimental Animal Farm (Guangzhou, China). The mice were maintained under controlled conditions (22 ± 2 ℃, 60% ± 5% humidity, 12 h/12 h day/night cycle) and given standard water or food ad libitum. The animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Traditional Chinese Medicine (reference No. 20211102).

The mice were randomly divided into four groups: (1) control group: given olive oil; (2) CRS group: given CRS procedure; (3) HF group: given CCl₄; (4) HF + CRS group: given CCl₄ and CRS procedure.

Mouse model of HF

to induce a liver fibrosis model, the mice were injected intraperitoneally with 15% CCl₄ (v/v, dissolved in olive oil) at a concentration of 5.0 mL/kg twice a week for 9 weeks continuously.

CRS procedure

the mice were assigned to restraint tubes (50 mL conical tubes) and restrained for 4 hours per day for 9 weeks. In the tubes, they could not move freely and did not have access to water or food, but they were not pressed and could breathe freely.

2.3 Evaluation of liver function and fibrosis

Liver function analysis

Serum of mice was collected by centrifugation at 3000 rpm, 4°C for 10 min. The serum levels of the ALT, AST, and ALP were measured by commercially available kit.

Hematoxylin and eosin staining

Liver tissues were fixed in 4% formaldehyde solution and then embedded in paraffin. Afterwards, 4 µm thick sections were cut by using a RM2016 microtome (Leica, Shanghai, China). stained with hematoxylin-eosin (H&E).

Fibrotic test

To detect liver fibrosis, the paraffin sections were stained with Masson’s Trichrome Staining, the blue-stained areas in the tissue sections were assessed using an Image-Pro Plus image analyzer (Media Cybernetics, Inc., Rockville, MD, USA) for semi-quantitative analysis. Hydroxyproline levels in mice serum were measured with hydroxyproline detection kit.

2.4 Behavioral studies

Sucrose preference test (SPT)

Animals were first trained to consume a 1% sucrose solution from two differing bottles (48 h before the formal experiment). Twenty-four hours later, the animals were allowed free access to 1% sucrose and water from two differing bottles. To avoid bottle side preference, the two bottles were switched. The amounts in the two bottles were measured after 24 h and sucrose preference was calculated.

Open field test (OFT)

We characterized mice behavior as they freely explored an open-field plastic chamber (40 cm width × 40 cm length × 30 cm height) using a video tracking system (Smart 3.0). The mice were placed in this arena for 10 min, and time spent in the center region during the final 5 min period was recorded.

Tail suspension test (TST)

The mice were suspended by their tails for 6 minutes from a metal rod (30 cm above the ground) and monitored using a video tracking system (Smart 3.0). Immobility duration period was recorded during the final 4 min.

Forced swimming test (FST)

Mice were placed in a container filled with water that eventually resulted in immobility, reflecting behavioral despair. Water (25 ± 1°C) was placed in a transparent acrylic cylinder bath (10 cm in diameter, 30 cm in height) filled to a depth of 15 cm. Mice were placed in the water for six minutes using a video tracking system (Smart 3.0); immobility duration within the final 4 min of testing was recorded.

2.5 Immunohistochemistry and immunofluorescence staining

Immunohistochemistry assay was performed according to method described before^[17], α-SMA antibody (Proteintech, 14395-1-AP), collagen type III (N-terminal) polyclonal antibody (Proteintech, 22734-1-AP), and p-AMPKα antibody (Cell Signaling Technology, #2535) were used.

2.6 Metabolomic profiling

Sample preparation: Sample extraction procedure were based on several previous studies with minor modifications^[18]. In brief, 50 mg liver tissue was mixed with 200 µL MeOH and the mixtures were vortexed for 3 min, then centrifuged at 14,000 rpm for 15 min at 4°C. Next, the supernatant was vacuum dried, reconstituted in 100 µL of MeOH:H₂O (1:1, v: v) with internal standards (500 nM reserpine and d4-CA) and centrifuged at 14000 rpm for 30 min at 4°C before analysis. Quality control (QC) samples were prepared by pooling and aliquoting sample extracts. The distribution of QC data in principal component analysis (PCA) and the standard deviation (STD) of retention times was used to evaluate data quality.

Chromatography and mass spectrometry conditions: LC-MS nontargeted analysis was performed using 1290 ultra-high-performance liquid chromatograph system (Agilent, California, USA) coupled to 6540 Q-TOF mass spectrometry (Agilent, California, USA), operated in positive and negative ion modes. The MS parameters were set as follows: nozzle voltage was set at 1500 V, capillary voltage at 3500 V, fragmentor voltage at 175 V, nebulizer pressure at 35 psi, sheath gas temperature at 350℃ and gas temperature at 320℃. The flow rate of sheath gas and gas were 11 L/min and 8 L/min, respectively. The analyses were separated with Waters BEH C18 column (150 mm, 2.1 x 1.7 µm) at 50℃ column temperature using constant flow rate of 0.4 mL/min. The LC conditions were as follows: 0.1% formic acid in deionized water (solvent A), and 0.1% formic acid in acetonitrile (solvent B); The gradient elution started with 2% B for 1 min, linearly increased to 100% B within 15 min and keep 100% of solvent B for 3 minutes. Afterwards, the composition was brought back to the initial ratio of 2% B within 1 min, followed by 1 min of re-equilibration.

Data processing and analysis: Raw data in both ionization modes was obtained by Mass Hunter Workstation software (Agilent, USA). The raw data format extracted by Profinder 10.0 (Agilent, USA) for selecting peaks, aligning, and then Mass Profiler Professional 15.0 (Agilent, USA) was used integrating to obtain three-dimensional information about the data of each sample, including all ion characteristics and their RT, m/z, and intensity. The output data was imported into SIMCA-P (version 14.1; Umetrics, Umea, Sweden) to conduct multivariate statistical analysis including the PCA and orthogonal partial least squares discriminate analysis (OPLS-DA) to obtain VIP value respectively of each feature. The features with p value below 0.05 and VIP above 1 was considered to be the potential differential metabolites. Accurate mass data and isotopic distribution for the precursor and product ion were compared to spectral data of the reference compounds in the databases, such as metlin, the Human Metabolome Database (HMDB, https://hmdb.ca/), MassBank (https://massbank.eu/MassBank/), Kyoto Encyclopedia of Genes and Genomes (KEGG, www.genome.jp/kegg/) and our own lab's proprietary database. Finally, the key metabolites were imported into MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) and Ingenuity Pathway Analysis (IPA, version 1.0, QIAGEN, DUS, Germany) for metabolic pathways analysis and network analysis.

2.7 Western blotting analysis

Liver tissues were homogenized in RIPA buffer, lysed on ice for 40 min, and then centrifuged at 4℃ at 12000 rpm for 10 min. The supernatant was qualified using BCA kit and boiled with loading buffer for 10 min. Thereafter, denatured protein was segregated by SDS polyacrylamide gel electrophoresis, and then transferred onto Immobilon-P-membranes (PVDF membrane). The membranes were blocked by 5% BSA, and then immunoblotted by various primary monoclonal antibodies. The horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and ECL were used to examine the expression of protein. Alpha-SMA antibody (Proteintech, 14395-1-AP), INSR antibody (Proteintech, 20433-1-AP), Phospho-PI3K p85 alpha (Tyr607) Antibody (Affinity, #AF3241), AKT Monoclonal antibody (Proteintech, 60203-2-Ig), p-AMPKα antibody (Cell Signaling Technology, #2535), and AMPKα antibody (Cell Signaling Technology, #2532) were used.

2.8 Real-Time PCR analysis

Total RNA was extracted from liver samples with TRIzol reagent (Accurate Biotechnology Co.,Ltd., Hunan, China) in accordance with the manufacturer’s protocol. First-strand cDNA was generated by using 1 µg of total RNA with a SYBR Green I reagent kit (Accurate Biotechnology Co.,Ltd., Hunan, China). The Real-Time PCR reaction was performed on a QuantStudio™ 5 (Applied Biosystems, USA) using QuantStudio™ design and analysis software v.1.5.1. The primers used are listed in Table S1, were purchased from Sangon Biotech (Shanghai, China). Target mRNA was normalized to Gapdh. Gapdh served as an endogenous control. Relative expression changes were determined using the 2^−ΔΔCt method.

2.9 Statistics

All data presented are expressed as arithmetic mean ± SEM. All statistical analyses were performed using SPSS software v22.0 (Chicago, IL, USA). For statistical comparisons between two groups, we used Student's T-test or Mann-Whitney U test for normally and non-normally distributed variables to obtain p value. For multiple comparisons, we used One-way ANOVA and Bonferroni correction or Kruskal Wallis H test for normally and non-normally distributed variables, respectively. And p value below 0.05 was considered statistically significant.

3.1 CRS mitigated CCl₄-induced liver injury

We established a CCl₄-induced liver injury model in 6-8-week-old mice. Following 9 weeks exposure of CCl₄, serological and histopathological examinations were performed with appropriate commercial kits. We measured the weekly weight change of the mice during the modeling periods (Fig. 2a), we found that the body weight of mice in the HF, CRS and HF + CRS groups decreased significantly compared with the normal group, but there was no significant difference between the liver fibrosis and HF + CRS group. Long-term and repeated injections of CCl₄ also caused noticeable increase in hepatic index, which indicated edema of the liver. However, compared with the HF group, the liver coefficient of the HF + CRS group instead decreased (Fig. 2b). Furthermore, the pathological observation also showed the same result of liver injury indicated by hepatic index, after CCl₄ induced, the liver was dull and less lustrous, and the liver surface showed obvious diffuse coarse granularity (Fig. 2c). The H&E staining pathological observation clearly showed hepatic morphological lesion with large areas of hepatocyte necrosis and increased inflammatory cells around the hepatic portal and sinusoid in HF (Fig. 2d), which were alleviated by CRS intervention. To further assess the liver function, we measured the serum levels of ALT, AST, and ALP. The results showed that the elevated levels of ALT, AST, and ALP in serum induced by CCl₄, which were diminished in CRS treatment (Fig. 2e). Collectively, all these results indicated that CRS ameliorated CCl₄-induced liver injury.

3.2 CRS attenuated CCl₄-induced HF

To investigate the progression of HF in mice, collagen fiber deposition and HSCs activation hall-marker α-SMA were further measured. Masson’s staining showed a significant increase in collagen fiber deposition around periportal areas with marked portal-portal and portal-central bridging in HF group, while it was reduced in the HF + CRS group (Fig. 3a). In addition, the immunohistochemical staining demonstrated α-SMA and type III collagen expression in liver tissues the HF group were remarkedly more than that in HF + CRS group (Fig. 3b, c). Semi-quantitative data was presented for better illustration (Fig. 3d). Also, we examined the protein level of α-SMA and the mRNA expression of α-SMA in mice liver tissue. As shown in Fig. 3e-g, CCl₄ induction enhanced the gene and protein expressions of α-SMA in mice, which were reversed in HF + CRS group. Hydroxyproline is a major component of fibrillar collagen of all types, we also measured the level of hydroxyproline in the serum and the results were consistent with the above (Fig. 3h).

3.3 CCl₄-induced liver fibrosis could cause depression-like behavior

Since CRS is a method of modeling mice depression, we did behavioral tests to determine whether CRS induces depression-like behavior and to observe whether CRS affects the behavior of HF mice in this experiment. Interestingly, experimental results show that mice in the HF group showed the most pronounced depression-like behavior. Compared with the control group, the rate of sucrose preference in the HF group decreased obviously, indicating that the mice in the HF group were deprived of pleasure (Fig. 4a). Moreover, mice showed a significant increase in immobility duration during TST and FST, indicating that the mice in the HF group were exhibit desperate behavior (Fig. 4b, c). The results of the OFT showed that the central dwell time of mice in the HF groups was reduced compared to the normal group, indicating a reduction in exploratory activity (Fig. 4d, e). Furthermore, the experimental results also showed that compared with the HF group, the TST, SFT and OFT indices of mice in the HF + CRS group did not change much. Surprisingly, the rate of sucrose preference was even improved in the HF + CRS group. This indicated that CRS did not worsen depression-like behavior in HF mice and might even play a positive role in liver fibrosis. By comparing the behavioral results of CRS and the control group, we could find that CRS increased the immobility time of FST and decreased the dwell time in the open field center of mice, while the other two indicators did not change significantly. These data suggested that although CRS had a negative effect on the behavior of normal mice, it has a protective effect on the mice with HF.

3.4 CRS altered metabolomic profiles in HF mice

We performed the metabolomic profiling of mouse liver tissue by using a LC-MS system. QC samples were used to evaluate the technical accuracy and reproducibility of the liver metabolomics profile, PCA scatter plots showed the CV of the QC samples was in the range of ± 3 STD (Fig. S1), indicating good reproducibility of the instrument throughout the batch analysis process. The PCA results (Fig. S2a, b) showed that the four groups could be separated to different degrees in positive and negative ion modes. To highlight the differences, we did OPLS-DA analysis. As shown in Fig. 5a and b, OPLS-DA showed an obvious separation between the Control, CRS, HF, and HF + CRS groups, indicating a difference in metabolic profiles between the four groups. At the same time, to ensure the validity and accuracy of the OPLS-DA data analysis model, it is necessary to carry out overfitting verification. The results (Fig. S2c, d) show that after 200 cross validations, the model had no overfitting phenomenon. To analyze the effects of CRS on the metabolic characteristics of HF mice, further analysis was undertaken for the HF and HF + CRS groups, the PCA (Fig. S3a, b) and OPLS-DA (Fig. 5c, d) revealed an obvious separation between the HF and HF + CRS groups. Moreover, after 200 permutation tests, the results show that the model is reliable (Fig. S3c, d). We eventually found 119 differential metabolites (Table S2). These differential metabolites were subjected to heatmap clustering analysis, as shown in Fig. 5e, these differential metabolites can distinguish the two groups. The pathway enrichment analysis of the differential metabolites revealed these following pathways were significantly perturbed in liver tissue: betaine metabolism; methionine metabolism; pyruvaldehyde degradation; pantothenate and CoA biosynthesis; purine metabolism (Fig. 5f). We then performed analysis with IPA and found some potential key targets associated with differential metabolites, such as, MAPK, AKT, ERK, AMPK, INSR, etc. (Fig. 5g).

3.5 CRS increased betaine levels in liver tissue

Through pathway enrichment analysis, we recognized that the most influential is betaine metabolism. Betaine is an alkaloid with the chemical name of N,N,N-trimethylglycine, the chemical structure (Fig. 6a). Many studies have shown that betaine has therapeutic effects on liver injury. In our study, compared to the HF group, betaine levels were remarkedly increased in the HF + CRS group (Fig. 6b). We purchased betaine standards as controls, the two metabolites could be further verified according to the retention time and the fragment ions (Fig. 6c, d). The secondary mass spectrometry of these two metabolites is shown in Fig. 7e and f. This provides evidence to identify the authenticity of betaine.

3.6 CRS regulated the INSR/PI3K/AKT/AMPK signaling pathway in HF mice

Combining the results of literature research and metabolomics IPA analysis, we proposed a hypothesis that CRS regulates the INSR/PI3K/AKT/AMPK signaling pathway through upregulation of betaine, thereby alleviate liver injury and HF (Fig. 7a). We then examined the liver tissue protein levels and mRNA expression of INSR, PI3K, AKT, and AMPK. The results showed that these protein levels and gene expression were significantly decreased in the HF group compared to the control and CRS groups and increased in the HF + CRS group compared to the HF group (Fig. 7b-d).

The immunofluorescence (Fig. 8a, b) results of p-AMPK in mouse liver tissue were also consistent with the previous. AMPK is a major regulator of lipid metabolism, and we further examined serum triglyceride (TC) and total cholesterol (TG) levels (Fig. 8c, d), which showed that although there was no statistical difference in triglyceride, total cholesterol expression increased in the HF group and decreased in the HF + CRS group. In conclusion, our results indicated that CRS might ameliorate HF through upregulation of the INSR/PI3K/AKT/AMPK pathway.

In our study, we established a CRS-involved HF mice model. We noticed that CRS was able to improve liver function and HF caused by CCl₄. Metabolomics analysis showed that CRS altered the metabolic profile of CCl₄-induced HF in mice, where the most significantly altered pathways were betaine metabolism. Betaine is an important nutrient that is synthesized through the choline metabolic pathway and is an endogenous metabolite^[19]. In recent years, a large amount of literature has reported that betaine has good preventive and therapeutic effects on a variety of liver diseases, in addition to maintaining normal physiological functions^[20–24]. It is worth mentioning that a number of studies have shown that betaine has significant antifibrotic effects on CCl₄-induced liver fibrosis in vitro and in vivo^[25–27].Our results showed that CRS increased betaine levels in liver tissue of mice with HF and that there was no significant difference in betaine levels between the normal and CRS groups (Fig. S4). Combining the results of IPA analysis and the literature on betaine treatment of HF, we hypothesize that CRS attenuates CCl₄-induced HF by upregulating the INSR/PI3K/AKT/AMPK pathway through the upregulation of betaine content in liver tissue. This conjecture was confirmed in the subsequent experiments.

Liver diseases are often accompanied by insulin resistance^[28–30], when insulin resistance occurs insulin receptor levels in the body are decreased and the ability of insulin to catabolize lipids is diminished, leading to an increase in free fatty acids in the serum, which leads to disorders of lipid metabolism^[31–32], which is consistent with our study in which insulin receptor levels were downregulated and TC levels were increased in liver fibrosis mice, whereas in liver fibrosis mice given CRS we observed upregulation of insulin receptor levels and downregulation of total cholesterol levels.

AMPK is an important molecule in maintaining energy metabolism homeostasis. AMPK activity is increased by 2–3 orders of magnitude by phosphorylation of Thr172 on the α-subunit^[33–34]. A close relationship between AMPK and liver fibrosis has been established by clinical studies. It was found that 28 patients with advanced fibrosis/cirrhosis had lower AMPK activity compared to healthy individuals^[35]. Some research were demonstrated that activation of AMPK inhibited the expression of TGF-β, tissue inhibitor of metalloproteinase I, collagen and α-SMA, all of which are important factors in promoting liver fibrosis in mice with carbon tetrachloride-induced HF^[36–38]. This is consistent with the results of our study. In our study, we found a decrease in p-AMPK levels in CCl₄-induced HF mice and an increase in p-AMPK in HF mice given CRS.

Feelings of stress are familiar to all of us, an encounter with a stressful challenge activates simultaneously the sympathetic nervous system and the hypothalamus-pituitary-adrenal axis^[39–40], and thereby provoke the releasing of glucocorticoid corticosterone in rodents and cortisol in humans and adrenal hormones, which are the most important regulatory hormone of the human stress response^[41–42]. In the field of neuroscience, the term "stress" has a negative connotation because of its potential to trigger or exacerbate psychopathology. However, in the face of stress, the more common response to stress is resilience, suggesting that resilience is the rule and stress-related pathology the exception^[43]. In fact, the physiological stress response has been selected as an important survival response throughout evolution^[44–45], stress adaptive response is considered to be the result of compensatory biological processes to restore homeostasis perturbed by stressors. There is accumulating evidence that most stress effects on health are bidirectional, proper stress can enhance memory and learning and improve cognitive function^[46–50]. We simulate stress with CRS, which is a milder form of stress. In studies of the relationship between stress and HF, it has also been shown that CRS can reduce HF^[12–14], these also provide support for our study.

In our study, behavioral experiments showed that CRS causes normal mice to perform negative behaviors but did not worsen the depression-like behavior of HF mice, and even attenuated the lack of pleasure in HF mice. It is indicating that CRS might be an appropriate stressor for mice with HF. The nature of the stress response is to restore homeostasis in vivo, and chronic restraint stress causes significant changes in the metabolic profile of HF mice, and these changes attenuate the symptoms of HF.

Our study, for the first time, demonstrated that CRS alleviates CCl₄-induced liver fibrosis in mice via upregulation of the INSR/PI3K/AKT/AMPK pathway. Moreover, proper stress might serve as a novel therapeutic strategy for HF.

α-SMA, alpha smooth muscle actin

Acta2, alpha smooth muscle actina

AKT, threonine Kinase

Akt1, threonine Kinase 1

ALP, alkaline phosphatase

ALT, alanine transaminase

AMPK, adenosine 5’-monophosphate (AMP)-activated protein kinase

AST, aspartate aminotransferase

CRS, chronic restraint stress

ECM, extracellular matrix

FST, forced swimming test

HF, hepatic fibrosis

HSCs, hepatic stellate cells

IPA, ingenuity Pathway Analysis

INSR, insulin receptor

Insr, insulin receptor

OFT, open field test

OPLS-DA, orthogonal partial least squares discriminate analysis

PCA, principal component analysis

PI3K, phosphoinositide 3-kinase

Pi3kr1, phosphoinositide-3-kinase regulatory subunit 1

Prkaa1, protein Kinase AMP-Activated Catalytic Subunit Alpha 1

QC, quality control

SPT, sucrose preference test

STD, standard deviation

TC, riglyceride

TG, total cholesterol

TST, tail suspension test

Acknowledgments and Funding Information

This study was supported in part by the National Natural Science Foundation of China (82104687, 82274226, 82074219), the Key Laboratory of Guangdong Drug Administration (2021ZDB03), the Key-Area Research and Development Program of Guangdong Province (2020B1111100004), and the 2020 Guangdong Provincial Science and Technology Innovation Strategy Special Fund (Guangdong-Hong Kong-Macau Joint Lab) (2020B1212030006).

Competing Interests

No competing financial interests exist.

Author Contributions

Shanshan Zhang: Performing the experiments, Data curation, Writing original draft. Binjie Liu: Validation, Investigation. Lan Huang: Experimental assistance. Rong Zhang: Writing-Review & Editing, Supervision. Lin An: Writing - Review & Editing, Methodology, Funding acquisition. Zhongqiu Liu: Conceptualization, Funding acquisition, Writing-Review & Editing.

Corresponding author

Correspondence to Zhongqiu Liu or Lin An.

Data availability

All data analyses during this study are included in this published article, and all data generated during the study are available from the corresponding author on request.

Ethics approval

The animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Traditional Chinese Medicine (reference No. 20211102).

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You are reading this latest preprint version

Metabolomics reveals that chronic restraint stress alleviates carbon tetrachloride-induced hepatic fibrosis through the INSR/PI3K/AKT/AMPK pathway

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Methods and Materials

2.1 Chemicals and reagents

2.2 Animals experiments

2.3 Evaluation of liver function and fibrosis

2.4 Behavioral studies

2.5 Immunohistochemistry and immunofluorescence staining

2.6 Metabolomic profiling

2.7 Western blotting analysis

2.8 Real-Time PCR analysis

2.9 Statistics

3 Results

3.1 CRS mitigated CCl₄-induced liver injury

3.2 CRS attenuated CCl₄-induced HF

3.3 CCl₄-induced liver fibrosis could cause depression-like behavior

3.4 CRS altered metabolomic profiles in HF mice

3.5 CRS increased betaine levels in liver tissue

3.6 CRS regulated the INSR/PI3K/AKT/AMPK signaling pathway in HF mice

4 Discussion

5 Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Metabolomics reveals that chronic restraint stress alleviates carbon tetrachloride-induced hepatic fibrosis through the INSR/PI3K/AKT/AMPK pathway

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Methods and Materials

2.1 Chemicals and reagents

2.2 Animals experiments

2.3 Evaluation of liver function and fibrosis

2.4 Behavioral studies

2.5 Immunohistochemistry and immunofluorescence staining

2.6 Metabolomic profiling

2.7 Western blotting analysis

2.8 Real-Time PCR analysis

2.9 Statistics

3 Results

3.1 CRS mitigated CCl4-induced liver injury

3.2 CRS attenuated CCl4-induced HF

3.3 CCl4-induced liver fibrosis could cause depression-like behavior

3.4 CRS altered metabolomic profiles in HF mice

3.5 CRS increased betaine levels in liver tissue

3.6 CRS regulated the INSR/PI3K/AKT/AMPK signaling pathway in HF mice

4 Discussion

5 Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

3.1 CRS mitigated CCl₄-induced liver injury

3.2 CRS attenuated CCl₄-induced HF

3.3 CCl₄-induced liver fibrosis could cause depression-like behavior