Ranscriptome Analysis Reveals the Molecular Mechanism of Yiqi Rougan Decoction in Reducing CCl4-induced Liver Fibrosis in Rats

Yu Xiong Chongqing Medical University Jinyuan Hu Chongqing Medical University Chen Xuan Chongqing Medical University Jiayu Tian Chongqing Medical University Kaiyue Tan Chongqing Medical University Zhiwei Chen Chongqing Medical University Yan Luo Chongqing Medical University Xuqin Du Chongqing Medical University Junxiong Cheng Chongqing Medical University Lanyue Zhang Chongqing Medical University Wenfu Cao (  caowenfu9316@163.com ) Chongqing Medical University

YQRG was purchased from the Department of Traditional Chinese Medicine at the First A liated Hospital of Chongqing Medical University (Chongqing, China). CCl 4 (batch no. c805329) and olive oil (batch no. Kits for assessing alanine aminotransferase (ALT; batch no. s03030) and aspartate aminotransferase (AST; batch no. s03040) kit were purchased from Shenzhen Redu Life Technology Co., Ltd. (Shenzhen, China), a kit for assessing hydroxyproline (HYP; batch no. a030-3-1) kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and colchicine (batch no. h20113208) was purchased from Guangdong Bidi Pharmaceutical Co., Ltd.  Table 1. According to the surface area conversion ratio between rats and humans (6.3), we calculated the low-, medium-, and high-dose YQRG concentrations for use in rats at 4.95 g/kg, 9.9 g/kg, and 19.8 g/kg, respectively. According to this dosage, the Chinese herbs described were submerged in distilled water for 30 min, decocted three times, ltered, and concentrated three times to create YQRG Decoction at low, medium, and high dosages [22]. The composition of YQRG was determined by UHPLC-QTOF-MS for quality control.

Animal experiments
Male Sprague-Dawley rats (n = 56; 180-220 g) were purchased from the Experimental Animal Center of Chongqing Medical University. Animal experiments were conducted in a speci c pathogen-free animal laboratory (syxk 2018-0003) at the Experimental Animal Center according to the Guidelines for the Care and Use of Experimental Animals from the National Institutes of Health (Bethesda, MD, USA). The experiments were approved by the Ethics Committee of Chongqing Medical University. After 1 week of adaptive feeding, rats were randomly divided into two groups: the control (n = 8) and the CCl 4 -treatment group (n = 48). Rats in the CCl 4 -treatment group received intraperitoneal (i.p.) injection of 50% CCl 4 -olive oil solution (1 mL/kg) twice weekly for 9 weeks. At the end of the 4 th week, rats in CCl 4 -treatment group were randomly divided into four groups: low, medium, and high-dose treatment groups (n = 12) received YQRG at 4.95 g/kg/day, 9.9 g/kg/day, and 19.8 g/kg/day, respectively; and the model group (n = 12) received saline for 5 weeks. At the end of the 9 th week, rats were anaesthetized by i.p. injection of 2% pentobarbital sodium and asphyxiated with high-concentration CO 2 . Blood was collected from the abdominal aorta, and the liver was removed immediately and the weight recorded. Liver tissue was quickly cut into pieces for subsequent liver haematoxylin and eosin (H&E) and Masson staining, immunohistochemistry (IHC), immuno uorescence staining, and transmission electron microscopy (TEM) analysis. The remaining tissues and serum were stored at -80°C until further use. liver index, Serum biochemical and liver HYP analyses During the animal experiments, all rats were weighed and recorded every 2 weeks. Following liver removal and weighing, the liver index was calculated according to the body weight and liver weight to evaluate liver injury, as follows: Liver index = (liver weight / body weight) × 100. According to their respective manufacturer instructions, Serum ALT, AST levels and liver HYP content were measured with kits, Con gure the working reagent, load the sample, set the parameters in an automatic biochemical analyzer (Shenzhen Redu Life Technology Co., Ltd.) and put it on the computer for detection.

Histologic analysis
Liver tissue was xed with 4% paraformaldehyde for 24 h, dehydrated, embedded in para n, and sliced (4 µm), followed by staining with H&E and Masson Staining. For H&E staining, the slices were dewaxed rst, soaked with hematoxylin dye solution for 10 min, then washed with distilled water. After differentiation, the slices were rinsed again, and then rinsed with blue-returning solution. Soak the slices in ethanol (85% and 95%) for 5 min, and infect the slices in eosin dye for 10 min. After dehydration for 5 min (ethanol and xylene). In addition, for Masson staining, the dewaxed slices were placed in Masson A for 15 h and then rinsed. The Masson mixture (Masson B:Masson C=1:1) was soaked for 1 min and washed. The mixture was differentiated with 1% hydrochloric acid alcohol for 1min and washed. The tissue turned red after Masson D treatment for 6 min. The slices were rinsed, differentiated, dehydrated and sealed with 1% glacial acetic acid. In the end,Tissue sections were then observed and images obtained using an optical microscope (BX53; Olympus, Tokyo, Japan).

IHC analysis
Para n sections were dewaxed, and citric acid antigen repair buffer was soaked to repair the antigen.
The antigen was incubated with 3% hydrogen peroxide solution for 25 min, washed with phosphatebuffered saline (PBS), and sealed with 3% bovine serum albumin (BSA) at room temperature for 30 min. The antibody was incubated with anti-α-SMA and HRP-conjugated secondary antibodies, followed by DAB staining, rinsed, sectioned with differentiation solution, dehydrated with ethanol and xylene, and sealed with neutral rubber. followed by washing, observation, and imaging using a microscope (BX53; Olympus).

Assessment of ultrastructural morphology
Liver tissue was cut into 1 mm 3 immediately upon collection of the liver (within 1 min -2 min), xed in 2.5% glutaraldehyde, and washed with 1 M PBS, 1% osmic acid was added to the sections for a 2 h incubation, washed, dehydrated, penetrated, dehydrated with ethanol and acetone, and then embedded with 812 embedding agent. The embedding plate was polymerized for 48 h, and the resin block was placed in the ultra-thin slicer (60 nm -80 nm). Stained, washed and dried with 2% uranium acetate saturated alcohol and 2.6% lead citrate solution. followed by observation of the ultrastructure of the liver and collection of images via TEM (JEM-1400plus; JEOL, Tokyo, Japan).

RNA-seq analysis
RNA-seq was performed and sequencing libraries were constructed using the Novaseq 6000 platform and the TruseqTM RNA sample prep kit (Illumina, San Diego, CA, USA). Total RNA was extracted with Trizol reagent, and mRNA was separated using oligo(DT) magnetic beads and randomly broken into small fragments. Six-base random primers were used to reverse transcribe cDNA, followed by end repair and sequencing. Changes in gene expression in liver tissue following YQRG treatment were assessed according to the identi cation of DEGs (fold changes >2 or <−2; false discovery rate < 0.05). DEGs were clustered and analysed using the GO database and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathway analysis.

Network construction and analysis
The STRING database (https://string-db.org/) is a database commonly used to predict protein-protein interaction (PPI), The main DEG was entered into the string database, the interaction score was set to ≥ 0.9 and the species was selected as "Homo sapiens" to create PPI network. Then, the results were saved and exported in TSV format. Cytoscape (v.3.6.0; https://cytoscape.org/) was used to visualize the PPI networks. The TSV format le is imported into Cytoscape for PPI visualization.
Double immuno uorescence staining Double immuno uorescence staining was performed to detect colocalization of TUNEL staining and a-SMA. Additionally, LC3-II and a-SMA were also examined. For the former staining, The slices were dewaxed into xylene, ethanol, and distilled water, respectively, and then circled into protease K working solution and incubated at 37℃ for 22 min. PBS was washed and 0.1% Triton was added for 20 min at room temperature. Then, the buffer was incubated at room temperature for 10 min. TUNEL kit reaction solution (TDT enzyme :dUTP: Buffer =1:5:50) was added into the ring and incubated for 2 h at 37℃, and sealed with 3% BSA for 30min. Sections were incubated with rabbit anti-α-SMA at 4℃ overnight, and then FITC-conjugated goat anti-rabbit IgG (Wuhan Service Biotechnology Ltd.) was used for 50 min at ambient temperature, dark. followed by nucleus staining with 4',6-diamino-2-phenylindole (DAPI; Wuhan Service Biotechnology Ltd.) for 10 min. Additionally, For the latter staining, sections were dewaxed, immersed in EDTA antigen repair buffer, circled, and sealed with 3% hydrogen peroxide and 3% BSA, respectively. Rabbit anti-LC3-II was added and incubated overnight, followed by FITC-Conjugated goat Anti-Rabbit IgG, then cy3-TSA was added for 10 min, washed, and antigen repair was performed again. In incubated with the primary antibodies (anti-α-SMA), followed by FITC-conjugated goat anti-Rabbit IgG, DAPI staining was used. Sections were then observed and images were obtained by uorescence microscopy (eclipse CI; Nikon, Tokyo, Japan).
ELISA ELISA was performed according to the manufacturer instructions for the ELISA kit. Levels of HA, LN, PC-III, IV-C, CASP12, CHOP, ATF6, IRE1, PERK and BiP in liver tissue were quanti ed. PBS was added to liver tissue for homogenization, followed by centrifugation at 4°C for 20 min. This process was repeated three times, and the supernatant was then used for ELISA detection. Add the sample and standard, wash the plate, add the antibody to be tested for incubation, wash the plate again, incubate with enzyme solution, wash the plate, develop the colour of TMB substrate, add 50 uL and 450um reading of termination solution, and nally calculate its concentration.

Western blot
Proteins were extracted from liver tissue with Radioimmunoprecipitation buffer containing a protease inhibitor and phosphatase inhibitor. Proteins were separated by electrophoresis, followed by transfer to polyvinylidene uoride membranes. Membranes were washed ve times with Tris-buffered saline

YQRG improves liver injury in vivo
To evaluate the effect of YQRG on liver brosis, we established a rat model of liver brosis according to the reported protocol [23]. During animal experiments, compared with the model group, the weight of rats receiving low, medium and high concentrations of YQRG (YQRG-L, YQRG-M, and YQRG-H) increased signi cantly (Fig. 2a). The liver weight ratio (LW: BW) used to evaluate liver injury showed that the LW: bw ratio decreased signi cantly in the YQRG treatment group (Fig. 2b). Next, we measured the levels of serum ALT and AST in rats to evaluate liver function. The results showed that the levels of ALT and AST in the model group increased signi cantly. Surprisingly, after YQRG treatment, the levels of ALT and AST decreased and were consistent with the normal levels ( Fig. 2c and d). Then, we evaluated the morphological changes of cells in liver tissue from different angles by renal appearance, H&E staining and TEM. In rats receiving CCl 4 , the appearance of the liver was enlarged, abnormal, severe swelling and degeneration, and in ammatory cell in ltration. In contrast, YQRG -H well improved this pathological change (Fig. 2e). Transmission electron microscopy analysis showed that the hepatocyte structure was damaged after CCl 4 -induced, including mitochondrial swelling, endoplasmic reticulum damage and bile duct dilatation. The tissue image of the YQRG H group showed an improvement in pathological damage (Fig. 2e). These results suggest that YQRG can effectively resist CCl 4 -induced liver injury in rats induced by carbon tetrachloride.

YQRG resists CCl 4 -induced liver brosis in vivo
Next, We then performed multiple methods to evaluate the effect of YQRG against liver brosis. Masson staining showed that collagen bres (blue) accumulated after CCl 4 -induced, resulting in more bre spacing, while collagen bres and bre spacing decreased in the YQRG-H group (Fig. 3a). Due to HSCs activation promotion α-SMA expression, we evaluated liver injury and after treatment Changes in α-SMA levels. IHC results showed that the YQRG-H group reduced the positive expression of the model group (Fig. 3a). In addition, the brosis markers in the model group showed that the levels of HA, LN, PC-III and IV-C were signi cantly higher than those in the YQRG-H group (Fig. 3b). In addition, as the main component of collagen, the level of HYP in the model group increased but decreased after treatment with YQRG-H (Fig. 3c). As shown in Fig. 3d-f shows that the results of α-SMA expression support the immunohistochemical results in Western blot and qPCR. In addition, hepatic brosis is accompanied by an imbalance in ECM synthesis and degradation. ECM is regulated by TIMPs (promote ECM synthesis) and MMPs (promote ECM degradation) [24]. We analyzed the mRNA expression of TIMP1 and MMP9 in the liver by qPCR. The results showed that compared with the model group, the level of MMP9 in the YQRF-H group increased signi cantly and the expression of TIMP1 decreased (Fig. 3g and h). These data show that YQRG-H processing reduces the accumulation of ECM. In conclusion, these results suggest that YQRG-H treatment reduces CCl 4 induced liver brosis in rats.

Gene expression analysis by RNA-seqp
To gain insight into the molecular mechanism associated with YQRG-mediated improvements in liver brosis in vivo, we performed RNA-seq analysis of samples from control, model, and YQRG-H-treated rats. We identi ed 2689 upregulated and 2545 downregulated DEGs in the control versus model group, whereas the model versus YQRG-H group revealed 2543 upregulated and 2476 downregulated DEGs (Fig.  4a). The model group both upregulated the genes of the normal control group and YQRG-H group, and both downregulated the genes of the normal control group and YQRG-H group, which are the DEGs of YQRG in the treatment of CCl 4 induced liver brosis, that is, 2141 YQRG upregulated DEGs and 2038 YQRG downregulated DEGs (Fig. 4b). Interestingly, these DEGs showed signi cant intergroup differences (Fig. 4c).

GO/KEGG enrichment analyses and construction of PPI network
GO enrichment analysis of the upregulated DEGs identi ed roles in biological processes (regulation of Lkynurenine metabolism and fatty acid oxidation, tryptophan metabolism, and branched-chain amino acid metabolism) (Fig. 5a). Downregulated DEGs were mainly involved in biological processes involving positive regulation of epithelial-to-mesenchymal transition, response to topological error protein, response to unfolded protein, regulation of ERS, autophagy, regulation of the apoptotic signalling pathway (Fig.  5b). KEGG pathway enrichment analysis showed that upregulated DEGs were associated with the peroxisome, complement, and coagulation cascade; cytochrome P450 metabolism of exogenous substances, and peroxisome proliferator-activated receptor signalling (Fig. 5c), whereas downregulated DEGs were associated with protein processing, MAPK signalling, PI3K/Akt signalling, tumour necrosis factor signalling in the ER (Fig. 5d). Notably, GO analysis identi ed biological processes related to YQRG treatment of liver brosis involved with ERS, apoptosis, and autophagy (Fig. 5e). The identi ed signal pathways and related genes of the rst 15 closely related to liver brosis were plotted (Fig. 4f). The PPI network and visualization were constructed involving DEGs of ERS, apoptosis, and autophagy ( Fig. 5g  and h). Furthermore, the construction of a PPI network identi ed 33 target DEGs in Table 3.

YQRG treatment alleviates ERS in vivo
The inhibition of ERS is related to the improvement of liver brosisN [25]. As shown in Fig. 6a, we can see the formation mechanism of ERS. Transcriptome analysis of go and KEGG showed that they were enriched in endoplasmic reticulum stress-related genes and pathways. BiP, PERK, ATF6, IRE1 and CHOP are considered to be important markers of ERS 25 . To evaluate the effect of YQRG treatment on ERS, we detected changes in the expression of ERS-related markers BiP, ATF6, PERK, IRE1, and CHOP by ELISA and qPCR. Consistent with the RNA-seq results, BiP, PERK, ATF6, IRE1, and CHOP protein and mRNA levels in the model group were signi cantly higher than those in the control group, indicating that CCl 4 administration induced ERS. However, YQRG-H treatment reversed these changes, resulting in levels similar to those in controls (Fig. 6b-k), and leading to reductions in ERS. These ndings suggested that YQRG-H treatment effectively alleviated ERS.

YQRG treatment regulates cell apoptosis
Cells apoptosis promote the occurrence of liver brosis, and this process is very complex (Fig. 7a.) We performed TUNEL and α-SMA (a marker for the activation of HSCs [26]) double immuno uorescence staining in liver tissues to evaluate the effect of YQRG treatment on cell apoptosis. The results showed an increased number of apoptotic cells in the model group relative to the control group along with a signi cant increase in the expression of α-SMA. however, YQRG-H treatment signi cantly reduced the number of apoptotic cells and a-SMA expression levers in the model group (Fig. 7b). Next, the expression of apoptosis-related proteins in liver tissue was detected by ELISA and qPCR. CASP12 and BCL-2 are apoptosis-related markers and play an important role in apoptosis [27]. According to ELISA and qPCR analysis, the expression of CASP12 in CCl 4 induced group increased signi cantly. In contrast, the YQRG-H treatment decreased the expression level of CASP12 (Fig. 7c-d). Similarly, compared with the model group, the expression of BCL-2 in the YQRG-H group increased (Fig. 7e). These results suggest that YQRG-H treatment inhibits apoptosis.

YQRG treatment regulates autophagy in vivo
Autophagy is a metabolic process that is closely related to liver brosis (Fig. 8a). To determine whether YQRG-H treatment affects autophagy, we evaluated autophagic ux by double immuno uorescence staining, western blot, and qPCR. LC3-II is a marker for autophagy [28], double immuno uorescence staining of LC3-II and α-SMA revealed that YQRG-H treatment reduced the uorescence signals of LC3-II and α-SMA in the model group (Fig. 8b). this result suggested autophagy activation was closely related to HSCs activation in the model group. Additionally, western blot and qPCR analyses con rmed the increased expression of LC3-II protein and mRNA in the model group, whereas these levels were decreased following YQRG-H treatment (Fig. 8c-e). ULK1 is also an important indicator regulating autophagy [29], qPCR analysis of Ulk1 in the model group was signi cantly lower than that in the control group and YQRG-H groups (Fig. 8f). These results suggested that CCl 4 can promote autophagy and that YQRG treatment inhibits this process.

Effects of YQRG treatment on cell signalling pathways
To further evaluate the mechanism associated with YQRG treatment of liver brosis, we evaluated markers related to the pathways identi ed by KEGG analysis. Western blot analysis of liver tissue samples from the control, model, and YQRG-H groups identi ed the expression of p38, p-p38, AMPK, and p-AMPK, with upregulated levels of p-p38 and downregulated levels of AMPK and p-AMPK observed in the model group relative to the control group. Following YQRG-H treatment, we found signi cantly decreased p-p38 levels and increased levels of AMPK and p-AMPK relative to those in the model group ( Fig. 9a and b). Moreover, qPCR results were consistent with those of western blot analysis ( Fig. 9c and   d). These results suggested that YQRG-H treatment inhibiting p38-MAPK signalling and activated AMPK signalling.

Discussion
Hepatic brosis is a repair response to chronic liver injury. Due to the proliferation of myo broblasts under the action of different stimuli, hepatocytes initiate the production of various chemokines, which promote myo broblast proliferation in the liver injury area and result in the formation of collagen and other bre components, ECM accumulation, and brosis. Liver injury from various causes leads to liver brosis, with this potentially followed by cirrhosis and liver cancer. There is currently a lack of therapeutic options for preventing hepatic brosis, making it necessary to research and actively develop effective drugs. YQRG is a traditional Chinese medicine comprised of eight herbs and a mixture of compounds. In this study, we identi ed YQRG compounds by UHPLC-QTOF-MS, most of which were previously identi ed as playing a role in improving liver brosis (i.e., Paeoni orin, Bisdemethoxycurcumin, Curcumin, Cryptotanshinone, Gallic acid, Protocatechualdehyde, Rosmarinic acid, Salvianolic acid A) [30][31][32][33][34][35][36][37] (Fig. 1c). This result suggests that most of the sampled compounds may be the components of YQRGs which mitigate against liver brosis.
The liver can be damaged by infection, excessive drinking or other stimuli [38], and various liver injuries can promote the expression of brogenic mediators and their receptors in response to liver brosis [39].
Because CCl 4 exerts hepatotoxicity, its administration can seriously damage hepatocytes [40]. Liver injury is reversible upon removal of the stimuli [41];therefore, numerous studies have evaluated the mechanisms associated with CCl 4 -induced liver injury. In this study, we evaluated the effect of YQRG on rat models of CCl 4 -induced liver injury and observed signi cant decreases in weight (Fig. 2a). and increases in the LW:BW ratio (Fig. 2b). in the CCl 4 -induced model rats, while weight increased and LW:BW decrease in the YQRG-H group. Moreover, we measured serum ALT and AST levels ( Fig. 2c and d) as markers of hepatocyte integrity [42], nding signi cantly increased levels of both in the model group, whereas these levels were restored to those similar to controls following YQRG treatment. Additionally, H&E staining and TEM analysis (Fig. 2e) identi ed evidence of pathological hepatocyte injury in the model group; however, these characteristics were signi cantly improved following YQRG treatment. These results indicate that YQRG treatment effectively Reduced CCl 4 -induced liver injury.
YQRG has been used to clinically treat liver brosis with good effect; however, there is currently a lack of experimental data supporting its e cacy. Here, we established a rat model of liver brosis to evaluate the e cacy and mechanism of YQRG against liver brosis induced by CCl 4 [43]. Masson staining (Fig. 3a) to evaluate liver brosis revealed brous septum in the model group, whereas the YQRG-treated group demonstrated signi cantly reduced brosis. Additionally, we evaluated serum levels of the brotic markers HA, LN, PC-III, and IV-C [44]. Hyp, an important component of collagen tissue, comprises up to 13% of collagen [45] and represents another marker of liver brosis [46]. In the present study, levels of serum HA, LN, PC-III, IV-C (Fig. 3c) and Hyp (Fig. 3d) in liver tissue in the model group were signi cantly higher than those in the control and YQRG-treatment groups. Moreover, in liver brosis, excessive accumulation of ECM is mainly due to HSC activation [47], we observed signi cant increases in α-SMA (a marker of HSCs activation [26]) levels detected by IHC (Fig. 3a), Western blot (Fig. 3d) and qPCR (Fig.   3f) in the model group, while decreased after YQRG-H treatment. ECM is regulated by TIMPs and MMPs [24]. A previous study reported that inhibiting HSC activity can reduce TIMPs secretion and increase MMPs activity to promote ECM degradation [48]. Consistent with these ndings, in the present study, we found that YQRG-H treatment signi cantly reduced Timp1 expression and increased Mmp9 expression in the model group ( Fig. 3g and h). These ndings suggested that YQRG treatment effectively improved CCl 4 -induced liver brosis in rats by attenuating HSCs activity and inhibiting ECM synthesis.
ER is the largest organelle in HPCs and has the functions of participating in protein folding and regulating calcium homeostasis [49,50]. The increased membrane hardness of ER leads to the damage of ER membrane energy, promotes the accumulation of misfolded proteins, and then activates the unfolded protein response (UPR) [51,52]. ATF6, IRE1 and PERK are transmembrane sensors of UPR [53]. ERS is closely related to the increased expression of UPR related genes. For example, Zhan et al. (2020) found that microcystin LR can up-regulate the gene levels of perk and IRE1 in the liver and ovary, causing ERS [54]. In this study, ELISA and mRNA expression levels of liver ATF6, IRE1 and PERK increased after CCl4 treatment (Fig. 5c-e and 5h-j). Under stress-free conditions, BiP, an important member of the heat shock protein 70 family, binds to URP. Under ERS, BiP binds to wrong or unfolded proteins and separates from URP, activates these sensors and their downstream signal cascade through dimerization and autophosphorylation, and promotes the increase of apoptosis regulator CHOP [55]. In our experiment, the ELISA and transcriptional gene levels of BiP and apoptotic transcription factor CHOP in the liver of rats with hepatic brosis were higher than those in the YQRG-H group (Fig. 5b and f, 5g and k). Studies have shown that inhibiting ERS can reduce liver brosis [56]. In this study, transcriptome analysis showed that ERS inhibition was an important mechanism of YQRG mediated reduction of CCl4 induced liver brosis ( Fig. 5b and d). This is consistent with our experimental data and transcriptome results, suggesting that YQRG treatment reduces CCl4 induced liver brosis in rats by inhibiting ERS.
HPCs apoptosis is an in ammatory stimulus that promotes HSCs and plays an important role in liver diseases [57,58]. Our data show that a signi cant increase of TUNEL and α-SMA positive cells can be seen in the liver of rats in the CCl 4 group (Fig. 7b).Under sustained ERS, pro-apoptotic signals are induced by activating several transcription factors [59][60][61]. CHOP is a key transcription factor related to ERS mediated activation of HPCs apoptosis [62]. Under continuous ERS, the pro-apoptotic factor CHOP inhibits the level of downstream anti-apoptotic protein BCL-2. In addition, Ca 2+ enters the cytoplasm, mcalpain and procaspase 12 are further cleaved and activated, and the caspase cascade induces apoptosis [63]. Apoptosis mainly includes endogenous and exogenous apoptosis. In the endogenous apoptosis pathway, mitochondrial permeability releases cytochrome c, Caspase 9 and Caspase 3 are activated in order. In the exogenous apoptotic pathway, Caspase 3 is activated by upstream bound Caspase 8 and by FADD. Death receptors such as trail bind to FADD to further activate downstream Caspase 8 and Caspase 3, and Caspase 3 is the co-executor of the two pathways [64][65][66][67]. In this study, we demonstrated the expression of CHOP, BCL2 and Caspase 12 in brotic rats (Fig. 7c-e, 6f and k), indicating that apoptosis occurs during the development of brosis. These results were combined with the signi cant increase of ERS related indexes in the model group, suggesting that CCl 4 may induce apoptosis by activating ERS.
Autophagy is a metabolic process in which autophagosomes formed by cell membranes phagocytize organelles and cell fragments for subsequent lysosomal degradation [68]. Studies have shown that ERS mediated HSCs autophagy promotes the occurrence of liver brosis. For example, men R et al. Found that after NOGO-B gene knockout, the levels of ERS and autophagy markers were down-regulated, and the autophagy level could be regulated byERS agonists and antagonists. The results showed that inhibiting ERS could reduce the autophagy of HSCs [69]. Overexpression of Ulk1 kinase death mutant can inhibit autophagy [29], and LC3-II represents autophagy marker [28]. Consistently, we found that prolonged exposure to CCl 4 increased the expression of two key autophagy genes (LC3 and ULK1). In this study, LC3-II and α-SMA positive cells increased signi cantly (Fig. 8b), indicating that the increased autophagy in the liver brosis model is related to HSCs activation. The protein and mRNA levels of LC3II increased in the model group and the mRNA expression of Ulk1 decreased in the model group (Fig. 8c-f). This result well supports our conclusion. In conclusion, these results suggest that YQRG improves liver brosis by inhibiting autophagy.
Pathway analysis suggested that YQRG treatment altered the MAPK and AMPK signalling pathways. p38 MAPK plays an important role in regulating in ammation, apoptosis, and liver brosis [70,71] and a previous study reported that ERS activates p38 MAPK signalling [72]. Additionally, ERS-mediated IRE1/p38 MAPK signalling promotes the activation of isolated HSCs in rats [73]. In the present study, we found that YQRG treatment inhibited p38 MAPK signalling ( Fig. 9a and d) along with the expression of ERS-related markers, suggesting that YQRG might alleviate liver brosis by inhibiting ERS through regulation of p38 MAPK signalling. Moreover, p38 MAPK promotes apoptosis [74] and is associated with autophagy induction. AMPK is an energy sensor that plays an important role in maintaining energy homeostasis [75]. A previous study showed that AMPK exerts an anti-apoptotic effect and can inhibit HPC apoptosis [76]. Additionally, AMPK activation inhibited autophagy in an HSC line (lx-2) [77]. In the present study, YQRG treatment promotes AMPK signalling ( Fig. 9a and d). Moreover, the observed regulation of apoptosis by YQRG suggests that activation of the AMPK pathway might inhibit apoptosis and autophagy, thereby alleviating the progression of liver brosis. However, the precise role of YQRG as a therapeutic agent for liver brosis requires further investigation.