Angelica sinensis polysaccharide attenuates CCl4-induced liver fibrosis via the IL-22/STAT3 pathway

Kaiping Wang, Junfeng Wang, Mengzi Song, Hanxiang Wang, Ni Xia, Yu Zhang

PII: S0141-8130(20)33607-2
DOI: https://doi.org/10.1016/j.ijbiomac.2020.06.166
Reference: BIOMAC 15923

To appear in: International Journal of Biological Macromolecules

Received date: 29 November 2019
Revised date: 18 June 2020
Accepted date: 18 June 2020

Please cite this article as: K. Wang, J. Wang, M. Song, et al., Angelica sinensis polysaccharide attenuates CCl4-induced liver fibrosis via the IL-22/STAT3 pathway, International Journal of Biological Macromolecules (2020), https://doi.org/10.1016/ j.ijbiomac.2020.06.166

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Angelica sinensis polysaccharide attenuates CCl4-induced liver fibrosis via the IL-22/STAT3 pathway
Author: Kaiping Wang1, Junfeng Wang1, Mengzi Song1, Hanxiang Wang3, Ni Xia2*, Yu Zhang3*
1Hubei Key Laboratory of Nature Chemistry and Resource Evaluation, Tongji Medical College of Pharmacy, Huazhong University of Science and Technology, Wuhan, China
2Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 3Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
*Corresponding authors: Ni Xia, Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; Yu Zhang, Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
E-mail: [email protected] (Ni Xia); [email protected] (Yu Zhang)

ABSTRACT

Angelica sinensis polysaccharide (ASP) has hepatoprotective effects in liver injury models. However, its role and mechanism in chronic liver fibrosis have not been fully elucidated. In this study, a carbon tetrachloride (CCl4)-induced chronic liver fibrosis mouse model was established. The results showed that ASP treatment reduced serum alanine aminotransferase by approximately 50% and liver fibrosis areas by approximately 70%. Hepatic stellate cell (HSC) activation was inhibited in ASP-treated mice. Furthermore, the mechanism was studied in-depth, focusing on the interleukin 22/signal transducer and activator of transcription 3 (IL-22/STAT3) axis. Concentrations of 50 mg/ml and 100 mg/ml ASP induced the secretion of IL-22 in vitro, which further increased at a concentration of 200 mg/ml. Moreover, in vivo data showed that ASP significantly promoted IL-22 production in splenocytes and liver tissues. The antifibrotic effects of ASP were abolished after IL-22 neutralization. In addition, ASP activated the STAT3 pathway in the liver, as demonstrated by a 2-fold increase compared to that of the CCl4 group, which was abrogated by the IL-22 antibody. Subsequently, we showed that the antifibrotic effects of ASP were abrogated by blocking STAT3 with S3I-201. In conclusion, ASP effectively alleviates chronic liver fibrosis by inhibiting HSC activation through the IL-22/STAT3 pathway. Keywords: Angelica sinensis polysaccharide; liver fibrosis; interleukin 22

Abbreviations
ALT, alanine aminotransferase; ASP, Angelica sinensis polysaccharide; CCl4, carbon tetrachloride; COLⅠ, collagen type Ⅰ; ECM, extracellular matrix; HSC, hepatic stellate cell; IL-22, interleukin 22; IL-22 Abs, IL-22neutralizing antibody; MMP2, matrix metalloproteinase 2; p-STAT3, phosphorylated STAT3; STAT3, signal transducer and activator of transcription 3; TIMP1, tissue inhibitor of matrix metalloproteinase 1; α-SMA, α-smooth muscle actin.

1. INTRODUCTION

Liver fibrosis is a significant worldwide health problem resulting from chronic liver disease, including hepatitis B and C, alcoholic liver disease, or nonalcoholic steatohepatitis [1]. Following liver injury, hepatocytes, Kupffer cells, T cells and other cells respond to create an inflammatory milieu, which stimulates hepatic stellate cells (HSCs) to transform from a quiescent vitamin A-storing cell lineage to activated myofibroblast-like cells that express α-smooth muscle actin (α-SMA) and proliferate to become fibrogenic and produce increased amounts of extracellular matrix (ECM) [1, 2]. Specifically, activated HSCs play a pivotal role in the disorder of matrix metalloproteinase (MMP) homeostasis, which is regulated by MMPs and tissue inhibitor of matrix metalloproteinases (TIMPs), and is an important cause of ECM accumulation [2]. To date, there are very few therapies for chronic liver fibrosis [3]. Liver cirrhosis, as the end-stage of liver fibrosis, has high mortality with liver transplantation as the only curative therapy [4]. Therefore, safe and effective antifibrotic drugs are urgently required.

Traditional Chinese medicine is a rich source of biologically activ substances that can be used to prevent or cure many types of human diseases. For the past few years, it has been shown that polysaccharides extracted from traditional Chinese medicines have beneficial pharmacological activity and low toxicity [5]. Angelica sinensis has been used as a Chinese herbal medicine and functional food in many Asian countries [6]. In our previous research, a novel water-soluble polysaccharide was isolated from the dry roots of Angelica sinensis, named as Angelica sinensis polysaccharide (ASP), and its repeating unit was obtained (Figure S1) [7]. Previously, it was reported that ASP has a protective effect in a mouse model of acute liver injury induced by concanavalin A or acetaminophen [8, 9]. However, the role and mechanism of ASP in chronic liver fibrosis have not been fully elucidated.

In recent years, the immune-inflammatory response has been shown to play an important role in the progression of liver fibrosis. Multiple immune cells affect the progression of liver fibrosis by secreting inflammatory mediators such as cytokines and chemokines [1]. Among them, interleukin 22 (IL-22) has been shown to have a significant hepatoprotective effect [10]. By binding to IL-10R2 and IL-22R1, IL-22 activates signal transducer and activator of transcription 3 (STAT3) to promote hepatocyte survival and inhibit HSC activation, resulting in liver repair and alleviated fibrosis [10, 11]. Additionally, IL-22 induces HSC senescence via activation of STAT3, which consequently enhances the in vivo clearance of senescent HSCs, thereby inhibiting liver fibrosis [12, 13].

In the current study, we focused on the protective effect of ASP on chronic liver fibrosis and the involvement of the IL-22/STAT3 axis. We used a mouse model of chronic liver fibrosis induced by carbon tetrachloride (CCl4) to demonstrate that ASP attenuated chronic liver fibrosis by inhibiting HSC activation. Notably, a further mechanistic study demonstrated that the antifibrotic effect of ASP was dependent on the IL-22/STAT3 pathways.

2. METHODS

2.1. Materials

Anti-STAT3 (Cat# 4904, RRID: AB_331269), anti-phospho (Tyr705)-STAT3 (Cat# 9145, RRID: AB_2491009), and anti-α-SMA
(Cat# 19245, RRID: AB_2734735) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH primary antibody (Cat# G9295, RRID: AB_1078992), anti-rabbit IgG (Cat# A0545, RRID: AB_257896) and anti-mouse IgG (Cat# A5278, RRID: AB_258232) secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). IL-22 neutralizing antibody (Cat# AF-582, RRID: AB_355457) and isotype antibody (Cat# AB-108-C, RRID: AB_354267) were purchased from R&D Systems (Minnesota, USA). The following compounds were also utilized: S3I-201 (MCE Biological Inc., Cat# HY-15146), CCl4 (Sigma-Aldrich, Cat# 48604), and DMSO (Sigma-Aldrich, Cat# D2650).

2.2. Preparation of Angelica sinensis polysaccharide

The dry roots of Angelica sinensis (Oliv.) Diels collected from Minxian (Gansu Province, China) were obtained from Union Hospital (Wuhan, China). Plant identification was performed by Professor Jinlan Ruan (Faculty of Pharmaceutical Science, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China) in compliance with the identification standard of the Pharmacopeia of the People’s Republic of China. The extraction and purification of the polysaccharide were performed as previously described [7]. Briefly, crude polysaccharides were obtained by boiling water extraction and alcohol precipitation with an equal volume of 95% alcohol. After repeated freeze-dissolution and dialysis, purified ASP was finally prepared by gel filtration chromatography with Sephadex G-50 and lyophilization. Determination of purity and structural characterization of ASP were performed by the phenol-sulfuric acid colorimetric method, GC-MS and high-performance gel permeation chromatography as described previously. The sugar content of ASP was approximately 95.1%, and the monosaccharide composition was glucuronic acid, glucose, arabinose and galactose, with a molar ratio of 1:1.7:1.85:5.02. The average molecular weight of ASP was 80kDa. The repeating unit of ASP is shown in Figure S1 (related data have been published previously) [7].

2.3. Animals

Animal studies are reported in accordance with the ARRIVE guidelines [14]. Male C57BL/6J mice (8-10 weeks old; 18-21 g) were purchased from Peking University (Beijing, China). All mice were housed under standard pathogen-free conditions and maintained on a chow diet in a 12 hour light/12 hour dark environment at 25°C in the Experimental Animal Center (Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China). All treatment and care of the animals were approved by the Animal Care and Utilization Committee of Huazhong University of Science and Technology, China. The experimental procedures used in the study were performed as humanely as possible.

2.4. Establishment of the CCl4-induced liver fibrosis mouse model and treatments
To study the antifibrotic effect of ASP, C57BL/6J mice were randomized into three groups (n=8 per group): normal control (NC) group, CCl4 group and CCl4+ASP group. Mice in the NC group were injected intraperitoneally (i.p.) with 2 ml/kg body weight olive oil three times per week for 4 weeks. Mice in the CCl4 and CCl4+ASP groups were i.p. administered 2 ml/kg body weight 10% CCl4 (dissolved in olive oil) three times per week for 4 weeks. Half an hour after the first CCl4 injection, daily oral gavage (i.g.) treatment with distilled water (the same volume as ASP) was started in the NC and CCl4 groups. Meanwhile, daily i.g. treatment with ASP (dissolved in distilled water, 200 mg/kg) was initiated in the CCl4+ASP group. To elucidate the important role of IL-22 in this process, C57BL/6J mice were randomly assigned to four groups (n=6 per group): isotype CCl4 group, isotype CCl4+ASP group, IL-22 Abs CCl4 group, and IL-22 Abs CCl4+ASP group. In these groups, 10% CCl4 (dissolved in olive oil, 2 ml/kg) was administered i.p. three times per week for 4 weeks. Isotype antibodies (dissolved in phosphate-buffered saline, 50 μg per mouse) or IL-22 neutralizing antibodies (IL-22 Abs) (dissolved in phosphate-buffered saline, 50 μg per mouse) were administered i.p. every other day each week. Half an hour after the first CCl4 injection, mice in the isotype CCl4 and IL-22 Abs CCl4 groups were daily administered distilled water by i.g. (the same volume as ASP). Meanwhile, mice in the isotype CCl4+ASP and IL-22 Abs CCl4+ASP groups were daily administered ASP by i.g. (dissolved in distilled water, 200 mg/kg).
To illustrate the important role of STAT3 in this process, mice were randomly assigned to four groups (n=6 per group): DMSO CCl4 group, DMSO CCl4+ASP group, S3I-201 CCl4 group and S3I-201 CCl4+ASP group. S3I-201, a STAT3 inhibitor, has been shown to effectively inhibit STAT3 phosphorylation in vivo and was dissolved in 0.05% DMSO [15]. In these groups, 10% CCl4 (dissolved in olive oil, 2 ml/kg) was administered i.p. three times per week for 4 weeks. Meanwhile, 0.05% DMSO (the same volume as S3I-201) or S3I-201 (5 μg/g) was administered i.p. three times per week. Half an hour after the first CCl4 injection, mice in the DMSO CCl4 and S3I-201 CCl4 groups were daily administered distilled water by i.g. (the same volume as ASP), while mice in the DMSO CCl4+ASP and S3I-201 CCl4+ASP groups were administered ASP by i.g. (dissolved in distilled water, 200 mg/kg).

On the third day after the last injection of CCl4, blood samples were rapidly collected from the orbital sinus, and serum samples were collected after centrifugation at 3500 rpm at 4 °C for 15 min. Then, the mice were sacrificed by cervical dislocation, and liver and spleen tissues were harvested. Partial liver tissues were fixed in 10% formalin solution for histological examination, and the remaining liver tissues were immediately frozen at -80 ℃. Partial spleen tissues were minced for splenocyte preparation, and the remaining spleen tissues were immediately frozen at -80 ℃.

2.5. Analysis of serum alanine aminotransferase (ALT) activity

Serum ALT activity was measured using commercially available biochemical kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

2.6. Histological assessment

Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 μm sections for staining with hematoxylin-eosin (H&E). For the quantitative assessment of fibrosis, sections were stained with Sirius red to analyze collagen content. To evaluate HSC activation, α-SMA levels were determined by staining with rabbit anti-α-SMA antibody and visualizing with HRP-labeled anti-rabbit antibody. The collagen area and α-SMA+ area were quantified by Image-Pro Plus version 6.0 software (Media Cybernetics, Inc.; RRID: SCR_007369).

2.7. Immunoblotting

Proteins were extracted from liver tissues using a total protein extraction kit (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors and phosphatase inhibitors according to the manufacturer’s instructions. The protein concentration was quantified by a BCA protein assay kit (Thermo, USA) with BSA as a standard. Total protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk in Tris-buffered saline (1x) for 2 hours, the membranes were probed with appropriate primary antibodies against STAT3, phosphorylated STAT3 (Tyr705) (p-STAT3), α-SMA and GAPDH at 4 °C overnight and then detected by HRP-labeled anti-rabbit or HRP-labeled anti-mouse secondary antibodies. Immunoreactive bands were visualized by an ECL immunoblot detection system (Thermo, USA) and were exposed by a ChemiDoc MP system (Bio-Rad, USA). Protein expression levels were defined as gray values (Image Lab 3.0 software, Bio-Rad, USA; RRID: SCR_014210).

2.8. RNA preparation, reverse transcription and real-time PCR (RT-PCR)
Total RNA was extracted from liver tissues, HSCs and splenocytes using TRIzol reagent (TaKaRa, Japan); samples (1 μg) of total RNA were reverse transcribed using PrimeScript RT Master Mix (TaKaRa, Japan), which was performed at 37 °C for 15 min and terminated by enzyme deactivation at 85 °C for 5 seconds. Real-time PCR was performed using SYBR Green (Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA). All primers were provided by TsingKe Biological Technology (Hubei, China). Primer sequences are shown in Supporting Information Table S1. The reverse transcription reaction products were amplified by RT-PCR with Premix Ex Taq (TaKaRa, Japan) and the respective primers. The RT-PCR cycling conditions were 95 °C for 3 min, followed by 40 cycles of a two-step amplification program (95 °C for 3 seconds and 60 °C for 30 seconds). The specificity of the amplification was confirmed by melting curve analysis. The data were collected and recorded by CFX Manager software (Bio-Rad), and are expressed as a function of the threshold cycle (Ct). The samples for RT-PCR analysis were evaluated using a single predominant peak as auality control. Relative expression levels of target genes were normalized to the housekeeping gene GAPDH, and the results were calculated by the Ct (2-△△Ct) method.

2.9. Splenocyte preparation and cell culture

Splenocyte suspensions were prepared from C57BL/6J mice by standard techniques as previously described [8]. Briefly, after the mice were sacrificed by cervical dislocation, the spleen tissues were removed and minced. After filtration through a nylon cell strainer (70 μm size; BD Falcon, Franklin Lakes, NJ, USA), the suspensions were separated by a Percoll (GE Healthcare, Sweden) step gradient. Cells were collected from the lymphocyte-rich fraction and cultured with RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37 °C. Then, the cells were cultured with 5 μg/ml concanavalin A, followed by treatment with 0, 50, 100, or 200 μg/ml ASP for 72 hours. The supernatant was stored at -80 °C for ELISA and subsequent experiments, and the cells were collected for total RNA extraction.

2.10. Isolation of hepatic stellate cells

HSCs were isolated from C57BL/6 mice after anesthesia as previously described [16]. Briefly, the liver was digested with collagenase after perfusion, and the digested liver was excised and incubated in a collagenase solution. The resulting suspension was filtered through a 100 μm nylon mesh and centrifuged in a Percoll (GE Healthcare, Sweden) step gradient, which produced an upper layer that was rich in HSCs. After isolation, the cells were cultured with DMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO2 at 37 °C. Then, the cells were cultured with 10 ng/ml transforming growth factor beta (TGF-β) for 24 hours, followed by treatment with 200 μg/ml ASP, splenocyte supernatant (SC), supernatant of splenocytes cocultured with 200 μg/ml ASP (SA) or SA+1 μg/ml IL-22 neutralizing antibody (SA+Abs) for 72 hours. The cells were then collected for total RNA extraction.

2.11. ELISA

Levels of IL-22 in the conditioned supernatant were quantified using commercial ELISA kits (Biolegend, US, Cat# 436307) according to the manufacturer’s instructions. The absorbance of each well was determined at 450 nm using a microplate reader (Elx800, Bio-Tek, USA).

2.12. Statistical analysis

All experiments were randomized and blinded. The data and statistical analysis in this study comply with the recommendations on experimental design and analysis in pharmacology [17, 18]. The data are expressed as the mean ± standard error of the mean (SEM). Differences between two groups were evaluated using Student’s unpaired t-tests. When comparing ≥3 different groups, one-way ANOVA followed by Tukey’s post hoc test was used for multiple comparisons, and Dunnett’s post hoc test was used when comparing each group with a certain group. P<0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism software (V6.01) (San Diego, CA, USA; RRID: SCR_002798).

3. RESULTS

3.1. ASP attenuates CCl4-induced chronic liver fibrosis

A CCl4-induced chronic liver fibrosis mouse model was established to explore the antifibrotic effect of ASP. As shown in Figure 1A and 1B, compared with the control mice, ASP-treated mice had fewer vacuolated and degenerated hepatocytes and a reduction in the area of fibrosis by approximately 70%. In addition, serum ALT levels in ASP-treated mice decreased by approximately 50% compared with that of control mice (Figure 1C). Moreover, the expression of collagen type I (COLⅠ) significantly decreased in ASP-treated mice as detected by quantitative PCR (Figure 1D). The transcript levels of MMP2 and TIMP1, which are involved in the homeostatic regulation of the ECM, were alleviated by ASP (Figure 1D). These results indicate that ASP has a significant antifibrotic effect.

3.2. ASP inhibits HSC activation in vivo

HSCs are the most important cells in liver fibrosis, and their activation significantly promotes the development of liver fibrosis [2]. To
understand the mechanisms underlying the reduction in fibrosis in the ASP-treated mice, HSC activation was examined. As illustrated in Figure 2A and 2B, the mRNA and protein levels of α-SMA, which is a marker of HSC activation, significantly decreased in mice treated with ASP compared with the control group, indicating that ASP inhibited the activation of HSCs. In addition, immunohistochemistry results showed lower α-SMA expression in ASP-treated mice than in control mice (Figure 2C and 2D). These findings suggest that the antifibrotic effect of ASP may be related to the inhibition of HSC activation.

3.3. ASP stimulates the production of IL-22 both in vitro and in vivo

IL-22 has been reported to inhibit HSC activation and restrict liver fibrosis [11]. To verify whether the antifibrotic effect of ASP is related to IL-22, we examined the effect of ASP on the production of IL-22. First, we cultured splenocytes for 72 hours with different concentrations of ASP. As shown in Figure 3A and 3B, ASP induced the secretion of IL-22 at concentrations of 50 mg/ml and 100 mg/ml, which further increased at a concentration of 200 mg/ml. Next, the results were verified in vivo. Splenocytes from mice treated with 200 mg/kg ASP expressed increased levels of IL-22, as detected by quantitative PCR and ELISA (Figure 3C and 3D). In addition, the expression of IL-22 was significantly increased in liver tissue from ASP-treated model mice with chronic liver fibrosis (Figure 3E).

3.4. IL-22 neutralizing antibodies block the hepatoprotective effect of ASP in vivo
To directly explore the role of IL-22 in the antifibrotic effect of ASP, we conducted an IL-22 neutralization study in the CCl4-induced liver fibrosis mouse model with or without ASP treatment. While ASP protected mice in the isotype group from liver injury, as indicated by a reduction in the number of vacuolated cells and decreased serum ALT levels, no significant difference was detected in the severity of liver injury between ASP-treated and untreated mice after IL-22 neutralization (Figure 4A and 4C). Although ASP significantly reduced the area of fibrosis in isotype group mice, this effect was not observed in IL-22 Abs-treated mice. In contrast, IL-22 Abs exacerbated fibrosis (Figure 4A and 4B). Moreover, ASP significantly reduced the expression of fibrosis-related genes (COLⅠ, MMP2 and TIMP1) in mice in the isotype group, but these effects were not observed in IL-22 Abs-treated mice (Figure 4D). These data indicate that IL-22 plays a vital role in the antifibrotic effect of ASP.

3.5. The inhibitory effect of ASP on HSC activation is related to IL-22 To explore the mechanism by which ASP inhibits HSC activation, primary HSCs were isolated and activated with 10 ng/ml TGF-b. Surprisingly, ASP had no effect on the expression of α-SMA or COLⅠin activated HSCs, indicating that ASP does not directly inhibit HSC significantly reduced after HSCs were exposed to the supernatant of ASP-treated splenocytes (Figure 5C and 5D). Moreover, this inhibitory effect disappeared upon the addition of IL-22 neutralizing antibodies
(Figure 5C and 5D).

3.6. ASP activates the STAT3 signaling pathway downstream of IL-22 In liver injury, IL-22 primarily activates the STAT3 pathway to inhibit HSC activation and exerts a hepatoprotective effect [11]. To further address the mechanism by which ASP mediated its antifibrotic effect, the downstream pathway of IL-22 was examined. The results showed that the ratio of p-STAT3 to total STAT3 increased approximately 2-fold in the livers of ASP-treated mice compared with control mice, indicating that ASP activated the STAT3 pathway (Figure 6A and 6B). However, these effects were not observed in IL-22 Abs-treated mice (Figure 6C and 6D). These results suggest that ASP activates the STAT3
signaling pathway through IL-22.

3.7. The hepatoprotective activity of ASP depends on the STAT3 pathway
To further show that the antifibrotic effect of ASP is due to the STAT3 signaling pathway, which is downstream of IL-22, the activation of STAT3 was inhibited in vivo by S3I-201, which inhibits STAT3 phosphorylation [15], in the presence of ASP. As expected, ASP-induced STAT3 phosphorylation in the liver was abolished by S3I-201 (Figure 7A and 7B). Importantly, the degree of liver injury and fibrosis were significantly reduced by ASP in the DMSO group but did not obviously change in the S3I-201 group (Figure 7C-E). ASP significantly reduced the expression of fibrosis-related genes (COLⅠ, MMP2 and TIMP1) in DMSO-treated mice, while no differences were found in S3I-201-treated mice (Figure 7F). These results demonstrate that the STAT3 pathway is required for the antifibrotic effect of ASP.

3.8. ASP inhibits HSC activation via the IL-22/STAT3 signaling pathway
To further clarify the role of the IL-22/STAT3 signaling pathway in ASP-induced inhibition of HSC activation, α-SMA, a direct indicator of HSC activation, was examined. As shown in Figure 8A, 8C and 8D, treatment with IL-22 Abs counteracted the inhibitory effect of ASP on the expression of α-SMA, suggesting that IL-22 is required for ASP-mediate inhibition of HSC activation. Moreover, the results suggested that ASP inhibited HSC activation via the STAT3 signaling pathway. After treatment with S3I-201, there was no significant change in the expression of α-SMA between the ASP and control groups (Figure 8B, 8E and 8F). These results indicate that the IL-22/STAT3 signaling pathway is required for ASP-mediated inhibition of HSC activation.

4. DISCUSSION

In recent years, polysaccharide drugs have received increasing attention due to their remarkable efficacy and nontoxic side effects. In our previous study, the novel ASP was extracted, and detailed identification and analysis of the structure of ASP were performed to determine its repeating unit [7]. In addition, we found that ASP has a potential hepatoprotective effect against liver diseases. Specifically, ASP alleviates oxidative stress in nonalcoholic fatty liver and suppresses the immune response against acute liver injury [8, 9], which is consistent with previous studies [19, 20]. In the current study, we established a potential antifibrotic effect of ASP in a CCl4-induced chronic liver fibrosis mouse model. Mechanistically, we revealed that ASP-mediated protection occurred by inhibiting HSC activation and was dependent on the IL-22/STAT3 pathway.
CCl4 has been widely used for the experimental induction of hepatic injury in animal models [21]. This compound is metabolized by cytochrome P450 2E1 to generate free radicals, which increase the permeability of the hepatocellular membrane, followed by the release of cytoplasmic contents, eventually damaging hepatocytes [22]. In our experiment, the level of serum ALT was significantly increased, and the structure of hepatocytes was disordered in the CCl4 group, reflecting the negative effect of chronic CCl4 administration on hepatocytes. ASP significantly reduced the level of serum ALT and ameliorated hepatocellular architecture, indicating an improvement in hepatic damage.

Liver fibrosis results from chronic damage to the liver in conjunction with excessive accumulation of ECM, especially the predominant ECM component collagen, which is a characteristic of chronic liver fibrosis [1]. Chronic CCl4 administration induced liver fibrosis and collagen deposition as indicated by Sirius red staining. Our present study demonstrated that ASP possesses potent antifibrotic activity, as confirmed by the significant reduction in collagen deposition and expression of liver fibrosis-related genes. In liver fibrosis, HSC activation is marked by increased α-SMA expression, which represents the primary pathophysiological event. Activated HSCs play a crucial role in the excessive synthesis and deposition of ECM by secreting collagen and unbalancing MMP homeostasis [2]. Therefore, inhibition of HSC activation represents an effective strategy for the prevention and treatment of liver fibrosis. Interestingly, our data demonstrated that ASP significantly reduced the expression of α-SMA in the livers of CCl4-treated mice, indicating that ASP effectively inhibited HSC activation, further confirming its antifibrotic effect.

A previous study reported that immune cells and cytokines affect the progression of liver fibrosis [1], and ASP has been shown to regulate the
inflammatory response in acute liver injury [8]. IL-22 is a cytokine that belongs to the IL-10 family [10], and importantly, IL-22 has been reported to be effective in inhibiting HSC activation to exert an antifibrotic effect [11]. Therefore, we explored the involvement of IL-22 in the antifibrotic effect of ASP. First, we showed that ASP significantly increased the expression of IL-22 in vivo and in vitro. Thereafter, neutralization of IL-22 blocked ASP-mediated protection against CCl4-induced chronic liver fibrosis, indicating an essential role of IL-22 in this process. Interestingly, the results showed that liver fibrosis was more severe in IL-22 Abs-treated mice than in isotype-treated mice. Other groups have demonstrated that an endogenous increase in IL-22 effectively alleviates liver fibrosis, which is consistent with our results, verifying that IL-22 plays an important role in the antifibrotic effect [12]. Furthermore, in vitro experiments demonstrated that ASP did not directly inhibit HSC activation. Instead, ASP-induced IL-22 was responsible for ASP-mediated inhibition of HSC activation, further supporting the in vivo results showing that IL-22 was critical for ASP-mediated hepatoprotection.

IL-22 activates STAT and mitogen-activated protein kinase pathways, among which the STAT3 pathway mediates the protective effect of IL-22 against liver fibrosis [10, 11]. After stimulation, STAT3 is phosphorylated and forms p-STAT3 dimers that translocate to the nucleus and function as transcription factors for downstream genes, including B cell lymphoma (bcl)-2, bcl-xl, c-myc, suppressor of cytokine signaling 3 (SOCS3), p53 and p21, which play important roles in promoting HSC senescence and inhibiting HSC activation [11, 12]. Our data demonstrated that ASP significantly activated the STAT3 pathway in the liver, which was attributed to IL-22. When treated with the STAT3 inhibitor S3I-201 [15], the antifibrotic effect of ASP was blocked, which directly verified the significant role of the STAT3 pathway in this process. Numerous studies reported that activation of IL-22/STAT3 in HSCs inhibits HSC activation [11, 12, 23]. Our data demonstrated that the inhibition of HSC activation by ASP was abolished in S3I-201-treated and IL-22 Abs-treated mice, indicating that this effect was directly related to the IL-22/STAT3 pathway.

In addition to HSCs, the development of liver fibrosis is associated with hepatocytes. Damaged hepatocytes release reactive oxygen species and fibrogenic mediators, which induce inflammatory recruitment. Apoptosis of damaged hepatocytes stimulates the fibrogenic actions of hepatic myofibroblasts [24]. Whether hepatocytes are also involved in the antifibrotic effect of ASP in liver fibrosis requires further exploration. In addition, how ASP affects the secretion of IL-22 in splenocytes needs to be addressed in the future.

5. CONCLUSION

Our study demonstrates that ASP exerts an antifibrotic effect during the development of liver fibrosis, which is related to the inhibition of HSC activation. Most importantly, we revealed the underlying pathway: the IL-22/STAT3 axis is critical for the antifibrotic effect of ASP. This finding indicates the potential clinical application of ASP as an antifibrotic drug.

AUTHOR CONTRIBUTORS K.W., J.W. and N.X. collected documents and designed the experiments; J.W., M.S. and H.W. conducted the experiments and performed data analysis; K.W., J.W. and N.X. wrote and reviewed the manuscript; N.X. and Y.Z. provided guidance during the study.

ACKNOWLEDGMENTS
We appreciate the staff from the Analysis and Testing Center of Huazhong University of Science and Technology for their technical assistance. The present research was supported by grants from the National Key Research and Development Program of China (2017YFC0909900).

CONFLICTS OF INTEREST
All the authors declared no conflicts of interest.

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