Abbreviations:
ECM, Extracellular matrix; HSC, hepatic stellate cells; MDP, Monomer derivative of paeoniflorin; MFC, myofibroblast-like cells; SA-β-Gal, senescence-associated beta-galactosidase; miRNA, MicroRNA; UTR, untranslated region (); HCC, hepatocellular carcinoma; IHC, immunohistochemical; ALT, Alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; E‐box, enhancer box; Ago2, Argonaute 2.
Introduction
Liver fibrosis is the diffuse ECM in the liver, including the excessive deposition of fibronectin and collagen (especially collagen) in the liver, which leads to cirrhosis, liver failure and portal hypertension in advanced liver fibrosis (Aydin & Akcali, 2018; Friedman, 2008). Under normal circumstances, hepatic stellate cells (HSC) are in a static state, but when the liver is stimulated, they are activated and transformed into myofibroblast-like cells (MFC) and secrete a large amount of cytokines (Anthony, Allen, Li & McManus, 2010; Duval, Moreno-Cuevas, Gonzalez-Garza, Rodriguez-Montalvo & Cruz-Vega, 2014). Therefore, inhibiting the proliferation and activation of HSC and promoting the degradation of ECM have become the treatment direction of anti-fibrosis (Gur et al., 2012; Zhang et al., 2015). In recent years, with the research, hepatic stellate cells can express almost all the key components required for matrix degradation. With the study of cell cycle, it is found that HSC undergoing cell senescence can reduce liver fibrosis (Krizhanovsky et al., 2008). The results of this study indicate that inducing HSC senescence may become another treatment for liver fibrosis.
Cellular senescence is a relatively stable cell cycle arrest that is implicated in many age-related diseases including cancer (Campisi & d’Adda di Fagagna, 2007). Although there are many factors that trigger cell senescence, the increase in senescence-associated β-galactosidase (SA-β-Gal) activity and changes in senescence-related molecular markers can be used as indicators of whether cells are aging (Braig et al., 2005; Calcinotto, Kohli, Zagato, Pellegrini, Demaria & Alimonti, 2019; Chen et al., 2005; Michaloglou et al., 2005). Senescent cells are characterized by the increase of SA-β-Gal activity, irreversible arrest of cell cycle, induction of senescence-related protein p53, p16, p21, and so on (Munoz-Espin & Serrano, 2014). Studies have shown that in the senescence stage, activated HSC cells could reduce the expression of ECM and increase the expression of ECM degrading enzymes while inhibiting cell proliferation (Krizhanovsky et al., 2008; Schnabl, Purbeck, Choi, Hagedorn & Brenner, 2003). Recently, some studies have shown that the tumor suppressor p53 can promote the senescence process of cells. Knockdown of the important cell cycle regulator p53 reduces HSC senescence, contributing to extensive liver fibrosis (Krizhanovsky et al., 2008). Hence, regulating p53 may be an effective therapeutic strategy for liver fibrosis.
Since the seed sequence of MicroRNA (miRNA) is partially or completely complementary to the target sequence in the 3’-untranslated region (UTR) of the mRNA (Peng & Croce, 2016). For example, miR-29 expression is downregulated in human and murine liver fibrosis, which is mediated by TGF-β and other inflammatory signals (Roderburg et al., 2011), suggesting that miRNA play a crucial role in the progression of liver fibrosis. In recent, some studies indicated that miR-708 was considered as one of the expression-silenced miRNAs in tumors including hepatocellular carcinoma (HCC) (Li, Yang, Xu, Yue, Fang & Liu, 2015; Li, Li, Wu & Gao, 2017). Interestingly, our previous study indicated that miR‐708 plays a role in repressing the progression of liver fibrosis through significantly inhibiting HSCs activation and proliferation. Moreover, miR-708 directly targeted ZEB1 (Yang et al., 2020). Meanwhile, one recent study reported that p53 promoter enhancer box (E‐box) could be regulated through ZEB1/2 (Deshpande et al., 2013; Roy, Beamon, Balint & Reisman, 1994), indicating that the miRNA-708 play a critical role in liver fibrosis progression. However, although to date, far less is known about its effect on HSCs senescence of in liver fibrosis.
TGP and Pae have physiological activities such as anti-inflammatory, anti-oxidation and immune regulation, while the content of Pae accounts for more than 90% of the total glycosides. It is the main active ingredient of a variety of solid preparations of traditional Chinese medicine. It can be used to treat a variety of liver diseases, but Pae has low bioavailability. It has become the biggest obstacle to its clinical use (Jia et al., 2016; Wang, Zhang, Wu & Wei, 2011; Zhu, Wei, Zheng & Jia, 2005). In order to improve the bioavailability of Pae, its chemical structure was optimized and modified to obtain MDP presented in Figure 1A, which improved absorption and reduced the sensitivity to p-glycoprotein (p-GP) (Yang et al., 2016). In addition, it still has the functions of regulating immunity and anti-inflammatory (Shu et al., 2019; Yang et al., 2019b). Supplementary Figure S1A shows the chemical construction of MDP. Studies have shown that MDP participates in the development of adjuvant arthritis rats mainly through immune response and regulation of inflammatory mediators, such as inflammation and bone damage (Tu et al., 2019; Wang et al., 2020). Nevertheless, the regulatory effect and molecular mechanisms of MDP on senescence of activated HSCs are not unveiled.
In this study, we clarified the role of MDP in senescence of activated HSCs from liver fibrosis and identified that MDP could regulate the Ago2/miR-708/p53 regulatory axis contributes to senescence of activated HSCs. These findings might provide novel therapeutic and diagnostic targets for activated HSCs from liver fibrosis.
Materials and methods
Animal studies
All male C57BL/6J mice were obtained from the Experimental Animal Center of Anhui Medical University. The mice model of liver fibrosis was established by three injections of carbon tetrachloride (CCl4, Sigma-Aldrich, St. Louis, MO, USA) as previously described (Yang et al., 2019a; Yang et al., 2020). All experimental protocols involving animals in this study were approved by the Laboratory Animal Research Committee of Anhui Medical University. For histological scoring of liver fibrosis, the liver was fixed in 4% buffered paraformaldehyde. The present study was reviewed and approved by the Animal Care and Use Committee (number: LLSC20150348).
Cell culture
The human HSC line (LX-2) was obtained from Shanghai Central Experiment Laboratory (China). The cells were cultured according to previous study (Yang et al., 2019a)
Immunofluorescence analysis
Immunofluorescence experiments were conducted as previously described (Yang et al., 2019), using fluorescein isothiocyanate (FITC)-conjugated anti-ZEB1 (1:300), anti-p53 (1:300). Alexa Fluor 594-conjugated anti-rabbit antibody was used as the secondary antibody. Counterstaining of the nuclei was performed with 4, 6-diamidino-2-phenylindole (Biyuntian, Shanghai, China). Images were captured using fluorescence microscopy.
Histopathology and immunohistochemical (IHC) staining
Liver tissues were preserved in 4% formalin (Solarbio, Beijing, China) and embedded in paraffin and cut into 50-µm thick sections. The section slides were stained with hematoxylin and eosin (H&E), Masson, Sirius red staining and immunohistochemical (IHC) staining according to standard methods. A laser scanning confocal microscope (Olympus, Tokyo, Japan) was used to examine the sections, and images were recorded.
Serum aminotransferase activity
Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) in serum were detected by using the commercial kits (Jiancheng Bio. Institute, Nanjing China) according to the manufacturer’s direction.
Flow cytometry
Flow cytometry were carried out according to the protocol described previously (Yang et al., 2017; Yang et al., 2019a).
Cell Transfection
miR-708 inhibitor, miR-708 mimics, ZEB1 and p53 siRNA were synthesized by GenePharma (Shanghai, China). The sequences are listed in Table 1. The transfection of cells was performed according to the method described previously (Yang et al., 2020). All experiments were performed in triplicate.
Dual luciferase reporter assay
The luciferase reporter plasmid ZEB1 and p53 (pYr‐PromDetect‐ZEB1 or TP53) was purchased from Changsha Yingrun Biotechnology Co. Ltd. (Hunan, China). Luciferase activity was detected by using a Dual‐Luciferase Reporter Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Luciferase activity was normalized by cotransfecting with pRL‐TK (Promega, Madison, WI). All experiments were repeated three times.
SA‐β‐Gal staining
The SA‐β‐Gal) assay was performed using a senescence staining kit (Beyotime, Shanghai, China). The transfection of cells was performed according to the method described previously (Yang et al., 2019a). All experiments were performed in triplicate.
RNA extraction and qRT‐PCR
Extraction of total RNA from liver LX-2 cells using TRIzol reagents (Invitrogen, USA) following the manufacturer’s instructions and then was reversely transcribed to cDNA with a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). The expression levels of the indicated genes were estimated by real-time PCR using SYBR® Green Master (BioRad, USA). The PCR results for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls. The primers used for the PCR are listed in Table 1.
Western blot analysis
Total proteins were extracted from tissues and cells with radioimmunoprecipitation assay and phenylmethylsulfonyl fluoride lysate buffer. The Western blot was carried out according to previous study (Yang et al., 2017; Yang et al., 2020) . Immunoreactive bands were visualized with ECL chromogenic substrate. The density of the immunoreactive bands was analyzed by using Image J computer software. β-actin was used as internal control for the protein.
Statistical analysis
All data analyses were conducted using SPSS 21.0 software (IBM Corp. Armonk, NY, USA). The measurement data were expressed as mean ± standard deviation, and Pearson correlation analysis was employed for correlation test. The unpaired t-test was performed for comparisons between two groups, one-way analysis of variance (ANOVA) for those among multiple groups and Tukey’s post hoc test for pairwise comparisons. Pvalue<0.05 was indicative of statistically significant difference.
Results
Hepato-protective effects of MDP on mice with CCl4-induced liver fibrosis
To determine the effects of MDP on liver fibrosis in vivo, the model of liver fibrosis was established by CCl4 in experimental animals. The HE, Masson and Sirius red staining showed that the liver tissues of mice showed more hepatic steatosis, necrosis, and fibrotic septa compared with normal groups (Figure 1B). Meanwhile, serum ALT, AST and TBIL activities were significantly increased by CCl4(Figure 1C). In addition, the expression of α-SMA and Col. I, which are widely accepted as fibrosis markers in activated HSCs, was examined. The IHC and Western Blot results indicated that the expression of α-SMA and Col. I was all significantly elevated in the fibrotic liver (Figure 1B and 1D). We further measured whether there is any change in the senescence marker in the development of liver fibrosis. Results from IHC and Western Blot showed that CCl4 impeded the expression of p16 and p21(Figure 1E and Supplementary Figure S1B). Next, we detected the effect of DPF-6 (25 mg/kg, 50 mg/kg and 100 mg/kg) on CCl4-induced liver fibrosis. The results indicated that MDP significantly inhibited the hepatic steatosis, necrosis, and fibrotic septa (Figure 1A). The upregulation of activities of ALT, AST and TBIL was reversed after the addition of MDP (Figure 1C). More importantly, the expression of α-SMA and Col. I reduced dose-dependently by MDP, concomitant with the upregulation of senescence marker (p16 and p21) (Figure 1B, 1D and 1E, Supplementary Figure S1B). These results consistently demonstrated that MDP attenuated CCl4-caused liver fibrosis in vivo.
MDP promoted the senescence in TGF-β1-induced LX-2 cells
The cytotoxicity test of MDP was achieved by MTT assay. LX-2 cells were treated with TGF-β1 (10 ng/ml, 24 h) in advance (Supplementary Figure S1C). In addition, the expression of α-SMA, Col. I, p16 and p21 was detected by using Western Blot. Compared with the control group, the expression of α-SMA and Col. I was upregulated and the expression of p16 and p21 was inhibited by TGF-β1 (10 ng/ml, 24 h) in LX-2 cells. Then, the LX-2 cells were treated with TGF-β1 (10 ng/ml, 24 h) and MDP (10-8, 10-7, 10-6, 10-5 mol/L, 24 h). The results indicated that MDP significantly inhibited the expression of α-SMA and Col. I as well as elevated p16 and p21 expression in LX-2 cells treated with TGF-β1 (10 ng/ml, 24 h) and MDP (10-8, 10-7, 10-6, 10-5 mol/L, 24 h) (Supplementary Figure S1D). More importantly, higher dose concentrations (10-5 mol/L) of MDP had obviously inhibition of cell viability in LX-2 cells treated with TGF-β1, so 10-5mol/L was chosen to be used in the following experiments. Next, we further test whether MDP had direct effects on HSCs senescence. SA‐β‐Gal staining showed that high numbers of SA‐β‐Gal‐positive cells were observed in activated LX-2 cells treat with MDP (10-5mol/L) (Supplementary Figure S1E). Immunofluorescence analysis revealed that TGF-β1promoted the α-SMA and Col. I expression and inhibited the expression of p16 and p21. However, MDP could inhibited the α-SMA and Col. I expression and increased the p16 and p21 expression (Figure 2A and 2B). Results from qRT-PCR and Western Blot showed that MDP increased the expression of senescence marker p16 and p21 in HSCs concomitant with the inhibition of α-SMA and Col. I expression (Figure 2C, 2D, 2E and 2F). These data indicated that MDP promoted senescence of activated HSCs in vitro.
MDP promoted miR-708 expression by targeting Ago2 in TGF-β1-induced LX-2 cells
Firstly, qRT-PCR was used to evaluate the level of miR-708 in liver tissue with liver fibrosis. As shown in Figure 3A, the expression of miR-708 was down-regulated in CCl4-treated group but significantly up-regulated in MDP-treated group, which treated with different concentrations of MDP. Then, the result of qRT-PCR also showed that MDP could induce miR-708 mRNA expression in activated LX-2 cells (Figure 3B). Ago2 is an effector of small RNA-mediated gene silencing. In addition, in recent years, continuous studies have shown that Ago2 could affect miRNA activity at many levels (Kretov, Walawalkar, Mora-Martin, Shafik, Moxon & Cifuentes, 2020; Zhang et al., 2019). Therefore, we hypothesized that MDP promoted miR-708 expression by targeting Ago2. IHC, immunofluorescence and Western Blot result showed that the expression of Ago2 was inhibited in CCl4-caused liver fibrosis and activated HSCs, and the decreased was reversed after the addition of MDP in liver fibrosis tissues and activated LX-2 cells (Figure 3C and 3D, Supplementary Figure S2A and S2B). More importantly, the qRT-PCR results showed that knockdown of Ago2 inhibited the expression of miR-708 in activated LX-2 cells (Supplementary Figure S2C). Overexpression of Ago2 increased the expression of miR-708 in activated LX-2 cells (Supplementary Figure S2D). Additionally, as shown in the Figure 3E, we using Discovery Studio 2017 R2 software to analyze the Ago2 X-ray crystal structure of MDP, it is found that MDP binds to the active pocket of the target protein in a folded manner (Rivas et al., 2005). Further examinations confirmed that knockdown of Ago2 elevated the expression of α-SMA and Col. I in activated LX-2 cells, which this increase was inhibited by MDP. In contrast, overexpression of Ago2 inhibited the expression of α-SMA and Col. I in activated LX-2 cells, and MDP further inhibited the expression of α-SMA and Col. I, indicating that MDP played a similar role to plasmid (Figure 3F and 3G). Collectively, these data demonstrated that MDP could upregulated the expression of miR-708 via targeting Ago2.
miR-708 promoted senescence of TGF‐β1‐induced LX‐2 cells
Firstly, we transfected activated LX-2 cells with miR-708 NC, miR-708 inhibitor and miR-708 mimics to investigating the role of miR-708 in senescence of activated HSCs, respectively. Flow cytometry results indicated that more cells were arrested in the G0/G1 phase, accompanied by a decrease in proportion of cells in the S or G2/M phase in miR-708 mimics group (Figure 4A). SA-β-gal was a senescence-associated marker. Therefore, it is used to detect the senescence of LX-2 cells induced by TGF-β1 after treatment with miR-708 NC, miR-708 inhibitor and miR-708 mimics. As expected, overexpression of miR-708 could lead to the production of SA-β-Gal positive cells (Figure 4B). Additionally, immunofluorescence analysis showed that overexpression of miR-708 increased the expression of p16 and p21 and inhibited the α-SMA and Col. I expression (Figure 4C). Meanwhile, Western Blot analyses of senescence-associated genes consistently showed that silence of miR-708 significantly downregulated the expression of senescence markers p16 and p21(Figure 4D). In contrast, overexpression of miR-708 increased the expression of senescence markers p16 and p21 (Figure 4E). Apart from these observations, we detected whether the expression of miR‐708 was involved in the severity of liver fibrosis. As shown in Figure 4F and 4G, the expression of α-SMA and Col. I was elevated by miR-708 inhibitor, and inhibited by miR-708 mimics. Combining the above results, we can find that mir-708 slows down liver fibrosis is related to inhibiting HSC activity by inducing senescence.
ZEB1 is identified as a directly target of miR-708
To explore the mechanism underlying miR-708 regulated HSCs senescence, we use TargetScan to predict the candidate targets of miR-708, and found a complementary site of miR-708 in ZEB1 (Figure 5A). Moreover, the Luciferase Reporter Assay was performed to further explore whether miR-708 directly binds to the 3’-UTR of ZEB1. The luciferase reporter vector containing wild-type (wt) or mutated (mt) 3′-UTR of ZEB1 were used. As predicted, the luciferase activity in the reporter vector of the wt 3’-UTR of ZEB1 was significantly decreased by miR-708 mimics. However, the luciferase activity of the reporter vector of the mt 3’-UTR of ZEB1 was not decreased (Figure 5B). Furthermore, we also detected the transcriptional and translational levels of ZEB1 after transfection by using immunofluorescence, qRT-PCR and Western Blot assays in LX-2 cell transfected with miR-708 inhibitor and miR-708 mimics. The results showed that ZEB1 expression was inhibited by miR-708 mimics and elevated by miR-708 inhibitor (Figure 5C, 5D and 5E), indicating that miR-708 repressed ZEB1 expression at the transcriptional and translational level. Next, we further tested whether changes in ZEB1 expression affect the senescence of HSC by induced miR-708, the protein expression of p16 and p21 were detected by using Western Blot in activated LX‐2 cells that was co-transfection of miR‐708 inhibitor and ZEB1‐siRNA. The results showed that the protein level of p16 and p21 was elevated compare with miR‐708 inhibitor (Figure 5F). Altogether, miR-708 influences ZEB1 expression by directly targeting the 3′-UTR of ZEB1 mRNA.
ZEB1 inhibited senescence of the TGF‐β1‐induced LX‐2 cells
Further, the alternation of ZEB1 expression in liver fibrosis was explored. The colabeling ZEB1 and α‐SMA was performed for colocalization. The results indicated that the expression of ZEB1 was remarkably increased by CCl4 (Figure 6A). The qRT-PCR and Western Blot analysis showed that the mRNA and protein level of ZEB1 was significantly augmented in CCl4-treated group compared to control group (Figure 6C and 6E). We further confirmed the expression of ZEB1 in TGF‐β1‐induced LX‐2 cells. Immunofluorescence analysis showed that TGF‐β1 could simultaneously increase the level of ZEB1 in LX‐2 cells (Figure 6B). There results were confirmed by qRT-PCR and Western Blot analysis (Figure 6D and 6F). Next, to further investigate the relationship between ZEB1 and senescence in activated HSCs, we transfected TGF‐β1‐induced LX‐2 cells with ZEB1 siRNA and p-CMV-ZEB1. Cell cycle assay revealed that ZEB1 knockdown reduced the cell number of S phase, and increased the cell number of G0/G1 phase (Figure 7A). In addition, SA‐β‐Gal staining suggested that SA-β-gal activity was decreased by overexpression of ZEB1 and was markedly increased by knockdown of ZEB1 (Figure 7B). Immunofluorescence analysis indicated that silence of ZEB1 could significantly increase the expression of p16 and p21 and inhibited the expression of α-SMA and Col. I in LX-2 cells (Figure 7C). At the same time, analyses of Western Blot displayed that knockdown of ZEB1 promoted the expression level of p16 and p21 in activated LX-2 cells (Figure 7D). In contrast, overexpression of ZEB1 inhibited the expression level of p16 and p21 in activated LX-2 cells (Figure 7E), suggesting that ZEB1 inhibited the senescence of activated HSCs in liver fibrosis. Then, we examined the expression of marker of HSC activation α-SMA and Col. I by using Western Blot. The results indicated that the protein level of α-SMA and Col. I was upregulated by ZEB1 siRNA (Figure 7F). Whereas, opposite results were obtained in LX‐2 cells treated with ZEB2 plasmid (Figure 7G). These findings suggested that ZEB1 might be involved in activated LX-2 cells senescence.
ZEB1 repressed activities of p53 promoter via binding to E‐box in LX‐2 cells
To further characterize the mechanism by which ZEB1 acted on HSCs senescence. We used luciferase reporter assays to identify the role of ZEB1 in the activities of the p53 promoter. The p53 promoter has an E‐box and can be potentially repressed by E‐box binding transcriptional repressors like ZEB1. Interestingly, the results showed that an increase of luciferase activity in the p53 promoter was observed upon downregulation of ZEB1 in LX‐2 cells compared with the empty vector (Figure 8A). In contrast, ZEB1 overexpression suppressed p53 promoter activity in activated LX‐2 cells (Figure 8B). Immunofluorescence analysis indicated that the expression of p53 was upregulated by ZEB1 siRNA (Supplementary Figure S2E). At the same time, the results from qRT‐PCR showed that the p53 mRNA level was significantly inhibited in cells transfected with ZEB1 plasmid than in the cells in the negative control group, whereas transfection with ZEB2‐siRNA increased the levels of p53 mRNA in cells (Figure 8C). There results were confirmed by using western blot analysis (Figure 8D). Altogether, the results supported that ZEB1 suppresses p53 promoter activity and thus inhibit the expression of p53.
P53 promoting senescence of the TGF‐β1‐induced LX‐2 cells
To disclose whether p53 displays a specific role in liver fibrosis progression, the expression level of p53 in development of liver fibrosis. Firstly, immunofluorescence double staining showed that p53 expression was inhibited by CCl4 in liver fibrosis tissues (Figure 8E). Concomitantly, the p53 mRNA and protein level was observed to be significantly decreased in liver fibrosis induced by CCl4 (Figure 8G, Supplementary Figure S2F). Apart from these observations, we also examined the expression of p53 in activated HSCs. Fluorescence staining showed that p53 was reduced in the nuclear upon TGF‐β1 stimulation in HSCs (Figure 8F). In agreement with the observed fluorescence changes, the p53 mRNA and protein levels were downregulated upon TGF‐β1 stimulation in HSCs (Figure 8H, Supplementary Figure S2G). Additionally, previous studies demonstrated that p53 plays a critical role in the induction of senescence (Rufini, Tucci, Celardo & Melino, 2013). Hence, we examined the effect of p53 on HSCs senescence. As illustrated in Figure 9A, HSCs treated with PEX-3-p53 showed significantly higher proportions of G1 cells and lower proportions of S cells compared with untreated HSCs. The evident activity of SA-β-gal was remarkably reduced by silencing p53 with p53 siRNA. In contrast, overexpression of p53 promoted the activity of SA-β-gal in activated LX-2 cells (Figure 9B). Furthermore, fluorescence staining showed that overexpression of p53 upregulated the p16 and p21 expression and downregulated α-SMA and Col. I expression (Figure 9C). Meanwhile, Results of Western Blot demonstrated that p16 and p21 expression was increased by PEX-3-p53. Whereas, silencing of p53 significantly reduced the expression level of p16 and p21 in activated LX-2 cells (Figure 9D and 9E). More importantly, we further detected the fibrotic markers α-SMA and Col. I expression in activated LX-2 cells. Western Blot analysis showed that overexpression of p53 could inhibit the expression of α-SMA and Col. I in activated LX-2 cells. In contrast, knockdown of p53 could increase the expression of α-SMA and Col. I in activated LX-2 cells (Figure 9F and 9G). Overall, these results indicated that p53 promotes senescence in activated HSCs and thus potentially slowing down liver fibrosis.
Discussion
Liver fibrosis is a precursor of liver cirrhosis. There are many causes of liver fibrosis. Various causes lead to the deposition of fibrous tissue in the liver, which is a passive and irreversible process. Nevertheless, accumulating evidence has demonstrated that fibrosis, and even cirrhosis, can be potentially reversed. Quiescent HSC activation is mainly characterized by differentiation into myofibroblasts, proliferation and production of extracellular matrix networks, and these characteristics are still the main driving forces of liver fibrosis (Chen et al., 2016; Duan et al., 2014; Troeger et al., 2012). Therefore, inhibiting HSC activation is an effective way to prevent liver fibrosis. Recent studies have also confirmed that promoting HSC senescence may also inhibit the occurrence and development of liver fibrosis (Jin et al., 2016; Yang et al., 2019a). Cells in the senescence stage have the characteristics of SA-β-gal (a lysosomal enzyme), p16 and p21 expression (Hayflick, 1965). Prior studies have discovered that promoting HSC senescence can inhibit the acute fibrotic response of tissues after external stimulus. With the reduction of activated HSC, the content of ECM also decreases (Poljak, Vidovic-Filipovic, Banovic, Gabric, Juric & Tadin, 1989; Schrader, Fallowfield & Iredale, 2009). Therefore, inducing HSC senescence can also be one of the ways to treat liver fibrosis.
Paeoniflorin, a monoterpene glycoside, is one of the main biologically active components of Paeonia lactiflora or Radix Paeoniae Alba . Previous studies have shown that paeoniflorin has been used to treat cerebrovascular diseases, cardiovascular diseases, nervous system diseases and liver diseases for more than 2,000 years (Ma et al., 2014). Interestingly, recent studies have shown that paeoniflorin has a variety of pharmacological effects in liver diseases (Zhao et al., 2013). For example, paeoniflorin modulates oxidative stress, inflammation and HSCs activation to alleviate CCl4-induced liver fibrosis by upregulation of heme oxygenase-1 in mice (Xiao, Wei, Yang, Peng & Zhang, 2011). On the basis of these strategies, our laboratory synthesized MDP to improve paeoniflorin water solubility, bioavailability. We previously found MDP exhibited well anti-inflammatory and immunomodulatory activity in several autoimmune animal models (Chang et al., 2016; Chen, Wang, Wu, Yan, Chang & Wei, 2018; Gu et al., 2018). However, the effects and detailed mechanism of MDP in senescence of activated HSCs was not fully understood. In this study, we found that the CCl4 could induce the expression of α-SMA and Col. I as well as inhibit the expression of p21 and p16 in vivo. However, MDP significantly reduced the expression of α-SMA and Col. I. More importantly, the expression level of p21 and p16 was increased by MDP. On the other hand. the results demonstrated that TGF-β1 could significantly inhibited the number of SA‐β‐gal‐positive cells in LX-2 cells compared with normal groups. However, MDP increased the number of SA‐β‐gal‐positive cells in LX-2 cells compared with the TGF-β1 group. Furthermore, the expression of p16 and p21 was inhibited by TGF-β1 and elevated by MDP in activated LX-2 cells. Hence, our results strengthened the observation of induction of HSC senescence by MDP, which could be a strategy for cell‐fate regulation in HSCs.
Most of miRNAs are highly conserved sequences due to their physiological functions. Therefore, the detection of miRNAs could provide detection indicators and new treatment options for related diseases (Yuan et al., 2015; Zhao et al., 2014). In previous studies, we have seen that miRNAs play their role in the cell cycle and liver, providing new ideas for studying the pathogenesis of liver fibrosis. (Hyun et al., 2016; Kim et al., 2018). Our previous research showed that miR-145 induces the senescence of activated HSCs (Yang et al., 2019a), indicating that miRNA may be involved in the senescence of activated HSCs. More importantly, we have also demonstrated that miR-708 represses HSCs activation and proliferation (Yang et al., 2020). However, there is no direct experiment proving the regulating effect of miR‐708 on HSCs senescence. In this study, wo showed that miR-708 was downregulated in vivo and in vitro. However, MDP could upregulate the expression of miR-708 in activated LX-2 cells. Importantly, overexpression of miR-708 effectively induced the G0/G1 arrest of activated HSCs and increased the number of SA‐β‐gal‐positive cells and the p16 and p21 expression and inhibited the α-SMA and Col. I expression in activated LX-2 cells. In contrast, silence of miR-708 obviously decreased the p16 and p21 expression and promoted the α-SMA and Col. I expression in activated LX-2 cells. These discoveries supported the possibility that induction of HSC senescence c ould treat liver fibrosis. Ago2 belongs to the mammalian Ago protein family, which is widely expressed and participates in post-transcriptional gene silencing (Meister, Landthaler, Patkaniowska, Dorsett, Teng & Tuschl, 2004). Ago2 recognizes specific targets through base-pairing of small interfering RNA or microRNA (miRNA) (Shen et al., 2013), resulting in degradation of mRNA (Huntzinger & Izaurralde, 2011) or repression of translation. In the current study, the results showed that the expression of Ago2 was inhibited by CCl4 and TGF–β1 and increased by MDP in vivo and in vitro. Interestingly, the result of LibDock program of Discovery Studio 2017 R2 software showed that MDP bound to the active pocket (ATP pocket) of the Ago2 protein in a folding manner. Further studies showed that silence of Ago2 inhibited the expression of miR-708 in activated LX-2 cells and the inhibitory effects were inhibited by MDP. In contrast, overexpression of Ago2 elevated the expression of miR-708 in activated LX-2 cells. MDP further promote the expression of miR-708 in activated LX-2 cells transfected Ago OE. More importantly, silence of Ago2 could promote the α-SMA and Col. I expression. However, the α-SMA and Col. I expression was increased by MDP in activated LX-2 cells treat with Ago2 siRNA. In contrast, upregulation of Ago2 inhibited the expression of α-SMA and Col. I in activated LX-2 cells. these data demonstrated that MDP regulated the expression of miR-708 via binding Ago2, and thus promoted senescence of activated HSCs.
ZEB1 is most often described as a zinc finger transcription factor and inhibits E-cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis(Larsen et al., 2016; Song et al., 2017). A large number of experimental data indicated that the increase in ZEB1 expression is related to the fibrosis of some organs, such as the lungs and kidneys (Qian et al., 2019; Wang et al., 2018). Furthermore, the results of our previous studies have shown that the expression level of ZEB1 is positively correlated with HSC (Yang et al., 2020). However, the role of ZEB1 in senescence of activated HSCs is still obscure. In current study, we found that the expression of ZEB1 was distinctly upregulated in vivo and in vitro. The result of flow cytometry showed that knockdown of ZEB1 leads to an increase in the proportion of LX-2 cells in the G0/G1 phase. Importantly, the SA-β-gal activity was downregulated by ZEB1 OE and upregulated by ZEB1 siRNA. We also found that overexpression of ZEB1 decreased the expression of p16 and p21 as well as increased the expression of α-SMA and Col. I. In contrast, knockdown of ZEB1 increased the expression of p16 and p21 as well as decreased the expression of α-SMA and Col. I. Additionally, our previous study and target screening have linked the miR‐708 and ZEB1. Our results demonstrated that miR‐708 could directly target ZEB1 mRNA through binding to the 3′‐UTR regions of ZEB1, and thus mRNA and protein expression of ZEB1 in LX‐2 cells were negatively regulated by alteration of miR‐708. It suggested that miR‐708 plays an essential role in liver fibrosis partly through directly targeting ZEB1. The underlying mechanism through which miR‐708 and ZEB1 regulate senescence of HSCs was examined in the subsequent experiment.
It is widely accepted that cell senescence has been widely used to inhibit tumor growth (Collado & Serrano, 2010). Recent research has shown that induction of cellular senescence could also play a crucial regulate role in other non-neoplastic diseases such as liver fibrosis (Huang, Chen, Guo, Chen, Chen & Wang, 2020). Our previous studies have found that p53 plays an important role in the regulation of senescence activation of HSCs (Yang et al., 2019a). Recently, Guo et al. reported that knockdown of p53 could inhibit HSCs senescence and fibrotic degradation (Guo et al., 2021; Jin et al., 2016). A growing number of studies show that the activity of SA-β-gal and expression levels of p21 and p16 could be elevated by p53 plasmid, and thus inducing cell cycle arrest (Okinaga, Ariyoshi, Akifusa & Nishihara, 2013). This study showed that the expression of p53 was decreased in fibrotic liver tissues and activated LX-2 cells. Meanwhile, the results of flow cytometry demonstrated that upregulation of p53 could induce activated HSCs cycle arrest and increase the activity of SA-β-gal and protein level of p16 and p21. However, the activity of SA-β-gal and protein level of p16 and p21 were inhibited by p53 siRNA. Further study confirmed that the expression of α-SMA and Col. I was reduced by PEX-3-p53 and promoted by p53 siRNA. Interestingly, the results revealed that ZEB1could interact with the p53 promoter E‐box, and thus suppress the expression level of p53 in LX-2 cells. In conclusion, above results suggested that MDP could induce activated HSCs senescence via p53-dependant manner to inhibiting liver fibrosis.
In conclusion, present study indicated that MDP induced senescence of activated HSCs to attenuate liver fibrosis (Figure 10). In brief, MDP treatment attenuated the CCl4-induced fibrosis via inducing accumulation of senescent activated HSCs in rats. MDP induced cell cycle arrest and senescence of activated HSCs via Ago2-miR-708-ZEB1-p53 signal pathway in cell model. In summary, our findings could provide a novel mechanism for the antifibrotic effects of MDP.