Polydatin inhibits ZEB1‐invoked epithelial‐mesenchymal transition in fructose‐induced liver fibrosis

Abstract High fructose intake is a risk factor for liver fibrosis. Polydatin is a main constituent of the rhizome of Polygonum cuspidatum, which has been used in traditional Chinese medicine to treat liver fibrosis. However, the underlying mechanisms of fructose‐driven liver fibrosis as well as the actions of polydatin are not fully understood. In this study, fructose was found to promote zinc finger E‐box binding homeobox 1 (ZEB1) nuclear translocation, decrease microRNA‐203 (miR‐203) expression, increase survivin, activate transforming growth factor β1 (TGF‐β1)/Smad signalling, down‐regulate E‐cadherin, and up‐regulate fibroblast specific protein 1 (FSP1), vimentin, N‐cadherin and collagen I (COL1A1) in rat livers and BRL‐3A cells, in parallel with fructose‐induced liver fibrosis. Furthermore, ZEB1 nuclear translocation‐mediated miR‐203 low‐expression was found to target survivin to activate TGF‐β1/Smad signalling, causing the EMT in fructose‐exposed BRL‐3A cells. Polydatin antagonized ZEB1 nuclear translocation to up‐regulate miR‐203, subsequently blocked survivin‐activated TGF‐β1/Smad signalling, which were consistent with its protection against fructose‐induced EMT and liver fibrosis. These results suggest that ZEB1 nuclear translocation may play an essential role in fructose‐induced EMT in liver fibrosis by targeting survivin to activate TGF‐β1/Smad signalling. The suppression of ZEB1 nuclear translocation by polydatin may be a novel strategy for attenuating the EMT in liver fibrosis associated with high fructose diet.

subsequently recruit their co-mediator Smad4 to nuclear translocation, and then regulate hepatocytic phenotype and function. 6,7 Survivin as a crucial inhibitor of apoptosis promotes bile duct ligation-induced rat liver fibrosis, 8 positively regulates TGF-β1 expression in adenoid cystic carcinoma cases 9 and provokes the EMT in glioblastoma. 10 Recently, excess fructose consumption is reported to increase collagen content of liver parenchyma in rodents. 3 In this regard, survivin may activate TGF-β1/Smad signalling to promote fructose-caused the EMT process in liver fibrosis.
Of note, survivin/baculoviral IAP repeat containing 5 (BIRC5) is identified as a target gene of microRNA-203 (miR-203). 11   inhibits the EMT of ovarian cancer cell line by targeting BIRC5 and blocking TGF-β signal pathway. 12 Moreover, miR-203 expression is decreased in hepatitis C virus core protein-simulated human hepatocyte cell line. 13 However, it remains unknown how fructose alters miR-203 expression and whether this event affects survivin-activated TGF-β1/Smad signalling in the process of the EMT in liver fibrosis.
Zinc finger E-box binding homeobox 1 (ZEB1) as a transcription factor suppresses the transcription of miR-203 in human cancer cells. 14,15 It up-regulates survivin gene expression and inhibits E-cadherin nuclear re-expression in thyroid papillary carcinoma cell line. 16 In addition, ZEB1 induces TGF-β1 expression in dimethylnitrosamine-induced liver fibrosis of rats. 17 However, it remains unclear whether ZEB1 nuclear translocation mediates miR-203 deregulation targeting survivin, which is required for TGF-β1/smad signalling activation in fructose-driven hepatocyte EMT.
Polydatin is a major active ingredient derived from the rhizome of Polygonum cuspidatum Siebold & Zucc, which alleviates liver fibrosis in patients and experimental animals. [18][19][20] Previous studies have shown that polydatin down-regulates TGF-β1, collagen and p-Smad3 in diet-induced fibrotic liver of mice, 21 up-regulates E-cadherin and represses radiation-induced EMT in lung tissues of mice. 22 However, whether polydatin inhibits ZEB1 nuclear translocation to augment miR-203 and block survivin-activated TGF-β1/Smad signalling in the alleviation of fructose-induced EMT and liver fibrosis remains mostly unexplored.
In this study, we found that ZEB1 nuclear translocation was sufficient to decrease miR-203 expression, this new action as a suitable alternative for targeting survivin-activated TGF-β1/Smad signalling in fructose-driven EMT and liver fibrosis. Additionally, we found that polydatin suppressed ZEB1 nuclear translocation to increase miR-203 expression, and then down-regulated survivin to block TGF-β1/Smad signalling activation, resulting in the alleviation of fructose-caused EMT and liver fibrosis.
Invitrogen TM TRIzol reagent was obtained from Thermo Fisher Scientific. The reverse transcription system kit and ChamQ SYBR qPCR master mix were got from Vazyme Biotechnology Co., Ltd.
The dual-luciferase reporter assay system kit was obtained from Promega Corporation. MiR-203 mimic, ZEB1 siRNA, survivin siRNA, TGF-β1 siRNA, the respective negative control and GP-miRGL0 reporter vector listed in Table 1 were provided by GenePharma Co., Ltd. The following antibodies were purchased from commercial sources: anti-survivin (sc-10811), anti-Histone H3 (sc-517576), anti-TGF-β1 (sc-146), anti-p-Smad3 (Ser208, sc-130218), anti-Smad3  Male Sprague-Dawley rats (180-220 g) were provided from Experimental Animal Center of Zhejiang Province (Hangzhou, China; SCXK 2014-0001). Each rat was given the drinking water or 100 mL drinking water containing 10% fructose (wt/vol) for 6 weeks. 23,24 Then, rats were randomly divided into six groups (n = 8/group): (a) normal control rats and (b) fructose control rats, which received saline; (c-e) 7.5, 15 and 30 mg/kg polydatin-and fructose-treated rats; (f) 4 mg/kg pioglitazone (positive drug)-and fructose-treated rats orally for the following 11 weeks. Doses of polydatin and pioglitazone used in this animal experiments were selected based on our previous studies 23,24 and other reports. 21,22 A schematic representation of the experiments performed and timeline with rats was provided in Figure 1A.

| Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)
OGTT and ITT were performed as previously described. 25 Briefly, for OGTT, rats received 1.5 g/kg glucose orally; for ITT, rats were given 0.3 IU/kg insulin intraperitoneally. Then, blood samples were collected from the rat tail veins to test glucose levels with blood glucose metre at 0, 30, 60, 90 and 120 minutes after the treatment of glucose or insulin, respectively.

| Serum and tissue collection
At the end of the animal experiments, rats were anaesthetized with 50 mg/kg sodium pentobarbital to collect blood samples from rat carotid artery as well as liver tissues. Blood samples were kept at room temperature for 1 hour and then centrifuged (3000 × g, 10 min) to obtain the serum samples for biochemical assays. Some liver samples were fixed with paraformaldehyde for histological study, while the others were stored at −80°C for protein or RNA extraction, biochemical assay, respectively.

| Histological study
Liver tissues fixed with paraformaldehyde were embedded in paraffin. Then, liver specimens (4 μm-thick) were cut and stained with Masson trichrome and Sirius red solution, respectively. These sections were observed and photographed under an optical microscope (Nikon Eclipse Ti-SR, Nikon), respectively.

| Gene expression analysis
Total RNA was extracted from rat liver tissues and the cultured BRL-3A cells using TRIzol reagent for analysis of survivin mRNA lev-  Table 1 were provided by Generay Biotechnology Co., Ltd.

| Western blot analysis
The whole, nuclear or cytoplasm proteins from rat liver tissues or the cultured BRL-3A cells were extracted using lysis buffer, followed by

| Statistical analysis
Data are presented as mean ± standard error of the mean (SEM).
Comparisons between two groups were performed by Student's t test. ANOVA with further analysed by post hoc Dunnett's test was used for comparison between more than two different groups. P < .05 was considered to be significant.

| Polydatin ameliorates liver fibrosis in fructosefed rats with metabolic syndrome
First, we examined whether polydatin ameliorated liver fibrosis in fructose-induced metabolic syndrome of rats. The data from biochemical analysis showed that polydatin significantly decreased serum concentrations of insulin ( Figure 1B), TG, IL-1β and TNF-α (Table 2), and alleviated insulin resistance in OGTT and ITT ( Figure 1C,D) in fructose-fed rats. Simultaneously, polydatin remarkably alleviated liver histologic changes including slight thickening of the central venous wall, perisinusoidal or portal/peripotal fibrosis and reduced fibrotic area in fructose-fed rats ( Figure 1E). In parallel, it significantly reduced serum levels of hyaluronic acid, laminin and type III procollagen, as well as liver levels of hyaluronic acid and hydroxyproline in this animal model (Table 2). Accordantly, polydatin remarkably decreased serum activities of ALT and AST in fructosefed rats ( Table 2). Pioglitazone exerted similar effects in this animal model ( Table 2 and Figure 1). These data suggest that polydatin and pioglitazone alleviate liver fibrosis to recover liver function in fructose-fed rats with metabolic syndrome.

| Polydatin attenuates fructose-induced EMT in rat liver fibrosis and BRL-3A cells
EMT plays an important role in liver fibrosis. 5 Next, we investigated whether polydatin attenuated fructose-induced EMT in liver fibrosis. As noted previously, polydatin effectively de-

| Polydatin augments miR-203 targeting survivin to inhibit TGF-β1/Smad signalling activation
Survivin is reported to positively regulate TGF-β1 expression in adenoid cystic carcinoma cases 9 and provoke the EMT in glioblastoma. 11 It is worth noticing that miR-203 inhibits the EMT of ovarian cancer cell line by targeting survivin/BIRC5 and blocking TGF-β signal pathway. 12 miR-203 is low-expression in carbon tetrachloride-induced rat liver fibrosis. 26 Therefore, we validated miR-203 expression change in fructose-caused liver fibrosis. In this study, we found that fructose significantly decreased miR-203 expression levels in rat livers ( Figure 4D) and BRL-3A cells ( Figure 4E).
To investigate whether miR-203 changed survivin expression, we carried out the luciferase assay. The results showed that the lu- Furthermore, we observed that polydatin significantly increased miR-203 expression ( Figure 4D,E), and down-regulated survivin mRNA and protein levels ( Figure 4A,B) in fructose-fed rat livers and fructose-exposed BRL-3A cells. Polydatin reversed the effect of survivin siRNA to significantly increase miR-203 expression in fructose-exposed BRL-3A cells ( Figure 4G). Of note, polydatin markedly decreased survivin mRNA and protein levels (24 h) in miR-203 mimic-transfected BRL-3A cells under fructose exposure ( Figure 4H,I). In addition, polydatin down-regulated TGF-β1 protein levels (24 h) in fructose-exposed BRL-3A cells transfected with survivin siRNA ( Figure 4C). Pioglitazone had similar effects in these animals and cell models (Figure 4). These results demonstrate that polydatin and pioglitazone augment miR-203 to down-regulate survivin, then inhibit TGF-β1/Smad signalling activation.  Figure 5C). In fact, fructose intake increased the nuclear ZEB1 protein levels and decreased the cytoplasm ZEB1 protein levels in rat livers ( Figure 5D,E). To investigate the role of ZEB1 in the change of miR-203 expression, we transfected ZEB1 siRNA into BRL-3A cells and found that ZEB1 siRNA significantly induced high-expression of miR-203 (12 h) in fructose-exposed BRL-3A cells ( Figure 5H). While miR-203 mimic was unable to affect the nuclear ZEB1 protein levels (1 h) in fructose-exposed BRL-3A cells ( Figure 5I). These data indicate that fructose may cause ZEB1 nuclear translocation to reduce miR-203 expression, leading to survivin-mediated the activation of TGF-β1/Smad signalling in EMT and liver fibrosis.

| Polydatin inhibits ZEB1 nuclear translocation to enhance miR-203 expression
More importantly, we found that polydatin was able to decrease the nuclear ZEB1 protein levels and increase cytoplasm ZEB1 protein levels in fructose-fed rat livers ( Figure 5D,E) and fructose-simulated BRL-3A cells ( Figure 5F,G). Polydatin increased miR-203 expression (12 h) in BRL-3A cells transfected with ZEB1 siRNA under fructose exposure condition ( Figure 5H). It was able to significantly decrease nuclear ZEB1 protein levels (1 h) in miR-203 mimic-transfected BRL-3A cells co-cultured with fructose ( Figure 5I). Pioglitazone had similar effects in these animals and cell models ( Figure 5). These results suggest that polydatin and pioglitazone may inhibit ZEB1 nuclear translocation to enhance miR-203 expression and then block survivin-activated TGF-β1/Smad signalling in fructose-induced EMT and liver fibrosis.

| D ISCUSS I ON
Clinically, excess fructose consumption is associated with the development of liver fibrosis. 2 Polydatin is a main constituent in P cuspidatum, which has potential utility in the treatment of liver fibrosis in patients. 20 To the best of our knowledge, we firstly find that ZEB1 nuclear translocation plays an essential role in fructose-induced EMT in liver fibrosis by targeting survivin to activate TGF-β1/Smad signalling. Moreover, polydatin represses ZEB1 nuclear translocation to increase miR-203 expression, subsequently, block survivin-activated TGF-β1/Smad signalling, attenuating fructose-induced EMT in liver fibrosis ( Figure 5J).
Generally, activated hepatic stellate cells are considered to be the main source of the extracellular matrix. 27 While, hepatocytes simulated by TGF-β1 not only lost the epithelial phenotype with the decrease of E-cadherin, and the increase of N-cadherin, vimentin and FSP1, but also produce the collagen, 4,6,7 indicating that these hepatocytes undergoing EMT induced by TGF-β1 could be another source of the extracellular matrix. Of note, long-term fructose intake causes massive collagen deposition of liver parenchyma in the cynomolgus monkeys. 3 In this study, we showed that fructose triggered EMT in hepatocytes, consistently, it caused rat liver fibrosis. These observations further demonstrated that fructose-induced hepatocyte EMT, at least to some extent, promoted liver fibrosis process.
In this study, we observed the decrease of E-cadherin with a relocation of E-cadherin from the membrane to the cytoplasm in fructose-exposed BRL-3A cells. β-catenin is reported to guide E-cadherin localization to the cell membrane in Madin-Darby canine kidney cells. 28 While, excessive fructose intake decreases β-catenin protein levels in fibrotic livers of mice. 29 Thus, we speculated that fructose-induced β-catenin reduction may obstruct the localization process of E-cadherin from the cytoplasm to the cell membrane, which may cause a relocation of E-cadherin from the membrane to the cytoplasm in fructose-exposed BRL-3A cells.
Of note, survivin positively regulates TGF-β1 in human adenoid cystic carcinoma cell line 9 and promotes the EMT occurrence with E-cadherin low-expression in glioblastoma. 10 In addition, the activation of TGF-β1/Smad signalling is detected in cirrhotic liver of patients 30 and carbon monoxide-induced liver fibrosis of mice. 5 In this study, fructose-induced survivin over-expression and TGF-β1/Smad signalling activation were also observed in rat livers and BRL-3A cells.
Furthermore, we found that fructose-induced survivin over-expression provoked the activation of TGF-β1/Smad signalling to develop the EMT, causing liver fibrosis. Therefore, we focused on the regulation of survivin in fructose-induced EMT in liver fibrosis. In relation to this, it is worth noting that miR-203 low-expression decreases E-cadherin in ovarian cancer cells line through a survivin-dependent manner. 12 We observed miR-203 low-expression in the animal and cell models, being consistent with these reports in carbon tetrachloride-induced fibrotic liver of rodents, 26 arecoline-induced fibrotic oral submucous of patients 31 and cirrhotic livers of patients. 32 We also showed that survivin was a target gene of miR-203 in BRL-3A cells. Importantly, miR-203 up-regulation nearly abrogated survivin over-expression in fructose-exposed BRL-3A cells. These results suggest that fructose-induced miR-203 low-expression may target survivin to activate TGF-β1/Smad signalling, causing the EMT.
Transcription factor ZEB1 suppresses miR-203 expression and then activates cancer cell epithelial differentiation. 14 High ZEB1 expression is observed in hepatocellular carcinoma patients with or without cirrhosis. 33 In this study, we found that fructose increased nuclear ZEB1 protein levels in the animal and cell models. This fruc-  34 Our previous study found that fructose up-regulated p-STAT3 in rat liver fibrosis. 35 Therefore, we speculated that fructose-induced up-regulation of p-STAT3 may transfer into the nucleus to recruit on ZEB1 promoter, causing ZEB1 nuclear translocation in rat liver fibrosis. However, the precise molecular mechanism by which fructose induces ZEB1 nuclear translocation needs further study.
Polydatin is reported to down-regulate TGF-β1 in unilateral ureter obstruction-induced fibrotic kidney of rats 36 and radiation-induced fibrotic lung of mice. 22 It also inhibits TGF-β1 and collagen, reduces TGF-β1-induced EMT in human alveolar epithelium A549 cells 37 and protects against methionine-choline deficient diet-induced mouse liver fibrosis. 21 Pioglitazone decreases hepatic TGF-β1 and COL1A1 in non-alcoholic steatohepatitis of mice. 38,39 In this study, polydatin  Figure 5J).

F I G U R E 5
Polydatin inhibits ZEB1 nuclear translocation to enhance miR-203 expression in fructose-exposed BRL-3A cells. (A) Images of fructose-exposed BRL-3A cells labelled with ZEB1 (red) at the indicated time points. (B) Western blot analysis of the nuclear ZEB1 protein levels at the indicated time points. (C) qRT-PCR analysis of miR-203 expression levels at the indicated time points. Western blot analysis of the nuclear and cytoplasm ZEB1 protein levels in rat livers (D and E) and BRL-3A cells (2 h) (F and G). (H) qRT-PCR analysis for expression of miR-203 (12 h) in transfected with 50 nM ZEB1 siRNA or NC BRL-3A cells treated with fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. (I) Nuclear ZEB1 protein levels in transfected with 50 nM miR-203 mimic or NC BRL-3A cells exposed to fructose in the presence or absence of 40 μM polydatin or 10 μM pioglitazone. (J) The mechanisms by which polydatin prevents fructoseinduced hepatocyte EMT in liver fibrosis. Histone H3 or Lamin A was as internal control for nuclear ZEB1. β-actin or GAPDH was as internal control for cytoplasm ZEB1. U6 was as internal control for miR-203. Each value is shown as mean ± SEM (n = 4-6). # P < .05, ## P < .01, ### P < .001 compared with the normal control; *P < .05, **P < .01, ***P < .001 compared with the fructose control; $ P < .05, $$ P < .01 compared with the fructose-negative control Therefore, the ability of polydatin and pioglitazone to inhibit ZEB1 nuclear translocation and increase miR-203 expression is the key to attenuate the EMT in the protection against liver fibrosis associated with high fructose intake through the blockade of survivin-mediated TGF-β1/Smad signalling activation.
In conclusion, this study demonstrates that fructose causes ZEB1 nuclear translocation to decrease miR-203 expression, and then target survivin to activate TGF-β1/Smad signalling, developing the EMT in liver fibrosis. ZEB1 nuclear translocation inhibition with high miR-203 expression may be the predictor of good prognosis in patients with liver fibrosis. Polydatin protects against fructose-induced hepatocyte EMT by suppressing ZEB1 nuclear translocation to up-regulate miR-203 expression and block survivin-activated TGF-β1/Smad signalling, exhibiting the potential anti-liver fibrosis activity. The present study also supports that the blockade of ZEB1 nuclear translocation by polydatin is a novel strategy for attenuating EMT in liver fibrosis associated with high fructose consumption.

This work was sponsored by Grants from National Key R&D
Program of China (2019YFC1711000), and National Natural Science Foundation of China (No. 81573667).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the finding of this study are available from the corresponding author upon reasonable request.