Val‐Val‐Tyr‐Pro protects against non‐alcoholic steatohepatitis in mice by modulating the gut microbiota and gut‐liver axis activation

Abstract Val‐Val‐Tyr‐Pro (VVYP) peptide is one of the main active components of Globin digest (GD). Our previous studies indicated that VVYP could protect against acetaminophen and carbon tetrachloride‐induced acute liver failure in mice and decrease blood lipid level. However, the effects and underlying mechanisms of VVYP in the treatment of non‐alcoholic steatohepatitis (NASH) have not been discovered. Our present study was designed to investigate the preventive effect of VVYP on NASH and its underlying specific mechanisms. We found that VVYP inhibited the cytotoxicity and lipid accumulation in L‐02 cells that were exposed to a mixture of free fatty acid (FFA). VVYP effectively alleviated the liver injury induced by methionine‐choline‐deficient (MCD) diet, demonstrated by reducing the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST)/triglycerides (TG)/non‐esterified fatty acids (NEFA) and improving liver histology. VVYP decreased expression levels of lipid synthesis‐related genes and reduced levels of the proinflammation cytokines in the liver of mice fed by MCD diet. Moreover, VVYP inhibited the increased level of LPS and reversed the liver mitochondria dysfunction induced by MCD diet. Meanwhile, VVYP significantly increased the abundance of beneficial bacteria such as Eubacteriaceae, coriobacteriacease, Desulfovibrionaceae, S24‐7 and Bacteroidia in high‐fat diet (HFD)‐fed mice, however, VVYP reduced the abundance of Lactobacillus. Moreover, VVYP conferred the protective effect of intestinal barrier via promoting the expression of the mucins and tight junction (TJ)‐associated genes and inhibited subsequent liver inflammatory responses. These results indicated that the protective role of VVYP on NASH is mediated by modulating gut microbiota imbalance and related gut‐liver axis activation. VVYP might be a promising drug candidate for NASH.


| INTRODUC TI ON
Non-alcoholic fatty liver disease (NAFLD), the most common form of adult chronic liver disease with a prevalence of approximately 25% ~ 30% of the worldwide, 1 consists of a broad spectrum of disease ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), causing liver fibrosis, and ultimately cirrhosis with a high risk of hepatocellular carcinoma (HCC). 2 NAFLD pathogenesis is associated with various types of insults that occur simultaneously and may work synergistically including enhanced accumulation of triglycerides, mitochondrial injury, elevated oxidative stress, autophagy and apoptosis imbalance, increased levels of lipotoxicity and liver inflammation. 3,4 Effective therapies for treating and preventing NASH are lacking, a single-targeted drug may not be sufficient to treat NASH which is a metabolic diseases involving multiple factors.
Given the complexity of the physiopathology of NASH, a multifunctional drug with two or more targets may provide a better therapeutic effect against NASH than a single-targeted one.
Stable-isotope studies showed that de novo lipogenesis (DNL) was increased in patients with NAFLD, which contributed to fat acids accumulation within the liver and the progression of NAFLD. 5 The most important pathway that adjusts the initiation of fatty acid biosynthesis in the liver involves activation of sterol regulatory element-binding protein 1 (SREBP-1c), an important transcription factor involved in hepatic lipid synthesis. 6 Overexpression of SREBP-1c in the liver lead to the onset of severe hepatic steatosis because of the increased lipogenesis. The active form of SREBP-1c promotes fatty acid biosynthesis by activating several downstream lipogenic enzymes, such as fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD)-1. 7,8 SCD-1 was responsible for catalysing fatty acid desaturation 9 and FASN played a crucial role in catalysing the synthesis of palmitate (16:0), which was used for both de novo biosynthesis of ceramide and triglyceride synthesis. 10 Therefore, the maintenance of normal levels of lipogenesis-related genes may provide therapeutic benefits in NASH.
Recently, many studies indicated that the progression of NAFLD was associated with the gut-liver axis. 11,12 The concept of gut-liver crosstalk in NAFLD development indicated a connection between increased intestinal epithelial barrier permeability and serum lipopolysaccharide (LPS) level which is a critical driver of hepatic inflammation. [13][14][15] In general , the composition of the intestinal microbiota reflects the diets, anti-biotic and the other environment factors of the host, 16 compelling evidence has demonstrated the gut microbiota played an important role in development of NAFLD to NASH. 17 Lower gut microbial richness and diversity were observed in NASH patients compared to healthy controls. 18 Gut microbiota dysbiosis promoted the influx of harmful substances, including LPS, bacterial DNAs and ethanol, into the liver through systemic circulation of portal vein circulation and accelerated the development of NASH. 19,20 Considerable evidence indicated that chronic inflammation 16 and intestinal barrier 21 played critical roles in metabolic diseases induced by gut microbiota disturbance. The combination of the mucus layers and epithelial tight junctions (TJs) formed a highly integrated barrier system that limited luminal contents contact with the host. 22 Mucins such as the secreted Mucs (Muc-2, Muc-3) and membrane associated (Muc-1) are the major components of the intestinal mucus layer, 23 which is responsible for maintaining the barrier function of the gut and protecting the epithelium from viruses, pathogenic bacteria and noxious agents present in the gastrointestinal tract. 24 The tight junction between epithelial cells is comprised of transmembrane proteins (junctional adhesion molecules, claudins, occludin) and scaffold proteins (zonula occludens-1(ZO-1), zonula occludens-2 (ZO-2), etc) that link the transmembrane proteins to the cytoskeleton, especially the tight junction at the top of the cells plays an important role in the regulation of mucosal permeability. 22 Recent studies reported that C57BL/6 mice fed by MCD diet 25 or HFD 26 impaired intestinal epithelial barrier function by decreasing expression of the TJ proteins in epithelial cells. Dysbiosis of intestinal barrier and ultimately bacterial translocation could trigger profibrogenetic and proinflammatory pathways, finally caused cirrhosis development. 27 Thus, targeting gut-liver crosstalk may be an effective approach to mitigate the development of NASH.
Globin digest (GD) is an edible oligopeptide mixture which is hydrolysed of porcine haemoglobin by acid protease. 28 GD has been used as a specific health food in Japan and it can improve hyperlipidaemia and hyperglycaemia in humans. 29 Moreover, GD inhibited the increase in serum transaminase activity and showed hepatoprotective effects in liver injury induced by galactosamine (GalN) in Sprague Dawley (SD) rats. 30 Val-Val-Tyr-Pro (VVYP) is one of the main active components of GD. It could promote the activity of triacylglycerol lipase and reduce blood triglyceride levels, 31,32 the lipid-lowering ability of VVYP is 7000 times than that of GD. 28 In our previous studies, VVYP had obvious protective effect against acetaminophen and carbon tetrachloride-induced acute liver failure in mice. 33 However, there is still little known about VVYP for the treatment of NASH. Therefore, the present study is aimed to investigate the protective effects of VVYP on models of NASH induced by MCD and HFD and its mechanisms of these effects.

| Animals
Forty-eight male 8-week-old C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. All animals were housed in a specific-pathogen-free barrier facility with controlled conditions (19-23°C, humidity 60%, 12-h light/dark cycle) and had free access to food and water. After 3 days of adaptive adaptation, the initial body weight was recorded. MCD diet was added to C57BL/6 mice to establish NASH mode for 2 weeks, 28 mice were randomly divided into four groups (n = 7 per group) as follows: (a) a control group (CTL) fed with a normal diet (b) a MCD group fed with a MCD diet (No. 11002900039337, Beijing Keao Xieli Feed Co.,Ltd.); (c) a VVYP 2 group fed with MCD diet and treated with VVYP (2 mg/kg daily by oral gavage); and (d) a VVYP 10 group fed with MCD diet and treated with VVYP (10 mg/kg daily by oral gavage). HFD was used to establish NASH mice model up to 8 week, 20 C57BL/6 mice were divided at random into four groups: CTL group, model(M) group, model +VVYP 10 mg/kg (M-VVYP), VVYP 10 mg/kg (VVYP). CTL group and VVYP group were fed with normal diet, M and M-VVYP group were fed with HFD. The body weights were recorded every 2 days. All animal experiments on mice were conducted according to the guidelines approved by the Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine (approval number JZLLSC 2018-0053).

| Oil red O staining for detecting lipid deposition in L-02 cells
Cells were processed by oil red O (Solarbio) staining to assess lipid content. The cells were washed three times with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 10 minutes, washed twice with ddH 2 O to remove paraformaldehyde. After once wash in 60% isopropanol, the cells were stained with oil red O for 10 minutes at 37°C.
Sixty per cent isopropanol were then added to each well and adjusted colour under the microscope. After three washes in ddH 2 O, the cells were synchronized with 60% isopropanol and then dyed with haematoxylin for 30 seconds. Finally, hydrochloric acid alcohol was used to differentiate the background for 3 seconds before microscopic examination. The results were statistically analysed using Image J software.

| Animal sacrifice and sample collection
After experimental period, faecal samples were collected from all mice upon defecation and stored at −80°C for further analysis. Mice were fasted for 12 hours, their final body weights were recorded. And then mice were killed. Freshly dissected liver was washed out in ice-cold physiological saline, and dried with filter paper. The liver was then weighed (in grams), the blood and liver tissues of all groups were collected for the following analysis. All serum samples were stored in a −80°C freezer. Liver and small intestine samples were snap-frozen in liquid nitrogen or kept in a −80°C freezer for further procedures.

| Biochemical analysis
Blood was collected at the end of study from each experimental animals and allowed to stand for 2 hours to clot. And then the blood samples were centrifuged (4500 rpm, 20 minutes) for serum separation. The biochemical indicators of mice in each group were measured using an auto-analyser (Hitachi). We determined ALT, AST, total cholesterol (CHOI), TG, NEFA.  Finally, the slices were sealed by cover glass and observed using a microscope (LEICA DM 1000). The lipid accumulation was statistically analysed using Image J software.
Then the sections were incubated with DAPI (1:1000) for 3 minutes and imaged using a fluorescent microscope (LEICA DMI300B). Visual fields were randomly selected and analysed with Image J software.

| Transmission electron microscopy (TEM) analysis of liver tissue
The liver specimens were fixed in 2.5% glutaraldehyde overnight at 4°C for 24 hours, washed three times in 0.1 mol/L phosphate buffer (pH 7.4) and then post-fixed in 1% osmium acid solution for 2 hours. Followed by secondary fixation, the specimens were washed briefly as mentioned above. Graded ethanol series dehydration and embedded in epoxy resin. Ultrathin sections (60-80 nm) were then cut, ultramicrostructure related to lipid droplets and mitochondrial morphology was examined with a transmission electron microscope (Hitachi, HT7700-SS).

| RNA extraction and real-time PCR analysis
The total RNA was extracted from pulverized frozen liver and small intestinal tissues using Trizol (CWBIO) according to the manufacturer's protocols. Then total RNA purity and content were measured by a spectrophotometer, total RNA (1 μg) from liver tissues and small intestine samples was reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientifi, USA). The mRNA expression levels were assessed by qRT-PCR using the SYBY Green PCR Master Mix (Thermo Fisher Scientific) and the ABI 7500 Real-time PCR system (Applied Biosystems). The relative expression of each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase (GADPH). Primer sequences used are listed in Table 1.

| Western blot analysis
Liver samples were randomly selected from each group, and total proteins were extracted from approximately 50 mg of liver with 500 μl radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, USA) containing phosphatase and protease inhibitors. Next, the mixture was centrifuged at 12 000 g for 5 minutes at 4°C and the supernatant was extracted. The protein content was estimated using a BCA Protein Assay Kit (Cwbiotech). Equal amount of proteins were separated on 7.5% or 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and sequentially transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocked with 5% skimmed milk powder for 2 hours, the membranes were incubated with the specific antibodies for FASN (1:800; Abcam), SCD-1 (1:1000; Cell Signaling Technology), SREBP-1c (1:800; Affinity Biosciences) and β-actin(1:1000; Abcam) at 4°C overnight. The next day, the membranes were washed and incubated with secondary antibodies for 1 hours at room temperature, goat anti-rabbit (N10429) IgG-HRP and the goat antimouse IgG-HRP (N10326) secondary antibodies were purchased from TransGen Biotech.
The membranes was placed in an electrochemiluminescence (ECL) Western blotting detection system (Bio-Rad), ECL detection reagent (Cwbiotech) was added, exposure and visualization were performed, and grey values of each band were analysed using Image J software.

| Statistical analysis
Statistical analysis was performed using GraphPad 8.0 statistical package (GraphPad Software) and the graphs were also generated with Prism. Results were expressed as the means ± SEM. Two-tailed Student's t-test was performed to measure the difference between two sets of data. The variance of three or more groups was determined by one-way ANOVA and the Bonferroni post-hoc analysis.
Kruskal-Wallis and Wilcoxon tests were used to perform LEfSe analysis associated with relative abundance of gut microbiota and the threshold on linear discriminant analysis (LDA) score was high than 3. Others were displayed using QIIME1 and R packages (V.2.15.3).
For all statistical tests, P values < .05 were considered to indicate statistically significant.

| VVYP improved cell viability and steatosis of L-02 cells induced by FFA
Previous studies have reported that treatment of L-02 cells with

| VVYP restored the diversity, richness of the gut microbiota in HFD-fed mice
Since intestinal microbial dysregulation is another factor in NASH and liver-gut axis plays a key role in various diseases such as obe-

| D ISCUSS I ON
NASH is a more severe form of NAFLD, it encompasses a range of distinct pathological features in the liver, including hepatocellular ballooning, hepatocyte injury, liver inflammation, steatosis and fibrosis and can further progress to liver cirrhosis and HCC. [37][38][39] Our previous study revealed that VVYP could reduce serum AST and ALT, improve the pathological state of liver tissue, and protect F I G U R E 4 VVYP improved intestinal barrier function in the small intestine.
A-E, Relative mRNA levels of the mucins (Muc-1, Muc-2) and TJ (tight junction)associated genes (claudin-1, occluding-1, ZO-1). F, Evaluation of representative small intestine histology by ZO-1 immunofluorescence (scale bar, 200 μm). F(a) Control group; F(b)MCD group; F(c) VVYP 2 mg/kg (VVYP-D group); F(d) VVYP 10 mg/kg (VVYP-H group). Each bar represents the mean ± SEM. #P < .05, ##P < .01, ###P < .001 vs Control; **P < .01, ***P < .001 vs MCD against liver injury caused by carbon tetrachloride or paracetamol in mice. 33 Elevated ALT level is a common marker of progressive NAFLD or NASH and has been correlated with insulin resistance and severity of hepatic steatosis. 40 In this studies, we found VVYP ameliorated MCD diet-induced liver injuries via reducing ALT/AST/ TG/NEFA levels, promoting lipid deposition, regulating expressions The gut-liver axis, a bidirectional interplay between intestinal and hepatic diseases, was recently under intense investigation as a critical factor in NAFLD progression. 11,12 It is well known that the abundance of gut microbiota plays a crucial role in maintaining the stability and efficiency of dynamic balance of micro-ecosystem. 60 Targeting the intestinal microbiota has been shown to be beneficial for the therapy of NAFLD. 3 The Bacteroidetes was decreased in HFD diet-fed mice, whereas the Firmicutes was increased, leading to a marked higher FB ratio.  25 It is well known that another important factor contributing to changes in the liver expression of genes is the interplay between gut microbiota and bile acid (BA) metabolism, protective gut microbiota(Desulfovibrionaceae and Coriobacteriaceae) associates with increased specific secondary BAs, which likely inhibit lipogenic pathways and enhance bile flow in the liver.. 79 On the other hand, short chain fatty acids (SCFAs) derived from gut microbiota are involved in the pathogenesis of NASH, SCFA-producing bacteria, such as S24-7 80 and Bacteroidaceae, 81   In the development of NAFLD, the endotoxin-TLR4-NF-κB pathway is considered as the critical factor to link intestinal microbiota dysbiosis and liver inflammation. 94 Interestingly, we also found MCD diet up-regulated LPS levels in the liver tissues of mice in MCD group while VVYP significantly reversed these effects. Elevated liver localization of LPS was recently displayed in the patients with NAFLD and experimental NAFLD, which was connected with liver inflammation via a transport of TLR4-mediated pathway. 95 In our study, VVYP inhibited the increased levels of TNF-α, IL-6 and LPS induced by MCD diet, which was possible linked to dysbiosis-mediated activation of the TLR4-NF-κB signalling pathway.
In conclusion, we have demonstrated that VVYP inhibited the cytotoxicity and lipid accumulation in L-02 cells exposed to FFA.
Noticeably, VVYP could protect against NASH modulate the gut microbiota imbalance by strongly enhancing the abundance of

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are in this paper.