Salvianic acid A alleviates chronic alcoholic liver disease by inhibiting HMGB1 translocation via down‐regulating BRD4

Abstract Alcoholic liver disease (ALD) is the major cause of chronic liver disease and a global health concern. ALD pathogenesis is initiated with liver steatosis, and ALD can progress to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Salvianic acid A (SAA) is a phenolic acid component of Danshen, a Chinese herbal medicine with possible hepatoprotective properties. The purpose of this study was to investigate the effect of SAA on chronic alcoholic liver injury and its molecular mechanism. We found that SAA significantly inhibited alcohol‐induced liver injury and ameliorated ethanol‐induced hepatic inflammation. These protective effects of SAA were likely carried out through its suppression of the BRD4/HMGB1 signalling pathway, because SAA treatment largely diminished alcohol‐induced BRD4 expression and HMGB1 nuclear translocation and release. Importantly, BRD4 knockdown prevented ethanol‐induced HMGB1 release and inflammatory cytokine production in AML‐12 cells. Similarly, alcohol‐induced pro‐inflammatory cytokines were blocked by HMGB1 siRNA. Collectively, our results reveal that activation of the BRD4/HMGB1 pathway is involved in ALD pathogenesis. Therefore, manipulation of the BRD4/HMGB1 pathway through strategies such as SAA treatment holds great therapeutic potential for chronic alcoholic liver disease therapy.

fibrosis and cirrhosis. 2 Alcohol can increase metabolic pressure and causes oxidative stress, lipid peroxidation and inflammation damage to the liver in 10%-40% of heavy drinkers. 3 Many studies, including ours, have confirmed that inflammation is a pivotal event in several types of liver injury, including ALD. 4,5 Therefore, an in-depth understanding of the mechanism of alcoholic liver inflammation may be clinically significant for the prevention and treatment of alcoholic liver disease.
BRD4, a member of the bromo and extraterminal (BET) family of genes, functions as a transcriptional coactivator through its bromodomain to carry out various pathophysiological activities. 6,7 Accumulating evidence indicates that BRD4 is a positive regulator that promotes inflammatory responses. For example, BRD4 inhibition attenuated the inflammatory response in microglia and facilitated recovery after spinal cord injury in rats. 8 In addition, BRD4 suppression alleviated cerebral ischaemia-induced brain injury via inhibiting inflammatory activities. 9 Mechanistically, BRD4 regulates expression of the inflammatory gene enhancer RNA (eRNA) and participates in its synthesis. 10,11 However, whether BRD4 is involved in regulating alcohol-induced inflammation during ALD is unclear.
High-mobility group box protein 1 (HMGB1), a highly conserved multifunctional nuclear protein, is related to regulating gene transcription and maintaining nucleosome structure. 12 Recent studies have shown that HMGB1 is a highly conserved multifunctional nuclear protein; however, nuclear HMGB1 is released into the extracellular space during inflammatory responses. [13][14][15] Acting as an inflammatory cytokine itself, HMGB1 suppression and translocation blockade were reported to protect against non-alcoholic steatohepatitis in our previous study. 16 More importantly, HMGB1 was recently reported to be an immediate object of BRD4 in osteoarthritis. 17 Therefore, we suggest that the BRD4/HMGB1 pathway is involved in the regulation of ALD pathogenesis and that the BRD4/ HMGB1 pathway represents a potential target for anti-inflammatory therapy in ALD.
Besides abstinence from alcohol and liver transplantation, there are no efficient therapies to prevent the pathogenesis of ALD. 18 In the absence of a reliable liver-protective drug in modern medicine, many herbal extracts and natural products have been found to be possible drugs to treat various chronic liver diseases with relatively high efficiency and low toxicity. 19 Danshen, a water-soluble bioactive distilled extract derived from the dried root of Salvia miltiorrhiza Radix, has been widely used in many Asian countries over thousands of years for the treatment of heart diseases and cerebrovascular diseases. 20,21 Salvianic acid A (SAA; Figure 1) is an abundant and structurally representative water-soluble active component of Danshen. 22 Recent research has suggested that SAA exhibits liver-protective effects in the treatment ALD 23,24 ; however, the underlying molecular mechanisms of these effects have not been reported.
In the recent research, we validated the protective effects of SAA on chronic alcoholic liver disease using a well-established rat ALD model and discovered that SAA exerts its liver-protective effects through, at least partially, suppressing alcohol-induced activation of the BRD4/HMGB1 inflammatory pathway in the rat liver.

| Chemicals
SAA (purity > 98%) was purchased from Guizhou Jingfeng Injection Co., Ltd. (Guizhou, China). MEM and foetal bovine serum were purchased from Life Technologies (Carlsbad, CA, USA). All biochemical indicator kits and other chemicals were commercially available.

| Animals and treatments
Male Wistar rats weighing 180 to 220 g (6 weeks old) were obtained from the Experimental Animal Center of Dalian Medical University (SCXK 2008-0002). All animal maintenance and treatment procedures were in concordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and had been authorized by the Institutional Animal Committee of Dalian Medical University. All animals with standard chow and water ad libitum were housed under standard laboratory conditions for approximately one week. The rats were nourished this way: (1) control, (2) control + SAA (40 mg/kg/d), (3) ethanol, (4) ethanol + SAA (20 mg/ kg/d) and (5) ethanol + SAA (40 mg/kg/d). Rats in the SAA group received SAA (20 and 40 mg/kg/d) by intragastric administration every day, and the same volume of normal saline was administered to rats in the control group. After exposure to the Lieber-DeCarli ethanol diet 25 for 8 weeks, all the rats were killed at the end of the experiment. Blood samples were obtained from the abdominal aorta, and liver tissues were gathered and snap-frozen on liquid nitrogen before being stored at −80°C until use.

| Biochemical assays
Serum was separated from the blood samples by centrifugation at 3000 rpm for 15 minutes. The serum levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using commercial kits (Nanjing F I G U R E 1 Chemical structure of salvianic acid A Jiancheng Bioengineering Institute, China) following the manufacturer's instructions. Liver tissues were stained with haematoxylin and eosin (H&E) and Oil Red O staining that was used to recognize tissue lipidosis.

| Liver histomorphology
Nile red solution (1 μg/mL), a selective fluorescent stain, was used to determine intracellular lipid droplets. Lipid-bound Nile red was assayed with a fluorescence microscope.

| Cell culture and treatment
The AML-12 mouse hepatocyte cell line was purchased from American Type Culture Collection (Rockefeller, USA). The cells were treated with 10 μmol/L SAA for 6 hours, followed by exposure to 100 mmol/L ethanol for 24 hours.

| Immunofluorescence staining
After fixed in 4% formaldehyde, the 1% bovine serum albumin in 0.1% Triton X-100 was used to block cells that were hatched with primary antibodies at 4°C overnight. The cells were hatched with the appropriate Cy3-or FITC-conjugated secondary antibodies for 2 hours at room temperature and then counterstained with DAPI

| Preparation of nuclear and cytosolic fractions
Nuclear and cytosol liver extracts were prepared with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Shanghai, China) in accordance with the manufacturer's protocols. All steps were carried out on ice or at 4°C unless stated otherwise.

| Western blot analysis
Equal amounts of protein were resolved by 8%-12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat dry milk in Tris-buffered saline, the membranes were incubated overnight with primary antibodies against BRD4, HMGB1, IL-1β, TLR4 and β-actin. Specific bands were detected by enhanced chemiluminescence (ECL) method using a Bio Spectrum gel imaging system (UVP, USA).

| RNA isolation and real-time PCR
After treatment, total RNA was extracted from liver samples and AML-12 cells using TRIzol reagent (Invitrogen, CA, USA) according to

| RNA silencing experiment
AML-12 cells were seeded onto 6-well plates at a density of 1 × 105 cells per well. When the cells had reached 50%-60% confluence, they were transfected with 100 nmol/L BRD4 siRNA,

| Statistical analysis
The data are expressed as the mean ± standard deviation (SD). Data were analysed with GraphPad Prism 5 software (GraphPad Software, Inc, San Diego, CA). The unpaired Student's t test or non-parametric Mann-Whitney U test was used for statistical analysis between two groups. For comparisons of multiple groups, one-way ANOVA and the non-parametric Kruskal-Wallis test were performed, followed by Dunnett's multiple comparison post hoc test. Differences were considered statistically significant when P < .05.

| SAA attenuates chronic alcohol-induced liver injury
We first investigated whether SAA treatment protects against chronic alcohol-induced liver injury in rats. As expected, the serum levels of ALT and AST were significantly increased in rats 8 weeks after an ethanol diet. SAA observably suppressed ALT and AST activities in a dose-dependent manner compared with those in the ethanol group (Figure 2A), indicating that SAA reveals a hepatoprotective effect against ALD. Similarly, while serum TC and TG levels were markedly up-regulated compared with the control group without alcohol feeding, SAA largely diminished this increase in TC and TG levels in a dose-dependent manner ( Figure 2B). Further histological evaluation by both H&E staining and Oil Red O staining of liver sections displayed that the control treatment produced no apparent abnormalities. Notably, SAA treatment significantly protected rats from ethanol-induced liver pathogenesis ( Figure 2C and D).
We then further validated the liver-protective effects of SAA using AML-12 mouse hepatocytes. The accumulation of lipid droplets in ethanol-treated AML-12 cells was detected by Oil Red O staining, and the number of these droplets was decreased by SAA treatment ( Figure 2E). Levels of the inflammatory mediator TNF-α and IL-1β are often elevated in liver tissues from individuals with chronic alcoholism. 26 Similarly, ethanol treatment induced a twofold to threefold increase in both IL-1β and TNF-α production. Importantly, to further support our belief that SAA protected rats from alcohol-induced liver damage, the addition of SAA significantly inhibited ethanolinduced inflammatory cytokine production ( Figure 2F). Hence, these consequences manifest that SAA is effective in protecting the liver from chronic ethanol-related injury, steatosis and inflammation.

| SAA suppresses hepatic inflammation induced by ethanol in vivo and in vitro through inhibiting BRD4 expression
BRD4 has latterly been found to be a vital inflammatory mediator, and its activation facilitates the pathogenesis of various diseases. 27,28 To estimate the remedial potential of targeting BRD4 in ALD, we first assessed the expression level of BRD4 in ALD. As shown in Figure 3A, BRD4 protein levels were significantly up-regulated in liver tissues from ALD patients compared with normal liver tissues (P < .01). We also analysed BRD4 expression in rats and AML-12 cells upon ethanol treatment. As shown in Figure 3B Figure 3B and C). Furthermore, SAA treatment neither affected BRD4 expression in the livers of rats without ethanol feeding used as a control nor altered their inflammatory responses ( Figure 3B and C), suggesting that SAA specifically inhibits alcohol-induced BRD4 expression and its associated inflammation. In addition, we analysed the effect of alcohol BRD4 mRNA level. As shown in Figure 3D, the mRNA level of BRD4 was significantly increased by ethanol, which is largely inhibited by SAA treatment. Furthermore, SAA treatment significantly blunted the alcohol-induced increase in BRD4 expression in a dose-dependent manner in AML-12 cells ( Figure 3E). Our results suggested that SAA suppresses hepatic inflammation in chronic alcoholic disease through down-regulating BRD4 expression.

| SAA suppresses HMGB1 nuclear translocation and liberates via the down-regulation of BRD4 to regulate inflammation in ALD
Our data suggested that BRD4 down-regulation was involved in SAA-mediated protection against inflammation in rats during ALD pathogenesis. Next, we further investigated the contribution of  = 3). B, Western blotting analysis of the hepatic BRD4, TLR4 and IL-1β protein levels in rats (n = 3). C, AML-12 cells were pre-treated with SAA (10 μmol/L) for 6 h before exposure to ethanol (100 mmol/L) for 24 h. BRD4, TLR4 and IL-1β protein expression (n = 3). D, The mRNA levels of BRD4 in rats were measured by real-time PCR (n = 3). E, AML-12 cells were pre-treated with 2.5, 5 or 10 µmol/L SAA for 6 h before exposure to ethanol (100 mmol/L) for 24 h. The figure shows independent response of BRD4 to SAA treatment (n = 3). * P <.05, ** P < .01 vs the control group; ## P < .01 vs the alcoholic group exposure ( Figure 7B)

| D ISCUSS I ON
The current study demonstrates that SAA had potent protective effects in the prevention and treatment of ethanol-induced liver injury in a rat ALD model and uncovered the novel underlying molecular mechanism of these effects, as documented by the following dis-  Figure 3D, the mRNA level of BRD4 was significantly increased by ethanol, which is largely inhibited by SAA treatment. These results indicate that BRD4 is induced in hepatocytes by alcohol for inflammatory cytokine production, which consequently leads to liver damage to promote ALD development and progression. SAA treatment protects rats from the disease through suppressing alcohol-induced BRD4 transcription. We are aware, however, as a protein expression is often regulated at both transcription and post-translational levels, the possibility that either alcohol or SAA, or both, regulates BRD4 protein stability cannot be fully excluded. Further studies are needed to dissect the molecular puzzles behind ALD development through, at least partially, BRD4-mediated inflammatory response as well as how SAA achieves its therapeutic efficacy.
HMGB1, a direct target of BRD4, 17 was originally depicted as a chromatin-associated protein functioning as a key endogenous danger signalling molecule that has a cytokine-like extracellular effect on immune cells by advancing pro-inflammatory signalling and the release of cytokines. In liver I/R and non-alcoholic liver disease, extracellular HMGB1 was conscientious for the inflammation to hepatic injury. 16,34,35 When excreted from cells, HMGB1 can also serve as a pro-inflammatory adjustor or alarmin. In addition, recent studies manifested that the level of HMGB1 was added in ALD, contributing to liver damage. 36  and TNF-α. 38 Given that IL-6, pro-IL-1β and TNF-α are abundant pro-inflammatory cytokines in the progression of liver injury, these pro-inflammatory cytokines were examined in BRD4/HMGB1-

ACK N OWLED G EM ENTS
This work was financially supported by grants from the Chinese National Natural Science Foundation (No. 81773799 and 81703771) and the Natural Science Foundation from the Department of Science and Technology of Liaoning Province (No. 20170540257).

CO N FLI C T S O F I NTE R E S T
The authors declare 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
Please contact the authors for data requests.