Withaferin A alleviates ethanol-induced liver injury by inhibiting hepatic lipogenesis

Abstract

Withaferin A (WA) is a natural steroidal compound with reported hepatoprotective activities against various liver diseases. Whether WA has therapeutic effects on alcoholic liver disease has not been explored. A binge alcoholic liver injury model was employed by feeding C57BL/6J mice an ethanol (EtOH) diet for 10 days followed by an acute dose of EtOH to mimic clinical acute-upon-chronic liver injury. In this binge model, WA significantly reduced the binge EtOH-induced increase of serum aminotransaminase levels and decreased hepatic lipid accumulation. Mechanistically, WA decreased levels of hepatic lipogenesis gene mRNAs in vivo, including Srebp1c, Fasn, Acc1 and Fabp1. In EtOH-treated primary hepatocytes in vitro, WA decreased lipid accumulation by lowering the expression of the lipogenesis gene mRNAs Fasn and Acc1 as well as decreasing hepatocyte death. In the established binge alcoholic liver injury model, WA therapeutically reduced the EtOH-induced increase of serum aminotransaminase levels as well as hepatic lipid accumulation. These results demonstrate that WA reduces EtOH-induced liver injury by inhibiting hepatic lipogenesis, suggesting a potential therapeutic option for treating alcoholic liver injury.

1. Introduction

Alcoholic liver disease (ALD) is a general term for liver diseases caused by excessive alcohol intake. ALD begins with lipodystrophy due to fat accumulation in the liver, which could progress to severe alcoholic steatohepatitis (Osna et al., 2017) or liver fibrosis due to abnormal accumulation of extracellular matrix proteins by activating stem cells (Smith et al., 2018). ALD at the early stage could be reversible in some patients upon cessation of alcohol consumption (Osna et al., 2017). However, long-term excessive alcohol consumption could result in chronic inflammation and progressive fibrosis finally leading to cirrhosis and ultimately even liver cancer (Fattovich et al., 2004).

The most effective measure to restrict ALD is abstinence from alcohol, which not only reduces hepatic fat accumulation, but also improves the survival rate of patients with cirrhosis (Verrill et al., 2009). Accordingly, FDA-approved disulfiram (also known chemically as tetraethylthiuram disulfide and commercially as Antabuse) has been widely used as an alcohol-aversive agent for alcoholism (Bernier et al., 2020). Liver transplantation is still a standard treatment for patients with the end-stage ALD (Im et al., 2019). When voluntary or pharmacological alcohol abstinence failes, pharmacological therapeutics for treating ALD becomes essential. However, until now, there are still no therapeutic options available for the treatment of ALD and therapeutic agents with superior therapeutic efficacy are urgently needed.

Traditional herbs have been extensively studied for the discovery of hepatoprotective drugs (Sun et al., 2017; Yan et al., 2016, 2018, 2020). Withaferin A (WA) is a steroidal natural product with a lactone skeleton isolated from Withania Somnifera (Mishra et al., 2000). WA possesses a wide range of pharmacological activities, including anti-obesity, anti--inflammation, anti-oxidation and anti-cancer activities (Vanden Berghe et al., 2012). In recent years, WA was found to have potent hepatoprotective effects in several liver injury models in rodents (Palliyaguru et al., 2016; Patel et al., 2019; Vanden Berghe et al., 2012; Xia et al., 2021a), among which WA was found to potently improve non-alcoholic steatohepatitis in mice through its leptin-independent hepatoprotective effects (Patel et al., 2019) and reduce D-galactosamine/lipopolysaccharide-induced fulminant hepatitis by inactivating the macrophage and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling (Xia et al., 2021a). However, whether WA has therapeutic effects on alcoholic liver injury has not been explored.

In this study, the preventive and therapeutic effects of WA were investigated in the treatment of ethanol (EtOH)-induced ALD by using the binge alcoholic liver injury model. The results demonstrated that WA significantly alleviated binge alcoholic liver injury mainly by inhibiting hepatic lipogenesis both in EtOH-treated mice in vivo and primary hepatocytes in vitro, suggesting a potential role for WA in treating ALD by inhibiting hepatic lipogenesis.

2. Materials and methods

2.1. Chemicals and reagents

WA was purchased from ChromaDex (Irvine, CA, USA). EtOH was purchased from MP Biomedicals (Solon, OH, USA). Primer oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). William E medium (Catalog #A1217601), primary hepatocyte culture supplement (Catalog #CM4000), HBSS (Catalog #14025092), collagenase type I (Catalog #17018029), collagenase type II (Catalog #17101015), and trypsin inhibitor (Catalog #17075029) were supplied by Gibco (Gaithersburg, MD, USA). Collagen I-coated plates (Catalog #356400) were purchased from Corning (Kennebunk, ME, USA). Percoll (Catalog #17544501) was purchased from Cytiva (Uppsala, Sweden). TRIzol reagent was purchased from Invitrogen (Waltham, MA, USA). qScript One-step SYBR Green qRT-PCR kit (Low Rox, Catalog# 95089) was obtained from Applied Biological Materials Inc (Richmond, BC, Canada). Cell-counting kit 8 (CCK8) was purchased from Dojindo Molecular Technologies Inc (Rockville, MD, USA). Alanine transaminase (ALT) and aspartate transaminase (AST) kits were obtained from Catachem (Oxford, CT, USA). The triglyceride (TG) kit was supplied by Wako chemical (Wako, Japan). Oil red O compound was purchased from Sigma Aldrich (St. Louis, MO, USA). Rodent liquid diet Lieber-DeCarli ‘82 (Catalog #F1258) and control diet (Catalog #F1259) were generated by Bioserv (Flemington, NJ, USA). Antibody against cytochrome P450 2E1 (CYP2E1, Catalog #1-98-1, RRID: AB_10628450) was purchased from Avantor (Wien, Wien, Austria). Antibodies against fatty acid synthase (FASN, Catalog #SC-271591, RRID: AB_627584) and fatty acid binding protein 1 (FABP1, Catalog #SC-271591, RRID: AB _10650273) were from Santa Cruz (Dallas, TX, USA). Antibody against β-actin (ACTB, Catalog #4970, RRID: AB_2223172), phosphorylated AMP-activated protein kinase (p-AMPK, Catalog #2535, RRID: AB_331250), AMP-activated protein kinase (AMPK, Catalog #2532, RRID: AB_330331), Lamin B1 (LMNB1, Catalog #13435, RRID: AB_2737428), Kelch like ECH associated protein 1 (KEAP1, Catalog #8047, RRID: AB_10860776) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against nuclear factor erythroid-related factor 2 (NRF2, Catalog #ab137550, RRID: AB_2687540) was purchased from Abcam (Waltham, MA, USA). Antibody against sterol regulatory element binding transcription factor 1 (SREBP1, Catalog #A15586, RRID: AB_2762992) was purchased from Abclonal (Woburn, MA, USA), antibody against sterol regulatory element binding transcription factor 2 (SREBP2, Catalog #NB100–74543, RRID: AB_1049752) was purchased from NOVUS (Centennial, CO, USA), while antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Catalog #60004-1-Ig, RRID: AB_2107436) was obtained from Proteintech (Rosemont, IL, USA).

2.2. Animal experiment designs

Wild-type mice on a C57BL/6J background were purchased from the Jackson Laboratory. All mice were housed in the National Cancer Institute animal facility with a pathogen-free environment controlled for temperature, light (25 °C, 12 h light/dark cycle) and humidity (45–65%). Age and body weight-matched 12-week-old female mice were randomized into groups and subject to treatments as indicated below. The National Cancer Institute Animal Care and Use Committee approved all animal experiments conducted in this study under protocol LM096. A heat pad was used to keep mice warm to avoid EtOH-induced hypothermia. All mice were killed by CO₂ inhalation 9 h after EtOH gavage and serum and tissues collected for further analyses.

To test whether WA prevented EtOH-induced binge chronic liver injury, 12-week-old body weight-matched female C57BL/6J mice were randomly divided into three groups (Control group, Vehicle + EtOH group, and WA + EtOH group) followed by EtOH treatment similar to that described previously (Bertola et al., 2013). Briefly, mice in the three groups were initially fed the control Lieber-DeCarli diet ad libitum for 5 days to acclimatize to the liquid diet and tube feeding. Subsequently, mice in the vehicle + EtOH group and WA + EtOH group were fed a EtOH Lieber-DeCarli diet containing 5% (vol/vol) EtOH for 10 days and dosed with control vehicle or 5 mg/kg of WA dissolved in saline containing 5% dimethyl sulfoxide and 5% Tween 80, once daily by intraperitoneal injection for 10 days. On day 11, the vehicle + EtOH group and WA + EtOH group were administered a single dose of EtOH (31%, 5 g/kg) by gavage in the early morning, 30 min after the last dose of WA. The dosage method of WA was designed and selected based on previous studies (Patel et al., 2019; Xia et al., 2021a).

To test whether WA has a therapeutic effect in treating EtOH-induced chronic liver injury, 12-week-old body weight-matched female C57BL/6J mice were randomly divided into three groups, Vehicle + Control group, Vehicle + EtOH group, and WA + EtOH group. Mice were treated with control diet for the initial 5 days and EtOH Lieber-DeCarli diet or control diet for the following 10 days. On day 11, mice in the EtOH groups were treated with a single dose of EtOH (31%, 5 g/kg) by gavage, while mice in the WA + EtOH group were intraperitoneally injected with WA (5 mg/kg) 3 h after EtOH dosing. All mice were killed 9 h after EtOH gavage and tissues harvested.

To test the effect of WA in treating a single dose of EtOH-induced acute liver injury, body weight and age-matched 12-week-old female C57BL/6J mice were randomly divided into three groups (Vehicle + Control group, Vehicle + EtOH group, and WA + EtOH group) as indicated in each experiment. In brief, the same volume (200 μL/20 g mice) of control vehicle or WA (5 mg/kg, dissolved in 5% dimethyl sulfoxide and 5% Tween 80-contained saline) was administered to mice via intraperitoneal injection, and at 30 min after WA dosing, the mice were treated with a single gavage of EtOH (31%, 5 g/kg) and killed 9 h after EtOH administration and tissues collected.

2.3. Measurement of ALT, AST and TG

Serum ALT and AST levels, serum and liver triglyceride (TG) levels were measured as described previously (Patel et al., 2019).

2.4. Primary hepatocyte isolation and culture

Isolation of primary hepatocytes was performed as described (Brocker et al., 2017). Adult male 8-12-week-old C57BL/6J mice were killed by CO₂ inhalation, and injected in the portal vein with a cannula and perfused with pre-perfusion solution (Hanks’ Balanced Salt Solution (HBSS) without CaCl₂, MgCl₂, MgSO₄, EDTA 1 mM) at 4 mL/min for 6 min. Then the pre-perfusion solution was changed to digestion solution (HBSS without CaCl₂, MgCl₂, MgSO₄, 0.037% CaCl₂ in H₂O, 0.005% Trypsin inhibitor, 0.0125% collagenase type I, and 0.0125% collagenase type II) at 4 mL/min for 6 min. The liver without gall bladder was collected in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, 10 mL). After filtration through a 70 μm mesh, the filtrated fraction was centrifuged at 500 rpm under 4 °C for 2 min and the precipitate (primary hepatocyte fraction) resuspended in 10 mL of cold Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) followed by the addition of 10 mL 90% Percoll solution (Percoll: 10 x PBS = 9:1) and centrifuged at 600 rpm under 4 °C for 10 min. The supernatant was carefully removed, and the pellet was resuspended with William E medium without phenol red containing primary hepatocytes culture supplement. After centrifugation at 500 rpm under 4 °C for 2 min, the pellet was resuspended and seeded into collagen I-coated plates.

2.5. Cell viability assay

Isolated primary hepatocytes were seeded in 96-well plates overnight and then treated with varying concentrations of EtOH (0, 200, and 400 mM) and WA (0, 0.1 and 0.25 μM) for 48 h. A sterile film was used to prevent the evaporation of EtOH during EtOH treatment. To test the cell viability of primary hepatocytes under treatment with EtOH and WA, 10 μL of CCK-8 solution was mixed with 90 μL of culture medium and added to each 96-well, followed by an incubation at 37 °C for 2 h. After incubation, O.D. values at 450 nm were measured and relative cell viabilities were calculated.

2.6. Oil red O staining of primary hepatocytes

For oil red O staining, the cells were seeded in the 12-well plates and cultured overnight. Then, the cells were treated with 0 and 0.25 μM WA and/or 200 mM EtOH for additional 48 h at 37 °C. A sterile film was used to prevent the evaporation of EtOH and oil red O staining was performed. Cultured primary hepatocytes were washed 3 times with 1x PBS and fixation performed using 4% paraformaldehyde solution for 15 min. While the cells were being fixed, the staining solution was prepared by dissolving 0.5% oil red O powder in isopropanol and then mixing it with water at a ratio of 6:4. After 10 min, the mixture was passed through filter paper to generate the final staining solution. The fixed cells were washed twice with water and a 60% isopropanol was added for 5 min. The cells were stained with oil red O for 15 min, washed five times with water, 1x PBS was added, and then the cells were analyzed by light microscopy. For quantification of the oil red O-stained primary hepatocytes, 100% isopropanol was added and the cells incubated for 5 min at room temperature to extract the oil red O dye. To quantity the concentration of oil red O dye, extracted isopropanol solution were measured absorbance at 492 nm by use of a SPECTRAmax (Molecular devices, San Jose, CA, USA).

2.7. Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted from frozen livers and primary hepatocytes using TRIzol reagent following the protocol of the manufacture. cDNA was synthesized from the extracted RNA using qScriptTM cDNA SuperMix. qPCR analyses were performed using qScript One-step SYBR Green qRT-PCR kit with Applied Biosystems 7500 (Fisher Scientific, Ottawa, Canada). The primer sequences are shown in Supplemental Table 1. The mRNA levels were calculated and normalized to the corresponding Gapdh or Actb mRNA.

2.8. Histological analysis

The mice were killed and livers collected and immediately fixed and embedded by use of Tissue-Tek^® O.C.T. Compound (Sakura finetek, CA, USA). The embedded livers were stained with hematoxylin and eosin (H&E) and oil red O staining at Histoserv (Germantown, MD, USA). Histological analyses were performed using a Keyence BZ-X800 microscope (Keyence, Osaka, Osaka, Japan). Quantitative analysis of oil red O staining was performed by ImageJ software (National Institutes of Health, Bethesda, MD).

2.9. Western blot

Liver proteins were prepared in radioimmunoprecipitation assay buffer lysis buffer containing protease inhibitors and phosphatase inhibitors. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed, and the bands transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% (w/v) bovine serum albumin in 0.1% Tween 20-containing tris buffered saline (pH 7.4) for 1 h. After blocking, primary and secondary antibodies were incubated. The membranes were visualized, and the signals normalized with LMNB1 for nuclear NRF2 protein and ACTB or GAPDH for the other proteins. To quantify the bands intensity in the western blot, the bands were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD).

2.10. Statistical analysis

Statistical analysis was performed using Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Two-tailed Student’s t-test between two groups or one-way ANOVA followed by Dunnett’s multiple comparisons test among multiple groups was used to compare the statistic difference for independent samples. The values were presented as mean ± SEM. A value of P< 0.05 was considered as statistically significant.

3. Results

3.1. WA improves binge EtOH-induced liver injury

To investigate whether WA reduces liver injury that results from chronic alcohol consumption, an experiment was designed following a method described previously (Bertola et al., 2013) as schemed (Fig. 1A). Age-matched female C57BL/6J mice were divided into three groups, and WA administered by intraperitoneal injection daily at 5 mg/kg/day for the WA + EtOH group. Food intake was measured, and no significant difference observed between the Vehicle + EtOH group and the WA + EtOH group (Fig. 1B). Both serum ALT and AST levels were increased by EtOH administration and decreased by WA treatment (Fig. 1C and D). Given that EtOH intake is known to cause hepatic lipid accumulation (Baraona and Lieber, 1979), hepatic lipid was quantified. Hepatic TG levels were increased in the EtOH-treated group, and reduced by WA administration (Fig. 1E). H&E staining also revealed significant hepatic lipid accumulation in the EtOH-dosed mice, which was ameliorated by WA treatment (Fig. 1F). Oil red O staining was carried out revealing that significant lipid accumulation in alcohol-treated livers was alleviated by WA administration (Fig. 1G and H). These results demonstrate that WA improves binge alcohol-induced liver injury and ameliorates hepatic lipid accumulation.

Fig. 1.

WA improves binge EtOH-induced liver injury. (A), Experimental time scheme. (B), Food intake in WA treated mice. (C and D), Serum ALT and AST values. (E), Liver TG values. (F), Representative hepatic H&E staining, scale bar 100 μm. (G), Representative oil red O staining, scale bar 100 μm. (H), Quantitation of oil red O staining area (N = 3). Data are expressed as mean ± SEM; N = 5 unless otherwise indicated. ***P< 0.001, **P< 0.01, and *P< 0.05 versus V + EtOH group (one-way ANOVA).

3.2. WA ameliorates EtOH-induced binge liver injury by affecting the expression of lipogenesis-related genes

CYP2E1, a key enzyme responsible for alcohol metabolism (Leung and Nieto, 2013; Xu et al., 2017), is induced by EtOH, which further accelerates EtOH-induced toxication (Cederbaum, 2010; Lu and Cederbaum, 2008). Cyp2e1 mRNA levels were slightly decreased by EtOH treatment compared to the control group, while there was no significant change in Cyp2e1 mRNA levels after WA administration to EtOH-dosed mice (Fig. 2A). However, the CYP2E1 protein was sharply increased by EtOH administration suggesting that CYP2E1 expression modulation by EtOH occurs at the posttranscriptional level consistent with earlier studies (Gonzalez et al., 1991; Song et al., 1989). No decrease of CYP2E1 protein levels by WA treatment was observed in EtOH-dosed mice (Fig. 2, B–C). These results indicate that the ameliorative effect of WA on alcoholic liver disease is not due to changes in CYP2E1 expression.

Fig. 2.

WA ameliorates EtOH-induced binge liver injury by inhibiting lipogenesis. (A), qPCR analysis of hepatic Cyp2e1 mRNA expression (N = 5). (B), Western blot analyses of CYP2E1 protein levels in the binge EtOH-injury model mice (N = 4). (C), Quantitation of CYP2E1 protein expression levels (N = 4). (D), qPCR analyses of hepatic proinflammatory and NLRP3 inflammasome signaling-related mRNAs. (E), qPCR analyses of hepatic oxidative stress and NRF2 signaling-related mRNAs. (F), Western blot analyses for NRF2, KEAP1, p-AMPK and AMPK levels in the binge EtOH-injury model mice (N = 3). (G) Quantitation of NRF2 and KEAP1 protein expression levels and ratio of p-AMPK/AMPK levels (N = 3). (H), qPCR analyses of fatty acid β-oxidation pathway-related mRNAs. (I), qPCR analyses of hepatic lipogenesis-related genes and Pparg target gene mRNAs (J), Western blot analyses for SREBP1, SREBP2, FASN and FABP1 in the livers of vehicle or WA-treated mice in binge model (N = 3). (K), Quantitation of protein expression levels (N = 3). Data are expressed as mean ± SEM; N = 5 unless otherwise indicated. ***P< 0.001, **P< 0.01, and *P< 0.05 versus V + EtOH group (one-way ANOVA for multiple groups, while two-tailed t-test for two groups). Acc1, acetyl-CoA carboxylase 1. Acca1, acetyl-CoA carboxylase carboxyltransferase. Acot1, acyl-CoA thioesterase 1. Acox, acyl-CoA oxidase. Acsl1, acyl-CoA synthetase long chain family member 1. Acadl, acyl-CoA dehydrogenase long chain. Acadm, acyl-CoA dehydrogenase medium chain. Adgre1, adhesion G protein-coupled receptor E1. Cat, catalase. Ccl2, C–C motif chemokine ligand 2. Casp1,Caspase 1. Cidec, cell death inducing DFFA like effector c. Cpt, carnitine palmitoyltransferase. Ehhadh, enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase. Gclc, glutamate-cysteine ligase catalytic subunit. Hmox1, heme oxygenase 1. IL, interleukin. Nqo1, NAD(P)H quinone dehydrogenase 1. Ppargc1a, Pparg coactivator 1α. Plin2, perilipin 2. Pycard, PYD and CARD domain containing. Sod, superoxide dismutase. Srebp, sterol regulatory element-binding protein. Tlr, toll-like receptor. Tnfa, tumor necrosis factor α.

In a previous study, WA was demonstrated to inhibit inflammation by inactivating NLRP3 inflammasome signaling (Xia et al., 2021a) and exert hepatoprotective effects via NRF2 activation (Kim et al., 2015; Palliyaguru et al., 2016). Therefore, NLRP3 signaling and NRF2 signaling were examined. Only Tlr9 and Casp1 mRNA levels were decreased by WA treatment, while the mRNA levels of the other tested genes including the classical proinflammatory effectors, Tnfa, Il6, Il1b and Ccl2, remained unchanged (Fig. 2D), indicating a minor contribution of the NLRP3 and inflammation pathways to the effects of WA. In addition, EtOH treatment induced the expression of most NRF2 target gene mRNAs including Hmox1, Gsta1, Nqo1, and Gclc, while WA treatment did not further increase, but decreased the expression levels of several NRF2 target gene mRNAs in EtOH-dosed mice (Fig. 2E). Western blot analyses demonstrated that NRF2 and KEAP1 showed no change in expression at the protein levels by WA treatment in EtOH-dosed mice (Fig. 2F and G), suggesting that the WA protection against EtOH-induced liver injury is not through NRF2 activation. In addition, the expression of AMPK and phosphorylated AMPK (p-AMPK) involved in intracellular lipid metabolism were found not to be altered by WA treatment in EtOH-dosed mice (Fig. 2F and G). Further analyses of genes involved in both mitochondrial and peroxisomal fatty acid β-oxidation pathways demonstrated that WA treatment decreased Ehhadh mRNA, and did not alter the expression of Acot1, Acsl1, Cpt1a, Cpt1b, Cpt2, Acox1, Acox2, Acadl, Acadm, Acaa1, and Ppargc1a mRNAs (Fig. 2H), suggesting that WA decreases hepatic lipid accumulation not through enhancing fatty acid β-oxidation. Levels of the lipogenesis-related mRNAs, Fasn, Acc1, Srebp1c, and Fabp1, were markedly decreased by WA, while Srebp2 remained unchanged, compared to the Vehicle + EtOH group (Fig. 2I). The mRNA expression of Pparg and its target genes was also analyzed, but no change was observed after WA treatment in EtOH-dosed mice (Fig. 2I). The protein expression levels of SREBP1, SREBP2, FASN and FABP1 in Vehicle + EtOH and WA + EtOH group were further examined. WA treatment significantly decreased SREBP1, FASN, and FABP1 proteins, while SREBP2 protein remained unchanged, compared to vehicle treatment in EtOH-dosed mice (Fig. 2J and K). These results indicate that WA ameliorates EtOH-induced binge liver injury via inhibiting the hepatic lipogenesis.

3.3. WA reduced lipid accumulation in acute EtOH-induced liver damage

To investigate whether WA could ameliorate acute liver injury caused by excessive alcohol intake, an experiment was performed as schemed (Fig. 3A). Serum ALT levels were slightly but significantly increased by a single dose of EtOH compared to the control group (Fig. 3B), and WA treatment had minimal effects on ALT levels in these mice. For serum AST levels, there was no statistically significant difference among all three groups (Fig. 3C). Hepatic TG levels were increased in the EtOH-treated group, which was reduced by WA administration (Fig. 3D). An acute EtOH gavage caused an increase in lipid droplet accumulation compared to the control group and WA treatment decreased the ethanol-induced lipid droplet accumulation compared to vehicle treatment based on the analyses of H&E staining (Fig. 3E) and oil red O staining (Fig. 3F and G). Consistent with the marked lipid-lowering effect of WA in the binge alcoholic liver injury model, these results suggest that WA holds the potential to ameliorate acute alcoholic liver injury by decreasing lipid accumulation prior to its significant effects in decreasing ALT and AST levels. Decreasing hepatic lipid accumulation by WA could at least partially contribute to its hepatoprotective effect as a causal factor at the early stage of ALD.

Fig. 3.

WA reduces lipid accumulation in acute EtOH-induced liver injury. (A), Experimental time scheme. (B and C), Serum ALT (B) and AST (C) values. (D), Liver TG values in EtOH treated mice followed by vehicle (V) or WA treatment (N = 6). (E), Representative hepatic H&E staining of V or WA-treated acute EtOH injury model mice (N = 3), scale bar 100 μm. (F), Effect of WA on lipid accumulation in liver determined by oil red O staining (N = 3). (G), Quantitation of the oil red O-stained areas (N = 3). scale bar 100 μm. Data are expressed as mean ± SEM, ***P< 0.001, **P< 0.01, and *P< 0.05 versus V + EtOH group (one-way ANOVA).

3.4. WA inhibits alcohol-induced cell death and lipogenesis in primary hepatocytes

Since excessive EtOH intake induces primary hepatocyte cell death (Gaitantzi et al., 2018), the effects of WA in EtOH-induced cell death were examined in primary hepatocytes. Cell viability of primary hepatocytes was reduced by 48-h treatment with EtOH in a dose-dependent manner (Fig. 4A). EtOH concentrations at 200 mM and 400 mM were further selected to test whether WA affected cell damage in EtOH-treated primary hepatocytes. CCK-8 assays demonstrated that WA treatment dose-dependently rescued EtOH-induced cell viability loss in primary hepatocytes (Fig. 4B and C). Since the in vivo data suggested that WA protected against ALD mainly by decreasing hepatic lipogenesis, oil red O staining was performed in EtOH-treated primary hepatocytes. EtOH treatment induced a significant accumulation of lipids compared to the control group, while WA treatment significantly reduced the EtOH-induced lipid accumulation (Fig. 4D and E). Unlike the in vivo results (Fig. 2I), western blot analysis of AMPK and p-AMPK revealed that WA significantly enhanced the AMPK and p-AMPK levels (Fig. 4F) and reduced the Cd36 mRNA levels (Fig. 4H) in EtOH-treated hepatocytes, suggesting different effects of WA under in vivo and in vitro conditions. Consistent with the in vivo data, the expression of mRNAs of Pparg and the PPARγ target gene Cidec and Plin2 were not changed by WA treatment (Fig. 4H). The expression levels of Srebp1c, Srebp2, Fasn, and Acc1 mRNAs were also decreased by WA treatment of EtOH-treated hepatocytes (Fig. 4I). These results further support the view that WA ameliorates EtOH-induced lipid accumulation by decreasing lipogenesis in vitro.

Fig. 4.

WA inhibits cell death and lipogenesis in EtOH-treated primary mouse hepatocytes. (A), Dose-dependent cell toxicity of EtOH was detected by the CCK-8 assay. (B and C), Effects of WA in cell viability of primary hepatocytes exposed to 200 mM EtOH and 400 mM EtOH treatment. (D), Representative oil red O staining of WA-treated (0, 0.25 μM) primary hepatocytes subjected to 200 mM of EtOH challenge for 48 h, scale bar 100 μm. (E), Quantitation of oil red O staining. (F), Western blot analyses of p-AMPK and AMPK protein levels. (G) Quantitation of AMPK and p-AMPK protein levels. (H), mRNA expression of Pparg and PPARγ target genes. (I), qPCR analyses of lipogenesis gene mRNAs in primary hepatocytes. N = 3. Data are expressed as mean ± SEM, ***P< 0.001, **P< 0.01, *P< 0.05 versus V + EtOH group (one-way ANOVA). Gene abbreviations were same as Fig. 2 legend.

3.5. WA shows a therapeutic effect in treating chronic binge alcoholic liver injury

The therapeutic effect of WA for treating chronic alcoholic liver injury was next evaluated as schemed (Fig. 5A). In brief, mice were put on a chronic dietary intake of EtOH for 10 days and then WA was administered once after the final dose of acute EtOH gavage (Fig. 5A). Serum ALT and AST levels as well as the hepatic TG levels were significantly reduced in the WA+EtOH-treated group as compared to the Vehicle + EtOH-treated group (Fig. 5, B–D). Histological analyses showed that EtOH-induced lipid droplet accumulation was remarkedly decreased in the WA therapy group compared to the vehicle-treated group (Fig. 5, E–G). These results support the view that WA potentially serves as a therapeutic agent for treating alcohol-induced liver injury.

Fig. 5.

WA shows a therapeutic effect in chronic binge alcoholic liver injury. (A), Experimental time scheme. (B, C), Serum ALT and AST values in binge EtOH-treated mice treated with or without WA in a therapy manner. (D), Liver TG values in binge EtOH-treated mice treated with or without WA as a therapeutic. (E), Representative hepatic H&E staining of V + EtOH or WA therapy group, scale bar 100 μm. (F), Representative hepatic oil red O staining of V or WA therapy group, scale bar 100 μm, (G), Quantitation of oil red O staining area (N = 3). (H), Graphic abstract of the major findings in this study. N = 6 unless otherwise indicated. Data are expressed as mean ± SEM, ***P< 0.001, **P< 0.01, *P< 0.05 versus V + EtOH (one-way ANOVA).

4. Discussion

ALD is a major cause of morbidity and mortality worldwide (Bataller, 2011) and pharmacotherapeutic discoveries are urgently needed for treating this disease. In previous studies, WA demonstrated hepatoprotective activities in various types of liver diseases (Jadeja et al., 2015; Palliyaguru et al., 2016; Patel et al., 2019; Xia et al., 2021a), while little was known about its affects and mechanism in treating EtOH-induced ALD. The binge alcoholic liver injury model that includes feeding mice an EtOH diet for 10 days followed by one acute bolus dose of EtOH by gavage mimics acute-on-chronic alcoholic liver injury in patients (Bertola et al., 2013). By using the binge ALD model and EtOH-treated primary hepatocytes, WA was found to improve ALD by inhibiting hepatic lipogenesis both in vivo and in vitro, while few effects were noted with WA on hepatic CYP2E1 expression, inflammation and oxidative stress signaling. The major findings of this study are summarized in Fig. 5H.

WA, as an active component of ayurvedic folk medicine Withania Somnifera, was initially found to be an anti-cancer agent (Hassannia et al., 2020) with broad activities in the preclinical treatment of tumors, inflammatory diseases and neurodegenerative diseases (Vanden Berghe et al., 2012). WA is also a leptin sensitizer and anti-obesity compound (Lee et al., 2016), and acts as a hepatoprotective component against various liver diseases including acute liver injury induced by D-galactosamine/lipopolysaccharide (Xia et al., 2021a), acetaminophen (Jadeja et al., 2015; Palliyaguru et al., 2016), bromobenzene (Vedi and Sabina, 2016), nonalcoholic steatohepatitis (Patel et al., 2019), liver fibrosis induced by bile duct ligation, platelet-derived growth factor BB and carbon tetrachloride (Gu et al., 2020) as recently reviewed (Xia et al., 2021b). The current study provides evidence that WA has hepatoprotective effects in the acute alcoholic liver injury model. It should be noted that orally administered WA has extensive first-pass metabolism in rats (Dai et al., 2019), which may explain the preferential route of intraperitoneal injection used in preclinical studies (Xia et al., 2021b). Since oral administration may obtain more patient compliance, hepatoprotective drug discovery from oral formulations of WA merits further study.

Ethanol is metabolized to acetaldehyde mainly by hepatic alcohol dehydrogenase and CYP2E1 (Edenberg, 2007), while CYP2E1 was shown to be induced in response to excessive ethanol intake as described previously (Cederbaum, 2010; Lu and Cederbaum, 2008), as well as in the present study, which could further accelerate ethanol toxication. Moreover, CYP2E1 inhibition is known to decrease ALD after administration of traditional herbs (Lu et al., 2008; Morimoto et al., 1995; Zeng et al., 2017). However, in the current study, WA was found to have no effects on the expression of CYP2E1, suggesting that the hepatoprotective effect of WA in treating ethanol-induced ALD is not through inhibiting CYP2E1 expression under the current experimental conditions. In line with this point, glycyrrhizin, a natural compound, was also found to protect against acute liver injury, independent of inhibiting CYP2E1-mediated xenobiotic metabolism (Yan et al., 2016).

Induction of hepatic NRF2 target genes and antioxidative response signaling was found to play a minor role in mediating the hepatoprotective effects of WA. Previously, WA was shown to alleviate acetaminophen-induced liver injury dependent on NRF2 induction (Palliyaguru et al., 2016). In contrast, in the present study, WA did not induce, but decreased the expression of NRF2 target genes at the mRNA level, and did not change NRF2 and KEAP1 expression at the protein level, suggesting that NRF2 activation is not involved in the protective effects of WA against binge ALD. This result is consistent with earlier studies showing that WA reduced nonalcoholic steatohepatitis (Patel et al., 2019) and D-galactosamine/lipopolysaccharide-induced fulminant hepatitis (Xia et al., 2021a) accompanied with decreased mRNA expression of NRF2 target genes. The differences found in the effects of WA on NRF2 signaling between the present study and earlier work (Jadeja et al., 2015; Palliyaguru et al., 2016) suggest that WA affects NRF2 signaling in a context-dependent manner. Given that WA failed to induce the mRNA expression of genes involved in the antioxidant response in this study, it is not likely that WA alleviates ALD through decreasing the oxidative stress.

Although WA was established as a potent anti-inflammation agent, in part by inhibiting IκκB activity (Heyninck et al., 2014; Kaileh et al., 2007) or NLRP3 inflammasome activation (Xia et al., 2021a), WA was only found to inhibit the expression of Casp1 and Tlr9 mRNA while it had no effects on the expression of effector proinflammatory gene mRNAs such as Tnfa, Il1b and Il6. Notably, the expression of the proinflammatory genes tested in this study was found not to be elevated in the binge ALD model when comparing the EtOH-treated group with the control vehicle-treated group thus indicating that the induction of inflammation did not occur in this binge model, which also supports fatty liver as a major symptom of clinical ALD at the early stages.

In line with the major phenotype mainly characterized by marked lipid accumulation induced by EtOH in the binge model, this study further revealed that hepatic lipogenesis suppressed by WA at least partially determined the hepatoprotective effect of WA. The most common hepatic alteration caused by alcohol intake is lipid accumulation (Baraona and Lieber, 1979), which could be due to decreased fatty acid β-oxidation or stimulation of hepatic lipogenesis (Grunnet and Kondrup, 1986; Jeon and Carr, 2020). Thus, enhancing fatty acid β-oxidation to accelerate the removal of lipids or decreasing lipogenesis could help relieve the lipid overloading burden in livers of EtOH-challenged mice. PPAR signaling regulates hepatic lipid homeostasis through modulating the expression genes involved in lipid transport, catabolism, synthesis and storage (Dixit and Prabhu, 2021; Wang et al., 2020) and is a potential therapeutic target for the treatment of alcoholic liver disease (Monroy-Ramirez et al., 2021; Zhang et al., 2016). In this study, WA decreased the expression of hepatic lipogenesis gene mRNAs including Srebp1c, Fasn, Acc1 and Fabp1, but was found not to enhance fatty acid β-oxidation and did not affect the mRNA expression of Pparg and PPARγ target genes in the current binge alcoholic liver injury model. Liver X receptor (LXR) activation is also known to contribute to the enhancement of hepatic lipogenesis in alcohol-induced liver injury (Na et al., 2015), while the inverse LXRα agonist SR9238 reduces ethanol-induced hepatotoxicity in alcoholic liver injury (Sengupta et al., 2018). Earlier studies revealed that WA had LXRα agonist activity (Dave et al., 2009; Mehrotra et al., 2011), increased the expression of LXRα target genes in HepG2 cells and QGY-7703 cells, and inhibited the growth of hepatocellular carcinoma cells (Shiragannavar et al., 2020). Whether the activities of WA toward alcoholic liver injury are mediated by LXRα has not been examined.

Further studies with the single-dose EtOH-induced acute liver damage model also revealed that WA reduced hepatic lipid accumulation prior to changing serum ALT and AST levels. Consistently, in EtOH-treated primary hepatocytes in vitro, WA was also found to decrease lipid accumulation by lowering the expression of lipogenesis genes. Previous studies also support the concept of targeting lipogenesis to alleviate alcoholic liver injury (Chen et al., 2018; Zhang et al., 2018) and pharmacotherapies for EtOH-induced liver injury via inhibiting lipogenesis (Nagappan et al., 2019; Tang et al., 2013). Notably, AMPK expression was suppressed by ethanol leading to enhancement of lipogenesis, while AMPK activation by natural product betulin decreased alcoholic liver injury as revealed in a previous publication (Bai et al., 2016). In the present study, no activation of AMPK by WA was observed in the livers of EtOH-treated mice in vivo, while WA significantly induced AMPK phosphorylation in EtOH-treated primary hepatocytes in vitro, suggesting the possibility that other in vivo factors could compromise the effect of WA in AMPK activation under EtOH treatment.

The limitation of the current study is that the mechanism by which WA inhibits hepatic lipogenesis in the ALD model was not fully elucidated, and thus how WA impedes hepatic lipogenesis under EtOH treatment conditions requires future study.

5. Conclusions

In conclusion, the current study demonstrates a novel role for WA in both preventing and therapeutically alleviating EtOH-induced ALD via inhibiting the hepatic lipogenesis both by using binge ALD model in vivo and by using EtOH-treated primary hepatocytes in vitro, which supports the potential for WA to be repurposed as a hepatoprotective agent against the clinical ALD, particularly featured with EtOH-related lipid accumulation.

Abbreviations

AMPK

AMP-activated protein kinase

ALD

alcoholic liver disease

ALT

alanine transaminase

AST

aspartate transaminase

CCK8

cell-counting kit 8

CYP2E1

cytochrome P450 2E1

EtOH

ethanol

FASN

fatty acid synthase

FABP1

fatty acid binding protein 1

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

H&E

hematoxylin and eosin

KEAP1

Kelch like ECH associated protein 1

LMNB1

Lamin B1

LXRα

liver X receptor α

NLRP3

NLR family pyrin domain containing 3

NRF2

nuclear factor erythroid-related factor 2

p-AMPK

phosphorylated AMP-activated protein kinase

PPAR

peroxisome proliferator activated receptor

qPCR

quantitative real-time polymerase chain reaction

SREBP

sterol regulatory element binding transcription factor

WA

withaferin A