Protective Effects of Korean Red Ginseng against Alcohol-induced Hepatosteatosis

알코올에 의해 유발된 지방변성증에서 홍삼의 보호효과

Abstract

Alcohol-induced fatty liver (steatosis) results from excessive generation of reducing equivalents by ethanol metabolism. Generally, chronic ethanol treatment causes hepatosteatosis by regulating sterol regulatory element-binding protein 1c (SREBP-1c), which increases the synthesis of hepatic lipids. The effect of ethanol on SREBP-1c is mediated through mammalian sirtuin-1 (SIRT-1), a NAD⁺-dependent protein deacetylase that regulates hepatic lipid metabolism. Ginseng is a widely used herbal medicine that is used in Asia for its anti-diabetes and anti-obesity effects. The pharmacological and therapeutic effects of ginseng are primarily produced by bioactive constituents known as ginsenosides. Here, we examined the regulatory effects of Korean red ginseng (KRG) extracts on SREBP-1c and SIRT-1 on lipid homeostasis in AML-12 mouse hepatocytes. AML-12 cells were treated with ethanol and/or KRG extracts (0 - 1,000 μg/ml). Lipid droplets were assayed using Oil red O staining, and western blotting was used to measure SIRT-1 and SREBP-1 expression. Treatment with KRG extracts restored SIRT-1 expression and reduced SREBP-1c expression in ethanol-treated cells. We also showed that KRG extract and ginsenosides Rb₂ and Rd significantly decreased SREBP-1 acetylation in ethanol-treated cells. These results show that treatment with KRG extract and its active ginsenoside constituents Rb₂ and Rd protected against alcohol-related hepatosteatosis via regulation of SIRT-1 and downstream acetylation of SREBP-1c, which altered hepatic lipid metabolism.

알코올에 의한 지방간(지방변성증)은 에탄올 대사에 의해 감소되는 당량의 과도한 발생에 의해 유발된다. 일반적으로 만성적인 에탄올 투여는 간 지질의 합성을 증가시키는 sterol regulatory element-binding protein 1c (SREBP-1c)를 조절함으로써 지방변성증을 유발시킨다. SPEBP-1c에서 에탄올의 영향은 간에서의 지방대사를 조절하는 NAD⁺ 의존적 단백질 탈아세틸화효소인 mammalian sirtuin-1 (SIRT-1)에 의해 조정된다. 홍삼은 항당뇨와 항비만 효과를 위해 아시아에서 광범위하게 사용되는 한약재이다. 홍삼의 약리학적 치료학적인 효과는 진세노사이드와 같은 생물활성 성분에 의해 주로 일어난다. 따라서 우리는 마우스 간세포주인 AML-12 세포에서 SREBP-1과 SIRT-1에대한 한국홍삼 추출물의 조절효과를 평가하였다. 알코올과 홍삼추출물(0-1,000 μg/ml)을 AML-12 세포주에 처리하고, 지방소립을 Oil red O 염색법으로 확인하고, western blots을 사용해 SIRT-1과 SREBP-1의 발현을 확인하였다. 에탄올을 처리한 세포에서 홍삼추출물은 SIRT-1과 SREBP-1c의 발현을 회복시켰다. 또한 에탄올이 처리된 세포에서 홍삼추출물과 진세노사이드 Rb₂와 Rd가 SREBP-1을 유의적으로 감소시키는 것으로 확인 되었다. 결과적으로 홍삼과 활성 진세노사이드 성분인 Rb₂와 Rd가 SIRT-1과 간 지질대사를 변화시키는 SREBP-1c의 아세틸화의 조절을 통해 알코올에 의한 간지방변성을 억제하는 것을 확인하였다.

Introduction

The liver is the main organ that metabolizes absorbed ethanol, and is therefore prone to diverse forms of liver damage, including fatty liver, steatohepatitis, hepatic fibrosis, and cirrhosis. Fatty liver (steatosis) is the most common form of hepatic disease that is caused by chronic alcohol consumption, as well as the earliest form to appear. Alcohol-induced fatty liver is believed to result from excessive generation of reducing equivalents from ethanol metabolism, which enhance fat accumulation. The production of reducing equivalents by hepatic lipid metabolism is controlled by levels of enzymes involved in fatty acid oxidation and synthesis, which are primarily regulated by 2 transcription factors: peroxisome proliferator-activated receptor-α (PPAR-α) and sterol regulatory element-binding protein 1c (SREBP-1c) [2]. The role of ethanol in the progression of fatty liver has been well documented and is mediated by alteration of NADH/NAD+ redox potential by ethanol metabolism, interference with PPAR-α, and induction of SREBP-1c. In addition, chronic ethanol administration impaired signaling via the hepatic sirtuin 1 (SIRT-1)-AMP-activated kinase (AMPK) axis, which is also involved in the control of lipid metabolism [1, 19, 22].

SREBP-1c is a transcription factor that regulates cholesterol and lipid synthesis and is involved in the development of alcoholic fatty liver in animals [15, 24]. SREBP-1c protein stability and activity are regulated by reversible acetylation, and many previous studies have suggested that SIRT-1, an NAD+-dependent class III protein deacetylase, regulates the acetylation of SREBP-1c [3, 15, 24]. The acetylation of SREBP-1c at Lys-289 and Lys-309, which increases the stability of SREBP-1c and prevents ubiquitination, is mediated by cAMP response element binding protein (CBP)/p300 [5]. SREBP-1c is a key lipogenic activator, and prolonged SREBP-1c activation stimulates lipid synthesis by inducing genes encoding lipogenic enzymes such as fatty acid synthase, stearoyl-coenzyme A desaturase, mitochondrial glycerol- 3-phosphate acyltransferase, and acetyl-CoA carboxylase [20].

Ginseng is a traditional Asian herbal medicine that contains bioactive constituents with therapeutic effects. Korean red ginseng (KRG) is a processed form of ginseng that has been reported to have more potent pharmacological effects than the unprocessed form. The main bioactive constituents of ginseng are ginsenosides, which are active saponins that are believed to be responsible for the pharmacological activity of Panax ginseng, including its hepatoprotective effect [9]. In several previous studies, ginsenosides reduced hepatic fat accumulation via regulation of AMPK [10]. Ginsenosides Rb1, Re, and compound K decreased hepatic lipid accumulation via AMPK activation in human hepatoma cells and obese rats [12, 16, 18].

Although the effects of KRG on alcohol-induced liver damage have been reported, and KRG is known to prevent fat accumulation in the liver, the mechanisms underlying the action of ginseng extracts and ginsenosides against alcohol-induced fatty liver are not clear [17]. The role of signaling via AMPK/SIRT-1 and SIRT-1/SREBP-1c should be clarified. Here, we examined the protective effect of ginsenosides against ethanol-induced hepatosteatosis in mouse hepatoma cells and the role of SREBP-1 acetylation in this effect.

 

Materials and Methods

Materials

KRG extract from the roots of 6-year-old specimens of Panax ginseng C.A. Meyer was purchased from the Korea Ginseng Corporation (Daejeon, Korea). Ginsenosides Rb1, Rb2, Rd, Rf, and Rg1 were obtained from Sigma-Aldrich (St. Louis, MO, USA). The SIRT-1 and SREBP-1 antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade and were used as received.

Cell culture and ethanol treatment

AML-12 cells were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were plated at 3×105 per well in 60-mm dishes, and cells were used at 70-80% confluency. Cells were maintained in Dulbecco's modified Eagle’s medium (Nutrient Mixture F-12; DMEM/F12) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 50 units/ml penicillin, 50 g/ml streptomycin, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone at 37℃ in a humidified 5% CO2 atmosphere. The KRG extract and ginsenosides were dissolved separately in phosphate-buffered saline (PBS) and added to the appropriate cells, which were incubated at 37℃ for the indicated time period. After incubation, cells were washed twice with ice-cold PBS before sample preparation.

Immunoblotting and immunoprecipitation

For western blot analysis, cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 8.0 containing 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 10 μl/ml protease inhibitor cocktail (Merck Millipore, Darmstadt, Germany) and cellular debris was cleared by centrifugation. The protein concentration in the lysate was determined using the BCATM protein assay kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. Total protein (40 μg) was separated by SDSPAGE on an 8% gel and transferred to PVDF membranes (Roche, Indianapolis, IL). After blocking in 5% BSA overnight, proteins were detected by incubation with primary antibodies overnight at 4℃, followed by incubation with HRP-conjugated secondary antibodies for 2 hr, and detection by an ImageQuant LAS-4000 mini (GE Healthcare, Little Chalfont, UK). Primary antibodies were used to detect SIRT1 (120 kDa), SREBP-1c (60-70 kDa, cleaved form), PPAR-α (52.5 kDa), and β-actin (42 kDa). For immunoprecipitation, the protein extracts were incubated overnight with the appropriate antibody at 4℃, followed by incubation overnight at 4℃ with a 50% slurry of protein G magnetic beads. After washing the immunoprecipitates 3 times with immunoprecipitation buffer, SDS-PAGE and western blotting were performed for the immunoprecipitated proteins as described above.

Oil red O staining

To analyze fat accumulation in the liver, cells were grown on a 6-well plate. After treatment, the cells were fixed in 4% formaldehyde in PBS for 1 hr and rinsed with 60% isopropanol, after which they were stained with Oil Red O solution.

SIRT1 activity assay

SIRT1 activity was measured using a SIRT1 fluorometric assay kit (Abcam, Cambridge, MA, USA) as recommended by the manufacturer. The cell lysate was mixed with SIRT1 assay buffer, fluoro-substrate peptide, and NAD+ at room temperature for 60 min. The reaction was stopped by the addition of 5 μl of developer reagent, and the fluorescence was subsequently monitored for 60 min at 350 nm (excitation) and 460 nm (emission), which was used to calculate the rate of the reaction.

Statistics

The mean value ± standard error (S.E.) was determined for each treatment group in a given experiment. Differences between the means of the individual groups were assessed by one-way analysis of variance (ANOVA) with Duncan’s multiple range test (SPSS 12.0; IBM Corp., Armonk, NY, USA). The threshold for significance was set at P <0.05.

 

Results and Discussions

Ethanol induced steatosis in hepatocytes has been associated with alcoholic fatty liver disease by in vitro and in vivo experiments. In this study, we used the AML-12 cell line, which are murine hepatocytes that express alcohol dehydrogenase and aldehyde dehydrogenase 2 proteins [8]. Therefore, ethanol can be metabolized in AML-12 cells, which are thus a model that can be used to investigate the protective effect of KRG against alcoholic fatty liver disease. Treatment of AML-12 cells with 100 mM ethanol for 5 days caused significant lipid accumulation in the cells, as revealed by Oil red O staining (Fig. 1A), and also down-regulated SIRT1 and up-regulated SREBP-1c in a time-dependent manner (Fig. 1B). This down-regulation of SIRT1 and up-regulation of SREBP-1c by ethanol exposure was similar to the results of previous studies [14, 24].

Fig. 1.Alcohol-induced lipid accumulation in AML-12 cells. AML-12 cells were treated with 100 mM ethanol for 5 days. Oil red O staining was performed after ethanol treatment (A), and the effects of ethanol on SIRT-1 and SREBP-1c expression were assayed via Western blotting (B).

Ethanol-induced lipid accumulation was significantly decreased by co-treatment with KRG extract at 100, 500, and 1,000 μg/ml (Fig. 2A). SIRT1 expression was significantly decreased by ethanol-treatment (100 mM), but ethanol-induced down-hrregulation of SIRT1 was dose-dependently restored by co-treatment with KRG extract at concentrations of 10-1,000 μg/ml (Fig. 2B). Moreover, acetyl-SREBP-1c was significantly and dose-dependently decreased by KRG extract, although this treatment did not change total SREBP-1c expression. KRG restored AMPK activity in AML-12 cells after it was reduced by ethanol treatment (Fig. 3). When KRG at a concentration of 500 μg/ml was co-administered with 100 mM ethanol for 24 hr, AMPK expression was increased and p-AMPK expression was restored in a time-dependent manner.

Fig. 2.The effect of KRG on acetyl-SREBP-1c in AML-12 cells. Oil red O staining (A) and Western blots for SIRT-1, SREBP-1c, and acetyl-SREBP-1c (B) after treatment with 100 mM ethanol for 3 days in combination with KRG extract at 0, 10, 100, or 1,000 μg/ml.

Fig. 3.The stimulatory effect of KRG on AMP-activated kinase (AMPK). Western blots for phospho-AMPK and AMPK after treatment with 100 mM ethanol and 500 μg/ml KRG extract for 24 hr.

AMPK is a molecular switch that can activate catabolic pathways and deactivate anabolic pathways [10]. The beneficial effects of ginseng and its active components on fat accumulation have been shown to be produced by activation of AMPK. For example, activation of the AMPK pathway mediated decreased hepatic fat accumulation produced by ginsenoside Rb1 in HFD-fed rats [18], and also reduced hepatic triglycerides and cholesterol levels in HepG2 cells produced by ginsenoside Rc [13]. In accordance with the activating effect of KRG extract on AMPK, KRG extract restored AMPK activation in ethanol-exposed hepatocytes. Moreover, AMPK, as a key regulator of cellular energy, controls the expression of genes involved in energy metabolism by acting in coordination with SIRT1 [1]. In AML-12 cells treated with 100 mM ethanol, restored SIRT1 expression might be mediated by activation of AMPK by KRG extract.

Among the bioactive saponins, polyacetylenes, sesquiterpenes, and polysaccharides found in ginseng, ginsenosides are known as the primary mediators of its pharmacological effects [6, 10]. Ginsenosides have been evaluated for their effects on molecular functions, but their effects on the SIRT1/SREBP-1c pathway have not been reported. Therefore, we investigated the effects of ginsenosides Rb2, Rd, and Rg1 at concentrations of 1 and 10 μg/ml in AML-12 cells treated with 100 mM ethanol for 3 days. As shown in Fig. 4A, Rb2 and Rd restored SIRT1 expression and reduced expression of SREBP-1c and acetyl-SREBP-1c in ethanol-treated AML-12 cells. The ethanol-induced expression of acetyl-SREBP-1c was significantly decreased by co-treatment with ginsenosides Rb2 and Rd, but not by treatment with Rg1. Additionally, SIRT1 activity was significantly increased by ginsenosides Rb2 and Rd (Table 1). The recovery of SIRT1 activity is an important mechanism in recovery from alcoholic liver steasteatosis, and deacetylation of SREBP-1c by SIRT1 reduces the stability of SREBP-1c. The hepatoprotective effects of ginsenoside Rb2 and Rd were confirmed by Oil red O staining in AML-12 cells, which showed that these compounds potently prevented lipid accumulation (Fig. 4B).

Table 1.SIRT-1 activity was calculated in AML-12 cells after treatment with ginsenosides Rb2, Rd, or Rg1 (10 μg/ml) and 100 mM ethanol for 3 days. The mean value ± standard error (S.E.) was determined for each treatment group (n = 3). The threshold for significance was p <0.05.

Fig. 4.The effects of individual ginsenosides on SIRT-1-mediated SREBP-1c deacetylation in AML-12 cells via Western blotting (A) and Oil red O staining (B). Western blots were used to assay SIRT-1, SREBP-1c, and acetyl- SREBP-1c expression in AML-12 cells after co-administration of 100 mM ethanol with ginsenoside Rb2, Rd, or Rg1 (1 and 10 μg/ml) for 3 days.

The pathology of alcoholic liver steatosis is initiated by ethanol-induced abnormal regulation of lipid metabolic processes involved in fatty acid oxidation and synthesis in the liver [4, 21, 23]. Several regulatory molecules are involved in this hepatotoxic effect of ethanol, including PPARα, SREBP-1c, and AMPK [11]. The preventive effects of KRG extract and ginsenosides Rb2 and Rd on lipid accumulation were mediated by decreased acetyl-SREBP-1c, which was caused by the activation of AMPK and SIRT1. Although the individual ginsenosides did not decrease SREBP-1c in a dose-dependent manner, the amount of acetyl-SREBP-1c was significantly decreased by treatment with Rb2 or Rd alone. Interestingly, while we tested the preventive effects of 3 ginsenosides, Rb2, Rd, and Rg1, only protopanaxadiol-type compounds Rb2 and Rd reduced fat accumulation in AML-12 cells; ginsenoside Rg1, a protopanaxatriol-type compound, did not produce this effect. Although the structural specificity of ginsenosides with regard to binding to AMPK and SIRT1 should be clarified by future studies, it is likely that the varying structures of individual ginsenosides produce a range of regulatory effects on SIRT1.

In this study, we investigated the hepatoprotective effects of ginsenoside Rd and Rb2 in the context of the molecular mechanisms underlying alcohol-induced fatty liver (Fig. 5). We found that ethanol may interfere with the activity of AMPK and SIRT1, and induce hyperacetylation of SREBP-1c as a result of decreased SIRT1 activity, which can increase SREBP-1c stability and enhance its function as a key lipogenic activator. KRG extract and ginsenosides Rd and Rb2 activate AMPK and SIRT1, which leads to destabilization of SREBP-1 via deacetylation, thus inhibiting the adverse effect of ethanol.

Fig. 5.Potential mechanisms of ginsenosides Rd and Rb2 underlying alcohol-induced fatty liver disease. Ethanol interferes with AMPK and SIRT-1 activity, which leads to hyperacetylation of SREBP-1c and accumulation of hepatic lipids. Ginsenosides Rd and Rb2 activate AMPK and SIRT-1, which destabilizes SREBP-1c via deacetylation, mitigating the adverse effect of ethanol.