Alisol A 24-acetate ameliorates nonalcoholic steatohepatitis by inhibiting oxidative stress and stimulating autophagy through the AMPK/mTOR pathway
Introduction
Nonalcoholic steatohepatitis (NASH) usually occurs as a consequence of nonalcoholic fatty liver disease (NAFLD), a condition associated predominantly with insulin resistance and metabolic syndrome [[1], [2], [3], [4], [5]]. Steatosis is thought to develop through the accumulation of free fatty acids (FFAs) that arise because of an imbalance between the synthesis and lipolysis of triglycerides [6]. In NASH, steatosis is combined with inflammation and fibrosis which over time can develop into cirrhosis of the liver [7]. At present, the only option for late-stage cirrhosis caused by NASH is liver transplantation because an effective therapeutic strategy is unavailable [8]. Therefore, a process that could regulate lipid homeostasis may provide a potential therapy.
A decrease in autophagy has been implicated in the development of NAFLD because autophagic pathways mediate the breakdown of intracellular lipids in hepatocytes and their activity is reduced under several conditions that predispose to NASH [9]. The possible mechanisms for the inhibition of autophagy in NAFLD include decreased expression of genes involved in autophagy, reduced levels of degradative lysosomal enzymes, hyperinsulinemia and impaired fusion of autophagosomes with lysosomes [[10], [11], [12]].
Rhizoma Alismatis is a common traditional herbal medicine derived from the dried rhizome of Alisma orientale (Sam.) Juzep. (Alismat aceae). It is mostly used as a diuretic in conditions such as oliguria and oedema but it is also used to control high cholesterol levels in hyperlipidemia [13,14]. Rhizoma Alismatis contains a number of triterpenoids including alisol A, B, and C, alisol A 24-acetate, alisol B 23-acetate, alismol, alismoxide, and epigalisol A [[15], [16], [17]]. Of these, alisols A, B and C and their monoacetates have demonstrated significant activity against hypercholesterolemia in rats [18]. Serum alanine transaminase (ALT) activity and triglyceride (TG) levels have indicated that they protect against carbon tetrachloride-induced liver damage in mice [19]. Recent research has found that alisol B 23-acetate significantly reduces hepatic triglyceride accumulation, inflammatory cell infiltration and hepatic fibrosis associated with NASH and dose-dependently decreased elevated activities of serum ALT and aspartate transaminase (AST) in mice through the activation of the farnesoid X receptor (FXR) [20]. FXR activation is thought to reduce liver steatosis and hyperlipidemia by suppressing lipogenesis and promoting the oxidation and hydrolysis of TGs [[21], [22], [23]]. AA has the molecular formula C32H52O6 and a molecular weight of 532.762 g/mol the chemical structure was first determined by chemical and spectral analysis using alisol as a reference (Fig. 1A) [24]. AA has three hydrogen donor bonds and six hydrogen acceptor bonds. Similarly, AA incubated with HepG2 cells, has resulted in significantly reduced lipid and intracellular TG levels [25].
In this study, we investigated the anti-NASH effect of AA and its underlying mechanisms in vitro using human hepatic stellate cell line LX-2 and in vivo using C57BL/6 mice fed a methionine and choline-deficient (MCD) diet to induce NASH. Liver oxidative stress, inflammatory indices and autophagy markers were examined. The results indicated that AA suppressed reactive oxygen species (ROS) and inflammation in the NASH mouse model and inhibited the expression of inflammatory cytokines and ROS in LX-2 cells grown in MCD medium. AMP-activated kinase (AMPK) has recently been found to reverse drug-induced hepatocellular damage through the regulation of autophagy [26]. An initiator of autophagy, Unc-51-like kinase-1 (ULK1), is thought to be phosphorylated by the mammalian target of rapamycin (mTOR) and AMPK which in turn activates downstream mediators to regulate autophagy [27]. In this work, we also assessed whether AA could influence the AMPK/mTOR/ULK1 pathway.
Section snippets
Chemicals and reagents
Alisol A 24-acetate and chenodeoxycholic acid (CDCA) with 98% purity were purchased from Shanghai Hitsanns Co., Ltd (Shanghai, China). Kits for analysis of alanine/aspartate aminotransferase (ALT/AST), malondialdehyde (MDA), myeloperoxidase (MPO), TG, total cholesterol (TCH) and FFA were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). H2DCFDA, RPMI1640, and fetal bovine serum (FBS) were purchased from Life Technology (Carlsbad, CA, USA). Pierce® BCA Protein Assay Kit
Liver injury induced by MCD is improved by AA
Mice fed an MCD diet were treated with 15, 30, and 60 mg kg−1 of AA daily. After 4 weeks, serum ALT and AST activities were assessed and liver ROS, MDA levels and MPO activity were evaluated (Fig. 1B–E). AA alleviated the effects of MCD in a dose-dependent manner. The addition of CDCA reduced this benefit slightly but not significantly. Images of H&E stained liver sections showed steatohepatitis with the macrovesicular accumulation of lipid deposits and inflammatory foci in the tissue of
Discussion
Autophagy has been implicated in the development of NASH because impaired autophagic function and defects in chaperone-mediated autophagy have been reported in a number of conditions that predispose to the disease [[30], [31], [32]]. In the present study, we found that AA may ameliorate NASH by inhibiting oxidative stress and stimulating autophagy and that this could involve the participation of the AMPK/mTOR/ULK1 pathway. We also found that dorsomorphin, a selective AMPK inhibitor, could
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81703879); the Budget Project of Shanghai University of Traditional Chinese Medicine (2016YSN60); Key specialties of Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (2016103A); Standardized training of specialists in Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (PTZP201605A).
References (44)
- et al.
Systematic review of risk factors for fibrosis progression in non-alcoholic steatohepatitis
J. Hepatol.
(2009) - et al.
A systematic review of follow-up biopsies reveals disease progression in patients with non-alcoholic fatty liver
J. Hepatol.
(2013) - et al.
Nonalcoholic fatty liver disease associated with metabolic syndrome: influence of liver fibrosis stages on characteristics of very low-density lipoproteins
Clin. Chim. Acta
(2017) - et al.
Current efforts and trends in the treatment of NASH
J. Hepatol.
(2015) - et al.
Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia
J. Biol. Chem.
(2009) Functions of autophagy in hepatic and pancreatic physiology and disease
Gastroenterology
(2011)- et al.
NF-κB activation represses tumor necrosis factor-α-induced autophagy
J. Biol. Chem.
(2006) - et al.
Carbon monoxide protects against hepatic steatosis in mice by inducing sestrin-2 via the PERK-eIF2α-ATF4 pathway
Free Radic. Biol. Med.
(2017) - et al.
Onconase induces autophagy sensitizing pancreatic cancer cells to gemcitabine and activates Akt/mTOR pathway in a ROS-dependent manner
Biochim. Biophys. Acta
(2015) - et al.
Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling
Cell. Signal.
(2010)
Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1
Free Radic. Biol. Med.
Biphasic effects of dietary antioxidants on oxidative stress-mediated carcinogenesis
Mech. Ageing Dev.
The structures of alisol B and alisol A monoacetate - occurrence of a facile acyl migration
Tetrahedron Lett.
Stability and structure studies on alisol a 24-acetate
Chem. Pharm. Bull. (Tokyo)
Astaxanthin prevents TGFβ1-induced pro-fibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells
Biochim. Biophys. Acta Gen. Subj.
The anti-hepatic fibrosis effects of dihydrotanshinone I are mediated by disrupting the yes-associated protein and transcriptional enhancer factor D2 complex and stimulating autophagy
Br. J. Pharmacol.
Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis
Nat. Rev. Gastroenterol. Hepatol.
Activation of insulin-PI3K/Akt-p70S6K pathway in hepatic stellate cells contributes to fibrosis in nonalcoholic steatohepatitis
Dig. Dis. Sci.
Molecular mediators of hepatic steatosis and liver injury
J. Clin. Invest.
From NAFLD to NASH to cirrhosis—new insights into disease mechanisms
Nat. Rev. Gastroenterol. Hepatol.
Function of autophagy in nonalcoholic fatty liver disease
Dig. Dis. Sci.
Altered lipid content inhibits autophagic vesicular fusion
Faseb. J.
Alisma orientalis (sam.) Juzep
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These authors contributed equally to this work.