| BackgroundNonalcoholic Fatty Liver Disease (NAFLD) which was coined by Ludwing in 1980 early is a clinical syndrome characterized by the excessive fat accumulation in the liver and hepatic cellular degeneration even in the absence of alcohol consumption. It encompasses a spectrum of disease including simple steatosis (triglyceride (TG) over-accumulation), nonalcoholic steatohepatitis (NASH, characterized by steatosis, hepatocellular damage, and lobular inflammation), cirrhosis and hepatocellular carcinoma. NAFLD becomes the most common liver disease, whose incidence is paralleling the increasing numbers of overweight and obese individuals worldwide. NAFLD affects 10% to 24% of the general population in various countries and the prevalence has even been up to 75 percent in obese people. In our country, NAFLD has become the second to the viral hepatitis liver disease, the incidence of liver fibrosis is as high as 25%, and about 1.5%～8.0% of the patients can develop liver cirrhosis, especially the incidence of NASH with obesity patients can rise to 58%～74%. However, NAFLD currently has not ideal therapeutic drug with stable outcomes and without side effect. Therefore, it has become an important topic for searching effective drugs for preventing and controling NAFLD.The pathogenesis of NAFLD is extremely complex, and the recognized theory is still the "two hits" hypothesis. The "first hit" is the deposition of liver free fatty acid and triglyceride in hepatocytes (steatosis). The second "hit", steatosis progresses to NASH and this progress is associated with factors such as oxidative stress, mitochondrial dysfunction, and cytokines capable of inducing inflammation, fibrosis, or necrosis. The elevated cytokine interactions with oxidative stress mediators and lipid peroxides have been postulated to play a role in induction of steatohepatitis. tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) are important cytokines in the development of many forms of liver injury.NAFLD begins with the accumulation of lipids in the liver. Therefore, the study of lipid metabolic mechanism become an important direction. In lipid metabolism, effects on lipid synthesis in NAFLD involve such as the Sterol Regulatory Element Binding Proteins (SREBPs), and fatty acid oxidation, through effects on Adiponectin, AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptors (PPARs). Adiponectin has fuction of regulating energy balance and fat metabolism, anti-steatotic and anti-inflammatory; AMPK is a key player in regulating energy metabolism and fatty acid oxidation; PPARa is a nuclear receptor highly expressed in the liver, which is able to exert its transcriptional effects on genes involved in fatty acid oxidation, uptake as well as inflammation. In addition, Adiponectin achieves its function in the liver likely via activating AMPK and PPARa pathways, and inhibition of SREBP-lc signaling. AMPK activation mediates suppression of lipogenic gene expression involved in triglyceride synthesis and accumulation (such as ACC) via decreasing the actions of transcriptional factors SREBP-lc. Adiponectin can also inhibit the production of TNF-a and IL-6, whereas TNF-a and IL-6 can suppress the expression of Adiponectin. Studies have repoted Adiponectin, AMPK and PPARa knockout mice developed hepatic steatosis, and patients with steatosis have decreased serum levels of Adiponectin, AMPK and PPARa.Currently there is no ideal therapeutic drug for NAFLD. Hugan Qingzhi Tablet (HQT), an empirical formula of a famous and old Chinese doctor for preventing and controlling fatty liver disease clinically in the Zhujiang hospital, Southern medical university, which consists of six traditional Chinese medicines, alisma, notoginseng, cattail pollen, the lotus leaf, hawthorn, dried tangerine and orange peel. HQT has a long history of use to alleviate NAFLD in clinical practice with the exact effect therapy in improving blood biochemistry and recovering body weight. Our previous study showed that HQT reduced the liver index and serum triglycerides, and increased the antioxidant capacity in a NAFLD rat model. Further mechanistic exploration revealed that this tablet ameliotates hepatic steatosis by activating AMPK and PPARa pathways in L02 cells and HepG2 cells. In order to clear and analyze the mechanism of HQT, the topic selected Alisol A 24-acetate, Ursolic acid, and Nuciferine as major active ingredients in Rhizoma Alismatis, Fructus Crataegi, and Folium Nelumbinis, respectively, which are major components in HQT, to explor their pharmacological effects in lipid metabolism and possible mechanisms. Alisol A 24-acetate has multiple biologic activities such as hypolipidemic, anti-inflammatory and anti-diabetic, but no reports of NAFLD; Ursolic acid has many kinds of pharmacological activities such as protect liver, anti-inflammatory, hypoglycemic, anti-oxidation and hypolipidemic, and studies have shown that it can improve steatosis and lipid metabolism disorders in NAFLD rats induced by high-fat-diet; Nuciferine can also prevent hepatic steatosis, lipid metabolic disorders and liver damage in NAFLD hamster induced by high-fat-diet. Thus we hypothesized that Alisol A 24-acetate, Ursolic acid, and Nuciferine would have lipid metabolic effects on NAFLD and possible related mechanisms.ObjectiveThis topic used the free fatty acid (FFA) (a mixture of oleic acid and palmitic acid, oleic acid:palmitic acid=2:1) induced HepG2 cells to establish hepatic steatosis cell model, which was designed to study the lipid metabolic effects and possible mechanisms of Alisol A 24-acetate, Ursolic acid, and Nuciferine as the main active ingredients of HQT on hepatic steatosis HepG2 cell model, so that it would further declare that the main material foundation which HQT play clinical curative effect.MethodsHepG2 cells cultured with medium containing different concentrations of Alisol A 24-acetate, Ursolic acid and Nuciferine, and MTT was performed to determine the cell viability to screen the best action concentration of drugs. This experiment was randomly divided into normal group (Con), model group (FFA), Alisol A 24-acetate group (FA), Ursolic acid group (FU), Nuciferine group (FN), Fenofibrate group (FF). HepG2 cells in normal group were incubated in culture medium containing 1% BSA for 24h, while in model group were treated with lmM FFA for 24h. HepG2 cells in drug groups separately were incubated in culture medium containing the final concentration of 10μuM Alisol A 24-acetate,80μM Ursolic acid and 50μM Nuciferine together with the final concentration of lmM for 24h. Oil red staining was performed to observe cellular morphology intracellular lipid droplets in different groups, and oil O red staining lipid contents were determinated. The kit was used to detect intracellular TG contents. Elisa was used to detect the levels of TNF-a, IL-6 and Adiponectin in the cell cuture supernatant. The mRNA expression of Adiponectin, AMPK, SREBP-lc, ACC and PPARa were determinated by RT-PCR; and the protein expression of p-AMPKa, SREBP-1, p-ACC and PPARa were determinated by Western blot.Results1. There was a dose-dependent relationship between the concentrations of Alisol A 24-acetate, Ursolic acid, Nuciferine and viability of HepG2 cells. During exposure to drugs for 24h, cell viability was reduced as the drug concentrations increased. When Alisol A 24-acetate was 10μM, Ursolic acid 80μM, Nuciferine 50μM, their viability were 91.5%,89.5% and 87.2%, respectively.2. Oil red O staining results showed that there were severe hepatic steatosis and a lot of red lipid droplets in model group, while the numbers of red lipid droplets significantly reduced in Alisol A 24-acetate, Ursolic acid and Nuciferine groups. Oil red O staining lipid content in model group was obviously higer than normal group (p<0.01), however, Alisol A 24-acetate, Nuciferine and Fenofibrate significantly reduced the lipid contents (p<0.05 or p<0.01), and Ursolic acid decreased lipid content but there was no statistical difference. In addition, intracellular TG content in model group was significantly higer than normal group (p<0.01),Alisol A 24-acetate, Ursolic acid, Nuciferine and Fenofibrate all significantly decreased intracellular TG contents (p<0.01).3. Enzyme-linked immunosorbent assay (Elisa) results showed that inflammatory factors TNF-a and IL-6 levels were increased significantly in model group (p<0.01), the increase was significantly reverted by Alisol A 24-acetate, Ursolic acid and Nuciferine (p<0.01). However, the results showed that Fenofibrate had no significant inhibition of TNF-α and IL-6 levels (p>0.05).4. Compared with normal group, Adiponectin mRNA and Adiponectin secretory protein expression were significantly lower in model group (p<0.01), Alisol A 24-acetate and Fenofibrate significantly increased the expression of Adiponectin at the two levels (p<0.05 or p<0.01); Ursolic acid and Nuciferine didn’t increased the Adiponectin mRNA expression, but raised its expression at the secretory protein level (p<0.05 orp<0.01).5. Compared with the normal group, AMPK mRNA expression reduced significantly, and ACC mRNA and SREBP-lc mRNA expression significantly increased in the model group (p<0.01); Alisol A 24-acetate, Ursolic acid and Nuciferine significantly down-regulated the ACC mRNA and SREBP-lc mRNA expression and up-regulated the AMPK mRNA expression (p<0.05 or p<0.01); Fenofibrate lowered the ACC mRNA and SREBP-lc mRNA expression without raising the AMPK mRNA expression. At the protein level, compared with normal group, p-AMPKa level was decreased significantly (p<0.01), and p-ACC and SREBP-1 levels were significantly increased (p<0.05 or p<0.01); Alisol A 24-acetate, Ursolic acid, Nuciferine and Fenofibrate obviously increased the levels of p-AMPKa (p<0.01), and decreased p-ACC and SREBP-lc levels (p< 0.05,p< 0.01).6. Compared with the normal group, PPARa mRNA and protein expression were significantly reduced in the model group (p<0.01); Ursolic acid, Nuciferine and Fenofibrate significantly increased the PPARa expression at mRNA and protein levels, while Alisol A24-acetate has no effect on the expression of PPARa (p>0.05).Conclusion1. The cell viabilities of 10μM Alisol A 24-acetate,80μM Ursolic acid and 50μM Nuciferine on HepG2 were about 90%, and they were the optimum action concentration.2. Alisol A 24-acetate, Ursolic acid and Nuciferine have effects on obviously reduing lipid accumulation and inhibiting the production of TNF-a and IL-6 in hepatic steatosis HepG2 cells. Fenofibrate could obviously decrease lipid accumulation but not suppress the production of TNF-a and IL-6 in hepatic steatosis HepG2 cells. To three major active components, Alisol A 24-acetate has the best effect.3. Alisol A 24-acetate up-regulated the expression of Adiponectin, and activated the AMPK signaling pathway thereby down-regulating SREBP-lc, ACC expressions; Ursolic acid, Nuciferine and Fenofibrate up-regulated the expression of Adiponectin, and activated AMPK signaling pathway thereby down-regulating SREBP-1c, ACC expressions, and activated PPARa signaling pathway, thereby to regulate the pharmacological effects of lipid metabolism, these might be the main material foundation and mechanisms that HQT play clinical curative effect on preventing and treating for NAFLD. |
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