Study On The Anti - Inflammatory And Anti - Oxidative Mechanisms Of Gastrodin In The Treatment Of Nonalcoholic Fatty Liver Disease

The nonalcoholic fatty liver disease (NAFLD) is a pathological syndrome which is characterized by excess fat accumulation in the liver caused by non-alcoholic factors. The major clinical features of NAFLD include liver steatosis, liver function damage, and so on. So far, there is no mono-therapy approved for the treatment of NAFLD in clinic. With the increase of obese people worldwide, the number of patients with NAFLD increased greatly in recent years. It’s urgent to find novel agents for the treatment of NAFLD. The Gastrodia elata BI. (Tianma) is an ancient Chinese medicinal herb that has been recorded to have pharmacological effects against headache, limb numbness, and convulsion. Gastrodin (GSTD, MW:295.3) is one of the major active components of Gastrodia elata BI.; it is now used in clinic to treat nervous system diseases. In our previous studies, we found that GSTD could suppress the oleic acid (OA)-induced steatosis in hepatocytes through activation of adenosine monophosphate-activated protein kinase (AMPK). In addition, the powder of Gastrodia elata BI. could ameliorate diet-induced liver steatosis greatly in rats. There were also reports suggested that GSTD had obvious antioxidative, anti-inflammatory effects in cardiomyocytes and neurons. The development and progression of NAFLD had close relationship with oxidative stress and proinflammatory response. So in the present study, we investigate the antioxidative and anti-inflammatory activities of GSTD in NAFLD and explore the possible signaling pathways.For the in vitro studies, HL-7702 cells were subjected to serum starvation for 24 h before experiment. The cells were then treated with 0.6 mmol/L of OA for 24 h to induce steatosis. At the same time, GSTD was added to treat the cells. Specific siRNA was used to transfect the cells in order to silence the expression of target genes; compound C (10μmol/L) was used to pretreat the cells for 1 h in the blocking experiments. After treatment, cells were harvested for the extraction of proteins and total RNAs for western blot and real-time reverse-transcription polymerase chain reaction (RT-PCR) experiments. In addition, oxidative stress and proinflammatory response-related parameters were determined. In one of the animal experiments, some of the male C57BL/6J mice were fed with regular rodent diet as the normal diet control group, while other mice were fed with a high-fat diet (HFD) with 60% kcal fat,20% kcal carbohydrate and 20% kcal protein. Mice fed with HFD were divided into 4 groups, which were left untreated (HFD control) or treated with 10 mg/kg,20 mg/kg or 50 mg/kg of GSTD at the same time of HFD-feeding, respectively. The duration of treatment was 10 weeks, during which body weights and food intake of the mice were recorded twice weekly. At the end of experiment, mice were fasted for 12h, blood samples were collected by retro-orbital puncture, serum cholesterol (CHO), triglyceride (TG), glucose and insulin levels were determined; liver and epididymal adipose tissues were collected and weighed, relative parameters were measured. In another set of animal experiment, male sprague-dawley (S.D.) rats were used. Some rats were fed with regular rodent diet as normal diet control group, while other rats were fed with a high-fat and high-CHO (HFHC, containing 10% lard,0.5% CHO,0.1% cholate and 89.4% regular rodent diet) diet to induce NAFLD. Eight weeks later, these rats were left untreated or treated with GSTD at 20 mg/kg or 50 mg/kg, respectively, for 10 weeks. Body weights and food intake of the rats were recorded twice weekly. At the beginning of the experiment, at the end of week 8 and then at the end of experiment, blood samples were collected by retro-orbital puncture after 12h of fasting for the measurement of serum CHO, TG, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels. At the end of the experiment, rats were sacrificed; their livers were harvested and weighed, relative parameters were measured.In the in vitro studies, our results showed that OA-treatment could induce oxidative stress and proinflammatory response in HL-7702 cells. GSTD could significantly increase the superoxide dismutase (SOD) activity but decrease reactive oxygen species (ROS)/malondialdehyde (MDA) in HL-7702 cells. Meanwhile, the mRNA expression levels of proinflammatory cytokines like tumor necrosis factor-a (TNF-α), interleukin-6 (IL-6), and cyclooxygenase-2 (COX2) were down-regulated by GSTD. In HL-7702 cells, GSTD could promote the phosphorylation of nuclear factor erythroid-2-related factor-2 (Nrf2) at serine 40 (Ser40) and enhance its nuclear translocation. The transcriptional activity of the antioxidant response element (ARE) and the expression level of heme oxygenase-1 (HO-1) was then stimulated or up-regulated. In addition, GSTD could activate AMPK by stimulating the liver kinase B1 (LKB1). Nrf2 siRNA could totally block the stimulating activities of GSTD on the nuclear translocation of Nrf2, the transcriptional activity of ARE, and the expression of HO-1. The inhibitory effects of GSTD on the OA-induced oxidative stress and proinflammatory response were abolished by Nrf2 siRNA as well. Furthermore, the stimulating activities of GSTD on the AMPK/Nrf2/HO-1 pathway and its antioxidative/anti-inflammatory activities were totally abolished by compound C, a specific inhibitor of AMPK. In the rat and mouse models of NAFLD, GSTD greatly suppressed liver steatosis and infiltration of inflammatory cells; reduced liver TG accumulation, liver weight and index; lowered serum TG, glucose, insulin, ALT and AST levels. GSTD also suppressed extra body weight gain and the weight of epididymal fat. In the liver, GSTD greatly increased SOD activity, reduced MDA content, and down-regulated the mRNA expression levels of proinflammatory cytokines like TNF-a, IL-6, and COX2. At the same time, GSTD significantly activated the AMPK/Nrf2/HO-1 pathway in the liver and up-regulated the expression of HO-1.In summary, our results demonstrate that GSTD up-regulates the hepatic HO-1 expression through activating the AMPK/Nrf2 signaling pathway. As a result, the oxidative stress/proinflammatory response in the NAFLD is ameliorated; meanwhile, lipid metabolism in the liver is improved. Our findings indicate that GSTD may be developed as a novel kind of agent for the treatment of NAFLD in clinic in the future.