泽泻提取物对油酸诱导建鲤脂肪肝细胞损伤中生化指标及CYP1A蛋白表达的影响

Effects of Rhizoma Alismatis Extract on Biochemical Index and CYP1A Protein Expression of Hepatocyte Steatosis Injury Induced by Oleic Acid in Jian Carp( Cyprinus carpio var. Jian)

为了研究中药泽泻提取物对油酸诱导建鲤脂肪肝变性细胞的保护作用,本实验以0.4 mmol·L-1油酸来构建建鲤原代脂肪肝细胞损伤模型,然后用不同浓度的泽泻提取物处理肝细胞24和48 h后,分别收集细胞和细胞培养液,然后测定谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、甘油三酯(TG)、总胆固醇(TC)、碱性磷酸酶(AKP)、胆碱酯酶(CHE)、细胞色素酶P4501A1(CYP1A1)和细胞色素酶P4502E1(CYP2E1)的含量等生化指标以及CYP1A的蛋白表达情况。结果显示,0.4 mmol·L-1的油酸与原代肝细胞共培养48 h可以显著提高肝细胞培养上清中GPT、GOT、TG、TC、AKP、CHE、LDH、CYP1A1和CYP2E1的含量(P<0.01或者P<0.05),可以诱导肝细胞中CYP1A的蛋白表达。将不同浓度的泽泻提取物(0、1、5、10、20、50μg·m L-1)与脂肪肝细胞共培养24~48 h发现,泽泻提取物能不同程度抑制油酸诱导的GOT、GPT、TG、TC、LDH、AKP、CHE水平和CYP1A1、CYP2 E1含量的升高(P<0.01或者P<0.05),不同程度降低肝细胞中CYP1 A蛋白表达,以作用时间48 h效果较好。研究证实了泽泻提取物对油酸诱导的鱼类脂肪肝细胞损伤具有保护作用,而泽泻提取物作为鱼类脂肪肝的防治药物还需要进一步的在体研究。

Fish fatty liver is one of increasingly serious diseases in aquaculture, it has become more and more serious in China aquaculture, In recent years, the incidence of fatty liver in fish had an increasing trend, but the occurrence mechanism of the fatty liver was still not very clear. Cytochrome P450 enzymes were the main oxidases metabolic enzymes in the liver microsome and played a very important role in the liver metabolism. However, no effective methods have been found for the prevention and treatment of this disease. The present study aimed to develop an in vitro model of hepatocyte steatosis using oleic acid as hepatotoxicant and evaluate the protective effects of Rhizoma Alismatis extracts against oleic acid induced hepatocyte steatosis in Jian carp. The primary cultured hepatocytes in Jian Carp were isolated and purified by trypsin digestion, cultured in vitro. The activities of glutamatepyruvate transaminase （GPT）, aspertate aminotransferase （GOT）, triglycerides （TG）, total cholesterol （TC）, alkaline phosphatase （AKP）, cholinesterase （CHE）, lactate dehydrogenase （LDH） were detected. The content of enzyme cytochrome P4501A1 （ CYP1A1 ） and enzyme cytochrome P4502E1 were detected by double antibody sandwichELISA method. CYP1A expression in the hepatocytes was analyzed by Western blot method. The results showed, the hepatocytes of Jian Carp grew well and had a good morphology, and they could be used for investigation of lipid metabolism. Exposure of the hepatocytes to 0.4 mmol/L oleic acid for 48 h, significantly elevated the levels of glutamatepyruvate trmlsaminase （ GPT） , aspertate aminotransferase （ GOT）, triglycerides （TG） , total cholesterol （ TC ） , alkaline phosphatase （AKP） ,cholinesterase （CHE） , lactate dehydrogenase （LDH） , the enzyme eytochrome P4501 A1 （CYP1A1） and the enzyme eytochrome P4502E1 （ P 〈 0.01 or P 〈 0.05 ）. After the primary hepatocytes treatment with five concentrations of Rhizoma Alismatis extract （0, 1, 5, 10, 20, 50 μg/mL） for 24 -48 hours, results showed that Rhizoma Alismatis extract could suppress the elevations of GOT, GPT, TG, TC, LDH, AKP, CI-IE, CYP1 A1, CYP2E1 （ P 〈 0. 01 or P 〈 0.05 ）, and reduced the protein expression of CYP1A to varying degrees, the most optimal action time is 48 h. It can be concluded that Rhizoma Alismatis extract exhibited protective effect against hepatocyte stcatosis in Jian carp. Further in vivo studies are needed to provide more evidence for using Rhizoma Alismatis extract as a hepatoprotective agent for the treatment of hepatocyte steatosis.