The Role Of PLA₂ On The Mechanism Of Hepatocytic Lipoapoptosis Of NAFLD In SD Rat

Background:Non-alcoholic Fatty Liver Disease(NAFLD) is a kind of clinical and pathological syndrome which was not caused by the damage of alchohol and other known factors. It's character is diffuse bullous adipose degeneration in which encompasses a spectrum of hepatic histological lesions ranging from hepatic steatosis, steatohepatitis, hepatic fibrosis to cirrhosis. Lipoapoptosis of hepatocyte is the most distinctive pathological features of the NAFLD. Studies have showed that the abnormal metabolism of Free Fatty Acide (FFA) is the leading cause of NAFLD, But the precise mechanism of NAFLD is still unclear. Recently some studies reported that most free fatty acides entered non-oxidative pathway and the metabolic products such as activated fatty acids probably were the crucial factors which resulted in hepatocytic lipoapoptosis. FFA metabolized by phosphatidic acid/diacylglycerol(DAG) pathway was more easily to make hepatocytic lipoapoptosis. DAG can synthetize phosphatidylcholine (PC), PC can active phosphatidase, then phosphatidase hydrolyzed PC to lysophosphatidyl choline (LPC), and LPC was the crucial factor which made many kinds of cells apoptosis. PhosphatidaseA2 (PLA2) is the unique enzyme among all phosphatidase which can hydrolyze glycerophospholipide to produce lysophosphatide and free fatty acide. However it is still unknown whether PLA2/LPC pathway activation play an important role in hepatocyte lipoapoptosis in NAFLD, and whether excessive activation of PLA2 was the crucial event in the mechanism of NAFLD.In this study, we used high-fat diet induced NAFLD model, in which the process was similar to that of human body. First of all, we detected the basic pathological changes of hepatocyte in vivo, including lipid aggregation, apoptosis level and appearance in electronic microscope. Secondly, we detected the gene levels of every subtype PLA2 and their activations and analyzed the relationship between the pathological changes and PLA2 level. Finally, we evaluated the pathological changes after PLA2 inhibitors were used, and explored the role of PLA2 or it's subtype in the mechanism of the lipoapoptosis of hepatocyte caused by FFA. This study may clarify the prime mechnism of the hepatocytic lipoapoptosis caused by FFA from molecule level.Objectives:To analyze the relationship of the basic pathological changes of hepatocyte and PLA2 or it's subtype in SD NAFLD model, and to evaluate the effect of PLA2 inhibitor (technique used medicine inhibitor) on the hepatocytic lipoapoptosis,as to clarify the role of PLA2 on the process of hepatocytic lipoapoptosis caused by FFA.Methods:52 male SD rats were randomly divided into six groups:Group I:Ordinary diet feeding for 18 weeks. Groupâ…¡:High-fat diet feeding group interfered with DMSO. Groupâ…¢:High-fat diet feeding group interfered with inhibitor of PLA2 (MAFP). Groupâ…£:High-fat diet feeding group interfered with inhibitor of Cytosolic PLA2 (ACCOCF3); Group V:High-fat diet feeding group interfered with inhibitor of calcium-independent phospholipaseA2(BEL); Groupâ…¥:High-fat diet feeding group interfered with inhibitor of secretary PLA2;â…¡-â…¥Groups:Feed High-fat diet for 18 weeks. From the 15th week, we gave Group II DMSO intraperitoneal injection qod, Groupâ…¢MAFP intraperitoneal injection qod, Groupâ…£ACCCOCF3 intraperitoneal injection qod, Groupâ…¤BEL intraperitoneal injection qod;,Group VIsPLA2 inhibitor intraperitoneal injection qod. Serum lipid biochemistry indexes,insulin resistant indexes and PLA2 levels and other indicators were measured and compared. Expression of PLA2 mRNA was also determined in each group. At same time the basic pathological changes, lipoapoptosis of hepatocyte and the pathological changes in Electronic microscope were detected.Results:Compared with groupâ… , the basic pathological changes of groupâ…¡was diffuse bullous adipose degeneration acompanied by balloon degeneration and inflammation cell infiltrating and local cellular necrosis. Dyeing with TUNEL, groupâ…¡has many apoptosis cells while groupâ… has fewer apoptosis cell. Electronic microscope study showed hepatocytic mitochondrion swelled and lophos obscure in groupâ…¡Compared with groupâ… , serum concentration of cPLA2 and iPLA2 and expression of cPLA2 and iPLA2 mRNA were higher in groupâ…¡,while sPLA2 had no obvious change. Among the groups treated with various PLA2 inhibitor, the basic pathological changes have no difference, however the lipoapoptosis of hepatocyte were decreased in groupâ…£and groupâ…¤, which were treated with cPLA2 and iPLA2 inhibitors, and the injury of mitochondrion also decreased in these two groups. The index of insulin resistance in groupâ…¤was decreased,and the level of serum leptin was decreased in groupâ…£.Conclusion:1. PLA2 plays an important role in the process of NAFLD, and the mainly subtypes of it are cPLA2 and iPLA2.2. Blocking iPLA2 and cPLA2 activity can improve lipoapoptosis of hepatocyte. Blocking iPLA2 can improve insulin resistance and blocking cPLA2 can degrade serum concentation of leptin on rats with NAFLD.