The Role And Mechanism Of Epoxyeicosatrienoic Acids In Nonalcoholic Steatohepatitis

[BACKGROUND&OB JECTIVE]Non-alcoholic fatty liver disease (NAFLD) is a common liver disease with high prevalence in developed countries. NAFLD are a broad spectrum of diseases, including simple fatty liver, steatohepatitis, liver fibrosis and cirrhosis.20%～30%of patients with nonalcoholic fatty liver disease will progress to nonalcoholic steatohepatitis (NASH), which is distinguished from pure steatosis by the presence of hepatocellular ballooning and inflammation in liver, and may ultimately progress to cirrhosis and hepatocellular carcinoma (HCC).At present, the exact mechanism of NASH is not fully understood. The commonly accepted hypothesis for the development of steatohepatitis is ’two-hit’ model.The "first hit" is peripheral and hepatic insulin resistance, which results in steatosis. The "second hit" involves a complex interaction of several factors, including oxidative stress, lipid peroxidation and overproduction of proinflammatory cytokines (such as TNF-a, IL-1and IL-6), which lead to inflammation and ultimately fibrosis.TNF-a is considered to be one of the most important cytokines responsible for the progression to NASH. TNF-a can1) combine with receptors on the liver cell membrane, activate Casepase-8, and then activate Casepase-3, leading to liver cell apoptosis;2) inhibit the mitochondrial respiratory function, increase ROS and lipid peroxide and cause liver damage and inflammation;3) activate other inflammatory cytokines such as IL-6, IL-8, and adhesion molecules, resulting in amplification of liver inflammation. It was reported that macrophage plays an important role in the pathogenesis of NASH. In the high-fat diet induced rats NASH model, the liver kupffer cells were largely activated, withgreat amounts of production of proinflammatory cytokines and ROS, causeing liver cell damage. In the MCD diet induced mice NASH model, mRNA expression of TNF a and MCP-1, the potent mediator of macrophage activity, increased significantly in the liver. The role of macrophages involved in the pathogenesis of NASH includes two aspects:on the one hand, macrophages is not able to correctly identify and eliminate risks, on the other hand, inflammation cannot be inhibited due to the overreaction of cytotoxicity.Epoxyeicosatrienoic acids belong to one of the arachidonic acid (AA) metabolites, and are generated through cytochrome P450epoxidation. The epoxygenase pathway results in the formation of four regioisomeric epoxyeicosatrienoic acids (EETs),5,6-EET,8,9-EET,11,12-EET, and14,15-EET. EETs are quickly metabolized by soluble epoxidehydrolase (she) to their less active dihydroxyeicosatrienoic acids (DHETs) in the body, making it difficult to study these lipid epoxides. It has been reported that she inhibitors are able to prevent EET degradation and subsequently increase endogenous levels of EETs.Several studies have found that one or more EETs may possess anti-inflammatory property in different disease entities, but their presence and functional role in non-alcoholic steatohepatitis have not been demonstrated.[METHODS]1. To establish NASH mice model, C57BL/6mice have been fed with MCD diet for6weeks. She inhibitor TPPUwas added into the feeding water for mice in the treatment group. The control mice were fed with MCS diet. The serum and liver tissue samples were collected at week1,2,4,5,6. Liver function (ALT/AST), liver and blood lipid levels (TC/TG) were tested by automatic biochemical analyzer; liver EETs level, peripheral and liver proinflammatory cytokines (TNF alpha, IL-6, IL-1) and chemokines (MCP-1, IL-8) expression levels were detected by ELISA; liver injury was observed by HE staining, and liver steatosis was observed by oil red staining. PPAR a, ICAM1, VCAM1mRNA expression in the liver were tested by Realtime-PCR assay. 2. HepG2and THP-1cells co-culture system was established using Transwell Chambers. The free fatty acids (oleic acid and palmitic acid) were added into the co-culture syste to creat the high fat environment,4kinds of EET isomers were respectively added into the system as an intervention treatment. The levels of inflammatory cytokines and chemokines were tested by ELISA assay. Morphological changes of HepG2cell were observed by Oil red staining.3. General expression of NF-kB in liver was dected by the immunohistochemistrytechnique in the MCD group, MCS group and MCD+TPPU group. Paraffin oil was intraperitoneally injected into C57BL/6mice, abdominal macrophages were collected3days later. Macrophages have been cultured with free fatty acid for12hours, four EET isomers (5,6-EET,8,9-EET,11,12-EET,14,15-EET) were respectively added into the treatment group. After the treatment, the expression of NF-kB in the cell nucleus was observed by laser confocal microscopy.[RESULTS]1. In mice fed MCD diet for6weeks, the body weight and liver weight were significantly decreased, with appearant fat deposition, hepatocellular ballooning degeneration and dot necrosis, accompanied by inflammatory cells infiltration in the liver. Meanwhile, the liver function (ALT, AST) and intrahepatic triglyceride level increased significantly, but serum triglyceride and cholesterol levels dropped. After TPPU treatment, liver EETs content significantly increased, hepatocyte injury and inflammatory cells infiltration were improved, liver function and liver triglyceride level decreased significantly, but the lipid levels and hepatic fat deposition had no significant changes.2. In MCD diet group, liver and serum levels of proinflammatory cytokines (TNF a, IL-1, IL-6) and related chemokines (IL-8, MCP-1) have increased, especially TNF a, IL-1and MCP-1. In TPPU treatment group, these indicators were dramatically declined. TPPU treatment also led to decreased expression of adhesion molecules (ICAM1, VCAM1), but increased expression of peroxisome roliferators-activated receptor (PPAR-a) in mice. 3. In order to further investigate whether EETs are able to reduce the release of proinflammatory cytokines induced by free fatty acid (FFA) and to alleviate liver damage in vitro, four EET isomers (5,6-EET,8,9-EET,11,12-EET and14,15-EET) were respectively added into the following:1) HepG2cells2) THP1cells3) HepG2and THP1cells co-culture system and incubated for24hour. It is found that11,12-EET significantly reduced the levels of TNF a, MCP-1and IL-lb in HepG2and THP-1cells co-culture system under FFA stimulation.5,6-EET and8,9-EET could also inhibit the release of TNF a and IL-lb, but the effect was weaker.14,15-EET did not show the inhibiting effect.11,12-EET was also found to have similar inhibiting effect for THP-1cells alone, but not so strong as in the HepG2and THP-1cell co-culture system. But this phenomenon was not observed in HepG2cells.4. The liver NF-κB expression was significantly increased in mice of MCD feeding group than in MCS feeding group, while TPPU treatment strongly downregulated NF-κB expression in mice fed MCD diet. It was observed through confocal laser microscopy that NF-κB expression in the nucleus of primary macrophages increased significantly under FFA stimulation, and11,12-EET treatment could suppress the phenomenon.[CONCLUSION]1. TPPU can increase the EETs content in mice, thus improve the liver inflammation, liver function, reduce liver triglyceride level in MCD diet induced steatohepatitis in mice, but had no obvious effect to hepatic steatosis.2. In MCD diet induced NASH mice model, TPPU can significantly reduce the serum and liver levels of proinflammatory cytokines and chemokines. It also downregulates the expression of inflammation related adhesion molecules, but raises the levels of PPAR-..which contributes to lipid metabolism.3. In the hepG2and THP1cells co-culture system,11,12-EET can significantly reduce FFA induced release of proinflammatory cytokines by THP1cells.5,6-EET and8,9- EET have a similar but weaker effect,whereas14,15-EET has no such inhibiting effect.4. Both in-vivo and in-vitro studies show that NF-kB is activated and translocates into the nucleus of macrophages in NASH mice and this pathway can be inhibited by11,12-EET.It is suggested that11,12-EET is likely to play a protective role in the development of NASH by inhibiting the activation of NF-kB in macrophages.