Cytochrome P450Epoxygenase2J2Overexpression Ameliorates Hyperlipidemia Via Increased Fatty Acid Oxidation

Hyperlipidemia, also known as lipid metabolism disorder, can be defined as a disease in which total triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels are increased while there is a decrease in high-density lipoprotein (HDL). Hyperlipidemia is a major risk factor for the development of hunman atherosclerosis and is associated with the morbidity and mortality of cardiovascular disease (CVD) such as coronary heart disease, myocardial infarction and renal disease, insulin resistance and fatty liver. Epidemiological studies have shown that with the improvement of our country’s economic development and people’s living standard, the incidence of hyperlipidemia also showed an increasing trend. If we could not make effective interventions, hyperlipidemia and its related diseases will be an important risk factor for human health and their quality of life.Arachidonic acid (AA) is polyunsaturated fatty acids which was the most widely distributed and abundant in vivo. Moreover, arachidonic acid is also the precursor of numerous important biological active substances. When the cells were exposed to various physiological or pathological stimuli, membrane phospholipids was lipolyzed by phospholipase A2(PLA2) and arachidonic acid (AA) was released from cell membrane into cytoplasm. Arachidonic acid (AA) was metabolized by three metabolic pathways, which were consisted of cyclooxygenase (COX) pathway, lipoxygenase (LOX) pathway, as well as cytochrome P450monooxygenases pathway. Cytochrome P450epoxygenase (CYP450) include2C and2J families in human body, which were expressed in many tissues. Cytochrome P450epoxygenase metabolize arachidonic acid (AA) to four epoxyeicosatrienoic acids, which were contained5,6-EET,8,9-EET,11,12-EET and14,15-EET. CYP450-EETs system has important biological effects on cardiovascular homeostasis. EETs could vasodilate blood vessels and decrease blood pressure by activation calcium-sensitive potassium channels, which were located in vascular smooth muscle cells; EETs could stimulate endothelial cells proliferation, as well as angiogenesis by activating mitogen activated protein kinase (MAPK) and PI3K/AKT pathways; EET could inhibit TNF-a-induced apoptosis of endothelial cells by activating PI3K/AKT signaling pathway and increasing expression of anti-apoptotic protein BCL-2. EETs could decrease vascular cell adhesion molecule-1(VCAM-1) expression, inhibit nuclear factor (NF-κB) and inhibit leukocyte adhesion to the vascular wall. In addition, EETs could not only promote the migration and invasion of tumor growth, but also play an important role in improving insulin resistance, diabetic nephropathy, obesity and some other metabolic diseases. EETs could be hydrolyzed by soluble epoxide hydrolase (sEH) into dihydroxyeicosatrienoic acids (DHETs), which have less biological activities.In this study, we hypothesize that CYP2J2overexpression and the resultant increase in EETs production increased fatty acids oxidation in blood vessel and liver tissues, and then ameliorated lipid metabolism disorders in mice induced by high fat diet. Therefore, we investigated the effects and possible molecular mechanisms of CYP2J2overexpression on hyperlipidemia in HFD-induced mice, and in vitro studies, we also investigated the effects and possible molecular mechanisms of CYP2J2overexpression or exogenous EETs on FFA-induced dyslipidemia in HepG2, LO2cells and HUVECs. The aim of this study is to explore whether CYP2J2epoxygenase-EETs system improve high-fat diet induced hyperlipidemia in mice. In Vivo Study CYP2J2Overexpression Ameliorate Hyperlipidemia in Mice induced by High Fat DietResearch aims:The effects and possible molecular mechanisms of CYP2J2overexpression on high-fat diet-induced hyperlipidemia in mice were investigated.Research methods:1. Forty male endothelial-specific CYP2J2overexpression transgenic (Tie2-CYP2J2-Tr) mice on a pure C57BL/6J genetic background and wild-type C57BL/6J mice (8weeks old) were randomly divided into four groups:Normal chow+Wild-type C57BL/6J mice group (Normal Chow+WT):n=10, wild-type C57BL/6J mice were fed with normal chow; Normal chow+Tie2-CYP2J2-Tr mice group (Normal Chow+Tie2-CYP2J2):n=10, endothelial-specific CYP2J2overexpression transgenic (Tie2-CYP2J2-Tr) mice were fed with normal chow; High-fat diet+Wild-type C57BL/6J mice group (HFD+WT):n=10, wild-type C57BL/6J mice were fed with high fat diet; High-fat diet+Tie2-CYP2J2-Tr mice group (HFD+Tie2-CYP2J2):n=10, endothelial-specific CYP2J2overexpression transgenic (Tie2-CYP2J2-Tr) mice were fed with high fat diet. According to the above experimental methods, all animals were fed by normal diet and high fat diet for16weeks.2. During the experiment, body weights of mice in each group were weighed. And blood samples of mice were collected and stored at-80℃;24-hour urine samples were collected with dimethylamine to prevent the decay of urine before the end of the experiment and stored at-80℃. All animals were anesthetized with pentobarbital (50mg/kg) and were sacrificed after the experiment, and then blood samples, liver, heart, aorta, subcutaneous and visceral fat tissues and kidney were collected and immediately snap-frozen in liquid nitrogen, and stored at-80℃. Some of the above samples were fixed in4%formaldehyde solution, embedded in paraffin after dehydration for reservation and then stored at room temperature (Subcutaneous and visceral fat tissues were weighed before fixed in formaldehyde solution).3. Triglyceride (TG), cholesterol (TC) and P-hydroxybutyrate levels in plasma and liver, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by related methods;4. GC-FID/MS were used to detect the levels of fatty acids in plasma and liver;5. H&E and Oil Red O staining were used to assess morphological changes in liver of mice in each group;6. Real-time quantitative PCR were performed to detect the expression of PPARa and CPT-1in liver and vascular tissues;7. Western blot were performed to detect the expression of fatty acid oxidation-related protein p-AMPKa, p-ACC, CPT-1and PPARa in livers and blood vessels.Results:1. Western blot results showed Tie2-CYP2J2-Tr mice have abundant expression of CYP2J2protein in livers and blood vessels, while no expression is observed in WT mice (P<0.05), indicating that CYP2J2is successful overexpression in Tie2-CYP2J2-Tr mice. Urine14,15-DHET results showed that urine14,15-DHET content of Normal chow+Tie2-CYP2J2-Tr mice group (Normal Chow+Tie2-CYP2J2) and High-fat diet+Tie2-CYP2J2-Tr mice group (HFD+Tie2-CYP2J2) significantly higher than that of Normal chow+Wild-type C57BL/6J mice group (Normal Chow+WT) and High-fat diet+Wild-type C57BL/6J mice group (HFD+WT)(P<0.05).2. The body weight, subcutaneous and visceral fat of wild-type C57BL/6J mice and Tie2-CYP2J2-Tr mice were no significant difference (P>0.05). The body weight, subcutaneous and visceral fat of High-fat diet+C57BL/6J mice group were significantly higher than that of normal chow+wild-type C57BL/6J mice group (P<0.05). However, these indexs of High-fat diet+Tie2-CYP2J2-Tr mice group were significantly lower than that of High-fat diet+wild-type C57BL/6J mice group (P<0.05).3. Results showed that HFD resulted in significantly increased plasma and liver triglycerides and cholesterol in WT mice (P<0.05); however, Tie2-CYP2J2-Tr mice with HFD had lower plasma and liver triglycerides than HFD-fed WT mice (P<0.05). Tie2-CYP2J2-Tr mice on HFD had lower liver cholesterol than HFD-fed WT mice (P<0.05). There was a reduction tendency in plasma cholestrol in Tie2-CYP2J2-Tr mice on HFD, it did not reach the significant difference (P>0.05). p-hydroxybutyrate was significantly decreased by HFD in both plasma and liver, but these effects were blocked by CYP2J2overexpression(P<0.05), demonstrating that there is an actual increase in fatty acid oxidation. The levels of triglycerides, cholesterol and β-hydroxybutyric acid in plasma and liver were no significant difference between Normal chow+Tie2-CYP2J2-Tr mice group (Normal Chow+Tie2-CYP2J2) and Normal chow+Wild-type C57BL/6J mice group (Normal Chow+WT)(P>0.05). These results indicate that CYP2J2overexpression attenuates metabolic dysfunction in hyperlipidemic mice.4. HFD resulted in significantly increased plasma ALT and AST in WT mice (P<0.05). CYP2J2overexpression also reversed ALT and AST abnormalities in mice caused by HFD (P<0.05). H&E staining showed that HFD induced deposition of numerous lipid droplets in liver compared to normal chow diet (P<0.05). In contrast, fewer and smaller lipid droplets were present in liver sections from Tie2-CYP2J2-Tr mice in HFD group than WT mice in the same group (P<0.05). Oil Red O staining for fatty acid further supported these finding. These results indicate that CYP2J2overexpression decreases triglyceride accumulation in liver by attenuating hepatic steatosis.5. GC-FID/MS results showed CYP2J2overexpression induced changes in fatty acids in plasma and liver samples. HFD caused widespread metabolic changes in fatty acid composition in plasma and liver (P<0.05). CYP2J2overexpression markedly attenuated liver fatty acids induced by HFD (P<0.05), but it had no significant effect on the fatty acids in plasma (P>0.05). In other words, the level of fatty acids in plasma has no significant difference between High-fat diet+Wild-type C57BL/6J mice group (HFD+WT) and High-fat diet+Tie2-CYP2J2-Tr mice group (HFD+Tie2-CYP2J2)(P>0.05). Similarly, under the condition of normal chow, the levels of fatty acids in plasma and liver have no significant difference between wild-type C57BL/6J mice and Tie2-CYP2J2-Tr mice (P>0.05).6. Real-time quantitative PCR showed the mRNA expression levels of PPARa and CPT-1in the mice livers and blood vessels have no significant difference between Normal chow+Wild-type C57BL/6J mice group (Normal Chow+WT) and Normal chow+Tie2-CYP2J2-Tr mice group (Normal Chow+Tie2-CYP2J2)(P>0.05). HFD resulted in significantly decreased the mRNA expression levels of PPARa and CPT-1in WT mice (P<0.05). However, the mRNA expression levels of PPARa and CPT-1in High-fat diet+Tie2-CYP2J2-Tr mice group (HFD+Tie2-CYP2J2) were higher than that in High-fat diet+Wild-type C57BL/6J mice group (HFD+WT)(P<0.05).7. Western blot results showed that the expression levels of p-AMPKa, p-ACC, CPT-1and PPARa in livers and blood vessels have no significant difference between Normal chow+Wild-type C57BL/6J mice group (Normal Chow+WT) and Normal chow+Tie2-CYP2J2-Tr mice group (Normal Chow+Tie2-CYP2J2)(P>0.05). HFD resulted in significantly decreased the expression levels of p-AMPKa, p-ACC, CPT-1and PPARa in WT mice (P<0.05). However, the expressions of p-AMPKa, p-ACC, CPT-1and PPARa in High-fat diet+Tie2-CYP2J2-Tr mice group (HFD+Tie2-CYP2J2) were higher than that in High-fat diet+Wild-type C57BL/6J mice group (HFD+WT)(P<0.05).In Vitro Study Effects of CYP2J2Overexpression or Exogenous EETs on Free Fatty Acids (FFA)-induced dyslipidemia in HepG2, LO2and HUVECsResearch aims:The effects and the possible molecular mechanisms of CYP2J2overexpression or exogenous EETs on free fatty acids (FFA)-induced dyslipidemia in HepG2, LO2and HUVECs.Research methods: 1. HepG2, L02and HUVECs were cultured in high glucose DMEM medium containing10%fetal bovine serum (FBS) at incubator with5%CO2at37℃. HepG2, LO2and HUVECs which were cultured in cell culture flasks grew to90%confluence were digested and passaged. When HepG2, LO2and HUVECs grew to70%-80%confluence, cells were cultured in DMEM medium containing0.5%fetal bovine serum (FBS) for starvation of12hours.2. The recombinant adeno-associated virus-mediated CYP2J2and GFP gene (rAAV-CYP2J2and rAAV-GFP) transfected into HepG2, LO2and HUVECs. After successful transfection, free fatty acid (FFA), AMPKa inhibitor (Compound C) and PPARa inhibitor (GW6471) were added into medium for24hours;3. After synchronization, AMPKa inhibitor (Compound C), PPARa inhibitor (GW6471),14,15-EET inhibitor (14,15-EEZE) and14,15-EET were also added into medium, and FFA was added to induce dyslipidemia in HepG2, LO2cells and HUVECs for24hours;4. The levels of triglyceride in HepG2, LO2and HUVECs were detected;5. After intervention, western blot were performed to detect the expression levels of p-AMPKa, p-ACC, CPT-1and PPARa in HepG2, LO2and HUVECs.Results:1. The triglyceride levels of FFA-treated HepG2, LO2and HUVECs were significantly higher than that of untreated cells (P<0.05). However, there was no significant difference during FFA group and FFA+rAAV-GFP group (P>0.05). The triglyceride levels of HepG2, LO2and HUVECs in FFA+rAAV-CYP2J2group were significantly lower than that in FFA group, which could be reversed by Compound C and GW6471(P<0.05). Similarly, triglyceride levels in FFA+14,15-EET were significantly lower than that in FFA group (P<0.05). The lowering triglyceride effect of14,15-EET could be reversed by14,15-EEZE and GW6471(P<0.05).2. Western blot was performed to detect the expression of fatty acid oxidation-related protein p-AMPKa, p-ACC and CPT-1in HepG2, L02and HUVECs. Results showed that compared to untreated cells, FFA significantly reduced the expression of p-AMPKa, p-ACC and CPT-1in HepG2, LO2and HUVECs (P<0.05). Compared to FFA-treated cells, rAAV-CYP2J2significantly increased the expression of p-AMPKa, p-ACC and CPT-1in FFA-treated cells and to some extent, this effect could be blocked by Compound C (P<0.05), suggesting that the hypolipidemic effect of CYP2J2could be dependent on AMPKa/ACC/CPT-1signaling pathway. Similarly, compared to FFA-treated cells,14,15-EET significantly increased the expression levels of p-AMPKa, p-ACC and CPT-1in FFA-treated cells, which could be blocked by14,15-EEZE (P<0.05). These results indicated that CYP2J2overexpression or exogenous14,15-EET could reduce intracellular triglyceride accumulation via increasing fatty acid P-oxidation related protein.3. Western blot showed that CYP2J2overexpression significantly increased the expression of PPARa which were reduced by FFA, and this effect was partly blocked by Compound C (P<0.05), indicating that PPARa, to some extent, was regulated by AMPKa. Compared to untreated cells, FFA could reduce the expression of PPARa and CPT-1(P<0.05). CYP2J2overexpression or exogenous14,15-EET significantly reversed the expression of PPARa and CPT-1which were decreased by FFA, and to some extent, this effect was blocked by GW6471(P<0.05). These results indicated that in addition to AMPKa/ACC/CPT-1signaling pathway, CYP2J2overexpression or14,15-EET could increase fatty acid P-oxidation by PPARa/CPT-1signaling pathway, and finally reduced intracellular triglyceride levels of HepG2, LO2and HUVECs.Statistical AnalysisAll data were showed as mean±S.E.M and were analyzed by one-way analysis of variance (ANOVA). Values of P<0.05was considered statistically significant.Conclusion1. CYP2J2overexpression improved dyslipidemia in high fat diet-fed mice, and ameliorated hyperlipidemia-induced fatty acids and lipid accumulation in liver.2. CYP2J2overexpression significantly increased the expression of fatty acid β oxidation related protein p-AMPKa, p-ACC, CPT-1and PPARa in liver and blood vessel.3. CYP2J2overexpression or exogenous EETs increased fatty acid β-oxidation through activation AMPKa/ACC/CPT-1and PPARa/CPT-1signaling pathways, and ultimately improved intracellular triglyceride accumulation. To some extent, PPARa was regulated by AMPKa.4. CYP epoxygenase-EETs system increased fatty acid β-oxidation, reduced plasma and liver triglyceride content, and reduced lipid accumulation in liver, ultimately improved high-fat diet-induced hyperlipidemia in mice.5. The results of this experiment provide a new strategy and a new research direction for prevention and treatment of hyperlipidemia in the future, and we expected gene therapy would be a new target for hyperlipidemia.