化瘀祛痰方对高脂血症大鼠肝脏CYP2J3/EETs介导脂肪酸代谢的影响

Effects of Huayu Qutan Prescription(化瘀祛痰方)on Liver CYP2J3/EETs Mediated Fatty Acid Metabolism in Hyperlipidemia Rats

目的基于CYP2J3/EETs介导脂肪酸代谢探讨化瘀祛痰方防治血脂异常的机制。方法32只SPF级SD大鼠分为空白对照组、模型组、化瘀祛痰方组、辛伐他汀组,每组8只。除空白对照组外,其他各组给予高脂饲料喂饲,建立高脂血症模型。于第13周开始,化瘀祛痰方组给予化瘀祛痰方灌胃,辛伐他汀组给予辛伐他汀灌胃,空白对照组、模型组给予相应体积的生理盐水灌胃,每日1次,灌胃4周。采用全自动生化分析仪检测各组大鼠血清中总胆固醇(Total cholesterol,TC)、甘油三酯(Triglyceride,TG)、低密度脂蛋白胆固醇(Low density lipoprotein cholesterol,LDL-C)及高密度脂蛋白胆固醇(High density lipoprotein cholesterol,HDL-C)水平;HE染色观察肝脏组织病理变化;Elisa检测各组大鼠肝脏14,15-环氧二十碳三烯酸(14,15-epoxyeicosatrienoic acid,14,15-EET)水平;Real time RT-PCR检测肝脏组织细胞色素P450表氧化酶2J3(Cytochrome P450 oxidase 2J3,CYP2J3)、肉毒碱棕榈酰转移酶1(Carnitine palmitoyltransferase-1,CPT-1)mRNA水平;Western blot法检测肝脏组织CYP2J3、腺苷酸活化蛋白激酶(AMP-actived protein kinase,AMPK)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、乙酰辅酶A羧化酶(Acetyl CoA Carboxylase,ACC)、磷酸化乙酰辅酶A羧化酶(p-ACC)、CPT-1蛋白水平。结果与空白对照组比较,模型组大鼠血清TC、TG和LDL-C水平显著升高(P<0.01),HDL-C水平显著降低(P<0.05);肝脏脂质沉积显著,可见大量脂滴;肝脏组织内14,15-EET含量显著降低(P<0.01);肝脏CYP2J3、CPT-1 mRNA和蛋白表达水平显著降低(P<0.01),p-AMPK/AMPK、p-ACC/ACC显著降低(P<0.01)。与模型组比较,经化瘀祛痰方及辛伐他汀干预后,大鼠血清TC、TG和LDL-C水平显著降低(P<0.05或P<0.01),HDL-C水平显著升高(P<0.05);肝脏脂质沉积情况明显改善,脂滴数明显减少;肝脏组织内14,15-EET水平显著升高(P<0.01);肝脏CYP2J3、CPT-1 mRNA和蛋白表达水平显著升高(P<0.05或P<0.01),p-AMPK/AMPK、p-ACC/ACC显著升高(P<0.05或P<0.01)。结论化瘀祛痰方可有效改善高脂血症大鼠血脂异常,减轻肝脏脂质沉积情况,其机制可能与CYP2J3/EETs介导脂肪酸代谢密切相关。

Objective To investigate the mechanism of Huayu Qutan Prescription(化瘀祛痰方)in the prevention and treatment of dyslipidemia based on cytochrome P4502J3(CYP2J3)/EETs mediated fatty acid metabolism.Methods Thirty-two SD rats were divided into normal group,model group,Huayu Qutan Prescription group and simvastatin group,with 8 rats in each group.Except the normal group,the other groups were given high-fat feed to establish the hyperlipidemia model.From the 13th week,the Huayu Qutan Prescription group was given Huayu Qutan Prescription by gavage,the simvastatin group was given simvastatin by gavage,and the normal group and the model group were given the corresponding volume of normal saline by gavage,once a day for 4 weeks.The serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)were detected.Observation of pathological changes in liver tissue by HE staining.The levels of 14,15-epoxyeicosatrienoic acid(14,15-EET)in liver of rats in each group were detected by ELISA.The mRNA levels of CYP2J3 and carnitine palmitoyltransferase-1(CPT-1)in liver tissue were detected by RT-PCR,and the protein levels of CYP2J3,phosphorylation AMP-actived protein kinase(p-AMPK)/AMP-actived protein kinase(AMPK),phosphorylation acetyl CoA carboxylase(p-ACC)/acetyl CoA carboxylase(ACC)and CPT-1 in liver tissue were detected by Western blot.Results Compared with those of the normal group,the contents of TC,TG and LDL-C in the serum in the model group were significantly increased(P<0.01),the HDL-C level was significantly decreased(P<0.05),the lipid deposition was observed,the lipid droplets in the liver were significantly increased,the 14,15-EET content in the liver tissue was significantly decreased(P<0.01),the mRNA expressions of CYP2J3 and CPT-1 in liver were significantly decreased(P<0.01),the protein expressions of CYP2J3 and CPT-1 were significantly decreased(P<0.01),and the levels of p-AMPK/AMPK and p-ACC/ACC were significantly decreased(P<0.01).Compared with those of the model group,the contents of TC,TG and LDL-C were significantly decreased(P<0.05 or P<0.01),the level of HDL-C was significantly increased(P<0.05),the lipid deposition was significantly improved and the lipid droplets in the liver were significantly decreased by HE staining,the level of 14,15-EET in the liver was significantly increased after the intervention of Huayu Qutan Prescription and simvastatin(P<0.01),the mRNA expressions of CYP2J3 and CPT-1 in liver were significantly increased(P<0.05 or P<0.01),the protein expressions of CYP2J3 and CPT-1 were significantly increased(P<0.05 or P<0.01)and the levels of p-AMPK/AMPK and p-ACC/ACC were significantly increased(P<0.05 or P<0.01).Conclusion Huayu Qutan Prescription can effectively improve the dyslipidemia of hyperlipidemia rats and reduce the liver lipid deposition.Its mechanism may be closely related to the CYP2J3/EETs mediated fatty acid metabolism.