化瘀祛痰方干预糖酵解途径对高脂血症大鼠肝脏脂质沉积的影响及机制研究

Effects of Huayu Qutan Formula(化瘀祛痰方)on Glycolytic Pathway on Lipid Deposition in Liver of Hyperlipidemia Rats and Its Mechanism

目的探讨化瘀祛痰方干预肝脏糖酵解途径对高脂血症大鼠肝脏脂质沉积的影响及机制。方法将24只SPF级SD大鼠随机分为正常组、模型组和化瘀祛痰方组,每组8只。正常组给予基础饲料喂饲,模型组和化瘀祛痰方组给予高脂饲料喂养制备高脂血症模型。造模时间为4周,造模结束后灌胃给药4周。化瘀祛痰方组给予化瘀祛痰方10 mL/(kg·d)灌胃,其余两组给予等量生理盐水。全自动生化分析仪检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)水平;HE及油红O染色观察大鼠肝脏组织形态学改变;ELISA法检测糖酵解关键酶己糖激酶-II(HK-II)、磷酸果糖激酶(PFKM)及丙酮酸激酶(PKM2)活性;用蛋白免疫印迹法(Western Blot法)检测肝脏HK-II、PFKM、PK、cleaved-caspase3、cleaved-PARP、Bax、Bcl2蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)法检测肝脏HK-II、PFKM、PKM2、cleaved-caspase3、cleaved-PARP、Bax、Bcl2 mRNA表达。结果与正常组比较,模型组大鼠血清TC、TG和LDL-C含量显著升高,HDL-C含量有下降趋势;HE染色显示大鼠的肝细胞肿胀明显,肝细胞脂肪变性;油红O染色显示胞浆内含有大量脂肪空泡蓄积;肝脏HK-II、PFK、PKM2活性降低;肝脏组织HK-II、PFKM、PKM2蛋白及mRNA表达降低,cleaved-caspase3、cleaved-PARP、Bax/Bcl2蛋白及mRNA的表达增高;与模型组比较,化瘀祛痰组大鼠血清TC、TG和LDL-C水平显著降低,HDL-C含量有升高趋势。肝脏HK-II、PFKM、PKM2活性升高。肝脏组织HK-II、PFKM、PKM2蛋白及mRNA表达升高,cleaved-caspase3、cleaved-PARP、Bax/Bcl2蛋白及mRNA的表达降低;肝细胞形态恢复或者部分恢复,胞浆内脂滴含量明显减少。结论化瘀祛痰方可能通过调控糖酵解途径,抑制肝脏细胞凋亡,减轻肝脏细胞脂质沉积,进而防治高脂血症。

Objective To investigate the effect and mechanism of Huayu Qutan Formula(化瘀祛痰方)intervention on liver glycolysis pathway on liver lipid deposition in hyperlipidemia rats.Methods Twenty-four SPF SD rats were randomly divided into normal group,model group and Huayu Qutan Formula group,with 8 rats in each group.The normal group was fed with basic feed,and the model group and Huayu Qutan Formula group were fed with high-fat feed to prepare a hyperlipidemia model.The modeling time was 4 weeks,and the rats were given the medicine intragastrically for 4 weeks after modeling.The Huayu Qutan Formula group was given Huayu Qutan Formula 10 mL/(kg·d)by gavage,and the other two groups were given the same amount of saline.Automatic biochemical analyzer was used to detect the levels of TC,TG,LDL-C and HDL-C.HE and Oil Red O staining methods were used to observe the morphological changes of rat liver.ELISA method was used to detect the key glycolytic enzymes hexokinase-II(HK-II),phosphofructokinase(PFKM)and Pyruvate kinase(PKM2)activity.Western blotting method was used to detect HK-II,PFKM,PK,cleaved-caspase3,cleaved-PARP,Bax and Bcl2 protein expressions in liver.RT-PCR was used to detect the expression of HK-II,PFKM,PKM2,cleaved-caspase3,cleaved-PARP,Bax and Bcl2 mRNA in liver.Results Compared with those of the normal group,the serum levels of TC,TG and LDL-C of the model group were increased significantly,while the HDL-C content was decreased.HE staining showed that the rats'liver cells were swollen and hepatocytes were fatty degeneration.The oil red O staining showed that the cytoplasm contained a large amount of fatty vacuoles accumulation.The activities of HK-II,PFK and PKM2 in liver were decreased.The expressions of HK-II,PFKM,PKM2 protein and mRNA in liver were decreased.The expressions of cleaved-caspase3,cleaved-PARP,Bax/Bcl2 protein and mRNA were increased.Compared with those of the model group,the serum levels of TC,TG and LDL-C of the rats in the Huayu Qutan Formula group were decreased significantly,and the HDL-C content had a tendency to increase.The activities of HK-II,PFKM and PKM2 were increased in the liver.The expressions of HK-II,PFKM,PKM2 protein and mRNA in liver were increased,and the expressions of cleaved-caspase3,cleaved-PARP,Bax/Bcl2 protein and mRNA were decreased.The morphology of liver cells recovered or partially recovered,and the lipid droplet content in the cytoplasm was significantly reduced.Conclusion Huayu Qutan Formula may regulate the glycolysis pathway,inhibit liver cell apoptosis,reduce liver cell lipid deposition and prevent hyperlipidemia.