化瘀祛痰方对载脂蛋白E基因敲除小鼠肝脏脂质过氧化及p53、GPX4、xCT表达的影响

Effect of Huayu Qutan Formula(化瘀祛痰方)on Liver Lipid Peroxidation and Expression of p53,GPX4 and xCT in ApoE^-/- Mice

目的探讨化瘀祛痰方治疗动脉粥样硬化(AS)的可能作用机制。方法24只载脂蛋白E基因敲除(ApoE^-/-)小鼠随机分为模型组、化瘀祛痰方组、辛伐他汀组,8只C57BL/6J小鼠作为空白组。除空白组外,其他各组给予高脂饲料喂养制备AS模型。化瘀祛痰方组给予化瘀祛痰方20 g/(kg·d)灌胃;辛伐他汀组给予辛伐他汀2.275 mg/(kg·d)灌胃,空白组与模型组均给予0.5 ml生理盐水灌胃。各组均灌胃4周后,检测血清甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)、超氧化物歧化酶(SOD)活性,丙二醛(MDA)、活性氧(ROS)水平,肝脏铁死亡相关因子p53、谷胱甘肽过氧化物酶4(GPX4)、胱氨酸/谷氨酸反向转运体(xCT)蛋白及mRNA表达;观察肝脏组织形态学改变。结果与空白组比较,模型组小鼠血清TC、TG和LDL-C含量显著升高,肝脏组织内ROS、MDA水平显著升高,SOD活性显著下降,肝脏组织p53蛋白及mRNA的表达增高,GPX4、xCT蛋白和mRNA水平显著降低(P<0.01),HE染色显示肝细胞肿胀明显且胞浆内含有大量脂肪空泡蓄积。与模型组比较,化瘀祛痰组及辛伐他汀组小鼠血清TC、TG和LDL-C水平显著降低,肝脏组织内ROS、MDA水平显著降低,SOD活性显著升高,p53蛋白及mRNA的表达降低,GPX4、xCT蛋白和mRNA水平显著升高(P<0.05或P<0.01);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p53/xCT通路影响肝细胞铁死亡,减轻肝脏脂质过氧化损伤,从而防治AS。

Objective To explore the mechanism of Huayu Qutan Fomula(化瘀祛痰方)in treating atherosclerosis(AS).Method A total of 24 ApoE^-/-mice were randomly divided into a model group,a Huayu Qutan Fomula group,and a simvastatin group.Eight C57BL/6J mice were used as the blank group.Except for the blank group,mice were given high-fat feed to make AS models.The mice in Huayu Qutan Fomula group was given Huayu Qutan Fomula 20 g/(kg·d)by gavage;the simvastatin group was given simvastatin 2.275 mg/(kg·d)by gavage,and the blank group and the model group were given 0.5 ml normal saline by gavage.After intragastric administration for 4 weeks,serum triglycerides(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C)and lowdensity lipoprotein cholesterol(LDL-C),superoxide dismutation(SOD)activity,malondialdehyde(MDA),reactive oxygen species(ROS)levels,liver ferroptosis related factor p53,glutathione peroxidase 4(GPX4),cystine/glutamate reverse transport Body(xCT)protein and mRNA expression were detected;and the morphological changes of liver tissue were observed.Results Compared with the blank group,the levels of serum TC,TG and LDL-C in the model group were significantly increased;the levels of ROS and MDA in the liver tissue were significantly increased;SOD activity was significantly decreased;the expression of p53 protein and mRNA in the liver tissue was increased;the levels of GPX4,xCT protein and mRNA were significantly reduced(P<0.01).HE staining showed that hepatocytes were swollen and the cytoplasm contained a large amount of fatty vacuoles.Compared with the model group,the serum TC,TG and LDL-C levels of the mice in the Huayu Qutan Fomula group and simvastatin group were significantly reduced;the levels of ROS and MDA in the liver tissue were significantly reduced;SOD activity was significantly increased;the levels of p53 protein and mRNA expression decreased;the levels of GPX4,xCT protein and mRNA increased significantly(P<0.05 or P<0.01);the morphology of liver cells recovered or partially recovered,and the lipid droplet content in the cytoplasm was significantly reduced.Conclusion Huayu Qutan Fomula affects ferroptosis in liver cells by p53/xCT pathways and alleviate the lipid peroxidation injury of ApoE-/-mice and prevents atherosclerosis.