健脾祛痰化瘀法对高脂血症大鼠肝脏脂质过氧化的影响及其可能机制

Effect and its possible mechanism of invigorating spleen and removing phlegm and blood stasis on lipid peroxidation in liver of hyperlipidemia rats

目的观察健脾祛痰化瘀法对高脂血症大鼠肝脏细胞铁死亡的影响,探讨其对肝脏脂质过氧化的影响及作用机制。方法将32只SPF级SD大鼠随机分为4组,分别为空白组、模型组、化瘀祛痰方组[13.846 mg/(kg·d)]以及辛伐他汀组[1.575 mg/(kg·d)]。空白组给予正常饮食;模型组、化瘀祛痰方组及辛伐他汀组大鼠给予高脂饲料。8周后,化瘀祛痰方组和辛伐他汀组进行灌胃给药;空白组和模型组给予等体积的生理盐水进行灌胃。末次给药后通过全自动生化分析仪检测血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)含量;HE染色观察肝脏病理变化;油红O染色观察各组大鼠肝脏脂质沉积情况;比色法检测肝脏铁离子含量;酶联免疫吸附测定(ELISA)检测肝脏组织中ROS、GSH含量;实时荧光定量PCR(RT-qPCR)与Western blotting法检测肝脏组织中GPx4、SLC7A11、TFR1 mRNA及蛋白的表达。结果与空白组相比,模型组大鼠血清TC、TG、LDL-C含量显著升高,HDL-C含量显著降低(P<0.01);肝脏脂肪空泡布满视野,肝细胞体积变大;并且可见明显红色脂滴分布;肝脏组织中ROS、Fe^(2+)含量显著升高,GSH含量显著下降(P<0.01);肝脏组织中TFR1 mRNA和蛋白的表达显著升高、GPx4和SLC7A11 mRNA和蛋白的表达显著降低(P<0.01)。化瘀祛痰方及辛伐他汀可使TC、TG、LDL-C含量显著降低,HDL-C含量显著升高(P<0.05或P<0.01);脂肪变性程度明显缓解,肝细胞脂肪空泡明显减少;脂滴分布明显减少;肝脏组织中ROS、Fe^(2+)含量显著降低,GSH含量显著升高(P<0.05或P<0.01);肝脏组织中TFR1 mRNA和蛋白的表达显著降低,GPx4和SLC7A11 mRNA和蛋白的表达显著升高(P<0.05或P<0.01)。结论健脾祛痰化瘀法改善高脂血症大鼠的作用机制可能与调控SLC7A11/GPx4/ROS信号通路介导的铁死亡的发生有关。

Objective To observe the effect of invigorating spleen and removing phlegm and blood stasis on iron death in liver cells of rats with hyperlipidemia,and to explore its effect and its mechanism on liver lipid peroxidation.Methods Thirty-two SPF SD rats were randomly divided into 4 groups:blank group,model group,Huayu Qutan recipe group and simvastatin group.The rats in the blank group were given normal diet,and the rats in the model group,Huayu Qutan recipe group and simvastatin group were given high fat diet.After 8 weeks,the rats in the Huayu Qutan recipe group and simvastatin group were given intragastric administration,while the blank group and model group were given the same volume of normal saline.After the last administration,the contents of serum total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)were detected by automatic biochemical analyzer.The pathological changes of liver were observed by hematoxylin-eosin staining;Liver lipid deposition was observed by oil red O staining;the content of iron ion in liver was detected by colorimetry;and the content of ROS and GSH in liver tissue was detected by enzyme-linked immunosorbent assay(Elisa).The expression of GPx4,SLC7A11,TFR1 mRNA and protein in liver tissue was detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting.Results Compared with the blank group,the contents of serum TC,TG and LDL-C in the model group increased significantly,while the content of HDL-C decreased significantly(P<0.01).Hepatic steatosis is full of fat vacuoles and hepatocytes become larger.The obvious distribution of red lipid droplets was observed.The contents of ROS and Fe^(2+)in liver tissue increased significantly,while the content of GSH decreased significantly(P<0.01).The expression of TFR1 mRNA and protein in liver tissue increased significantly,while the expression of GPx4 and SLC7A11 mRNA and protein decreased significantly(P<0.01).Huayu Qutan recipe and simvastatin can significantly reduce the content of TC,TG,LDL-C and increase the content of HDL-C(P<0.05 or P<0.01).The degree of steatosis was significantly alleviated and the fatty vacuoles in hepatocytes were significantly reduced.The distribution of lipid droplets decreased significantly.The content of ROS and Fe^(2+)in liver tissue decreased significantly,while the content of GSH increased significantly(P<0.05 or P<0.01).The expression of TFR1 mRNA and protein decreased significantly,while the expression of GPx4 and SLC7A11 mRNA and protein increased significantly in liver tissue(P<0.05 or P<0.01).Conclusion The mechanism of invigorating spleen and removing phlegm and blood stasis in improving hyperlipidemia rats may be related to the regulation of iron death mediated by SLC7A11/GPx4/ROS signal pathway.