基于PCR Array技术探讨健脾化瘀祛痰方对动脉粥样硬化家兔主动脉线粒体能量代谢相关基因mRNA表达的影响

Effect of Jianpi Qutan Huayu Recipe on Mitochondrial Energy Metabolism Related Gene Expression in Atherosclerotic Rabbits Based on PCR Array Technology

目的采用PCR Array技术检测健脾化瘀祛痰方对动脉粥样硬化(atherosclerosis,AS)家兔主动脉线粒体能量代谢相关基因mRNA表达的影响,从线粒体能量角度初探健脾化瘀祛痰方防治AS的潜在靶点。方法将45只新西兰家兔随机分为对照组、模型组和中药组,每组15只。对照组喂饲普通饲料;模型组和中药组给予免疫性内皮损伤合并高脂饲料喂饲干预8周,制备家兔AS模型;造模成功后,中药组给予健脾化瘀祛痰方,对照组及模型组给予同等体积生理盐水,灌胃给药,每日1次,给药4周后取材。采用全自动生化分析仪检测各组家兔血脂水平;油红O染色和天狼星红染色观察主动脉斑块脂质沉积和血管胶原成分;PCR Array技术检测主动脉线粒体能量代谢相关基因的mRNA表达。结果血脂结果显示,与对照组比较,模型组血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)水平升高,高密度脂蛋白(HDL)水平降低;与模型组比较,中药组血脂水平均有所改善。油红O染色可见,对照组动脉管壁未见脂质形成;模型组动脉管壁动脉粥样硬化区域可见大面积红色脂滴阳性染色;中药组鲜红色脂质沉积区域明显减少。天狼星染色结果显示,对照组家兔主动脉血管内胶原分布致密、均匀、排列整齐,呈红色;模型组家兔主动脉血管胶原成分减少,呈疏松网状分布,且有部分断裂;中药组家兔主动脉血管胶原分布较规则,胶原之间的腔隙较少,血管结构部分发生改变。PCR Array结果显示中药组与模型组相比下调≥4倍的基因有ARRDC3、ATP5C1、COX4I1LOC100343255、NDUFA6、NDUFS2、NDUFS3、NDUFS4、NDUFV2、SDHB、SDHC、SDHD、SLC25A25、UQCRQ等21个基因。结论健脾化瘀祛痰方可改善AS家兔主动脉线粒体能量代谢相关基因mRNA的表达,这可能是其降低AS家兔血脂含量,减少主动脉斑块沉积的潜在机制之一。

Objective To detect the changes of mitochondrial energy metabolism related gene expression in atherosclerosis(AS)rabbits by using PCR Array techniqueandexplore the potential target of Jianpi Qutan Huayu Formula in the prevention and treatment of AS from the perspective of mitochondrial energy.Methods Forty-five New Zealand rabbits were randomly divided into control group,model group and traditional Chinese medicine group(Jianpi Qutan Huayu Recipe group),15 rabbits in each group.The control group was fed with common diet.The model group and the Chinese medicine group were given immune endothelial injury combined with high-fat diet for 8 weeks to prepare atherosclerosis model.After the modelwas successfully set up,the traditional Chinese medicine group was given Jianpi Qutan Huayu Recipe once a day,and the control group and the model group were given the corresponding volume of saline.Four weeks afterintervention,the levels of serum triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL-C)and low density lipoprotein(LDL-C)were detected by automatic biochemical analyzer.The morphological changes of aorta were observed by Sirius stainingand the expressions of mitochondrial energy metabolism related genes was detected by PCR Array.Result The results of blood lipid showed that the levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein(HDL)in the model group were significantly higher than those in the control group,and the levels of TC,TG,LDL and HDL in the traditional Chinese medicine group were significantly improved compared with those in the model group.Oil red O staining showed that there was no lipid formation in the arterial wall of the control group.The large area of red lipid droplets positive staining in the atherosclerotic area of the arterial wall of the model group,and bright red lipid deposition area in the traditional Chinese medicine group were significantly reduced.The results of Sirius staining showed that the collagen in aorta of rabbits in the control group was dense,uniform,well-arranged and red.The collagen composition in aorta of rabbits in the model group decreased,showing loose reticular distribution and partial rupture.The collagen distribution in aorta of rabbits in the traditional Chinese medicine group was regular,and there were fewer lacunae between collagen.The vascular structure changed.The results of PCR Array showed that 21 genes,such as ARRDC3,ATP5 C1,COX4 I1 LOC100343255,NDUFA6,NDUFS2,NDUFS3,NDUFS4,NDUFV2,SDHB,SDHC,SDHD,SLC25 A25 and UQCRQ,were down-regulated more than 4 times as compared with those of the model group.Conclusion Jianpi Qutan Huayu Recipe can participate in the expressions of mitochondrial energy metabolism related genes in AS rabbit aorta,thereby improving the deposition of AS rabbit aortic plaque.