基于内质网自噬的豁痰解毒通络饮干预兔颈动脉粥样硬化机制研究

Mechanism research of Huotan Jiedu Tongluo Decoction interventional treatment of rabbit carotid atherosclerosis based on endoplasmic reticulum autophagy

目的:研究豁痰解毒通络饮对兔颈动脉硬化模型的疗效,探讨其是否通过内质网自噬信号通路干预颈动脉硬化的进展。方法:雄性日本大耳兔随机分为4组,假手术组、模型组、中药组、西药组,每组6只。除了假手术组均用球囊损伤颈总动脉并蛋氨酸饲料喂养,模型组给予正常饮用水灌胃,中药组给予豁痰解毒通络饮灌胃(6.67g·kg-1·d-1),西药组给予阿托伐他汀灌胃(1.42mg·kg-1·d-1)。取损伤侧颈总动脉,HE染色检测病理结构;酶联免疫吸附测定法(ELISA)检测各组血清中同型半胱氨酸(Hcy)的含量;实时荧光定量聚合酶链反应(Real-time PCR)检测动脉组织中的Atg12 mRNA水平;蛋白免疫印记法(Western Blot)测GRP78、DDIT3、Atg12在颈总动脉组织中的蛋白分子表达水平。结果:①HE染色结果表明与假手术组比较,模型组有明显的内膜增生;与模型组比较,中药组内膜增生与损伤程度明显减轻。②与假手术组比较,模型组血清中Hcy的含量显著升高(P<0.01);与模型组比较,中药组能够降低血清中的Hcy的含量(P<0.01)。③RT-PCR和Western Blot结果显示:与假手术组比较,模型组中的Atg12 mRNA水平以及GRP78、DDIT3、Atg12的蛋白表达水平显著升高(P<0.01);与模型组比较,中药组能够降低Atg12 mRNA水平(P<0.01)以及GRP78、DDIT3、Atg12的蛋白表达水平(P<0.01)。结论:豁痰解毒通络饮可以明显改善兔颈动脉粥样硬化,降低兔血清中Hcy的含量,其机制可能是通过抑制内质网自噬通路的过度激活。

Objective:To study the effect of Huotan Jiedu Tongluo Decoction on rabbits carotid atherosclerosis model and to explore whether it interferes with the progression of carotid atherosclerosis through endoplasmic reticulum autophagy signaling pathway.Methods:Male Japanese rabbits were randomly divided into 4 groups:sham operation group,model group,Chinese medicine group and western medicine group.Except for the sham operation group,the carotid artery was injured by balloon and fed with the methionine diet.The model group was given normal drinking water,the Chinese medicine group was given Huojie Jiedu Tongluo Decoction(6.67 g·kg-1·d-1),and the western medicine group was given atorvastatin(1.42 mg·kg-1·d-1).The common carotid artery of the injured side was taken and the pathological structure was detected by HE staining.The content of homocysteine(Hcy)in the serum of each group was detected by enzyme linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the level of Atg12 mRNA in arterial tissue.The expression levels of protein molecules in the common carotid artery tissues of GRP78,DDIT3 and Atg12 were detected by Western Blot.Results:①The results of HE staining showed that the model group had obvious intimal hyperplasia compared with the sham operation group.The degree of intimal hyperplasia and injury was significantly reduced compared with the model group.②Compared with the sham operation group,the serum Hcy content in the model group was significantly increased(P<0.01).Compared with the model group,the Chinese medicine group was able to reduce the serum Hcy content(P<0.01).③The results of RT-PCR and Western Blot showed that compared with sham-operated group,the levels of Atg12 mRNA and the protein expression levels of GRP78,DDIT3 and Atg12 protein expression in the model group were significantly increased(P<0.01).Compared with model group,Huotan Jiedu Tongluo Decoction could decrease the expression of Atg12 mRNA(P<0.01)and the protein expression levels of GRP78,DDIT3 and Atg12(P<0.01).Conclusion:Huotan Jiedu Tongluo Decoction can significantly improve rabbit carotid atherosclerosis and reduce the content of Hcy in rabbit serum.The mechanism may be through inhibiting the over-activation of endoplasmic reticulum autophagy pathway.