同型半胱氨酸通过YAP通路调控大鼠冠状动脉血管平滑肌细胞高反应性的作用机制研究

Action Mechanism of Homocysteine in the Regulation of Hyperresponsiveness of Coronary Artery Smooth Muscle Cells through YAP Pathway in Rats

背景冠状动脉血管平滑肌细胞(VSMC)高反应性是导致冠状动脉痉挛的关键,而高同型半胱氨酸(Hcy)能诱导冠状动脉痉挛,但其具体作用机制尚不完全清楚。目的探讨Hcy通过YAP通路调控冠状动脉VSMC高反应性的作用机制。方法 2018年1—10月,将SD大鼠实施安乐死后在无菌环境中迅速去除心脏并制成长1.0~1.5 mm血管环。将新鲜的冠状动脉血管环作为A组;在37℃、5%CO2条件下,将在DMEM低糖培养基中培养的冠状动脉血管环作为B组,将在Hcy+DMEM低糖培养基中培养的冠状动脉血管环作为C组,将在维替泊芬(VP)+DMEM低糖培养基中培养的冠状动脉血管环作为D组,将在Hcy+VP+DMEM低糖培养基中培养的冠状动脉血管环作为E组;除A组外冠状动脉血管环均培养24 h。采用微血管张力检测仪检测内皮素B型受体激动剂蛇毒类似物(S6c)和内皮素1(ET-1)诱导的冠状动脉血管张力,采用Western blot法检测内皮素A型(ETA)受体、内皮素B型(ETB)受体、YAP、磷酸化ERK1/2(p-ERK1/2)、ERK1/2、磷酸化mTOR(p-mTOR)、mTOR、磷酸化AMPK(p-AMPK)、AMPK及Sirt1的表达水平。结果 (1)Hcy可增强S6c和ET-1呈浓度依赖方式介导的冠状动脉血管收缩。C组冠状动脉血管环的ETA受体/β-actin、ETB受体/β-actin及YAP/β-actin高于A组和B组,B组冠状动脉血管环的ETB受体/β-actin及YAP/β-actin高于A组(P<0.05)。(2)VP能有效抑制由Hcy上调的S6c和ET-1呈浓度依赖方式介导的冠状动脉血管收缩。C组大鼠冠状动脉血管环ETA受体/β-actin、ETB受体/β-actin及YAP/β-actin高于B、D、E组(P<0.05)。(3)C组冠状动脉血管环p-ERK1/2/ERK1/2、p-mTOR/mTOR高于B、D、E组(P<0.05);B组冠状动脉血管环p-ERK1/2/ERK1/2高于D组,p-AMPK/AMPK及Shirt1/β-actin高于C、D、E组(P<0.05)。结论 Hcy可能通过YAP通路激活ERK1/2和mTOR信号通路,上调VSMC内皮素受体,进而增强冠状动脉VSMC高反应性,这将为高Hcy致冠状动脉痉挛的具体作用机制研究提供新的理论依据。

Background Hyperresponsiveness of coronary artery smooth muscle cells(VSMC)is the key to result in coronary artery spasm,however hyperhomocysteinemia(HHcy)may lead to coronary artery spasm,but its specific mechanism is not yet clear.Objective To investigate action mechanism of homocysteine(Hcy)in the regulation of hyperresponsiveness of coronary artery VSMC through YAP pathway in rats.Methods From January to October 2018,some SD rats were euthanized and rapidly removed the hearts in a aseptic condition to make a 1.0-1.5 mm vascular rings.The fresh coronary vascular ring was served as A group,that cultured for 24 hours in DMEM medium with low sugar content(37℃and 5%CO2)as B group,that cultured for 24 hours in DMEM medium with low sugar content and Hcy(37℃and 5%CO2)as C group,that cultured for 24 hours in DMEM medium with low sugar content and verteporfin(37℃and 5%CO2)as D group,and that cultured for 24 hours in DMEM medium with low sugar content,Hcy and verteporfin(37℃and 5%CO2)as E group.Vascular tension of coronary artery mediated by S6c and ET-1 was detected by Microvascular tension detector,respectively,moreover Western blot method was used to detect expressions of ETA receptor,ETB receptor,YAP,p-ERK1/2,ERK1/2,p-mTOR,mTOR,p-AMPK,AMPK and Sirt1.Results(1)Hcy could enhance the vasoconstriction of coronary artery mediated by S6c and ET-1 with concentration-dependent mode.ETA receptor/β-actin,ETB receptor/β-actin and YAP/β-actin of coronary vascular ring in C group were statistically significantly higher than those in groups A and B,meanwhile ETB receptor/β-actin and YAP/β-actin of coronary vascular ring in B group were statistically significantly higher than those in A group(P<0.05).(2)Verteporfin could effectively inhibit the vasoconstriction of coronary artery mediated by S6c and ET-1 with concentration-dependent mode.ETA receptor/β-actin,ETB receptor/β-actin and YAP/β-actin of coronary vascular ring in C group were statistically significantly higher than those in groups B,D and E(P<0.05).(3)p-ERK1/2/ERK1/2 and p-mTOR/mTOR of coronary vascular ring in C group were statistically significantly higher than those in groups B,D and E(P<0.05);p-ERK1/2/ERK1/2 of coronary vascular ring in B group was statistically significantly higher than that in D group,meanwhile p-AMPK/AMPK and Shirt1/β-actin of coronary vascular ring in B group were statistically significantly higher than those in groups C,D and E(P<0.05).Conclusion Hcy may enhance the hyperresponsiveness of coronary artery VSMC through activating the ERK1/2 and mTOR signaling pathway via YAP pathway and up-regulating the endothelin receptors of VSMC,which may provide a new theoretical basis for the studies about specific action mechanism of HHcy-induced coronary artery spasm.