辣椒素激活TRPV1/钙蛋白酶-1通路改善血管再狭窄的研究

Capsaicin ameliorates vascular restenosis via activating TRPV1/Calpain1 pathway

目的:探讨辣椒素在血管再狭窄中的作用及其机制。方法:将20只SPF雄性小鼠随机分为空白组和辣椒素组各10只,颈动脉结扎后分别用标准饲料和含0.01%辣椒素的饲料喂养14 d;另将20只SPF辣椒素预处理雄性小鼠随机分为标准组和抑制剂组两组各10只,分别给予生理盐水或瞬时受体电位香草素受体1(TRPV1)抑制剂5′-碘树脂毒素(iRTX)干预,采用苏木精-伊红染色测定并计算小鼠颈动脉内膜厚度/中膜厚度(I/M)值,蛋白质印迹法检测细胞钙蛋白酶亚型1(钙蛋白酶-1)的表达与活性,免疫组化法测定增殖细胞核抗原(PCNA)阳性细胞数。分离培养小鼠血管平滑肌细胞(VSMCs),将其分为空白对照组和使用血小板源性生长因子(PDGF-BB)预处理的生长因子组,再分组分别加入生理盐水或1 mmol·L^(-1)辣椒素培养,形成标准对照组、辣椒素对照组、生长因子组和生长因子辣椒素组,用细胞计数试剂盒-8(CCK-8)、细胞迁移法检测细胞增殖和迁移情况。结果:辣椒素组颈动脉的I/M值和PCNA阳性细胞数明显降低(P<0.01)。PDGF-BB可诱导VSMCs增殖和迁移,增加钙蛋白酶-1的表达和活性(P<0.01),而辣椒素可部分逆转上述效应(P<0.01)。TRPV1抑制剂iRTX与辣椒素同时干预的小鼠颈动脉I/M值和PCNA阳性细胞数均增加(P<0.01),同时组织中钙蛋白酶-1表达及活性也明显增加(P<0.01)。结论:辣椒素可改善血管再狭窄,该过程可能由TRPV1/钙蛋白酶-1通路介导实现。

Objective:To investigate the role and mechanism of capsaicin in vascular restenosis.Methods:Twenty male SPF mice were randomly divided into vehicle group(n=10)and capsaicin group(n=10).After carotid artery ligation,mice of the two groups were respectively fed with standard diet and diet supplemented with 0.01%capsaicin for 14 days.For the study of its internal mechanism,twenty capsaicin pretreated male mice were randomly divided into standard group and inhibitor group,which were respectively given normal saline and 5′-iodo-resiniferatoxin.The intima/media thickness(I/M)was measured by hematoxylin-eosin staining.The expression and activity of calpain-1 were detected by Western blot.The number of proliferating cell nuclear antigen(PCNA)positive cells was determined by immunohistochemical staining.The vascular smooth muscle cells(VSMCs)were isolated from the aorta and cultured in DMEM medium.Then,VSMCs were divided into blank control group and platelet-derived growth factor-BB(PDGF-BB)group(PDGF-BB pretreated),which then were subdivided into standard comparison group,capsaicin control group,growth factor group and growth factor capsaicin group by adding saline or capsaicin(1 mmol·L^(-1))for culture.Cell proliferation and migration were detected by Cell Counting Kit-8(CCK-8)and Transwell.Results:In the capsaicin group,both I/M and PCNA positive cells of carotid arteries decreased significantly(P<0.01).PDGF-BB induced VSMCs proliferation and migration(P<0.01),and increased the expression and activity of calpain1(P<0.01).However,capsaicin partially reversed all these effects(P<0.01).Both I/M and the number of PCNA positive cells of the carotid artery increased in mice treated concomitantly with iRTX(TRPV1 inhibitor)and capsaicin(P<0.01),meanwhile the expression and activity of calpain1 in tissues also increased significantly(P<0.01).Conclusion:Capsaicin can ameliorate vascular restenosis,which may be mediated by the TRPV1/Calpain1 pathway.