摘要
目的探究瞬时受体电位阳离子通道蛋白6(TRPC6)基因对过氧化氢(H_(2)O_(2))诱导大鼠脑血管平滑肌细胞(VSMCs)损伤的影响。方法于2022年3—12月完成研究。分离大鼠脑基底动脉VSMCs,采用随机数字表法分为五组:空白组、H_(2)O_(2)组(500μmol/L H_(2)O_(2)处理6 h)、H_(2)O_(2)+空转染组(500μmol/L H_(2)O_(2)处理6 h,并转染空载脂质体)、H_(2)O_(2)+TRPC6 siRNA组(500μmol/L H_(2)O_(2)处理6 h,并转染TRPC6 siRNA)和H_(2)O_(2)+pcDNA3.1 TRPC6组(500μmol/L H_(2)O_(2)处理6 h,并转染pcDNA3.1 TRPC6)。采用人胆囊收缩素/缩胆囊素八肽(CCK-8)法检测细胞增殖活性,流式细胞术检测细胞凋亡,酶联免疫吸附测定检测细胞中白细胞介素(IL)-6、IL-1β、单核细胞趋化蛋白1(MCP-1)、超氧化物歧化酶(SOD)、丙二醛,DCFH-DA荧光探针法检测细胞中活性氧水平。结果与H_(2)O_(2)组比较,H_(2)O_(2)+TRPC6 siRNA组D(λ)450 nm值明显升高,细胞凋亡率明显降低(P<0.05),H_(2)O_(2)+pcDNA3.1 TRPC6组D(λ)450 nm值明显降低,细胞凋亡率明显升高(P<0.05)。H_(2)O_(2)组、H_(2)O_(2)+TRPC6 siRNA组、H_(2)O_(2)+pcDNA3.1 TRPC6组细胞中IL-6含量分别为(159.24±21.65)ng/L、(52.95±12.43)ng/L、(257.48±23.44)ng/L,IL-1β含量分别为(126.09±19.85)ng/L、(41.67±9.06)ng/L、(248.77±23.32)ng/L,MCP-1含量分别为(89.25±11.73)ng/L、(27.73±5.18)ng/L、(187.61±15.42)ng/L,SOD活性分别为(53.40±8.92)U/mL、(172.33±14.52)U/mL、(23.75±5.63)U/mL,丙二醛含量分别为(91.35±10.41)nmol/mL、(8.29±2.17)nmol/mL、(169.37±14.37)nmol/mL,活性氧水平分别为2.68±0.27、0.76±0.18、5.89±0.34;与H_(2)O_(2)组比较,H_(2)O_(2)+TRPC6 siRNA组IL-6、IL-1β、MCP-1、丙二醛和活性氧明显降低、SOD活性明显升高(P<0.05),H_(2)O_(2)+pcDNA3.1 TRPC6组与之相反。结论TRPC6基因低/过表达可促进/抑制H_(2)O_(2)诱导VSMCs增殖活性和抑制/促进细胞凋亡,可能与调节炎症反应和氧化应激损伤有关。
Objective To explore the effects of transient receptor potential cationic channel protein 6(TRPC6)gene on hydrogen peroxide(H_(2)O_(2))-induced cerebrovascular smooth muscle cells(VSMCs)injury in rats.Methods The study was performed between March 2022 and December 2022.VSMCs of cerebral basilar artery in rats were isolated and divided into blank group,H_(2)O_(2) group(treatment with 500μmol/L H_(2)O_(2) for 6 h),H_(2)O_(2)+empty transfection group(treatment with 500μmol/L H_(2)O_(2) for 6 h,transfection with empty liposomes),H_(2)O_(2)+TRPC6 siRNA group(treatment with 500μmol/L H_(2)O_(2) for 6 h,transfection with TRPC6 siRNA)and H_(2)O_(2)+pcDNA3.1TRPC6 group(treatment with 500μmol/L H_(2)O_(2) for 6 h,transfection with pcDNA3.1TRPC6)according to random number table method.The activity of cells proliferation was detected by human cholecystokinin/octapeptide(CCK)-8,cells apoptosis was detected by flow cytometry,levels of interleukin(IL)-6,IL-1β,monocyte chemotactic protein 1(MCP-1),superoxide dismutase(SOD)and malondialdehyde(MDA)were detected by enzyme-linked immunosorbent assay,levels of reactive oxygen species(ROS)were detected by DCFH-DA fluorescent probe.Results Compared with H_(2)O_(2) group,D(λ)450 nm was significantly increased in H_(2)O_(2)+TRPC6 siRNA group,while apoptosis rate was significantly decreased(P<0.05).Compared with H_(2)O_(2) group,D(λ)450 nm was significantly decreased in H_(2)O_(2)+pcDNA3.1 TRPC6 group,while apoptosis rate was significantly increased(P<0.05).In H_(2)O_(2) group,H_(2)O_(2)+TRPC6 siRNA group and H_(2)O_(2)+pcDNA3.1 TRPC6 group,IL-6 levels were(159.24±21.65)ng/L,(52.95±12.43)ng/L and(257.48±23.44)ng/L,IL-1βlevels were(126.09±19.85)ng/L,(41.67±9.06)ng/L and(248.77±23.32)ng/L,MCP-1 levels were(89.25±11.73)ng/L,(27.73±5.18)ng/L and(187.61±15.42)ng/L,SOD activities were(53.40±8.92)U/mL,(172.33±14.52)U/mL and(23.75±5.63)U/mL,malonaldehyde(MDA)levels were(91.35±10.41)nmol/mL,(8.29±2.17)nmol/mL and(169.37±14.37)nmol/mL,and ROS levels were 2.68±0.27,0.76±0.18 and 5.89±0.34.Compared with H_(2)O_(2) group,IL-6,IL-1β,MCP-1,MDA and ROS were significantly decreased,while SOD activity was significantly increased in H_(2)O_(2)+TRPC6 siRNA group(P<0.05),and all the above indexes were contrary in H_(2)O_(2)+pcDNA3.1 TRPC6 group.Conclusion The low/over expression of TRPC6 gene can promote/inhibit proliferative activity of H_(2)O_(2)-induced VSMCs and inhibit/promote cells apoptosis,which may be related to regulating inflammatory response and oxidative stress injury.
作者
高寒冰
张开创
黄林轲
郑静
GAO Hanbing;ZHANG Kaichuang;HUANG Linke;ZHENG Jing(Department of Neurosurgery,Sanmenxia Hospital of Traditional Chinese Medicine,Sanmenxia,He'nan 472000,China;Department of Brain Intervention Surgery,Sanmenxia Hospital of Traditional Chinese Medicine,Sanmenxia,He'nan 472000,China;Department of Encephalopathy,Sanmenxia Hospital of Traditional Chinese Medicine,Sanmenxia,He'nan 472000,China)
出处
《安徽医药》
2025年第3期541-545,共5页
Anhui Medical and Pharmaceutical Journal
基金
2019年度河南省医学科技攻关计划联合共建项目(LHGJ20190592)。
