摘要
目的探讨瞬时受体电位阳离子通道(transient receptor potential canonical,TRPC)促进大鼠肾小球系膜细胞(HBZY-1)细胞外基质(extracellular matrix,ECM)沉积的作用机制。方法免疫荧光染色方法观察TRPC1和TRPC6在HBZY-1细胞上的定位及表达;给予AngⅡ干预HBZY-1细胞后,应用qRT-PCR和Western blotting方法检测Gαq/PLCβ4/TRPC信号通路关键蛋白和ECM沉积指标[α-SMA、CollagenⅢ(ColⅢ)和Fibronectin(Fn)]的mRNA和蛋白表达;siRNA技术沉默TRPC1和TRPC6表达后,检测ECM沉积指标的表达;Fluo-4AM Ca^(2+)成像技术测定细胞内Ca^(2+)内流的变化情况。结果TRPC1和TRPC6在HBZY-1细胞中均有表达,主要定位在细胞膜和胞质。AngⅡ刺激后,可以激活Gαq/PLCβ4/TRPC信号通路,表现为Gαq、PLCβ4、TRPC1和TRPC6的mRNA和蛋白表达均升高(P<0.05)。细胞内的Ca^(2+)内流增加(P<0.01),ECM沉积指标α-SMA、ColⅢ和Fn的mRNA和蛋白表达上调(P<0.05)。RNA干扰技术沉默TRPC1和TRPC6表达后,导致胞内Ca^(2+)内流减少(P<0.05),HBZY-1细胞ECM沉积指标的mRNA和蛋白表达均下调(P<0.05)。结果表明,沉默TRPC表达可以抑制AngⅡ诱导的HBZY-1细胞ECM沉积,且可能与减少[Ca^(2+)]i内流有关。结论TRPC可能是肾脏纤维化新的治疗靶点。
Objective To explore the role and mechanism of TRPC in promoting extracellular matrix(ECM)deposition in rat glomerular mesangial cells(HBZY-1).Methods Immunofluorescence staining was performed to observe the distribution and expression of TRPC1 and TRPC6 in HBZY-1 cells.After AngⅡstimulation,qRT-PCR and Western blotting were used to detect the mRNA and protein expressions of Gαq/PLCβ4/TRPC signaling pathway main proteins and ECM deposition indicators(α-SMA,collagenⅢand fibronectin).By silencing the expressions of TRPC1 and TRPC6 by RNA interference,the expressions of ECM deposition indicators were detected.Changes in[Ca^(2+)]i influx were determined through Fluo-4AM Ca^(2+)imaging.Results Both TRPC1 and TRPC6 were expressed in HBZY-1,and were mainly located in cell membrane and cytoplasm.After AngⅡstimulation,Gαq/PLCβ4/TRPC signaling pathway was activated,and the mRNA and protein expressions of Gαq,PLCβ4,TRPC1 and TRPC6 were all increased(P<0.05).[Ca^(2+)]i influx also increased(P<0.01),and the mRNA and protein expressions of ECM deposition indicators(α-SMA,ColⅢand Fn)were upregulated(P<0.05).Silencing the expressions of TRPC1 and TRPC6 by RNA interference led to decreased[Ca^(2+)]i influx(P<0.05),and downregulated mRNA and protein expressions of ECM deposition indicators in HBZY-1 cells(P<0.05).The results suggested that inhibition of TRPC expressions could inhibit AngⅡinduced ECM deposition in HBZY-1 cells,which might be associated with decreased[Ca^(2+)]i influx.Conclusion TRPC may be a novel therapeutic target of renal fibrosis.
作者
魏琳婷
葛蓬勃
李柯
李艳
王引红
赵纬昊
崔晨凯
董静
高洁
王莉
付荣国
WEI Linting;GE Pengbo;LI Ke;LI Yan;WANG Yinhong;ZHAO Weihao;CUI Chenkai;DONG Jing;GAO Jie;WANG Li;FU Rongguo(Department of Nephrology,The Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004;Department of General Surgery,The First Affiliated Hospital of Xi’an Medical College,Xi’an 710077,China)
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2023年第4期518-524,共7页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.82170697),陕西省自然科学基金资助项目(No.2022JM-472),西安交通大学第二附属医院院基金[No.YJ(QN)202024]。
作者简介
共同第一作者:葛蓬勃;共同第一作者魏琳婷;通信作者:付荣国,博士,教授.E-mail:fu_rongguo@sina.com。
