TRPV4对脑血管平滑肌增殖及凋亡的影响

Effect of TRPV4 on the proliferation and apoptosis of basilar arterial smooth muscle cells

目的探究新型钙离子通道香草醛受体4(transient receptor potential vanilloid receptor,TRPV4)对自发性高血压(spontaneously hypertensive rats,SHR)大鼠脑血管平滑肌增殖及凋亡的影响。方法分离并培养大鼠脑基底动脉平滑肌细胞(basilar arterial smooth muscle cells,BASMCs),采用实时定量聚合酶链式反应(qRT-PCR)及免疫印迹(Western blot)法检测细胞中TRPV4的表达。转染TRPV4 siRNA沉默TRPV4基因,CCK-8检测细胞增殖、流式细胞术检测细胞周期及凋亡;Western blot检测cyclin D1、p-Rb、Bcl-2、cleaved-caspase-3/caspase-3、p-AKT/AKT、p-GSK3β/GSK3β蛋白表达。结果 qRT-PCR及Western blot结果显示自发性高血压大鼠BASMCs中TRPV4的表达明显高于对照组大鼠。而在自发性高血压大鼠BASMCs中,沉默TRPV4可抑制细胞增殖,阻滞细胞周期并诱导其凋亡;此外还可下调cyclin D1、p-Rb、Bcl-2、p-AKT、p-GSK3β蛋白的表达,同时上调cleaved-caspase-3蛋白的表达。结论沉默TRPV4能够抑制自发性高血压大鼠BASMCs细胞增殖,同时促进细胞凋亡,机制可能与抑制AKT/GSK3β信号通路的激活有关,所以新型钙离子通道TRPV4可为今后靶向诊疗高血压脑血管疾病提供依据。

Objective To study the expression and effect of a novel calcium channel TRPV4 on the proliferation and apoptosis of BASMCs in spontaneous hypertension rat（ SHR）. Methods The expression of TRPV4 in BASMCs was checked by qRT-PCR and Western blot assay. TRPV4 siRNA was used to block TRPV4 channel in BASMCs. Cell survival was determined by the CCK-8 assay,while cell cycle and apoptosis was detected by flow cytometry. The expressions of cyclin D1,p-Rb,bcl-2,cleaved-caspase-3/caspase-3,p-AKT/AKT,p-GSK3β/GSK3β proteins were detected by Western blot. Results qRTPCR and Western blot showed that the expression of TRPV4 was markedly increased in BASMCs of SHR compared to（ WKY）. Cell viability decreased after transfected with TRPV4 shRNA in BASMCs of SHR,while cell cycle arrest and apoptosis were induced. Furthermore,the expression of cyclin D1,p-Rb,Bcl-2,p-AKT,p-GSK3β protein were down-regulated and the expression of cleaved-caspase-3 protein was up-regulated. Conclusion Block of TRPV4 in BASMCs of SHR could inhibit cell proliferation andpromote cell apoptosis. The mechanism may be related to the inhibition of AKT/GSK3 β pathway. So the new calcium ion channel TRPV4 provided a new target for diagnosis and therapy of hypertensive encephalopathy.