miR-328在运动改善高血压大鼠胸主动脉L型钙通道表达中的作用

Effect of miR-328 in the exercise-induced improvement of the L-type calcium channel expression in thoracic aorta from hypertensive rats

目的探讨microRNA-328(miR-328)在有氧运动改善高血压大鼠胸主动脉平滑肌细胞L型电压门控钙通道(voltage-gated L-type Ca2+channel,LTCC)表达的中促进作用。方法选取12周龄雄性正常血压大鼠(Wistar Kyoto,WKY)与自发性高血压大鼠(spontaneously hypertensive rat,SHR)随机分为正常血压对照组(WKYC)、正常血压运动组(WKY-EX)、高血压对照组(SHR-C)、高血压运动组(SHR-EX),运动组进行12周中等强度跑台训练。12周后采用股动静脉置管术检测大鼠心血管反应;取大鼠胸主动脉,采用HE染色检测胸主动脉形态,qPCR检测LTCCα1C和β1亚基mRNA、miR-328表达,Western Blot检测α1C和β1亚基蛋白表达。离体实验,培养WKY胸主动脉平滑肌细胞,脂质体转染miR-328 mimic和miR-328 inhibitor使之过表达或沉默,检测LTCCα1C及β1亚基mRNA和蛋白表达变化。结果(1)有氧运动可显著降低SHR体重、血压和心率;(2)运动可显著降低SHR胸主动脉壁厚;(3)运动可显著降低SHR对去甲肾上腺素的升压反应和硝苯地平Nifedipine(LTCC阻断剂)的降压反应;(4)SHR-C组的LTCCα1C及β1亚基mRNA、蛋白表达显著高于WKY-C组,而SHR-EX组的α1C及β1亚基mRNA、蛋白表达显著低于SHR-C组;(5)SHR-C组中miR-328的表达显著低于WKY-C组,12周有氧运动后miR-328表达量显著增加;(6)离体实验中,与对照组相比,转染miR-328 mimic组α1C和β1亚基mRNA、蛋白表达显著下调,而转染miR-328 inhibitor组α1C和β1亚基mRNA、蛋白表达显著上调。结论规律有氧运动可有效降低SHR血压,调控miR-328靶向抑制LTCCα1C和β1亚基的表达,可能是运动改善高血压胸主动脉LTCC重构的机制之一。

Objective The present study investigated the effect of microRNA-328(miR-328)during the aerobic exercise-induced improvement of voltage-gated L-type Ca2+channel(LTCC)expression in thoracic aorta smooth muscle cells from spontaneously hypertensive rats(SHRs).Methods Twelve-week-old male normotensive Wistar Kyoto rats(WKYs)and SHRs randomly were separated into a control group(SHR-C and WKY-C)and an exercise group(SHR-EX and WKY-EX).Rats in exercise were subjected to moderate-intensity treadmill training for 12 weeks.The cardiovascular responses of rats were then detected by femoral arterial and venous cannulation.The thoracic aorta was stained with hematoxylin and eosin(HE)for histological assays.The mRNA expression levels of LTCCα1C andβ1 subunits and the expression of miR-328 were detected by qPCR.The protein expression levels of LTCCα1C andβ1 subunits were detected by Western Blot.For in vitro analysis,primary vascular smooth muscle cells(VSMCs)were isolated and cultured from the thoracic aorta of WKYs.Both miR-328 mimic and miR-328 inhibitor were transfected into cultured arterial myocytes to produce miR-328 overexpression or silencing.The mRNA and protein expression levels of LTCCα1C andβ1 subunits were detected.Results Aerobic exercise significantly reduced body weight,blood pressure,and heart rate in SHRs.Aerobic exercise significantly reduced the wall thickness of thoracic aorta in SHRs.Exercise significantly reduced the pressor response to norepinephrine and the depressor response to nifedipine(LTCC blocker)in SHRs.The mRNA and protein expression levels of LTCCα1Candβ1 subunits were significantly upregulated in the SHR-C group,compared with the WKYC group,while the mRNA and protein expression levels of LTCCα1C andβ1 subunits were downregulated in the SHR-EX group,compared with the SHR-C group.miR-328 expression was significantly lower in the SHR-C group than that in the WKY-C group;this expression significantly increased after aerobic exercise.In the transfection experiment,compared with the negative control(NC)group,the mRNA and protein expression levels ofα1C andβ1 subunits were significantly downregulated in the miR-328 mimic transfected group;these expression was significantly upregulated in the miR-328 inhibitor transfected group.Conclusions Regular aerobic exercise may effectively reduce blood pressure in SHR and enable miR-328 to inhibit LTCCα1C andβ1 subunit expression.This mechanism might contribute to exercise-mediated improvement of LTCC channel remodeling in hypertensive thoracic aorta.