运动通过微小RNA-181b/磷酸酶和张力蛋白同源物调控平滑肌表型缓解糖尿病大鼠血管损伤的研究

Exercise alleviates diabetic vascular injury by regulating smooth muscle phenotype through microRNA-181b/phosphatase and tensin homologue

目的:观察有氧运动对2型糖尿病大鼠血管损伤的作用,探讨潜在的分子调控机制。方法:高糖高脂喂养联合链脲佐菌素注射建立大鼠2型糖尿病模型。选模型鼠20只随机分为糖尿病对照组(DC)和糖尿病运动组(DE),同龄正常饮食大鼠为正常对照组(NC),每组10只。DE组进行8周中等强度的跑台运动,其它组不运动。无创血压仪测试大鼠的心率、血压,HE染色观察血管壁病理学变化,电镜观察血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的超微结构,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测血管微小RNA-181b(microRNA-181b,miR-181b)的表达,Western Blot检测血管α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、波形蛋白(Vimentin)、磷酸肌醇3激酶(phospho inositide-3 kinase,PI3K)、磷酸酶和张力蛋白同源物(phosphatase and tensin homologue,PTEN)、蛋白激酶B(protein kinase B,AKT)等蛋白表达。结果:相比NC组,DC组大鼠心率(heart rate,HR)、收缩压(systolic blood pressure,SBP)、舒张压(diastolic blood pressure,DBP)、平均血压(mean blood pressure,MBP)显著降低(P<0.001);VSMCs失去典型的长梭形特征,表现为菱形,核膨大而不规则,细胞膜边界不清晰,密体、密斑少见;腹主动脉中膜显著增厚(P<0.05);收缩型特异蛋白α-SMA表达降低而合成型特异蛋白Vimentin表达升高(P<0.01);血管miR-181b的表达升高(P<0.001),其靶蛋白PTEN的表达降低(P<0.01),PI3K、AKT表达升高(P<0.01)。相比DC组,8周有氧运动使DE组的SBP、DBP、MBP显著升高(P<0.05或P<0.001);VSMCs呈长方形,核呈长椭圆形,细胞膜清晰完整,可见少许密体和密斑;收缩型特异蛋白α-SMA表达升高而合成型特异蛋白Vimentin表达降低(P<0.05);血管miR-181b的表达显著降低(P<0.001),PTEN蛋白表达升高而AKT表达下降(P<0.01或P<0.05)。结论:有氧运动具有缓解2型糖尿病大鼠血管损伤的作用,且可能机制为运动激活miR-181b/PTEN通路抑制下游AKT信号从而逆转糖尿病大鼠动脉VSMCs向合成表型转化。

Objective:To observe the effect of aerobic exercise on vascular injury in type 2 diabetic rats,and to explore its potential molecular regulation mechanism.Method:A rat model of type 2 diabetes was established by high-sugar and high-fat feeding combined with streptozotocin injection.Twenty model rats were randomly divided into diabetic control group(DC)and diabetic exercise group(DE),and rats of the same age with normal diet were included in normal control group(NC),with 10 rats in each group.The DE group underwent 8 weeks of moderate-intensity treadmill exercise,the other groups of rats did not exercise.Non-invasive blood pressure monitor was used to test the heart rate and blood pressure,HE staining was used to observe the pathological changes of vascular wall,the ultrastructure of vascular smooth muscle cells(VSMCs)was observed by electron microscope,the expression of vascular miR-181b was detected by qRT-PCR,and theα-SMA,Vimentin,PI3K,PTEN,AKT were detected by Western Blot.Result:Compared with the NC group,the heart rate,systolic blood pressure(SBP),diastolic blood pressure(DBP),and mean blood pressure(MBP)of the DC group were significantly decreased(P<0.001);the VSMCs lost the typical long-fusiform feature and appeared as diamond-shaped,nuclei were enlarged and irregular,cell membrane boundaries are not clear,dense bodies and dense plaques were rare;abdominal aorta media was significantly thickened(P<0.05);the expression of contractile specific proteinα-SMA was decreased and the synthetic specific protein Vimentin was increased(P<0.051);the expression of vascular miR-181b was increased(P<0.001),but its target protein PTEN was decreased(P<0.01),and the expressions of PI3K and AKT were increased(P<0.01).Compared with DC group,8 weeks of aerobic exercise significantly increased SBP,DBP and MBP of DE group(P<0.05 or P<0.001);the VSMCs were rectangular,with oval nucleus,clear and complete cell membrane,and a few dense bodies and dense plaques were seen;the expression ofα-SMA was increased and Vimentin was decreased(P<0.05);the expression of miR-181b in blood vessels was significantly decreased(P<0.001),and PTEN protein was increased while AKT was decreased(P<0.01 or P<0.05).Conclusion:Aerobic exercise can alleviate vascular damage in type 2 diabetic rats,and it may be produced by activating miR-181b/PTEN pathway and inhibiting downstream AKT signaling,thereby reversing the transformation of arterial VSMCs to synthetic phenotype in diabetic rats.