骨形成蛋白2通过上调磷酸化Smad1/5促进大鼠颅内动脉瘤血管平滑肌细胞凋亡

Bone morphogenetic protein 2 facilitates the apoptosis of smooth muscle cells of intracranial aneurysms in rats through up-regulation of Smad1/5 phosphorylation

目的探讨骨形成蛋白2(BMP2)信号通路在颅内动脉瘤中的表达情况及其介导血管平滑肌细胞(VSMCs)凋亡的作用机制.方法将健康成年雄性SD大鼠按随机数字表法分为模型组(n=32)和假手术组(n=32).模型组通过结扎左侧颈外动脉、翼颚动脉及右侧颈总动脉制备颅内动脉瘤模型,假手术组不结扎血管.两组于术后3个月处死,采用电镜和HE染色法判断成模情况;采用免疫组织化学染色和蛋白免疫印迹法分别检测BMP2及下游磷酸化Smad1/5(p-Smad1/5)在两组大鼠前交通动脉复合体VSMCs中的表达情况.进一步离体培养大鼠脑动脉的VSMCs,分为对照组、BMP2组(加入BMP2100 ng/ml)以及Smad6+BMP2过表达组(转染Smad6过表达载体+BMP2100 ng/ml).采用TUNEL法检测各组细胞的凋亡情况,进一步采用蛋白免疫印迹法检测各组细胞中BMP2、Caspase3及p-Smad1/5的表达情况.结果电镜和HE染色结果显示,与假手术组比较,模型组大鼠前交通动脉复合体的血管壁均发生不同程度的瘤样病变,且VSMCs发生明显的重构和凋亡,说明建模成功.免疫组织化学染色结果显示,模型组大鼠前交通动脉复合体中的Caspase3(0.25±0.03)和BMP2(0.12±0.03)相对表达量均较假手术组(均为0.06±0.01)增加(均P<0.05).蛋白免疫印迹法结果显示,与假手术组比较,模型组大鼠BMP2(分别为:0.75±0.07、0.50±0.04)和p-Smad1/5(分别为:0.73±0.07、0.27±0.04)的相对表达量均明显增高(均P<0.05).TUNEL法检测离体VSMCs,结果显示BMP2组[(74.63±6.05)%]和Smad6+BMP2组[(45.92±1.08)%]的细胞凋亡率均较对照组增加[(4.35±0.65)%,均P<0.01];但Smad6+BMP2组的细胞凋亡率较BMP2组低(P<0.01).蛋白免疫印迹法结果显示,BMP2组和Smad6+BMP2组的p-Smad1/5、Caspase3的相对表达量均较对照组增加(均P<0.01);但Smad6+BMP2组的p-Smad1/5、Caspase3相对表达量均较BMP2组低(均P<0.05).结论BMP/Smad信号通路在颅内动脉瘤中被激活,且可能通过BMP2上调p-Smad1/5进而促进VSMCs凋亡.

Objective To investigate the role of bone morphogenetic protein 2(BMP2)signaling pathway in the development of intracranial aneurysms and its mechanism in regulating the apoptosis of vascular smooth muscle cells(VSMCs).Methods The healthy male SD rats were divided into 2 groups according to the random number table method,which were the model group(n=32)and the sham operation group(n=32).In the model group,the intracranial aneurysm model was established by ligating the left external carotid artery,the left pterygopalatine artery and the right common carotid artery of the rat.The vessels were not ligated in sham operation group.The rats in both groups were sacrificed at 3 months after operation,and the model establishment was judged by electron microscopy observation and hematoxylin-eosin(HE)staining.BMP2 and downstream phosphorylated Smad1/5(p-Smad 1/5)expression in VSMCs of the rats′anterior communicating artery complex in both groups were detected by immunohistochemical staining and Western blotting respectively.VSMCs of the rat′s cerebral arteries were further cultured in vitro and divided into 3 groups:control group,BMP2 group(add BMP2100 ng/ml)and Smad6+BMP2 group(trasfect Smad6 overexpression vector+add BMP2100 ng/ml).The apoptosis of VSMCs in each group was detected by TUNEL(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling),and the expression of Caspase3,BMP2 and downstream phosphorylated Smad1/5 of VSMCs in each group was detected by Western blotting.Results Compared with the sham group,the rat′s cerebral arteries had different degrees of lesions in the model group,and VSMCs of the blood vessel wall had obvious remodeling and apoptosis by electron microscopy observation and HE staining,indicating that the disease model was established successfully.The immunohistochemical staining showed that the relative expression levels of Caspase3(0.25±0.03 vs.0.06±0.01)and BMP2(0.12±0.03 vs.0.06±0.01)were increased significantly in the model group compared with the sham group(both P<0.05).Compared with the sham group,the Western blotting results showed that BMP2(0.50±0.04 vs.0.75±0.07)and p-Smad1/5(0.27±0.04 vs.0.73±0.07)relative expression in the model group was increased significantly(both P<0.05).The TUNEL detection showed that the apoptosis rates of VSMCs in BMP2 group(4.35%±0.65%vs.74.63%±6.05%)and Smad6+BMP2 group(4.35%±0.65%vs.45.92%±1.08%)were significantly increased compared with those in the control group(both P<0.01),while the VSMCs apoptosis rate in the Smad6+BMP2 group was less than that in the BMP2 group(P<0.01).Western blotting results showed that the relative expression levels of p-Smad1/5 and Caspase3 in the BMP2 group and Smad6+BMP2 group were both increased compared with those in the control group(both P<0.01),while the expression levels of p-Smad1/5 and Caspase3 in the Smad6+BMP2 group were lower than those in the BMP2 group(both P<0.05).Conclusions BMP2 and downstream p-Smad1/5 are closely related to the apoptosis of VSMCs in intracranial aneurysms.The apoptosis of VSMCs mediated by BMP/Smad signaling pathway may be one of the mechanisms of the occurrence and development of intracranial aneurysms.