山豆根醇提取物对大鼠脑动脉瘤血管内皮细胞的作用机制

Mechanism of Subprostrate sophora alcohol extract in the vascular endothelial cells of rat brain aneurysms

目的 探讨山豆根醇提取物(Subprostrate sophora alcohol extract, SSAE)对大鼠脑动脉瘤(cerebral aneurysm, CA)血管内皮细胞(endothelial cells, ECs)结构蛋白的影响及其可能的作用机制。方法 随机选取15只7周龄雄性Wistar大鼠作为假手术组,另外60只通过结扎左肾动脉和左颈总动脉联合饲喂含氯化钠(质量浓度80 mg·mL^(-1))及3-氨基丙腈(质量浓度1.2 mg·mL^(-1))的食物以诱导构建CA大鼠模型。将建模成功的大鼠随机分为模型组、SSAE低剂量组、SSAE高剂量组及西药组,每组15只,其中SSAE低剂量组、SSAE高剂量组CA大鼠的给药量分别为1、2 g·kg^(-1)·d^(-1),西药组喂服硝苯地平(缓释片)120 mg·kg^(-1)·d^(-1),假手术组及模型组大鼠均灌服等量生理盐水,每日1次,连续20 d。用HE检测大脑Willis环动脉组织形态学变化,用Elastic Van Gieson (EVG)染色检测动脉壁弹性纤维完整性以评估动脉壁血管重构情况;用TUNEL染色检测各组大鼠动脉壁ECs凋亡指数;用ELISA法检测血清基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)、白介素17A(interleukin-17A,IL-17A)、一氧化氮和内皮素-1(endothelin-1,ET-1)的含量;用Western blot法检测活化素受体样激酶1(activin receptor-like kinase 1, ALK1)、细胞信号转导分子Smad^(-1)/5(Smad1/5)、磷酸化Smad1/5(p-Smad1/5)、平滑肌肌动蛋白-α(α-SMA)和Ⅲ型胶原蛋白(ColⅢ)的相对表达水平。结果 与假手术组大鼠比较,模型组大鼠的CA明显膨出,动脉壁较薄,结构不完整,血管壁弹性纤维出现断裂,波状结构消失,大量ECs凋亡。血清MMP9、IL-17A和ET-1的含量升高,一氧化氮的含量降低,Willis动脉ALK1、p-Smad1/5、α-SMA和ColⅢ蛋白的相对表达水平降低(P<0.05);与模型组比较,SSAE低剂量组、高剂量组和西药组大鼠CA的体积明显减小,动脉壁组织病理损伤有显著改善,血清中MMP9、IL-17A和ET-1的含量降低,一氧化氮的含量升高,Willis动脉ALK1、p-Smad1/5、α-SMA和ColⅢ蛋白的相对表达水平升高(P<0.05);药物对于CA大鼠的作用效果表现为西药组较强,SSAE高剂量组次之,SSAE低剂量组最弱(P<0.05)。结论 SSAE可减轻CA大鼠动脉壁组织的病理变化,减少ECs凋亡,改善血管内皮及结构蛋白损伤,促进CA大鼠ECs功能障碍恢复及动脉结构蛋白相关因子的表达,其作用机制可能与激活Smad1/5通路有关。

Objective To investigate the possible mechanism of Subprostrate sophora alcohol extract(SSAE) in the structural proteins of vascular endothelial cells(ECs) of rat brain aneurysm(CA).Methods Fifteen 7-week-old male Wistar rats were randomly selected as sham operation group, and another 60 rats were fed with sodium chloride(80 mg·mL^(-1)) and 3-aminoponitrile(1.2 mg·mL^(-1)) by ligation of left renal artery and left common carotid artery to induce the CA rat model. They were randomly divided into model group, SSAE low dose group, SSAE high dose group and western medicine group, with 15 rats in each group. The CA rats in SSAE low dose group and high dose group were given SSAE 1 and 2 g·kg^(-1)·d^(-1), respectively. The western medicine group was fed nifedipine(sustained release tablet) 120 mg·kg^(-1)·d^(-1), the sham operation group and model group were given the same amount of normal saline, once a day, for 20 days. HE was used to detect the arterial histomorphology of the circle of Willis, and Elastic Van Gieson(EVG) staining was used to detect the integrity of elastic fibers in the arterial wall to evaluate the vascular remodeling of the arterial wall. TUNEL staining was used to detect the apoptosis index of ECs in each group. The contents of matrix metalloproteinase 9(MMP9), interleukin-17A(IL^(-1)7A), nitric oxide and endothelin-1(ET-1) in serum were determined by ELISA. Western blot was used to detect the relative expression levels of activin receptor-like kinase 1(ALK1), Smad1/5, phosphorylated Smad1/5(p-Smad1/5), smooth muscle actin-α(α-SMA) and type Ⅲ collagen(Col Ⅲ) protein.Results Compared with sham operation group, the CA in model group was significantly expanded, the artery wall was thinner, the structure was incomplete, the elastic fiber of the vascular wall was broken, the wavy structure disappeared, and more ECs apoptosis was observed.In addition,the contents of MMP9,IL^(-1)7Aand ET-1in serum were increased,and the content of nitric oxide was decreased.The relative expression levels of ALK1,p-Smad1/5,α-SMA and ColⅢ protein in Willis artery were decreased(P<0.05).Compared with model group,the CA volume decreased significantly in SSAE low-dose,high-dose and western medicine groups,and pathological injury of arterial wall was significantly improved,MMP9,IL^(-1)7Aand ET-1contents in serum decreased,while nitric oxide content increased,the relative expression levels of ALK1,p-Smad1/5,α-SMA and ColⅢprotein in Willis artery increased(P<0.05).The effect of drugs on CA rats was stronger in western medicine group,followed by SSAE high-dose group,and the weakest in SSAE low-dose group(P<0.05).Conclusion SSAE could reduce the histopathological changes of arterial wall,reduce ECs apoptosis,improve vascular endothelial and structural protein injury,promote the recovery of ECs dysfunction and the expression of arterial structural protein related factors in CA rats.Its mechanism may be related to the activation of Smad1/5pathway.