黄芪甲苷抑制转化生长因子β1诱导肾小管上皮细胞凋亡的实验研究  

Effects of Astragaloside Ⅳ on the apoptosis of renal tubular epithelial cells induced by transforming growth factor-β1

作　　者：田磊[1] 徐维佳[2] 张珍[1] 邵兴华[1] 谢园园[1] 王琴[1] 王玲[1] 车霞静[1] 倪兆慧[1] 牟姗[1] 

机构地区：[1]上海交通大学医学院附属仁济医院肾脏科,分子细胞(肾病)实验室,上海200127 [2]南京军区福州总医院肾内科

出　　处：《肾脏病与透析肾移植杂志》2015年第2期139-144,共6页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金："973"课题(2012CB517602);港澳台科技合作专项项目(2014DFT30090);国家自然科学基金(81102700;81373865);上海市高级中西医结合人才培养项目(ZYSNXD012-RC-ZXY017);上海市科委项目资金(12401906400;14140903200)

摘　　要：目的:探讨黄芪甲苷(AS-IV)抑制转化生长因子β1(TGF-β1)诱导肾小管上皮细胞凋亡的机制。方法:体外采用TGF-β1(10 ng/m1)刺激人近端小管上皮细胞(HK-2),采用不同浓度的AS-IV(50μg/rl、100μg/ml、200μg/ml)进行干预治疗24h收集细胞。体内建立小鼠单侧输尿管梗阻(UUO)模型,C57BL/6小鼠随机分为假手术组(Sham组)、模型组(UUO组)和治疗组(AS-IV组)。采用单侧(右)输尿管结扎建立UUO模型,Sham组仅游离输尿管但不结扎,AS-IV组连续7天给予AS-Ⅳ[20mg/(kg·d)]灌胃,UUO组和Sham给予等剂量溶剂灌胃,术后第14天采集肾脏组织。TUNEL法检测凋亡,CCK法观察细胞活性,Western印迹检测TGF-β1和被切割的半胱氨酸蛋白酶3(cleaved-caspase 3)表达,并观察c-Jun氨基末端激酶(JNK)丝裂原活化蛋白激酶(MAPK)磷酸化情况。结果:AS-Ⅳ改善UUO诱导的肾组织TGF-β1表达及细胞凋亡(P<0.01),抑制TCF-β1诱导的HK-2细胞活性下降及细胞凋亡,200μg/mlAS-Ⅳ作用最明显(P<0.01)。体内体外实验均可见,AS-IV减少caspase 3活化,抑制JNK MAPK磷酸化。结论:AS-IV可抑制TGF-β1的表达及其诱导的肾小管上皮细胞凋亡,其机制可能与JNK MAPK通路有关。Objective：To investigate the effects of Astragaloside IV （AS-IV） on the apoptosis of renal tubular epithelial cells induced by transforming growth factor-β1 （TGF-β1）. Methodology：In vitro, normal human renal tubular epithelial ceils （HK 2） were stimulated with recombinant TGF-β1 （ 10 ng/ml） and simuhaneously treated with different concentrations of AS-IV （50, 100, and 200 μg/ml） for 24 h. In vivo, the unilateral ureteral obstruction （UUO） model was constructed. Mice in AS-IV group were orally administrated AS-IV 20 mg·kg^-1·d^-1 for 7 days after operation, and mice in other groups were administrated the equal volume vehicle. Bilateral kidneys were collected on the 14th day after operation. Western blot was used to detect the expression levels of cleaved easpase 3, c-Jun N-terminal kinase （JNK） and p-JNK. Cell viability was determined by a cell count kit and apoptosis was estimated by TUNEL staining. Results：Cell viability was significantly inhibited and cell apoptosis was increased by TGF-β1 stimulating for 24h, while those phenomenon were notably attenuated by AS-IV treatment, and the 200 μg/ml AS-IV was the optimal effect （P〈0. 01 ）. The proteins expression of TGF-β1 and cleaved caspase 3 were increased significantly in UUO kidney tissues （P〈0. 01 ） , which was also reversed by AS-W administration （P〈0. 01）. AS-IV inhibited JNK MAPK signals both in vivo and in vitro （ P〈 0. 01）. Conclusion： AS-IV could attenuate the apoptosis of renal tubular epithelial cells induced by TGF-β1. The mechanism of AS-IV protective effect might be associated with inhibition of JNK MAPK phosphorylation.

关 键 词：黄芪甲苷 转化生长因子Β1 细胞凋亡 C-JUN氨基末端激酶 

分 类 号：R285[医药卫生—中药学]