从自噬机制探讨加味黄芪赤风汤含药血清对阿霉素诱导的肾小球足细胞损伤的保护作用

Protective Effect of Modified Huangqi Chifeng Decoction Containing Serum on Adriamycin Induced Glomerular Podocytes Injury from Autophagy Mechanism

目的研究加味黄芪赤风汤对肾小球足细胞自噬及足细胞损伤相关蛋白nephrin、podocin及desmin表达的影响。方法采用体外培养小鼠肾小球足细胞、以阿霉素(adriamycin,ADR)诱导的方法制作足细胞损伤模型。随机分为空白组、模型组、替米沙坦组及加味黄芪赤风汤高、中、低剂量组(简称中药高、中、低剂量组)。采用Western blot法检测各组足细胞相关蛋白nephrin、podocin、desmin和自噬相关蛋白Beclin-1、LC3-Ⅱ的表达水平,透射电镜下观察各组足细胞自噬超微结构变化。结果 (1)与空白组比较,模型组nephrin、podocin表达明显下降(P<0.01),desmin表达明显升高(P<0.01);与模型组比较,中药高、中剂量组及替米沙坦组nephrin、podocin表达明显升高(P均<0.01),desmin表达明显下降(P均<0.01),中药低剂量组无明显改善(P>0.05)。(2)模型组较空白组自噬小体数量明显增多,体积增大,细胞器减少;中药高、中剂量组及替米沙坦组较模型组自噬超微结构均有不同程度改善,而中药低剂量组无明显改变。(3)与空白组比较,模型组Beclin-1、LC3-Ⅱ表达明显上升(P<0.01);与模型组比较,中药高、中剂量及替米沙坦组Beclin-1、LC3-Ⅱ表达明显下降(P<0.01),中药低剂量组无明显下降(P>0.05)。结论加味黄芪赤风汤对ADR诱导的足细胞损伤具有保护作用,调节足细胞自噬活性可能是其足细胞保护作用的内在机制之一。

Objective To observe the effect of Modified Hangqi Chifeng Decoction （MHCD） on autophagy of glomerular podocytes and the expressions of nephrin, podocin, desmin podocyte related proteins. Methods Podocytes were in vitro cultured. Podocyte injury model was prepared by adriamycin （ADR） induction. Podocytes were intervened with MHCD. Podocytes were then divided into 6 groups, the blank group, the ADR model group, the Telmisartan group, and the high, middle and low dose MHCD groups. Western blot was used to test the expression levels of podocin, nephrin, desmin, Beclin-1 and LC3- 11. Transmission electron microscope was used to observe micro-ultrastructural changes of autophagosome. Results （1） Compared with the blank group, the expressions of nephrin and podocin significantly decreased, and desmin expression significantly increased in the ADR model group （all P 〈0.01 ）. Compared with the ADR model group, the expressions of nephrJn and podocin increased significantly, desmin expression obviously decreased in high and middle MHCD groups and the Telmisartan group （all P 〈0.01 ）. However, they were not obviously changed in the low MHCD group （P 〉0.05）. （2） Compared with the blank group, the number of autophagosome increased significantly, the volume was significantly enlarged, organelles were reduced in the ADR model group. Compared with the ADR model group, micro- ultrastructural changes of autophagosome were improved to some degrees in high and middle MHCD groups and the Telmisartan group. However, they were not obviously changed in the low MHCD group. （3） Compared with the blank group, the expressions of Beclin-1 and LC3-1 was significantly increased in the ADR model group （P 〈0.01 ）. Compared with the ADR model group, the expressions of Beclin-1 and LC3-1 protein significantly decreased in high and middle MHCD groups and the Telmisartan group （all P 〈0.01）. But they were not significantly decreased in the low dose MHCD group （P 〉0.05）. Conclusions MHCD could protect ADR induced podocyte injury. Regulating the activities of podocyte autophagy might be one of its mechanisms for protecting the podocytes.