基于活性氧簇-自噬通路调控的加味黄芪赤风汤对肾小球足细胞的保护机制研究

Study on the Protective Mechanism of Modified Huangqi Chifeng Decoction Based on the Regulation of ROS-Autophagy Pathway on Glomerular Podocytes

目的观察加味黄芪赤风汤基于活性氧簇(ROS)-自噬通路对AngⅡ诱导肾小球足细胞损伤的保护机制。方法制备低、中、高剂量加味黄芪赤风汤(分别应用生药浓度为0.57、1.15、2.29 g/mL的加味黄芪赤风汤灌胃小鼠)含药血清及替米沙坦(0.83 mg/mL灌胃小鼠)含药血清,采用AngⅡ(1×10^-7mol/L)诱导足细胞损伤模型,分为空白组,模型组,加味黄芪赤风汤低、中、高剂量组以及替米沙坦组;为验证自噬与模型的关系,设置3MA与氯喹组。采用Western Blot检测足细胞损伤相关蛋白nephrin、podocin及自噬相关蛋白LC3、Beclin-1、P62表达,溶酶体荧光探针检测自噬流活性,透射电镜观察足细胞内自噬体的形成,流式细胞仪检测细胞内ROS平均荧光强度(MFI)。结果与空白组比较,模型组足细胞nephrin、podocin、P62表达下调(P<0.01),LC3Ⅱ/LC3Ⅰ、Beclin-1表达上调(P<0.01),ROS的MFI增强(P<0.01)。透射电镜及溶酶体荧光探针结果显示模型组自噬体数量增多,溶酶体表达增强。与模型组比较,加味黄芪赤风汤中、高剂量组及替米沙坦组nephrin、podocin、P62表达上调(P<0.01,P<0.05),LC3Ⅱ/LC3Ⅰ、Beclin-1表达下调(P<0.01,P<0.05);3MA及氯喹组足细胞nephrin、podocin表达下调(P<0.01,P<0.05);加味黄芪赤风汤低剂量组Beclin-1表达下调(P<0.05);各给药组ROS MFI减弱(P<0.01,P<0.05)。透射电镜及溶酶体荧光探针结果显示加味黄芪赤风汤组及替米沙坦组自噬体数量减少,溶酶体表达减弱。结论AngⅡ通过下调nephrin、podocin表达损伤足细胞;加味黄芪赤风汤可上调AngⅡ诱导的nephrin、podocin表达,缓解足细胞损伤;加味黄芪赤风汤可能基于ROS-自噬通路,下调AngⅡ诱导的自噬流,拮抗肾小球足细胞损伤。

Objective To observe the mechanism of Modified Huangqi Chifeng Decoction(MHCD)allevi-ating the injury of glomerular podocytes induced by angiotensin Ⅱ(Ang Ⅱ)based on the regulation of reactive oxygen species(ROS)-autophagy pathway.Methods The low,middle,high dosage drug-containing serum of MH-CD(0.57 g/mL,1.15 g/mL,2.29 g/mL,respectively)and telmisartan(0.83 mg/mL)was prepared,the injury model of podocytes was induced by Ang Ⅱ(1×10^-7mol/L).Podocytes were divided into control group,model group,low,middle and high dosage MHCD group and telmisartan group.To verify the relationship between autophagy and the model,the 3MA and chloroquine groups were set up.The expressions of nephrin,podocin,LC3,Beclin-1 and P62 were analyzed by Western Blot;the activity of autophagy was analysed by lysosomes fluorescent probe;the formation of autophagosomes were observed by transmission electron microscope(TEM);intracelluar ROS levels were determined by flow cytometer.Results Compared with the control group,the expressions of nephrin,podocin and P62 of podocytes were decreased(P<0.01),the expressions of LC3Ⅱ/LC3Ⅰ and Beclin-1 were increased(P<0.01),MFI of ROS was increased(P<0.01)in the model group.The results of TEM and lysozyme fluorescence probe showed that the expressions of autophagosomes and lysosomes increased in the model group.Compared with the model group,the expressions of nephrin,podocin and P62 of podocytes were increased(P<0.01,P<0.05),the expressions of LC3Ⅱ/LC3Ⅰ and Beclin-1 were decreased(P<0.01,P<0.05)in the middle dose MHCD group,high dose MHCD group and telmisartan group;the expressions of nephrin,podocin were decreased in the 3MA and chloroquine group(P<0.01,P<0.05);the expressions of Beclin-1 was decreased(P<0.05)in the low dose MHCD group;MFI of ROS were decreased(P<0.01,P<0.05)in the MHCD groups and telmisartan group.The results of TEM and lysozyme fluorescence probe showed that the expressions of autophagosomes and lysosomes decreased in the intervention groups.Conclusions Ang Ⅱ induced injury of podocytes by decreasing the expressions of nephrin and podocin of podocytes;MHCD alleviates injury of podocytes by increasing the expressions of nephrin and podocin of podocytes.Based on the regulation of ROSautophagy pathway,MHCD could alleviate the injury of glomerular podocytes by downregulating autophagy flux induced by Ang Ⅱ.