归肾经方含药血清对阿霉素诱导的足细胞损伤及自噬相关蛋白的影响

Effect of medicated serum of composing prescription with kidney-meridian tropismon adriamycin-induced podocyte damage and autophagy-related proteins

目的:研究归肾经方对阿霉素(adriamycin,ADR)诱导的肾小球足细胞损伤标志蛋白nephrin及自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ、p-mTOR等表达的影响。方法:以ADR体外诱导的方法制作足细胞损伤的细胞模型。除空白组外均以ADR诱导足细胞,分为5组:空白组(control group,10%空白血清)、模型组(model group,10%空白血清+ADR 0.5μg·mL-1)、中药高剂量组(H-TCM group,10%高剂量归肾经方含药血清+ADR 0.5μg·mL-1)、中药低剂量组(L-TCM group,10%低剂量归肾经方含药血清+ADR 0.5μg·mL-1)、替米沙坦组(TMST group,10%替米沙坦含药血清+ADR 0.5μg·mL-1)。CCK-8检测各组细胞活性改变,Western blot法检测各组足细胞标志蛋白nephrin、desmin及自噬相关蛋白beclin-1、LC3Ⅱ/Ⅰ等表达。结果:①与空白组比较,模型组细胞活力显著降低(P<0.01),与模型组比较,中药高剂量组及替米沙坦组细胞活性显著增高(P<0.05);②Western blot检测结果显示,与control组比较,model组nephrin显著降低(P<0.01),desmin、LC3Ⅱ/Ⅰ、Beclin-1、p-Akt、p-mTOR显著增高(P<0.01);与Model组比较,H-TCM组及TMST组nehprin表达显著增高(P<0.05),desmin表达显著降低(P<0.05),H-TCM、L-TCM及TMST组Beclin-1、LC3Ⅱ/Ⅰ、p-Akt、p-mTOR表达显著降低(P<0.01);与H-TCM组比较,L-TCM组LC3Ⅱ/Ⅰ表达显著增高(P<0.05)。结论:归肾经方可以修复ADR诱导的足细胞损伤,改善细胞内的自噬水平,与上调细胞内nephrin、Beclin-1、p-Akt、p-mTOR的表达有关。

OBJECTIVE To study the effect of the prescription of kidney meridian on the expression of nephrin, Beclin-1, LC3Ⅱ/Ⅰ and p-mTOR in glomerular podocyte injury induced by adriamycin(ADR).METHODS The podocyte injury cell model was established by ADR induction in vitro.Podocytes were induced by ADR in all but the blank group and divided into five groups: blank group(control group, 10% blank serum), model group(model group, 10% blank serum+ADR 0.5 μg·mL-1), high-dose group(H-TCM group, 10% high-dose Guishen Jingfang medicated serum+ADR 0.5 μg·mL-1), low-dose group(L-TCM group, 10% low-dose Guishen Jingfang medicated serum+ADR 0.5 μg·mL-1), and telmisartan group(TMST group, 10% telmisartan medicated serum+ADR 0.5 μg·mL-1).CCK-8 was used to detect changes in cell viability in each group, and Western blot was used to detect the expression of podocyte marker proteins nephrin, desmin and autophagy-related proteins beclin-1 and LC3 Ⅱ/Ⅰ in each group.RESULTS ①Compared with control group, the cell viability of model group was significantly reduced(P<0.01). Compared with model group, cell activity was significantly increased in the high-dose group and the telmisartan group(P<0.05).②Western blot showed expression of nephrin of model group was reduced significantly(P<0.01), and the expression of desmin, LC3Ⅱ/Ⅰ,Beclin 1, p-Akt,p-mTOR increased significantly(P<0.01) compared with the control group. Nehprin expression of H-TCM group and TMST group was significantly increased(P<0.05), desmin expression was decreased significantly(P<0.05), Beclin-1, LC3Ⅱ/Ⅰ,p-Akt, p-mTOR expression of H-TCM group, L-TCM group and TMST group were reduced significantly(P<0.01) compared with the model group. LC3Ⅱ/Ⅰexpression of L-TCM group increased significantly(P<0.05) compared with H-TCM group.CONCLUSION The prescription of kidney meridian could repair ADR-induced podocyte injury and improve the level of autophagy in cells, which was related to the up-regulation expressions of nephrin, Beclin-1, LC3Ⅱ/Ⅰ,p-Akt and p-mTOR in cells.