温阳活血利水法对足细胞骨架及相关蛋白影响

Effect of Wenyang-Huoxue-Lishuiformula on Cytoskeleton and Relatedproteins of Podocytes

目的:观察温阳活血利水方含药血清对嘌呤霉素损伤小鼠永生系足细胞组织蛋白酶L(cathepsin L,Cat L)的转录因子Dendrin、膜相关鸟苷酸激酶转化蛋白2(membrane-associated guanylate kinase inverted2,MAGI-2)、下游底物synaptopodin、Rho A mRNA与蛋白表达变化以及足细胞骨架蛋白actin、中间丝分布情况的影响,探讨温阳活血利水方的作用改善肾病综合征蛋白尿及足突融合的机制。方法:体外培养小鼠足细胞,采用嘌呤霉素45 mg/L、中药含药血清或地塞米松与足细胞孵育12 h,荧光定量PCR检测Cat L、synaptopodinmRNA表达变化; western blotting测定MAGI-2、synaptopodin、Cat L、Rho A的蛋白表达;细胞免疫荧光观察Dendrin的分布以及细胞骨架蛋白微丝和中间丝的荧光分布变化。结果:嘌呤霉素作用足细胞12 h后,Dendrin转核比例增多(与正常组对比P <0. 01),MAGI-2、Rho A、synaptopodin蛋白的表达显著性下降(与正常组对比,均P <0. 01),Cat L mRNA与蛋白的表达显著性升高(与正常组对比,均P <0. 01),synaptopodin mRNA表达没有变化(与模型组比较P> 0. 05),足细胞伪突回缩变短,中间丝和微丝结构紊乱,杂乱无章排列,结构不清晰。地塞米松、10%与20%含药血清干预后Cat L mRNA与蛋白表达减少,MAGI-2、Rho A、synaptopodin蛋白的表达有所增多,Dendrin转核比例减少(与模型组对比,均P <0. 01),均改善了足细胞骨架结构。结论:温阳活血利水方能减少足细胞受损后Dendrin核内转移,降低胞质cat L水平,从而减少synaptopodin降解,使Rho A表达增加,改善微丝(F-actin)和中间丝的重构,稳定细胞骨架结构,减少足突融合,从而减少蛋白尿。

Objective:This study aimed to investigate the effect of warming yang serum on correlative proteins of podocytes in podocytes of mouse with puromycin-induced injury,including the CathepsinL,transcription factor Dendrinon,membrane-associated guanylate kinase inverted 2,synaptopodin and RhoA as well as the distribution of cytoskeletal protein actin and intermediate filament in the podocyte.Methods:Mouse podocytes were challenged with puromycin aminonucleoside(PAN)alone in the present of dexamethasone or serum containing traditional Chinese drugs at 10%or 20%concentration respectively for 12h.The protein expression of MAGI-2,synaptopodin,CatL and RhoA was quantitatively measured by western blotting,and the mRNA expression of CatL and synaptopodin were detected by fluorescence quantitative PCR.Indirect cell immunofluorescence staining was used to display the location of dendrin in podocytes.FITC-conjugated phalloidinand Cy3-conjugated rabbit-anti-vimentin were used to observe the distribution of F-actin and intermediate filament.Results:Afterp PAN-induced podocyte injury for 12 hours,the percentage of nuclear translocation of dendrin was elevated(compared with that of normal group,P<0.01).The protein expression of MAGI-2,RhoA,and synaptopodin were decreased significantly(compared with normal group,all P<0.01).However,there was no change in the mRNA expression of synaptopodin(compared with model group,P>0.05).The mRNA and protein expression of CatL were significantly increased(compared with normal control group,all P<0.01).Podocyte pseudopodia were retracted and shortened;the structure and arrangement of microfilament and intermediate filament were almost completely disrupted.After treatment with dexamethasone,10%and 20%of Chinese medicine,the mRNA and protein expression of CatL were decreased(compared with model group,all P<0.01).The protein expression of MAGI-2,RhoA,and synaptopodinwere increased(compared with model group,all P<0.01).The percentage of nuclear translocation of dendrin was declined(compared with model group,P<0.01).The cytoskeleton structure was restored in each treatment group.Conclusion:Wenyang-Huoxue-Lishui formula is able to upregulate the expression of MAGI-2,and then to decrease nuclear import of dendrin,in turn todecrease CatL level,consequently to increase synaptopodin and RhoAandstabilize the cytoskeletal structure,ultimately to decrease foot process fusion and proteinuria.