摘要
目的:雷公藤甲素对Heymann肾炎和嘌呤霉素肾病大鼠的疗效及其对足细胞病变的影响提示,雷公藤甲素有可能直接作用于足细胞,从而改善其病变状况。本研究借助小鼠足细胞系,体外观察了雷公藤甲素对足细胞损伤的保护和修复作用,并对其作用机制进行了研究。方法:体外采用氨基核苷嘌呤霉素(puromycin aminonucleo-side,PAN)致足细胞损伤模型。观察雷公藤甲素对足细胞损伤的保护作用研究中,雷公藤甲素与足细胞预孵育30min后,再加入含PAN的培养基,继续作用24h;观察雷公藤甲素对足细胞损伤的修复作用研究中,PAN作用足细胞24h,造成足细胞明显损伤后,再加入含或不含雷公藤甲素的培养基,继续培养3天。免疫荧光法和流式细胞仪分析足细胞骨架相关蛋白F-actin和Synaptopodin的变化,并进一步观察了细胞内调节骨架装配的Rho/ROCK信号通路的活性,即采用Western Blot检测ROCK-I底物——肌球蛋白磷酸酶结合亚单位(MBS)磷酸化水平,以及Rho/ROCK信号通路选择性阻断剂Y-27632对雷公藤甲素作用的影响。同时采用免疫荧光法和流式细胞仪分析足细胞裂孔膜相关分子Nephrin和Podocin在足细胞的分布及表达量的变化。结果:正常分化足细胞F-actin呈丝状结构,密度大,丝强劲有力,沿细胞极性分布;Synaptopodin呈颗粒状沿细胞骨架分布。裂孔膜蛋白Nephrin沿足细胞膜和细胞骨架分布,Podocin呈线状均匀分布于胞浆内,PAN作用24h后,细胞足突回缩,F-actin几乎完全解聚,Synaptopodin表达消失。Nephrin和Podocin表达减弱,正常丝状结构消失。而经过雷公藤甲素的预处理,PAN诱导的足细胞损伤明显减弱,足细胞未表现出上述骨架结构的改变,Synaptopodin表达没有明显减弱。同时我们的结果显示,在PAN造成足细胞明显损伤后再加入雷公藤甲素,足细胞损伤能够得到一定修复,细胞内重新出现极性分布的微丝,同时,Synaptopod-in的表达也得以修复。Rho/ROCK信号通路研究显示,100μg/mlPAN明显降低Rho/ROCK通路活性。雷公藤甲素能有效遏制MBS磷酸化水平的降低,维持足细胞Rho/ROCK通路的活性状态。Rho/ROCK信号通路选择性抑制剂Y-27632可阻断雷公藤对足细胞骨架结构的保护作用。此外,对裂孔膜蛋白Nephrin,Podocin的研究表明,不论是在预防组和治疗组,PAN对Nephrin和Podocin的损伤可在一定程度上被雷公藤甲素预防和修复。结论:雷公藤甲素可稳定足细胞的细胞骨架,拮抗PAN对足细胞的损伤。在足细胞损伤已经发生后,雷公藤甲素可逆转足细胞的损伤,修复足细胞骨架结构和相关分子的表达。雷公藤甲素的上述作用与细胞内Rho/ROCK信号通路密切相关。雷公藤甲素对足细胞的影响可能是其降低肾小球肾炎患者尿蛋白的重要机制之一。
Objective：To explore the protection and therapeutic effect of triptolid on podocyte injury induced by puromycin aminonucleoside （PAN） in vitro. Methodology：Conditionally immortalized mouse pedocytes were treated with PAN. For the protective experiments, the pedocytes were pretreated for 30mins with triptolid （3 ng/ml） before PAN （100 μg/ml） exposure for 24hrs, while for the recovery experiments, after 24hr of PAN （ 100 μg/ml） treatment, the culture medium was changed to medium with or without triptolide （3 ng/ml）. The cytoskeleton distribution of pedocyte as indicated by F-actin and synaptopedin was observed by fluorescence microscopy and flow cytometry. At the same time, the activity of Rho signal pathway that mediates actin filament polymerization was analyzed by measure the extent of phosphorylation of the myosin-binding subunit of myosin phosphatase （ MBS）, one of the major substrates of Rho-kinase. In addition, the slit diaphragms proteins, podocin and nephrin, were observed by fluorescence microscopy and flow cytometry. Results： We found that PAN almost completely disrupted the podocyte actin cytoskeleton, and made the disappearance of synaptopodin expression, while triptolide stabilized the actin filaments, and improved the synaptopodin expression. Rho signaling was not activated in PAN-induced podocyte injury. Addition of triptolide induced a strong increase in phosphorylated MBS. Y-27632, a specific kinase inhibitor for Rho, can block triptolide protective effect. We also observed that PAN decreased the expression of nephrin and podocin, and disrupt filamentous pattern of podocin in podocytes, which was ameliorated by the treatment with Triptolid. Conclusion：Triptolide can not only provide direct protection of podocytes against PAN-induced injury, but also enhance recovery from prior podocytes injury. The protective effect of triptolide may partially contribute to the restoration of myosin phosphatase activity mediated by Rho kinase. The antiproteinuria effect of triptolid observed in vivo may be due, at least in part, to its direct effects on podocyte.
出处
《肾脏病与透析肾移植杂志》
CAS
CSCD
2007年第2期119-126,200,共9页
Chinese Journal of Nephrology,Dialysis & Transplantation
基金
全军"十一.五"重点课题(06G040)
南京军区"十一.五"重点课题(06Z35)
