论文部分内容阅读
目的:体外观察雷公藤甲素对膜攻击复合物C5b-9介导的足细胞损伤的影响,探讨雷公藤甲素治疗膜性肾病的疗效机制。方法:以纯化的C5b6及C7~C9体外组装C5b-9,建立足细胞亚溶破模型。免疫荧光染色分析C5b-9对足细胞骨架相关蛋白F-actin表达和分布的影响;在观察雷公藤甲素对C5b-9导致的足细胞损伤的治疗机制研究中,免疫荧光观察和流式细胞仪定量分析雷公藤甲素对C5b-9在足细胞膜上组装和活性的影响。并进一步在C5b-9组装完成后加入雷公藤甲素,观察雷公藤甲素对C5b-9诱导的足细胞内活性氧(ROS)和丝裂原活化蛋白激酶(MAPK)信号通路的影响。流式细胞仪分析荧光探针CM-H2DCFDA标记的细胞内ROS,MAPK信号通路的活性采用W estern b lot检测p-38,ERK 1/2和JNK MAPK磷酸化水平,以及MAPK信号通路选择性阻断剂SB202190,U0126和SP600125对C5b-9作用的影响。结果:免疫荧光和LDH释放试验显示C5b-9在足细胞膜组装成功,亚溶解剂量C5b-9对足细胞膜完整性没有明显影响,但可呈时间依赖性的破坏足细胞骨架结构,表现为细胞骨架的极性消失,排列紊乱,部分足细胞F-actin的丝状结构完全消失。C5b-9可诱导足细胞内ROS的明显增加,激活p-38 MAPK信号通路,但C5b-9对ERK MAPK和JNK MAPK信号通路没有明显的影响。抗氧化剂N-乙酰半胱氨酸(NAC)、p-38 MAPK通路特异性抑制剂SB202190均能够拮抗C5b-9对足细胞的损伤,而ERK MAPK和JNKMAPK通路抑制剂U0126和sp600125对C5b-9诱导的足细胞损伤没有明显的保护作用。雷公藤甲素(10 ng/m l)对C5b-9复合物在足细胞膜上组装量和活性没有明显的影响。当C5b-9在足细胞膜上组装完成后加入雷公藤甲素,雷公藤甲素对C5b-9诱导的细胞内ROS的产生没有明显的抑制作用,但能够有效遏制p-38 MAPK信号通路的活化,p-38 MAPK的磷酸化水平显著降低。结论:雷公藤甲素的疗效机制除了免疫抑制和抗炎作用外,还可通过抑制C5b-9激活的细胞内p-38 MAPK信号通路发挥直接的足细胞保护作用。雷公藤甲素对足细胞的上述作用可能部分参与了其对膜性肾病的疗效。
OBJECTIVE: To observe the effect of triptolide on C5b-9-mediated podocyte injury in vitro and to explore the therapeutic mechanism of Triptolide on membranous nephropathy. Methods: C5b-9 was assembled with purified C5b6 and C7-C9 in vitro to establish a podocyte sub-lysis model. The effect of C5b-9 on the expression and distribution of F-actin in podocyte cytoskeleton was analyzed by immunofluorescence staining. In the study of therapeutic mechanism of triptolide-induced podocyte injury by triptolide, immunofluorescence and flow cytometry The effect of triptolide on the assembly and activity of C5b-9 on podocyte membranes was quantified. Triptolide was further added after C5b-9 assembly, and the effects of triptolide on C5b-9-induced intracellular reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signaling pathway were observed. Flow cytometric analysis of intracellular ROS and MAPK signaling pathways labeled with fluorescent probe CM-H2DCFDA The phosphorylation of p38, ERK1 / 2 and JNK MAPK was detected by Western blot and Western blotting of MAPK signaling pathway Effect of Disintegrants SB202190, U0126 and SP600125 on the Effect of C5b-9. Results: Immunofluorescence and LDH release assay showed that C5b-9 was successfully assembled in the podocyte membrane. C5b-9 had no significant effect on the integrity of podocytes, but it could destroy the podocyte cytoskeleton in a time-dependent manner. The polarity disappeared, disordered arrangement, partial podocyte F-actin filamentous structure completely disappeared. C5b-9 induced a significant increase of ROS in podocytes, activating p-38 MAPK signaling pathway, but C5b-9 had no obvious effect on ERK MAPK and JNK MAPK signaling pathway. Antioxidant N-acetylcysteine (NAC) and p-38 MAPK pathway-specific inhibitor SB202190 both could antagonize C5b-9 injury to podocytes, but ERK MAPK and JNKMAPK pathway inhibitor U0126 and sp600125 inhibited C5b-9 Induction of podocyte injury did not have a significant protective effect. Triptolide (10 ng / ml) had no significant effect on the amount and activity of C5b-9 complex assembly on the podocytes. Triptolide did not significantly inhibit the production of intracellular ROS induced by C5b-9, but inhibited the activation of p-38 MAPK signaling pathway when C5b-9 was added to the podocyte membrane after the assembly of triptolide , p-38 MAPK phosphorylation levels were significantly reduced. Conclusion: The mechanism of action of triptolide in addition to immunosuppressive and anti-inflammatory effects, but also by inhibiting C5b-9 activation of intracellular p-38 MAPK signaling pathway to play a direct podocyte protective effect. The above effects of triptolide on podocytes may be partially involved in its efficacy in membranous nephropathy.