Triptolide ameliorates podocyte injury induced by the terminal complement factor C5b-9 in vitro

Objective:To explore the protective effects and mechanism of triptolide on podocytes injury induced by sublytic complement complex C5b-9. Methodology:Terminal complement complex C5b-9 was formed on podocytes using purified C5b-6 and C7-C9.Complement lysis was determined by measuring release of lactate dehydrogenase,similarly(LDH).C5b-9 deposition on the membrane of podocyte was detected by LSM and flow cytometry using the human C5b-9-specific mAb aE11.F-actin was observed by fluorescence microscopy.The podocytes were treated with triptolide before or after C5b-9 has been assembled,and then observed the effect of triptolide on the reactive oxygen species(ROS) gerneration and MAPK activation induced by C5b-9.ROS was assayed using the fluoroprobe CM-H2DCFDA,while MAPK activation was analyzed by western blot. Results:Intact C5b-9 was formed on the surface of podocyte as detected by imunofluorescence.Sublytic C5b-9 induced marked actin cytoskeleton alteration in a time-dependent manner.C5b-9 significantly increased ROS generation in podocytes.This effect was observed rapidly at 10min and maintained for 90min.Furthermore,C5b-9 stimulated a strong increase in phosphorylated p38 in podocytes after 1h incubation.In contrast,C5b-9 treatment had no significant effect on phospho-ERK1/2 and phosphor c-Jun MAPK levels.In addition,antioxidant NAC and SB202190,a specific kinase inhibitor for p-38,can block C5b-9 induced podocyte injury as showed by cytoskeleton staining,indicating that ROS generation and MAPK activation mediated C5b-9-induced podocyte injury.Triptolide did not interfere with the formation of C5b-9 on the membrane of podocytes.When podocytes were treated with triptolide after C5b-9 has been assembled for 30min,it is observed that triptolide has no significant inhibiting effect on C5b-9 induced ROS generation.However,the activation of p38 MAPK decreased. Conclusion:Triptolide had a protective effect in C5b-9 induced podocyte injury,which might partially due to the inhibition of p38 MAPK activation.