Brucine Ameliorates Podocyte Injury Induced By The Terminal Complement Factor C5b-9 In Vitro

Objective To explore the effects and mechanism of brucine on podocytes injury induced by sublytic complement complex C5b-9,and provide experimental basis for its clinical application.Methods ①Podocytes were cultured in vitro.Remove the podocyte from liquid nitrogen,rapid recovery of the cells.Then induce the cells propagated by INF-γ at 33℃ with 5%CO2,and subsequently culture them at 37℃ for differentiation for 10-14 days..②To detect the toxicity with the LDH kit in different doses of brucine on podocyte for choosing the Appropriate intervention dose.Cell morphology was observed at 8 h,24 h and 48 h,respectively,and collected for subsequent experiments.③C5b-9 was assembled to establish the model of the cell sub dissolution with different concentrations of C5b6,C7,C8,C9 incubated in the cultured cells,.Observed the deposition of C5b-9 of the model by indirect immunofluorescence.④Grouping and intervention:the normal control group cells;C5b-9 effect of podocyte group;C5b-9 and brucine effect of podocyte.⑤ To detect the expression of nephrin,podocin protein of podocytes in Western Blot method.Results LDH test results:compared with NC group,when the brucine concentration was 80 and 160ug/ml in 48h,the cell activity of LDH was significantly increased(P<0.05),and no significant differences in others(P>0.05).Indirect immunofluorescence showed in C5b-9 group cell surface could be fould the deposition of fine particles,and the normal group and negative control group could not be found.The results of Western blot:in the C5b-9 intervention group the protein expression of nephrin and podocin was decreased(P<0.05).All the time the nephrin and podocin protein expression in high brucine concentration group was higher than that of C5b-9 intervention group(P<0.05),and the effect was in a centration-dependent characteristiccon.From the time piont,the brucine in 8h increased the protein expression of Nephrin relatively obviously,and the protein expression of Podocin increased more significantly in 24h.Conclusion LDH test result teld us that the suitable intervention dose of brucine were 10,20,40ug/ml.Immunofluorescence result showed that C5b-9 was successfully assembled in the membrane of the podocytes and then we could go on the experimentation.Brucine within a certain dosage could up regulate the expression of nephrin and podocin protein in C5b-9 induced cells,and showed that brucine had a protective effect in C5b-9 induced podocyte injury.The brucine intervention to the protein expression of the two podocyte molecular had a time effect,which the obvious up regulation of nephrin was at 8h,and for podocin was at 24h.