The Study Of Possible Mechanism Of Autophagy In Microglial Inflammation Which Was Induced By The Omp31 Of Brucella Melitensis

1.ObjectiveThe objective of this study is as follows:(1)The BV-2 cells were treated with different concentrations of Omp31 for 6h(Reported to induce autophagy time),the purpose is to confirm whether Omp31 could induce autophagy of BV-2 cells and determine the appropriate concentration of Omp31,which can induce autophagy.(2)BV-2 cells were treated with Omp31 at different time,the purpose is to determine the appropriate concentration and time of Omp31-induced autophagy in order to further study the mechanism of autophagy in BV-2 cell inflammation.(3)BV-2 cells were treated with different concentrations of Rapa and 3-MA for 24 h to determine the proper concentration of Rapa to induce autophagy and the appropriate concentration of 3-MA to inhibit autophagy.(4)BV-2 cells were treated with Omp31,Rapa and 3-MA,in order to determine whether autophagy can regulate the expression of NF-?B(P65)pathway on TNF-? expression.2.Methods(1)Omp31 of Brucella melitensis induced BV-2 microglia autophagy?BV-2 cells were treated with(0,0.17,0.5,1.5,4.5,13.5)?g/m L Omp31 for 6 h,the expression of LC3 B was detected by Western blot.RT-q PCR was used to detect the expression of LC3 B m RNA.The autophagy in BV-2 cells was observed by transmission electron microscopy.The expression of TNF-?,IL-6 and IL-10 was detected by ELISA;?BV-2 cells were treated with 0.5 ?g/m L Omp31 for(0,6,12,24)h,the expression of LC3 B was detected by Western blot.RT-q PCR was used to detect the expression of LC3 B m RNA.The autophagy in BV-2 cells was observed by transmission electron microscopy.The expression of TNF-?,IL-6 and IL-10 was detected by ELISA;? Effects of Omp31-induced autophagy on NF-?B(P65)pathway in BV-2 cells?(2.5,5,10)?M Rapa,(2.5,5,10)m M 3-MA pretreated BV-2 cells for 24 h,and the content of autophagy marker LC3 B was detected by immunoblotting;??The expression of autophagy related proteins Beclin-1,LC3 B,P62 and NF-?B(P65)pathway-related proteins I?B?,p-P65 and t-P65 were detected by Western blot.The expression of TNF-? was detected by ELISA and the relative expression of(Beclin-1,LC3 B,TNF-?)m RNA was detected by RT-PCR.The intracellular autophagy was observed by transmission electron microscopy.The p-P65 protein was detected by immunofluorescence.3.Results(1)The BV-2 cells were treated with different concentrations of Omp31 for 6h,and it was shown that 0.5?g/m L Omp31 could increase the expression of LC3 B protein and m RNA,which was significantly different from that of other groups(P <0.05),and LC3 B Protein ?content increased(P <0.05),and more autophagy was observed under transmission electron microscope;(2)The expression of TNF-? and IL-6 in the 0.5 ?g / m L group was lower than that in the other experimental groups(P <0.01).The expression of IL-10 in each exp erimental group was lower than that in the control group(P <0.05),and it was not in a concentration-dependent manner;(3)The expression of LC3 B protein and the content of LC3 B protein in the experiental ?groups were significantly higher than that in the control group(P <0.05).The expression of LC3 B protein and LC3 B m RNA in the 6h group were significantly higher than that in the ?control group(P <0.01),and more autophagy was observed under transmission electron microscop;(4)The expression of TNF-? and IL-6 in each time group was higher than that in the control group(P <0.01),and the non time-dependent expression of IL-10 was lower than that of the control group(P <0.01);(5)The content of LC3 B protein in 5??M Rapa pretreated BV-2 cells was higher than that in each group(P <0.01).The pretreatment of BV-2 cells with 2.5m M 3-MA could decrease the content of LC3 B protein,which was statistically different from those of other ?groups(P <0.01);(6)BV-2 cells were treated with Omp31,Rapa and 3-MA.The results showed that both Omp31 and Rapa could promote the expression of LC3 B and Beclin-1 protein,and could promote the expression of both m RNAs and increase the content of LC3 B protein.The ?above results compared with the control group were statistically significant(P <0.05).3-MA had no effect on the expression of LC3 B protein and m RNA and the expression of Beclin-1 protein,but it could decrease the content of LC3 B protein and inhibit the expression of ?Beclin-1 m RNA(P <0.05).Omp31 and Rapa could decrease the content of P62 protein,while 3-MA resulted in an increase in P62 protein content compared with the control group(P <0.05).Omp31 and Rapa were able to induce autophagosome formation under transmission electron microscope,while 3-MA inhibited the formation of autophagosomes;(7)Omp31,Rapa and 3-MA could decrease the content of I?B? and t-P65 protein,which was significantly different from that of the control group(P <0.01).Omp31 could increase the p-P65 protein level,but Rapa and 3-MA could decrease the p-P65 protein level,which was significantly different from the control group(P <0.05).Omp31 could increase the content of p-P65 protein in the nucleus,which was detected by immunofluorescence technique.But Rapa and 3-MA could reduced the content of p-P65 protein in the nucleus.(8)Omp31 could increase the expression of TNF-?(P <0.01)compared with the control group,but Rapa and 3-MA decreased the expression of TNF-?(P <0.01).Compared with the control group,Omp31,Rapa and 3-MA could decrease the expression of TNF-? m RNA(P <0.01).4.Conclusion(1)Omp31 of Brucella melitensis can induce BV-2 microglia autophagy,and promoting the expression of TNF-? and IL-6,but inhibiting the expression of IL-10.(2)Autophagy can inhibit TNF-? expression by negatively regulating NF-?B(P65)signaling pathway.