| HIV,a pathogen causing global AIDS epidemic,is one of the most serious diseases that threaten human health.HIV-1 membrane glycoprotein gp120 and gp41 play an important role in the process of HIV infection.Gp120 is a toxic protein when the virus enters cells and interacts with the target cells.It is toxic to nerve cells also.Therefore,the aim of this study is to determine the effect of HIV-gp120 on the expression of BDNF in microglia and its possible mechanism.Firstly,MTT assay was used to determine the appropriate concentration of gp120 on microglia BV2.Compared to the control,the viability of cells treated with 2 ng/m L,10 ng/mL and 50 ng/mL gp120 for 12 h was 80.1%,79.7% and 80.5% respectively.However,gp120 at a dose of 1000 ng/m L reduced the viability to 29.2%.Thus,a dose of 10 ng/m L was chosen in the subsequent experiments only to induce appropriate toxic effects on the cells.Secondly,BV2 cells were incubated with HIV-gp120 for 1 h,3 h,6 h,9 h,12 h and 24 h,and the intracellular proteins were extracted from the cells and analyzed by Western blotting,to check the expression of BDNF.The results showed that proBDNF and mBDNF protein were the highest in gp120-stimulated BV2 cells for 3 h,which were 1.9 times and 2.2 times higher than that of the control group.In addition,we also found that the BV2 cells were activated by gp120,which induced the expression of CD11 b increasing 1.62 times than that of the control group.At the same time,the accumulation of Wnt3 a and ?-catenin occurred in gp120-treated BV2 cells,but the expression of Wnt5 a did not change significantly.Therefore,it is speculated that gp120 activates BV2 cells via the classical Wnt/?-catenin signaling pathway and promotes the expression of BDNF.To further investigate the possible mechanism,the signal pathway was activated with different concentrations of Wnt3 a protein(25 ng/m L,50 ng/mL,100 ng/m L).Different concentrations of DKK1(10 ng/m L,50 ng/m L,100 ng/m L)and IWR-1(0.1 ?M,1 ?M,10 ?M)were used to block the classical Wnt/?-catenin signaling.The results showed that Wnt3a(100 ng/mL)increased the expression of ?-catenin in BV2 cells,which was 24 times of that in the control group.This indicated the activation of classical Wnt signaling pathway.Interestingly,the expression of BDNF in the microglia after Wnt3 a incubation was 1.81 times higher.In contrast,the expression of BDNF was significantly inhibited by DKK1(100 ng/m L)and IWR-1(10 ?M),and the expression was decreased by 64.6% and 79.8% respectively.After exploring the optimal concentrations of DKK1 and IWR-1,this study continued to investigate whether DKK1 or IWR-1 could inhibit the increase in BDNF expression induced by gp120.The results showed that the expression of BDNF in BV2 cells treated with DKK1 or IWR-1 alone was 68.6% and 62.6% lower than that of gp120 treated cells.Compare with gp120-treated group,the expression of BDNF in DKK1 and gp120 treated group or IWR-1 and gp120 simultaneously treated group were reduced by 70% and 72.6%.These results suggest that DKK1 and IWR-1 can significantly inhibit the up-regulation of BDNF expression induced by gp120,indicating that blocking the Wnt/?-catenin signaling pathway can inhibit the expression of BDNF.The effect of DKK1 on the up-regulation of BDNF expression by Wnt3 a was investigated by blocking the classical signal pathway in BV2 with DKK1.It was found that the expression of BDNF in DKK1 and Wnt3a-treated cells decreased by 73% compared with Wnt3a-treated cell group,but its expression was still higher than that of control.This experiment showed that DKK1 can significantly inhibit the expression of BDNF induced by Wnt3 a.This further demonstrated that blocking the Wnt/?-catenin signaling pathway can inhibit the expression of BDNF.The results of the above studies showed that HIV-gp120 can activate BV2 cells and up-regulate the expression of BDNF by Wnt/?-catenin classical signaling pathway. |
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