Inhibitory effects of special siRNA targeting TLR4 gene on the TNF-α expression of BV-2 cells induced by hypoxia-reoxygenation / 中华急诊医学杂志

ABSTRACT

Objective To study the inhibition of tumor necrosis factor-alpha(TNF-α)cytokine expression of BV-2 cells induced by hypoxia-reoxygenation injury by siRNA targeting TLR4 gene via the RNAi mechanisms.Method BV-2 mouse microglial cell line was cultured in six-well plates and randomly divided into group N(nor-mal group),group H(hypoxia-reoxygenation),group T(hypoxia-reoxygenation+TLR4-siRNA transfected group),group C(hypoxia-reoxygenation+pEGFP-H1/control-siRNA transfected group)and group B(hypox-ia-reoxygenation+pEGFP-H1 transfected group).Group H,group T,group C and group B were cultured in hy-poxia condition for 3 h followed by reoxygenation for 24 h.The plasma was transfected into BV-2 cells mediated by lipofectamine 2000.The efficiency of transfection were detected by flow cytometry to observe the expression of EGFP.RT-PCR method was used to detect the level of mRNA of TLR4 or NF-кB p65.Westem blot methed was used to test the expression of TLR4 protein.and ELISA was used to test the level of TNF-α in the supernatants.Analysis of variances was used for statistical analysis.Results The expression of EGFP gene waa;(67.58±7.16)% after transfection by flow cytometry analysis.Compared to group N,the TLR4 mRNA,NF-кB p65 mR-NA,TLR4 protein level and the TNF-α quantity in group H,group T,group C and group B increased after the hy-poxia-reoxygenation treatment(P＜0.01).While the expression of the TLR4 mRNA,NF-кB p65 mRNA,TLR4 protein level and the TNF-α quantity in the group T down-regulated compared to group H,group C and group B(P＜0.01).And there were no changes in group C,group B and group H about observation index(P＞0.05).Conclusions The siRNA targeting TLR4 mRNA could inhibit the inflammatory reaction released by BV-2 cells in-duced by hypoxia-reoxygenation stimulation.