Experimental Study Of Baicalein On The Mechanism In Inhibiting The Exprssion Of Human Cytomegalovirus IE-86

Objective:1. To study the anti-infection mechanism of chinese medicine baicalein(BAI) in inhibiting the expression of human cytomegalovirus(HCMV) IE86 in vitro by observing the histone acetylation expression in IE-86 promoter region and the cellular(hsa-mi R-200) and viral(hcmv-mi R112-1) micro RNAs expression. 2.To explore the action mechanism of Chinese Medicine baicalein which inhibit human cytomegalovirous(HCMV) IE86 and further study the anti-HCMV infection mechanism of Chinese medicine baicalein(BAI)Methods:1.The human primary astrocytes cells were derived from the primary human neural stem cells,the cells were cultured and passaged to 3-4 generations and then infected with the human cytomegalovirus. 2.HCMV was propagated in human embryo lung cells;The empty spot filamentous experiment was chosen to detect the degree of virus drops. 3.The immediate early(IE) m RNA of HCMV group,HCMV+BAI group and BAI+HCMV group were detected by Real-time PCR. 4.Expression changes of histone acetylation in IE-86 promoter region were detected by chomatin immunoprecitation(CHIP). 5.Real-time PCR were used to test the expression changes of the cellular(hsa-mi R-200) in BAI group,HCMV group,HCMV+BAI group,BAI+HCMV group and control group. 6.The expression changes of the viral(hcmv-mi R112-1) mi RNAs in BAI group,HCMV group,HCMV+BAI group, BAI+HCMV group and control group were detected by the Real-time PCR.Results:1.Cytopathic effects(CPE) appeared at 96 hours post inoculation in more than 80% human embryonic lung fibroblasts.The infected cells were consonant with changes of cytomegalovirus infection cells in morphology.Determination results of plaque showed the plaque forming unit(pfu) of HCMV was 3.5×108pfu/m L.2.Real-time PCR found the expression of IE m RNA in HCMV+BAI group and BAI+HCMV group were lower than HCMV group at 48h、72h post-infection(P<0.05). 3.The results of chomatin immunoprecitation indicated that in 20umol/LBAI+HCMV group the expression of histone acetylation in IE-86 promoter region were apparently lower than HCMV group(P<0.05). 4.Real-time PCR results showed that the expression of cellular micro RNAs(mi R-200b/c/429)in HCMV+BAI group and BAI+HCMV group were obviously higher than HCMV group.And that the relative expression were higher at 24 h post infection,then reduced at 48 h post infection, the levels of 72 h between the two group.(P<0.05). 5.Real-time PCR results indicted that the expression of cellular micro RNAs(mi R-200a/141)in HCMV+BAI group and BAI+HCMV group were obviously higher than HCMV group.And that the relative expression were higher at 72 h post infection. 6.Real-time PCR results showed that the expression of viral micro RNAs(hcmv-mi R112-1) in HCMV+BAI group and BAI+HCMV group were obviously higher than HCMV group.Conclusions:1.Baicalein at appropriate concentration can inhibit the expression of histone acetylation in IE-86 promoter region. 2.Baicalein at appropriate concentration can upregulate the expression of micro RNAs(hsa-mi R-200). 3.Baicalein at appropriate concentration can upregulate the expression of micro RNAs(mi R-UL112-1). 4.Baicalein at appropriate concentration can downregulate the expression of HCMV-IE m RNA.