Experimental Study Of Baicalein Inhibits Human Cytomegalovirous Infection In Human Astrocyte

Objective:1. To investigate the inhibitory effects of baicalein on abnormal changes of IE and pp65mRNA and protein expression caused by HCMV in astrocyte (AS).2. To explore the action mechanism of Chinese Medicine baicalein inhibit human cytomegalovirous (HCMV) infection in astrocyte.Methods:1. Primary human astrocytes were extracted by anchorage velocity-dependent separation method. Immunofluorescence technique were used to calculate the purity of human primary astrocytes by analyze the expression of the astrocytes marker protein-GFAP.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) and late (pp65) mRNA and protein of HCMV were detected by Real-time PCR and immunofluorescence staining to verify that the human astrocytes are permissive for HCMV replication.4. MTT assay were chosen to detect the inhibitory effects of BAI on abnormal changes of cell viability caused by HCMV in astrocyte cells.5. IE-. pp65mRNA expression in different treatment groups (BAI+HCMV group, HCMV group) was detected by Real-time PCR.6. Changes of IE、UL84、pp65and Acetyl-Histone H3K9protein in different groups were detected by western blot and immunofluorescence.Results:1. Human primary astrocytes were isolated and cultured successfully in vitro; Specific bright red fluorescence was observed in the plasmas of the cells by immunofluorescence staining and the GFAP-positive cells came up to90%.2. Cytopathic effects (CPE) appeared at5day post inoculation in more than80%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 was1×108pfu/ml.3.The Real-time PCR and immnuofluorescence staining technique confirmed that the mRNA and protein of IE and pp65were positive in infected astrocytes whereas uninfected cells were negative.4. In20μM BAI+HCMV group MTT values were higher than HCMV group at24h、48h、72h post-infection(P<0.05).5. In HCMV group, Cytopathic effects (CPE) were observed at48h post-infection. In20μM BAI+HCMV group, CPE can not be observed obviously and most cells kept normal forms.6. Real-time PCR found the expression of IE、pp65mRNA in20μM BAI+HCMV group were lower than HCMV group at48h、72h post-infection(P<0.05).7. Immunofluorescence and western blot assay indicated that in20μM BAI+HCMV group the expression of IE、UL84、pp65and Acetyl-Histone H3K9protein were lower than HCMV group (P<0.05)..Conclusions:1. Human primary astrocytes are permissive to HCMV infection.2. Baicalein at appropriate concentration can inhibit abnormality cell viability3. Baicalein at appropriate concentration can inhibit expression of IF、pp65mRNA and protein in HCMV infection.4. The mechanism of baicalein anti-HCMV infection may be through the inhibition of IE2acetylation.