Inhibitory Effects Of Genistein On Abnormal Changes Of Proliferation And Apoptosis Caused By HSV-1 In Human Glioma Cells

Objective:1 To establish an in vitro Herpes simplex virus-1 (HSV-1) infection model of human glioma cells(U251) and investigate the effects of HSV-1 on proliferation, apoptosis and celll cycle of U251 cells.2 To investigate the inhibitory effects of Genistein(GST) on abnormal changes of proliferation and apopptosis in U251 cells caused by HSV-1 infection.Materials and methods:1 HSV-1 group cells were infected by HSV-1 at a multiplicity of infection(MOI) of 5, control group was set up in the meantime. Inverted microscope was used to detect the morphologic changes. Cell proliferation was measured by MTT. Flow cytometry along with Guava Nexin(?) Reagent were used to detect cells apoptosis and cell cycle was measured.2 U251 cells were infected by HSV-1 while GST group, GST+HSV-1 group, HSV-1 group and control group were set up. Cell proliferation were measured by MTT assay at 24,48,72,96 h.p.i. Flow cytometry along with Guava Nexin(?) Reagent were used to detect cells apoptosis(15μg/ml GST) while cell morphologic changes were observed in the meantime. U251 cells in GST+HSV-1 group(15μg/ml GST), HSV-1 group and control groupwere collected at 6,12,24,36,48 h.p.i to analyze GD mRNA products via RT-PCR.Results:1 Cells in control group kept normal shape all along. Infected cells started to merge at 24 h.p.i, and typical cytopathic effects were observed at 48 h.p.i.MTT assay showed cell proliferation in HSV-1 group was reduced compared to control group (p<0.05). Annexin V/7-AAD double staining technique showed cell apoptosis rates in HSV-1 group were remarkable increased compared to control group at 12,24,36 h.p.i (p <0.05). Cell cycle analysis showed that percent of G0/G1 phase cells were reduced(p <0.05), while those of S phase and G2/M phase increased significantly(p<0.05) in HSV-1 infected cells.2 MTT assay showed the great differences between different groups(F=510.743, P <0.05) and an abvious time effect (F=2243.2, P<0.05). MTT values in 15μg/ml GST+HSV-1 group were higher than HSV-1 group(P<0.05). HSV-1 group showed typical morphologic changes at 36 h.p.i, while most of the cells in 15μg/ml GST+HSV-1 group kept their normal shape. Cell apoptosis indicated the great differences between different groups(F=1788.58, P<0.05) and an abvious time effect (F=766.478, P<0.05). Apoptosis rates in 15μg/ml GST+HSV-1 group were lower than HSV-1 group(P< 0.05). The mRNAs of gD was first detected at 6h p.i and 24h p.i in HSV-1 group and 15μg/ml GST+HSV-1 group respectively,Conclusions:1 U251 cells are permissive for HSV-1.2 HSV-1 could inhibit cell proliferation, accelerate apoptosis and perturb cell cycle in infected U251 cells.3 15μg/ml GST has nothing to do with proliferation or apoptosis of normal U251 cells, but it could inhibit abnormality of U251 cells proliferation and apoptosis caused by HSV-1 infection.4 15μg/ml GST could interfere expression of gD mRNA in early HSV-1 infection.