The Effects Of Allitridin On The Expression Of Caspase-3 In Human Cytomegalovirus Infected HELs

Objectives:To investigate the effect of allitridin on the expression of caspase-3 in HCMV infected HELs, in order to explore partial mechanism of allitridin against HCMV from the point of view of apoptosis.Methods:①Establishing HCMV-infected cell model: Human embryo lung fibroblast cells (HELs) were infected by HCMV AD169 strain with high multiply of infection (MOI=2.5) and low MOI (0.25), resepectively. And two kinds of HCMV infected cell models were established.②T he dosage of allitridin and the groups of experiment: According to the maximum tolerance concentration (MTC) of allitridin to HELs (9.6μg/mL), 9μg/mL, 6μg/mL and 3μg/mL were determined as the high, medium and low dosage of the drug in this experiment. Based on the viral load of infection and the dosage of the drug, the experiments were grouped as the followings: low MOI infected cells treated with low, medium or high dosage of allitridin; high MOI infected cells treated with low, medium or high dosage of drug; and normal cells, low MOI infected cells and high MOI infected cells served as controls.③Caspase-3 protein levels in every group were analyzed by using western blot assay at 72h after viral infection and/or drug treatment.④The integral density values (IDV) of the desired bands were measured by the auto-analyzer for medicine colorful pictures (TYPE HMIAS-2000) and normalized with the IDV of internal control bands (A value=IDV of desired band/IDV of actin band). A value was used for the semi-quantitative analysis of experimental results. Results:①The influence of HCMV infection on the expression of Caspase-3 protein in HELs: Normal HELs only expressed Caspase-3 P32; while after HCMV infection, no matter what kind of MOI was used, the expression of Caspase-3 P32 increased, moreover, active fragment of P17 of Caspase-3 appeared, and the expression of P17 in high MOI group was obviously stronger than that of the low MOI.②The influence of allitridin on the expression of Caspase-3 protein in low MOI infected HELs: Compared with low MOI control, the active fragment of P17 was up-regulated in low MOI HELs treated with allitridin, and the ratio of P17 versus P32 also increased significantly.③The influence of allitridin on the expression of Caspase-3 protein in high MOI infected HELs: Compared with high MOI control, the active fragment of P17 was down-regulated in high MOI HELs treated with allitridin, and the ratio of P17 versus P32 decreased also.Conclusions:HCMV infection, no matter high or low MOI, could both increase the apoptosis of HELs, and this up-regulation was more intensive in high MOI group than that in low MOI.Allitridin could up-regulate the apoptosis of low MOI HELs, while down-regulate that of high MOI cells. It was supposed that the former was inclined to eliminate the virus from the infected cells, and the latter meant to relieve the damage of the cells and protect them. The regulation of allitridin on HCMV-infected cells was dualism.