Molecular Mechanisms Of Tanshinone ⅡA Sulfonate Againsts Marek’s Disease Virus In Vitro

Objective:Sodium Tanshinone IIA sulfonate (STS) is a Traditional Chinese Medicine, the aim of the study is to investigate the molecular mechanism of STS on Marek’s disease virus (MDV) on Chick embryo fibroblast (CEFs) in vitro and investigate the effect of STS on the expression of Meq and UL49gene of MDV.Methods:Using Real-time PCR to determine the optimal concentration in concentration gradient assay; constructing the standard curves of Meq and UL49gene to quantify the copy numbers of the two genes on DNA level and mRNA level after STS was mixed with MDV or was added on CEFs which was infected by MDV; Using the methods of western blot and Immunofluorescence assay (IFA) to test the effects of STS on VP22protein.Results:1. The virus titer was1×105(pfu/ml)and the number of virion which can infected CEFs succeedly was105per mililiter;2. The inhibition ratio of MDV load reached92.24%when STS was0.25mg/ml and in concentration-dependent manners.3. On DNA level, STS could inhibite the expression of Meq and UL49gene from24h to96h ather STS was added at CEFs. The copy numbers of Meq and UL49in STS group was significantly different from virus group at24h.48h.72h and96h when MDV premixed with STS (P<0.05); They were least at24h and was2.82×103copies and8.00x102copies respeceivly. When CEFs pre-incubated with MDV, the copy numbers of Meq in STS group was significantly different from virus group at48h.72h and96h (P<0.05) but not24h (P>0.05); The copy numbers of UL49gene in STS group was significantly different from virus group at24h.48h.72h and96h (P<0.05); The copy numbers of Meq and UL49were least at96h and they were1.21×104copies and1.42x104copies respeceivly.4. On mRNA level, STS could inhibite the expression of Meq and UL49gene from24h to96h ather STS was added at CEFs.The copy numbers of Meq in STS group was significantly different from virus group at48h.72h and96h (P<0.05) but not24h (P>0.05) when MDV premixed with STS; The copy numbers of UL49gene in STS group was significantly different from virus group at24h.48h.72h and96h (P<0.05); The copy numbers of Meq and UL49were least at48h and they were19.1copies and21.4 copies respeceivly. When CEFs pre-incubated with MDV, The copy numbers of Meq in STS group was significantly different from virus group at24h48h.and72h (P<0.05) but not96h (P>0.05); The copy numbers of UL49gene in STS group was significantly different from virus group at24h.48h.72h and96h (P<0.05); The copy numbers of Meq and UL49were least at48h and they were1.72×102copies and5.01x102copies respeceivly.5. In western blot and Immunofluorescence assay, STS could inhibite the expression of VP22protein when when MDV premixed with STS and CEFs pre-incubated with MDV.Conclusion:STS could against MDV and the inhibition ratio was92.24%on CEFs, STS could effectively inhibit the expression of Meq. UL49gene and VP22protein, which provided a novel alternative mechanism of how STS could against MDV.