The Yuantong Mixture In Vitro Anti-hsv-2 The Role Of Experimental Research

Objective: To probe the inhibitory effect of Chinese herbs of Yantong Heji (YT) on replication of herpes simplex virus (HSV)-2in Vero cells in vitro.Methods:①The cytotoxic effects of YT in Vero cells were examined by CPE method. Monolayers of Vero cells in 96-well plates were added in YT at a range of concentrations in medium.Acyclovir group and normal Vero cells served simultaneously as positive controlled group and negative controlled group, respectively.Cell cultures were incubated at 37℃ in a humidified atmosphere of 5%CO₂-95% air and cytopathic effects (CPE) were observed under a microscope day by day.The 0% toxic concentration (TC₀) to YT and ACV in Vero cells were respectively evaluated and the 50% toxic concentration (TC₅₀) were calculated by Reed - Muench method, respectively. (2) Stocks of HSV-2 333 strains were diluted by phosphate-buffered saline (PBS) from 10^-1 to 10^-7. Confluent cell monolayers in 96-well plates were infected with different concentration of virus, in normal Vero cells served as negative controlled group. Infected cultures were incubated at 37℃ in a humidified atmosphere of 5%CO₂-95% air and CPEs were observed under a microscope day by day. The degree and number of CPE was enregistered until cytopathic effects did not aggravate. Pathological changes of Vero cells exceeded 50% were determinanted positive well. Tissue cell infection median dose (TCID₅₀) were calculated by Reed - Muench method. (3) Stocks of HSV-2 333 strains were diluted by PBS to 100 times of TCID₅₀. The activity of YT of different drug addition methods against HSV-2 333 was studied in vitro by MTT colorimetry, in acyclovir served as positive reference drug. untreated Vero cells and infected Vero cells served as normal cell control group and virus control group, respectively.Different drug addition methods as following: Method Ⅰ ,inhibiting virus multiplication -Confluent cell monolayers in 96-well plates were infected with HSV-2333 in medium. After adsorption at 37℃ for 1.5 h, residual inoculum was replaced with 0.2ml of medium containing test drug. Method Ⅱ,interdicting cells infection - Cell cultures were treated with the drug medium for 24h,washed thrice with PBS, 100 TCID₅₀ of virus were added to give 0.1 ml per well and subsequently reincubated in drug-free medium. Method Ⅲ ,directly killing virus - Virus were incubated in drug medium for 24h at 37℃ in a humidified atmosphere of 5%CO₂-95% air, virus-drug mixed medium was added to give 0.2 ml per well. After adsorption at 37℃ for 1.5 h, residual inoculum was removed and cell monolayers were washed thrice with PBS.All cells were incubated...