清气解毒方含药血清干预呼吸道合胞病毒感染人喉癌上皮细胞Hep-2的体外实验研究

Research on Qingqi Jiedu Decoction(清气解毒方) Containing Serum to Intervene Respiratory Syncytial Virus Infected Human Laryngeal Epithelial Cell Hep-2:In Vitro Experimental Study

目的探讨清气解毒方抗呼吸道合胞病毒(RSV)感染的作用及可能机制。方法 3只新西兰白兔分为空白血清组、利巴韦林组和清气解毒方组,每组1只。清气解毒方组灌胃量为36. 32 mg/(kg·d),利巴韦林组给药量为18. 24 mg/(kg·d),空白血清组给予50 ml生理盐水灌胃,均连续灌胃3天后制备含药血清。检测RSV半数感染量(TCID50)并筛选清气解毒方、利巴韦林药物最大无毒浓度用于后续实验。Hep-2细胞以1×10~5/ml种于96孔板中,分为细胞组、病毒组、利巴韦林组和清气解毒方组,每组设6个复孔。利巴韦林组加入利巴韦林血清,清气解毒方组加入清气解毒方血清,细胞组和病毒组加入细胞维持液。分别培养48、72、96 h后检测各组细胞病毒抑制情况,96 h后观察各组细胞周期及凋亡率。结果呼吸道合胞病毒A亚型(Long株) TCID50为10-4. 581/100μl。最终筛选出浓度为40%的清气解毒方含药血清作为其最大细胞无毒浓度,即0. 363 g/ml;浓度为50%的利巴韦林含药血清作为其最大细胞无毒浓度,即0. 228 mg/ml。与细胞组比较,感染后48、72、96 h病毒组OD值明显降低(P <0. 01);与病毒组比较,利巴韦林组和清气解毒方组在48、72、96 h时OD值均明显升高(P <0. 05或P <0. 01)。清气解毒方组在48、72、96 h病毒抑制率分别为42. 1%、57. 0%、66. 6%,低于同时间利巴韦林组的51. 2%、69. 6%、77. 3%。与细胞组比较,病毒组G1期明显升高,S期明显降低,凋亡率亦升高(P <0. 05或P <0. 01);与病毒组比较,利巴韦林组与清气解毒方组G1期明显降低,S期和G2期明显升高,凋亡率亦降低(P <0. 05或P <0. 01)。结论清气解毒方具有一定的体外抗RSV作用,效果略差于利巴韦林。

Objective To study the effects and possible mechanism of Qingqi Jiedu Decoction(清气解毒方)against respiratory syncytial virus(RSV).Methods Three New Zealand white rabbits were divided into blank serum group,ribavirin group and Qingqi Jiedu Decoction group,with 1 rabbit in each group.The Qingqi Jiedu Decoction group was administered with 36.32 mg/(kg · d) Qingqi Jiedu Decoction,the ribavirin group was given18.24 mg/(kg·d) ribavirin,and the blank serum group was given 50 ml normal saline.The drug-containing serum was prepared after continuous administration for 3 days.The half-infection of RSV(TCID50) was detected and the maximum non-toxic concentration of Qingqi Jiedu Decoction and ribavirin was screened for subsequent experiments.Hep-2 cells were seeded in 96-well plates at 1×10~5/ml,and were divided into cell group,virus group,ribavirin group and Qingqi Jiedu Decoction group,with 6 duplicate wells in each group.The ribavirin group was added with ribavirin serum,the Qingqi Jiedu Decoction group was added with Qingqi Jiedu Decoction serum,and the cell group as well as virus group were added with the cell maintenance solution.After 48,72,and 96 hours of culture,the virus inhibition of each group was detected.After 96 hours,the cell cycle and apoptosis rate of each group were observed.Results The respiratory syncytial virus A subtype(Long strain) had a TCID50 of 10-4.581/100 μl.The concentration of 40% Qingqi Jiedu Decoction drug-containing serum was screened out as its maximum cell non-toxic concentration(0.363 g/ml),and the concentration of 50% ribavirin-containing serum was screened out as its maximum cell nontoxic concentration(0.228 mg/ml).Compared with the cell group,the OD value of the virus group was significantly decreased at 48,72 and 96 hours after infection(P<0.01).Compared with the virus group,the OD values of the ribavirin group and the Qingqi Jiedu Decoction group were significantly increased at 48,72,and 96 h(P<0.05 or P<0.01).The virus inhibition rates of Qingqi Jiedu Decoction group were 42.1%,57.0%,and 66.6% at 48,72,and 96 h,respectively,which were lower than 51.2%,69.6%,and 77.3% of the ribavirin group.Compared with the cell group,the G1 phase of the virus group was significantly increased,the S phase was significantly decreased,and the apoptotic rate was also increased(P<0.05 or P<0.01).Compared with the virus group,the G1 phase of the ribavirin group and the Qingqi Jiedu Decoction group was significantly decreased,the S phase and the G2 phase were significantly increased,and the apoptotic rate was also decreased(P<0.05 or P<0.01).Conclusion Qingqi Jiedu Decoction has a certain anti-RSV effect in vitro,and the effect is slightly worse than ribavirin.