多种受体酪氨酸激酶抑制剂舒尼替尼减轻流行性乙型脑炎病毒感染的研究

Alleviating Japanese encephalitis virus infection with multiple receptor tyrosine kinase inhibitor sunitinib

目的评价多种受体酪氨酸激酶抑制剂舒尼替尼(sunitinib)在体内和体外治疗流行性乙型脑炎病毒(Japa⁃nese encephalitis virus,JEV)感染的效果。方法4周龄BALB/c小鼠腹腔注射JEV。感染小鼠以两种给药策略进行治疗,分别为连续5 d和连续10 d给与舒尼替尼治疗,后观测其体重变化和生存率;通过qRT-PCR技术检测小鼠脑内病毒RNA载量的变化;利用免疫组织化学染色技术检测小鼠脑内CD4^(+)T淋巴细胞和CD8^(+)T淋巴细胞的数量及分布;利用免疫荧光染色技术观察药物治疗前后小鼠脑内JEV病毒E蛋白的表达。此外,通过JEV体外感染Vero细胞并给与一定浓度舒尼替尼进行干预,观察细胞存活状态;采用间接免疫荧光染色观察治疗前后细胞内JEV病毒E蛋白的表达。结果小鼠感染JEV后连续给与舒尼替尼可显著提高其生存率,并且连续给药5 d的生存率和体重变化结果优于连续给药10 d。舒尼替尼治疗后减少了脑内CD4^(+)T淋巴细胞和CD8^(+)T淋巴细胞浸润,小鼠脑内血管套袖样改变减轻;但治疗前后小鼠脑内的JEV病毒载量和脑内E蛋白的表达变化不太明显;感染JEV的Vero细胞在给与舒尼替尼后细胞病变效应稍有减轻,E蛋白表达少量变化。结论舒尼替尼治疗能够显著降低JEV感染小鼠死亡率,并且连续给与舒尼替尼5 d的治疗效果更好,JEV感染小鼠接受舒尼替尼治疗后可减少脑内CD4^(+)T淋巴细胞和CD8^(+)T淋巴细胞浸润,改善脑内血管袖套样炎性炎症状,延长小鼠生命。

Objective To evaluate the effects of sunitinib on Japanese encephalitis virus(JEV)infection in vitro and vivo.Methods The 4-week-old BALB/c mice infected with JEV by intraperitoneal injection.The infected mice were treated with sunitinib for 5 days and 10 days respectively.After that,the change of weight and survival rate were evaluated continuously.The viral load variation in mice brain were detected by qRT-PCR.Indirect immunohistochemical staining assay(IFA)was used to detect the number and distribution of CD4^(+)/CD8^(+)T cells in mouse brain.IFA was also used to observe the expression of virus E protein in the brain of mice.Vero cells were infected with JEV in vitro and given a certain concentration of sunitinib to observe the cell survival status.The expression of virus E protein in cells was detected by IFA.Results Continuous administration of sunitinib significantly improved the survival rate of infected mice.Survival rate and body weight changes showed that the 5-day's administration strategy was significantly better than the 10-day's administration strategy.The treatment of sunitinib decreased the infiltration of CD4^(+)/CD8^(+)T cells in the brain and reduced the changes of vascular sleeve.However,the variation of viral load and E protein expression in brain were not obvious.The cytopathic effect(CPE)of infected Vero cells were slightly relieved after giving sunitinib,and the expression of E protein was also slightly changed.Conclusion Sunitinib can significantly reduce the mortality of infected mice,and the 5-day's administration strategy is significantly better than the 10-day's administration strategy.Sunitinib decrease T lymphocyte infiltration in brain of mice,relieve the encephalitis symptoms,and prolonged the life of mice.