百里香药茶对人冠状病毒OC43体内外增殖的影响

Effect of Thyme Herbal Tea on Proliferation of Human Coronavirus OC43 in vitro and in vivo

摘要:

目的 探究百里香药茶（BLX）对人冠状病毒OC43（HCoV-OC43）的体内外抗病毒活性及作用机制。方法 通过超高效液相色谱-质谱（UPLC-MS）分析BLX化学成分，利用HCoV-OC43细胞模型检测BLX的体外毒性及其对该病毒的抑制作用，采用时间差异给药法检测药物作用阶段，实时荧光定量聚合酶链式反应（Real-time PCR）法检测病毒基因拷贝数，利用HCoV-OC43小鼠模型检测BLX的体内毒性及其对小鼠体质量和生存周期变化的影响，通过免疫组化技术检测脑和肺组织中病毒蛋白表达。结果 从BLX中鉴定出11种化合物；在HCoV-OC43感染的HRT-18细胞中，BLX的半数毒性浓度（CC₅₀）为（13 859.56±319） mg·L^-1，半数抑制浓度（IC₅₀）为（1 439.09±200） mg·L^-1，其选择指数范围为8.26~11.44。与病毒组比较，BLX在1 500、1 000、500 mg·L^-1剂量下均可明显抑制病毒复制（P<0.05，P<0.01），该药物在病毒复制早期即可发挥抗病毒作用，且对病毒吸附过程的干扰大于对病毒入胞过程的干扰（P<0.05）。在HCoV-OC43感染的小鼠模型中，BLX在1 200、600 mg·kg^-1·d^-1剂量下可缓解被感染小鼠的症状、延长生存期并降低死亡率，有效抑制脑组织中（P<0.01）和肺组织（P<0.01）中病毒核衣壳mRNA表达水平；BLX在1 200 mg·kg^-1剂量下可抑制脑组织（P<0.01）和肺组织（P<0.01）中病毒核衣壳蛋白的表达。结论 BLX含有多种具有抗病毒活性的化学成分，可在体内外抑制HCoV-OC43复制，该药物可能通过干扰病毒吸附等方式发挥其抗病毒活性。该研究为冠状病毒感染治疗提供了候选药物，为BLX的进一步开发应用提供了理论参考。

Abstract:

Objective To investigate the effects of thyme herbal tea （BLX） on the proliferation of human coronavirus OC43 （HCoV-OC43） in vitro and in vivo.Method The chemical composition of BLX was analyzed by UPLC-MS. The cytotoxicity of BLX in HRT-18 cells and the effect of BLX treatment on the proliferation of HCoV-OC43 in cells were analyzed. Copies of viral gene were detected by real-time PCR. The effect of BLX treatment on the life cycle of HCoV-OC43 was detected by time-of-addition assay. The maximum tolerated dose of BLX and the influences of BLX on the body weight and survival time of suckling mice infected with HCoV-OC43 were determined. The expression of viral protein in the brain and lung tissue was analyzed by immunohistochemistry.Result There were 11 chemical components identified in BLX by UPLC-MS. BLX showed the 50% cytotoxic concentration （CC₅₀） of （13 859.56±319） mg·L^-1， the median inhibitory concentration （IC₅₀） of （1 439.09±200） mg·L^-1， and the selection index of 8.26-11.44 for HCoV-OC43 in HRT-18 cells. Compared with the cells infected with HCoV-OC43， BLX at the concentrations of 1 500， 1 000， 500 mg·L^-1 inhibited the proliferation of this virus （P<0.05， P<0.01）. BLX exhibited antiviral effect in the early stage of virus infection， and the inhibition role in the attachment stage was more significant than that in the entry stage （P<0.05）. In the suckling mice infected with HCoV-OC43， BLX at 1200 and 600 mg·kg^-1·d^-1 alleviated the symptoms， prolonged the survival period， reduced the death rate， and down-regulated the mRNA level of nucleocapsid protein in the mice. Moreover， BLX at 1 200 mg·kg^-1·d^-1 down-regulated the expression of nucleocapsid protein in the brain （P<0.01） and the lung （P<0.01）.Conclusion BLX contained multiple antiviral ingredients. It inhibited the proliferation of HCoV-OC43 both in vitro and in vivo by interference with viral attachment. This study provides theoretical reference for the treatment of acute respiratory tract infection with HCoV-OC43 and for further development and application of BLX.