蜘蛛香不同提取物抗轮状病毒的作用研究

Study on the anti-rotavirus effect of different extracts of Valeriana jatamansi Jones

关键词:  轮状病毒  蜘蛛香  环烯醚萜类  肠上皮细胞  紧密连接蛋白

Key words:  rotavirus  Valeriana jatamansi Jones  cyclenyl ether terpenes  intestinal epithelial cells  tight junction protein

摘要:

目的　研究蜘蛛香不同提取物抗轮状病毒（Rotavirus，RV）的作用。方法　采用RV-Wa株感染MA104 细胞 模型，MTT染色法测定病毒抑制率，考察蜘蛛香水提物、70%乙醇提取物（ZZX-1）及 90%乙醇提取后经大孔吸附树脂 法纯化所得提取物（ZZX-2）体外抗RV的作用；采用免疫荧光和实时荧光定量PCR（qRT-PCR）检测RV结构蛋白VP6 的 表达变化，验证蜘蛛香抗RV作用；采用RV-Wa株感染Caco-2 细胞，qRT-PCR检测蜘蛛香对神经营养因子GDNF、紧密连 接蛋白ZO-1 和促炎因子[肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）]的影响。结果　蜘 蛛香水提物无抗RV的作用；蜘蛛香醇提物ZZX-1 和ZZX-2 具有抗RV生物合成作用。免疫荧光和qRT-PCR实验结果表 明，ZZX-1 和ZZX-2 均能够抑制VP6 蛋白的合成以及降低VP6 mRNA的表达，抑制RV的合成。与RV组相比，ZZX-1 和 ZZX-2 能够显著提高GDNF、ZO-1 mRNA的相对表达量（P<0.001 或 0.01），降低TNF-α、IL-1β、IL-6 mRNA的相对表达 量（P<0.0001 或 0.001）。结论　蜘蛛香醇提物ZZX-1 和ZZX-2 具有抗RV生物合成作用，可能通过提高GDNF、ZO-1 基 因表达，降低炎症因子 IL-1β、IL-6、TNF-α的基因表达发挥抗RV感染作用。

Abstract:

Objective The aim of this study is to examine the anti-rotavirus (RV) effects of different extracts of Valeriana jatamansi Jones. Methods The RV-Wa strain infected MA104 cell model was used, and the virus inhibition rate was determined by using MTT staining. The in vitro anti-RV activity of spider perfume extract, 70% ethanol extract (ZZX-1) and 90% ethanol extract (ZZX-2) was purified by the macroporous adsorption resin method. Immunofluorescence and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expression of RV structural protein VP6, and to verify the anti-RV effect of spide. Caco-2 cells were infected with RV-Wa strain and the effects of arachnid on the expressions of neurotrophic factor GDNF, tight junction protein ZO-1 and pro-inflammatory factors (TNF-α), interleukin1β (IL-1β), interleukin-6 (IL-6) were detected by qRT-PCR. Results The extract of Valeriana jatamansi Jones did not have any anti-RV effect; Valeriana jatamansi Jones extracts ZZX-1 and ZZX-2 have anti-RV biosynthesis effects. The results of immunofluorescence and qRT-PCR indicate that both ZZX-1 and ZZX-2 can reduce the fluorescence and mRNA expression of VP6, and inhibit RV synthesis. Compared with the RV group, ZZX-1 and ZZX-2 significantly increased the expression levels of neurotrophic factor GDNF and tight junction protein ZO-1 mRNA (P<0.001 or 0.01), and reduced the relative expression levels of tumor necrosis factor alpha (TNF alpha), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA (P<0.0001 or 0.001). Conclusion The alcohol extracts ZZX-1 and ZZX-2 from the traditional Chinese medicine Valeriana jatamansi Jones have anti-RV biosynthesis effects, which may exert anti-RV infection effects by increasing the expression of GDNF and ZO-1 and reducing the expression of inflammatory factors IL-1 β, IL-6, and TNF-α.