天山堇菜总黄酮抗流感病毒作用及机制研究

Study on the Mechanism Anti-Influenza Virus of Total Flavonoids from Viola Tianshanica Maxim

目的研究天山堇菜总黄酮(TFVM)的体内外抗流感病毒作用及其机制。方法体外实验采用细胞病变效应(CPE)法观察TFVM对流感病毒A/FM/1/1947(H1N1)致犬肾细胞(MDCK)病变的影响;设置细胞对照组,病毒感染组,阳性药金刚烷胺(AH)对照药组(10μmol/L),TFVM 100、50、25μg/mL 3个浓度组,以H1N1分别感染MDCK和人髓系白血病单核细胞(THP-1),Western Blot法检测基质蛋白2(M2)、非结构蛋白(NS1)蛋白表达,检测THP-1细胞中p65蛋白表达及核转位;鸡血球细胞凝集实验检测TFVM对血凝素(HA)的抑制作用,荧光法检测神经氨酸酶(NA)活性。体内实验将小鼠随机分为病毒感染对照组,阳性药达菲(11.375 mg/kg)组,TFVM高、低剂量(0.4、0.2 g/kg)组,以流感病毒鼠肺适应株FM1感染小鼠建立病毒性肺炎模型,取小鼠肺组织研磨液接种MDCK细胞,CPE法观察对小鼠肺组织病毒增殖量的影响;qRT-PCR检测M2蛋白及肺组织中炎症因子TNF-α、IL-1β、IL-6、IL-10、干扰素诱导蛋白10 (IP-10)mRNA表达水平。结果 TFVM明显抑制流感病毒致MDCK细胞病变,其IC50为79.1μg/mL;其100μg/mL浓度对MDCK和THP-1细胞中流感病毒M2、NS1蛋白表达均有明显抑制作用;TFVM还可抑制THP-1细胞内p65蛋白表达及其核转位。TFVM对流感病毒的两个膜蛋白NA和HA功能无明显影响;TFVM以0.2、0.4 g/kg剂量给药可明显降低流感病毒感染小鼠肺组织内病毒载量(P<0.05,P<0.01),并降低炎症因子TNF-α、IL-1β、IL-6、IL-10、IP-10 mRNA表达水平(P<0.05,P<0.01),在一定程度上能降低M2 mRNA表达水平(P>0.05)。结论 TFVM在体内外均有明显的抗流感病毒作用,其机制可能是通过抑制M2蛋白和NS1蛋白表达而直接抑制流感病毒的复制;还可能通过抑制宿主细胞内p65蛋白表达及其核转位,从而干预NF-κB信号通路下游炎症因子的表达,发挥间接抗病毒作用。

Objective To investigate the effect and mechanism against influenza virus of total flavonoids from Viola tianshanica Maxim(TFVM). Methods CPE method was used to detect the cytotoxicity of TFVM on madin-darby canine kidney(MDCK) cells and the anti-influenza efficacy. In vitro, normal cell control group, virus control group, positive Amantadine(AH)control group(10 μmol/L), TFVM 100, 50, 25 μg/mL groups were set. Western Blot experiment was used to detect the effects on M2, NS1 expression in MDCK or THP-1 cells which were infected with influenza virus A/FM/1/1947(H1 N1). The effects of TFVM on protein expression and nuclear translocation of p65 in THP-1 cells were detected by Western Blot. The inhibitory effect of TFVM on hemagglutinin(HA)was detected by agglutination assay of chicken erythrocyte. The effect of TFVM on neuraminidase(NA)activity of influenza virus was detected by fluorescence. In vivo, mice were randomly divided into virus infection control group, Tamiflu positive(11.375 mg/kg)group, TFVM(0.4, 0.2 g/kg)groups.The mice were infected with virus FM1, then, the lung tissue lapping solution were collected to inoculate MDCK cells, the virus proliferation in the lung tissue was detected by CPE method;RNA was extracted from lung tissue of the mice, the expression levels of influenza virus M2, TNF-α, IL-1β, IL-6, IL-10 and IP-10 in lung tissues were detected by qRT-PCR. Results TFVM significantly inhibited pathological changes in MDCK cells infected with influenza virus, the IC50 against influenza virus was 79.1μg/mL. At the dose of 100 μg/mL, TFVM showed a good inhibitory effect on the protein expression of M2, NS1 in MDCK or THP-1 cells which were infected with influenza virus. TFVM could significantly inhibit p65 protein expression and nuclear translocation of p65 in THP-1 nucleus. TFVM had no effect on NA and HA. For in vivo investigation, at the dose of 0.2 g/kg and 0.4 g/kg, TFVM could significantly decrease the virus proliferation(P<0.05, P<0.01)and the mRNA expression of TNF-α, IL-1β,IL-6,IL-10 and IP-10(P<0.05, P<0.01)in the lung tissue of mice infected with influenza virus, TFVM could also decrease M2 mRNA expression to some extent(P >0.05). Conclusions TFVM has obvious anti-influenza virus effect in vivo and in vitro. The mechanism of its antiviral effect may be directly inhibiting the replication of influenza virus by inhibiting the expression of M2 and NS1 protein, on the other hand, TFVM may also has an indirect anti-influenza virus effect by inhibiting p65 protein expression in host cells and nuclear translocation of p65, thereby interfering with the expression of inflammatory factors of the NF-κB signaling pathway.