基于TLR3/TRIF通路探讨栀子苷抗流感病毒引起的病毒性肺炎的实验研究

Regulatory Effect of Geniposide on Influenza Virus-Induced Lung Injury in Mice Based on the TLR3/TRIF Pathway

中药在预防和治疗流感病毒方面具有独特的优势,栀子苷具有抗炎抗病毒作用,但栀子苷保护流感病毒引起的肺损伤的作用机制尚不明确。为了基于Toll样受体3(Toll-like receptor 3,TLR3)/β干扰素TIR结构域衔接蛋白(TIR-domain-containing adaptor inducing interferon-β,TRIF)信号传导途径探讨栀子苷对流感病毒诱导的肺损伤的调节作用,本研究按照随机数字表法将108只小鼠分为空白对照组、模型组、阳性对照组(50mg/kg利巴韦林)、低剂量栀子苷组(5mg/kg栀子苷)、中剂量栀子苷组(10mg/kg栀子苷)、高剂量栀子苷组(20mg/kg栀子苷)、TLR3激动剂组、TLR3激动剂+阳性药物组、TLR3激动剂+高剂量栀子苷组。采用流感病毒亚甲型鼠肺适应株A/FM/1/47(H1N1)建立流感病毒性肺炎小鼠模型,摘取肺组织,测定小鼠肺指数,HE染色观察肺组织病理学变化,酶联免疫法检测肺组织白介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和干扰素-β(Interferon-β,IFN-β)水平,qRT-PCR检测肺组织TLR3和TRIF mRNA表达,Western Blot检测肺组织TLR3、TRIF和p-NF-κB65蛋白表达。结果显示,与空白对照组相比,模型组小鼠肺指数、IL-6、TNF-α水平显著升高(P<0.05),TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白水平均显著升高(P<0.05)。与模型组比,中、高剂量栀子苷组和阳性对照组小鼠肺指数、IL-6和TNF-α水平均显著降低(P<0.05),TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白水平均显著降低(P<0.05)。与阳性对照组比,高剂量栀子苷组小鼠肺指数、IL-6、TNF-α、TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白均无显著差异(P>0.05)。与空白对照组相比,TLR3激动剂组小鼠肺指数显著升高(P<0.05),与TLR3激动剂组相比,TLR3激动剂+高剂量栀子苷组小鼠肺指数显著降低(P<0.05),TLR3激动剂+阳性药物组小鼠肺指数无显著差异(P>0.05)。本研究揭示,栀子苷可能通过调节TLR3/TRIF通路介导的p-NF-κB65活性增强机体的抗病毒能力。

Traditional Chinese medicine has unique advantages in the prevention and treatment of influenza virus. Geniposide has anti-inflammatory and antiviral effects,but the mechanism of geniposide in protecting lung injury caused by influenza virus is not very clear. To investigate the regulatory effect of geniposide on influenza virus-induced lung injury based on the Toll-like receptor 3/TIR domain binding protein of interferon β(TLR3/TRIF)signal transduction pathway,one hundred and eight mice were divided into nine groups:blank control,model,positive control(50 mg/kg palamivir),low-dose geniposide(5 mg/kg geniposide),medium-dose geniposide(10 mg/kg geniposide),high-dose geniposide(20 mg/kg geniposide),TLR3 agonist,TLR3 agonist+positive drug,and TLR3 agonist+high-dose geniposide. A mouse model of influenza-virus pneumonia was established using the rat lung-adaptive strain A/FM/1/47(H1 N1). After killing,pulmonary tissue was extracted,and the lung index of mice was measured. Histopathologic changes in the lung were observed by hematoxylin&eosin staining. Levels of interleukin(IL)-6,tumor necrosis factor(TNF)-α and interferon(IFN)-β in lung tissue were detected by enzyme-linked immunosorbent assay(ELISA). mRNA expression of TLR3 and TRIF in lung tissue was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR). Western Blot was used to detect the protein expression of TLR3,TRIF and phosphorylated nuclear factor-kappa B(p-NF-κB)65 in lung tissue. Results showed that compared with the blank control group,the lung index as well as levels of IL-6 and TNF-α of mice in the model group increased significantly(P<0.05);also,levels of TLR3 and TRIF mRNAs and proteins,and p-NF-κB65 protein,were increased significantly(P<0.05). Compared with the model group,the lung index,levels of IL-6 and TNF-α in the middle-and highdose geniposide groups and positive control group were significantly lower(P< 0.05);also,the levels of TLR3 and TRIF mRNAs and proteins,and p-NF-κB65 protein were decreased significantly(P< 0.05).Compared with the positive control group,there were no significant differences in the lung index,levels of IL-6,TNF-α,TLR3 and TRIF mRNAs and proteins,or p-NF-κB65 protein in the high-dose geniposide group(P> 0.05). Compared with the blank control group,the lung index in the TLR3 agonist group increased significantly(P< 0.05);compared with the TLR3 agonist group,the lung index in the TLR3 agonist+highdose geniposide group decreased significantly(P< 0.05),but the lung index in the TLR3 agonist+positive drug group showed no significant differences(P> 0.05).This study suggested that geniposide may enhance the antiviral activity of the body by regulating the activity of p-NF-κB65 after mediation by the TLR3/TRIF pathway.