白及提取物小鼠体内抗流感病毒药效研究

Study on the Anti-influenza Virus Efficacy of the Bletilla Striata Extracts in Mice

[目的]评价白及提取物小鼠体内抗流感病毒的药效。[方法]流感病毒A/Sydney/5/97(H3N2),经BALB/C小鼠和鸡胚体内增毒建立鼠肺适应株,测定其流感病毒半数致死剂量(mouse influenza virus median lethal dose,MLD50)。雌性BALB/C小鼠84只,适应性培养后随机分为7组,其中模型组,白及提取物高、中、低剂量组和达菲组以0.1MLD50的鼠肺适应株病毒90μL滴鼻感染,二甲基亚砜(dimethyl sulfoxide,DMSO)组和正常对照组以等量0.9%氯化钠溶液滴鼻。感染后2h,白及提取物高、中、低剂量组和达菲组分别以白及提取物高、中、低剂量(8g/kg·d、4g/kg·d、2g/kg·d)和达菲0.03g/kg·d灌胃,模型组和正常对照组以等量0.9%氯化钠溶液、DMSO组给予等量DMSO灌胃,灌胃剂量均为200μL/只,1次/d,连续7d。灌胃第1、3、5、7天小鼠摘除眼球取血,ELISA法测定血清中白细胞介素-2(interleukin-2,IL-2)、干扰素-α(interferon-α,IFN-α)和IFN-β的含量;取肺脏称重并计算肺指数和肺指数抑制率;荧光定量PCR检测肺组织中病毒载量;HE染色观察肺组织的病理变化,综合评价白及提取物体内抗流感病毒药效。[结果]模型组小鼠肺组织病理切片显示肺组织间质性炎症明显,可见大量炎性细胞浸润,病毒载量不断增加;正常对照组和DMSO组小鼠肺组织中无流感病毒检出,无炎症改变。经白及提取物治疗,肺组织病理切片结果显示,炎症改善、炎性浸润细胞数量下降,高剂量组尤其明显。白及提取物肺指数抑制率最高达35.8%(高剂量组,第7天),达菲组最高可达47.2%。与模型组比较,各时间点白及提取物各剂量组与达菲组Ct值增加,病毒载量逐渐减少。与模型组比较,感染后第1、3、5、7天,白及提取物各剂量组和达菲组小鼠血清中细胞因子IL-2含量均升高,除白及提取物中剂量组第3天外,其余时点白及提取物高、中剂量组与达菲组差异均有统计学意义(P<0.01,P<0.05),低剂量组无统计学差异(P>0.05)。感染后第1~3天,模型组、白及提取物各剂量组和达菲组小鼠血清中IFN-α、IFN-β含量明显升高,到第5~7天又逐渐下降。与模型组比较,白及提取物高剂量组和达菲组第1、3、5天IFN-α含量升高,差异具有统计学意义(P<0.01);中、低剂量组第1、3天IFN-α含量升高,差异有统计学意义(P<0.01,P<0.05)。与模型组比较,白及提取物高、中剂量组和达菲组第1、3、5、7天IFN-β含量升高,差异均有统计学意义(P<0.01);白及提取物低剂量组第5天IFN-β含量升高,差异具有统计学意义(P<0.05)。[结论]白及提取物在小鼠体内具有显著的抗流感病毒作用,可能与促进IL-2、INF-α、INF-β生成,进而增强小鼠免疫功能有关。

[Objective] To evaluate the efficacy of anti-influenza virus in mice of Bletilla striata extract.[Methods] Influenza virus A/Sydney/5/97 (H3N2) was inactivated into BALB/C mice and chicken embryos to determine the mouse influenza virus median lethal dose(MLD50).Eighty-four female BALB/C mice were randomly divided into 7 groups: model group,Bletilla striata extract high,medium and low dose groups,and Tamiflu group were intranasally administrated with 90μL 0.1MLD50 mouse lung adaptation strain virus,while dimethyl sulfoxide(DMSO) group,normal control group were intranasally administrated with the same amount of sodium chloride solution.Two hours after infection,Bletilla striata extract high,medium and low dose groups were intragastric administrated with high,medium and low dose of Bletilla striata extract(8g/kg·d,4g/kg·d,2g/kg·d);Tamiflu group was intragastric administrated with Tamiflu 0.03g/kg·d;model group and normal control group were given same amount of sodium chloride solution;DMSO group was given same amount of DMSO,and the dose was 200μL per mice,once a day for 7 days.The blood specimen was taken on the 1st,3rd,5th and 7th days. The contents of interleukin-2 (IL-2),interferon-α(IFN-α) and IFN-β in serum were determined by ELISA.The lungs were weighed and the lung index and lung index inhibition rate were calculated.Viral load in lung tissue was detected by Real-time PCR.The pathological changes of lung tissue were observed by HE stain.The anti-influenza virus efficacy of Bletilla striata extract was comprehensively evaluated.[Results] The lung tissue of model group showed obvious interstitial inflammation,with a large number of inflammatory cell infiltration;the viral load of model group increased continuously;no virus was detected in lung tissue of normal control group and DMSO group,and there was no inflammation.After treated with Bletilla striata extract,the inflammatory symptoms of lung tissue were relieved,the number of infiltrating inflammatory cells decreased,especially in Bletilla striata extract high dose group.The lung index inhibition rate was up to 35.8%(Bletilla striata extract high dose group,the 7th day),and the Tamiflu group was up to 47.2%. Compared with model group,the Ct value of Bletilla striata extract high,medium and low dose groups and Tamiflu group at each time point increased,the viral load decreased gradually.Compared with model group,the content of IL-2 of Bletilla striata extract groups and Tamiflu group increased on the 1st, 3rd,5th and 7th days after infection,especially in Bletilla striata extract high and medium dose groups and Tamiflu group(P<0.01,P<0.05),except Bletilla striata extract medium dose group on the 3rd day,and there was no significant difference in Bletilla striata extract low dose group(P>0.05).The IFN-α and IFN-β contents of model group,Bletilla striata extract high,medium and low dose groups and Tamiflu group increased significantly on the 1st to 3rd day after infection,and gradually decreased on the 5th to 7th day.Compared with model group,the content of IFN-α increased in Bletilla striata extract high dose group and Tamiflu group on the 1st,3rd and 5th day after infection(P<0.01);the content of IFN-α increased in Bletilla striata extract medium and low dose groups on the 1st and 3rd days(P<0.01,P<0.05).Compared with model group,the content of IFN-β increased in Bletilla striata extract high and medium dose groups and Tamiflu group on the 1st,3rd,5th and 7th days after infection(P<0.01);the content of IFN-β increased in Bletilla striata extract low dose group on the 5th day after infection(P<0.05).[Conclusion] Bletilla striata extract has significant anti-influenza virus effect in mice,which may be related to the increase of IL-2,INF-α,INF-β and thus enhance the immune function of mice.