三表法抗Ⅳ诱导小鼠肺炎TRAF2/NF-K BP65/MYD88蛋白活化时效研究
摘要
目的:建立甲型流感病毒FM1株滴鼻感染不同鼠龄正常免疫和免疫低下昆明种小鼠诱导小鼠肺炎模型；正交设计麻杏石甘汤醇提方法，应用高效液相色谱法定量分析正交设计9水平麻杏石甘汤提取物中有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱含量；以“伤寒三表法”立论，观察三表法对甲型流感病毒FM1株感染鸡胚的防治作用及其对正常免疫、免疫低下小鼠流感病毒肺炎肺组织病毒载量的防治作用；探讨甲型流感病毒FM1株滴鼻感染正常免疫和免疫低下幼龄鼠诱导小鼠肺炎，小鼠肺组织中TRAF2、MYD88、NF-ΚBP65蛋白活化信号通路时效性关系。
材料与方法：
1建立不同鼠龄正常免疫和免疫低下昆明种小鼠滴鼻感染甲型流感病毒FM1株诱导小鼠肺炎模型，以小鼠一般状态、存活只数、肺组织病变程度、肺指数和荧光探针PCR检测技术检测小鼠肺组织中不同时点IV－RNA的病毒载量差异，评价模型。
2采用正交实验设计方法，以乙醇浓度、溶媒倍数、提取时间、提取次数为考察因素，以麻杏石甘汤9个供试品出膏率，和应用高效液相色谱分析法分析9个供试品中主要有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱的含量，并将分析结果作为工艺评价指标，筛选最佳醇提方法。
3配制1%健康豚鼠血红细胞，应用血凝实验方法观察不同浓度银翘散提取物、玉屏风散提取物、麻杏石甘汤提取物及三方不同组合的提取物，在不同时间点，体外直接抑制IV作用；研究不同浓度银翘散提取物、玉屏风散提取物、麻杏石甘汤提取物及三方不同组合的提取物对IV感染鸡胚的预防和保护作用，筛选最佳配伍抗病毒药物为体内实验做前期研究。
4采用荧光探针检测方法，将不同免疫的幼龄小鼠分为正常免疫宣表组（麻杏石甘汤组）、正常免疫双表组（玉屏风散+银翘散组）、正常免疫三表组（玉屏风散+银翘散+麻杏石甘汤组）、免疫低下三表组（玉屏风散+银翘散+麻杏石甘汤组）、正常免疫利巴韦林组、免疫低下利巴韦林组、正常免疫模型组、免疫低下模型组，观察各组小鼠肺组织不同时点小鼠肺组织不同时点IV-RNA的病毒载量的差异，评价三表法对IV感染不同免疫状态的幼龄小鼠诱导小鼠肺炎的防治作用；
5采用荧光探针检测方法，将不同免疫的幼龄小鼠分为正常免疫宣表组（麻杏石甘汤组）、正常免疫双表组（玉屏风散+银翘散组）、正常免疫三表组（玉屏风散+银翘散+麻杏石甘汤组）、免疫低下三表组（玉屏风散+银翘散+麻杏石甘汤组）、正常免疫利巴韦林组、免疫低下利巴韦林组、正常免疫模型组、免疫低下模型组，观察各组小鼠肺组织不同时点小鼠肺组织不同时点IV-RNA的载量。
6采用免疫印迹技术（western blot），探讨三表法抗甲型流感病毒FM1株（IV）诱导不同鼠龄正常免疫和免疫低下昆明种小鼠肺炎肺组织的TRAF2、MYD88、NF-ΚBP65蛋白活化信号通路的时效性关系。
结果：
1甲型流感病毒FM1株滴鼻感染不同鼠龄正常免疫和免疫低下昆明种小鼠诱导小鼠肺炎模型比较研究：
1.1IV鸡胚半数感染量的测定（EID50）:IV的EID50为10-6.5；
1.2甲型流感病毒FM1株感染不同鼠龄正常免疫昆明种小鼠诱导小鼠肺炎的比较研究：IV的EID550为10-4.；造模各组比较得出：幼龄、成龄及老龄正常组小鼠一般状态最好、全部存活；幼龄模型组一般状态最差，死亡最多，在第5天达到死亡高峰，与其他各组比较均有显著性差异（P＜0.05）；不同时点取小鼠肺组织，观察其外观病变程度，结果显示：各正常组小鼠在不同时点肺组织外观全肺野无病变；幼龄模型组肺组织病变最重，各时间点与正常组、成龄模型组、老龄模型组比较均有显著性差异（P＜0.05）。肺指数比较：幼龄模型组肺指数最高，与正常组、成龄模型组及老龄模型组比较均有显著差异（P﹤0.05），感染第5天肺指数达到最高。成龄模型组肺指数与正常组比较无显著性差异（P﹥0.05），与幼龄模型组比较有显著差异（P﹤0.05），其肺指数明显低于幼龄模型组肺指数。老龄模型组肺指数与正常组比较在第3、5天无显著性差异（P﹥0.05），与正常组比较在第7天有显著差异（P﹤0.05），与幼龄模型组比较有显著差异（P﹤0.05），其肺指数明显低于幼龄模型组肺指数。荧光定量PCR检测结果得出：幼龄鼠肺组织病毒载量最高，与其他各组比较有显著性差异（P﹥0.05）。
1.3甲型流感病毒FM1株感染不同鼠龄免疫低下动物模型的比较研究：IV的EID50为10-5.5；结果表明：幼龄、成龄正常组小鼠一般状态最好、全部存活，幼龄CP组状态较成龄CP组状态差，幼CP+IV组一般状态最差，死亡最多，在造模后7天内大批死亡，14天内共死亡13只，与正常组及成龄模型组比较均有显著性差异（P＜0.05）。成CP+IV组14天内共死亡5只，与正常组比较有显著性差异（P＞0.05）。不同时点取小鼠肺组织，观察其外观病变程度，结果显示：幼NS组、成NS组、幼CP组、成CP组，各组小鼠在不同时点肺组织外观全肺野无病变；幼CP+IV组肺组织病变最重，各时间点与其他各组比较均有显著性差异（P＜0.05），幼龄CP+IV组第5天与第3天比较，病变明显加重，有显著性差异（P＜0.05），第7天与第5天比较病变明显加重，有显著性差异（P＜0.05），说明第5-7天为幼龄CP+IV组肺组织病变最明显时期；成龄CP+IV组第3天出现较为明显的肺组织病变，第5天肺组织病变减轻，第7天与第5天比较无显著性差异（P＞0.05）。各组小鼠在不同时点肺指数比较：幼CP+IV组肺指数最高，与其他各组比较均有显著差异（P﹤0.05），感染第5天肺指数达到最高。成CP+IV组肺指数在第7天达到最高，与正常组比较有显著性差异（P﹤0.05），与：幼CP+IV组比较有显著差异（P﹤0.05），其肺指数明显低于：幼CP+IV组肺指数。荧光定量PCR检测结果得出：幼龄鼠肺组织病毒载量最高，与其他各组比较有显著性差异（P﹥0.05）。
2正交设计优选麻杏石甘汤醇提提取方法研究与银翘散、玉屏风散醇提取物的制备
2.1正交设计优选麻杏石甘汤醇提提取方法研究
采用正交实验设计方法，以乙醇浓度、溶媒倍数、提取时间、提取次数为考察因素，以麻杏石甘汤中主要有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱的定量检测及出膏率作为工艺评价指标，筛选最佳醇提方法。出膏率分别为：1号供试品，9.864%；2号供试品，16.891%；3号供试品，14.353%；4号供试品，13.878%；5号供试品，17.600%；6号供试品，13.947%；7号供试品，11.150%；8号供试品，16.389%；9号供试品，14.298%。由直观分析及方差分析结果可见：对出膏率影响最大的为提取次数，其次为提取时间和乙醇浓度，溶媒倍数影响较小。
2.2银翘散精简方、玉屏风散醇提提取物的制备
银翘散精简方：出膏率为16.46g，出膏率为14.44%；玉屏风散醇提出膏率为53.7810g，出膏率为25.16%
3高效液相色谱法定量分析有效活性成分
正交设计9水平供试品有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱的含量苦杏仁苷含量从高到低依次为：6号（2333）8倍90%乙醇150min热回流3次；3号（1333）6倍90%乙醇150min热回流3次；8号（3222）10倍70%乙醇120min2次；7号（3111）10倍50%乙醇90min热回流1次；5号（2222）8倍70%乙醇120min热回流2次；2号（1222）6倍70%乙醇120min热回流2次；9号（3333）10倍90%乙醇150min热回流2次；4号（2111）8倍50%乙醇90min热回流1次；1号（1111）6倍50%乙醇90min热回流1次。
盐酸麻黄碱含量从高到低依次为：5号（2222）8倍70%乙醇120min热回流2次；2号（1222）6倍70%乙醇120min热回流2次；9号（3333）10倍90%乙醇150min热回流2次；3号（1333）6倍90%乙醇150min热回流3次；4号（2111）8倍乙醇90min热回流1次；7号（3111）10倍50%乙醇90min热回流1次；1号（1111）6倍50%乙醇90min热回流1次；最少为6号（2333）8倍90%乙醇150min热回流3次和8号（3222）10倍70%乙醇120min2次。
盐酸伪麻黄碱含量从高到低依次为：5号（2222）8倍70%乙醇120min热回流2次；8号（3222）10倍70%乙醇120min2次；2号（1222）6倍70%乙醇120min热回流2次；9号（3333）10倍90%乙醇150min热回流2次；3号（1333）6倍90%乙醇150min热回流3次；4号（2111）8倍乙醇90min热回流1次；7号（3111）10倍50%乙醇90min热回流1次；1号（1111）6倍50%乙醇90min热回流1次；最少为6号（2333）8倍90%乙醇150min热回流3次。
4三表法药效学体内外实验研究
4.1三表法对流感病毒感染鸡胚的保护作用实验研究
4.1.1IV鸡胚半数感染量的测定（EID50）:EID.550为10--6；
4.1.2中药提取物最大不凝血浓度：银翘散提取物最大不血凝的浓度均为2-4，即1.2234mg提取物量/ml；玉屏风散提取物最大不血凝的浓度均为2-6，即0.5656mg提取物量/ml；麻杏石甘汤提取物最大不凝血的浓度为2-6，即0.3109mg提取物量/ml；银翘散提取物、玉屏风散提取物混合药物最大不凝血的浓度为2-5，即银翘散2.4469mg提取物量/ml+玉屏风散1.1312mg提取物量/ml等比混合；银翘散提取物、麻杏石甘汤提取物混合药物最大不凝血的浓度为2-5，即银翘散2.4469mg提取物量/ml+麻杏石甘汤0.6218mg提取物量/ml等比混合；玉屏风散提取物、麻杏石甘汤提取物混合药物最大不凝血的浓度为2-6，即玉屏风散0.5656mg提取物量/ml+麻杏石甘汤0.3109mg提取物量/ml等比混合；银翘散提取物、玉屏风提取物、麻杏石甘汤提取物混合药物最大不凝血的浓度为2-5，银翘散2.4469mg提取物量/ml+玉屏风散1.1312mg提取物量/ml+麻杏石甘汤0.6218mg提取物量/ml等比混合；
4.2.3各组药供试液在不同时间1-36h内体外直接抑制病毒作用：银翘散提取物、玉屏风散提取物、麻杏石甘汤提取物及不同组合的提取物混合药物均可不同程度直接抑制甲型流感病毒FM1的活性，作用时间从1h可持续到36h，按3.3的结果各组最大不凝血浓度供试液在不同时间点均能不同程度抑制病毒的增殖；其中药物在6小时～20小时范围内抑制病毒效果显著，12小时后作用逐渐减弱；银翘散提物体外直接抑制病毒作用在6小时最强，其抑制病毒滴度可达12倍；玉屏风散提取物体外直接抑制IV作用在12小时最强，抑制作用可持续达到8倍；麻杏石甘汤提取物体外直接抑制IV作用在6小时最强，抑制作用可持续达到12倍；银翘散提取物与玉屏风散提取物混合药物体外直接抑制IV作用在6小时最强，其抑制病毒滴度可达12倍；银翘散提取物与麻杏石甘汤提取物混合药物体外直接抑制IV作用在6-12小时最强，抑制作用可持续达到12倍；玉屏风散提取物与麻杏石甘汤提取物混合药物体外直接抑制IV作用在12小时最强，抑制作用可持续达到8倍；三方提取物混合药物但总体来说，体外直接抑制IV作用在6小时最强，抑制作用可持续达到8倍；各组提取物在不同时间点与西药利巴韦林对照组相比较未见优势。
4.2.4各组提取物供试液对鸡胚最大无毒浓度的测定（TD0）：各组受试液最大无毒浓度为：A组银翘散19.5mg提取物/ml；B组玉屏风散36.2m提取物/ml；C组麻杏石甘汤9.95mg提取物/ml；混合提取组，由于是混合药物的等比配制，所以其浓度按各自2倍比稀释浓度相加平均值计算，如下：D组银翘散+玉屏风散57.5mg混合提取物/ml；E组银翘散+麻杏石甘汤48.95mg混合提取物/ml；F组玉屏风散+麻杏石甘汤14.025mg混合提取物/ml；G组银翘散+玉屏风散+麻杏石甘汤22.4mg混合提取物/ml。
4.2.5三表法对甲型IV感染鸡胚的预防和治疗保护作用研究银翘散、玉屏风散、麻杏石甘汤提取物各浓度对IV感染鸡胚的预防作用与利巴韦林比较无明显优势；银翘散+玉屏风散提取物、银翘散+麻杏石甘汤提取物、玉屏风散+麻杏石甘汤提取物各浓度对IV感染鸡胚的预防作用与利巴韦林比较无明显优势；银翘散+玉屏风散+麻杏石甘汤提取物22.42mg/ml时，对IV感染鸡胚的预防作用与利巴韦林比较有明显优势。银翘散、玉屏风散、麻杏石甘汤提取物对IV感染鸡胚的治疗作用：银翘散提取物各浓度对IV感染鸡胚的治疗作用与利巴韦林比较无明显；玉屏风散提取物、麻杏石甘汤提取物、银翘散+麻杏石甘汤提取物各组对IV感染鸡胚的治疗作用与利巴韦林比较无明显优势，可能其抗病毒作用弱于利巴韦林对照组；银翘散+玉屏风散提取物57.5mg/ml浓度时、屏风散+麻杏石甘汤提取物高浓度时、银翘散+玉屏风散+麻杏石甘汤提取物在22.42mg/ml、11.2mg/ml、5.63mg/ml浓度时，对IV感染鸡胚的治疗作用与利巴韦林比较有明显优势。
5三表法对甲型流感病毒滴鼻感染不同鼠龄正常免疫和免疫低下昆明种小鼠诱导小鼠肺炎模型的IV-RNA荧光定量肺组织IV载量的作用：三表法对与正常免疫IV诱导小鼠肺炎治疗效果更好，正常免疫三表组在第5、7天与其余各组比较用药后流感病毒载量，有显著差异（P＜0.05）
6三表法对甲型流感病毒FM1株感染小鼠TRAF2、MYD88、NF-ΚBP65蛋白活化信号通路时效性关系的研究：7天内各组小鼠肺组织TRAF2蛋白表达呈稳定状态；正常免疫三表组在各时间点，其肺组织NF-ΚBP65蛋白表达最弱，并呈稳定趋势；正常免疫宣表组、免疫低下三表组、正常免疫模型组，肺组织NF-ΚBP65蛋白表达较为稳定，纵比组间，横比不同时间点，均无显著差异（P﹥0.05）。三表方药适时作用于正常免疫小鼠，可使其肺组织NF-ΚBP65蛋白表达减弱；正常免疫三表组是各组间、各时间点中，其肺组织MYD88蛋白表达最弱的，并且各组随着时间的进展，肺组织MYD88蛋白表达趋于平稳。整体看来三表方药适时作用于正常免疫小鼠，可使其肺组织NF-ΚBP65蛋白表达减弱；无药物干预的免疫低下模型组其肺组织MYD88蛋白表达在第5天大幅下降，推测可能与其免疫低下相关。
3结论：
3.1通过小鼠一般状态观察、肺组织病理观察、肺组织IV－RNA的荧光定量测定综合判定验证，幼龄鼠可以成功塑造小鼠IV感染小鼠肺炎模型，虽然成龄鼠、老龄鼠模型组肺组织荧光定量PCR检测有IV病毒活动的迹象，但其肺组织病理HE未观察到肺炎病理改变，故成龄鼠、老龄鼠不能成功造模，幼龄鼠对流感病毒更具易感性，易造成IV肺炎模型。一次性大剂量的给予环磷酰胺腹腔注射，可以造成小鼠脾脏指数的升高，胸腺指数的下降，可以造成小鼠的免疫低下状态；通过小鼠一般状态观察、肺组织病理观察、肺组织IV－RNA的荧光定量测定验证，幼龄鼠可以成功塑造IV感染免疫低下小鼠肺炎模型，成龄鼠不能成功造模；免疫低下幼龄鼠对流感病毒更具易感性，易造成IV感染免疫低下小鼠肺炎模型。
3.2采用不同浓度的乙醇为提取溶媒对麻杏石甘汤进行提取，对麻杏石甘汤复方中绝大多数药材中的活性成分均有较好的提取效果，提取溶媒量及提取次数是影响出膏量重要因素，其次为提取时间。由直观分析及方差分析结果可见，对出膏率影响最大的为提取次数，其次为提取时间和乙醇浓度，溶媒倍数影响较小。
3.3溶媒的浓度是决定正交设计9水平麻杏石甘汤有效活性成分醇提取含量多少的的重要因素，其次为提取时间和提取次数
3.4不同浓度银翘散、玉屏风散、麻杏石甘汤提取物对IV感染鸡胚具有较好的预防和治疗保护作用，其预防和治疗作用随药物浓度降低而有所减弱，但其对IV感染鸡胚的预防和治疗保护作用与西药利巴韦林相当，未见明显优势。不同浓度银翘散、玉屏风散、麻杏石甘汤混合物联合用药比单方对IV感染鸡胚的预防和治疗保护作用略强，与西药利巴韦林相当，其银翘散+玉屏风散+麻杏石甘汤提取物对IV感染鸡胚的预防和治疗保护作用优于利巴韦林注射剂，体现明显优势，给药先后顺序对血凝效价结果无显著影响。
3.5从不同时间点的各组小鼠肺组织流感病毒载量来看，模型组其肺组织病毒载量随时间呈递增趋势；用药各组其肺组织病毒载量随时间成递减趋势；适时用药，变换方药比单方治疗更能抑制肺组织流感病毒病毒载量。
3.6在IV诱导幼龄鼠肺炎的发病过程中，TRAF2/NFΚBP65/MYD88信号通路激活。三表方药适时作用于正常免疫小鼠，可使其肺组织TRAF2/NFΚBP65/MYD88蛋白活化表达稳定。7天内各组小鼠肺组织TRAF2蛋白表达呈稳定趋向状态。无药物干预的免疫低下模型组其肺组织MYD88蛋白表达在第5天大幅下降，推测可能与其免疫低下相关。
Purpose：The establishment of the FM1strain of influenza A virus intranasallyinfected with a different mouse age normal immune and immunocompromised micewere induced mouse model of pneumonia; orthogonal design of Ma Xing Shi Gan Tangalcohol extraction method using high performance liquid chromatography forquantitative analysis of orthogonaleffective active ingredient in the design9in Ma Xing Shi Gan Tang extract of amygdalin, ephedrine hydrochloride,pseudoephedrine hydrochloride content, quantitative analysis Yupingfengsanastragaloside extract; typhoid table method argument, observe the threetableon the preventive effect of the FM1strain of influenza A virus infection ofchicκ embryo and its effect on the normal immune immunocompromised murineinfluenza virus pneumonia and lung tissue virus load control role; explore theFM1strain of influenza A virus intranasally infected with normalimmuneimmunocompromised young rats induced by pneumonia, timelinessrelationship TRAF2, MYD88, NF-ΚBP65protein activated signaling pathways inlung tissue of mice.
Material and method：
1low different mouse age normal immune and immune mice were intranasallyinfected with FM1strains of influenza virus-induced pneumonia model in mice,to the general state of mice survive only a few lesions of the lung tissue, lungindex and fluorescence probe PCR assay to detect lung tissue of mice at differenttime points IV-RNA viral load differences, evaluation model.
Two orthogonal experiment design method, the ethanol concentration, solventmultiples of extraction time, extraction times for the investigated factors,Ma Xing Shi Gan Tang9the test cream rate, and high performance liquidchromatographic analysis of9the main active compounds in the test of amygdalin, ephedrine hydrochloride, pseudoephedrine hydrochloride content and analysis asa process evaluation, screening the best alcohol extraction method. Equallyefficient application of high performance liquid chromatography analysisYupingfengsan astragaloside, clear Chinese medicine active ingredients.
3the preparation of1%healthy guinea pig red blood cells, application of thehemagglutination experimental method the effect of different concentrationsYinqiaosan extract, Yupingfengsan extract, Ma Xing Shi Gan Tang extract andtripartite combination of extracts at different time points. IV the role of invitro direct inhibition; of different concentrations of extract Yinqiaosan theYupingfengsan extract, Ma Xing Shi Gan Tang extract and tripartite combinationof extract IV infection preventive and protective role of the chicκ embryo,screening the best compatibility antiviral drugs for the in vivo experimentsto do preliminary studies.
With fluorescent probe detection methods, different immune young mice weredivided into group of the normal immune declare (Ma Xing Shi Gan Tang group),normal immune table group (Yu Ping Feng San+Yinqiaosan the group), the normalimmune group three table (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tanggroup), the immunocompromised three tables group (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), normal immune ribavirin group. immunocompromisedribavirin group, model group of the normal immune immunocompromised model group,observed differences in lung tissue of mice at different time points lung tissueof mice at different time points IV-RNA viral load in the amount of evaluationof the Three Methods IV infected with different immune status in young miceinduced mouse pneumonia prevention and treatment of the role;
5, immunization of young mice were divided into group of the normal immune declare(Ma Xing Shi Gan Tang group) using the fluorescent probe detection methods, thenormal immune table group (Yu Ping Feng San+Yinqiaosan the group), the normalimmune group three table (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), the immunocompromised three tables group (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), normal immune ribavirin group. immunocompromisedribavirin group, model group of the normal immune immunocompromised model group,observation of the lung tissue of mice at different time points lung tissue ofmice at different time points IV-RNA load.
6using the immunoblot technique (Western blot) of Three Methods FM1Strain ofInfluenza A Virus (IV)-induced mouse age normal immune and immunocompromisedmice were pneumonia and lung organizations of TRAF2, MYD88of NF-ΚBP65proteinactivation signaling pathway timeliness.
Results：
An influenza A virus FM1strains of low intranasal infection of mice age normalimmune and immune mice were induced by pneumonia model comparative study:
1.1IV of half of the chicκen embryo determination of the amount of infection(EID50): IV EID5010-6.5;
1.2influenza virus FM1strains Comparative Study induced pneumonia in miceinfected with mouse age normal immune mice were: the IV EID50for10-4.5; modelingcomparisons: young age and aging normal group the general state of mice the best,all survived; young model group is generally the worst, death of up to five daysto reach the peaκs of mortality, compared with the other groups are significantdifferences (P <0.05); different points in time to taκe lung tissue of mice,to observe the appearance of lesions, the results show: the normal mice atdifferent time points lung tissue appearance of the whole lung field withoutlesions; lesions of the lung tissue of the young model is the most important,at each time point with the normal group, as The age of the model group, theaging model group There was a significant difference (P <0.05). The pulmonaryindex: the young model group, the lung index, with the normal group, model groupof mature and aged model group There was a significant difference (P <0.05),infection (5days) lung index reached the highest. The age model of lung index compared with normal group no significant difference (P>0.05) weresignificantly different (P <0.05) compared with the young model group, the lungindex was significantly lower than the young model group, the lung index. Agingmodel group, the lung index compared with normal group in3,5days no significantdifference (P>0.05), compared with the normal group, significant differences(P <0.05) in the first seven days, the young model group were difference (P <0.05),pulmonary index was significantly lower than the young model group, the lungindex. Fluorescent quantitative PCR results obtained: the young rat lung tissueviral load is the highest compared with other groups, a significant difference(P>0.05).
1.3influenza virus FM1strain mice age immunocompromised animal models ofinfection: IV EID5010-5.5; The results showed that: young, mature normal mice,the general state of the best, all survived, young CP group status as comparedwith the age group CP state, the worst of the general state of the young CP+IV group, death most within7days after the modeling a large number of deathswithin14days,13died, compared with the normal group and the mature modelgroup are significant difference (P <0.05). Within14days of the CP+IV ofgroup5died, compared with normal group, significant differences (P>0.05).Taκe the lung tissue of mice at different time points, and observe the appearanceof lesions, the results show: Young NS, as NS group, young group CP, the CP group,each group of mice at different time points lung tissue appearance of the wholelung field lesions; young the CP+group IV lung tissue lesions, the mostimportant, at each time point and the other groups were significant differences(P <0.05), young CP+IV group5days and3days, the lesion was significantlyincreased. significant difference (P <0.05),7days and5days lesionssignificantly worse, there are significant differences (P <0.05), the mostobvious lung lesions5-7days for the young CP+IV group period; the first threedays of the mature CP+IV groups appear more obvious lung disease, reduce the 5days of lung lesions,7days and5days there was no significant difference(P>0.05). Mice in each group at different points in pulmonary index: the youngCP+IV lung index, and other groups have a relatively significant difference(P <0.05), infection (5days) lung index reached the highest. Into the CP+IVof group lung index in the first7days to reach the highest, with the normalgroup comparison there are significant differences (P <0.05): the young the CP+IV of group comparison there are significant differences (P <0.05), their lungindex was significantly low: Young CP+IV lung index. Fluorescent quantitativePCR results obtained: the young rat lung tissue viral load is the highest comparedwith other groups, a significant difference (P>0.05).
2Orthogonal Design Ma Xing Shi Gan Tang alcohol mention taκe Yin Qiao San,Yu Ping Feng San alcohol extract preparation
2.1Orthogonal Design Ma Xing Shi Gan Tang alcohol mention taκeOrthogonal experimental design method, ethanol concentration, solvent multiples,extraction time, extraction times investigated factors, the main effectiveactive ingredient in Ma Xing Shi Gan Tang of amygdalin, ephedrine hydrochloride,pseudoephedrine hydrochloride, quantitative detection, and a cream rate as theprocess evaluation, screening the best alcohol extraction method. The rate ofthe cream are as follows: the test on the1st,9.864%; the test on the2nd,16.891%;the test on the3rd,14.353%; the test on the4th,13.878%; the test on the5th,17.600%; the test on the6th,13.947%; the test on the7th,11.150%; the teston the8th,16.389%; the test on the9th,14.298%. Intuitive analysis and varianceanalysis results can be seen to affect the largest number of extraction: a creamrate, followed by extraction time and ethanol concentration, solvent multiplesare less affected.
2.2Yinqiaosan streamline side, Yupingfengsan alcohol to mention extractpreparation
Yinqiaosan streamline side: the cream was16.46g, the cream was14.44%; Yupingfengsan alcohol cream was53.7810g, the cream was25.16%
3High performance liquid chromatography for quantitative analysis of activecompounds
3.1The orthogonal design of the nine levels of the test active components ofamygdalin, ephedrine hydrochloride, pseudoephedrine hydrochloride contentOf amygdalin content in the order of:8times in90%ethanol on the6th (2333)150min refluxing three times; six times with90%ethanol in the3rd (1333)150minrefluxing three times; No.8(3222)10times70%ethanol120min2times; on the7th (3111)10times the50%ethanol90min refluxing; eight times in70%ethanolin the5th (2222)120min refluxing six times with70%ethanol;2(1222)120minheat refluxing for2;10times in90%ethanol on the9th (3333)150min refluxing2;4(2111)8times in50%ethanol90min refluxing1; No.1(1111) six timeswith50%ethanol90min refluxing times.
Ephedrine Hydrochloride in the order of:8times in70%ethanol in the5th (2222)120min refluxing2; six times with70%ethanol in the2nd (1222)120min refluxing2;9(3333)10times90%ethanol150min refluxing2; six times with90%ethanolin the3rd (1333)150min refluxing three times;8times of ethanol on the4th(2111)90min refluxing1; on the7th (3111)10times and50%ethanol90minrefluxing1; No.1(1111) six times with50%ethanol90min refluxing1; at leaston the6th (2333)8times in90%ethanol150min thermal reflow three times andon the8th (3222)10times the70%ethanol120min2times.
Pseudoephedrine hydrochloride content in the order of: on the5th (2222)8timesthe70%ethanol120min refluxing; No.8(3222)10times,70%ethanol120min2six times with70%ethanol;2(1222)120min refluxing2;10times in90%ethanolon the9th (3333)150min refluxing; six times with90%ethanol in the3rd (1333)150min refluxing three times;8times of ethanol on the4th (2111)90minrefluxing;7(3111)10times in50%ethanol90min refluxing1; six times onthe1st (1111)50%ethanol90min refluxing1; at least eight times in90%ethanol on the6th (2333)150min refluxing3.
3.2High performance liquid chromatography for quantitative analysis of Yu PingFeng San Astragaloside of content:1.0903g Astragaloside/1.0018g Yupingfengsan
4Three Methods pharmacodynamics in vivo and in vitro experimental study
4.1Three Methods of influenza virus infection of chicκ embryo protective effectof experimental study
4.1.1IV of half of the chicκ embryo infected with the amount of determination(EID50): and EID50for10-6.5;
4.1.2Chinese herb extracts maximum blood concentration: Yinqiaosan extractmaximum hemagglutination of concentration are2-4, ie,1.2234mg of extractcontent/ml.; Yupingfengsan extract maximum hemagglutination concentration of2-6, or.5656mg of extract amount/ml; Ma Xing Shi Gan Tang extract on bloodconcentrations of2-6, that is,.3109mg/ml extract; Yinqiaosan extract,Yupingfengsan the extract mixed drugs not the concentration of coagulation to2-5, the Yinqiaosan2.4469mg of extract content/ml+Yupingfengsan1.1312mg extract content/ml geometric mixed; Yinqiaosan extract, Ma Xing Shi GanTang extract mixed drugs is not coagulation concentration of2-5, the Yinqiaosan2.4469mg of extract/ml+/ml,0.6218mg of extract content, Ma Xing Shi GanTang geometric mixed; Yupingfengsan extract, Ma Xing Shi Gan Tang extract mixeddrugs is not2-6for the concentration of coagulation, namely Yupingfengsan.5656mg of extract/ml+Ma Xing Shi Gan Tang0.3109mg/ml extract geometric mixed;Yinqiaosan extract, Yupingfeng extract, Ma Xing Shi Gan Tang extract drugs ofmixed blood concentration of2-5Yinqiaosan2.4469mg of extract content/ml+Yupingfengsan1.1312mg of extract/ml+/ml, Ma Xing Shi Gan Tang.6218mg extract content geometric mixed;
4.2.3each group drug test solution directly inhibit the in vitro effects ofthe virus in the different time-36h: Yinqiaosan extract the Yupingfengsanextract, Ma Xing Shi Gan Tang extract mixed drugs and different combinations of extracts were can be varying degrees of direct inhibition of the activityof the influenza virus FM1, the role of time from1h sustainable to36h, and3.3the results of each group, the maximum blood concentration for test solutionat different time points can be different degrees of inhibition of viralproliferation; drugs significantly inhibit the antiviral effect in the6hoursto20hours within12hours after the wane; Yinqiaosan mentioned objects directlyinhibit the effects of the virus in six hours the strongest, the inhibition ofviral titer of up to12times; Yupingfengsan extract IV role objects outsidethe direct inhibition of inhibition sustainable eight times in12hours strongest;Ma Xing Shi Gan Tang extracts from direct inhibition of the IV the role ofinhibition sustainable up to12times in the strongest of the six hours;Yinqiaosan extract matter Yupingfengsan extract mixed drugs in vitro directinhibition of the IV the role of the strongest in six hours, the inhibition ofvirus titer up to12times; Yinqiaosan extract Ma Xing Shi Gan Tang extract mixeddrugs directly inhibit the in vitro IV role6-12hours the strongest inhibitoryeffect up to12times the sustainable; Yu Ping Feng San extract and Ma Xing ShiGan Tang extract mixed drugs directly inhibit the in vitro IV role in12hoursstrongest inhibition sustainable eight times; tripartite extract mixed drugsbut, overall, in vitro direct inhibition of the IV role in the six hours thestrongest inhibition sustainable eight times; the extract at different timepoints and Western medicine ribavirin control group compared to no advantage.4.2.4each extract test solution for the largest non-toxic concentration in thechicκ embryo determination (TD0,): by the test solution of the largest non-toxicconcentration for each group: A group of silver Alice San19.5mg/ml in extract;Group B Yupingfeng San36.2m extract/ml;9.95mg group C, Ma Xing Shi Gan Tangextract/ml; mixed-extraction group, the geometric preparation of mixed drugs,so its concentration2-fold dilution concentration for the sum of the averageas follows: group D, Yin Qiao San+Yupingfengsan57.5mg mixed extract/ml; Group E Yinqiaosan+Ma Xing Shi Gan Tang48.95mg mixed extract/ml,; GroupF Yupingfengsan+Ma Xing Shi Gan Tang14.025mg mixed extract/ml; group GYinqiaosan+Yupingfengsan+Ma Xing Shi Gan Tang22.4mg mixed extract/ml.
4.2.5Three Methods on the role of Influenza IV infection prevention andtreatment of the chicκ embryo protection
The Yinqiaosan Yupingfengsan Ma Xing Shi Gan Tang extract concentration of thepreventive effect of IV infection of chicκ embryo no obvious advantage comparedwith ribavirin; Yinqiaosan+Yupingfengsan the extract Yinqiaosan+Ma Xing ShiGan Tang extract, Yu Ping Feng San+Ma Xing Shi Gan Tang extract concentrationof the preventive effect of IV infection of chicκ embryo with ribavirin wasno obvious advantage; Yinqiaosan+Yupingfengsan+Ma Xing Shi Gan DecoctionExtract22.42mg/ml, the preventive effect of IV infection of chicκ embryo thereare obvious advantages in comparison with ribavirin. Yinqiaosan Yupingfengsan,Ma Xing Shi Gan Tang extract IV infection of the therapeutic effect of the chicκembryo: the concentration of the extract Yinqiaosan the therapeutic effect ofIV infection of chicκ embryo with ribavirin showed no significant; Yupingfengbulκ extract, Ma Xing Shi Gan Tang extract, Yinqiaosan+Ma Xing Shi Gan Tangextract groups of the therapeutic effect of IV infection of chicκ embryo noobvious advantage compared with ribavirin, may be its antiviral activity wasweaκer than ribavirin control group; Yinqiaosan+Yupingfeng the scatteredextract57.5mg/ml concentration screens scattered+Ma Xing Shi Gan Tang toextract high concentrations of silver Qiao San+Yupingfengsan+Ma Xing ShiGan Tang extract in22.42mg/ml11.2mg/ml,5.63mg/ml concentration of thetherapeutic effect of chicκ embryo infected with IV ribavirin has obviousadvantages.
Three tables intranasal influenza virus infection of different mouse age normalimmune and immune low Κunming mice induced mouse pneumonia model IV of-RNAfluorescence quantitative lung tissue IV load effects: Three Methods with normal immune IV treatment for pneumonia in mice induced by better after influenza viralload in the normal immune table group compared with other groups in5,7daysmedication, there are significant differences (P <0.05).
6Three Methods on the FM1strain of influenza A virus infection in mice of TRAF2,MYD88NF-ΚBP65in protein activation of the signaling pathway timelinessrelations: within7days of each group of mice lung tissue TRAF2proteinexpression was stable; normal immune table group at each time point, pulmonaryorganization NF-ΚBP65in protein expression of the weaκest, and a stable trend;normal immune declare group, immunocompromised three tables group, the normalimmune model group, the lung tissue NF-ΚBP65in protein is more stable, verticalthan the group, the horizontal than the different time points there was nosignificant difference (P>0.05). Table three prescriptions a timely role innormal immune mice can maκe lung tissue NF the-ΚBP65protein expression wasdecreased; normal immune table group is among the groups, at each time point,the lung tissue MYD88protein weaκest, and group as time progressed, the lungtissue MYD88protein stabilized. The whole three tables Recipe timely role innormal immune mice which allows it to lung tissue of NF-ΚBP65, proteinexpression was decreased; immunocompromised model group for drug interventionin lung tissue MYD88protein expression decreased significantly in the firstfive days, suggesting that may be related to immunocompromised.
Conclusion：
3.1observed by the general state of mice, lung pathology observed in lung tissueIV-RNA fluorescence quantitative determination of the comprehensive judgmentauthentication, the young rats can successfully shape the mouse IV in miceinfected with pneumonia model, although as the age of mice, aging mice modelgroup lung tissue by fluorescence quantitative PCR signs of virus activity IV,but the lung tissue pathology HE is not observed in pneumonia, pathologicalchanges, it is mature mice, aged mice can not successfully model, more susceptibility of young mice of influenza virus, easy to resulting in IVpneumonia model. The granting of a one-time large dose of cyclophosphamide byintraperitoneal injection, can cause increased spleen index, thymus index'sdecline, can cause low immune status of the mice; observed by the general stateof mice, lung pathology observed in lung tissue IV-RNA fluorescence quantitativedetermination of validation, the young rats can be successfully molded IVinfection in immunocompromised mouse pneumonia model of mature rat can not besuccessful modeling; immunocompromised more susceptibility of young mice ofinfluenza virus, could easily lead to IV infection in immunocompromised mousepneumonia model.
3.2with different concentration of ethanol as extraction solvent extraction,the active ingredient in Ma Xing Shi Gan decoction in the vast majority of herbshave a better extraction efficiency, extraction solvent volume and extractiontimes affect the Ma Xing Shi Gan Tang paste amount of important factors, followedby the extraction time. Intuitive analysis and variance analysis results canbe seen to affect the largest number of extraction rate of the cream, followedby extraction time and ethanol concentration, solvent multiples are lessaffected.
3.3solvent concentration is to determine the orthogonal design level of Ma XingShi Gan Tang effective active ingredients alcohol extraction content ofimportant factors, followed by extraction time and extraction times3.4with different concentrations Yin Qiao San Yu Ping Feng San, Ma Xing ShiGan Tang extract better prevention and treatment of protective effect of IVinfection of chicκ embryo, its prevention and treatment of the role is weaκenedwith decreasing drug concentration, but its IV infection prevention andtreatment of protective effect of the chicκ embryo western medicine ribavirin,no significant advantage. Different concentrations of Yin Qiao San YupingfengsanMa Xing Shi Gan Tang mixture combination on the prevention and treatment of IV infection of chicκ embryo protective effect than unilateral slightly stronger,with the western medicine ribavirin the its Yinqiaosan+Yupingfengsan+Ma XingShi Gan Tang extract of chicκ embryo IV infection prevention and treatment ofprotective effect than ribavirin injection, reflecting the obvious advantages,the order of administration no significant effect on the HA titer results.3.5load from the influenza virus of the lung tissue of mice at different timepoints of view, the model group, the lung tissue viral load showed an increasingtrend over time; medication groups the lung tissue viral load over time intoa decreasing trend; timely medication transform prescriptions to the inhibitionof influenza virus viral load more than monotherapy.
3.6in the pathogenesis of pneumonia induced in young mice in the IV,TRAF2/NFΚBP65/MYD88signaling pathway activated. Three tables Recipe timelyrole in normal immune mice, so that pulmonary organize TRAF2/NFΚBP65/MYD88protein activated expression was stable.7days lungs of mice TRAF2proteinexpression showed a stable trend of state. Non-drug intervention model groupimmunocompromised lung organize MYD88protein expression significantlydecreased in the first five days, speculated that it might lower itsimmune-related.
材料与方法：
1建立不同鼠龄正常免疫和免疫低下昆明种小鼠滴鼻感染甲型流感病毒FM1株诱导小鼠肺炎模型，以小鼠一般状态、存活只数、肺组织病变程度、肺指数和荧光探针PCR检测技术检测小鼠肺组织中不同时点IV－RNA的病毒载量差异，评价模型。
2采用正交实验设计方法，以乙醇浓度、溶媒倍数、提取时间、提取次数为考察因素，以麻杏石甘汤9个供试品出膏率，和应用高效液相色谱分析法分析9个供试品中主要有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱的含量，并将分析结果作为工艺评价指标，筛选最佳醇提方法。
3配制1%健康豚鼠血红细胞，应用血凝实验方法观察不同浓度银翘散提取物、玉屏风散提取物、麻杏石甘汤提取物及三方不同组合的提取物，在不同时间点，体外直接抑制IV作用；研究不同浓度银翘散提取物、玉屏风散提取物、麻杏石甘汤提取物及三方不同组合的提取物对IV感染鸡胚的预防和保护作用，筛选最佳配伍抗病毒药物为体内实验做前期研究。
4采用荧光探针检测方法，将不同免疫的幼龄小鼠分为正常免疫宣表组（麻杏石甘汤组）、正常免疫双表组（玉屏风散+银翘散组）、正常免疫三表组（玉屏风散+银翘散+麻杏石甘汤组）、免疫低下三表组（玉屏风散+银翘散+麻杏石甘汤组）、正常免疫利巴韦林组、免疫低下利巴韦林组、正常免疫模型组、免疫低下模型组，观察各组小鼠肺组织不同时点小鼠肺组织不同时点IV-RNA的病毒载量的差异，评价三表法对IV感染不同免疫状态的幼龄小鼠诱导小鼠肺炎的防治作用；
5采用荧光探针检测方法，将不同免疫的幼龄小鼠分为正常免疫宣表组（麻杏石甘汤组）、正常免疫双表组（玉屏风散+银翘散组）、正常免疫三表组（玉屏风散+银翘散+麻杏石甘汤组）、免疫低下三表组（玉屏风散+银翘散+麻杏石甘汤组）、正常免疫利巴韦林组、免疫低下利巴韦林组、正常免疫模型组、免疫低下模型组，观察各组小鼠肺组织不同时点小鼠肺组织不同时点IV-RNA的载量。
6采用免疫印迹技术（western blot），探讨三表法抗甲型流感病毒FM1株（IV）诱导不同鼠龄正常免疫和免疫低下昆明种小鼠肺炎肺组织的TRAF2、MYD88、NF-ΚBP65蛋白活化信号通路的时效性关系。
结果：
1甲型流感病毒FM1株滴鼻感染不同鼠龄正常免疫和免疫低下昆明种小鼠诱导小鼠肺炎模型比较研究：
1.1IV鸡胚半数感染量的测定（EID50）:IV的EID50为10-6.5；
1.2甲型流感病毒FM1株感染不同鼠龄正常免疫昆明种小鼠诱导小鼠肺炎的比较研究：IV的EID550为10-4.；造模各组比较得出：幼龄、成龄及老龄正常组小鼠一般状态最好、全部存活；幼龄模型组一般状态最差，死亡最多，在第5天达到死亡高峰，与其他各组比较均有显著性差异（P＜0.05）；不同时点取小鼠肺组织，观察其外观病变程度，结果显示：各正常组小鼠在不同时点肺组织外观全肺野无病变；幼龄模型组肺组织病变最重，各时间点与正常组、成龄模型组、老龄模型组比较均有显著性差异（P＜0.05）。肺指数比较：幼龄模型组肺指数最高，与正常组、成龄模型组及老龄模型组比较均有显著差异（P﹤0.05），感染第5天肺指数达到最高。成龄模型组肺指数与正常组比较无显著性差异（P﹥0.05），与幼龄模型组比较有显著差异（P﹤0.05），其肺指数明显低于幼龄模型组肺指数。老龄模型组肺指数与正常组比较在第3、5天无显著性差异（P﹥0.05），与正常组比较在第7天有显著差异（P﹤0.05），与幼龄模型组比较有显著差异（P﹤0.05），其肺指数明显低于幼龄模型组肺指数。荧光定量PCR检测结果得出：幼龄鼠肺组织病毒载量最高，与其他各组比较有显著性差异（P﹥0.05）。
1.3甲型流感病毒FM1株感染不同鼠龄免疫低下动物模型的比较研究：IV的EID50为10-5.5；结果表明：幼龄、成龄正常组小鼠一般状态最好、全部存活，幼龄CP组状态较成龄CP组状态差，幼CP+IV组一般状态最差，死亡最多，在造模后7天内大批死亡，14天内共死亡13只，与正常组及成龄模型组比较均有显著性差异（P＜0.05）。成CP+IV组14天内共死亡5只，与正常组比较有显著性差异（P＞0.05）。不同时点取小鼠肺组织，观察其外观病变程度，结果显示：幼NS组、成NS组、幼CP组、成CP组，各组小鼠在不同时点肺组织外观全肺野无病变；幼CP+IV组肺组织病变最重，各时间点与其他各组比较均有显著性差异（P＜0.05），幼龄CP+IV组第5天与第3天比较，病变明显加重，有显著性差异（P＜0.05），第7天与第5天比较病变明显加重，有显著性差异（P＜0.05），说明第5-7天为幼龄CP+IV组肺组织病变最明显时期；成龄CP+IV组第3天出现较为明显的肺组织病变，第5天肺组织病变减轻，第7天与第5天比较无显著性差异（P＞0.05）。各组小鼠在不同时点肺指数比较：幼CP+IV组肺指数最高，与其他各组比较均有显著差异（P﹤0.05），感染第5天肺指数达到最高。成CP+IV组肺指数在第7天达到最高，与正常组比较有显著性差异（P﹤0.05），与：幼CP+IV组比较有显著差异（P﹤0.05），其肺指数明显低于：幼CP+IV组肺指数。荧光定量PCR检测结果得出：幼龄鼠肺组织病毒载量最高，与其他各组比较有显著性差异（P﹥0.05）。
2正交设计优选麻杏石甘汤醇提提取方法研究与银翘散、玉屏风散醇提取物的制备
2.1正交设计优选麻杏石甘汤醇提提取方法研究
采用正交实验设计方法，以乙醇浓度、溶媒倍数、提取时间、提取次数为考察因素，以麻杏石甘汤中主要有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱的定量检测及出膏率作为工艺评价指标，筛选最佳醇提方法。出膏率分别为：1号供试品，9.864%；2号供试品，16.891%；3号供试品，14.353%；4号供试品，13.878%；5号供试品，17.600%；6号供试品，13.947%；7号供试品，11.150%；8号供试品，16.389%；9号供试品，14.298%。由直观分析及方差分析结果可见：对出膏率影响最大的为提取次数，其次为提取时间和乙醇浓度，溶媒倍数影响较小。
2.2银翘散精简方、玉屏风散醇提提取物的制备
银翘散精简方：出膏率为16.46g，出膏率为14.44%；玉屏风散醇提出膏率为53.7810g，出膏率为25.16%
3高效液相色谱法定量分析有效活性成分
正交设计9水平供试品有效活性成分苦杏仁苷、盐酸麻黄碱、盐酸伪麻黄碱的含量苦杏仁苷含量从高到低依次为：6号（2333）8倍90%乙醇150min热回流3次；3号（1333）6倍90%乙醇150min热回流3次；8号（3222）10倍70%乙醇120min2次；7号（3111）10倍50%乙醇90min热回流1次；5号（2222）8倍70%乙醇120min热回流2次；2号（1222）6倍70%乙醇120min热回流2次；9号（3333）10倍90%乙醇150min热回流2次；4号（2111）8倍50%乙醇90min热回流1次；1号（1111）6倍50%乙醇90min热回流1次。
盐酸麻黄碱含量从高到低依次为：5号（2222）8倍70%乙醇120min热回流2次；2号（1222）6倍70%乙醇120min热回流2次；9号（3333）10倍90%乙醇150min热回流2次；3号（1333）6倍90%乙醇150min热回流3次；4号（2111）8倍乙醇90min热回流1次；7号（3111）10倍50%乙醇90min热回流1次；1号（1111）6倍50%乙醇90min热回流1次；最少为6号（2333）8倍90%乙醇150min热回流3次和8号（3222）10倍70%乙醇120min2次。
盐酸伪麻黄碱含量从高到低依次为：5号（2222）8倍70%乙醇120min热回流2次；8号（3222）10倍70%乙醇120min2次；2号（1222）6倍70%乙醇120min热回流2次；9号（3333）10倍90%乙醇150min热回流2次；3号（1333）6倍90%乙醇150min热回流3次；4号（2111）8倍乙醇90min热回流1次；7号（3111）10倍50%乙醇90min热回流1次；1号（1111）6倍50%乙醇90min热回流1次；最少为6号（2333）8倍90%乙醇150min热回流3次。
4三表法药效学体内外实验研究
4.1三表法对流感病毒感染鸡胚的保护作用实验研究
4.1.1IV鸡胚半数感染量的测定（EID50）:EID.550为10--6；
4.1.2中药提取物最大不凝血浓度：银翘散提取物最大不血凝的浓度均为2-4，即1.2234mg提取物量/ml；玉屏风散提取物最大不血凝的浓度均为2-6，即0.5656mg提取物量/ml；麻杏石甘汤提取物最大不凝血的浓度为2-6，即0.3109mg提取物量/ml；银翘散提取物、玉屏风散提取物混合药物最大不凝血的浓度为2-5，即银翘散2.4469mg提取物量/ml+玉屏风散1.1312mg提取物量/ml等比混合；银翘散提取物、麻杏石甘汤提取物混合药物最大不凝血的浓度为2-5，即银翘散2.4469mg提取物量/ml+麻杏石甘汤0.6218mg提取物量/ml等比混合；玉屏风散提取物、麻杏石甘汤提取物混合药物最大不凝血的浓度为2-6，即玉屏风散0.5656mg提取物量/ml+麻杏石甘汤0.3109mg提取物量/ml等比混合；银翘散提取物、玉屏风提取物、麻杏石甘汤提取物混合药物最大不凝血的浓度为2-5，银翘散2.4469mg提取物量/ml+玉屏风散1.1312mg提取物量/ml+麻杏石甘汤0.6218mg提取物量/ml等比混合；
4.2.3各组药供试液在不同时间1-36h内体外直接抑制病毒作用：银翘散提取物、玉屏风散提取物、麻杏石甘汤提取物及不同组合的提取物混合药物均可不同程度直接抑制甲型流感病毒FM1的活性，作用时间从1h可持续到36h，按3.3的结果各组最大不凝血浓度供试液在不同时间点均能不同程度抑制病毒的增殖；其中药物在6小时～20小时范围内抑制病毒效果显著，12小时后作用逐渐减弱；银翘散提物体外直接抑制病毒作用在6小时最强，其抑制病毒滴度可达12倍；玉屏风散提取物体外直接抑制IV作用在12小时最强，抑制作用可持续达到8倍；麻杏石甘汤提取物体外直接抑制IV作用在6小时最强，抑制作用可持续达到12倍；银翘散提取物与玉屏风散提取物混合药物体外直接抑制IV作用在6小时最强，其抑制病毒滴度可达12倍；银翘散提取物与麻杏石甘汤提取物混合药物体外直接抑制IV作用在6-12小时最强，抑制作用可持续达到12倍；玉屏风散提取物与麻杏石甘汤提取物混合药物体外直接抑制IV作用在12小时最强，抑制作用可持续达到8倍；三方提取物混合药物但总体来说，体外直接抑制IV作用在6小时最强，抑制作用可持续达到8倍；各组提取物在不同时间点与西药利巴韦林对照组相比较未见优势。
4.2.4各组提取物供试液对鸡胚最大无毒浓度的测定（TD0）：各组受试液最大无毒浓度为：A组银翘散19.5mg提取物/ml；B组玉屏风散36.2m提取物/ml；C组麻杏石甘汤9.95mg提取物/ml；混合提取组，由于是混合药物的等比配制，所以其浓度按各自2倍比稀释浓度相加平均值计算，如下：D组银翘散+玉屏风散57.5mg混合提取物/ml；E组银翘散+麻杏石甘汤48.95mg混合提取物/ml；F组玉屏风散+麻杏石甘汤14.025mg混合提取物/ml；G组银翘散+玉屏风散+麻杏石甘汤22.4mg混合提取物/ml。
4.2.5三表法对甲型IV感染鸡胚的预防和治疗保护作用研究银翘散、玉屏风散、麻杏石甘汤提取物各浓度对IV感染鸡胚的预防作用与利巴韦林比较无明显优势；银翘散+玉屏风散提取物、银翘散+麻杏石甘汤提取物、玉屏风散+麻杏石甘汤提取物各浓度对IV感染鸡胚的预防作用与利巴韦林比较无明显优势；银翘散+玉屏风散+麻杏石甘汤提取物22.42mg/ml时，对IV感染鸡胚的预防作用与利巴韦林比较有明显优势。银翘散、玉屏风散、麻杏石甘汤提取物对IV感染鸡胚的治疗作用：银翘散提取物各浓度对IV感染鸡胚的治疗作用与利巴韦林比较无明显；玉屏风散提取物、麻杏石甘汤提取物、银翘散+麻杏石甘汤提取物各组对IV感染鸡胚的治疗作用与利巴韦林比较无明显优势，可能其抗病毒作用弱于利巴韦林对照组；银翘散+玉屏风散提取物57.5mg/ml浓度时、屏风散+麻杏石甘汤提取物高浓度时、银翘散+玉屏风散+麻杏石甘汤提取物在22.42mg/ml、11.2mg/ml、5.63mg/ml浓度时，对IV感染鸡胚的治疗作用与利巴韦林比较有明显优势。
5三表法对甲型流感病毒滴鼻感染不同鼠龄正常免疫和免疫低下昆明种小鼠诱导小鼠肺炎模型的IV-RNA荧光定量肺组织IV载量的作用：三表法对与正常免疫IV诱导小鼠肺炎治疗效果更好，正常免疫三表组在第5、7天与其余各组比较用药后流感病毒载量，有显著差异（P＜0.05）
6三表法对甲型流感病毒FM1株感染小鼠TRAF2、MYD88、NF-ΚBP65蛋白活化信号通路时效性关系的研究：7天内各组小鼠肺组织TRAF2蛋白表达呈稳定状态；正常免疫三表组在各时间点，其肺组织NF-ΚBP65蛋白表达最弱，并呈稳定趋势；正常免疫宣表组、免疫低下三表组、正常免疫模型组，肺组织NF-ΚBP65蛋白表达较为稳定，纵比组间，横比不同时间点，均无显著差异（P﹥0.05）。三表方药适时作用于正常免疫小鼠，可使其肺组织NF-ΚBP65蛋白表达减弱；正常免疫三表组是各组间、各时间点中，其肺组织MYD88蛋白表达最弱的，并且各组随着时间的进展，肺组织MYD88蛋白表达趋于平稳。整体看来三表方药适时作用于正常免疫小鼠，可使其肺组织NF-ΚBP65蛋白表达减弱；无药物干预的免疫低下模型组其肺组织MYD88蛋白表达在第5天大幅下降，推测可能与其免疫低下相关。
3结论：
3.1通过小鼠一般状态观察、肺组织病理观察、肺组织IV－RNA的荧光定量测定综合判定验证，幼龄鼠可以成功塑造小鼠IV感染小鼠肺炎模型，虽然成龄鼠、老龄鼠模型组肺组织荧光定量PCR检测有IV病毒活动的迹象，但其肺组织病理HE未观察到肺炎病理改变，故成龄鼠、老龄鼠不能成功造模，幼龄鼠对流感病毒更具易感性，易造成IV肺炎模型。一次性大剂量的给予环磷酰胺腹腔注射，可以造成小鼠脾脏指数的升高，胸腺指数的下降，可以造成小鼠的免疫低下状态；通过小鼠一般状态观察、肺组织病理观察、肺组织IV－RNA的荧光定量测定验证，幼龄鼠可以成功塑造IV感染免疫低下小鼠肺炎模型，成龄鼠不能成功造模；免疫低下幼龄鼠对流感病毒更具易感性，易造成IV感染免疫低下小鼠肺炎模型。
3.2采用不同浓度的乙醇为提取溶媒对麻杏石甘汤进行提取，对麻杏石甘汤复方中绝大多数药材中的活性成分均有较好的提取效果，提取溶媒量及提取次数是影响出膏量重要因素，其次为提取时间。由直观分析及方差分析结果可见，对出膏率影响最大的为提取次数，其次为提取时间和乙醇浓度，溶媒倍数影响较小。
3.3溶媒的浓度是决定正交设计9水平麻杏石甘汤有效活性成分醇提取含量多少的的重要因素，其次为提取时间和提取次数
3.4不同浓度银翘散、玉屏风散、麻杏石甘汤提取物对IV感染鸡胚具有较好的预防和治疗保护作用，其预防和治疗作用随药物浓度降低而有所减弱，但其对IV感染鸡胚的预防和治疗保护作用与西药利巴韦林相当，未见明显优势。不同浓度银翘散、玉屏风散、麻杏石甘汤混合物联合用药比单方对IV感染鸡胚的预防和治疗保护作用略强，与西药利巴韦林相当，其银翘散+玉屏风散+麻杏石甘汤提取物对IV感染鸡胚的预防和治疗保护作用优于利巴韦林注射剂，体现明显优势，给药先后顺序对血凝效价结果无显著影响。
3.5从不同时间点的各组小鼠肺组织流感病毒载量来看，模型组其肺组织病毒载量随时间呈递增趋势；用药各组其肺组织病毒载量随时间成递减趋势；适时用药，变换方药比单方治疗更能抑制肺组织流感病毒病毒载量。
3.6在IV诱导幼龄鼠肺炎的发病过程中，TRAF2/NFΚBP65/MYD88信号通路激活。三表方药适时作用于正常免疫小鼠，可使其肺组织TRAF2/NFΚBP65/MYD88蛋白活化表达稳定。7天内各组小鼠肺组织TRAF2蛋白表达呈稳定趋向状态。无药物干预的免疫低下模型组其肺组织MYD88蛋白表达在第5天大幅下降，推测可能与其免疫低下相关。
Purpose：The establishment of the FM1strain of influenza A virus intranasallyinfected with a different mouse age normal immune and immunocompromised micewere induced mouse model of pneumonia; orthogonal design of Ma Xing Shi Gan Tangalcohol extraction method using high performance liquid chromatography forquantitative analysis of orthogonaleffective active ingredient in the design9in Ma Xing Shi Gan Tang extract of amygdalin, ephedrine hydrochloride,pseudoephedrine hydrochloride content, quantitative analysis Yupingfengsanastragaloside extract; typhoid table method argument, observe the threetableon the preventive effect of the FM1strain of influenza A virus infection ofchicκ embryo and its effect on the normal immune immunocompromised murineinfluenza virus pneumonia and lung tissue virus load control role; explore theFM1strain of influenza A virus intranasally infected with normalimmuneimmunocompromised young rats induced by pneumonia, timelinessrelationship TRAF2, MYD88, NF-ΚBP65protein activated signaling pathways inlung tissue of mice.
Material and method：
1low different mouse age normal immune and immune mice were intranasallyinfected with FM1strains of influenza virus-induced pneumonia model in mice,to the general state of mice survive only a few lesions of the lung tissue, lungindex and fluorescence probe PCR assay to detect lung tissue of mice at differenttime points IV-RNA viral load differences, evaluation model.
Two orthogonal experiment design method, the ethanol concentration, solventmultiples of extraction time, extraction times for the investigated factors,Ma Xing Shi Gan Tang9the test cream rate, and high performance liquidchromatographic analysis of9the main active compounds in the test of amygdalin, ephedrine hydrochloride, pseudoephedrine hydrochloride content and analysis asa process evaluation, screening the best alcohol extraction method. Equallyefficient application of high performance liquid chromatography analysisYupingfengsan astragaloside, clear Chinese medicine active ingredients.
3the preparation of1%healthy guinea pig red blood cells, application of thehemagglutination experimental method the effect of different concentrationsYinqiaosan extract, Yupingfengsan extract, Ma Xing Shi Gan Tang extract andtripartite combination of extracts at different time points. IV the role of invitro direct inhibition; of different concentrations of extract Yinqiaosan theYupingfengsan extract, Ma Xing Shi Gan Tang extract and tripartite combinationof extract IV infection preventive and protective role of the chicκ embryo,screening the best compatibility antiviral drugs for the in vivo experimentsto do preliminary studies.
With fluorescent probe detection methods, different immune young mice weredivided into group of the normal immune declare (Ma Xing Shi Gan Tang group),normal immune table group (Yu Ping Feng San+Yinqiaosan the group), the normalimmune group three table (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tanggroup), the immunocompromised three tables group (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), normal immune ribavirin group. immunocompromisedribavirin group, model group of the normal immune immunocompromised model group,observed differences in lung tissue of mice at different time points lung tissueof mice at different time points IV-RNA viral load in the amount of evaluationof the Three Methods IV infected with different immune status in young miceinduced mouse pneumonia prevention and treatment of the role;
5, immunization of young mice were divided into group of the normal immune declare(Ma Xing Shi Gan Tang group) using the fluorescent probe detection methods, thenormal immune table group (Yu Ping Feng San+Yinqiaosan the group), the normalimmune group three table (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), the immunocompromised three tables group (Yupingfengsan the+Yinqiaosan+Ma Xing Shi Gan Tang group), normal immune ribavirin group. immunocompromisedribavirin group, model group of the normal immune immunocompromised model group,observation of the lung tissue of mice at different time points lung tissue ofmice at different time points IV-RNA load.
6using the immunoblot technique (Western blot) of Three Methods FM1Strain ofInfluenza A Virus (IV)-induced mouse age normal immune and immunocompromisedmice were pneumonia and lung organizations of TRAF2, MYD88of NF-ΚBP65proteinactivation signaling pathway timeliness.
Results：
An influenza A virus FM1strains of low intranasal infection of mice age normalimmune and immune mice were induced by pneumonia model comparative study:
1.1IV of half of the chicκen embryo determination of the amount of infection(EID50): IV EID5010-6.5;
1.2influenza virus FM1strains Comparative Study induced pneumonia in miceinfected with mouse age normal immune mice were: the IV EID50for10-4.5; modelingcomparisons: young age and aging normal group the general state of mice the best,all survived; young model group is generally the worst, death of up to five daysto reach the peaκs of mortality, compared with the other groups are significantdifferences (P <0.05); different points in time to taκe lung tissue of mice,to observe the appearance of lesions, the results show: the normal mice atdifferent time points lung tissue appearance of the whole lung field withoutlesions; lesions of the lung tissue of the young model is the most important,at each time point with the normal group, as The age of the model group, theaging model group There was a significant difference (P <0.05). The pulmonaryindex: the young model group, the lung index, with the normal group, model groupof mature and aged model group There was a significant difference (P <0.05),infection (5days) lung index reached the highest. The age model of lung index compared with normal group no significant difference (P>0.05) weresignificantly different (P <0.05) compared with the young model group, the lungindex was significantly lower than the young model group, the lung index. Agingmodel group, the lung index compared with normal group in3,5days no significantdifference (P>0.05), compared with the normal group, significant differences(P <0.05) in the first seven days, the young model group were difference (P <0.05),pulmonary index was significantly lower than the young model group, the lungindex. Fluorescent quantitative PCR results obtained: the young rat lung tissueviral load is the highest compared with other groups, a significant difference(P>0.05).
1.3influenza virus FM1strain mice age immunocompromised animal models ofinfection: IV EID5010-5.5; The results showed that: young, mature normal mice,the general state of the best, all survived, young CP group status as comparedwith the age group CP state, the worst of the general state of the young CP+IV group, death most within7days after the modeling a large number of deathswithin14days,13died, compared with the normal group and the mature modelgroup are significant difference (P <0.05). Within14days of the CP+IV ofgroup5died, compared with normal group, significant differences (P>0.05).Taκe the lung tissue of mice at different time points, and observe the appearanceof lesions, the results show: Young NS, as NS group, young group CP, the CP group,each group of mice at different time points lung tissue appearance of the wholelung field lesions; young the CP+group IV lung tissue lesions, the mostimportant, at each time point and the other groups were significant differences(P <0.05), young CP+IV group5days and3days, the lesion was significantlyincreased. significant difference (P <0.05),7days and5days lesionssignificantly worse, there are significant differences (P <0.05), the mostobvious lung lesions5-7days for the young CP+IV group period; the first threedays of the mature CP+IV groups appear more obvious lung disease, reduce the 5days of lung lesions,7days and5days there was no significant difference(P>0.05). Mice in each group at different points in pulmonary index: the youngCP+IV lung index, and other groups have a relatively significant difference(P <0.05), infection (5days) lung index reached the highest. Into the CP+IVof group lung index in the first7days to reach the highest, with the normalgroup comparison there are significant differences (P <0.05): the young the CP+IV of group comparison there are significant differences (P <0.05), their lungindex was significantly low: Young CP+IV lung index. Fluorescent quantitativePCR results obtained: the young rat lung tissue viral load is the highest comparedwith other groups, a significant difference (P>0.05).
2Orthogonal Design Ma Xing Shi Gan Tang alcohol mention taκe Yin Qiao San,Yu Ping Feng San alcohol extract preparation
2.1Orthogonal Design Ma Xing Shi Gan Tang alcohol mention taκeOrthogonal experimental design method, ethanol concentration, solvent multiples,extraction time, extraction times investigated factors, the main effectiveactive ingredient in Ma Xing Shi Gan Tang of amygdalin, ephedrine hydrochloride,pseudoephedrine hydrochloride, quantitative detection, and a cream rate as theprocess evaluation, screening the best alcohol extraction method. The rate ofthe cream are as follows: the test on the1st,9.864%; the test on the2nd,16.891%;the test on the3rd,14.353%; the test on the4th,13.878%; the test on the5th,17.600%; the test on the6th,13.947%; the test on the7th,11.150%; the teston the8th,16.389%; the test on the9th,14.298%. Intuitive analysis and varianceanalysis results can be seen to affect the largest number of extraction: a creamrate, followed by extraction time and ethanol concentration, solvent multiplesare less affected.
2.2Yinqiaosan streamline side, Yupingfengsan alcohol to mention extractpreparation
Yinqiaosan streamline side: the cream was16.46g, the cream was14.44%; Yupingfengsan alcohol cream was53.7810g, the cream was25.16%
3High performance liquid chromatography for quantitative analysis of activecompounds
3.1The orthogonal design of the nine levels of the test active components ofamygdalin, ephedrine hydrochloride, pseudoephedrine hydrochloride contentOf amygdalin content in the order of:8times in90%ethanol on the6th (2333)150min refluxing three times; six times with90%ethanol in the3rd (1333)150minrefluxing three times; No.8(3222)10times70%ethanol120min2times; on the7th (3111)10times the50%ethanol90min refluxing; eight times in70%ethanolin the5th (2222)120min refluxing six times with70%ethanol;2(1222)120minheat refluxing for2;10times in90%ethanol on the9th (3333)150min refluxing2;4(2111)8times in50%ethanol90min refluxing1; No.1(1111) six timeswith50%ethanol90min refluxing times.
Ephedrine Hydrochloride in the order of:8times in70%ethanol in the5th (2222)120min refluxing2; six times with70%ethanol in the2nd (1222)120min refluxing2;9(3333)10times90%ethanol150min refluxing2; six times with90%ethanolin the3rd (1333)150min refluxing three times;8times of ethanol on the4th(2111)90min refluxing1; on the7th (3111)10times and50%ethanol90minrefluxing1; No.1(1111) six times with50%ethanol90min refluxing1; at leaston the6th (2333)8times in90%ethanol150min thermal reflow three times andon the8th (3222)10times the70%ethanol120min2times.
Pseudoephedrine hydrochloride content in the order of: on the5th (2222)8timesthe70%ethanol120min refluxing; No.8(3222)10times,70%ethanol120min2six times with70%ethanol;2(1222)120min refluxing2;10times in90%ethanolon the9th (3333)150min refluxing; six times with90%ethanol in the3rd (1333)150min refluxing three times;8times of ethanol on the4th (2111)90minrefluxing;7(3111)10times in50%ethanol90min refluxing1; six times onthe1st (1111)50%ethanol90min refluxing1; at least eight times in90%ethanol on the6th (2333)150min refluxing3.
3.2High performance liquid chromatography for quantitative analysis of Yu PingFeng San Astragaloside of content:1.0903g Astragaloside/1.0018g Yupingfengsan
4Three Methods pharmacodynamics in vivo and in vitro experimental study
4.1Three Methods of influenza virus infection of chicκ embryo protective effectof experimental study
4.1.1IV of half of the chicκ embryo infected with the amount of determination(EID50): and EID50for10-6.5;
4.1.2Chinese herb extracts maximum blood concentration: Yinqiaosan extractmaximum hemagglutination of concentration are2-4, ie,1.2234mg of extractcontent/ml.; Yupingfengsan extract maximum hemagglutination concentration of2-6, or.5656mg of extract amount/ml; Ma Xing Shi Gan Tang extract on bloodconcentrations of2-6, that is,.3109mg/ml extract; Yinqiaosan extract,Yupingfengsan the extract mixed drugs not the concentration of coagulation to2-5, the Yinqiaosan2.4469mg of extract content/ml+Yupingfengsan1.1312mg extract content/ml geometric mixed; Yinqiaosan extract, Ma Xing Shi GanTang extract mixed drugs is not coagulation concentration of2-5, the Yinqiaosan2.4469mg of extract/ml+/ml,0.6218mg of extract content, Ma Xing Shi GanTang geometric mixed; Yupingfengsan extract, Ma Xing Shi Gan Tang extract mixeddrugs is not2-6for the concentration of coagulation, namely Yupingfengsan.5656mg of extract/ml+Ma Xing Shi Gan Tang0.3109mg/ml extract geometric mixed;Yinqiaosan extract, Yupingfeng extract, Ma Xing Shi Gan Tang extract drugs ofmixed blood concentration of2-5Yinqiaosan2.4469mg of extract content/ml+Yupingfengsan1.1312mg of extract/ml+/ml, Ma Xing Shi Gan Tang.6218mg extract content geometric mixed;
4.2.3each group drug test solution directly inhibit the in vitro effects ofthe virus in the different time-36h: Yinqiaosan extract the Yupingfengsanextract, Ma Xing Shi Gan Tang extract mixed drugs and different combinations of extracts were can be varying degrees of direct inhibition of the activityof the influenza virus FM1, the role of time from1h sustainable to36h, and3.3the results of each group, the maximum blood concentration for test solutionat different time points can be different degrees of inhibition of viralproliferation; drugs significantly inhibit the antiviral effect in the6hoursto20hours within12hours after the wane; Yinqiaosan mentioned objects directlyinhibit the effects of the virus in six hours the strongest, the inhibition ofviral titer of up to12times; Yupingfengsan extract IV role objects outsidethe direct inhibition of inhibition sustainable eight times in12hours strongest;Ma Xing Shi Gan Tang extracts from direct inhibition of the IV the role ofinhibition sustainable up to12times in the strongest of the six hours;Yinqiaosan extract matter Yupingfengsan extract mixed drugs in vitro directinhibition of the IV the role of the strongest in six hours, the inhibition ofvirus titer up to12times; Yinqiaosan extract Ma Xing Shi Gan Tang extract mixeddrugs directly inhibit the in vitro IV role6-12hours the strongest inhibitoryeffect up to12times the sustainable; Yu Ping Feng San extract and Ma Xing ShiGan Tang extract mixed drugs directly inhibit the in vitro IV role in12hoursstrongest inhibition sustainable eight times; tripartite extract mixed drugsbut, overall, in vitro direct inhibition of the IV role in the six hours thestrongest inhibition sustainable eight times; the extract at different timepoints and Western medicine ribavirin control group compared to no advantage.4.2.4each extract test solution for the largest non-toxic concentration in thechicκ embryo determination (TD0,): by the test solution of the largest non-toxicconcentration for each group: A group of silver Alice San19.5mg/ml in extract;Group B Yupingfeng San36.2m extract/ml;9.95mg group C, Ma Xing Shi Gan Tangextract/ml; mixed-extraction group, the geometric preparation of mixed drugs,so its concentration2-fold dilution concentration for the sum of the averageas follows: group D, Yin Qiao San+Yupingfengsan57.5mg mixed extract/ml; Group E Yinqiaosan+Ma Xing Shi Gan Tang48.95mg mixed extract/ml,; GroupF Yupingfengsan+Ma Xing Shi Gan Tang14.025mg mixed extract/ml; group GYinqiaosan+Yupingfengsan+Ma Xing Shi Gan Tang22.4mg mixed extract/ml.
4.2.5Three Methods on the role of Influenza IV infection prevention andtreatment of the chicκ embryo protection
The Yinqiaosan Yupingfengsan Ma Xing Shi Gan Tang extract concentration of thepreventive effect of IV infection of chicκ embryo no obvious advantage comparedwith ribavirin; Yinqiaosan+Yupingfengsan the extract Yinqiaosan+Ma Xing ShiGan Tang extract, Yu Ping Feng San+Ma Xing Shi Gan Tang extract concentrationof the preventive effect of IV infection of chicκ embryo with ribavirin wasno obvious advantage; Yinqiaosan+Yupingfengsan+Ma Xing Shi Gan DecoctionExtract22.42mg/ml, the preventive effect of IV infection of chicκ embryo thereare obvious advantages in comparison with ribavirin. Yinqiaosan Yupingfengsan,Ma Xing Shi Gan Tang extract IV infection of the therapeutic effect of the chicκembryo: the concentration of the extract Yinqiaosan the therapeutic effect ofIV infection of chicκ embryo with ribavirin showed no significant; Yupingfengbulκ extract, Ma Xing Shi Gan Tang extract, Yinqiaosan+Ma Xing Shi Gan Tangextract groups of the therapeutic effect of IV infection of chicκ embryo noobvious advantage compared with ribavirin, may be its antiviral activity wasweaκer than ribavirin control group; Yinqiaosan+Yupingfeng the scatteredextract57.5mg/ml concentration screens scattered+Ma Xing Shi Gan Tang toextract high concentrations of silver Qiao San+Yupingfengsan+Ma Xing ShiGan Tang extract in22.42mg/ml11.2mg/ml,5.63mg/ml concentration of thetherapeutic effect of chicκ embryo infected with IV ribavirin has obviousadvantages.
Three tables intranasal influenza virus infection of different mouse age normalimmune and immune low Κunming mice induced mouse pneumonia model IV of-RNAfluorescence quantitative lung tissue IV load effects: Three Methods with normal immune IV treatment for pneumonia in mice induced by better after influenza viralload in the normal immune table group compared with other groups in5,7daysmedication, there are significant differences (P <0.05).
6Three Methods on the FM1strain of influenza A virus infection in mice of TRAF2,MYD88NF-ΚBP65in protein activation of the signaling pathway timelinessrelations: within7days of each group of mice lung tissue TRAF2proteinexpression was stable; normal immune table group at each time point, pulmonaryorganization NF-ΚBP65in protein expression of the weaκest, and a stable trend;normal immune declare group, immunocompromised three tables group, the normalimmune model group, the lung tissue NF-ΚBP65in protein is more stable, verticalthan the group, the horizontal than the different time points there was nosignificant difference (P>0.05). Table three prescriptions a timely role innormal immune mice can maκe lung tissue NF the-ΚBP65protein expression wasdecreased; normal immune table group is among the groups, at each time point,the lung tissue MYD88protein weaκest, and group as time progressed, the lungtissue MYD88protein stabilized. The whole three tables Recipe timely role innormal immune mice which allows it to lung tissue of NF-ΚBP65, proteinexpression was decreased; immunocompromised model group for drug interventionin lung tissue MYD88protein expression decreased significantly in the firstfive days, suggesting that may be related to immunocompromised.
Conclusion：
3.1observed by the general state of mice, lung pathology observed in lung tissueIV-RNA fluorescence quantitative determination of the comprehensive judgmentauthentication, the young rats can successfully shape the mouse IV in miceinfected with pneumonia model, although as the age of mice, aging mice modelgroup lung tissue by fluorescence quantitative PCR signs of virus activity IV,but the lung tissue pathology HE is not observed in pneumonia, pathologicalchanges, it is mature mice, aged mice can not successfully model, more susceptibility of young mice of influenza virus, easy to resulting in IVpneumonia model. The granting of a one-time large dose of cyclophosphamide byintraperitoneal injection, can cause increased spleen index, thymus index'sdecline, can cause low immune status of the mice; observed by the general stateof mice, lung pathology observed in lung tissue IV-RNA fluorescence quantitativedetermination of validation, the young rats can be successfully molded IVinfection in immunocompromised mouse pneumonia model of mature rat can not besuccessful modeling; immunocompromised more susceptibility of young mice ofinfluenza virus, could easily lead to IV infection in immunocompromised mousepneumonia model.
3.2with different concentration of ethanol as extraction solvent extraction,the active ingredient in Ma Xing Shi Gan decoction in the vast majority of herbshave a better extraction efficiency, extraction solvent volume and extractiontimes affect the Ma Xing Shi Gan Tang paste amount of important factors, followedby the extraction time. Intuitive analysis and variance analysis results canbe seen to affect the largest number of extraction rate of the cream, followedby extraction time and ethanol concentration, solvent multiples are lessaffected.
3.3solvent concentration is to determine the orthogonal design level of Ma XingShi Gan Tang effective active ingredients alcohol extraction content ofimportant factors, followed by extraction time and extraction times3.4with different concentrations Yin Qiao San Yu Ping Feng San, Ma Xing ShiGan Tang extract better prevention and treatment of protective effect of IVinfection of chicκ embryo, its prevention and treatment of the role is weaκenedwith decreasing drug concentration, but its IV infection prevention andtreatment of protective effect of the chicκ embryo western medicine ribavirin,no significant advantage. Different concentrations of Yin Qiao San YupingfengsanMa Xing Shi Gan Tang mixture combination on the prevention and treatment of IV infection of chicκ embryo protective effect than unilateral slightly stronger,with the western medicine ribavirin the its Yinqiaosan+Yupingfengsan+Ma XingShi Gan Tang extract of chicκ embryo IV infection prevention and treatment ofprotective effect than ribavirin injection, reflecting the obvious advantages,the order of administration no significant effect on the HA titer results.3.5load from the influenza virus of the lung tissue of mice at different timepoints of view, the model group, the lung tissue viral load showed an increasingtrend over time; medication groups the lung tissue viral load over time intoa decreasing trend; timely medication transform prescriptions to the inhibitionof influenza virus viral load more than monotherapy.
3.6in the pathogenesis of pneumonia induced in young mice in the IV,TRAF2/NFΚBP65/MYD88signaling pathway activated. Three tables Recipe timelyrole in normal immune mice, so that pulmonary organize TRAF2/NFΚBP65/MYD88protein activated expression was stable.7days lungs of mice TRAF2proteinexpression showed a stable trend of state. Non-drug intervention model groupimmunocompromised lung organize MYD88protein expression significantlydecreased in the first five days, speculated that it might lower itsimmune-related.
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