【作者】 吴振起；

【导师】 王雪峰；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2008， 博士

【摘要】 目的:依据“性味—功效”相关性理论和医家的临证思维方法,从性味配伍对银翘散进行拆方研究。通过银翘散及其性味分组提取物抗IV感染MDCK细胞、小鼠实验,从抑制IV、诱生干扰素、抑制细胞凋亡等角度来探讨银翘散抗IV主要作用机制、主要生物效应和方中药物的最佳剂量配比关系,为银翘散多层次、多环节、多靶点抗IV作用提供理论支持和实验依据。材料与方法:1.采用乙醇提取方法制备银翘散及其性味分组提取物。2.用IV滴鼻造成小鼠感染模型,以小鼠肺组织中病毒的血凝滴度作为观察指标,运用正交设计方法,观察银翘散各药物配伍与药效之间的关系。3.建立IV感染细胞模型,通过先感染病毒后给药、先给药后感染病毒和药物病毒同时作用三种给药方式,采用CPE和MTT法观察银翘散及其性味分组提取物对IV感染MDCK细胞的作用。4.采用DNA琼脂糖凝胶电泳和TUNEL法检测细胞凋亡;通过细胞爬片,采用免疫组化法检测凋亡细胞Fas/FasL的表达,观察银翘散及其性味分组提取物对IV感染MDCK细胞诱导细胞凋亡的影响。5.建立小鼠IV肺炎模型,应用RT-PCR和ELISA法检测IV感染量及IFN-γ的含量,结合小鼠一般状态、肺组织病理切片、形态学、肺指数等指标,观察银翘散及其性味分组提取物对IV感染小鼠的作用。6.采用TUNEL法和免疫组化法观察小鼠肺组织细胞凋亡及Fas/FasL的表达,观察银翘散及其性味分组提取物对IV感染小鼠诱导细胞凋亡的作用。结果:1.通过直观分析和正交分析,银翘散必要因素有连翘、金银花、牛蒡子、荆芥穗、竹叶、生甘草、桔梗、薄荷(P<0.05),而淡豆豉的作用不明显(P>0.05)。2.银翘散及其性味分组对IV感染具有不同程度的治疗作用,银翘散组作用与药物浓度呈正向关。银翘散(1:1～1:4)及清热解毒(1:1～1:2)组治疗效果与病毒唑组比较无显著性差异(P>0.05)。银翘散及其性味分组对IV具有较好的预防作用,银翘散(1:1～1:8)、清热解毒(1:1～1:8)、辛凉解表(1:1～1:4)、辛温解表(1:1～1:8)组与病毒唑组比较有显著差异(P﹤0.05),银翘散、清热解毒、辛温解表组作用与药物浓度呈正向关。银翘散及其性味分组对IV具有直接灭杀作用(P﹤0.05),其中银翘散、清热解毒组作用与药物浓度呈正向关。银翘散(1:1～1:2)、清热解毒(1:1～1:4)、辛凉解表(1:1)组直接灭杀IV作用优于病毒唑组(P﹤0.05),而辛温解表组与病毒唑组比较无显著差异(P﹥0.05)。3.银翘散及其性味分组和病毒唑组未出现凋亡“梯状”图谱,能降低凋亡指数(AI),减少细胞凋亡,增强Fas的吸光度(P﹤0.05)。与病毒唑组相比,银翘散、清热解毒组无显著性差异(P﹥0.05),而辛凉解表、辛温解表组有显著性差异(P﹤0.05)。与病毒组相比,银翘散、清热解毒、辛凉解表组和病毒唑组能升高FasL的吸光度,有显著性差异(P﹤0.05);与病毒唑组相比,银翘散、清热解毒组无显著性差异(P﹥0.05),而辛凉解表组有显著性差异(P﹤0.05)。4.小鼠IV肺炎模型复制:肺组织中检测到IV总RNA大量表达,肺指数增高,病理改变呈间质性肺炎病变:肺泡间隔水肿、炎细胞浸润,肺泡腔内浆液性渗出。5.小鼠肺组织IV总RNA的表达:与病毒组比较,银翘散及其性味分组和病毒唑组均有显著差异(P﹤0.05),与病毒唑组比较,银翘散、清热解毒、辛凉解表组无显著差异(P﹥0.05),辛温解表组有显著差异(P﹤0.05)。6.RT-PCR法测IFN-γ表达:与病毒组比较,银翘散、清热解毒、辛温解表组及病毒唑组有显著差异(P﹤0.05),辛凉解表组无显著差异(P﹥0.05);与病毒唑组比较,银翘散、清热解毒、辛凉解表组有显著差异(P﹤0.05),辛温解表组无显著差异(P﹥0.05)。ELISA法:IV感染小鼠,其肺组织中的IFN-γ含量明显增多(P﹤0.05);与病毒组比较,银翘散、清热解毒组及病毒唑组有显著差异(P﹤0.05),辛凉解表、辛温解表组无显著差异(P﹥0.05);与病毒唑组比较,银翘散及其性味分组均有显著差异(P﹤0.05)。7.IV感染能够诱导小鼠肺组织细胞凋亡,Fas及FasL蛋白大量表达(P<0.05),银翘散组、清热解毒组能够降低凋亡指数(AI),抑制IV感染诱导小鼠肺组织的细胞凋亡(P<0.05),同时能下调肺组织中Fas/FasL蛋白表达(P<0.05)。与病毒唑组FasL相比,银翘散组无显著性差异(P﹥0.05),清热解毒组有显著性差异(P﹤0.05)。辛凉解表、辛温解表组,与病毒组AI比较,无显著差异(P﹥0.05);与病毒唑组Fas相比,有显著性差异(P﹤0.05);与正常组FasL比较,无显著性差异(P﹥0.05)。结论:1.从降低IV滴鼻感染小鼠肺组织匀浆血凝滴度的角度看,银翘散以连翘2金银花2牛蒡子2荆芥穗1竹叶2生甘草2桔梗1薄荷2淡豆豉1治疗效果好。2.银翘散及其性味分组对IV感染MDCK细胞具有预防、治疗和直接灭杀作用。辛温解表组对银翘散直接灭杀IV作用的贡献率不如其它二组。在不同的给药方式中,“先用药后感染病毒”抑制病毒作用效果最好。3. IV感染MDCK细胞能够诱导细胞凋亡。银翘散及其性味分组能够抑制IV诱导MDCK细胞凋亡,其作用机制可能是通过Fas/FasL途径来实现,其中清热解毒组起了主要作用。4.银翘散具有直接抑制IV、减少肺病理损伤和诱生干扰素作用,前二者是清热解毒组、辛凉解表组、辛温解表组三合方“整合”的结果,后者主要是通过清热解毒组而奏效。5.IV感染能够诱导小鼠肺组织发生细胞凋亡。银翘散能够抑制IV诱导小鼠肺组织细胞凋亡,可能是通过Fas/FasL途径来实现,其中清热解毒组起了主要作用。更多还原

【Abstract】 Objective:Do the study on the separately groups of Honeysuckle Forsythia Powder on the basis of compatibility of medicine flavor-nature according to the theory of correlation between nature-flavor of herbs and their efficacy and to the doctors’thinking methods on clinical symptom-complex. Through observing MDCK cell which anti-IV infects of Honeysuckle Forsythia Powder and extract of it’s separately groups. Explore the mechanism of how Honeysuckle Forsythia Powder can anti IV﹑main organism effect and excellent proportion of each herb in powder from the angles of anti IV、leading to produce Interferon and restrain apoptosis. Offer theory support and experimental basis for the anti IV effect of Honeysuckle Forsythia Powder from every target and segment.Methods:1. Prepare extract of Honeysuckle Forsythia Powder and it’s separately groups by use of alcohol solution .2. Make infected mice model by use of inoculating IV in cavitasnasi, make the hemagglutination inhibition titer of virus in mice lung tissue observe target. Observe correlation between compatibility of every groups and the drug efficacy3. Make infecting vitro models in three ways which are infect first-take medicine then, take medicine first-infect then and take medicine when infect. Use CPE and MTT to observe the effect Honeysuckle Forsythia Powder and extract of it’s separately groups made by nature-flavor of herbs take on apoptosis induced by infection of influenza virus4. Use DNA- agarose gel electrophoresis, TUNEL to examine apoptosis. Examine expression of apoptosis Fas/Fasl by use of Immunity group reduction. Observe the effect Honeysuckle Forsythia Powder and extract of it’s separately groups made by nature-flavor of herbs take on apoptosis induced by infection of influenza virus5. Make infected mice model. Examine quantity of IV and content of IV by RT-PCR and ELISA, Observe the effect Honeysuckle Forsythia Powder and extract of it’s separately groups made by nature-flavor of herbs take on apoptosis induced by infection of influenza virus with observing external features of mice, lung tissue pathological section, morphology and lung index.6. Examine expression of apoptosis Fas/Fasl by use of TUNEL and Immunity group reduction. Observe the effect Honeysuckle Forsythia Powder and extract of it’s separately groups made by nature-flavor of herbs on apoptosis induced by infection of influenza virus. Result:1. To employ intuition analyze and orthogonal analyze, know the essential ingredients in Honeysuckle Forsythia Powder are Flos lonicerae ,forsythia suspense ,arctium lappa, bamboo leaf ,jingjiesui,gancao,jiegeng,mint(p<0.05),expect dandouchi(>0.05)2. Honeysuckle Forsythia Powder and it’s separately groups can take treatmental measure on IV, It’s effect is in direct proportion to concentration of medicine .The drug efficacy of Honeysuckle Forsythia Powder(1:1-1:4)and clear heat-toxic medicine(1:1-1:2 ) is similar to Ribavirin(p>0.05)Honeysuckle Forsythia Powder and it’s separately groups can take prevention measure on IV .The drug efficacy of Honeysuckle Forsythia Powder(1:1-1:8), clear heat-toxic medicine(1:1-1:8 ) ,formulae that release the exterie with pungent -cool herbs (1:1-1:4 ) and formulae that release the exterie with pungent -warm herbs(1:1-1:8) is markly different to Ribavirin(p<0.05). Concentration of medicine is in direct proportion to ER. Honeysuckle Forsythia Powder and it’s separately groups can directly kill IV. The drug efficacy of Honeysuckle Forsythia Powder, clear heat-toxic medicine is in direct proportion to concentration of medicine .The effect of Honeysuckle Forsythia Powder(1:1-1:2), clear heat-toxic medicine (1:1-1:4), formulae that release the exterie with pungent -cool herbs (1:1)which directly kill IV is better then Ribavirin (p<0.05),while formulae that release the exterie with pungent -warm herbs is similar to Ribavirin (p>0.05)3. Honeysuckle Forsythia Powder and it’s separately groups and Ribavirin do not appear Apoptosis trapezoid atlas. It can reduce AI, apoptosis and light absorption of Fas (p<0.05). Compare to Ribavirin, the effect of Honeysuckle Forsythia Powder and clear heat-toxic medicine is similar to Ribavirin(p<0.05). But formulae that release the exterie with pungent–cool and warm herbs are quite different(p<0.05). Compare to IV, Honeysuckle Forsythia Powder, clear heat-toxic medicine, formulae that release the exterie with pungent -cool herbs and Ribavirin can be more induced the light absorption degree, but are different from each other (p<0.05) . Compare to Ribavirin, Honeysuckle Forsythia Powder and clear heat-toxic medicine are similar to it (p>0.05),but more different from formulae that release the exterie with pungent -cool herbs(p<0.05).4. Reconstructed model of mice IV pneumonia: the IV RNA examined in lung is expressed in bulk .Lung index increase. Pathological change is internior pneumonia:dropsy between pulmonary alveolei, smash by inflammatory cell .5. The expression of the total IV RNA examined in lung: Compare to IV,Honeysuckle Forsythia Powder and it’s separately groups and Ribavirin are more different(p<0.05). Compare to Ribavirin, the effect of Honeysuckle Forsythia Powder is the same as clear heat-toxic medicine and formulae that release the exterie with pungent -cool herbs(p>0.05),is markedly different from formulae that release the exterie with pungent -warm herbs(p<0.05)6. Use RT-PCR to examine the expression of IFN-γ: Compare to virus infected groups, the effect of Honeysuckle Forsythia Powder is markedly different from clear heat-toxic magician , formulae that release the exterie with pungent -warm herbs and Ribavirin (p<0.05), is similar to formulae that release the exterie with pungent -warm herbs (p>0.05).Compare to Ribavirin.Honeysuckle Forsythia Powder, clear heat-toxic medicine and formulae that release the exterie with pungent -cool herbs are more effectual but formulae that release the exterie with pungent -cool herbs.Use ELISA to examine the expression of IFN-γ: it is increased in virus infected mice lung tissue (p<0.05). Compare to virus infected groups, The effect of Honeysuckle Forsythia Powder and clear heat-toxic medician, and Ribavirin (p<0.05) is more effectual, but formulae that release the exterie with pungent -warm herbs and with pungent -cool herbs (p>0.05): Compare to Ribavirin, Honeysuckle Forsythia Powder, clear heat-toxic medicine and formulae that release the exterie with pungent -cool herbs are more effectual but formulae that release the exterie with pungent -cool herbs. Compare to Ribavirin, Honeysuckle Forsythia Powder it’s separately groups are more effectual (p<0.05)7. Infected can induce apoptosis in mice lung, more Fas/Fasl express(p<0.05). Honeysuckle Forsythia Powder and clear heat-toxic medicine can reduceAI, control apoptosis in lung(p<0.05), reduce expression of protein (p<0.05).Compare to Ribavirin Fasl, the effect of Honeysuckle Forsythia Powder is similar (p>0.05) but clear heat-toxic medicine(p<0.05). Compare to virus infected groups AI, formulae that release the exterie with pungent -warm herbs and with pungent -cool herbs are no different (p>0.05). Compare to Aciclovir Fas, formulae that release the exterie with pungent -warm herbs and with pungent -cool herbs are more different (p<0.05) and compare to normal groups, similar(p>0.05).Conclusion:1.From the angel of reducing the hemagglutination inhibition titer of the virus in lung , know the essential ingredient in Honeysuckle Forsythia Powder are Flos lonicerae ,forsythia suspense ,arctium lappa,jingjiesui,bamboo leaf,gancao,jiegeng ,dandouchi2. Honeysuckle Forsythia Powder and it’s separately groups can take treatmenal and prevention measure on IV in MDCK cell and directly kill it. They can directly kill IV expect the less effectual formulae that release the exterie with pungent -warm herbs. In different ways of take medicine: the groups that first-take medicine then. Take medicine is more effectual on control virus3. MDCK cell Infected by IV can induce apoptosis .Honeysuckle Forsythia Powder can control it main through Fas/Fasl, and clear heat-toxic medicine make more act on it.4. Honeysuckle Forsythia Powder can directly kill IV, reduce damnification in lung and induce produce of Interferon. The former two are got from integration of clear heat-toxic medicine, formulae that release the exterie with pungent -warm herbs and with pungent -cool herbs ,the later one is main got from clear heat-toxic medicine5. Infected by IV can induce apoptosis in mice lung tissue .Honeysuckle Forsythia Powder can control it main through Fas/Fasl ,and clear heat-toxic medicine make more act on it.更多还原