【作者】 陈创荣；

【导师】 刘小虹；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2015， 博士

【摘要】 研究目的:为了进一步探讨中医药对于流行性感冒的有效性和作用机制,本研究建立了 H1N1流感病毒FM1株感染小鼠模型,以具有岭南特色的岗藿抗感汤为方,从细胞学、分子生物学和病理学等角度研究,观察岗藿抗感汤对病毒感染模型小鼠肺指数、死亡保护、湿干重比例、血气分析、病毒载量、肺组织形态学的变化,以及血清细胞因子含量及肺泡灌洗液中炎症细胞计数和分类的影响等的含量,进而探讨岗藿抗感汤抗流感的药效作用机制,为中医药治疗流感提供有效可靠的实验理论依据。研究方法:实验一流感病毒FM1株感染小鼠LD50的测定KM种小鼠91只,体重13.0-15.0g。随机分为7组,每组13只。在乙醚轻度麻醉下,选用7个病毒滴度的流感病毒液,滴鼻感染小鼠,每鼠4滴,约0.05ml以死亡和生存为观察指标,24h内死亡的不予统计。每天观察,记录小鼠死亡情况,直至感染后第15天为止。数据处理后用Reed—Muench两氏法,计算出病毒的半数致死量(LD50)。实验二岗藿抗感汤对流感病毒感染小鼠死亡保护研究KM小鼠50只,13-15g,雌雄各半。将KM小鼠随机分为正常组、模型组、利巴韦林组、岗藿抗感汤高剂量组、岗藿抗感汤低剂量组,共5组,每组10只,在乙醚轻度麻醉下,除正常组外,其余各组采用10LD5oH1N1病毒液滴鼻感染小鼠造模,正常组以等体积的无菌生理盐水滴鼻。感染4h后各组开始分别给药,各组给药体积均为0.2mL/10g,连续给药15天,观察小鼠一般状态,对小鼠的活动度、精神状况、气喘、有无咳嗽、皮毛光泽、体重变化等情况进行记录,并记录其生存天数,从而计算死亡保护率和延长生命率。实验三岗藿抗感汤对流感病毒感染小鼠的药效作用机制1 KM小鼠30只,13-15g,雌雄各半。随机分为正常组、模型组、利巴韦林组、岗藿抗感汤高剂量组、岗藿抗感汤低剂量组,共5组,每组6只。除正常组外,其余各组采用10LD50H1N1病毒液滴鼻感染小鼠造模,正常组以等体积的无菌生理盐水滴鼻。感染4h后各组开始分别给药,各组给药体积均为0.2mL/l0g,连续给药5天后进行实验。2岗藿抗感汤对流感小鼠肺指数、湿干重比例的影响末次给药后30min,将小鼠称重后摘眼球放血处死,仰位固定于小鼠板上。经酒精消毒后打开胸腔取出全肺,肉眼观察肺部病变,记录肺部病变的程度,用生理盐水洗净,后用干净滤纸吸掉残余盐水,称量全肺湿重,后再测量左肺湿重,将左肺放于60℃烘箱中干燥72h,取出称量干重,计算肺指数及湿干重比例。3岗藿抗感汤对流感小鼠肺组织形态学的影响末次给药后30min,将小鼠称重后摘眼球放血处死,仰位固定于小鼠板上。经酒精消毒后打开胸腔取出右肺,肉眼观察肺部病变,记录肺部病变的程度,用生理盐水洗净,后用干净滤纸吸掉残余盐水。保存于装有4%的多聚甲醛溶液5ml的EP管中,固定24小时,用于HE染色。4岗藿抗感汤对流感病毒感染小鼠血气分析的影响末次给药后30min,将小鼠轻微麻醉,进行仰卧固定(注意确保小鼠呼吸不受影响),腹部经酒精消毒后,剪开腹部皮肤和肌肉,分离腹主动脉,用采血针采取肝素抗凝血0.4ml,注射器针头抽出后立即插入小橡皮塞内,用手轻轻搓动注射器,使肝素与血液充分混合。使用全自动血气分析仪立即测定血气指标。5岗藿抗感汤对流感小鼠肺病毒载量的影响末次给药后30min,将小鼠称重后摘眼球放血处死,仰位固定于小鼠板上。经酒精消毒后打开胸腔取出左肺,肉眼观察肺部病变,记录肺部病变的程度,用生理盐水洗净,后用干净滤纸吸掉残余盐水。取小鼠左肺肺组织样本(200-300mg),测定肺组织中病毒含量。6岗藿抗感汤对流感小鼠血清细胞因子含量及肺泡灌洗液中炎症细胞计数和分类的影响末次给药后30min,取各组小鼠,眼静脉丛取血1.5mL,按CBA试剂盒方法测定血清中IL-6、IL-10、IL-12、IFN-γ、TNF-α、MCP-1含量。取肺泡灌洗液测定炎症细胞计数和分类。研究结果:实验一流感病毒FM1株感染小鼠LD5。的测定各浓度病毒观察15天结束后,统计各组小鼠死亡数目,按Reed和Muench氏法计算LD50,结果LD50为每0.05ml病毒液中含有10-2.57浓度的病毒。实验二岗藿抗感汤对流感病毒感染小鼠死亡保护研究正常组和利巴韦林组小鼠无死亡,模型组有9只小鼠死亡,死亡天数在4-7天之间,第6天达到死亡高峰。岗藿抗感汤高剂量组有6只小鼠存活,平均存活天数与模型组比较有显着统计学差异(P<0.05),岗藿抗感汤低剂量组有4只小鼠存活,平均存活天数与模型组比较无统计学差异,但有延长存活时间的趋势。实验三岗藿抗感汤对流感病毒感染小鼠的药效作用机制1岗藿抗感汤对流感小鼠肺指数、湿干重比例的影响病毒感染小鼠后,小鼠肺指数和肺湿干重比明显升高。和模型组比较,利巴韦林组肺指数和肺湿干重比明显降低(P<0.01)。岗藿抗感汤高剂量组肺指数明显降低(P<0.01);岗藿抗感汤低剂量组肺指数降低,但无显着性差异。岗藿抗感汤高、低剂量组与正常组和模型组相比,能降低病毒感染小鼠肺湿干重比(P<0.01),表明岗藿抗感汤具有抑制炎性介质的生成和渗出的作用。2岗藿抗感汤对流感小鼠肺组织形态学的影响正常组肺细胞完整,肺细胞间隔小,无炎性介质渗出,淋巴细胞少。模型组肺泡壁明显增厚,炎性介质生出严重,肺泡间隔明显增厚,肺泡毛细血管扩张,淋巴细胞明显增多,并形成实变。利巴韦林组肺泡明显,细胞相对完整,炎性介质渗出少,肺细胞间隔增厚不明显。和模型组相比岗藿抗感汤高、低组小鼠肺组织的形态结构基本恢复,炎性介质渗出少,肺细胞间隔增厚少。3岗藿抗感汤对流感小鼠血气分析的影响血气分析表明,病毒感染后小鼠PCO2明显升高,PO2、SpO2明显降低。利巴韦林组小鼠PCO2降低,PO2、SO2升高,与正常组及模型组相比有显着性变化(P<0.01或P<0.05)。岗藿抗感汤高剂量组能明显降低病毒感染后小鼠的PCO2、升高PO2、SpO2,,与正常组、模型组比较有显着差异(P<0.01或P<0.05),而低剂量组也可降低PCO2、升高PO2、SpO2,但没有显着性差异,效果弱于高剂量组。4岗藿抗感汤对流感小鼠肺病毒载量的影响病毒感染小鼠后,肺组织中病毒含量显着升高。和模型组比较,利巴韦林组小鼠病毒载量明显降低(P<0.01);岗藿抗感汤高剂量组能明显降低病毒感染小鼠肺组织中的病毒含量(P<0.01);岗藿抗感汤低剂量组能降低病毒感染小鼠肺组织中的病毒含量,但无统计学差异。提示岗藿抗感汤能抑制病毒在小鼠肺中的扩增,有抑制病毒复制的功效,且岗藿抗感汤高剂量组效果优于低剂量组。5岗藿抗感汤对流感小鼠血清细胞因子含量及肺泡灌洗液中炎症细胞计数和分类的影响利巴韦林组均能降低促炎性因子的含量及提高抗炎性因子的含量,可降低白细胞总数、淋巴细胞百分数、中性粒细胞百分比及提高巨噬细胞百分比,且与正常组、模型组对比均有显着性变化,P<0.01或P<0.05。岗藿抗感汤高、低剂量组均能降低小鼠血清中IL-6、TNF-α、MCP-1等促炎因子的水平,升高IL-10、IFN-Y在血清中的浓度,而IL-12水平改变不大,和正常组相比差异显着,P<0.01或P<0.05;与模型组相比,除岗蕾抗感汤低剂量组降低IL-6和岗藿抗感汤高、低剂量组降低IL-12没有显着性变化外,岗蕾抗感汤高、低剂量组对各细胞因子的影响均有显着性变化,P<0.01或P<0.05。岗藿抗感汤高剂量组能明显降低病毒感染小鼠肺泡灌洗液中白细胞总数、淋巴细胞百分数、中性粒细胞百分比(P<0.01或P<0.05),明显升高病毒感染小鼠肺泡灌洗液中巨噬细胞百分比(P<0.05);岗藿抗感汤低剂量组能明显降低病毒感染小鼠肺泡灌洗液中白细胞总数和中性粒细胞百分比(P<0.01),也可降低淋巴细胞百分数和提高巨噬细胞百分比,但无统计学意义。研究结论:1经岗藿抗感汤干预之后,岗藿抗感汤高、低剂量组能明显改善病毒感染小鼠的生存状态,提高小鼠生活质量,延长小鼠存活时间,岗藿抗感汤高剂量组效果优于低剂量组。2岗藿抗感汤可抑制炎性介质的生成和渗出,降低病毒感染小鼠肺指数和湿干重比,减轻肺水肿,改善小鼠的呼吸功能,减轻病毒对肺组织的病理损伤,岗藿抗感汤高剂量组效果优于低剂量组。3岗藿抗感汤可直接抑制H1N1流感病毒的复制,降低病毒感染小鼠的病毒载量,高剂量岗藿抗感汤效果优于低剂量组。4岗藿抗感汤通过调节体内抗炎、促炎因子水平,促进IL-10、IFN-γ等抗炎细胞因子的分泌;抑制IL-6、IL-10、TNF-α、MCP-1等促炎因子的合成和分泌来调节病毒感染小鼠免疫损伤机制;同时可影响白细胞的分类和比例,抑制流感病毒引致的小鼠肺部炎性介质的生成和渗出,减轻病毒引致的小鼠肺部炎症而实现其体内抗病毒作用。岗藿抗感汤高剂量组效果优于低剂量组。更多还原

【Abstract】 Objective:With a view To further investigate the effectiveness and mechanism of Chinese medicine for influenza,this study established infected model mice of H1N1 influenza virus FM1 strain,using Ganghuo anti-influenza Soup which is characteristic of the south of the five ridges,to observe the effects of Ganghuo anti-influenza Soup on viral infected mice during the experimental study,the variation of the lung index,death-protection、virus replication ratio in lung tissue,arterial blood gas analysis,viral load、cytokine levels in serum and inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF)from the aspect of cytology,molecular biology and pathology.The study has explored the mechanism of Ganghuo anti-influenza Soup on anti-influenza in order to provide effective and reliable theoretical foundation for traditional Chinese treatment for influenza.Methods:Experiment 1 Determination of LD50 in mice infected with influenza virus FM1 strainThere were ninety-one KM mice,weighing 13.0-15.Og,half male and half female.The KM mice were randomly divided into 7 groups,9 mice in each group.Chose 7 virus titer of influenza virus liquid and get mice in each group infected with one kind of virus titer of influenza virus through nasal infection with 4 drops about 0.05ml under ether mild anesthesia.Death and survival was observed indicators except which died within 24h.Daily mortality of mice were observed and recorded until 15 days after infection so far.Calculate the median lethal dose of the virus by using the Reed-Muench method after the data Processing.Experiment 2 Research on death of protection of mice infected with influenza virusThere were fifty KM mice,weighing 13.0-15.Og,half male and half female.KM mice were randomly divided into five groups on average,namely normal group,Ribavirin group,Ganghuo anti-influenza Soup(large and small)dose group,10 mice per group.Except the normal group,each group was under ether mild anesthesia infected with influenza virus with 101d50 of flu virus droplets with nasal infection,while normal group were with the same amount of saline nose drops.Since infection on the day of the continuous observation for 15 days,mice daily observation records of deaths,and calculate the mortality and death of protection.Four hours after infection each group began to be administrated separately and the dosage of each group were 0.2mL/10g.Continuous observation had been made for general condition,activity on mice,mental condition,wheezing,coughing,shiny fur,weight changes of each mice and deaths were observe and recorded,and calculation the mortality and death of protection.Experiment 3 Mechanism of pharmacodynamic actions of Ganghuo anti-influenza Soup against mice infected with influenza virus1 There were thirty KM mice,weighing 13.0-15.0g,half male and half female.KM mice were randomly divided into five groups on average,Normal group,Ribavirin group,Ganghuo anti-influenza Soup(large and small dose)group,and there were six mice in each group.Except the normal group,each group was under ether mild anesthesia in mice infected with influenza virus with 10 1d50 of flu virus droplets nasal infection,normal group was with the same amount of saline nasal drops.Four hours after infection each group began to be administered separately and the dosage of each group were 0.2mL/10g.Administered for 5 consecutive days and begun to experiment.2 Determination of lung index and lung wet-dry weight ratio of mice infected with with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and were killed after weighing,supine position fixed in mice board.Opened the chest and removed the whole lung after alcohol disinfection.visual inspection and recording of lung lesions.Weighed wet weight after abstersion with physiological saline and siphoning off residual brine with a clean filter paper.And Weighed dry weight after dried in an oven at 60℃for 72h.At last calculate lung index,inhibition rate of lung index and wet to dry weight ratio.3 The changes of lung morphology of mice infected with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and were killed after weighing,supine position fixed in mice board.Opened the chest and removed the right lung after alcohol disinfection.Visual inspection and recording of lung lesions.Abstersion the lung with physiological saline and siphoning off residual brine with a clean filter paper.Fixed the lung in 4%polyformaldehyde solution with 5ml of EP tube for 24 hours and then used it for HE staining.4 The Effect of Ganghuo anti-influenza Soup on blood gas analysis of mice infected with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and were killed after weighing,supine position fixed in mice board.Cut the skin and muscle of abdomenby by using scissor after alcohol disinfection,then the abdominal aorta was separated.0.4ml heparin anti-coagulating blood were taken by blood taking needle.Syringe needle was inserted into the small rubber stopper immediately after extraction and gently rubbing syringe to make heparin thoroughly mixed with the blood.Determination of blood gas immediately by automatic blood gasanalyzer.5 Effect on the viral load of mice infected with influenza virus.Half an hour after the last administration,the mice were drawn blood from eyeball and killed after weighing,with supine position fixed in mice board.Opened the chest and removed the left lung after alcohol disinfection.Visual inspection and recording of lung lesions and absterge the lung with physiological saline and siphoning off residual brine with a clean filter paper.Tissue samples of left lung of mice were taken to measure the levels of virus.6 Effect on cytokine levels in serum and inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF)of mice infected with influenza virus.Half an hour after the last administration,blood samples were collected from ophthalmic venous plexus.Determination of serum IL-6,IL-10,IL-12,IFN-γ,TNF-α,MCP-1 content were made with CBA kit methods.Observation was taken on inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF).Results:Experiment 1 Determination of LD50 in mice infected with influenza virus FM1 strainCounted the number of deaths in each group of mice after observed 15 days and Calculate the median lethal dose of the virus by using the Reed-Muench method.The result was each 0.05ml virus solution containing 10-2.57 concentration of virus.Experiment 2 Research on death of protection of mice infected with influenza virusThere were no death on Normal group and Ribavirin group.However there were nine mice died in model group,that begun at the fourth day and peak at the sixth day.Ganghuo anti-influenza Soup large dose group had six survived mice,the average living days compared to model group,the chi-square test,P<0.05,has the obvious death protection.Ganghuo anti-influenza Soup small dose group had survived four mice,but the average living days wasn’t significant difference compared with model group,it had tendency to prolong the survival time.Experiment 3 Mechanism of pharmacodynamic actions of Ganghuo anti-influenza Soup against mice infected with influenza virus1 Determination of lung index and lung wet-dry weight ratio of mice infected with influenza virus.Lung index and lung wet-dry weight ratio of mice markedly increased after infected with Influenza A virus.Lung index and lung wet-dry weight ratio of Ribavirin group reduced obviously compared with model group(P<0.01).Ganghuo anti-influenza Soup large dose group could reduce lung index obviously.Ganghuo anti-influenza Soup small dose group also had reduce lung index,but it wasn’t significant difference compared with model group.Lung wet-dry weight ratio of Ganghuo anti-influenza Soup(large and small dose)group reduced obviously compared with normal and model group(P<0.01).It showed that Ganghuo anti-influenza Soup could inhibited the production of inflammatory mediators and oozing.2 The changes of lung morphology of mice infected with influenza virus.The lung cells and alveolar septum of normal group was in good condition.Inflammatory cell infiltration could not be found in the lung cells and there was few lymphocytes in it.In the model group,thickening of alveolar walls and alveolar septum,the lung tissue of mice with inflammatory cell infiltration,dilation of alveolar capillaries,a large number of lymphocyte can be seen and formation of lung consolidation.Ribavirin group restored alveolar walls and alveolar septum and reduced inflammatory cell infiltration obviously.Ganghuo anti-influenza Soup(large and small dose)group recovered the morphological structure of lung tissue basically and had reduced inflammatory cells compared with model group.3 The effect of Ganghuo anti-influenza Soup on blood gas analysis of mice infected with influenza virus.The blood gas analyses showed that virus infected can elevate carbon dioxide partial pressure,reduce partial pressure of oxygen and sulfur dioxidearterial.Carbon dioxide partial pressure were reduced,partial pressure of oxygen and sulfur dioxidearterial were elevated in the mice of the Ribavirin group significantly compared with the normal and model group(P<0.01,F<0.05).Ganghuo anti-influenza Soup large dose group could elevate partial pressure of oxygen and sulfur dioxidearterial,reduce carbon dioxide partial pressure,showing a remarkable change compared with the normal and model group(P<0.01,P<0.05)while the small dose group could not.4 Effect on the viral load of mice infected with influenza virus.Levels of virus of lung tissue increase significantly after infected with virus.Viral load of Ribavirin group decreased significantly compared with model group(P<0.01);Ganghuo anti-influenza Soup large dose group also could decreased the viral load significantly(P<0.01);Viral load of Ganghuo anti-influenza Soup small dose decreased but not significantly,there was no statistical difference compared with the model group.The result suggested that Ganghuo anti-influenza Soup(large and small dose)groupcan inhibit influenza virus replication in the lung tissue,and the large dose was superior to the small dose.5 Effect on cytokine levels in serum and inflammatory cell count and classification in bronchoalveolar lavage fluid(BALF)of mice infected with influenza virus.Ribavirin could lower pro-inflammatory cytokine levels,improve anti-inflammatory cytokine,reduced leukocyte count,lymphocyte percentage,neutrophils percentage and increased the percentage of macrophages,showing a remarkable change compared with the normal and model group(P<0.01,P<0.05).Ganghuo anti-influenza Soup(large and small dose)group could lower IL-6,TNF-α,MCP-1 and other pro-inflammatory cytokine levels,improve anti-inflgammatory cytokine IL-10,IFN-γ levels,showing a remarkable change compared with the normal group(P<0.01,P<0.05),except IL-12 levels.Compared with the model group,every kind of cytokines had significant changes except IL-6 after using Ganghuo anti-influenza Soup small dose and IL-12 after using Ganghuo anti-influenza Soup(large and small dose),with a statistical significance(P<0.01,P<0.05).Ganghuo anti-influenza Soup large dose group could reduce bronchoalveolar lavage fluid leukocyte count,lymphocyte percentage,neutrophils percentage of infected with virus strain mice significantly(P<0.01,P<0.05),increased the percentage of macrophages(P<0.05);Ganghuo anti-influenza Soup small dose group also could reduce bronchoalveolar lavage fluid leukocyte count and neutrophils percentage of infected with virus strain mice significantly(P<0.01),reduced lymphocyte percentage and increased the percentage of macrophages.However there were no statistical difference.Conclusion:1 Ganghuo anti-influenza Soup could improve living condition and quality of Life of influenza virus infected mice,and prolong the survival time of influenza virus infected mice.Ganghuo anti-influenza Soup large dose group was superior to the small dose group.2 Ganghuo anti-influenza Soup could inhibite the production of inflammatory mediators and oozing,reduce lung index and lung wet-dry weight ratio,attenuate lung edema,restore respiration function,relieve pathological injury of lung tissue of influenza virus infected mice.Ganghuo anti-influenza Soup large dose group was superior to the small dose one.3 Ganghuo anti-influenza Soup could inhibit influenza virus replication in the lung tissue directly,decrease viral load significantly of influenza virus infected mice.Ganghuo anti-influenza Soup large dose group was superior to the small dose one.4 Ganghuo anti-influenza Soup could regulate the proinflammatory cytokines and anti-inflammatory cytokine of influenza virus infected mice.By promoting the secretion of IL-10,IFN-γ and other anti-inflammatory cytokines and inhibiting the synthesis and secretion of IL-6,IL-10,TNF-a,MCP-1 and other pro-inflammatory cytokines,Ganghuo anti-influenza Soup could modulate the immune mechanisms of injury in mice infected with influenza virus.At the same time,Ganghuo anti-influenza Soup may regulate leukocyte classification and counting,scale inhibition of influenza virus production and had exudation caused by inflammatory mediators in the lungs of mice,relieved the production of inflammatory mediators and oozing caused by infection of influenza virus as to achieve its anti-virus action.The ideal anti-viral effect concentration was high-dose.更多还原