以“肺常不足”立论双表法抗流感病毒感染TLR信号通路时效关系研究

【作者】 董丹；

【导师】 王雪峰；

【作者基本信息】 辽宁中医药大学 ， 中医儿科学， 2010， 博士

【摘要】 目的:通过幼龄鼠与成龄鼠建立流感病毒(IV)感染动物模型对照实验研究以及流感病毒感染小鼠肺组织TLR4-NF-κB P65信号通路改变情况,探讨幼龄鼠与成龄鼠对流感病毒的易感性,在动物实验角度阐述“肺常不足”立论机制;将辛凉解表经典方剂银翘散与益气固表方剂玉屏风散进行比较研究,通过双表法对流感病毒感染鸡胚的保护作用以及小鼠肺组织TLR4-NF-κB P65信号通路活化程度的影响,探讨解表法与固表法在流感病毒感染防治中的作用;对辛凉解表法代表方剂银翘散采用高效液相色谱法建立指纹图谱,以期进一步明确其抗流感病毒物质基础。材料与方法:1.采用鼻腔接种甲型流感病毒鼠肺适应株A/FM1/47诱导昆明种小鼠,将实验小鼠分为幼龄鼠和成龄鼠两组,观察幼龄鼠和成龄鼠在一般状态、存活只数、肺组织病变程度等方面的差异,计算肺指数,应用RT-PCR技术检测两组小鼠肺组织中不同时点IV-RNA的病毒载量的差异。为“肺常不足”理论实质提供实验验证。2.采用免疫组织化学和RT-PCR技术检测幼龄鼠和成龄鼠肺组织中TLR4和NF-κB P65的蛋白及mRNA在不同时点的表达,比较幼龄鼠和成龄鼠感染流感病毒后TLR4-NF-κB P65信号通路的改变情况,为“肺常不足”立论提供实验佐证。3.采用血凝实验方法观察不同浓度银翘散提取物、玉屏风颗粒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、玉屏风颗粒对IV感染鸡胚的预防和保护作用。4.采用免疫组织化学方法,检测银翘散提取物、玉屏风颗粒及银翘散合玉屏风颗粒给药各组小鼠肺组织不同时点TLR4、NF-κB P65蛋白表达情况。5.采用RT-PCR方法,检测银翘散提取物、玉屏风颗粒及银翘散合玉屏风颗粒给药各组小鼠肺组织不同时点TLR4、NF-κB P65mRNA表达情况。6.应用高效液相色谱法建立银翘散全方指纹图谱,同时测定银翘散及单味药五种有效成分;分析银翘散指纹峰分布特征及归属情况。结果:1.接种流感病毒后,成龄模型组基本保持较好状态,幼龄模型组一般状态最差,正常组小鼠一般状态最好。接毒后14天,正常组全部存活。幼龄模型组死亡最多,在第5天达到死亡高峰,与正常组及成龄模型组比较均有显著性差异(P<0.05)。成龄模型组与正常组比较无显著性差异(P>0.05)。2.肺组织外观病变程度显示: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,各时间点与正常组及成龄模型组比较均有显著性差异(P<0.05),第5天为幼龄模型组肺组织病变最明显时期;成龄模型组各时间点与正常组比较无显著性差异(P>0.05),与幼龄模型组有显著性差异(P<0.05)。3.肺指数测定结果:幼龄和成龄正常组小鼠在不同时点肺指数最低。幼龄模型组肺指数最高,与正常组及成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)。4.肺组织不同时点IV-RNA的半定量测定结果:正常组小鼠各时点肺组织中未检测到IV病毒核酸,幼龄模型组在各时点均检测到大量的病毒核酸,在第5天病毒核酸量最大,为2.1238±0.2774,幼龄模型组各时间点与成龄模型组比较有显著性差异(P﹤0.05)。成龄模型组各时点IV病毒核酸只有微量表达,与幼龄模型组比较有显著性差异(P﹤0.05),与正常组比较,无显著性差异(P﹥0.05)。5.肺组织TLR4蛋白表达情况:幼龄正常组和成龄正常组均可以检测到少量的TLR4蛋白表达,且各个时点比较,蛋白表达无显著性差异(P﹥0.05)。幼龄模型组TLR4蛋白表达最强,且随着时间推移表达逐渐增强,在第5天达到峰值;幼龄模型组与正常组和成龄模型组比较,均具有显著性差异(P﹤0.05)。成龄模型组TLR4蛋白表达高于各正常组,但低于幼龄模型组。6.肺组织NF-κB P65蛋白表达情况:幼龄正常组和成龄正常组均可以检测到少量的NF-κB P65蛋白表达,且各个时点比较,蛋白表达无显著性差异(P﹥0.05)。幼龄模型组NF-κB P65蛋白表达最强,且随着时间变化表达逐渐增强,每一时间点与前一时间点比较具有显著性差异(P﹤0.05);成龄模型组NF-κB P65蛋白表达高于各正常组,但低于幼龄模型组(与正常组和幼龄模型组比较,P﹤0.05),成龄模型组蛋白表达各个时间点无显著性差异(P﹥0.05)。7.肺组织TLR4 mRNA表达情况:幼龄正常组和成龄正常组肺组织均可以检测到少量的TLR4 mRNA表达,且各个时点比较,mRNA表达无显著性差异(P﹥0.05)。幼龄模型组TLR4 mRNA表达最强,且随着病程进展表达逐渐增强,在第5天达到峰值,TLR4mRNA含量检测强度与内参比值达到1.8351±0.0110;幼龄模型组TLR4 mRNA明显高于正常组和成龄模型组,具有显著性差异(P﹤0.05)。成龄模型组TLR4mRNA表达高于各正常组,但低于幼龄模型组。8.肺组织NF-κB P65 mRNA表达情况:幼龄正常组和成龄正常组肺组织均可以检测到少量的NF-κB P65 mRNA表达,在观察第1、3、5、7天各个时点比较,mRNA表达无显著性差异(P﹥0.05)。幼龄模型组NF-κB P65 mRNA表达最强,且随着病程进展表达逐渐增强;与正常组和成龄模型组比较,具有显著性差异(P﹤0.05)。成龄模型组NF-κB P65 mRNA表达高于各正常组,但低于幼龄模型组。9.药物体外直接抑制病毒作用:银翘散提取物、玉屏风颗粒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24h,其中药物在8小时～18小时范围内抑制病毒效果显著,18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8h时最强,其抑制病毒滴度可达16倍;玉屏风颗粒体外直接抑制IV作用在8-18h最强,抑制作用可持续达到8倍;银翘散提取物和玉屏风颗粒在不同时间点与西药利巴韦林对照组相比较未见优势。10.银翘散提取物、玉屏风颗粒对IV感染鸡胚的预防和治疗作用比较:银翘散提取物、玉屏风颗粒在400g/ml浓度时无论是预防给药还是治疗给药,血凝效价均比病毒对照组有明显降低,具有显著性差异(P﹤0.05);联合应用时,其血凝效价比病毒对照组均有明显降低,具有显著性差异(P﹤0.05);给药各组之间比较表明,两方合用组比单药组血凝效价低,但各组之间血凝效价因数并无显著性差异(P﹥0.05)。11.各组小鼠肺组织TLR4、NF-κB P65蛋白表达情况:各中药组TLR4蛋白表达均低于利巴韦林组,具有显著性差异(P﹤0.05)。在同一时间点,各中药组间比较,具有显著性差异(P﹤0.05),双表法组TLR4蛋白表达最低。在各个时点,中药组TLR4蛋白表达均高于正常组,但明显低于模型组,与模型组和正常组比较,均具有显著性差异(P﹤0.05)。各中药组间各时间点TLR4蛋白表达有显著性差异(P﹤0.05),双表法组在各时点蛋白表达最低。银翘散提取物组、双表法组在各个时点NF-κB P65蛋白表达均低于利巴韦林组,具有显著性差异(P﹤0.05)。玉屏风颗粒组在第1、3天蛋白表达低于利巴韦林组,具有显著性差异(P﹤0.05),第5、7天蛋白表达水平与利巴韦林组相当,无显著性差异(P﹥0.05)。各中药组间各时间点NF-κB P65蛋白表达有显著性差异(P﹤0.05),双表法组在各时点蛋白表达最低。12.各组小鼠肺组织TLR4、NF-κB P65mRNA表达情况:银翘散提取物组在第1、5天TLR4mRNA表达高于利巴韦林组,具有显著性差异(P﹤0.05),在第3、7天TLR4mRNA表达与利巴韦林组相当,无显著性差异(P﹥0.05)。玉屏风颗粒组在1、3天TLR4mRNA表达高于利巴韦林组,具有显著性差异(P﹤0.05),在第5、7天TLR4mRNA表达与利巴韦林组相当,无显著性差异(P﹥0.05)。双表法组在各时点TLR4mRNA表达均低于利巴韦林组,具有显著性差异(P﹤0.05)。各中药组间各时间点TLR4mRNA表达有显著性差异(P﹤0.05),双表法组在各时点mRNA表达最低。利巴韦林组及各中药组在各时间点NF-κB P65 mRNA表达低于模型组,高于正常组,具有显著性差异(P﹤0.05)。银翘散提取物组等各中药组在各时点NF-κB P65mRNA表达均低于利巴韦林组,具有显著性差异(P﹤0.05)。在各中药组中双表法组的NF-κB P65mRNA表达最低,与其他各组比较有显著性差异(P﹤0.05)。13.银翘散全方不同提取方法提取物色谱图共有26个共有指纹峰; 26个共有指纹峰都分别归属于9个单味药材。从色谱峰归属的数量上来看,牛蒡子、金银花、连翘所属峰占主要峰;从色谱峰的归属面积上来看,牛蒡子、甘草、金银花、荆芥占主要峰值。结论:1.通过小鼠一般状态观察、肺组织病理观察、肺组织IV-RNA的半定量测定验证,幼龄鼠可以成功塑造小鼠IV感染模型,成龄鼠不能成功造模,可以佐证“肺常不足”理论。2.幼龄鼠与成龄鼠IV感染后肺组织TLR4-NF-κB P65信号通路活化程度不同,幼龄鼠TLR4、NF-κB P65表达显著升高,成龄鼠轻度升高,二者差异显著;TLR4-NF-κB P65信号通路的过度活化,可能是幼龄鼠肺脏免疫病理损伤严重的机制之一,可以为“肺常不足“理论立论提供佐证。3.不同浓度银翘散提取物、玉屏风颗粒对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;银翘散提取物、玉屏风颗粒联合应用比单药对IV感染鸡胚保护作用略强,但无显著性差异,且给药先后顺序对血凝效价因数结果无显著影响。4.银翘散提取物、玉屏风颗粒对IV感染鸡胚的预防和治疗保护作用与西药利巴韦林相当,未体现明显优势。5.银翘散提取物、玉屏风颗粒及双表法组在不同时点均能不同程度阻断流感小鼠肺组织中TLR4和NF-κB P65蛋白的高表达、抑制TLR4和NF-κB P65 mRNA的高表达。6.银翘散提取物、玉屏风颗粒及双表法组在病变过程中大部分时点对TLR4和NF-κB P65高表达的抑制作用强于西药利巴韦林,双表法抑制作用最强。7.辛凉解表法、益气固表法及双表法对流感病毒感染小鼠的防治作用与抑制流感病毒感染小鼠肺组织TLR4-NF-κB P65信号通路的过度活化有关。8.高效液相色谱法同时测定银翘散全方不同提取方法提取物中五种活性成分,方法简便,重现性好。银翘散全方指纹图谱共有26个共有指纹峰,分别归属银翘散全方中九味药材;共有峰的相对保留时间及峰面积比值,可以为明确银翘散复方抗IV作用物质基础提供重要依据。更多还原

【Abstract】 ObjectiveThrough establishing young and mature mice model with mouse influenza virus (IV) infection and changes of TLR4-NF-κB P65 signaling pathway in lungs of mice infected with influenza virus to discuss the situation of young mice and mature mice against influenza virus susceptibility and Lung often inadequate mechanism under animal experiments. Compare the classic cold-pungent diaphoresis relieving exterior syndrome prescription Yinqiaosan and consolidating superficies prescription Yupingfeng, discuss the role of the two methods in the prevention of influenza virus infection through the effects of the protection of the two methods in chick embryo study infected with Influenza virus and the activation of TLR4-NF-κB P65 signaling pathway in the lung tissues of mice. And thus provide experimental basis for the Chinese medicine to protect children from influenza virus infections. Obtained the finger printings of classical cold-pungent diaphoresis prescription yinqiaosan by high efficiency liquid chromatography.Materials and Methods:1. Using influenza A virus A/FM1/47 to infect mice .The mice were divided into two groups: young mice and mature mice, observed their general status, survival, the differences of lung lesions, calculated lung index, applied RT-PCR method to detect IV-RNA viral load differences at two different time points in lung tissues of mice , which was to provide experimental verification for Lung often inadequate theory mechanism.2. Immunohistochemistry and RT-PCR was applied to detect the expressions of lung TLR4 and NF-κB P65 protein and mRNA at different time points in young mice and mature rat, comparing the changes of the TLR4-NF-κB P65 signaling pathway after young mice and mature mice infected with influenza virus, as providing experimental evidence for the“Lung often inadequate”theory mechanism.3. Used Hemagglutination test to observe the role of direct inhibition IV effects with different concentrations of Yinqiaosan extract, Yupingfeng particles at different time points in vitro; studied different concentrations of Yinqiaosan extract and Yupingfeng particles on IV infection prevention and protective effects of chick embryo.4. Used Immunohistochemical methods to detect TLR4, NF-κB P65 protein expression in lungs of mice in each group administered at different time points with Yinqiaosan extract, Yupingfeng particle and particle of both.5. Using RT-PCR method to detect TLR4, NF-κB P65mRNA expression lungs of mice in each group administered at different time points with Yinqiaosan extract, Yupingfeng particle and particle of both.6. The Yinqiaosan and nine herbs finger printings were established by the high efficiency liquid chromatography and analysis the distribution features and focuses of finger printing capyluses of Yinqiaosan. Simultaneous determination of five components and discuss the relationship of finger printing capyluses.Result:1. Inoculated with influenza virus, the mature model group basically maintained normal conditions, young model group had worse general states, and normal mice had better ones. 14 days after inoculation, the normal group all survived. Most died in young model group, and death reached the peak at the 5th day. The normal group and the mature model group showed significantly difference (P <0.05). Mature model group and normal group showed no significant difference (P> 0.05).)2. The appearance of lung lesions showed: The normal lung tissue of mice at different times showed no pathological changes in the appearance of the whole lung field; young model group showed the worst changes, at each time point the normal group and model group mature showed significant differences (P <0.05), the 5th day the young model group showed the most significant appearances of disease; mature model group at different time points compared with the normal group had no significant difference (P> 0.05), and there was a significant group difference compared with young model group(P <0.05).)3. Lung index determination results: lung indexes of young and mature normal mice groups at different time points were the lowest. Young model group were the highest and the comparisons with the normal group and the mature model group were significantly different (P <0.05), lung infection on day 5 reached the highest index. Mature model group and normal group showed no significant difference (P> 0.05), and there was a significant group difference compared with young model group(P <0.05).)4. The semi-quantitative determination results of IV-RNA in lung tissue at different time points : In normal lung tissue of mice at each time point did not detected any virus nucleic acid of IV, young model group detected at each time point a large number of viral nucleic acid, at day 5 there were the largest viral nucleic acid as 2.1238±0.2774, In the model groups at different time points in young and mature model groups, there was a significant difference (P <0.05). Mature model group at all time points the expression of viral nucleic acid showed only traces, and there was a significant group difference compared with young model group(P <0.05), no significant difference compared with the normal group.(P> 0.05).)5. TLR4 protein expression in lung tissues: the young normal group and mature normal group were able to detect small amounts of TLR4 protein expression, and compared at each time point, protein expressions had no significantly differences (P> 0.05).Young model group showed the strongest expression of TLR4 protein, and the expression increased gradually over time, peaked at day 5; young model group compared with normal group and the mature model group showing significant differences (P <0.05).TLR4 protein expressions in mature model group were higher than the normal group but lower than the young model group.6. Lung tissue NF-κB P65 protein expression: the normal group and mature young normal group were able to detect small amounts of NF-κB P65 protein expression, and compared at each time point, protein expression was not significantly different (P> 0.05).In young model group, the NF-κB P65 protein expression was the strongest, and the expression increased gradually over time, each time point compared with the previous point in time showing a significant difference (P <0.05); NF-κB P65 protein expressed more than the normal group in the mature model group but lower than the young model group (compared with young normal group and model group, P <0.05), expression of mature model group at each time point there was no significant difference (P> 0.05).7. TLR4 mRNA expression in lung tissue: the young normal group and mature normal group were able to detect small amounts of TLR4 mRNA expression, and compare each time points, mRNA expression was not significantly different (P> 0.05).Young model group showed the strongest expression of TLR4 mRNA, and expression gradually increased with the progression in the first 5 days to reach the peak, TLR4 mRNA and the internal reference intensity ratio was 1.8351±0.0110; TLR4 mRNA of young model group was significantly higher than the normal group and the mature model group, with significant difference (P <0.05).TLR4mRNA in mature model group was higher than the normal group but lower than the young model group.8. Lung tissue NF-κB P65 mRNA expression: the young normal group and mature normal group were able to detect small amounts of NF-κB P65 mRNA expression,at each day point 1,3,5 and 7, the comparisons of mRNA expressions were not significantly different (P> 0.05).In young model group, the NF-κB P65 mRNA expression was the strongest, and gradually increased with the progression of expression; compared with the normal group and the mature model group, there were no significant differences (P <0.05).In mature model group, NF-κB P65 mRNA expression was higher than the normal group but lower than the young model group.9. The effects of medicines directly inhibiting the virus in vitro: Yinqiaosan extract, Yupingfeng particles can directly inhibit the different levels of activity of influenza A virus FM1, the sustainable time lasted from 1h to 24h, the medicine showed significant effect from 8 to 18 hours after the effects wore off gradually after that; Yinqiaosan Extract showed a role of direct inhibition of viruses in 8h when the strongest inhibition titers up to 16 times; Yupingfeng particle took strongest inhibition effects in vitro of IV in 8 -18h when the strongest inhibition titers up to 8 times; Yinqiaosan extract and Yupingfeng particles at different time points compared with western medicine ribavirin showed no advantages.10. Comparison of Yinqiaosan extract and Yupingfeng particles on IV infection prevention and treatment of chick embryo : When Yinqiaosan extract and Yupingfeng particle concentrations were at 400g/ml, either prophylaxis or treatment , blood clotting virus titer significantly decreased than the control group, there were significant differences (P <0.05); When combined together, the hemagglutination titer virus control were significantly lower, with significant difference (P <0.05); Comparing the groups with one anther showed that combined group showed lower blood clotting, but the factors of HI titer between the groups had no significant difference (P> 0.05).11. The lung TLR4, NF-κB P65 protein expression in different groups: The TLR4 protein expression were lower than ribavirin group in each TCM groups, with significant difference (P <0.05).At the same time point, there were significant differences between each TCM groups (P <0.05), the group using double relieving exterior syndrome methods showed the lowest TLR4 expression. At each time point, the TLR4 protein expression in TCM groups were higher than the normal group, but significantly lower than the model group, there were significantly differences compared with model group and normal group (P <0.05).Within the TCM groups at all time points, TLR4 expression were significantly different (P <0.05), the group using double relieving exterior syndrome methods showed the lowest TLR4 expression.Yinqiaosan extraction group and the group using double relieving exterior syndrome methods at each time point the expression of NF-κB P65 protein were lower than ribavirin group, with significant difference (P <0.05).Yupingfeng particle group at day 1 and 3, protein expressed lower than ribavirin group, with significant difference (P <0.05),day 5 and 7, protein expressed similar to ribavirin, with no significant difference(P> 0.05).The TCN groups at all time points, NF-κB P65 protein expression were significantly different (P <0.05), the group using double relieving exterior syndrome methods showed the lowest expression at all time points.12. The lung TLR4, NF-κB P65mRNA expression in different groups: Yinqiaosan extract group showed higher TLR4mRNA expression than ribavirin group at day 1 and 5, with significant difference (P <0.05), similar at day 3 and 7, with no significant difference (P> 0.05).Yupingfeng particle group was higher than ribavirin group at day 1 and 3, with significant difference (P <0.05), similar at day 5 and 7, with no significant difference (P> 0.05).the group using double relieving exterior syndrome methods showed lower expression than ribavirin group, with significant difference (P <0.05).The TCM group at all time points had significant differences in expression (P <0.05), the group using double relieving exterior syndrome methods had the lowest expression at all time points.Ribavirin group and the TCM groups at all time points showed lower NF-κB P65 mRNA expression compared with model group, higher than the normal group, with significant difference (P <0.05).Yinqiaosan extract group and other TCM groups at all time points were lower than the ribavirin group, with significant difference (P <0.05).InTCM groups the group using double relieving exterior syndrome methods was the lowest compared with other groups ,with significant difference (P <0.05).13. Finger printings of different extraction artworks of Yinqiaosan have 26 fingerprint capylus, which belong to 9 single herbs.Acorrding to the quantity of chromatographic peak groups, Fructus arctii,Flos Lonicerae,Forsythia Suspense Radix Glycyrrhizae,and Spica Schizonepetae were the main herbs.Conclusion:1. With the general states observed in mice, lung pathology and the IV-RNA semi-quantitative determination in lung tissue, in young rats can be successfully shaped as the IV infection model in mice while mature rats can not, which can support the Lung often inadequate theory.2. TLR4-NF-κB P65 signaling pathway of lung tissue of young mice and mature rat after IV infection activated in varying degrees, young rat TLR4, NF-κB P65 expression was significantly increased while mature rats increased slightly, with significant difference; the over activation of TLR4-NF-κB P65 signaling pathway may be one lung immune pathological mechanism of serious injury in the young rat, which can provide evidence for the“Lung often inadequate”theory.3. Different concentrations of Yinqiaosan extracts and Yupingfeng particles have good effects on the prevention and treatment of chick embryo infected with IV. its role in the prevention and treatment decreases with lower drug concentrations; combination of Yinqiaosan extract and Yupingfeng particles has a slightly better effect than use either alone, but has no significant difference, And the delivery sequence showed no significant effects on the HA titer factor.4. Yinqiaosan extracts and Yupingfeng particles have good effects on the prevention and treatment of chick embryo infected with IV as ribavirin did, but shows no clear advantage over the former one.5. Yinqiaosan extract group, Yupingfeng particle group and the group using double relieving exterior syndrome methods can at different levels block TLR4 and NF-κB P65 protein over expression and inhibit TLR4 and NF-κB P65mRNA over expression at different time points.6. Yinqiaosan extract group, Yupingfeng particle group and the group using double relieving exterior syndrome methods showed higher effects on the inhibit of TLR4 and NF-κB P65mRNA over expression in the disease process at most of the time points than ribavirin group, and the group using double relieving exterior syndrome methods was the strongest.7. Cold-pungent diaphoresis ,consolidating superficies and double relieving superficies methods are related to the prevention and treatment of mice infected with influenza virus and the inhibition of lung TLR4-NF-κB P65 signaling pathway over-activation.8. Simultaneous determination of five components in the classic yinqiaosan was a simple and reasonably with well reproducibility. the finger printing of classic yinqiaosan complex has 26 generous fingerprint capyluses belong to the single herbs in the formula; The relative retention time and peak area ratio of generous capyluses could being as one of the quality control of formula.更多还原