【作者】 李佳曦；

【导师】 汪受传；

【作者基本信息】 南京中医药大学 ， 中医儿科学， 2012， 博士

【摘要】 小儿肺炎是儿科最常见疾病之一,严重危害着儿童的健康,目前世界卫生组织(WHO)将本病列为全球重要的三种儿科疾病之一,我国卫生部也将其列为重点防治的儿科四大疾病之一。其中病毒性肺炎占小儿肺炎的一半左右,而呼吸道合胞病毒(respiratory syncytial viral, RSV)又是病毒性肺炎中最常见的病原体,可以引起婴幼儿毛细支气管炎、支气管肺炎、间质性肺炎等疾病。RSV感染的发病机制目前尚未完全明确,且缺乏安全有效的疫苗及治疗手段,而中医药在治疗病毒感染性疾病方面有着独到的优势。金欣口服液是临床治疗小儿病毒性肺炎痰热闭肺证的常用有效制剂,具有开肺化痰解毒的功效,且前期大量实验研究已证实其具有一定的抗RSV活性及免疫调节作用。白藜芦醇是金欣口服液组成药物虎杖的主要有效成分之一,大量文献报道证实其具有一定的抗炎抗病毒活性。Toll样受体(toll-like receptors, TLRs)属于Ⅰ型跨膜受体蛋白,是重要的模式识别受体(pattern recognition receptors, PRRs)之一,可以识别不同微生物的病原相关分子模式(pathogen associated molecule patten, PAMP)。RSV是单股负链RNA病毒,在复制及转录过程中会合成大量双链RNA (dsRNA),故能被识别dsRNA的TLR3所特异性识别,通过一系列信号传导途径,引起下游细胞因子的大量分泌,引起系统性炎症反应。鉴于中医药作用的多靶点性,本实验从Toll样受体3及其介导的信号通路入手,探讨了金欣口服液及其有效组分白藜芦醇的抗RSV感染机理。目的：研究金欣口服液及白藜芦醇对RSV活化诱导的TLR3及其信号传导通路的调控作用,探讨其治疗RSV肺炎的可能免疫学机制。方法：体外实验：RSV感染体外培养的RAW264.7细胞,采用金欣口服液含药血清及白黎芦醇进行干预,24h后收集细胞,Real time RT-PCR法测定TLR3及其信号通路关键分子TRIF、 TRAF6mRNA的表达变化；激光其聚焦显微镜观察免疫荧光细胞化学染色法处理的各组细胞TLR3蛋白表达情况；ELISA法检测各组细胞培养上清中IFN-β、IL-1β、IL-6、TNF-α含量。体内实验：RSV滴鼻感染BALB/c小鼠,金欣口服液及白藜芦醇进行干预,并于首次滴鼻后24、72、144h,取各组小鼠肺组织,分别行病理组织切片评价肺部病变情况,Real time RT-PCR法检测肺组织中TLR3、TRIF及TRAF6mRNA表达情况,蛋白免疫印迹法(western blot)检测各组小鼠肺组织TLR3蛋白表达情况；取各组小鼠支气管肺泡灌洗液(BALF), ELISA法检测IFN-β、IL-1β、IL-6、TNF-α表达情况。结果：体外实验：1.RSV感染RAW264.7细胞后24h, TLR3mRNA表达量明显升高(与正常组比较,P<0.01),金欣口服液含药血清组TLR3mRNA表达量较RSV感染组显著降低(P<0.01),各浓度白藜芦醇组TLR3mRNA表达量较RSV感染组均有不同程度降低(P<0.01)；RSV感染RAW264.7细胞后24h, TRIF、TRAF6mRNA表达量较正常组均略有升高,但差异无统计学意义(P>0.05),金欣口服液含药血清及各浓度白藜芦醇组TRIF、TRAF6mRNA表达量与RSV感染组比较无显著性差异(P>0.05)。2.RSV感染RAW264.7细胞后24h,TLR3蛋白表达量较正常组明显升高(P<0.01)；金欣口服液含药血清组TLR3蛋白表达量较RSV感染组明显降低(P<0.01)；各浓度白藜芦醇组无论是预防给药还是治疗给药TLR3蛋白表达量较RSV组均有不同程度降低(P<0.01)。3.RSV感染RAW264.7细胞后24h,细胞培养上清中IL-1p未检测出；IL-6、TNF-α表达量明显升高(与正常组比较,P<0.01),金欣口服液含药血清组IL-6(P<0.05)、TNF-α(P<0.01)表达量较RSV感染组显著降低,各浓度白藜芦醇组IL-6、TNF-α表达量较RSV感染组均不同程度降低；IFN-表达量有所升高(与正常组比较,P<0.01),金欣口服液含药血清及各浓度白黎芦醇组IFN-β表达量较RSV组比较均不同程度增高。体内试验：1.RSV感染BALB/c小鼠后肺部病理改变主要表现为肺间质炎,肺泡壁血管扩张充血,水肿增厚,肺泡壁及间质有炎细胞浸润；肺泡腔基本清晰,无明显渗出物；支气管上皮细胞无明显变性、坏死,管腔内无明显炎性渗出物。肺部病变程度在24h最轻,随着感染时间的推移,病变程度逐渐加重。金欣口服液及白黎芦醇组小鼠肺内炎症程度均有不同程度减轻,病理评分结果显示治疗组各时间店与模型比较均有统计学显著并性差异。2.RSV感染BALB/c小鼠后24h,肺组织中TLR3mRNA表达量显著增高(与正常组比较,P<0.01),随着感染时间的延长,TLR3mRNA的农达量逐渐下降。RSV感染后24h,金欣口服液和白黎芦醇组TLR3mRNA表达量较RSV感染组不同程度降低(P<0.01),但随着感染时间的延长,金欣口服液等效剂量组TLR3mRNA表达量较RSV感染组有所增高,而金欣高剂量组和白藜芦醇组TLR3mRNA表达量较RSV感染组无差异(P>0.05)。3.RSV感染BALB/c小鼠后24、72、144h,肺组织中TRIF、TRAF6mRNA表达量与正常组比较差异无统计学意义(P>0.05),金欣口服液和白藜芦醇组小鼠肺组织TRIF、 TRAF6mRNA表达量与RSV感染组织比较差异无统计学意义(P>0.05)。4.RSV感染BALB/c小鼠后24h,肺组织内TLR3蛋白表达量较正常组高(P<0.01),72h表达达到高峰,后逐渐下降。金欣口服液及白藜芦醇组TLR3表达量于24h和72h均较RSV感染组不同程度降低(P<0.01),感染144h时金欣口服液等效剂量及白藜芦醇组TLR3表达量较RSV组差异无统计学意义(P>0.05)。5.RSV感染BALB/c小鼠后24h,BALF中IL-1β、IL-6β、TNF-α表达量均明显升高(与正常组比较,P<0.01),随着感染时间的延长,BALF中IL-1β、IL-6、TNF-α表达量均逐渐下降；RSV感染后24h,金欣口服液和白藜芦醇组小鼠BALF中IL-1β、IL-6βTNF-α表达量均较感染组显著下降,随着感染时间的延长,金欣等效剂量组IL-1β、IL-6、TNF-α表达较感染组有所上升,而金欣高剂量组和白藜芦醇组无上升趋势。RSV感染BALB/c小鼠后24h,BALF中IFN-β表达量与正常组比较无显著性差异(P>0.05),随着感染时间的延长,RSV组IFN-β表达量逐渐上升。RSV感染后24h,金欣口服液组和白藜芦醇组IFN-β表达量较RSV组显著上升(P<0.01),随着感染时间的延长各给药组IFN-β表达量与RSV组差异无统计学意义(P>0.05)。结论：1.金欣口服液及白藜芦醇能够显著改善RSV感染小鼠的肺部病变；2.金欣口服液及白藜芦醇能够从核酸和蛋白层面显著抑制RSV感染早期过度活化的TLR3表达；3.金欣口服液及白藜芦醇对TLR3信号通路关键分子TRIF、TRAF6mRNA表达无明显的直接调控作用；4.金欣口服液及白藜芦醇能够显著调控RSV感染初期TLR3介导的信号通路下游IL-1β、IL-6、TNF-α等炎症细胞因子的过量表达；随着感染时间的延长,当上述炎症因子表达下降或不足时,金欣口服液能适当上调其表达,具有一定双向动态调节作用,而白藜芦醇无此作用；金欣口服液和白藜芦醇能迅速诱导RSV感染初期IFN-β的表达,协同机体发挥一定的抗病毒作用；5.金欣口服液和白藜芦醇是治疗RSV感染的有效药物,其作用机制可能是通过调节RSV活化诱导的TLR3及其介导的信号通路实现的。更多还原

【Abstract】 Pneumonia, one of the most common diseases in pediatrics, is harmful to children’s health. It has been one of three important global pediatric diseases by WHO and also been one of four imprtant diseases by the Ministry of health of PRC. Viral pneumonia is accounted for about half of the children’s pneumonia, and respiratory syncytial virusis (RSV) is the most commom pathogen in viral pneumonia, which can cause infant capillary bronchitis, bronchial pneumonia, interstitial pneumonia and so on.It is not fully understood, so far, the pathogenesis about RSV infection, and there are nearly no safe and effective vaccines and treatments in the medical market. As it is know to all that Chinese medicine has its unique advantage in treating viral infections, and Jinxin oral liquid, which have the function of opening lung reducing phlegm and detoxification is an efficacy prescription in treating viral pneumonia with the syndrome of obstruction of the lung by phlegm heat. Many scientific research have also shown that it has some certain anti-RSV activity. Resveratrol, which is reported having the effect of anti-inflammatory and anti-viral, is one of the effective components of Polygonum cuspidatum in Jinxin oral liquid.Toll-like receptors (TLRs), the proteins belone to type I across membrane, are very important pattern recognition receptors (PRRs), which could identify different Pathogens associated molecule pattens expressed by infectious agents. RSV, an enveloped, negative-stranded RNA virus, can induces the synthesis of dsRNA during its replication and transcription, so TLR3can produce a marked effect during the infection by recognizing the dsRNA, and then the signal transduction pathways are activatded accompanyed by the expression of cytokine, finally cause systematic inflammatory response. In view of multiple targets of traditional Chinese medicine, this experiment are developed from the Toll like receptor3and its signal pathway, in order to discuss the antiviral mechanism of Jinxin oral liquid and its effective component resveratrol.Objective:Study on Jinxin oral liquid and resveratrol on regulating TLR3and its downstream signaling transduction pathway activated by RSV, in order to demostrate the possible immunological mechanism in treating RSV pneumonia.Methords:In vitro experiment:RAW264.7cell line was infected with RSV, and then treated with the drug serum of Jinxin oral liquid and resveratrol.24hours later, detected the level of TLR3、 TRIF、TRAF6mRNA using Realtime RT-PCR assey; observe the expression of TLR3protein using laser confocal microscopy by immunofluorescence (IF) assay; collected the supernatant and measured the level of interleukin6(IL-6)、interleukin1β (IL-1β)、tumor necrosis factor alpha (TNF-α) and interferon β (IFN-β)In vivo experiment:BALB/c mice were inoculated with RSV, and treated with Jinxin oral liquid in different dosage and resveratrol, delivery method. After24、72and144hours of the first inoculated with RSV, killed the mice and collected the lung tissue of each mouse, evaluate pulmonary lesions by Histological; detected the level of TLR3、TRIF、TRAF6mRNA using Realtime RT-PCR assey; observe the expression of TLR3protein using western blot assey; and collected the bronchoalveolar lavage fluid, measured the level of IL-6、IL-1β、TNF-α、IFN-β.Results:In vitro experiment:1. The expression of TLR3mRNA in RAW264.7was up-regulated significantly compared with the control group (P<0.01) after24h of infection with RSV, but it was typically down-regulated in Jinxin oral liquid group and resveratrol groups with different degree compared with RSV infected groups (P<0.01). The expression of TRIF and TRAF6mRNA in RAW264.7were slightly higher compared with the control group after24h of infection with RSV, but no statistical difference (P>0.05), and there were no statistical difference among Jinxin oral liquid group, resveratrol groups and RSV infected groups also with the expression of TRIF and TRAF6mRNA (P>0.05)2. The expression of TLR3protein in RAW264.7was up-regulated significantly compared with the control group (P<0.01) after24h of infection with RSV, but it was typically down-regulated in Jinxin oral liquid group and resveratrol groups in different degree compared with RSV infected groups (P<0.01)3. There was no expression of IL-1P in the supernatant after24h of infection with RSV. But the expression of IL-6and TNF-a were up-regulated significantly compared with the control group (P<0.01), but IL-6(P<0.05) and TNF-α (P<0.01) were typically down-regulated in Jinxin oral liquid group and resveratrol groups in different degree compared with RSV infected groups. The expression of IFN-β was up-regulated compared with the control group (P<0.01), and it was typically up-regulated in Jinxin oral liquid group and resveratrol groups in different degree compared with RSV infected groups.In vivo experiment:1. The pulmonary pathological changes of BALB/c mice that infected RSV were mainly pulmonary interstitial inflammation, dilatated and congested for the vascular of alveolar wall, and with inflammatory cell infiltration in interstitia; The alveolar cavity was clear, without obvious exudation; and there were no apprent degeneration and necrosis in bronchial epithelial cells, and without inflammatory exudate in the lumen. Pulmonary lesions was lighe after24h of infection with RSV, and with the extention of infection time, the lesions were gradually increased. The pulmonary inflammation degree were relieved in Jinxin oral liquid and resveratrol groups and the pathological score of treatment group showed statistically significant difference at each time point compared with the model groups.2. The expression of TLR3mRNA in the lung tissue was up-regulated significantly compared with the control group (P<0.01) after24h of infection with RSV, and it was declined gradually with the extention of infection time. The expression of TLR3mRNA was typically down-regulated in Jinxin oral liquid and resveratrol groups with different degree compared with RSV infected group (P<0.01), but it was slightly higher in Jinxin group with equivelent dosage than RSV infected groups with the extention of infection time, and the high dosage group of Jinxin oral liquid and resveratrol group had no difference with model groups on TLR3mRNA expression.3. The expression of TRIF and TRAF6mRNA in lung tissue of BALB/c mice had no statistical difference (P>0.05) compared with the control group after24h、72h and144h of infection with RSV, and there were no statistical difference among Jinxin oral liquid group, resveratrol groups and RSV infected groups also with the expression of TRIF and TRAF6mRNA (P>0.05)4. The expression of TLR3protein in the lung tissue was up-regulated compared with the control group (P<0.01) after24h of infection with RSV, and it reached to the peak at72h after infection (P<0.01), then declined gradually with the extention of infection time. The expression of TLR3protein was typically down-regulated in Jinxin oral liquid and resveratrol group at24h and72h after infection, but there was no difference at144h (P>0.05)5. The expression of IL-lβ、IL-6and TNF-α were up-regulated significantly compared with the control group (P<0.01) in BALF at24h after infection with RSV, and then declined gradually with the extention of infection time. They were typically down-regulated in Jinxin oral liquid and resveratrol group in different degree compared with RSV infected groups at24h after the infection. With the extension of infection time, the expression of IL-1β、IL-6and TNF-α in Jinxin oral liquid group with equivelent dosage were up-regulated compared with RSV group. There was no difference between control and model group on the expression of IFN-β (P>0.05) at24h after infection with RSV. But with the extention of infection time, the expression of IFN-P was up-regulated gradually in RSV group. The expression of IFN-β were up-regulated typically in Jinxin oral liquid and resveratrol group in different degree at24h after infection, but there was no difference between them with the extention of infection time (P>0.05)Conclusion:1. Jinxin oral liquid and resveratrol can typically improve the lung lesions after the infection with RSV in BALB/c mouse.2. Jinxin oral liquid and resveratrol can inhibit the activation of TLR3expression by RSV at the early stage from nucleic acid and protein levels significantly.3. Jinxin oral liquid and resveratrol have no direct effect on regulating the expression of the critical molecular TRIFn TRAF6mRNA in TLR3signaling pathways.4. Jinxin oral liquid and resveratrol can obviously control the over-expression of the inflammatory cytokines like IL-1β、IL-6and TNF-α mediated by TLR3signaling pathways at the early stage of RSV infection. Along with the extension of time, Jin Xin oral liquid can increase the expression of that inflammatory cytokines moderately, with the effect of two-way regulation, expect of resveratrol. Jinxin oral liquid and resveratrol can induce the expression of IFN beta rapidly after RSV infection, to play certain antiviral effect coordinated with the body.5. Jinxin oral liquid and resveratrol are the effective drugs to cure RSV infection, and the mechanism may be the regulation of TLR3and its signal transduction pathway activated by RSV.更多还原