金欣口服液及白藜芦醇对RSV活化诱导的TLR3及其信号通路的调控作用
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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。
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.
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。
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.
引文
[1]Ghildyal R, H.A., Jans DA. Central role of the respiratory syncytial virus matrix protein in infection [J]. FEMS Microbiol Rev.2006,30(5):692-705.
[2]Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity [J]. Nat.Immunol.2001,2(8):675-680.
[3]Akira, S. Toll-like receptors and innate immunity [J].Adv.Immunol.2001,78:1-56.
[4]Kopp E, Medzhitov R. Recognition of microbial infection by Toll-like receptors [J]. Curr Opin Immunol. 2003;15(4):396-401.
[5]Takeda K, Kaisho T, Akira S. Toll-like receptors [J]. Annu Rev Immunol,2003,21:335-376
[6]Vasselon T, Detmers PA. Toll receptors:a central element in innate immuneresponses [J]. Infect Immun. 2002,70(3):1033-1041.
[7]Sabroe I, Read RC, Whyte MK, et al. Toll-like receptors in health and disease:complex questions remain [J].J Immunol.2003,171(4):1630-1635.
[8]Janssen R, Pennings J, Hodemaekers H, et al. Host transcription profiles upon primary respiratory syncytial virus infection [J]. J Virology.2007,81(11):5958-5967.
[9]Xiao-hong Xie, Helen K. W. Law, Li-Jia Wang, et al. Lipopolysaccharide Induces IL-6 Production in Respiratory Syncytial Virus-Infected Airway Epithelial Cells Through the Toll-Like Receptor 4 Signaling Pathway [J]. Pediatric Research.2009,65(2):156-162.
[10]Martinez I, Melero JA.A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome:predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA [J] J.Gen.Virol.2002,83:1445-1455.
[11]汪受传,韩新民,任现志,徐玲,朱先康,马融,王孟清,刘光陵.小儿病毒性肺炎痰热闭肺证治疗方法研[J].南京中医药大学学报,2004,20(2)：72-75.
[12]廖辉,汪受传,徐建亚,李华丽.金欣口服液含药血清对呼吸道合胞病毒黏附,膜融合影响的实验研究[J].实用中西医结合临床,2008,8(1)：3-4.
[13]陈彩霞,汀受传.金欣口服液对呼吸道合胞病毒感染人胚肺成纤维细胞胞内钙离子的影响[J].江西中医学院学报,2009,21(2)：52-54.
[14]王萍芬,朱大年.中医儿科学[M].上海科技出版社,1997,1：43-45.
[15]赵宇红,申昆玲,刘亚谊,等.鱼腥草注射液治疗呼吸道合胞病毒感染的体外实验研究[J].首都医科大学学报,2005,26(5)：571-573.
[16]王桂亭,宋艳艳,任桂杰,等.注射用银黄体外抗呼吸道合胞病毒[J].中国新药与临床杂志,2005,24(11)：887-889.
[17]冯旰珠,周锋,黄茂,等.热毒宁抗呼吸道合胞病毒(RSV, Long株)作用体外实验研究[J].南京医科大学 学报(自然科学版),2007,27(9)：1009-1012.
[18]王毅军,奚肇庆,冯旰珠.痰热清对呼吸道合胞病毒体外抑制作用研究[J].2010,32(24)：3447-3448.
[19]张正军,肖敏.痰热清抗呼吸道合胞病毒作用体外实验研究[J].医药论坛杂志,2011,22(5)：23-26.
[20]王雪峰,吴振起,崔振泽.定喘汤及宣降清分解剂对RSV感染Hela细胞的实验研究[J].中华实验和临床病毒学杂志,2005,19(2)：168.
[21]莫红缨,赖克方,江永南,等.双黄连及其拆方抗呼吸道合胞病毒作用的药效学研究[J].中国中医丛础医学杂志,2005,11(3)：194-196.
[22]肇丽梅,张岩,韩国柱.清热合剂体外抗呼吸道合胞病毒作用[J].中国实验方剂学杂志,2006,12(1)：53-55.
[23]杨京华,刘妮,刘政,等清肺祛瘀汤体外抗呼吸道合胞病毒作用的研究[J].辽宁中医杂志,2010,37(5)：814-815
[24]李珊珊,李洪源,朴英爱.败酱草抗病毒有效部位(AP3)体外抑制呼吸道合胞病毒作用研究[J].中华流行病学杂志,2004,25(2)：150-153.
[25]张凤梅,刘璐,李鑫,等.败酱草多糖提取、纯化、鉴定及其体外抗RSV作用研究[J].中药材,2008,31(12)：1879-1881.
[26]苏妙贤,李药兰,周艳晖,等.瑶药“野葛花”的抗病毒和抑菌活性[J].暨南大学学报(医学版),2006,27(2)：204-213.
[27]李药兰,李克明,苏妙贤,等.猴耳环抗病毒有效成分研究[J].中国中药杂志,2006,31(5)：397-400.
[28]张建军,李洪源,王吉锡,等.甘草活性成分抗呼吸道合胞病毒作用[J].中国公共卫生,2007,23(6)：723-724.
[29]张丽亚,董琳,陈小芳,等.黄岑苷抗呼吸道合胞病毒感染作用的体外研究[J].浙江临床医学,2008,10(12)：1528-1530.
[30]刘钊,杨古秋,肖红.大青叶有效单体抗呼吸道合胞病毒作用的实验研究[J].时珍国医国药,2009,20(8)：1977-1979.
[31]李佳曦,汀受传,徐建亚,等.金欣口服液含药血清及其有效单体白藜芦醇对RSV的RNA聚合酶转录活性的影响[J].南京中医药大学学报,2011,27(1)：36-39.
[32]何雅慧,姜春英,柳晓琳,等.中药苦碟子抗呼吸道病毒药效观察[J].中国公共卫生,2006,22(8)：980-981.
[33]戴开金,罗奇志,候连兵.桑叶体外抗会洗道合胞病毒作用研究[J].中药材,2009,32(8)：1276-1278.
[34]李美玉.金银花体外抗呼吸道合胞病毒的作用研究[J].热带医学杂志,2010,10(4)：420422.
[35]李江权,任现志,汪受传.清肺口服液对常见呼吸道病毒作用的研究[J].辽宁中医学院学报,2002,4(2)：153-155.
[36]汪受传,王霖,陈超,等.清肺口服液含药血清对呼吸道合胞病毒抑制作用的实验研究[J].南京中医药大学学报,2008,24(1)：25-27.
[37]李江全,任现志,汪受传.清肺口服液对免疫功能影响的实验研究[JJ.中国实验方剂学杂志报,2002,4(2)：153-154.
[38]王霖,陈超,汪受传.清肺口服液对呼吸道合胞病毒感染人胚肺成纤维细胞肿瘤坏死因子-α转化生长因子-β1蛋白表达的影响[J].辽宁中医杂志,2010,37(4)：618-620.
[39]陈彩霞,汪受传.金欣口服液对呼吸道合胞病毒感染人胚肺成纤维细胞胞内钙离子的影响[J].江西中医学院学报.2009,21(2)：52-54.
[40]赵霞,汪受传,胡钰,等.金欣口服液含药血清对呼吸道合胞病毒感染细胞早期凋亡的影响[J].中华中医药杂志,2010,25(2)：225-227.
[41]廖辉,汪受传,徐建亚,等.金欣口服液阻断呼吸道合胞病毒入侵的实验研究[J].南京中医药大学学报2008,24(3)：168-170.
[42]赵霞,汪受传,张沛,等.金欣口服液对呼吸道合胞病毒感染大鼠Ⅰ型干扰素表达的影响[J].中华中医药杂志,2011,26(7)：1508-1510.
[43]夏晨,陈超,汪受传.金欣口服液对感染RSV大鼠血清IL-6、IL-8的影响[J].中医药信息,2011,8(1)：44-46.
[44]夏晨,陈超,汪受传,等.金欣口服液对感染RSV大鼠血清IL-12表达水平的影响[J].南京中医药大学学报,2011,27(3)：247-249.
[45]陈超,夏晨,徐建亚,等.金欣口服液对呼吸道合胞病毒肺炎大鼠模型TNF-α、分泌型IgA的影响[J].四川中医,2011,29(3)：28-31.
[46]Fremont L. et, al Biological effects of resveratrol [J]. Life Sci,2000,66(8):663-673.
[47]冯永红,许实波.白藜芦醇药理作用研究进展[J].国外医药·植物药分册,1996,11(4)：155-157.
[48]Chen G-H, Liu W, Li J-X, et al. Novel 1,2-diphenylethene derivatives for treatment of immune diseases.2002:28.
[49]郝捍东,何立人,徐清.白藜芦醇抗柯萨奇病毒感染性小鼠心肌炎的实验研究[J].上海医药,2003,24(8)：373-376.
[50]Birrell M A, McCluskie K, Wong S, et al. Resveratrol, an extract of red wine, inhibits lipopolysaccharide induced airway neutrophilia and inflammatory mediators through an NF-KB-independent mechanism [J]. FASEB,2005,19(7):840-841.
[51]Youn H S, Lee J Y, Fitzgerald K A, et al. Specific inhibition of myD88-independent signaling pathways of TLR3 and TLR4 by resveratrol:Molecular Targets Are TBKI and RIP1 in TR1F Complex [J]. Immun, 2005,175(5):3339-3346.
[52]朴花子,张成镐,朴日龙.白藜芦醇对脂多糖诱导一氧化氮和肿瘤坏死因子生成的影响[J].中药药理与临床,2006,22(5)：12-14.
[53]董玉,杨爱雄,戚好文,等.白藜芦醇对小鼠肺泡巨噬细胞释放IL-6、TNF-a的影响[J].陕西医学杂志,2007,36(9)：1131-1133.
[54]Mitsuhiro Iyori, Hideo Kataoka, Haque Mohammad Shamsul, et al. Resveratrol Modulates Phagocytosis of Bacteria through and NF-KB-Dependent Gene Program [J]. Antimicrobial agents and Chemotherapy, 2008,52(1):121-127.
[55]张中,李树平,卫涛涛.白藜芦醇对脂多糖诱导小鼠腹腔巨噬细胞炎性因子生成的调控[J].卫生研究,2009,38(4)：465-467.
[56]臧娜,谢晓虹,邓昱,等.白藜芦醇对呼吸道合胞病毒感染小鼠肺部病毒滴度及其气道炎症的影响[J].第三军医大学学报,2011,33(18)：1924-1927.
[57]关文达,杨子峰,刘妮,等.白藜芦醇对多种呼吸道病毒作用体外实验研究[J].中药材,2008,31(9)：1388-1390.
[58]周崇俊,冯劲立,张俊丽,等.白藜芦醇对Friend鼠白血病病毒抑制作用及其机制研究[J].新中医,2008,40(9)：98-100.
[59]高璐璐,陈清,李建栋,等.白藜芦醇体外抗肠道病毒71型的作用[J].南方医科大学学报,2011,31(4)：665-668.
[60]Jin H, Zhou H.Cheng X, et al. Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in, vivo [J]. Virology,2000,273(1):210-218.
[61]Takeda K, Kaisho T, Akira S, Toll-like receptors [J]. Annu Rev Immunol,2003,21:335-376
[62]Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity [J]. Nat Immunol,2001,2:675-680.
[63]Akira S. Toll-like receptors and innate immunity [J]. Adv Immunol,2001,78:1256.
[64]云云,黄升海.TLR在呼吸道合胞病毒感染中的作用研究进展[J].国际生物制品学杂志,2006,29(5)：196-199.
[65]Kawai T, Akira S. TLR signaling [J]. Cell Death Differ,2006,13(5):816-825.
[66]Muzio M, Mantovani A. Toll-like receptors (TLRs) signalling and expression pattern [J].J Endotoxin Res,2001,7(4):297-300.
[67]Kurt-Jones EA, Popova L, Kwinn L, et al. Pattern recognition receptors TLR4 and CD 14 mediate response to respiratory syncytial virus [J]. Nat Immunol,2000,1:398-401.
[68]Haynes LM. Moore DD. Kurt-Jones EA, et al. Involvement of Toll-like receptor 4 in innate immunity to respiratory syncytial virus [J]. J Virol,2001,75:10730-10737.
[69]Haeberle HA, Takizawa R, Casola A, et al. Respiratory syncytial virus-induced activation of nuclear factor-kappa B in the lung involves alveolar macrophages and Toll-like receptor 4-dependent pathways [J]. J Infect Dis,2002,186:1199-1206.
[70]Ehl S, Bischoff R, Ostler T, et al. The role of Toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus [J]. Eur J Immunol,2004,34:1146-1153.
[71]Monick M M, Yarovinsky T O, Powers L S, et al. Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin [J]. J Biol Chem,2003,278:53035-53044.
[72]Mandelberg A. Tal G. Naugolny L, etal. Lipopolysaccharide hyporesponsiveness as a risk factor for intensive care unit hospitalization in infants with respiratory syncitial virus bronchiolitis [J]. Clin Explmmunol,2006,144(1):48-52.
[73]谢晓红.呼吸道合胞病毒感染后气道上皮细胞株Toll样受体表达及其功能研究[B].2009：22-71.
[74]Rock FL, Hardiman G, Timans JC, et al. Proc Natl Acad Sci USA,1998,95(2):588-593.
[75]Groskreutz DJ, Monick MM, Powers LS, et al. Respiratory syncytial virus induces TLR3 protein and protein kinase R. leading to increased double-stranded RNA responsiveness in airway epithelial cells [J]. J Immunol,2006,176:1733-1740.
[76]黄升海.Toll样受体3介导呼吸道合胞病毒感染所致免疫反应的机制及褪黑素的作用[B].2007：8-11
[77]云云,黄升海.Toll样受体3介导呼吸道合胞病毒感染人肺上皮细胞的炎性反应及其信号转导通路[J].中国组织工程研究与临床康复.2007,11(21)：4119-4122
[78]Diebold SS, Kaisho T, Hemmi H, et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA [J]. Science.2004,303:1529-1531.
[79]Hornung V, Schlender J, Guenthner Biller M, et al. Replication dependent potent IFN-ainduction in human plasmacytoid dendritic cells by a single-stranded RNA virus [J]. J Immunol,2004,173:5935-5943.
[80]Schlender J, Hornung V, Finke S, et al. Inhibition of Toll-like receptor 7 and 9 mediated alpha/beta interferon production inhuman plasmacytoid dendritic cells by respiratory syncytial virus and measles virus [J]. J Virol,2005,79:5507-5515
[81]Ulevitch RJ. Therapeutics targeting the innate immune system [J]. Nat Rev Immunol,2004,4:512-520.
[82]Robert C, Franklin BS, et al. Role of TLRs/MyD88 in host resistance and pathogenesis during protozoan infection:lessons grommalaria [J].Semin Immunopathol,2008,30(1):41-51.
[83]揭志军,郭雪君,徐卫国,等.呼吸道合胞病毒对小鼠骨髓细胞Ⅰ型干扰素信号通路的抑制作[J].上海交通大学学报,2008,28(10)：1262-1265.
[84]Matthew R, Murawski, Glennice N. et al. Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2 [J]. Journal of Virology,2009,83(3):1492-1500.
[85]Kreso Bendelja, Valerija Vojvoda, Neda Aberle, et al Decreased Toll-like receptor 8 expression and lower TNF-alpha synthesis in infants with acute RSV infection [J]. Respiratory Research.2010,11:143.
[86]刘菊,杨春,唐安芬,等.呼吸道合胞病毒Long株的培养及应用[J].微生物学杂志,2004,24(5)：47-49.
[87]陈奇.中药药理研究方法学[M].人民卫生出版社,2006：33.
[88]Matsushima H, Yamada N, Matsue H, et al. TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. Journal of Immunology,2004,173(1): 531-541.
[89]赵保胜,李兰芳,马悦颖,等.参苏饮含药血清对小鼠巨噬细胞Toll样受体及其下游信号转导通路的影响[J].中国中药杂志,2007,32(4)：327-332.
[90]Winer J, Jung C K, Shackel I, et al Development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro [J]. Anal Biochem,1999,270(1):41.
[91]Schmittgen T D, Zakrajsek B A, Mills A G, et al. Quantitative reverse transcription polymerase chain reaction to study mRNA decay:comparison of endpoint and real time methods [J]. Anal Biochem,2000,285(2):194.
[92]Singh BP, Chauhan RS, Singhal LK. Toll-like receptors and their role in innate immunity [J]. Current Science,2003,85(8):1156-1164
[93]Bowie AG, Haga IR. The role of Toll-like receptors in the host response to viruses [J]. Mol Immunol,2005, 42(8):859-867
[94]徐文飞,刘恩梅.中性粒细胞中Toll样受体的表达与功能[J].现代免疫学,2007,27：426-429.
[95]Hasan S. Jafri, Susana Chavez-Bueno, Asuncion Mejias, et al. Respiratory Syncytial Virus Induces Pneumonia, Cytokine Response, Airway Obstruction, and Chronic Inflammatory Infiltrates Associated with Long-Term Airway Hyperresponsiveness in Mice [J]. The Journal of infectious diseases,2004, 189(10):1856-65.
[96]Shah PS. Current perspectives on the prevention and management of chronic lung disease in preterm infants [J]. Pediatric Drugs,2003,5(7):463-480.
[97]金奇.医学分子病毒学[M].北京：科学出版社,2001：475
[98]Plata A G, S Baron, Poas J S, et al. Activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental Infections [J]. J Virol,2005,79(16):10190-10199.
[99]YoneyamaM, SuharaW, FukuharaY,etal. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300 [J]. EMBO J,1998,17(4): 1087-1095.
[100]刘清泉.肿瘤坏死因子变化水平在小儿呼吸道合胞病毒感染中的意义[J].中医院感染学杂志,2002,9(12)：669-670.
[101]Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination:the control of NF-κB activity [J]. Annu Rev Immunol,2000,18(5):621-663.
[102]骆亚丽、季伟,季正华,等.人偏肺病毒与呼吸道合胞病毒感染患儿外周血细胞因子变化及意义[J].江苏医药,2011,2(37)：174-177.
[103]Ware LB, Matthay MA. Medical progress:The acute respiratory distresssyndrome [J]. New England Journal of Medicine,2000,342:1334-1349.
[104]Kishinoto T. The biology of interleukin-6 [J]. Blood,1989,74(1):1.
[2]Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity [J]. Nat.Immunol.2001,2(8):675-680.
[3]Akira, S. Toll-like receptors and innate immunity [J].Adv.Immunol.2001,78:1-56.
[4]Kopp E, Medzhitov R. Recognition of microbial infection by Toll-like receptors [J]. Curr Opin Immunol. 2003;15(4):396-401.
[5]Takeda K, Kaisho T, Akira S. Toll-like receptors [J]. Annu Rev Immunol,2003,21:335-376
[6]Vasselon T, Detmers PA. Toll receptors:a central element in innate immuneresponses [J]. Infect Immun. 2002,70(3):1033-1041.
[7]Sabroe I, Read RC, Whyte MK, et al. Toll-like receptors in health and disease:complex questions remain [J].J Immunol.2003,171(4):1630-1635.
[8]Janssen R, Pennings J, Hodemaekers H, et al. Host transcription profiles upon primary respiratory syncytial virus infection [J]. J Virology.2007,81(11):5958-5967.
[9]Xiao-hong Xie, Helen K. W. Law, Li-Jia Wang, et al. Lipopolysaccharide Induces IL-6 Production in Respiratory Syncytial Virus-Infected Airway Epithelial Cells Through the Toll-Like Receptor 4 Signaling Pathway [J]. Pediatric Research.2009,65(2):156-162.
[10]Martinez I, Melero JA.A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome:predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA [J] J.Gen.Virol.2002,83:1445-1455.
[11]汪受传,韩新民,任现志,徐玲,朱先康,马融,王孟清,刘光陵.小儿病毒性肺炎痰热闭肺证治疗方法研[J].南京中医药大学学报,2004,20(2)：72-75.
[12]廖辉,汪受传,徐建亚,李华丽.金欣口服液含药血清对呼吸道合胞病毒黏附,膜融合影响的实验研究[J].实用中西医结合临床,2008,8(1)：3-4.
[13]陈彩霞,汀受传.金欣口服液对呼吸道合胞病毒感染人胚肺成纤维细胞胞内钙离子的影响[J].江西中医学院学报,2009,21(2)：52-54.
[14]王萍芬,朱大年.中医儿科学[M].上海科技出版社,1997,1：43-45.
[15]赵宇红,申昆玲,刘亚谊,等.鱼腥草注射液治疗呼吸道合胞病毒感染的体外实验研究[J].首都医科大学学报,2005,26(5)：571-573.
[16]王桂亭,宋艳艳,任桂杰,等.注射用银黄体外抗呼吸道合胞病毒[J].中国新药与临床杂志,2005,24(11)：887-889.
[17]冯旰珠,周锋,黄茂,等.热毒宁抗呼吸道合胞病毒(RSV, Long株)作用体外实验研究[J].南京医科大学 学报(自然科学版),2007,27(9)：1009-1012.
[18]王毅军,奚肇庆,冯旰珠.痰热清对呼吸道合胞病毒体外抑制作用研究[J].2010,32(24)：3447-3448.
[19]张正军,肖敏.痰热清抗呼吸道合胞病毒作用体外实验研究[J].医药论坛杂志,2011,22(5)：23-26.
[20]王雪峰,吴振起,崔振泽.定喘汤及宣降清分解剂对RSV感染Hela细胞的实验研究[J].中华实验和临床病毒学杂志,2005,19(2)：168.
[21]莫红缨,赖克方,江永南,等.双黄连及其拆方抗呼吸道合胞病毒作用的药效学研究[J].中国中医丛础医学杂志,2005,11(3)：194-196.
[22]肇丽梅,张岩,韩国柱.清热合剂体外抗呼吸道合胞病毒作用[J].中国实验方剂学杂志,2006,12(1)：53-55.
[23]杨京华,刘妮,刘政,等清肺祛瘀汤体外抗呼吸道合胞病毒作用的研究[J].辽宁中医杂志,2010,37(5)：814-815
[24]李珊珊,李洪源,朴英爱.败酱草抗病毒有效部位(AP3)体外抑制呼吸道合胞病毒作用研究[J].中华流行病学杂志,2004,25(2)：150-153.
[25]张凤梅,刘璐,李鑫,等.败酱草多糖提取、纯化、鉴定及其体外抗RSV作用研究[J].中药材,2008,31(12)：1879-1881.
[26]苏妙贤,李药兰,周艳晖,等.瑶药“野葛花”的抗病毒和抑菌活性[J].暨南大学学报(医学版),2006,27(2)：204-213.
[27]李药兰,李克明,苏妙贤,等.猴耳环抗病毒有效成分研究[J].中国中药杂志,2006,31(5)：397-400.
[28]张建军,李洪源,王吉锡,等.甘草活性成分抗呼吸道合胞病毒作用[J].中国公共卫生,2007,23(6)：723-724.
[29]张丽亚,董琳,陈小芳,等.黄岑苷抗呼吸道合胞病毒感染作用的体外研究[J].浙江临床医学,2008,10(12)：1528-1530.
[30]刘钊,杨古秋,肖红.大青叶有效单体抗呼吸道合胞病毒作用的实验研究[J].时珍国医国药,2009,20(8)：1977-1979.
[31]李佳曦,汀受传,徐建亚,等.金欣口服液含药血清及其有效单体白藜芦醇对RSV的RNA聚合酶转录活性的影响[J].南京中医药大学学报,2011,27(1)：36-39.
[32]何雅慧,姜春英,柳晓琳,等.中药苦碟子抗呼吸道病毒药效观察[J].中国公共卫生,2006,22(8)：980-981.
[33]戴开金,罗奇志,候连兵.桑叶体外抗会洗道合胞病毒作用研究[J].中药材,2009,32(8)：1276-1278.
[34]李美玉.金银花体外抗呼吸道合胞病毒的作用研究[J].热带医学杂志,2010,10(4)：420422.
[35]李江权,任现志,汪受传.清肺口服液对常见呼吸道病毒作用的研究[J].辽宁中医学院学报,2002,4(2)：153-155.
[36]汪受传,王霖,陈超,等.清肺口服液含药血清对呼吸道合胞病毒抑制作用的实验研究[J].南京中医药大学学报,2008,24(1)：25-27.
[37]李江全,任现志,汪受传.清肺口服液对免疫功能影响的实验研究[JJ.中国实验方剂学杂志报,2002,4(2)：153-154.
[38]王霖,陈超,汪受传.清肺口服液对呼吸道合胞病毒感染人胚肺成纤维细胞肿瘤坏死因子-α转化生长因子-β1蛋白表达的影响[J].辽宁中医杂志,2010,37(4)：618-620.
[39]陈彩霞,汪受传.金欣口服液对呼吸道合胞病毒感染人胚肺成纤维细胞胞内钙离子的影响[J].江西中医学院学报.2009,21(2)：52-54.
[40]赵霞,汪受传,胡钰,等.金欣口服液含药血清对呼吸道合胞病毒感染细胞早期凋亡的影响[J].中华中医药杂志,2010,25(2)：225-227.
[41]廖辉,汪受传,徐建亚,等.金欣口服液阻断呼吸道合胞病毒入侵的实验研究[J].南京中医药大学学报2008,24(3)：168-170.
[42]赵霞,汪受传,张沛,等.金欣口服液对呼吸道合胞病毒感染大鼠Ⅰ型干扰素表达的影响[J].中华中医药杂志,2011,26(7)：1508-1510.
[43]夏晨,陈超,汪受传.金欣口服液对感染RSV大鼠血清IL-6、IL-8的影响[J].中医药信息,2011,8(1)：44-46.
[44]夏晨,陈超,汪受传,等.金欣口服液对感染RSV大鼠血清IL-12表达水平的影响[J].南京中医药大学学报,2011,27(3)：247-249.
[45]陈超,夏晨,徐建亚,等.金欣口服液对呼吸道合胞病毒肺炎大鼠模型TNF-α、分泌型IgA的影响[J].四川中医,2011,29(3)：28-31.
[46]Fremont L. et, al Biological effects of resveratrol [J]. Life Sci,2000,66(8):663-673.
[47]冯永红,许实波.白藜芦醇药理作用研究进展[J].国外医药·植物药分册,1996,11(4)：155-157.
[48]Chen G-H, Liu W, Li J-X, et al. Novel 1,2-diphenylethene derivatives for treatment of immune diseases.2002:28.
[49]郝捍东,何立人,徐清.白藜芦醇抗柯萨奇病毒感染性小鼠心肌炎的实验研究[J].上海医药,2003,24(8)：373-376.
[50]Birrell M A, McCluskie K, Wong S, et al. Resveratrol, an extract of red wine, inhibits lipopolysaccharide induced airway neutrophilia and inflammatory mediators through an NF-KB-independent mechanism [J]. FASEB,2005,19(7):840-841.
[51]Youn H S, Lee J Y, Fitzgerald K A, et al. Specific inhibition of myD88-independent signaling pathways of TLR3 and TLR4 by resveratrol:Molecular Targets Are TBKI and RIP1 in TR1F Complex [J]. Immun, 2005,175(5):3339-3346.
[52]朴花子,张成镐,朴日龙.白藜芦醇对脂多糖诱导一氧化氮和肿瘤坏死因子生成的影响[J].中药药理与临床,2006,22(5)：12-14.
[53]董玉,杨爱雄,戚好文,等.白藜芦醇对小鼠肺泡巨噬细胞释放IL-6、TNF-a的影响[J].陕西医学杂志,2007,36(9)：1131-1133.
[54]Mitsuhiro Iyori, Hideo Kataoka, Haque Mohammad Shamsul, et al. Resveratrol Modulates Phagocytosis of Bacteria through and NF-KB-Dependent Gene Program [J]. Antimicrobial agents and Chemotherapy, 2008,52(1):121-127.
[55]张中,李树平,卫涛涛.白藜芦醇对脂多糖诱导小鼠腹腔巨噬细胞炎性因子生成的调控[J].卫生研究,2009,38(4)：465-467.
[56]臧娜,谢晓虹,邓昱,等.白藜芦醇对呼吸道合胞病毒感染小鼠肺部病毒滴度及其气道炎症的影响[J].第三军医大学学报,2011,33(18)：1924-1927.
[57]关文达,杨子峰,刘妮,等.白藜芦醇对多种呼吸道病毒作用体外实验研究[J].中药材,2008,31(9)：1388-1390.
[58]周崇俊,冯劲立,张俊丽,等.白藜芦醇对Friend鼠白血病病毒抑制作用及其机制研究[J].新中医,2008,40(9)：98-100.
[59]高璐璐,陈清,李建栋,等.白藜芦醇体外抗肠道病毒71型的作用[J].南方医科大学学报,2011,31(4)：665-668.
[60]Jin H, Zhou H.Cheng X, et al. Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in, vivo [J]. Virology,2000,273(1):210-218.
[61]Takeda K, Kaisho T, Akira S, Toll-like receptors [J]. Annu Rev Immunol,2003,21:335-376
[62]Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity [J]. Nat Immunol,2001,2:675-680.
[63]Akira S. Toll-like receptors and innate immunity [J]. Adv Immunol,2001,78:1256.
[64]云云,黄升海.TLR在呼吸道合胞病毒感染中的作用研究进展[J].国际生物制品学杂志,2006,29(5)：196-199.
[65]Kawai T, Akira S. TLR signaling [J]. Cell Death Differ,2006,13(5):816-825.
[66]Muzio M, Mantovani A. Toll-like receptors (TLRs) signalling and expression pattern [J].J Endotoxin Res,2001,7(4):297-300.
[67]Kurt-Jones EA, Popova L, Kwinn L, et al. Pattern recognition receptors TLR4 and CD 14 mediate response to respiratory syncytial virus [J]. Nat Immunol,2000,1:398-401.
[68]Haynes LM. Moore DD. Kurt-Jones EA, et al. Involvement of Toll-like receptor 4 in innate immunity to respiratory syncytial virus [J]. J Virol,2001,75:10730-10737.
[69]Haeberle HA, Takizawa R, Casola A, et al. Respiratory syncytial virus-induced activation of nuclear factor-kappa B in the lung involves alveolar macrophages and Toll-like receptor 4-dependent pathways [J]. J Infect Dis,2002,186:1199-1206.
[70]Ehl S, Bischoff R, Ostler T, et al. The role of Toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus [J]. Eur J Immunol,2004,34:1146-1153.
[71]Monick M M, Yarovinsky T O, Powers L S, et al. Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin [J]. J Biol Chem,2003,278:53035-53044.
[72]Mandelberg A. Tal G. Naugolny L, etal. Lipopolysaccharide hyporesponsiveness as a risk factor for intensive care unit hospitalization in infants with respiratory syncitial virus bronchiolitis [J]. Clin Explmmunol,2006,144(1):48-52.
[73]谢晓红.呼吸道合胞病毒感染后气道上皮细胞株Toll样受体表达及其功能研究[B].2009：22-71.
[74]Rock FL, Hardiman G, Timans JC, et al. Proc Natl Acad Sci USA,1998,95(2):588-593.
[75]Groskreutz DJ, Monick MM, Powers LS, et al. Respiratory syncytial virus induces TLR3 protein and protein kinase R. leading to increased double-stranded RNA responsiveness in airway epithelial cells [J]. J Immunol,2006,176:1733-1740.
[76]黄升海.Toll样受体3介导呼吸道合胞病毒感染所致免疫反应的机制及褪黑素的作用[B].2007：8-11
[77]云云,黄升海.Toll样受体3介导呼吸道合胞病毒感染人肺上皮细胞的炎性反应及其信号转导通路[J].中国组织工程研究与临床康复.2007,11(21)：4119-4122
[78]Diebold SS, Kaisho T, Hemmi H, et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA [J]. Science.2004,303:1529-1531.
[79]Hornung V, Schlender J, Guenthner Biller M, et al. Replication dependent potent IFN-ainduction in human plasmacytoid dendritic cells by a single-stranded RNA virus [J]. J Immunol,2004,173:5935-5943.
[80]Schlender J, Hornung V, Finke S, et al. Inhibition of Toll-like receptor 7 and 9 mediated alpha/beta interferon production inhuman plasmacytoid dendritic cells by respiratory syncytial virus and measles virus [J]. J Virol,2005,79:5507-5515
[81]Ulevitch RJ. Therapeutics targeting the innate immune system [J]. Nat Rev Immunol,2004,4:512-520.
[82]Robert C, Franklin BS, et al. Role of TLRs/MyD88 in host resistance and pathogenesis during protozoan infection:lessons grommalaria [J].Semin Immunopathol,2008,30(1):41-51.
[83]揭志军,郭雪君,徐卫国,等.呼吸道合胞病毒对小鼠骨髓细胞Ⅰ型干扰素信号通路的抑制作[J].上海交通大学学报,2008,28(10)：1262-1265.
[84]Matthew R, Murawski, Glennice N. et al. Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2 [J]. Journal of Virology,2009,83(3):1492-1500.
[85]Kreso Bendelja, Valerija Vojvoda, Neda Aberle, et al Decreased Toll-like receptor 8 expression and lower TNF-alpha synthesis in infants with acute RSV infection [J]. Respiratory Research.2010,11:143.
[86]刘菊,杨春,唐安芬,等.呼吸道合胞病毒Long株的培养及应用[J].微生物学杂志,2004,24(5)：47-49.
[87]陈奇.中药药理研究方法学[M].人民卫生出版社,2006：33.
[88]Matsushima H, Yamada N, Matsue H, et al. TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. Journal of Immunology,2004,173(1): 531-541.
[89]赵保胜,李兰芳,马悦颖,等.参苏饮含药血清对小鼠巨噬细胞Toll样受体及其下游信号转导通路的影响[J].中国中药杂志,2007,32(4)：327-332.
[90]Winer J, Jung C K, Shackel I, et al Development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro [J]. Anal Biochem,1999,270(1):41.
[91]Schmittgen T D, Zakrajsek B A, Mills A G, et al. Quantitative reverse transcription polymerase chain reaction to study mRNA decay:comparison of endpoint and real time methods [J]. Anal Biochem,2000,285(2):194.
[92]Singh BP, Chauhan RS, Singhal LK. Toll-like receptors and their role in innate immunity [J]. Current Science,2003,85(8):1156-1164
[93]Bowie AG, Haga IR. The role of Toll-like receptors in the host response to viruses [J]. Mol Immunol,2005, 42(8):859-867
[94]徐文飞,刘恩梅.中性粒细胞中Toll样受体的表达与功能[J].现代免疫学,2007,27：426-429.
[95]Hasan S. Jafri, Susana Chavez-Bueno, Asuncion Mejias, et al. Respiratory Syncytial Virus Induces Pneumonia, Cytokine Response, Airway Obstruction, and Chronic Inflammatory Infiltrates Associated with Long-Term Airway Hyperresponsiveness in Mice [J]. The Journal of infectious diseases,2004, 189(10):1856-65.
[96]Shah PS. Current perspectives on the prevention and management of chronic lung disease in preterm infants [J]. Pediatric Drugs,2003,5(7):463-480.
[97]金奇.医学分子病毒学[M].北京：科学出版社,2001：475
[98]Plata A G, S Baron, Poas J S, et al. Activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental Infections [J]. J Virol,2005,79(16):10190-10199.
[99]YoneyamaM, SuharaW, FukuharaY,etal. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300 [J]. EMBO J,1998,17(4): 1087-1095.
[100]刘清泉.肿瘤坏死因子变化水平在小儿呼吸道合胞病毒感染中的意义[J].中医院感染学杂志,2002,9(12)：669-670.
[101]Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination:the control of NF-κB activity [J]. Annu Rev Immunol,2000,18(5):621-663.
[102]骆亚丽、季伟,季正华,等.人偏肺病毒与呼吸道合胞病毒感染患儿外周血细胞因子变化及意义[J].江苏医药,2011,2(37)：174-177.
[103]Ware LB, Matthay MA. Medical progress:The acute respiratory distresssyndrome [J]. New England Journal of Medicine,2000,342:1334-1349.
[104]Kishinoto T. The biology of interleukin-6 [J]. Blood,1989,74(1):1.
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