【作者】 黄升海；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 博士

【摘要】 Toll样受体（Toll-like receptors,TLRs）是近年来发现的重要天然免疫受体,为Ⅰ型跨膜信号蛋白,能识别不同的病原微生物或其组分,在启动天然免疫反应、激活核转录因子-κB（NF-κB）和介导炎症反应中发挥关键作用,可能是治疗多种急、慢性疾病的新的药物靶标。研究发现TLR3（Toll-like receptor 3）能特异性地识别双链RNA（dsRNA）。呼吸道合胞病毒（respiratory syncytial virus,RSV）是世界范围内婴幼儿病毒性下呼吸道感染最重要的病毒病原,也是中老年和免疫缺陷成年人呼吸道感染的重要病原。其核酸类型为单负链RNA,但在病毒复制和转录过程中可合成大量dsRNA,因此,TLR3可通过识别RSV而发挥作用。TLR3和dsRNA结合后可经由髓样分化因子（myeloid differentiation factor 88,MyD88）依赖性和非MyD88依赖性两条途径来诱导NF-κB的激活,促进其转位进入细胞核内和DNA片段结合,启动相应的促炎细胞因子、趋化因子和协同刺激因子表达上调,引起系统性炎性反应。可见TLRs的发现,为研究RSV感染的炎症免疫反应机理提供了新的思路,可能在RSV肺炎的发生发展过程中发挥重要作用。RSV感染的机制十分复杂,目前尚未完全明了,也无理想的治疗药物。因此,深入研究RSV感染的分子机制具有重要医学意义。研究发现,褪黑素（melatonin,MT）具有抗炎、调节免疫和强大的抗氧化作用,能显著降低炎症性损伤,抑制iNOS的产生,与抑制NF-кB有关。MT可能通过直接作用于免疫细胞上的MT受体而发挥作用。在用MT治疗由细菌内毒素诱导的损伤时,能使炎症反应明显减轻。但MT能否对RSV所致炎症免疫反应具有调节作用,其在TLR3信号通路中的作用靶点和作用机制如何,尚不得而知。本文体外和体内实验结合,研究了TLR3活化的信号转导通路参与RSV感染所致炎症免疫反应的分子机制;通过MT对TLR3活化信号转导途径的影响,探讨了药理浓度的MT对RSV所致炎症反应的干预作用及其作用靶点。目的:研究RSV感染后TLR3的表达变化及其介导的下游信号分子改变,探讨TLR3介导RSV感染所致炎症免疫反应的分子机制。用MT预先给药进行干预,通过比较TLR3的表达变化及其介导的下游信号分子改变,研究MT作用的机制;同时观察MT膜和核受体的表达,明确MT是通过与何种受体的结合而发挥作用。方法:1. TLR3介导RSV感染所致炎症免疫反应的体外实验用RSV感染体外培养的RAW264.7细胞,收集不同感染时间点（设RSV感染0,1,4,8,16,24小时组）的细胞和培养上清,用半定量逆转录聚合酶链反应（RT-PCR）检测TLR3、MyD88、TNF-α和iNOS mRNA的表达变化,用Western blot检测TLR3蛋白和核内活性NF-κBp65蛋白的表达变化,用酶联免疫吸附试验（ELISA）检测细胞培养上清中TNF-α蛋白变化。2. TLR3介导RSV感染所致炎症免疫反应的体内实验用RSV滴鼻感染昆明小鼠,收集不同感染时间点（设RSV感染0,1,4,8,16,24小时组或48,72,96小时组）的小鼠血清和肺,用半定量RT-PCR检测肺TLR3、MyD88、TNF-α和iNOS mRNA的表达变化,用Western blot检测TLR3蛋白和核内活性NF-κBp65蛋白的表达变化,用ELISA检测小鼠血清中TNF-α表达变化。3. MT干预的体外实验MT分10-7M、10-6M、10-5M三个浓度组,预先处理RAW264.7细胞,分别收集上述不同感染时间点的细胞和培养上清,用半定量RT-PCR检测TLR3、MyD88、TNF-α和iNOS mRNA的表达变化,并对MT膜受体MT1和MT2以及核受体RORα进行mRNA测定。用Western blot检测TLR3蛋白和核内活性NF-κBp65蛋白的表达变化,用ELISA方法检测细胞培养上清中TNF-α蛋白变化。4. MT干预的体内实验MT分5mg·kg^-1、10 mg·kg^-1、20 mg·kg^-1三个剂量组,预先灌胃给药,连续七天。分别收集上述不同感染时间点的小鼠血清和肺,用半定量RT-PCR检测肺TLR3、MyD88、TNF-α和iNOS mRNA的表达变化,并对MT膜受体MT1、MT2以及核受体RORα进行mRNA测定。用Western blot检测TLR3蛋白和核内活性NF-κBp65蛋白的表达变化,用ELISA方法检测小鼠血清中TNF-α表达变化。结果:1. TLR3介导RSV感染所致炎症免疫反应的体外实验结果RSV感染4 h后即上调RAW264.7细胞TLR3、TNF-α和iNOS mRNA的转录水平;RSV感染8h后即可诱导活化TLR3蛋白和核内活性NF-κB蛋白的表达,并升高细胞培养上清中TNF-α的分泌量。各指标的变化与对照组相比差异均有显著性,并且随着RSV感染时间的延长而表达量增加。但MyD88 mRNA表达未见明显变化。2. TLR3介导RSV感染所致炎症免疫反应的体内实验结果RSV感染小鼠4 h后即能上调肺TLR3 mRNA的转录水平,诱导活化核内NF-κB蛋白的表达;RSV感染48 h后上调TNF-αmRNA的转录水平;感染72 h上调iNOS mRNA的转录水平。并于RSV感染48 h后观察到小鼠血清中TNF-α的分泌量也增加。各指标的变化较对照组差异均有显著性。但在感染24 h内未检测到肺组织中TLR3蛋白的表达;而MyD88 mRNA表达也未见明显变化。3. MT干预的体外实验结果MT三个浓度组（10-7M、10-6M、10-5M）在RSV感染RAW264.7细胞后4 h,即可下调RSV诱导的TNF-αmRNA的转录水平,其下调作用随着MT浓度的增加更加明显。而对于iNOS mRNA,10-7M的MT未能起到下调作用,10-6M和10-5M的MT则从RSV感染4 h开始就能下调iNOS表达,10-5M的作用更明显。结果还显示:MT在10-7M浓度时未能起到下调NF-κB的作用,10-6M和10-5M浓度MT则从RSV感染4 h开始就能下调其表达,10-5M的MT作用更明显。但各浓度组的MT对TLR3mRNA和蛋白的表达均没有明显的下调作用,而对MyD88 mRNA表达也未见明显影响。对MT受体的检测结果显示,RAW细胞均未检测到膜受体MT1、MT2的表达,而核受体RORα在各组均有表达且均匀一致。MT在10-7M浓度时未能起到下调细胞培养上清TNF-α蛋白的作用,10-6M和10-5M浓度MT则从RSV感染8 h开始就能下调其表达,其中10-5M的MT作用更明显。4. MT干预的体内实验结果MT在10 mg·kg^-1和20 mg·kg^-1剂量组时,均能在RSV感染72 h后下调RSV诱导的肺组织TNF-α和iNOS mRNA的转录水平。20 mg·kg^-1剂量的MT组在RSV感染4 h而10 mg·kg^-1剂量MT在RSV感染8 h,即可下调NF-κB的活性表达。MT在10 mg·kg^-1剂量时,从RSV感染的48 h开始可降低血清中TNF-α的产生,其中20 mg·kg^-1剂量的下调作用更明显。但各剂量组的MT对TLR3、MyD88和RORα的表达均不影响。我们也未检测到MT1、MT2的表达。结论:1. RSV感染可上调TLR3、TNF-α和iNOS mRNA的表达,诱导活化TLR3蛋白和核内NF-κB的表达,升高细胞培养上清和小鼠血清中TNF-α的分泌量。表明病毒诱导的炎症免疫反应与TLR3活化的信号转导途径有关,TLR3可能参与了RSV诱导的炎症免疫反应,推测是通过MyD88非依赖或部分依赖途径而活化NF-κB。2. MT在一定浓度和剂量范围内,能降低RSV诱导的RAW264.7细胞和肺的核内活性NF-κB的表达,下调RSV诱导的TNF-α和iNOS mRNA的转录水平,降低细胞培养上清和小鼠血清中TNF-α的分泌量。但MT对TLR3 mRNA和蛋白,MyD88和RORα的mRNA表达均无明显影响。3. MT抑制RSV诱导的NF-кB活化、下调TNF-α和iNOS的表达随着药物剂量（或浓度）的增加及感染时间的延长而更加明显。4. MT可能通过与其核受体结合,抑制NF-кB活化,发挥抗RSV感染所致的炎症反应。5.在RSV感染所致炎症免疫反应中,MT可能对TLR3、MyD88等上游信号分子无抑制作用。更多还原

【Abstract】 Toll-like receptors （TLRs）, type I across membrane proteins, very important receptors in innate immune responses in the recent studies, which could identify difference pathogen-associated molecular patterns expressed by infectious agents, play critical roles in activating the innate immune response and mediating the inflammatory reaction, now they are considered as the new targets of the drugs to cure the acute and chronic diseases. Recent studies indicate that Toll-like receptor 3（TLR3） is the specific receptor for double-stranded RNA （dsRNA）. Respiratory syncytial virus （RSV） is the major cause of serious lower respiratory tract infections in infancy and early childhood and is also becoming widely recognized as an important pathogen in the elderly and in immunosuppressed adults. The nucleic acid of RSV is single- stranded RNA （ssRNA）, but the virus induces the synthesis of dsRNA during its replication and transcription, so the TLR3 can produce a marked effect during the infection by recognizes the dsRNA of RSV. After the combination between TLR3 and dsRNA, the nuclear factor kappa B （NF-κB） would be activated through MyD88-dependent or MyD88-independent pathway, which can up-regulates the proinflammatory factor, chemotatic factor and collaborative stimulating factor, caused systematic inflammatory response. Therefore, the discovery of TLRs may give a significant revolution in the pneumonia caused by the infection of RSV, and it will provide a new method for the research about the mechanism of RSV-induced inflammatory and immune reaction. Up to now, its mechanism is unknown since it was so complex that there is no satisfactory medicine or vaccine. Accordingly, it has very important medical significance to deep research the mechanism of RSV infection.Studies showed that melatonin （MT） had the effects on anti-inflammation, regulating the immune response and great anti-oxidation, cutting down the inflammatory injury significantly and down-regulating the iNOS through inhibiting the activation of NF-кB. Melatonin may produce an effect through combining with the melatonin receptor directly in the immune cells. Some previous data showed that MT could reduce the inflammatory injury caused by bacterial endotoxin. But whether melatonin could give a regulatory effect on RSV–induced inflammatory and immune response, or its target and mechanism in the TLR3 signaling pathway, these questions are unknown and will be a focus in our present work. We here study the TLR3 activated signaling pathway involvement of the mechanism of RSV-induced inflammatory and immune response, in vitro and in vivo, and the effect of intervention and target of pharmacological profile of MT in RSV-caused inflammation through observation the MT’s influence on TLR3 signaling pathway.Objective:To investigate the expression changes of TLR3 and its downstream signaling molecules, and the relationships between TLR3 signal transduction pathway and its mediated inflammatory and immune response to RSV. Using MT as an intervention, to study the potential target of MT through compared with the change of TLR3 and its downstream signaling molecules. Meanwhile, observe the expression of membrane melatonin receptor and nuclear receptor, explain which receptor can combine with the melatonin and take effect.Methods1. The TLR3-mediated inflammatory and immune response to RSV in vitroRAW264.7 cells infected with RSV in vitro were used to collect cells and cell culture supernatants. Semiquantitative RT-PCR was used to evaluate the expression of TLR3, MyD88, TNF-αand iNOS mRNA. The protein expression of TLR3 and NF-κB p65 activity was detected by Western-Blot using total protein and nuclear protein, respectively. The expression levels of TNF-αprotein were measured by ELISA assay in cell culture supernatant.2. The TLR3-mediated inflammatory and immune response to RSV in vivo The Kung Ming mice inoculated intranasally with RSV were used to collect lung and mouse serum. Semiquantitative RT-PCR was used to evaluate the expression of TLR3, MyD88, TNF-αand iNOS mRNA. The protein expression of TLR3 and NF-κB p65 activity was detected by Western-Blot using total protein and nuclear protein, respectively. The expression level of TNF-αprotein in mouse serum was measured by ELISA assay.3. The effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vitroThe concentration of MT at 10-7, 10-6, and 10-5M was given as an intervention in three levels before the infection of RSV in RAW264.7 macrophages, respectively. The same works were done to compare with the groups which not used MT, and also the analysis of membrane melatonin receptor（MT1、MT2） and nuclear receptor（RORα） mRNA expression were tested at same time.4. The effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vivoThe concentration of MT at 5mg·kg^-1, 10mg·kg^-1, and 20mg·kg^-1 was given as an intervention in three levels before the infection of RSV in Kung Ming mice, respectively, and lasting seven days. The same works were done to compare with the groups which not used MT, and also the analysis of melatonin membrane receptor（MT1、MT2） and nuclear receptor（RORα） mRNA expression were tested at same time.Results:1. The results of TLR3-mediated inflammatory and immune response to RSV in vitro RSV infection could up-regulate the transcriptional levels of TLR3, TNF-αand iNOS mRNA since 4 hours in RAW264.7 cells. RSV stimulation also induced the TLR3 protein and the activation of NF-κB p65 since 8 hours, and enhanced TNF-αproduction in cell culture supernatants. The changes of each index varied significantly and in a time-dependent manner, which compared with the control group. But the MyD88 mRNA had no evident change.2. The results of TLR3-mediated inflammatory and immune response to RSV in vivo RSV infection can up-regulates the transcription of TLR3 since 4 hours in mouse lungs. In accordance with the expression of TLR3, RSV stimulation induced the activation of NF-κB p65. The up-expression of TNF-αmRNA was detected since 48 hours, and the iNOS mRNA was detected since 72 hours. The TNF-αin the mouse serum was also increased after 48 hours. The changes were significant compared with the control group. But the MyD88 mRNA had no evident change, and the protein of TLR3 was not detected within 24 hours.3. The results of effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vitroMelatonin could down-regulate the elevated transcriptional levels of TNF-αmRNA which induced by RSV infection, and the down-regulation had a dose-dependent manner. Result showed that 10-7M concentration of MT did not have an evident effect on down regulation the transcription of iNOS mRNA, but 10-6M and 10-5M of MT could do since 4 hours. The effect of melatonin on down regulation the activation of NF-κB was similar to that of iNOS mRNA. There was no change in the protein of TLR3 and the MyD88 mRNA treated with MT. The membrane melatonin receptors MT1 and MT2 were not detected, but nuclear receptor RORαwas opposite and the level in each group was uniformity. TNF-αproduction in cell culture supernatant could decrease treated with MT in 10-6M and 10-5M since 8 hours, and the effect of 10-5M on down regulation TNF-αproduction was obvious, but there is no down-regulation role at the level of 10-7M.4. The results of effect of melatonin on TLR3-mediated inflammatory and immune response to RSV in vivoMT could down-regulate the transcriptional levels of TNF-αand iNOS mRNA in lung following 72h of RSV infection, but the down-regulation of NF-κB was detected at 4h of large dose and 8h of middle dose melatonin, respectively. Moreover, MT cut down the production of TNF-αin the mouse serum at the dose of 10mg·kg^-1 since 48 hours of RSV infection, 20 mg·kg^-1 per mouse can greatly reduce the TNF-αproduction. But MT had no effect on the expression of TLR3, MyD88 and RORα. Also we did not detect the MT1 and MT2 receptor expression. Conclusion:1. RSV infection significantly up-regulates the transcriptional levels of TLR3, iNOS, and TNF-αmRNA. Meanwhile, RSV stimulation induces the activation of NF-κB, and enhances TNF-αproduction not only in cell culture supernatants but also in mouse serum. The inflammatory and immune reaction induced by virus is related with the TLR3 signal transduction pathway, and TLR3 might take effect in the inflammatory and immune response to RSV and activate the NF-κB through MyD88-independent or partial-dependent pathway.2. Melatonin at a range of levels and dose could down-regulate the activation of NF-κB and the transcription of iNOS and TNF-αmRNA, decrease the TNF-αproduction both in cell culture supernatants and mouse serum caused by RSV infection. But there were no evident effects of melatonin on suppressing the expression levels of TLR3 mRNA and protein,as well as MyD88 and RORαmRNAexpression both in RAW264.7 cells and mouse lung.3. The inhibitory effects of melatonin on RSV-induced increasing NF-κB, TNF-αand iNOS expression have a relationship with RSV-infected times and melatonin′s dose.4. The effect of melatonin on the anti-RSV infection probably is that melatonin may be combining with its nuclear receptor and suppress the activation of NF-κB.5. Some of melatonin′s effects on the immune and inflammatory response caused by RSV infection probably do not inhibit the TLR3 or MyD88 signaling molecules.更多还原