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。
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.
Methods
1. The TLR3-mediated inflammatory and immune response to RSV in vitro
RAW264.7 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 vitro
The 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 vivo
The 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 vitro
Melatonin 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 vivo
MT 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.
引文
1. Hall CB. Respiratory syncytial virus and parainfluenza virus. N Engl J Med. 2001,344(25):1917–1928
2. Haeberle HA, Kuziel WA, Dieterich HJ, Casola A, Gatalica Z, Garofalo RP. Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1a in lung pathology. J Virol, 2001;75(2): 878–890
3. Leclerc F, Scalfaro P, Noizet O, Thumerelle C, Dorkenoo A, Fourier C. Mechanical ventilatory support in infants with respiratory syncytial virus infection. Pediatr Crit Care Med. 2001; 2(3):197–204
4. Dowell, SF, LJ. Anderson, HE. Gary, Erdman DD, Plouffe JF, File TM Jr, Marston BJ, Breiman RF. Respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults. J. Infect. Dis. 1996; 174(3):456–462
5. Falsey, AR, RM. McCann, WJ. Hall, Criddle MM, Formica MA, Wycoff D, Kolassa JE. The“common cold”in frail older persons: impact of rhinovirus and coronavirus in a senior daycare center. J. Am. Geriatr. Soc. 1997; 45(6):706–711
6. Glezen, WP, SB. Greenberg, RL. Atmar, Piedra PA, Couch RB. Impact of respiratory virus infections on persons with chronic underlying conditions. JAMA 2000; 283(4):499–505
7. Garofalo R, Welliver R, Ogra PL. Clinical aspects of bronchial reactivity and cell–virus interaction. Mucosal Immunology, 2nd Edition, Academic Press, San Diego, 1998; pp. 1223-1238
8. Graham BS, Johnson TR, Peebles RS. Immune-mediated disease pathogenesis in respiratory syncytial virus infection. Immunopharmacology. 2000; 48(3):237–247
9. Zhang Y, Luxon B, Casola A, Garofalo RP, Jamaluddin M, Brasier AR. JamaluddinM, Brasier AR. Expression of RSV-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays. J Virol, 2001;75(19):9044–9058
10. Lugo, RA, and MC. Nahata. Pathogenesis and treatment of bronchiolitis. Clin. Pharm. 1993, 12(2):95-116
11. Wissel H, Schulz C, Koehne P, Richter E, Maass M, Rüdiger M. Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-αand MIP-2 via an NF-κB pathway in rat type II pneumocytes. Respir Res. 2005; 6(1): 51-65
12. Basu S, Fenton MJ. Toll-like receptors:function and roles in lung disease. Am.J.Physiol Lung Cell Mol Physiol. 2004; 286:L887-L892
13. Power MR, Peng Y, Maydanski E, Marshall JS, Lin TJ. The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on MyD88 in mice. J.Biol.Chem. 2004; 279(47):49315- 49322
14. Jeyaseelan S, Chu HW, Young SK, Freeman MW, Worthen GS. Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury. Infect Immun. 2005; 73(3):1754-1763
15. Isogawa M, Robek MD, Furuichi Y, Chisari FV. Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo. Journal of Virology. 2005; 79(11):7269-7272
16. Akira, S., K. Takeda, and T. Kaisho. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2001,2(8): 675–680
17. Akira, S. Toll-like receptors and innate immunity. Adv. Immunol. 2001; 78: 1–56
18. Kopp E, Medzhitov R. Recognition of microbial infection by Toll-like receptors. Curr Opin Immunol. 2003; 15(4):396-01
19. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol, 2003; 21: 335-376
20. Vasselon T, Detmers PA. Toll receptors: a central element in innate immune responses. Infect Immun. 2002, 70(3): 1033-1041
21. Sabroe I, Read RC, Whyte MK, Dockrell DH, Vogel SN, Dower SK. Toll-like receptors in health and disease: complex questions remain. J Immunol. 2003, 171(4):1630-1635
22. Yamamoto M, Takeda K, Akira S. TIR domain-containing adaptors define the specificity of TLR signaling. Molecular Immunol, 2004, 40(12):861-868
23. Rudd BD, Burstein E, Duckett CS, Li X, Lukacs NW. Differential role for TLR3 in respiratory syncytial virus-induced chemokine expression. J Virol, 2005, 79(6):3350-3357
24. Wang H, Wei W, Zhang SY, Shen YX, Wang NP, Yue L, Xu SY. Melatonin-selenium nanoparticles protects liver against immunological injury induced by bacillus Calmette-Guerin and lipopolysaccharide. Acta Pharmacol Sin. 2005; 26(6):745-752
25. Li JH, Yu JP, Yu HG, Xu XM, Yu LL, L J. Melatonin reduces inflammatory injury through inhibiting NF-kappaB activation in rats with colitis. Mediators Inflamm. 2005;4(2005):185-193
26. Bruck R, Aeed H, Avni Y, Shirin H, Matas Z, Shahmurov M, Avinoach I, Zozulya G, Weizman N, Hochman A. Melatonin inhibits nuclear factor kappa B activation and oxidative stress and protects against thioacetamide induced liver damage in rats. J Hepatol. 2004;40(1):86-93
27. Bernasconi D, Amici C, Frazia SL, Ianaro A, Santoro MG. The IκB kinase is a key factor in triggering influenza A virus-induced inflammatory cytokine production in airway epithelial cells. J Biol Chem. 2005; 280(25):24127-24134
28.刘菊,杨春,唐安芬,陈霄,何敏,雍雪飞.呼吸道合胞病毒Long株的培养及应用.微生物学杂志, 2004; 24(5):47-49
29. Heilman CA. Respiratory and parainflunenza viruses. J Infect Dis, 1990; 161(3):402-406
30. Soloski MJ. Recognition of tumor cells by the innate immune system. Current Opinion in Immunology, 2001, 13(2): 154-162
31. Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, Ghosh S. A Toll-like receptor that prevents infection by uropathogenic bacteria. J Science, 2004, 303(5663):1552-1526
32. Tabeta K, Georgel P, Janssen E, Du X, Hoebe K, Crozat K, Mudd S, Shamel L, Sovath S, Goode J, Alexopoulou L, Flavell RA, Beutler B. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci U.S.A. 2004; 101(10):3516-3521
33. Singh BP, Chauhan RS, Singhal LK. Toll-like receptors and their role in innate immunity. Current Science, 2003; 85(8):1156-1164
34. Bowie AG, Haga IR. The role of Toll-like receptors in the host response to viruses. Mol Immunol, 2005; 42(8):859-867
35.李睿,刘恩梅,杨锡强,王莉佳.小鼠呼吸道合胞病毒感染肺TLR4表达与气道高反应性.重庆医科大学学报, 2006; 31(4):518-520
36.李睿,刘恩梅,杨锡强,王莉佳,王建军.新生期小鼠卡介苗接种和呼吸道合胞病毒感染对肺部炎症和免疫状态的影响.临床儿科杂志, 2006, 24(1): 28-30
37.鲍一笑,董晓艳,吕婕.呼吸道合胞病毒诱导气道上皮粘液分泌及调节机制研究.临床儿科杂志, 2006; 24(1):25-27.
38. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent Toll-Like receptor signaling pathway. Science, 2003; 301 (5633):640-643.
39. Dunne A, O’Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci Stke, 2003, (171): re3.
40. Sato S, Sugiyama M, Yamamoto M, Watanabe Y, Kawai T, Takeda K, Akira S. Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-kappa B and IFN-regulatory factor-3, in the Toll-like receptor signaling. J Immunol, 2003; 171(8):4304-4310
41. Aderem A, Ulevitch RJ. Toll-like ceceptors in the induction of the innate immune response. Nature, 2006; 406(6769):782-787
42. Groskreutz DJ, Monick MM, Powers LS, Yarovinsky TO, Look DC, Hunninghake GW. Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells. J Immunol, 2006; 176(3):1733-1740.
43. Monick MM, Yarovinsky TO, Powers LS, Butler NS, Carter AB, Gudmundsson G, Hunninghake GW. Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin. J Biol Chem, 2003; 278(52):53035-53044
44.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.中华医院感染学杂志, 2002; 9(12)669-670。
45. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol, 2000; 18 (5):621-663
46. Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992; 6(12): 3051–3064
47. Xie, Q. and Nathan, C. The high-output nitric oxide pathway: role and regulation. J Leukoc Biol. 1994; 56(5): 576–582
48. Stark, JM, Khan, AM, Chiappetta, CL, Xue, H, Alcorn, JL, Colasurdo, GN. Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection. J Infect Dis. 2005; 191(3): 387–395
49. Ricciardolo, FL. Multiple roles of nitric oxide in the airways. Thorax. 2003; 58(2): 175–182
50. Steer, SA, JM Moran, BS Christmann, LB Maggi, Jr and JA Corbett. Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages. J Immunol. 2006, 177(5):3413-3420
51. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA. Involvement of Toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol, 2001, 75(22):10730-10737
52. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science, 2004, 303(5663): 1529– 1531
53. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol, 2004, 4(7):499–511
54. Hornung V, Schlender J, Guenthner-Biller M, Rothenfusser S, Endres S, Conzelmann KK, Hartmann G. Replication dependent potent IFN-αinduction in human plasmacytoid dendritic cells by a single-stranded RNA virus. J Immunol, 2004, 173(10):5935-5943
55. Schlender J, Hornung V, Finke S, Günthner-Biller M, Marozin S, Brzózka K, Moghim S, Endres S. Inhibition of Toll-Like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic dells by respiratory syncytial virus and measles virus. J Virol, 2005, 79(9): 5507-5515
56. Ulevitch RJ. Therapeutics targeting the innate immune system. Nat Rev Immunol, 2004, 4(7):512–520
57. Dubocovich, ML, Markowska, M. Functional MT1 and MT2 melatonin receptors in mammals. Endocrine 2005, 27(2):101–110
58. Benitez-King G, Huerto-Delgadillo L, Anton-Tay F. Binding of 3H-melatonin to calmodulin. Life Sci 1993, 53(3):201–207
59. Chen Q, Wei W. Effects and mechanisms of melatonin on inflammatory andimmune responses of adjuvant arthritis rats. Int Immunopharmacol. 2003, 2(10):1443-1449
60. Wei W, Shen YX, Dai M,Chen Q. Effects and mechanisms of melatonin on immune responses in mice of different months. Acta Pharmacol Sin. 2003, 24(7):719-723
61. Naji L, Carrillo-Vico, A, Guerrero, JM. Calvo, JR. Expression of membrane and nuclear melatonin receptors in mouse peripheral organs. Life Sci, 2004; 74(18):2227-2236
62. Carrillo-Vico A, Garcia-Perganeda A, Naji L, Calvo JR, Romero MP, Guerrero JM. Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system. Cell Mol Life Sci. 2003; 60(10):2272-2278
63. Smirnov, A.N. Nuclear melatonin receptors. Biochemistry. 2001, 66(1):19-26
64. Jarzynka MJ, Passey DK, Ignatius PF, Melan MA, Radio NM, Jockers R, Rasenick MM, Brydon L, Witt-Enderby PA. Modulation of melatonin receptors and G-protein function by microtubules. J Pineal Res. 2006, 41(4):324-36
65. Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappaB activation. Mutat Res, 2001, 480-481 (1～2) :243-268
66. Gilad, E, Wong, HR, Zingarelli, B, Virág, L, O’Connor, M, Salzman, AL, Szabó, C. 1998. Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in murine macrophages: role of inhibition of NFkB activation. FASEB J.1998; 12(9), 685–693
67. Crespo E, Macias M, Pozo D, Escames G, Martin M, Vives F, Guerrero JM, Acuna-Castroviejo D. Melatonin inhibits expression of the inducible NO synthaseⅡin liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats. FASEB J. 1999, 13(12):1537-1546
68. Escames G, Leon J, Macias M, Khaldy H, Acuna-Castroviejo D. Melatonincounteracts lipopolysaccharide-induced expression and activity of mitochondrial nitri oxide synthase in rats. FASEB J. 2003, 17(8):932-934
69 Silva SO, Rodrigues MR, Ximenes VF, Bueno-da-Silva AE, Amarante-Mendes GP, Campa A. Neutrophils as a specific target for melatonin and kynuramines: effects on cytokine release. J Neuroimmunol. 2004;156(1-2):146-152
70. Valero, N, Meleán, E, Bonilla, E, Arias, J, Espina, LM, Chacín -Bonilla, L, Larreal, Y, Maldonado, M, and A?ez, F. In vitro, melatonin treatment decreases nitric oxide levels in murine splenocytes cultured with the venezuelan equine encephalomyelitis virus. Neurochemical Research, 2005, 30(11): 1439–1442
71. Negrette B, Bonilla E, Valero N, Pons H, Tamayo JG, Chacín-Bonilla L, Medina-Leendertz S and A?ez F. Melatonin treatment enhances the efficiency of mice immunization with venezuelan equine encephalomyelitis virus TC-83. Neurochemical Research, 2001, 26(7):767–770
72. Lissoni, P, Barni, S, Tancini, G, Brivio, F, Tisi, E, Zubelewicz, B and Braczkowski. Role of pineal gland in the control of macrophage functions and its possible implications in cancer: a study of interactions between tumor necrosis factor-alpha and the pineal hormone melatonin. J Biol Reg Homeost Agents. 1994, 8(4):126-129
73. Carrillo-Vico A, Garcia-Maurino S, Calvo JR, Guerrero JM. Melatonin counteracts the inhibitory effect of PGE2 on IL-2 production in human lymphocytes via its mt1 membrane receptor. FASEB J. 2003, 17(6):755– 757
74. Tomas-Zapico C, Coto-Montes A. A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. J Pineal Res. 2005;39(2):99-104
75. Rodriguez C, Mayo JC, Sainz RM, Antolin I, Herrera F, Martin V, Reiter RJ. Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res. 2004;36(1):1-9
1. Reiter RJ. Remembrance: growing up with the pineal gland. Endocrinology. 1992; 131 (5) :2039-2041
2. Mocchegiani E, Bulian D, Santarelli L, Tibaldi A, Muzzioli M, Lesnikov V, Pierpaoli W, Fabris N. The zinc pool is involved in the immune-reconstituting effect of melatonin in pinealectomized mice. J Pharmacol Exp Ther. 1996; 277(3):1200-1208
3. Wang H, Wei W, Zhang SY, Shen YX, Wang NP, Yue L, Xu SY. Melatonin-selenium nanoparticles protects liver against immunological injury induced by bacillus Calmette-Guerin and lipopolysaccharide. Acta Pharmacol Sin. 2005; 26(6):745-752
4. Li JH, Yu JP, Yu HG, Xu XM, Yu LL, Liu J, Luo HS. Melatonin reduces inflammatory injury through inhibiting NF-kappaB activation in rats with colitis. Mediators Inflamm. 2005;4(2005):185-193
5. Bruck R, Aeed H, Avni Y, Shirin H, Matas Z, Shahmurov M, Avinoach I, Zozulya G, Weizman N, Hochman A. Melatonin inhibits nuclear factor kappa B activation and oxidative stress and protects against thioacetamide induced liver damage in rats. JHepatol. 2004;40(1):86-93
6. Chen Q, Wei W. Effects and mechanisms of melatonin on inflammatory and immune responses of adjuvant arthritis rats. Int Immunopharmacol. 2003;2(10):1443-1449
7. Wei W, Shen YX, Dai M,Chen Q. Effects and mechanisms of melatonin on immune responses in mice of different months. Acta Pharmacol Sin. 2003; 24(7):719-723
8. Marstroni GJM, Conti A, Pierpaoli W. Role of the pineal gland in immunity.Ⅱ. Melatonin enhances the antibody response via an opeatergic mechanism. Clin Exp Immunol. 1997; 68(2):384-391
9. Alicelebic S, Mornjakovic Z, Susko I, Cosovic E, Beganovic-Petrovic A. The role of pineal gland and exogenous melatonin on the irradiation stress response of suprarenal gland. Bosn J Basic Med Sci. 2006; 6(4):18-21
10. Baltaci AK, Mogulkoc R, Bediz CS, Pekel A. Effects of zinc deficiency and pinealectomy on cellular immunity in rats infected with Toxoplasma gondii. Biol Trace Elem Res. 2005; 104(1):47-56
11. Dimitrov S, Lange T, Tieken S, Fehm HL, Born J. Sleep associated regulation of T helper 1/T helper 2 cytokine balance in humans. Brain Behav Immun. 2004; 18(4):341-348
12. Carrillo-Vico A, Reiter RJ, Lardone PJ, errera JL, Fernandez-Montesinos R, Guerrero JM, Pozo D. The modulatory role of melatonin on immune responsiveness. Curr Opin Investig Drugs. 2006; 7(5):423-431
13. Zimecki M. The lunar cycle: effects on human and animal behavior and physiology. Postepy Hig Med Dosw (online). 2006; 60:1-7
14. Maestroni GJ. T-helper-2 lymphocytes as a peripheral target of melatonin. J Pineal Res. 1995; 18(2):84-89
15. Naji L, Carrillo-Vico, A, Guerrero, JM. Calvo, JR. Expression of membrane and nuclear melatonin receptors in mouse peripheral organs. Life Sci. 2004; 74(18):2227-2236
16. Carrillo-Vico A, Garcia-Perganeda A, Naji L, Calvo JR, Romero MP, Guerrero JM. Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system. Cell Mol Life Sci. 2003; 60(10):2272-2278
17. Lissoni, P, Barni, S, Tancini, G, Brivio, F, Tisi, E, Zubelewicz, B and Braczkowski. Role of pineal gland in the control of macrophage functions and its possible implications in cancer: a study of interactions between tumor necrosis factor-alpha and the pineal hormone melatonin. J Biol Reg Homeost Agents. 1994; 8(4):126-129
18. Carrillo-Vico, A, Garcia-Maurino, S, Calvo, JR, Guerrero, JM. Melatonin counteracts the inhibitory effect of PGE2 on IL-2 production in human lymphocytes via its mt1 membrane receptor. FASEB J. 2003; 17(6),755– 757
19. Smirnov AN. Nuclear melatonin receptors. Biochemistry. 2001; (Mosc) 66(1):19-26
20. Kokkola T, Laitinen JT. Melatonin receptor genes. Ann Med. 1998; 30(1):88-94
21. Tomas-Zapico C, Coto-Montes A. A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. J Pineal Res. 2005;39(2):99-104
22. Rodriguez C, Mayo JC, Sainz RM. Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res. 2004; 36(1):1-9
23. Dubocovich ML, Markowska M. Functional MT1 and MT2 melatonin receptors in mammals. Endocrine, 2005; 27(2):101-110
24. Torres-Farfan C, Richter HG, Rojas-García P, Vergara M, Forcelledo ML. Mt1 melatonin receptor in the primate adrenal gland: inhibition of adrenocorticotropin-stimulated cortisol production by melatonin. J Clin Endocrinol Metab. 2003; 88(1):450-458
25. Torres-Farfan C, Richter HG, Germain AM, Valenzuela GJ, Campino C. Maternal melatonin selectively inhibits cortisol production in the primate fetal adrenal gland. J Physiol. 2004; 554(Pt 3):841-856
26. Rivera-Bermudez MA, Masana MI, Brown GM, Earnest DJ, Dubocovich ML. Immortalized cells from the rat suprachiasmatic nucleus express functional melatoninreceptors. Brain Res. 2004; 1002(1-2):21-27
27. Al-Ghoul WM, Herman MD, Dubocovich ML. Melatonin receptor subtype expression in human cerebellum. Neuroreport. 1998; 9(18):4063-4068
28. Moore CB, Siopes TD. Melatonin can produce immunoenhancement in Japanese quail (Coturnix coturnix japonica) without prior immunosuppression. Gen Comp Endocrinol . 2002; 129(2):122-126
29. Silva SO, Rodrigues MR, Ximenes VF, Bueno-da-Silva AE, Amarante-Mendes GP, Campa A. Neutrophils as a specific target for melatonin and kynuramines: effects on cytokine release. J Neuroimmunol. 2004; 156(1-2):146-152
30. Labunets IF, Butenko GM, Khavinson VKh, Magdich LV, Dragunova VA, Pishel' IN, Azarskova MV. Regulating effect of pineal gland peptides on development of T-lymphocytes in CBA aging mice: role of microenvironment of immune system organs and neuroendocrine factors. Adv Gerontol. 2003; 12:111-120
31.梅俏,许建明,赵宗豪,等.褪黑素影响免疫性结肠炎的几种炎性细胞因子表达检测.中华微生物学和免疫学杂志. 2004; 24 (9): 711-714
32. Kriegsfeld LJ, Drazen DL, Nelson RJ. In vitro melatonin treatment enhances cell-mediated immune function in male prairie voles (Microtus ochrogaster). J Pineal Res. 2001; 30(4):193-198
33. Garcia-Maurino S, Pozo D, Carrillo-Vico A, Calvo JR, Guerrero JM. Melatonin activates Th1 lymphocytes by increasing IL-12 production. Life Sci. 1999; 65(20):2143-2150
34. Sacco S, Aquilini L, Ghezzi P, Pinza M, Guglielmotti A. Mechanism of the inhibitory effect of melatonin on tumor necrosis factor production in vivo and in vitro. Eur J Pharmacol, 1998; 343(2-3):249-255
35. Carrillo-Vico A, Lardone PJ, Naji L, Naji L, Fernandez-Santos JM, Martin-Lacave I, Guerrero JM, Calvo JR. Beneficial pleiotropic actions of melatonin in an experimental model of septic shock in mice: regulation of pro-/anti-inflammatorycytokine network, protection against oxidative damage and anti-apoptotic effects. J Pineal Res. 2005; 39(4):400-408
36. Imamoglu M, Cay A, Cobanoglu U, Bahat E, Karahan C, Tosun I, Sarihan H. Effects of melatonin on suppression of renal scarring in experimental model of pyelonephritis. Urology, 2006; 67(6):1315-1319
37.薛桥,王士雯,卢才义,李泱,马路.褪黑素对肺炎小鼠心肌的保护作用.中国临床康复. 2004; 8(24):5011-5013
38. Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: downregulation of COX-2and iNOS through suppression of NF-kappaB activation. Mutat Res, 2001; 480-481 (1~2) :243-268
39. Crespo E, Macias M, Pozo D, Escames G, Martin M, Vives F, Guerrero JM, Acuna-Castroviejo D. Melatonin inhibits expression of the inducible NO synthaseⅡin liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats. FASEB J. 1999; 13(12):1537-1546
40. Escames G, Leon J, Macias M, Khaldy H, Acuna-Castroviejo D. Melatonin counteracts lipopolysaccharide-induced expression and activity of mitochondrial nitri oxide synthase in rats. FASEB J. 2003; 17(8):932-934
41. Deng WG, Tang ST, Tseng HP, Wu KK. Melatonin suppresses macrophage cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation and binding. Blood, 2006;108(2):518-524
42.王亚亭,陈胜利,徐叔云.褪黑素对哮喘大鼠核因子-κB表达的影响及抗炎作用.中华儿科杂志. 2004; 42 (2): 94-97
43. Bonilla E, Valero N, Chacin-Bonilla L, Medina-Leendertz S. Melatonin and viral infections. J Pineal Res, 2004; 36(2):73-79
44. Negrette B, Bonilla E, Valero N, Pons H, Garcia Tamayo J, Chacin-Bonilla L, Medina-Leendertz S, Anez F. Melatonin Treatment Enhances the Efficiency of MiceImmunization with Enezuelan Equine Encephalomyelitis Virus TC283. Neurochem Res. 2001; 26(7): 767-770
45. Bonilla E, Rodon C, Valero N, Pons H, Chacin-Bonilla L, Garcia Tamayo J, Rodriguez Z, Medina-Leendertz S, Anez F. Melatonin prolongs survival of immunodepressed mice infected with the Venezuelan equine encephalomyelitis virus. Trans R Soc Trop Med Hyg. 2001; 95(2):207–210
46. Valero N, Bonilla E, Pons H, Chacin-Bonilla L, Anez F, Espina LM, Medina-Leendertz S, Garcia Tamayo J. Melatonin induces changes to serum cytokines in mice infected with the Venezuelan equine encephalomyelitis virus. Trans R Soc Trop Med Hyg. 2002; 96(3):1–4
47. Finocchiaro LM, Arzt ES, Fernandez S, Criscuolo M, Finkielman S, Nahmod VE. Serotonin and melatonin synthesis in peripheral blood mononuclear cells: stimulation by interferon-gamma as part of the immunomodulatory pathway. J Interferon Res. 1988; 8(6):705–716
48. Bonilla E, Valero N, ChacI′n-Bonilla L, Pons H, Larreal Y, Medina-Leendertz S, Espina LM. Melatonin increases interleukin-1βand decreases tumor necrosis factor alpha in the brain of mice infected with the venezuelan equine encephalomyelitis virus. Neurochem Res. 2003; 28(5):687–692
49. Ben-Nathan D, Maestroni GJM, Lustig S, Conti A. Protective effects of melatonin in mice infected with encephalitis viruses. Arch Virol. 1995; 140(2):223–230
50. Shiu SYW, Reiter RJ, Tan DX, Pang SF. Urgent search for safe and effective treatments of severe acute respiratory symdrome: is melatonin a promising candidate drug. J Pineal Res, 2003; 35(1):69–70