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Toll样受体阻断剂对内毒素血症小鼠肠黏膜损伤的影响
Effects of Toll-like receptor blockers on intestinal mucosal injury in mice with endotoxemia
目的 探究Toll样受体（TLR）阻断剂对小鼠肠黏膜上皮细胞间紧密连接蛋白ZO-1的影响及对核转录因子-κB （NF-κB）和肿瘤坏死因子-α（TNF-α）的影响。方法 将32只BALB/C小鼠分为对照组、模型组、TLR4处理组、TLR2处理组（n=8），腹腔注射LPS建立内毒素血症小鼠模型，TLR4处理组、TLR2处理组在腹腔注射LPS同时分别给予TLR4抗体和TLR2抗体（10 μg/只）腹腔注射，对照组以生理盐水替代。取各组小鼠远端小肠组织，采用RT-PCR及免疫组化法检测ZO-1、NF-κBp65及TNF-α的mRNA和蛋白定位表达。结果 模型组ZO-1 mRNA及蛋白表达明显低于对照组（P < 0.05），NF-κBp65、TNF-α mRNA及蛋白表达明显高于对照组（P < 0.05）；TLR4处理组及TLR2处理组ZO-1 mRNA及蛋白表达明显高于模型组（P < 0.05），NF-κBp65、TNF-α mRNA及蛋白表达明显低于模型组（P < 0.05）；TLR4处理组与TLR2处理组ZO-1、NF-κBp65、TNF-α mRNA及蛋白表达比较差异无统计学意义（P > 0.05）。结论 抗TLR2、TLR4单克隆抗体可减少核转录因子的激活，抑制炎症因子大量分泌，保护紧密连接蛋白，有望对治疗肠源性感染疾病提供新的思路。
Objective To investigate the effects of Toll-like receptor blockers TLR2-Ab and TLR4-Ab on the tight junction protein ZO-1 in intestinal epithelial cells in mice, as well as their effects on nuclear factor-kappa B (NF-κB) and tumor necrosis factor-α (TNF-α).Methods A total of 32 BALB/C mice were divided into control group, model group, TLR4 treatment group, and TLR2 treatment group, with 8 mice in each group. A mouse model of endotoxemia was established by intraperitoneal injection of lipopolysaccharide. The mice in the TLR4 treatment group and the TLR2 treatment group were given intraperitoneal injection of TLR4 antibody and TLR2 antibody (10 μg each mouse), respectively, and those in the control group were given normal saline. The distal small intestinal tissue was collected, and RT-PCR and immunohistochemistry were used to measure the mRNA and protein expression of ZO-1, NF-κBp65, and TNF-α.Results Compared with the control group, the model group had significantly lower mRNA and protein expression of ZO-1 and significantly higher mRNA expression of NF-κBp65 and TNF-α (P < 0.05). Compared with the model group, the TLR4 treatment group and the TLR2 treatment group had significantly higher mRNA and protein expression of ZO-1 and significantly lower mRNA and protein expression of NF-κBp65 and TNF-α (P < 0.05). There were no significant differences in the mRNA and protein expression of ZO-1, NF-κBp65, and TNF-α between the TLR4 treatment group and the TLR2 treatment group (P > 0.05).Conclusions Anti-TLR2 and anti-TLR4 monoclonal antibodies can reduce the activation of nuclear transcription factors, inhibit the secretion of inflammatory factors, and protect tight junction protein, which is expected to provide new ideas for the treatment of enterogenous infectious diseases.
肠黏膜屏障 / Toll样受体 / ZO-1 / 核转录因子-κB / 肿瘤坏死因子-α / 小鼠
Intestinal mucosal barrier / Toll-like receptor / ZO-1 / Nuclear factor-κB / Tumor necrosis factor-α / Mice
[1] 罗兵, 李涛, 徐元宏. Toll样受体的信号转导及抗感染免疫研究进展[J]. 免疫学杂志, 2011, 27(2):165-169.
[2] 李维朝, 蒋电明, 朱凤臣, 等. 内毒素耐受的分子机制研究进展[J].中国免疫学杂志, 2010, 26(8):764-768.
[3] 王丽杰, 刘春英, 许玲芬, 等. 血小板活化因子受体拮抗剂对幼年大鼠肠黏膜上皮细胞间紧密连接蛋白的影响[J]. 世界华人消化杂志, 2006, 14(4):392-397.
[4] 白小武, 嵇武, 丁博文, 等. 大黄素对肠黏膜屏障损伤的保护作用及机制研究[J]. 东南国防医药, 2012, 14(1):12-15.
[5] Shindou H, Ishii S, Yamamoto M, et al. Priming effect of lipopolysaccharide on acetyl-coenzyme A:lyso-platelet-activating factor acetyltransferase is MyD88 and TRIF independent[J]. Immunol, 2005, 175(2):1177-1183.
[6] 刘志华, 秦环龙. 肠上皮细胞紧密连接基因及信号调控的研究进展[J]. 中华临床营养杂志, 2010, 18(5):310-316.
[7] Zareie M, Riff J, Donato K, et al. Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function[J]. Cell Microbiol, 2005, 7(12):1782-1797.
[8] Lendemans S, Kreuzfelder E, Rani M, et al. Toll-like recepor 2 and 4 expression after severe injury is not involved in he dysregulation of the innate immune system[J]. J Trauma, 2007, 63(4):740-746.
[9] 牛海刚. LPS-CD14-TLR传导通路的研究进展[J]. 山西医科大学学报, 2004, 35(1):76-78.
[10] Yuki T, Yoshida H, Akazawa Y, et al. Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes[J]. J Immunol, 2011, 187(6):3230-3237.
[11] Boulard O, Asquith MJ, Powrie F, et al. TLR2-independent induction and regulation of chronic intestinal inflammation[J]. Eur J Immunol, 2010, 40(2):516-524.
[12] González-Mariscal L, Betanzos A, Nava P, et al. Tight junction proteins[J]. Prog Biophys Mol Biol, 2003, 81(1):1-44.
[13] Huang B, Zhao J, Unkeless JC, et al. TLR signaling by tumor and immune cells:a double-edged sword[J]. Oncogene, 2008, 27(2):218-224.
[14] Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway[J]. Cytokine, 2008, 42(2):145-151.
[15] Kim TW, Staschke K, Bulek K, et al. A critical role for IRAK 4 kinase activity in Toll-like receptor-mediated innate immunity[J]. J Exp Med, 2007, 204(5):1025-1036.
[16] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity[J]. Cell, 2006, 124(4):783-801.
[17] Mazzon E, Sturniolo GC, Puzzolo D, et al. Effect of stress on the paracellular barrier in the rat ileum[J]. Gut, 2002, 51(4):507-513.
[18] Fukata M, Michelsen KS, Eri R, et al. Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis[J]. Am J Physiol Gastrointest Liver Physiol, 2005, 288(5):G1055-G1065.
[19] Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, et al. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis[J]. Cell, 2004, 118(2):229-241.
[20] Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function[J]. Gastroenterology, 2007, 132(4):1359-1374.
[21] Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier[J]. Am J Physiol Gastrointest Liver Physiol, 2006, 290(3):G496-G504.
[22] 李春艳, 肖福大, 于明. 幼鼠肠缺血再灌注损伤时TNF-α的产生及作用[J]. 中国医科大学学报, 2002, 31(4):241-243.
[23] Andonegui G, Goyert SM, Kubes P. Lipopolysaccharide-induced leukocyte-endothelial cell interactions:a role for CD14 versus toll-like receptor 4 within microvessels[J]. J Immunol, 2002, 169(4):2111-2119.
[24] 高志光, 秦环龙. 肠上皮细胞紧密连接的生物学功能及在肠屏障中的作用[J]. 肠外与肠内营养, 2005, 12(5):299-302.
[25] Han X, Fink MP, Delude RL. Proinflammatory cytokines cause NO*-dependent and -independent changes in expression and localization of tight junction proteins in intestinal epithelial cells[J]. Shock, 2003, 19(3):229-237.
[26] 崔巍, 周莹, 刘沛. 谷氨酰胺对肿瘤坏死因子-α引起的肠上皮紧密连接损害的保护作用[J]. 山西医药杂志, 2009, 38(6):491-493.
[27] Kuo IH, Carpenter-Mendini A, Yoshida T, et al. Activation of epidermal toll-like receptor 2 enhances tight junction function:implications for atopic dermatitis and skin barrier repair[J]. J Invest Dermatol, 2013, 133(4):988-998.
[28] Ragupathy S, Esmaeili F, Paschoud S, et al. Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1[J]. Tissue Barriers, 2014, 2:e29166.
