Toll样受体9依赖的p38MAPK信号通路在原发性舍格伦综合征中的作用机制

摘要

目的：在动物模型NOD鼠中，研究Toll样受体9（Toll like receptor 9,TLR9）依赖的p38MAPK信号通路在原发性舍格伦综合征发病机制中的作用，从而寻找疾病药物治疗的新靶点。方法：选取4、5、8、10、15周龄的NOD雌性小鼠，利用流式细胞学技术检测小鼠外周血单个核细胞中TLR9、p-p38 MAPK 双阳性细胞的比率。利用免疫组化检测小鼠下颌下腺TLR9及p-p38 MAPK的表达情况。同时，观察小鼠刺激性唾液流率的改变以及下颌下腺的病理学改变。结果：TLR9、p-p38MAPK双阳性细胞在4、15周龄NOD鼠外周血单个核细胞中的表达，相对于正常对照组Balb/c小鼠无显著性差异。而自第5周开始，NOD鼠中双阳性细胞的比率逐渐升高，到第8周达到最高，第10周后逐渐下降。 TLR9在NOD鼠下颌下腺的浸润淋巴细胞和部分腺上皮细胞中呈阳性表达，p-p38在NOD鼠下颌下腺的浸润淋巴细胞和周围少量腺上皮细胞中呈阳性表达。 NOD鼠刺激性唾液流率自第5周起逐渐减少，相较于正常小鼠降低50%~60%。结论：从第5周到第10周，TLR9、p-p38MAPK双阳性细胞在NOD鼠中显著升高，同时伴随着刺激唾液流率的降低以及下颌下腺TLR9、p-p38MAPK阳性的淋巴细胞浸润。结果提示，外周血单个核细胞中TLR9依赖的p38MAPK信号通路的激活，可能在原发性舍格伦综合征发病早期起到重要作用，NOD鼠可用于p38 MAPK 或TLR9抑制实验的动物模型。%Objective: The objective of this study was to investigate the potential role of Toll-like receptor 9-dependent p38 MAPK signaling pathway in the pathogenesis of primary Sjögren's syndrome in NOD/Ltj mouse, aiming to identify an ideal target therapy model for human primary Sjögren's syndrome (pSS). Methods:NOD/Ltj mice were chosen as a model of primary Sjögren's syndrome. The Toll-like receptor 9 and p-p38 MAPK double positive peripheral blood mononuclear cells From 4-, 5-, 8-, 10- and, 15-week-old NOD/Ltj mouse were analyzed by flow cytometry. The expressions of Toll-like receptor 9 and p-p38 MAPK in the submandibular gland were also examined by immunohistochemistry. The change of stimulated salivary flow rate was dynamically measured, and the histopathology of submandibular gland was evaluated by hematoxylin and eosin stain. Results: The stimulated salivary flow rate in NOD was reduced to 50%~60% as compared to the flow rate of control mice since the fifth week onwards. The Toll-like receptor 9 and p-p38 MAPK double positive pe-ripheral blood mononuclear cells in both groups increased gradually from 5 weeks, peaked at 8 weeks and then gradually decreased at 10 weeks, yet the percentage of Toll-like receptor 9 and p-p38MAPK double positive peripheral blood mononuclear cells in 5-, 8-, and 10-week-old NOD mouse was significantly increased compared with that in control sub-jects. After the tenth week onwards, there were no significant difference in the Toll-like receptor 9 and p-p38 MAPK dou-ble positive peripheral blood mononuclear cells between NOD mouse and controls. Immunohistochemical staining showed that Toll-like receptor 9 were positive in the acinar epithelium cells and infiltrating lymphocytes in NOD mouse. P-p38 MAPK was detected in infiltrating lymphocytes and few ductal or acinar epithelium cells adjacent to infiltrating lympho-cytes in NOD mouse. Conclusions: From the fifth week till the tenth week, Toll-like receptor 9 and p-p38 MAPK double positive peripheral blood mononuclear cells was significantly increased in NOD mouse, accompanied with reduced stimulat-ed salivary flow rate and Toll-like receptor 9 or p-p38 MAPK positive infiltrating lymphocytes observed in the submandibu-lar gland of NOD mouse. Our results indicate that activation of Toll-like receptor 9-depended p38 MAPK signal pathway in peripheral blood mononuclear cells was an early event in primary Sj ögren's syndrome which made NOD as an ideal therapy model to test the treatment effects of p38 MAPK or Toll-like receptor 9 inhibitors on primary Sjögren's syndrome.