【作者】 黄绥心；

【导师】 范永升； 吴启富；

【作者基本信息】 浙江中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的：观察益气养阴祛瘀方对NOD (non-obese diabetic)小鼠TLR-IFN-BAFF信号通路的调控作用,探讨益气养阴祛瘀方对干燥综合征的治疗作用及其机制,为其在临床上应用提供理论依据。方法：将30只8周龄NOD小鼠随机分为3组,即模型组、羟氯喹组、益气养阴祛瘀方组(中药组),每组10只。ICR小鼠10只做为正常对照组。中药组每天按照27.4g/kg灌胃益气养阴祛瘀方；羟氯喹组按照每天75mg/kg灌胃羟氯喹。正常组和模型组正常饲养。实验期间每日记录小鼠平均饮水量,每周测定一次唾液分泌量。灌胃饲养12周后取血并处死小鼠,解剖分离脾脏、胸腺和颌下腺,计算脾脏、胸腺和颌下腺指数,光镜下观察各组小鼠颌下腺病理变化,酶联免疫法检测血清肿瘤坏死因子-α (Tumor necrosis factor-a, TNF-a)、白细胞介素-6(Interleukin-6, IL-6)、脱氢表雄酮(Dehydroepiandrosterone, DHEA)、雌二醇(Estrodio, E2)、免疫球蛋白G (Immunoglobulin G, IgG)水平,RT-PCR法检测外周血单个核细胞(Peripheral blood mononuclear cell, PBMC)中Toll样受体5(Toll like receptor5, TLR5)、 a-干扰素(Interferon-a, IFN-a)、 B淋巴细胞刺激因子(B-cell activating factor, BAFF) mRNA的表达,Western blot法检测脾脏和颌下腺组织TLR5、IFN-α、 BAFF蛋白的表达。结果：①NOD小鼠发病后每日饮水量增加,唾液分泌量减少,脾脏指数增高,颌下腺指数降低,并伴颌下腺组织淋巴细胞浸润等病理损害。益气养阴祛瘀中药能增加NOD小鼠唾液分泌量,减少其每日饮水量,调节脾脏、胸腺和颌下腺指数,缓解颌下腺病理损害。②NOD小鼠血清TNF-α、IL-6、IgG水平升高,血清DHEA、E2水平降低。益气养阴祛瘀中药能降低NOD小鼠血清TNF-a. IL-6. IgG水平,升高血清DHEA. E2水平,对自身免疫性炎症有抑制作用,并能调节性激素水平。③NOD小鼠PBMC中TLR5、IFN-a. BAFFmRNA表达量增加；脾脏及颌下腺组织TLR5、IFN-a、BAFF蛋白表达水平升高。中药益气养阴祛瘀方能降低干燥综合征样NOD小鼠PBMC中TLR5、IFN-a、BAFFmRNA及脾脏、颌下腺组织TLR5、IFN-a、BAFF蛋白的表达。结论：①益气养阴祛瘀方具有较好的改善NOD小鼠口干多饮症状及颌下腺组织病理损害、促进其分泌功能的作用。②益气养阴祛瘀方具有抑制炎症、调节免疫及性激素水平的作用。③益气养阴祛瘀方能下调TLR5、IFN-α、BAFFmRNA及蛋白的表达,调控TLR-IFN-BAFF信号通路,可能是治疗SS的作用机制之一④益气养阴祛瘀方是治疗NOD小鼠SS样病变较为有效的方法,值得进一步深入研究。更多还原

【Abstract】 Objective To observe the effect of Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula on regulating TLR-IFN-BAFF signal pathway in NOD mice, and to explore the therapeutic effect and mechanism of Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula in treating Sjogren’s syndrome, and offer experimental evidence for its clinncal application.Methods Thirty NOD mice were randomized into three groups with10in each,i.e.model group,hydroxychloroquine group, Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula group(Chinese Herbs group). Take10ICR mice as normal control group. Chinese Herbs group were given27.4g·kg-1of Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula decoction every day. Hydroxychloroquine group were given hydroxychloroquine75mg-kg-1by gavage. Mice of normal group and model group were given nomal saline. Average drinking amount of water every mouse were recorded per day and salivary secretory volume were determined per weeks.Mice were sacrificed after drawing blood from the femoral artery after12weeks, taking thymus, submaxillary glands, spleen and calculating organs index. Pathological changes of submaxillary glands were observed under light microscope.The levels of TNF-a, IL-6, DHEA, E2, IgG in serum were detected by ELISA.Expression of TLR5, IFN-a, BAFF mRNA in peripheral blood mononuclear cells were detected using reverse transcriptase polymerase chain reaction(RT-PCR).Expression of TLR5, IFN-a, BAFF protein in spleen and submaxillary glands were detected by western blot.Results①The amount of daily water intake increase, salivary secretory volume decrease, Spleen index rise, thymus and submaxillary glands index was reduced in NOD mice, with pathological damage in submaxillary glands, ie lymphocytic infiltration. Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula can increase the salivary secretory volume, decrease the amount of daily water intake of NOD mice, regulat the index of Spleen, and thymus and submaxillary glands, alleviate the pathological damage in submaxillary glands.②The levels of TNF-a, IL-6, IgG in serum rise, the levels of DHEA, E2in serum decrease in NOD mice. Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula can reduce the levels of TNF-a, IL-6, IgG in serum, promote the levels of DHEA, E2in serum, which can inhibition autoimmune inflammation, regulae the levels of sex steroids.③Expression of TLR5, IFN-a, BAFF mRNA in peripheral blood mononuclear cells and TLR5, IFN-a, BAFF protein in spleen and submaxillary glands in NOD mice increase. Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula can reduce expression of. TLR5, IFN-a, BAFF in PBMC, spleen and submaxillary glands.Conclusions①Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula can improve the symptom and pathological damage in submaxillary glands, promote secretion function of submaxillary glands.②Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula can depress inflammation, regulate immunologic function and the levels of sex steroid.③Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula can decrease expression of TLR5, IFN-a, BAFF mRNA and protein, regulate and control TLR-IFN-BAFF signal pathway, which may be one of the mechanisms in treatment of Sjogren’s syndrome.④Strengthening Qi, Nourishing Yin and Removing Blood Stasis Formula is an effective measure in treatment of Sjogren’s syndrome in NOD mice and is worth further studying.更多还原