从白介素17-水通道蛋白-血清AQPs抗体探讨“针刺润燥”治疗干燥综合征增效作用机制

With no radical therapy, Sjogren's syndrome (SS) affects quality of life of patients. Relieving clinical symptoms is the core of treatment in most cases without multiple organs involvement, in which structural destroy and secreting functional decrease of exocrine glands should be improved. As the key factor in chronic inflammation among the SS glands, interleukin 17 affects the expression and function of aquaporin (AQP). SS was first named by Professor Lu Zhizheng as "Dry Bi" in TCM. And we treat SS with acupuncture for a long time following the wisdom and experiences of Professor Lu. A number of clinical studies we did show that the acupuncture has a good effect on SS in improving the symptoms, and slows down the structure destroy of salivary glands. However, the mechanism awaiting to study. On one hand, meridian blocking, very essential in the onset of dry Bi in TCM theory, is closely related to inflammatory factors showed by many studies. On the other hand, acupuncture has a noted function of dredging meridians. Then the possible mechanism of acupuncture for SS is that it inhibits IL-17, improves AQPs expression and function in glands, while reduces serum AQPs antibodies, thereby promotes glandular secretion. In this study, by the combination of human and animal experiments, with the application of monoclonal antibodies and multicolor flow cytometry, ELISA, semi-quantitative RT-PCR, PA-HRP indirect method, Western blot method, etc. we will detect the above factors to explore the molecular mechanism of acupuncture for SS treatment, which is meaningful for clinical practice and need to be supported.

干燥综合征(SS)是一种自身免疫性疾病，改善临床症状是大多数无内脏受损的病例治疗核心。这需要通过抑制患者外分泌腺结构破坏、改善分泌功能来实现。白介素17正是SS外分泌腺体慢性炎症关键因子，还可影响水通道蛋白(AQP)表达及功能。“针刺润燥”疗法传承我院路志正教授针药并用治疗“燥痹”学术思想，前期扎实的临床研究示其改善SS症状增效作用明显、延缓外分泌腺结构破坏，而缺少其机制研究。“针刺润燥”临床疗效实质是针刺疏通经络而润燥，其机制可能为：针刺抑制IL-17，改善局部AQP表达和功能，同时降低血清AQP抗体，进而改善腺体分泌功能。本研究结合人体和动物实验，应用单克隆抗体和多色流式细胞术检测、ELISA、半定量RT-PCR、PA-HRP间接法、Western blot等法检测上述成分，探讨针刺治疗SS增效作用的分子机制。为临床实践“针刺润燥”提供理论依据和科学内涵，以丰富中医治疗SS理论和方法。

本课题基于水通道蛋白作为外分泌腺体的终点功能分子，研究其抗体在 SS 患者血清，以及 SS 模型动物体内的表达，及其和相关上游因子 IL-17 之间的关系，以揭示“针刺润燥”机制的科学内涵。 通过临床试验，观察 SS 患者血清中抗 AQP1、5、8、9 抗体表达谱以及血清 IL-17 分子的表达比较在针刺治疗前后的差异。通过 SS 动物模型为非肥胖型糖尿病小鼠（Non-ObeseDiabetic mice，NOD），进行病理形态学观察，观察各组大鼠颌下腺：颌下腺指数、组织学观察、免疫组织化学；唾液流量；进行比较。并对各组 NOD 小鼠颌下腺 AQP1 和 AQP5 及其 mRNA 的表达，外周血抗 AQP1、5、8、9 抗体的表达。观察各组小鼠外周血 PBMCs、脾脏和颌下腺中 TLR9、IFN-α 、BAFF mRNA 表达的影响作用，以及外周血 IL-17 的表达，研究针刺对其作用和影响。结果表明：第一、针刺能明显降低SS患者血清炎性因子白介素17水平，减轻唾液腺炎症，改善唾液腺的结构及功能，有利于缓解口干症状。第二、针刺廉泉穴和颊车穴能够改善NOD小鼠干燥综合征样局部口干症状，抑制颌下腺淋巴细胞浸润，减轻唾液腺炎症，增加颌下腺水通道蛋白的表达，为针刺治疗干燥综合征的基础和临床研究提供了理论依据。第三、针刺对NOD小鼠血清炎性因子的表达无明显改善，可以考虑将针刺作为干燥综合征的有效对症治疗，切实解决局部口干燥症，结合其他全身受累情况合并相应药物系统治疗，进一步研究与推广。