从“M3R/AQP5”研究酸甘化阴法促进干燥综合征唾液分泌的作用机制

Compared with western medicine, the treatment over Sjogren syndrome with traditional Chinese medicines shows the remarkable advantage that improves the dryness of the mouth and eyes, and the SuanGanHuaYin therapy can nourish yin and blood as well as engender liquid. The sweet herb can nourish spleen that controls the saliva and the sour herb can nourish liver that controls tears. The clinical manifestation shows that the SuanGanHuaYin therapy can promote the secretion of the saliva and tear of the Sjogren syndrome patients. As shown by animal experiment, this therapy that improves the dryness of the mouth is associated with the increase in the expression of the AQP5 in salivary glands. Saliva secretion is under the control of membrane receptor and its effector protein as well as the intracellular transduction signal. Sjogren syndrome patients show positive reaction to member receptor antibody. This study is to prove that this therapy plays a role in various saliva secretion links such as M3R (membrane receptor), AQP5 (effector protein) and cAMP/PKA (intracellular transduction signal), able to control the expression of the specific antibody of Sjogren syndrome to treat both symptoms and root causes. In the in-vivo experimental study, traditional Chinese medicine played a role in M3R and AQP5 in the salivary glands of rat model, and the expression of the specific antibody of peripheral blood was also tested. In in-vitro experiment, the cell strain of labial gland acinus was used to study not only the effect of traditional Chinese medicine on the gene of M3R and AQP5 as well as protein expression in acinus cell but also the phosphorylation of cAMP/PKA signal pathway. The study will expand the connotations of SuanGanHuaYin therapy, laying a foundation for the formula optimization and clinical application of this therapy.

与西药相比，中药治疗干燥综合征的突出优势在于改善口干眼干症状，酸甘化阴法能滋阴养血生津，且甘入脾，脾主涎；酸入肝，肝主泪。临床发现酸甘化阴法能增加干燥综合征患者唾液和泪液分泌。动物试验表明：该法改善口干症状与增加唾液腺水通道蛋白5（AQP5）的表达有关。唾液分泌受膜受体及其效应蛋白、胞内转导信号控制，干燥综合征患者存在膜受体抗体阳性。本研究拟证明该法可能在M3R（膜受体）、AQP5（效应蛋白）、cAMP/PKA（胞内转导信号）等多个唾液分泌的环节起作用，并且能控制干燥综合征特异性抗体的表达，起到标本兼治的作用。体内试验研究中药对模型鼠唾液腺中的M3R及AQP5作用；并检测外周血特异性抗体的表达。体外试验采用人唇腺腺泡细胞株，研究药物对腺泡细胞中M3R、AQP5的基因和蛋白表达的影响，研究cAMP/PKA信号通路的磷酸化情况。研究将拓宽酸甘化阴法的内涵，为该法组方优化和临床应用奠定基础。

与西药相比，中药治疗干燥综合征的突出优势在于能明显改善患者口干眼干的症状。酸甘化阴法能滋阴、养血、生津，且甘入脾，脾主涎；酸入肝，肝主泪。临床发现酸甘化阴法能增加干燥综合征患者唾液和泪液的分泌。唾液分泌受膜受体及其效应蛋白、胞内转导信号控制，已证实干燥综合征患者体内存在膜受体抗体。本课题分为体内实验及体外实验。体内实验研究芍药甘草汤对模型鼠唾液腺中的M3R（膜受体）及AQP5（效应蛋白）的作用，并检测外周血特异性抗体的表达。结果显示，小鼠外周血中芍药甘草汤高剂量组SSA、SSB、α-Fodrin的表达水平最低，M3R的表达水平最高；中剂量组AQP5 mRNA、M3R mRNA的表达水平最高；中、高剂量组TNF-α、TNFR1、NF-κB的mRNA表达水平均较生理盐水组及毛果芸香碱组低；高剂量组PKA蛋白表达高于其他各组；高剂量组P56(NF-κB)蛋白表达最低。体外实验采用人唇腺腺泡细胞株，从转录水平和翻译水平研究药物对腺泡细胞中M3R、AQP5表达的影响。结果显示，芍药甘草汤（5:1）组细胞顶膜的荧光强度最强，说明中药有促进AQP5蛋白重新分布，提高细胞水转运的作用。芍药甘草汤（5:1）组AQP5、M3R的蛋白及mRNA表达水平最高。芍药甘草汤（5:1）组对AQP5“短时调节”及“长时调节”的效果最为明显。研究将拓宽酸甘化阴法的内涵，为该法组方优化和临床应用奠定基础。