基于“酸入肝”“肝开窍于目”探讨酸味中药及五味间的作用机制

This research is on the basis of traditional Chinese medicinal theory that sour herbs in taste enter the liver and enrich liver and the liver has its aperture in the eyes, so we propose the hypothesis that “Chinese medicinal materials that are sour in taste could promote the combination with the Vitamin A correlated proteins and (or) promote the transportation of Vitamin A correlated proteins, improve the expression of a series of proteins during deficiency syndrome, ameliorate the structural and functional alterations in the eye region of the deficiency syndrome experimental animals and inhibit the inflammation reactions. Chinese medicinal materials that are salty and bitter in taste have the similar and weaker effects compared with sour herbs in taste. While the acrid and sweet herbs in taste reveal opposite effectiveness in contrast with sour herbs in taste”. Through using animal models of ovariectomized rats cause of dry eye, we apply the morphology observation, PCR, western blotting, HPLC, chromatin immunoprecipitation assay (ChIP) and laser scanning confocal microscope techniques to observe the influences of five kinds of flavors on the structure of lachrymal gland, apoptosis and local inflammatory factors. By means of analyzing the expression, combination and (or) transportation effects of Vitamin A binding protein (RBP) and the receptor of STRA6 as well as analyzing the influences of five kinds of flavors on the expressions of mucoprotein (MP) and prealbumin (PA) to testify the hypothesis. This investigation aims to partial reveal the molecular mechanism that sour herbs in taste enter the liver and the liver has its aperture in the eyes. Furthermore, this research explores the relationships among Chinese medicinal materials of five flavors and mutual promotion and mutual restriction relationships among the five elements in depth. It is of great significance in promoting the modern studies and application.

本课题在前期工作基础上，根据传统酸入肝，酸补肝，肝开窍于目的中医学理论，我们提出“补肝的酸味中药能促进转运维生素A的相关蛋白的表达和/或结合，改善虚证相关的系列蛋白表达和虚证实验动物眼组织结构和功能变化，降低炎症反应，咸、苦中药有类似酸味但弱于酸味作用，辛、甘中药与酸味中药作用相反”的假说。利用去势大鼠导致干眼症的动物模型，通过形态学观察、聚合酶链式反应、免疫印迹法、高效液相色谱法、免疫共沉淀技术和激光共聚焦方法等来观察五味中药对实验动物泪腺组织结构、细胞凋亡和局部炎症因子的影响；分析对维生素A结合蛋白（RBP）及其受体STRA6的表达、结合和/或转运作用，检测对粘蛋白和泪液前蛋白的表达影响来证明假说。目的在于部分地揭示酸入肝，肝开窍于目的分子机制，并进一步探讨中药五味间、五行生克制化间的关系，推动中药性味学说的现代研究，促进性味学说在理论上的发展和实践中的运用。

实验选用健康1月龄雄性小鼠90只，采用随机数字法分为9组，正常组，假手术空白组，模型组，假手术加酸味组，酸味中药治疗组，苦味中药治疗组，甘味中药治疗组，辛味中药治疗组，咸味中药治疗组，每组10只。去势造模后，各干预组连续给药四个月后全部处死，取材，并观察试验动物的生存状态。采用泪液分泌试纸测定了各组大鼠泪液分泌量，Schirmer I试验（SIT）和泪膜破裂时间（BUT）检查法测定了各组小鼠泪膜稳定性。采用HE染色法，观察了各组小鼠泪腺、角膜及结膜的病理改变。采用电镜的方法观察了小鼠泪腺、角膜的超微结构。免疫组织化学法检测了角膜、泪腺组织中TNF-α、IL-1β、IL-6、TGF-β、IL-8、IL-10的表达水平。ELISA法检测了泪液组织中黏蛋白的含量。免疫印迹及PCR法检测了泪腺组织中黏蛋白、bax、bcl-2、RBP及STRA6 mRNA及蛋白表达水平。免疫荧光及免疫共沉淀法检测了RBP及STRA6的表达。.实验结果表明：苯扎氯铵连续滴眼6周后，模型组小鼠泪液分泌量及泪膜破裂时间均显著减少和缩短，提示模型复制成功。药物干预后，各干预组小鼠泪液分泌量不同程度增加，泪膜破裂时间不同程度延长，以高剂量山茱萸干预效果最为明显。各干预组小鼠结膜、角膜以及泪腺组织中IL-1β、IL-6、TNF-α的表达水平均有不同程度的下调，且高中低剂量山茱萸煎剂对IL-1β、IL-6、TNF-α表达水平的抑制程度具有量效关系。酸味药能够显著抑制干眼小鼠泪腺及角膜细胞凋亡的发生。各干预组小鼠泪腺组织中bax表达水平显著降低，而bcl-2、RBP及STRA6表达水平显著升高，泪液中PALB含量显著增加。完成了合同书中所有的规划指标。.依托本项目，完成科研论文发表5篇，培养研究生2名。.本项目部分地揭示酸味入肝，肝开窍于目的分子机制，并进一步探讨中药五味间的关系，推动中药传统性味学说的现代研究，促进性味学说在理论上的发展和实践中的运用，并结合现代实验技术验证五行理论。但酸味药调控炎性因子表达的更深层次的分子机制尚需进一步研究。