糖皮质激素引发药源性证候及补肾中药的调控机制研究

The primary syndromes of drug-origin in the treatment of various stubborn and chronic diseases by glucocorticoid is a new problem that affects the efficacy of traditional Chinese medicine. In the past period of time, the study of kidney deficiency syndrome induced by glucocorticoid in human and experimental animals concentrating on hormones secretion and metabolites, however, the profound mechanism exploration is not enough. The hypothesis has been proposed in this project, hypocorticalism is the root cause of drug-induced syndrome induced by glucocorticoid, tonifying kidney herbs maybe improving the efficiency of the conversion from cholesterol to steroid hormones in adrenocortical cells, thus, the function of adrenal cortex can be protected or self recovery. Therefore, we will carry out animal experiments and expect to develop standardized animal modeling scheme by imitating clinical glucocorticoid medication way. Then, the evaluation index system of primary syndromes of drug-origin by hydrocortisone will be formulated by mice characterization informations and adrenal cortex function indexs. Then, the efficiency of cholesterol transformation and steroid synthesis in the adrenal tissue and primary cultured adrenal cortical cells of mice induced by hydrocortisone was observed with the intervention of tonifying kidney herbs. In order to reveal the new targets of molecular pharmacology of different tonifying kidney herbs, and to find the new mechanism of primary syndromes of drug-origin by glucocorticoid and enrich the theory of kidney deficiency syndrome in TCM.

糖皮质激素在治疗临床多种难治病、慢性病过程中引发的药源性证候，是影响中医药疗效的新问题。既往学术界对皮质激素诱发人类肾虚证及其动物模型的研究停留于激素分泌及代谢产物层面，缺乏深入的机制探索。本研究提出“肾上腺皮质功能减退是糖皮质激素引起的药源性证候的根源，补肾中药通过提高肾上腺皮质细胞内胆固醇转化为类固醇激素的效率，从而发挥保护与恢复肾上腺皮质功能的作用”的科学假说。为此，我们将开展有关动物实验，模拟临床糖皮质激素用药方式制定标准化的动物造模方案，通过小鼠表征信息与反映肾上腺皮质功能客观指标，建立氢化可的松药源性证候的评价指标体系。进而通过常用补肾中药干预，观察对氢化可的松药源性证候小鼠肾上腺组织与原代培养的肾上腺皮质细胞胆固醇转化与合成类固醇激素的效率，以揭示不同补肾中药的分子药理作用新靶标，发现糖皮质激素药源性证候在肾上腺皮质功能层面的新机制，丰富中医肾虚证候理论。

本项目围绕临床慢性难治病及危重症状态下需要使用糖皮质激素药物，进而可能诱发的激素药源性证候的现象，开展了一系列的研究，主要包括：1）观察了不同品系小鼠对泼尼松龙诱发药源性证候的差异性，结果提示ICR小鼠对激素造成的小鼠药源性虚证更稳定，表现为肾虚、脾虚，以垂体-肾上腺皮质轴功能被抑制为主。2）分别评价了氢化可的松、泼尼松龙、地塞米松诱发小鼠药源性虚证的特征，结果发现采用3.3 mg/(kg·d)氢化可的松给药14天、2.5mg/(kg·d)泼尼松龙用药7天、0.25mg/(kg·d)地塞米松用药7天，均可建立稳定的药源性脾肾虚证小鼠模型，其物质基础以类固醇激素合成酶分子表达下降、以及垂体-肾上腺皮质轴功能受抑制为主。3）观察了激素不同给药方案对小鼠证候的影响，发现大于25 mg/(kg·d)氢化可的松短期给药5天可显著抑制小鼠肾上腺皮质功能，其证候表现以药源性气阴两虚证为主；小于1.32 mg/(kg·d)氢化可的松长期给药可诱发小鼠气阴两虚证表象，且肾上腺皮质功能被抑制，药物减停后小鼠外观表现自我恢复快而皮质酮分泌功能恢复慢。4）观察了不同补肾中药对氢化可的松药源性虚证小鼠的药效，发现地黄、知母、黄柏单独及其配伍治疗可通过调节胆固醇稳态平衡及恢复部分受抑制的皮质激素合成酶，配伍给药更有助于保护受损伤的肾上腺皮质功能；淫羊藿、仙茅、杜仲、菟丝子等温补肾阳中药可促进由氢化可的松诱导的虚证小鼠肾上腺皮质激素合成能力；四君子汤、六味地黄丸、桂附地黄汤对氢化可的松诱发的肾上腺皮质功能抑制均有缓解作用，桂附地黄汤的药效更明显。5）研究了补肾中药对氢化可的松诱导的肾上腺Y1细胞皮质酮合成的作用，发现淫羊藿对模型细胞的皮质酮合成具有促进作用，可上调Star和Hsd3b1基因表达，其中淫羊藿苷是最主要的药效成分；而黄柏则具有抑制作用，可下调StAR、CYP11A1和3β-HSD2蛋白表达，降低皮质酮含量。6）规范了糖皮质激素诱发小鼠药源性证候模型的造模方法，并建立起量化的疗效评价标准，为中药及复方药效研究提供实用的小鼠证候模型。