基于海马(杏仁核)-HPA轴研究艾条悬灸治疗皮质酮肾阳虚大鼠的作用机制

Based on the efficacy of moxibustion treating syndrome of kidney-YANG deficiency in clinic and the influences of acupuncture on kidney-YANG deficiency rats in early research, taking hippocampus(amygdala)- HPA axis as keypoint, this research expect to find out the mechanism of suspension moxibustion on Guanyuan(RN4) and Shenshu(BL23) with moxa stick in treating kidney-YANG deficiency rats. This research aims: To observe the influence of suspension moxibustion in treating kidney-YANG deficiency rats by detecting the mRNA and polypeptide express of MR and MG in hippocampus and in amygdale,CRF and CRH in amygdale and in hypothalamus,ACTH in pituitary and 11β-HSD1 in those organs mentioned above using RT-PCR, Western blot and immunohistochemistry of hybridization in situ；by detecting the content of CRH in amygdale and in hypothalamus,ACTH in pituitary,CORT in adrenal gland and those indexes mentioned above in serum using radioimmunity； To discuss the key effect component element of suspension moxibustion in influencing hippocampus (amygdala)-HPA axis; To expect to confirm that suspension moxibustion can up-regulate the mRNA and polypeptide express of MR in hippocampus, up-regulate the express of MR and GR in amygdale, down-regulate GR express in hippocampus, promote the express of 11β-HSD1 in hippocampus and amygdale, increase the express and the content of CRH in amygdale and hypothalamus, ACTH in pituitary, CORT in adrenal gland, and that this is the important molecule mechanism of suspension moxibustion to improve syndrome of deficiency of kidney-YANG, therefore provide theory evidence for moxibustion treating kidney-YANG deficiency patients.

基于临床艾灸治疗肾阳虚证的有效性及针灸对肾阳虚大鼠影响的前期研究，本研究以海马(杏仁核)-HPA轴为切入点，以冀探明艾条悬灸关元、肾俞治疗肾阳虚大鼠的作用机制。本研究拟应用RT-PCR、Western blot和原位杂交方法检测悬灸对肾阳虚大鼠海马和杏仁核中MR和GR、杏仁核和下丘脑中CRF和CRH、垂体ACTH以及上述器官中11β-HSD1mRNA及多肽表达的影响，用放免法检测杏仁核和下丘脑CRH、垂体ACTH、肾上腺CORT以及血清中上述指标含量的影响，探讨悬灸影响海马(杏仁核)-HPA轴的关键作用环节，拟证实悬灸能上调海马中MR mRNA及多肽的表达，上调杏仁核中MR及GR的表达、下调海马中GR的表达，促进海马和杏仁核中11β-HSD1的表达，增加杏仁核和下丘脑CRH、垂体ACTH、肾上腺CORT的表达及含量是改善肾阳虚证的重要分子机制，为艾灸治疗肾阳虚证提供理论依据。

艾灸对肾阳虚证具有较好的疗效，但其机制值得研究。研究证实下丘脑-垂体-肾上腺轴(HPA轴)的功能紊乱是肾阳虚证的主要物质基础，而海马、杏仁核等是影响HPA轴的上位调控中枢。糖皮质激素(GC)在海马(杏仁核)-HPA轴调控中起到信使作用，海马和杏仁核中的盐皮质激素受体(MR)、糖皮质激素受体(GR)通过浓度依赖性的与GC结合，从而对HPA轴进行调控。而GC的浓度又主要受11β-羟类固醇脱氢酶(11β- HSD)的调控。基于课题组的前期研究，我们提出艾条悬灸可能通过调控海马(杏仁核)-HPA轴相关因子治疗肾阳虚证的假设。.本研究采用氢化可的松注射液肌注14天建立肾阳虚大鼠模型,通过观察造模后大鼠一般情况、兴趣指数、体重，检测血清睾酮、雌二醇的水平，且经右归丸反证确认模型成功后，80只大鼠如上方法造模，分为模型组、艾灸组、甘珀酸组和艾灸+甘珀酸组，另设立空白对照组。根据分组方案予以相应治疗14天，提取相应组织进行检测。ELISA方法检测血清CRH、ACTH、CORT的含量；RT-PCR、原位杂交和WB技术分别检测海马和杏仁核中MR和GR、杏仁核和下丘脑中CRH、垂体ACTH 以及上述器官中11β-HSD1 基因和蛋白的表达。研究结果显示：造模后，血清CRH、ACTH、CORT的含量下降，海马和杏仁核中MR和GR、杏仁核和下丘脑中CRH、垂体ACTH 以及上述器官中11β-HSD1 基因和蛋白的表达下调。治疗后，与模型组比较，除PCR检测海马MR、GR和下丘脑11β-HSD1基因表达外，其它上述指标艾灸组都明显升高；艾灸+甘珀酸组大鼠上述大部分指标较模型组明显升高；甘珀酸组大鼠上述小部分指标较模型组明显升高。除原位杂交检测海马和杏仁核MR基因外，其它所有指标艾灸组与艾灸+甘珀酸组比较都无明显差异；艾灸组上述绝大部分指标较甘珀酸组明显升高；艾灸+甘珀酸组只有部分上述指标明显高于甘珀酸组。.本研究阐明了艾灸改善肾阳虚证的可能作用机制，即艾灸肾俞、关元穴可有效改善海马(杏仁核)-HPA轴的功能。本研究为艾灸治疗肾阳虚证的机制研究提供了实验依据和理论基础。考虑到本研究肾阳虚证的实验模型在病理上与人类有一些相似之处，因此，MR、GR和11β-HSD 1有可能成为以肾阳虚证为基础的多种疾病或慢病治疗的新靶点。