生地-山茱萸调控睾丸细胞糖酵解改善糖尿病雄性生殖损伤的作用及机制研究

With the increase of the incidence of Diabetes and younger age of onset, it has become an important cause of male reproductive disorders. Western medicine treatment has problems such as poor long-term efficacy and great side effects, while Chinese medicine has a strong advantage from the overall syndrome differentiation. Our previous work showed that the “enriching yin and nourishing kidney” medicine pair Radix Rehmanniae and Cornus Officinalis could improve the diabetic nephropathy and at the same time promote the glycolysis of testicular cells, increase the production of lactic acid, ameliorate the apoptosis of spermatogenic cells and the reduction of sperm motility. On the basis, our research innovatively proposed that “kidney yin deficiency” is the core TCM pathogenesis of male reproductive injury in diabetes. AGEs-mediated inhibition of testicular glycolysis and reduction of lactic acid in diabetic environment is the modern pathological mechanism. Radix Rehmanniae and Cornus Officinalis can regulate the testicular glycometabolism by intervention with AGEs-RAGE/HIF-1α pathway in diabetic environment, thus improve diabetic reproductive injury. The research intends to induce the reproductive damage model of spontaneous diabetic male mice, AGEs-induced Sertoli cell and sperm cell co-culture damage model to explore the mechanism of the drug pair on the prevention of male reproductive injury in diabetes, clarifying the modern scientific essence of “enriching yin and nourishing kidney”, providing a theoretical basis for clinical treatment.

随着糖尿病发病率的升高及发病年龄的年轻化，已成为男性生育障碍的重要病因，西医治疗存在长期疗效不佳、副作用大等问题，中医药从整体辨证施治，具有较强的优势。我们的工作显示滋阴补肾药对生地-山茱萸改善糖尿病肾病的同时，通过促进睾丸细胞糖酵解，增加能量底物乳酸的生成，而改善精细胞凋亡、精子活力降低等损伤。结合中医肾主生殖理论和文献报道，本项目创新性提出肾阴亏虚是糖尿病雄性生殖损伤的核心中医病机，AGEs介导抑制睾丸细胞糖酵解是生殖损伤肾阴亏虚的现代病理机制，生地-山茱萸可通过对AGEs-RAGE/HIF-1α通路的干预调控睾丸细胞糖酵解改善生殖损伤，拟通过自发性糖尿病雄性小鼠生殖损伤模型，AGEs致支持细胞与精细胞共培养损伤模型，聚焦睾丸细胞糖酵解，深入探讨生地-山茱萸改善生殖损伤的作用机制，从动物、细胞和分子水平，阐明滋阴补肾治法的现代科学实质，为临床从肾阴亏虚论治糖尿病生殖损伤提供理论依据。

近年来，随着糖尿病发病率的升高及发病年龄的年轻化，已成为男性生殖损伤的重要病因，中医认为糖尿病生殖损伤是因肾阴亏虚所致。生地清热凉血、养阴生津，山茱萸补益肝肾，涩精固脱，两药配伍加强补益肝肾之功能，是滋阴补肾治法的代表药对。项目从整体、细胞及分子水平开展了生地-山茱萸水提液及代表效应单体成分梓醇、马钱苷对糖尿病生殖损伤的改善研究，聚焦睾丸细胞糖酵解，阐明药效学作用机制。实验表明生地-山茱萸及效应成分梓醇、马钱苷在改善糖尿病症状的同时，对小鼠睾丸病理形态、精子数目、活力、性激素水平等生殖指标具有不同程度的改善作用，配伍组的效应要优于单用组。此外，生地-山茱萸可上调糖尿病小鼠睾丸及支持细胞株TM4的糖酵解关键葡萄糖膜转运蛋白（GLUT1, GLUT3和MCT4）和糖酵解关键限速酶己糖激酶(HK)，乳酸脱氢酶（LDH）和磷酸果糖激酶（PFK）等的表达水平。药对组以及单用组可不同程度降低AGEs在糖尿病小鼠血液中和睾丸中的沉积，可逆转模型组RAGE蛋白表达的增加和HIF-1α蛋白表达的降低。AGEs抑制剂氨基胍（Ami）、RAGE抑制剂FPS-ZM1也可降低血液和睾丸中的AGEs的水平，下调模型组睾丸中RAGE的表达，升高HK、PFK的表达，FPS-ZM1还可上调GLUT1,GLUT3和MCT4的表达。为进一步验证RAGE在睾丸糖酵解中的作用，通过腺病毒尾静脉注射建立RAGE过表达小鼠模型，过表达组RAGE蛋白在睾丸中表达进一步上调，加重了AGEs在睾丸组织中的沉积，加剧空腹血糖的升高，降低转运体GLUT1、GLUT3、PFK、LDHC蛋白的表达，生地-山茱萸可以逆转过表达组RAGE表达的上调、AGEs的沉积，逆转转运蛋白GLUT1、GLUT3等表达的降低。体外细胞实验构建了TM4和小鼠精母细胞株GC2条件培养损伤模型，在此模型的基础上进一步确证了梓醇、马钱苷可通过AGEs-RAGE/HIF-1α通路影响TM4的糖酵解而改善精母细胞损伤。综上所述，滋阴补肾药对生地-山茱萸可通过调控AGEs-RAGE/HIF-1α轴上调糖尿病环境下睾丸细胞糖酵解改善生殖损伤，该项目确证睾丸细胞糖酵解在糖尿病诱导的生殖损伤中的病理介导作用，阐明生地-山茱萸改善糖尿病雄性生殖损伤的作用机制。