GLP-1受体激动剂经cAMP-PKA通路影响调节性T细胞作用骨代谢的机制研究

With the progress of the aging society, osteoporosis has a serious impact on the quality of life for the elderly. Our preliminary study confirmed that GLP-1R can exert a bone protective effect in both diabetic and non-diabetic osteoporotic rats in vivo, while can facilitate osteoblastogenesis during BMSC and osteoblast lineage differentiation in vitro. However, the exactly signaling pathways involved in the bone effect of GLP-1RA were not fully reported at home and abroad. We found that the population of CD4+CD25+ regular T cells was increased in peripheral blood mononuclear cells after treated with GLP-1R, which were collected from normal humans. While the level of cAMP was closely in relation with the proliferation and immunosuppressive function of regular T cells, we speculated that GLP-1RA may benifit bone metabolism via an effect in the activity of regulatory t cell through the cAMP-PKA pathways. Our research aims to investigate the effects of pathway when GLP-1 receptor agonists on the bone metabolism in vivo and in vitro using the approaches for molecular biology and cell biology. Potentially, our findings may provide a basis for the design of new therapeutic strategy for the intervention of osteoporosis in the clinic.

骨质疏松症越发影响老年人的生活质量,课题组前期研究发现肠道激素胰高血糖素样肽-1受体激动剂（GLP-1R）在体内可改善糖尿病及非糖尿病大鼠的骨结构损伤，体外可促进骨髓间充质干细胞及成骨细胞成骨分化，但具体信号通路和作用机制尚未完全阐明。前期结果显示，GLP-1R处理正常人外周血单个核细胞（PBMC）后，CD4+CD25+调节性T细胞（Treg）比例明显升高，而cAMP水平也与CD4+CD25+ Treg细胞的增殖及免疫抑制功能相关，继而推测GLP-1R可能通过cAMP-PKA通路影响体内Treg细胞最终影响骨代谢。本课题拟选用重排活化基因缺失（Rag-1敲除）小鼠作为动物模型，并采用骨质疏松患者PBMC运用细胞培养、分子生物学和细胞移植等技术进一步探讨GLP-1R如何通过cAMP-PKA通路促进Treg细胞增殖分化，并由调节性T细胞改善骨代谢作用机制，为骨质疏松症临床防治提供新的靶点。

胰高血糖素样肽 -1 受体激动剂 (glucagon-like peptide-1 receptor agonist，GLP-1RA) 是目前治疗 2 型糖尿病的有效药物，前期研究表明GLP-1RA除降糖外，还可作用于骨骼，但目前关于GLP-1RA如何参与肠道抗炎作用研究甚少。本研究在RAG-1敲除小鼠中，通过尾静脉注射Treg细胞，micro CT结果初步确定Treg细胞可联合GLP-1RA在骨代谢中起保护作用；并采用3%葡聚糖硫酸钠盐(dextran sulfate sodium，DSS) 诱导构建小鼠炎症性肠病（Inflammatory bowel disease, IBD）模型，通过腹腔注射利拉鲁肽 0.6 mg/kg予以干预。实验发现，与模型组相比，干预组小鼠粪便稀松、血便等症状出现时间推迟且症状较轻，结肠长度缩短程度减轻（P<0.05）;病理学评分上，H-E 染色显示其结肠内炎症细胞浸润显著减少，糖原染色显示黏液增多。进一步通过流式细胞术分析结肠固有层细胞显示，与模型组相比，干预组小鼠结肠内嗜酸性粒细胞 (CD11b+ SiglecF+) 、DC细胞比例明显降低（P<0.05）；2 型固有淋巴细胞 (group 2 innate lymphoid cell，ILC2）比例降低，3 型 ILC（ILC3）比例升高，但在亚型分析中ILC3三群细胞未见明显比例差异；ILC3 分泌的细胞因子白介素 -22 、GMCSF水平明显增加，且主要由NCR+ILC3分泌IL-22增多为主。进一步检测血清发现干预组IL-23显著增加，考虑药物作用于肠道上皮刺激产生IL-23，进而诱导ILC3数量及功能的改变。综上，GLP-1 受体激动剂利拉鲁肽可通过影响肠道上皮细胞分泌IL23进而影响 ILC 亚群比例，尤其是NCR+ILC3分泌IL-22的功能，进而延缓 IBD 的发生与发展。GLP-1 受体激动剂有望成为辅助治疗炎症性肠病新型药物。