II型蛋白激酶G通过下调PLC/IP3/Ca2+轴拮抗内质网应激改善糖尿病种植体骨结合的研究

2型糖尿病(T2DM)易导致骨再生不良而严重影响种植牙骨结合，提高糖尿病患者种植牙的成功率是亟待解决的临床难题。有研究表明II型蛋白激酶G(PKGII)与骨代谢的调控密切相关；本课题组前期研究发现高糖环境下小鼠骨髓间充质干细胞(BMSCs)的PKGII表达降低，增加PKGII表达可显著改善T2DM小鼠种植体骨结合，但机制尚不清楚。本课题组利用蛋白组学技术检测出靶向增强PKGII后磷酸脂酶Cβ(PLCβ)的表达在cGMP/PKG与钙离子信号通路中明显下调，推测PKGII可能通过PLCβ作用于钙离子通路并影响内质网应激来发挥对骨代谢的调控。基于上述结果，本项目拟通过体内外实验来揭示PKGII/PLC/IP3/Ca2+通路、内质网应激与高糖环境下BMSCs成骨分化之间内在联系的分子机制，行临床观测验证PKGII对T2DM种植体骨结合的影响，为寻找提高T2DM牙种植成功率的新思路、新方法提供依据。

Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia with a high incidence rate. Various studies have proved that T2DM have bad effects on bone metabolism and could inhibit bone regeneration. Dental implant is an important method to treat dentition defects, while the success rate of it in diabetic patients is significantly lower than that in the normal population. Therefore, to improve the osseointegration of dental implant in diabetic patients is a urgent clinical problem to be solved. Type II protein kinase G (PKGII) is a kind of serine/threonine protein kinase that is related to the regulation of bone metabolism. Our previous study found that the expression of PKGII decreased in mouse bone marrow mesenchymal stem cells (BMSCs) which were cultured in high glucose environment, and upregulating the expression of PKGII can significantly improve the osseointegration of implants in T2DM mice. These results suggested PKGII may be an important regulator of osteogenic differentiation in high glucose environment, but the inner regulatory mechanism is still unknown. Using comparative proteome analysis, our team found that the expression of phospholipase C β (PLCβ) was decreased when PKGII was upregulated. PLC is an important enzyme that could regulate the growth and differentiation of cells, and several studies reported PLC participated in the IP3/Ca 2+ signaling pathway which could affect endoplasmic reticulum stress. Therefore, we speculated that the regulation of PKGII in high glucose environment is related to PLCβ and endoplasmic reticulum stress. Based on the above results, this project intends to prove that PKGII improves the osseointegration of dental implants in T2DM through inhibiting PLC/IP3/Ca2 + mediated endoplasmic reticulum stress through in vivo, in vitro and clinical experiments: First, explore the effects of PKGII on the osteogenic differentiation of BMSCs in in high glucose environment; next, verify the direct phosphorylation and phosphorylation site of PKGII on PLCβ; then, clarify the regulation of PKGII on PLC/IP3/Ca2 + mediated endoplasmic reticulum stress; in addition, explore the effect of Cinaciguat, the agonist of PKGII, on the osseointegration of implants in T2DM mice; finally, analyze the connection of PKGII and the osseointegration of implants in clinic. We hope the result could provide new ideas, new approaches and new research basis for improving the success rate of dental implant in T2DM patient.