Objective: To investigate the effects of neuropeptide Y (NPY) Y1 receptor antagonist PD160170 in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and accelerating healing of femoral defect in rats.
Methods: The third generation of rat BMSCs were treated with PBS (control) or 10-6, 10-7, or 10-8 mol/L NPY Y1 receptor antagonist PD160170. After 7 and 14 days of treatment, the cells were examined for osteogenic differentiation with alkaline phosphatase (ALP) and alizarin red staining. At 7 and 21 days of treatment, the mRNA and protein expressions of collagen type I (COLI), osteocalcin (OCN) and Runt-related transcription factor 2 (Runx2) in the cells were detected using q-PCR and Westem Blotting. In a male SD rat model (body weight 300∓20 g) of bilateral femoral condyle defects (2.5 mm in diameter), the effect of daily local injection of 0.2 mL PD160170 (10-6 and 10-8 mol/L, for 28 consecutive days) in promoting bone defect repair was evaluated with micro-CT scans.
Results: ALP and alizarin red staining showed that the BMSCs treated with PD160170, at the optimal concentration of 10-8 mol/L, contained more intracellular cytoplasmic brown particles and mineralized nodules in extracellular matrix than PBS-treated cells. PD160170 (10-8 mol/L) significantly up-regulated the mRNA and protein expressions of COLI at day 7 and those of OCN and Runx2 at day 21 (P<0.05). In the rat models of femoral bone defect, the volume/tissue volume ratio, bone mineral density and the number of bone trabeculae were significantly greater in 10-6 mol/L PD160170 group than in the control group (P<0.05), but the bone trabecular thickness (P=0.07) and bone volume (P=0.35) were similar between the two groups.
Conclusion: NPY Y1 receptor antagonist PD160170 can promote osteogenic differentiation of BMSCs and healing of femoral defects in rats, suggesting the potential of therapeutic strategies targeting NPY Y1 receptor signaling in the prevention and treatment of bone fracture and osteoporosis.
目的: 探索神经肽Y Y1受体拮抗剂（PD160170）对大鼠骨髓间充质干细胞（BMSCs）成骨分化以及骨缺损修复的影响。
方法: 第三代大鼠BMSCs成骨分化诱导培养，随机分为4组，对照组（等体积PBS），终浓度分别为10-6、10-7、10-8 mol/L Y1受体拮抗剂三浓度梯度组，在干预7、14 d时，分别进行碱性磷酸酶（ALP）和茜素红染色；在干预7、21 d时，采用q-PCR和Westen blot检测Ⅰ型胶原（COLI）、骨钙素（OCN）和Runt相关转录因子2（Runx2）基因和蛋白的表达。采用300±20 g雄性SD大鼠构建股骨髁上骨缺损模型，直径为2.5 mm，随机分为3组，每组6只，分别每日局部注射0.2 mL溶液等体积PBS（对照组）、10-6 mol/L和10-8 mol/L Y1受体拮抗剂溶液，28 d后处死，Micro-CT检测观察骨缺损修复情况。
结果: ALP和茜素红染色发现，Y1受体拮抗剂组细胞胞浆内棕色颗粒、细胞外周基质矿化结节均高于对照组，且具有浓度特异性，以10-8 mol/L最为明显；q-PCR及Westem blot检测发现，在成骨分化诱导第7天，10-8 mol/L Y1受体拮抗剂组COLI mRNA和蛋白表达水平均显著高于对照组，差异具有统计学意义（P < 0.05）；在第21天，10-8 mol/L Y1受体拮抗剂组OCN、Runx2 mRNA和蛋白表达明显高于对照组（P < 0.05）。通过micro-CT分析发现，在局部注射10-6 Y1受体拮抗剂28 d后，骨缺损BV/TV、骨密度、骨小梁数量高于对照组，差异均具有统计学意义（P < 0.05）；而骨小梁厚度（P=0.07）、骨体积（P=0.35）在各组之间差异无统计学意义。
结论: 神经肽Y Y1受体拮抗剂可促进BMSCs成骨分化以及骨缺损的修复，提示阻断Y1受体信号传导具有预防或治疗骨折、骨质疏松症等潜力，为临床药物研究提供一定的实验依据。