阿司匹林防治绝经后骨质疏松的最新用途及其分子机制研究

Postmenopausal osteoporosis has a high incidence in elder women. The current prevention and treatment methods are mainly orally administration of active vitamin D, bisphosphonates and other drugs, yet with their slow effect, long course of treatment and high cost, the clinical effects are unsatisfactory. Epidemiological investigation and preliminary studies of our group proved that aspirin had the effect of anti-postmenopausal osteoporosis. Nevertheless, its molecular mechanism is totally not clear. Based on all these results, we propose this study project. Firstly, the postmenopausal osteoporosis model will be established by ovariectomy of female rats, and different doses of aspirin treatment will be given to them at the same time. By detection of bone formation indicators, the effect of aspirin on the prevention and treatment of postmenopausal osteoporosis and its best dosage will be investigated. We hypothesize that the key molecular mechanism of aspirin inhibiting osteoclast maturation and bone resorption through the inhibition of OPG/ RANKL/RANK axis system: utilizing qPCR, ELISA and western-blot methods, RANK expression from osteoclast, OPG and RANKL expression from osteoblast will be detected; the direct effect of aspirin on the NF-κB pathway in osteoclast will be investigated; and finally its impact on inflammatory factors and its effect on osteoclast differentiation as well as the bone resorption activity will be explored. As a result, the molecular mechanism hypothesis of aspirin for the treatment of postmenopausal osteoporosis will be revealed, which may provide the fundamental evidence for the feasible novel clinical indication of aspirin - for the prevention and treatment of postmenopausal osteoporosis.

女性绝经后骨质疏松发病率高，目前防治以口服活性维生素D、双磷酸盐等药物为主，起效慢、疗程长、费用高，临床效果并不理想。流行病学调查及本课题组前期研究证实，阿司匹林具有抗绝经后骨质疏松作用，但其分子作用机制尚未明了。本研究拟首先建立雌性大鼠去势后骨质疏松模型，同时给予不同剂量阿司匹林治疗，通过检测成骨指标，明确阿司匹林对骨质疏松防治的确切效果及最佳量效。提出阿司匹林通过抑制OPG/RANKL/RANK轴系统而抑制破骨细胞成熟分化及骨吸收功能的分子机制假设：利用qPCR、ELISA、western-blot等检测各组成骨细胞/破骨细胞OPG、RANKL及RANK表达的差异，及其对破骨细胞内NF-κB分子通路的影响和直接作用靶点;观察其对炎性因子的表达及破骨细胞成熟分化和骨吸收活性的影响。最终明确阿司匹林逆转绝经后骨质疏松的分子机制，为阿司匹林这一老药创新用途- - 防治绝经后骨质疏松提供理论依据。

女性绝经后骨质疏松发病率高，目前防治以口服活性维生素D、双磷酸盐等药物为主，起效慢、疗程长、费用高，临床效果并不理想。流行病学调查及本课题组前期研究证实，阿司匹林具有抗绝经后骨质疏松作用，但其分子作用机制尚未明了。本研究首先建立雌性大鼠去势后骨质疏松模型，同时给予不同剂量阿司匹林治疗，通过骨密度仪、显微CT、生物力学测定、骨组织微观结构及检测成骨指标，明确阿司匹林对骨质疏松防治的确切效果及最佳量效。采用RANKL和 M-CSF 诱导大鼠骨髓单核巨噬细胞破骨分化模型，给予不同浓度的阿司匹林处理，而后进行TRAP染色 观察细胞形态特征，扫描电镜观察骨磨片破骨细胞骨吸收陷窝，RT-PCR技术检测破骨细胞表面RANK基因的表达，ELISA法检测RANK蛋白的表达，结果表明阿司匹林可使大鼠破骨细胞成熟分化程度和骨吸收活性降低，抑制破骨细胞RANK基因和蛋白的表达。通过体外培养RAW 264.7细胞，以100 ng/ml NF-KB受体配体(RANKL)诱导培养，并同时添加不同溶度的阿司匹林培养。在不同时间点，通过TRAP染色的方法来观察破骨细胞诱导生成能力，用实时荧光PCR方法检测其破骨细胞系标志基因，包括CTSK、TRAP、MMP-9 和CTR mRNA的表达，裂解不同培养条件的细胞 并提取蛋白上样，行western印迹检测NF-KB通道蛋白的表达以及MAPKS通道蛋白的表达，通过免疫荧光的方法分析确定NF-KB的P65的核易位，结果表明阿司匹林抑制RANKL诱导的破骨细胞生成，其标志性基因TRAP、CTSK、MMP9及CTR的mRNA表达下调，磷酸化的P65、P50、IKB-a、P38以及c-JNK和ERK蛋白表达均有所减少，对NF-KB P65的核易位也表现出抑制效果。最终明确阿司匹林逆转绝经后骨质疏松的分子机制，为阿司匹林这一老药创新用途——防治绝经后骨质疏松提供理论依据。