壮骨止痛方通过RANKL/RANK信号通路抑制骨吸收的机制研究

Study of the mechanism of inhibition of bone resorption with the strong-bone and analgesic prescription through RANKL/RANK signaling pathway

目的探讨壮骨止痛方调控骨吸收及破骨活动干预去势大鼠骨质疏松症的疗效和作用机制,为临床治疗绝经后骨质疏松症提供实验依据。方法40只SD大鼠按照完全随机原则分为ZGZTF组、EV组、OVX组、SHAM组。除SHAM组之外余下均按国内外常用PMOP模型构建方法造模。术后第7天分别给予中药、雌二醇和蒸馏水灌胃。末次取材后检测大鼠右侧股骨骨密度,HE染色法观察各组大鼠胫骨骨组织病理形态变化,ELISA检测血清E_(2)、RANKL水平,免疫组化法分析RANK、TRAP、CTSK的蛋白表达。结果与OVX组相比,ZGZTF组大鼠骨密度有所升高,胫骨股骨骨组织微结构得到改善,骨小梁连续性稍恢复,骨髓腔缩小,脂肪空泡减少。血清中E2表达上调,RANKL水平下调(P<0.05),骨组织RANK、TRAP、CTSK蛋白表达下调(P<0.05)。结论壮骨止痛方可以降低去势大鼠骨组织相关的破骨因子的表达。抑制破骨细胞过度分化,改善骨耦联失衡,达到治疗绝经后骨质疏松症的疗效。

Objective To explore the curative effect and mechanism of the strong-bone and analgesic prescription on interfering with osteoporosis in castrated rats by regulating bone resorption and osteoclastic activity,and to provide a feasible strategy for the clinical treatment of postmenopausal osteoporosis.Methods Forty SD rats were randomly divided into the following groups:the strong-bone and analgesic prescription group,estradiol group,model group,and sham surgery group.All groups,except for the sham surgery group,were constructed according to the internationally recognized postmenopausal osteoporosis(PMOP)model.Seventh day after modeling,the rats were respectively given Chinese medicine liquid,estradiol,and distilled water by gavage.After the final collection,bone mineral density of the right femur of the rats was measured.The pathological morphology of the tibial bone tissue of each group was observed using HE staining.The serum levels of E_(2) and RANKL were detected with ELISA.The protein expressions of RANK,TRAP,and CTSK were analyzed using immunohistochemistry.Results Compared with the castrated rat model group,the group treated with the strong-bone and analgesic prescription showed an increase in bone mineral density,improvement in the microstructure of the tibial and femoral bone tissue,slight restoration of trabecular bone continuity,reduction in the size of the bone marrow cavity,and decrease in the number of adipose vacuoles.The expression of E2 in serum was up-regulated.The level of RANKL was down-regulated.The protein expressions of RANK,TRAP,and CTSK in the bone tissue were also downregulated(P<0.05).Conclusion The strong-bone and analgesic prescription reduces the expressions of relevant osteoclast factors in castrated rat.It inhibits over-differentiation and excessive bone absorption activity of osteoclasts,restores bone-coupling imbalance,thereby exerting therapeutic effect on postmenopausal osteoporosis.