冬凌草甲素对卵巢切除大鼠骨量丢失的作用及机制

Effects and mechanisms of oridonin on ovariectomy-induced bone loss in rats

目的探讨冬凌草甲素(ORI)对卵巢切除(OVX)大鼠骨质疏松模型骨组织病理学改变、骨微结构影响及其作用机制。方法选取由徐州医科大学动物实验室提供的8周龄雌性SD大鼠30只,参照随机化数字表分假手术(Sham)组、卵巢切除+溶剂(Veh)(OVX+Veh)组、卵巢切除+冬凌草甲素(OVX+ORI)组。OVX+ORI组予0.1%冬凌草甲素2 mg/kg腹腔注射,OVX+Veh组予相同体积的灭菌注射用水,均为每周2次,持续12周。获取各组大鼠血浆、骨组织标本。骨组织染色切片观察病理改变。微型计算机断层扫描成像行骨微结构扫描和骨密度测定。聚合酶链反应法检测骨组织中骨保护素-核因子-κB受体活化因子-核因子-κB受体活化因子配体(OPG-RANKL-RANK)信号通路和Wnt/β-连环蛋白(Wnt/β-Catenin)信号通路相关蛋白信使RNA相对水平。蛋白质印迹法检测获得信号通路相关蛋白表达情况。酶联免疫吸附试验方法检测血浆中骨代谢指标。两组间均数比较采用独立样本t检验。结果与OVX+Veh组比较,OVX+ORI组骨小梁断裂明显减少,完整性改善。RANKL表达量、骨硬化蛋白表达量、Ⅰ型胶原C端交联肽水平、抗酒石酸酸性磷酸酶水平OVX+Veh组高于OVX+ORI组[1.62±0.04比1.02±0.06,t=26.766,P<0.01;1.60±0.04比1.08±0.04,t=29.898,P<0.01;(61.68±6.88)ng/ml比(28.97±4.07)ng/ml,t=12.937,P<0.01;(759.44±32.47)pg/ml比(435.71±31.43)pg/ml,t=22.654,P<0.01]。骨密度、OPG表达量、wnt3a表达量、β-Catenin表达量、低密度脂蛋白受体相关蛋白5表达量、碱性磷酸酶水平、Ⅰ型原胶原N端前肽水平OVX+Veh组低于OVX+ORI组[(96.75±8.44)mg/cm3比(195.84±13.16)mg/cm3,t=-20.036,P<0.01;0.56±0.04比1.00±0.05,t=-21.267,P<0.01;0.31±0.01比0.93±0.04,t=-52.286,P<0.01;0.31±0.01比1.02±0.07,t=-30.535,P<0.01;0.32±0.03比1.00±0.03),t=-50.398,P<0.01;(15.71±3.44)ng/ml比(53.21±6.99)ng/ml,t=-15.229,P<0.01;(23.83±3.54)ng/ml比(63.95±4.17)ng/ml,t=-23.217,P<0.01]。结论冬凌草甲素可能通过抑制OPG-RANKL-RANK信号通路,同时上调Wnt/β-Catenin信号通路,改善雌激素缺乏大鼠骨量丢失,减少骨微结构破坏。

Objective To investigate the effects of Oridonin(ORI)on bone tissue pathology,microstructure,and potential mechanisms in an ovariectomized(OVX)rat model of osteoporosis.Methods Female Sprague-Dawley(SD)rats aged 8 weeks were randomly assigned to Sham,OVX+vehicle(Veh),and OVX+ORI groups.The OVX+ORI group received intraperitoneal injections of 0.1%Oridonin at 2 mg/kg twice weekly,and the OVX+Veh group received an equivalent volume of sterile water injections,both for a duration of 12 weeks.Blood plasma and bone tissue specimens were collected from all groups.Bone tissue staining sections were used to observe pathological changes.Micro computed tomography(Micro-CT)scans were conducted to assess bone microstructure and bone mineral density(BMD).The mRNA levels of Osteoprotegerin-Receptor Activator of NF-κB-Receptor Activator of NF-κB Ligand(OPG-RANKL-RANK)signaling pathway and Wnt/β-Catenin signaling pathway-related proteins in bone tissue were measured using polymerase chain reaction(PCR).The expression of these signaling pathway-related proteins was evaluated using Western blotting.Bone metabolism markers in blood plasma were assessed using the enzyme linked immunosorbent assay(ELISA).Independent sample t-test wasused for comparisons between two groups.Results Compared to the OVX+Veh group,the OVX+ORI group exhibited significantly reduced trabecular fractures and improved integrity.The levels of RANKL,sclerostin(SOST),c-terminal telopeptide of typeⅠcollagen(CTX)and tartrate resistant acid phosphatase(TRAP)in the OVX+Veh group were higher than those in the OVX+ORI group[1.62±0.04 vs.1.02±0.06,t=26.766,P<0.01;1.60±0.04 vs.1.08±0.04,t=29.898,P<0.01;(61.68±6.88)vs.(28.97±4.07)ng/ml,t=12.937,P<0.01;(759.44±32.47)vs.(435.71±31.43)pg/ml,t=22.654,P<0.01].The levels of bone mineral density(BMD),OPG,wnt3a,β-Catenin,low-density lipoprotein receptor-related protein 5(LRP5),alkaline phosphatase(ALP)and procollagenⅠN-terminal propeptide(PINP)in the OVX+Veh group were significantly lower than those in the OVX+ORI group[(96.75±8.44)vs.(195.84±13.16)mg/cm3,t=-20.036,P<0.01;0.56±0.04 vs.1.00±0.05,t=-21.267,P<0.01;0.31±0.01 vs.0.93±0.04,t=-52.286,P<0.01;0.31±0.01 vs.1.02±0.07,t=-30.535,P<0.01;0.32±0.03 vs.1.00±0.03,t=-50.398,P<0.01;(15.71±3.44)vs.(53.21±6.99)ng/ml,t=-15.229,P<0.01;(23.83±3.54)vs.(63.95±4.17)ng/ml,t=-23.217,P<0.01].Conclusion Oridonin may exert its effects by inhibiting the OPG-RANKL-RANK signaling pathway,while concurrently upregulating the Wnt/β-Catenin signaling pathway.These combined effects contribute to the mitigation of ovariectomy-induced bone loss,reduction of bone microstructural damage in rats.