摘要
目的 从骨-软骨代谢及低氧诱导因子(HIFs)/骨保护素(OPG)/核因子κB受体激活因子配体(RANKL)通路探讨温肾通络止痛方治疗骨质疏松症和骨关节炎共病的可能作用机制。方法 35只SD大鼠随机分为假手术组、模型组、阿仑膦酸钠组、温肾通络止痛方低剂量组及高剂量组,每组7只。除假手术组外,其余各组大鼠采用双侧卵巢去除术+改良式Hulth术构建骨质疏松症和骨关节炎共病大鼠模型。于造模结束后2周开始干预,温肾通络止痛方低剂量组、高剂量组分别给予温肾通络止痛方14.76、29.52 g/(kg·d)灌胃,每日1次;阿仑膦酸钠组给予6.3 mg/kg灌胃,每周1次;假手术组与模型组均以10 ml/(kg·d)生理盐水灌胃,每日1次。干预10周后,观察大鼠膝关节大体形态并进行Collins评分;采用Micro-CT检测股骨远端骨密度和胫骨平台软骨下骨骨微结构[包括骨体积分数(BV/TV)、骨小梁分离度(Tb. Sp)];HE染色和番红固绿染色观察膝关节病理变化,并进行Mankin评分;采用ELISA法测定血清炎症因子水平[包括肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)]和HIFs/OPG/RANKL信号通路相关因子[包括缺氧诱导因子1α(HIF-1α)、缺氧诱导因子2α(HIF-2α)、OPG、RANKL]水平,并计算OPG/RANKL比值;免疫荧光检测膝关节HIF-1α、HIF-2α蛋白表达;Western Blot法测定膝关节骨-软骨代谢[包括Ⅱ型胶原α1链(COL2A1)、Ⅰ型胶原α2链(COL1A2)、基质金属蛋白酶13(MMP13)、基质金属蛋白酶9(MMP9)、Runt相关转录因子(RUNX2)]和HIFs/OPG/RANKL蛋白[包括OPG、RANKL、HIF-1α、HIF-2α、Egl九同源蛋白(EGLN)]水平。结果 与假手术组比较,模型组大鼠膝关节Collins评分、Mankin评分显著升高;骨密度、BV/TV显著下降,Tb. Sp显著上升;血清TNF-α、IL-1β、RANKL、HIF-1α、HIF-2α水平升高,OPG/RANKL水平降低;HIF-1α蛋白平均免疫荧光强度降低,HIF-2α蛋白平均免疫荧光强度升高;膝关节COL2A1、COL1A2、HIF-1α、OPG/RANKL水平降低,MMP9、MMP13、RUNX2、RANKL、HIF-2α、EGLN蛋白水平升高(P<0.05)。与模型组比较,温肾通络止痛方高剂量组和阿仑膦酸钠组上述各指标均改善(P<0.05)。温肾通络止痛方高剂量组在改善血清HIF-1α、HIF-2α、OPG水平,HIF-2α蛋白平均免疫荧光强度,COL2A1、MMP9、RUNX2、COL1A2、OPG、OPG/RANKL、EGLN水平方面优于温肾通络止痛方低剂量组(P<0.05);阿仑膦酸钠组在降低血清TNF-α、IL-1β、HIF-1α、HIF-2α水平和膝关节HIF-1α、HIF-2α蛋白平均荧光强度方面优于温肾通络止痛方低剂量组(P<0.05)。结论 温肾通络止痛方可能通过调控膝关节HIFs/OPG/RANKL通路,平衡骨代谢和改善软骨退变,进而缓解骨质疏松和骨关节炎共病发展进程。
Objective To explore the potential mechanism of Wenshen Tongluo Zhitong Formula(温肾通络止痛方,WTZF)in treating comorbidity of osteoporosis(OP)and osteoarthritis(OA)from the perspectives of osteo-cartilage metabolism and the hypoxia-inducible factors(HIFs)/osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand(RANKL)pathway.Methods Thirty-five Sprague-Dawley(SD)rats were randomly divided into 5 groups,sham operation group,model group,alendronate sodium group,low-and high-dose WTZF group,7 rats in each group.Except for the sham operation group,rats were subjected to bilateral ovariectomy combined with modified Hulth surgery to establish a rat model of comorbidity of OP and OA.Interventions were initiated 2 weeks after model establishment.The low-and high-dose WTZF groups were given WTZF by gavage at doses of 14.76 g/(kg·d)and 29.52 g/(kg·d),respectively,once a day;the alendronate sodium group was given alendronate sodium by gavage at 6.3 mg/kg once a week;the sham operation group and the model group were given normal saline by gavage at 10 ml/(kg·d)once a day.After 10 weeks of intervention,the gross morphology of the rat knee joint was observed and scored using the Collins scoring system;micro-computed tomography(Micro-CT)was used to detect the bone mineral density of the distal femur and the subchondral bone microstructure of the tibial plateau including bone volume fraction(BV/TV)and trabecular separation(Tb.Sp);HE staining and safranin O-fast green staining were performed to observe the pathological changes of the knee joint,and the Mankin scoring system was used for evaluation;enzyme-linked immunosorbent assay(ELISA)was used to determine the serum levels of inflammatory factors such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and factors related to the HIFs/OPG/RANKL signaling pathway including hypoxia-inducible factor-1α(HIF-1α),hypoxia-inducible factor-2α(HIF-2α),OPG,and RANKL,and the OPG/RANKL ratio was calculated.Immunofluorescence was used to detect the protein expression of HIF-1αand HIF-2αin the knee joint;Western Blotting was used to determine the levels of proteins related to knee joint osteo-cartilage metabolism,including collagen typeⅡα1 chain(COL2A1),collagen typeⅠα2 chain(COL1A2),matrix metalloproteinase 13(MMP13),matrix metalloproteinase 9(MMP9),and Runt-related transcription factor 2(RUNX2),and also the HIFs/OPG/RANKL pathway including OPG,RANKL,HIF-1α,HIF-2α,Egl nine homolog(EGLN)was determined.Results Compared with the sham operation group,the model group showed significantly increased Collins score and Mankin score of the knee joint,significantly decreased BMD and BV/TV,significantly increased Tb.Sp,increased serum levels of TNF-α,IL-1β,RANKL,HIF-1α,and HIF-2α,decreased serum OPG/RANKL ratio,average immunofluorescence intensity of HIF-1αprotein,increased average immunofluorescence intensity of HIF-2αprotein,decreased levels of COL2A1,COL1A2,HIF-1α,and OPG/RANKL in the knee joint,and increased protein levels of MMP9,MMP13,RUNX2,RANKL,HIF-2α,and EGLN(all P<0.05).Compared with the model group,the high-dose WTZF group and the alendronate sodium group showed improvements in all the above indicators(all P<0.05).The high-dose WTZF group was superior to the low-dose WTZF group in improving serum levels of HIF-1α,HIF-2α,and OPG,the average immunofluorescence intensity of HIF-2αprotein,and the levels of COL2A1,MMP9,RUNX2,COL1A2,OPG,OPG/RANKL,and EGLN(all P<0.05);the alendronate sodium group was superior to the low-dose WTZF group in reducing serum levels of TNF-α,IL-1β,HIF-1α,and HIF-2α,and the average immunofluorescence intensity of HIF-1αand HIF-2αproteins in the knee joint(all P<0.05).Conclusion The Wenshen Tongluo Zhitong Formula may regulate the HIFs/OPG/RANKL pathway in the knee joint,balance bone metabolism,and improve cartilage degeneration,thereby alleviating the progression of comorbid osteoporosis and osteoarthritis.
作者
傅璟玥
马勇
周勤峰
孙杰
潘娅岚
王礼宁
李沐哲
黄项阳
郭杨
FU Jingyue;MA Yong;ZHOU Qinfeng;SUN Jie;PAN Yalan;WANG Lining;LI Muzhe;HUANG Xiangyang;GUO Yang(New Technology Laboratory of Bone Injury Repair and Reconstruction,Nanjing University of Chinese Medicine,210023;Yancheng Affiliated Hospital of Nanjing University of Chinese Medicine;School of Integrated Chinese and Western Medicine,Nanjing University ofChinese Medicine)
出处
《中医杂志》
北大核心
2025年第24期2578-2587,共10页
Journal of Traditional Chinese Medicine
基金
国家自然科学基金(82174411)
江苏省自然科学基金(BK20221351)
国家中医药管理局高水平中医药重点学科建设项目(国中医药人教函[2023]85号)
盐城市基础研究计划(YCBK2024087)
江苏省研究生科研创新计划(KYCX24_2190)。
关键词
骨质疏松症
骨关节炎
低氧诱导因子
骨保护素
核因子κB受体激活因子配体
骨代谢
温肾通络止痛方
osteoporosis
osteoarthritis
hypoxia-inducible factors
osteoprotegerin
receptor activator of nuclear factor-κB ligand
bone metabolism
Wenshen Tongluo Zhitong Formula(温肾通络止痛方)
