基于OPG/RANKL及Notch1信号通路探讨温针灸对膝骨性关节炎兔软骨下骨损伤的影响

Effect of Needle-Warming Moxibustion on Subchondral Bone Injury in Rabbits with KOA Based on OPG/RANKL and Notch1 Signaling Pathways

目的:观察温针灸对膝骨性关节炎(KOA)兔软骨下骨的骨保护素(OPG)/核因子κB受体活化子配体(RANKL)及Notch1信号通路的影响,探讨温针灸治疗KOA的作用机制。方法:选取新西兰雌雄兔共50只(6个月),随机分为空白组、模型组、药物组和温针灸组。采用管型石膏伸直位固定法建立兔KOA模型。经6周实验干预后采用HE染色观察各组软骨下骨形态结构,蛋白质印迹法检测OPG、RANKL及Notch1蛋白表达,实时荧光定量PCR检测各组软骨下骨中OPG、RANKL及Notch1 mRNA含量。结果:与模型组比较,药物组和温针灸组软骨表面相对光滑完整,结构较为清晰,软骨下骨处细胞分布较为整齐均匀。与空白组比较,模型组Lequesne MG评分、Mankin评分、RANKL mRNA、Notch1 mRNA、RANKL及Notch1蛋白表达水平均明显升高,差异具有统计学意义(P<0.01)。与模型组比较,药物组和温针灸组Lequesne MG评分、Mankin评分、RANKL mRNA、Notch1 mRNA、RANKL及Notch1蛋白表达水平均明显降低,差异具有统计学意义(P<0.01),而OPG mRNA、OPG蛋白、OPG/RANKL及OPG/Notch1比值均明显升高,差异具有统计学意义(P<0.01)。温针灸组Lequesne MG评分及Mankin评分均明显低于药物组,差异具有统计学意义(P<0.01),而OPG蛋白、OPG/RANKL及OPG/Notch1水平均高于药物组,差异具有统计学意义(P<0.01)。结论:温针灸可促进OPG表达,抑制RANKL及Notch1表达,通过调控OPG/RANKL及Notch1信号通路,减轻KOA兔软骨下骨的损伤,从而起到治疗KOA的作用。

Objective:To observe the effects of needle-warming moxibustion on Osteoprotegerin(OPG)/receptor activator of NF-κB lignand(RANKL)and Notch1 signaling pathway in rabbits with knee osteoarthritis(KOA),and to explore the mechanism of needle-warming moxibustion in treatment of KOA.Methods:50 New Zealand female and male rabbits(6 months old)were randomly divided into the blank group,the model group,the medication group and the needle-warming moxibustion group.The KOA model was established by tube plaster extension fixation in the rabbits.After 6 weeks of intervention,the morphological structure of subchondral bone was observed by HE staining,the protein expressions of OPG,RANKL and Notch1 were detected by Western blot,and the mRNA contents of OPG,RANKL and Notch1 in subchondral bone were detected by real-time fluorescence quantitative PCR in each group.Results:Compared with those in the model group,the cartilage surface was relatively smooth and complete,the structure was relatively clear,and the cell distribution at the subchondral bone was relatively uniform in the medication group and the needle-warming moxibustion group.Compared with those in the blank group,the scores of Lequesne MG and Mankin,the mRNA expressions of RANKL and Notch1,as well as the proteins of RANKL and Notch1 were significantly increased in the model group(P<0.01).Compared with those in the model group,the scores of Lequesne MG and Mankin,the mRNA expressions of RANKL and Notch1,as well as the proteins of RANKL and Notch1 were significantly decreased(P<0.01),whereas OPG mRNA,OPG protein,OPG/RANKL and OPG/Notch1 were significantly increased in the medication group and the needle-warming moxibution group(P<0.01).The scores of Lequesne MG and Mankin were lower(P<0.01),whereas the levels of OPG protein,OPG/RANKL and OPG/Notch1 were higher in the needle-warming moxibustion group than those in the medication group(P<0.01).Conclusion:Needle-warming moxibustion can promote the expression of OPG,inhibit the expressions of RANKL and Notch1,regulate OPG/RANKL and Notch1 signaling pathways,and reduce the injury of subchondral bone in KOA rabbits,so as to play a role in treatment of KOA.