内热针疗法对兔膝骨性关节炎软骨下骨中骨保护素及核因子κB受体活化因子表达的影响

Effect of internal heat-type acupuncture needle on the expression of osteoprotegerin, receptor activator of NF-κB ligand and receptor activator of NF-κB of subchondral bone in knee osteoarthritis rabbits

目的:观察内热针疗法对兔膝骨性关节炎(KOA)软骨下骨中骨保护素(OPG)、核因子κB受体活化因子配体(RANKL)、核因子κB受体活化因子(RANK)表达的影响,探讨内热针治疗KOA的作用机制。方法:将30只新西兰兔随机分为空白组、模型组和治疗组,每组10只。采用Hulth法建立兔KOA模型。治疗组给予兔内热针治疗,1次/周,每次20 min,连续4周。干预前后采用改良版Lequesne量表对各组兔进行行为学评价。取各组兔左后膝关节标本行甲苯胺蓝染色,观察软骨下骨病理变化并分析骨组织形态计量学参数的差异;分别采用Western blot和实时荧光PCR法检测软骨下骨中OPG、RANKL、RANK蛋白和mRNA的表达。结果:与空白组比较,模型组Lequesne评分升高(P<0.05),软骨下骨骨小梁面积百分比、骨小梁数目降低(P<0.05),骨小梁分离度升高(P<0.05),软骨下骨组织中OPG蛋白表达和mRNA均降低(P<0.01,P<0.05),RANKL、RANK蛋白表达和mRNA均升高(P<0.01)。与模型组比较,治疗组Lequesne评分降低(P<0.05),干预前后Lequesne评分差值升高(P<0.05),软骨下骨骨小梁面积百分比、骨小梁数目升高(P<0.05),骨小梁分离度降低(P<0.05),软骨下骨组织中OPG蛋白表达和mRNA均升高(P<0.05),RANKL、RANK蛋白表达和mRNA均降低(P<0.05)。结论:内热针疗法治疗KOA的机制可能是通过上调兔KOA软骨下骨组织中OPG表达,下调RANKL和RANK的表达,从而抑制了骨吸收。

Objective To investigate the effect of internal heat-type acupuncture needle on the expression of osteoprotegerin(OPG), receptor activator of NF-κB ligand(RANKL), and receptor activator of NF-κB(RANK) in knee osteoarthritis(KOA) rabbits, so as to explore its mechanisms in relieving KOA. Methods Thirty New Zealand rabbits were randomly divided into control, model and treatment groups, with 10 rabbits in each group. The KOA model was established by using Hulth method. The rabbits of the treatment group received internal heat-type acupuncture needles(42 ℃) on the left hind limb 20 min, once a week for 4 weeks. The behavioral scores were assessed according to the pain severity, gait, joint motion range and articular swelling severity in reference to the modified Lequesne’s methods. Toluidine Blue staining was performed to observe the structure of the subchondral bone and to analyze the difference of morphometric parameters. The protein and mRNA expressions of OPG, RANKL and RANK were detected by Western blot and real-time PCR, respectively. Results Compared with the control group, the Lequesne total score, the separation degree of trabecular bone, the protein and mRNA expressions of RANKL and RANK in subchondral bone tissues were significantly increased in the model group, while the percentage of trabecular bone area, number of trabecular bone, the expression of OPG protein and mRNA were decreased(P<0.05, P<0.01). The above indexes were all reversed in the treatment group relevant to those of the model group(P<0.05). Conclusion The internal heat-type acupuncture needle therapy can improve the motor function of rabbits with KOA, which may be related to its effects in up-regulating the expression of OPG and down-regulating the RANKL and RANK in subchondral bone tissue.