电针对去卵巢大鼠关节软骨退变及软骨下骨骨质疏松的影响

Electroacupuncture Intervention Improves Cartilage Degeneration and Subchondral Bone Osteoporosis of Knee-joint Possibly by Adjusting OPG/RANK/RANKL Signaling in Ovariectomized Rats

目的:通过观察去卵巢大鼠关节软骨及软骨下骨的变化,探讨电针对骨保护素/核因子κB受体活化因子/核因子κB受体活化因子配体(OPG/RANK/RANKL)信号通路及基质金属蛋白酶(MMP)-13表达的影响。方法:雌性3月龄SD大鼠随机分为假手术组、模型组、电针组,每组8只。模型组及电针组均切除双侧卵巢复制模型,假手术组切除卵巢周围同等大小脂肪组织。电针组电针大鼠双侧"三阴交""阳陵泉""阴陵泉""足三里",频率3Hz/15Hz,强度1mA,持续30min,每日1次,每周5次,共12周。番红染色及Mankin’s评分观察软骨全层结构;显微CT及骨组织定量分析软骨下骨变化;ELISA法检测尿液Ⅰ型胶原C端肽(CTX-Ⅰ)、Ⅱ型胶原C端肽(CTX-Ⅱ)及血清雌激素(E 2)含量;RT-PCR法检测OPG、RANKL及MMP-13mRNA表达水平。结果:模型组Mankin’s评分显著高于假手术组(P<0.01);电针组Mankin’s评分显著低于模型组(P<0.01)。模型组骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)均显著低于假手术组(P<0.01),骨小梁分离度(Tb.Sp)显著高于假手术组(P<0.01);电针组BV/TV、Tb.N、Tb.Th显著高于模型组(P<0.01),Tb.Sp显著低于模型组(P<0.01)。模型组血清E 2含量显著低于假手术组(P<0.01);电针组血清E 2含量显著高于模型组(P<0.01)。模型组尿液CTX-Ⅰ、CTX-Ⅱ含量显著高于假手术组(P<0.01);电针组尿液CTX-Ⅰ、CTX-Ⅱ含量显著低于模型组(P<0.05)。模型组RANKL、MMP-13mRNA表达水平显著高于假手术组(P<0.01),OPG mRNA表达水平显著低于假手术组(P<0.01);电针组RANKL、MMP-13mRNA表达水平显著低于模型组(P<0.05),OPG mRNA表达水平显著高于模型组(P<0.01)。结论:电针可通过调节OPG/RANK/RANKL信号通路抑制去卵巢大鼠软骨下骨骨质疏松,通过下调MMP-13的表达抑制关节软骨退变。

Objective To observe the effect of electroacupuncture(EA)on histopathological changes of cartilage and subchondral bone and osteoprotegerin(OPG),receptor activator of nuclear factor-κB(RANK),and RANK ligand(RANKL)(OPG/RANK/RANKL)signaling and the expression of matrix metalloproteinase-13(MMP-13)in ovariectomized(OVX)rats,so as to explore its mechanism underlying improvement of osteoporosis.Methods Three-month female SD rats were randomly divided into sham operation,model and EA groups(n=8 in each group).The ovoariectomy model was established by resection of bilateral ovaries.Rats of the sham group were treated by simple removal of a piece of adipose tissue around the bilateral ovaries.EA(3 Hz/15 Hz,1 mA)was applied to bilateral"Sanyinjiao"(SP 6),"Yanglingquan"(GB 34),"Yinlingquan"(SP 9)and"Zusanli"(ST 36)for 30 min,once daily(except the weekends)for 12 weeks.The histopathological changes of the subchondral bone of the right knee-joint were observed after Saffron O dyeing and evaluated by Mankins score,and its anatomical structure including the bone volume fraction(bone volume/tissue volume,BV/TV),trabecular bone number(Tb.N)and trabecular thickness(Tb.Th),trabecular separation(Tb.Sp)was observed by using Micro CT imaging.The urine C-terminal cross-linking telopeptide of typeⅠcollagen(CTX-Ⅰ),CTX-Ⅱ(two bone resorption markers)and serum estrogen(E 2)contents were assayed by ELISA,and the expression levels of OPG,RANKL and MMP-13 mRNAs in the cartilage tissue of the left knee-joint were detected by quantitative RT-PCR.Results Following modeling,the BV/TV,Tb.N and Tb.Th levels were significantly decreased(P<0.01)and Tb.Sp and Mankin’s score obviously increased in the model group compared with the sham group(P<0.01),suggesting a formation of osteoporosis and degeneration of the cartilage tissue.The serum E 2 content and OPG mRNA level in the cartilagetissue were significantly down-regulated(P<0.01),and urine CTX-Ⅰ and CTX-Ⅱcontents and RANKL mRNA and MMP-13 mRNA expression levels cartilagetissue were considerably up-regulated in the model group relevant to the sham group(P<0.01).After EA intervention,modeling-induced decrease of BV/TV,Tb.N,Tb.Th,E 2 and OPG mRNA levels and OVX-induced increase of Tb.Sp,Mankin’s score,CTX-Ⅰ,CTX-Ⅱ,RANKL mRNA and MMP-13 mRNA levels were all completely reversed in the EA group relevant to the model group(P<0.01,P<0.05).Conclusion EA intervention can inhibit subchondral bone osteoporosis and articular cartilage degeneration of knee-joint in OVX rats,which is closely associated with its effects in inhibiting the downregulation of serum E 2 and OPG mRNA expression and up-regulation of CTX-Ⅰ,CTX-Ⅱ,RANKL mRNA and MMP-13 mRNA levels,including adjusting OPG/RANK/RANKL signaling.