软骨下骨局部注射常山酮抑制转化生长因子β1信号通路缓解犬骨关节炎

Local injection of halofuginone into the subchondral bone relieves canine osteoarthritis by inhibiting transforming growth factor beta 1 signaling pathway

背景:研究表明随着破骨细胞骨吸收的增加,过度激活了的转化生长因子β1使骨吸收和骨形成解偶联,最终导致骨关节炎动物模型中软骨下骨的硬化表型,且可通过抑制转化生长因子β1信号传导减弱骨关节炎的进展。目的:检测前十字韧带横切比格犬骨关节炎模型中局部注射常山酮是否可以延缓骨关节炎进展。方法:将18只雄性比格犬分为假手术(对照组)、骨性关节炎组及治疗组,后两组通过前十字韧带横切构建骨关节炎模型,治疗组造模后于软骨下骨局部注射常山酮37.8 ng。在造模术后4,8,12,16周纵向测量血清Ⅱ型胶原C端肽(CTX-Ⅱ)和X型胶原蛋白α1链血清标志物的水平;术后16周Micro-CT扫描观察软骨下骨微结构;番红固绿染色及OARSI-Modified Manking评分评估关节软骨退变程度;免疫组织化学染色对比转化生长因子β1、基质金属蛋白酶13的表达情况。实验方案经新疆医科大学第一附属医院动物实验伦理委员会批准(批准号为IACUC20160304-07)。结果与结论:①造模后8,12周,骨关节炎组X型胶原蛋白α1链水平高于治疗组和对照组(均P<0.01);②造模后8,12,16周,骨关节炎组CTX-Ⅱ水平均高于对照组和治疗组(均P<0.05);③骨关节炎组软骨下骨的骨体积分数较治疗组和对照组增加,骨小梁分离度降低,骨小梁模式因子减小(均P<0.05);④骨关节炎组OARSI-Modified Manking评分较对照组和治疗组更高(均P<0.01);⑤骨关节炎组基质金属蛋白酶13及转化生长因子β1的表达量较对照组和治疗组更高(均P<0.01);⑥对照组与治疗组上述各指标比较差异均无显著性意义(均P>0.05);⑦结果说明,局部注射常山酮可通过抑制软骨下骨中异常升高的转化生长因子β1,阻断异常骨重塑来减轻前十字韧带横切诱导的骨关节炎,表明这可能是骨关节炎的一种新的治疗选择。

BACKGROUND:Studies have shown that the increase of bone resorption in osteoclasts over-activates transforming growth factor beta 1(TGF-β1),and uncouples bone resorption and bone formation,ultimately leading to a hardened phenotype of the subchondral bone in an animal model of osteoarthritis.Progression of osteoarthritis can be attenuated by inhibiting TGF-β1 signaling pathway.OBJECTIVE:To detect whether osteoarthritis progression can be delayed by the local injection of halofuginone in Beagle models of osteoarthritis.METHODS:Eighteen male Beagle dogs were randomized into a sham(control)group,a model(osteoarthritis)group,or a treatment(halofuginone)group.Animal models of osteoarthritis were made by anterior cruciate ligament transection in the latter two groups.Animals in the treatment group were given local injection of halofuginone(37.8 ng)into the subchondral bone.Serum levels of type II collagen C-terminal peptide(CTX-II)and type X collagenα1 chain(COL10 A1)were measured at 4,8,12,and 16 weeks after modeling.The Beagle dogs were sacrificed at the 16 th week after surgery,and the alterations of microarchitecture of the subchondral bone were detected by micro-CT.Articular cartilage degeneration was graded using safranin O and fast green staining combined with the Osteoarthritis Research Society International(OARSI)-modified Manking criteria.Immunostaining analyses were conducted to detect the expression levels of TGF-β1 and matrix metalloproteinase 13.The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University,with the approval No.IACUC20160304-07.RESULTS AND CONCLUSION:The COL10 A1 level in the model group was higher than that in the control group and the treatment group at 8 and 12 weeks after modeling(P<0.01).The levels of CTX-II in the model group were higher than that in the control group and the treatment group at 8,12,and 16 weeks after modeling(P<0.05).Micro-CT examination showed that compared with the control and treatment groups,the bone volume fraction of the subchondral bone was increased in the model group,while the subchondral bone trabecular separation and the trabecular model factor were decreased significantly(all P<0.05).The OARSI-modified Manking score was significantly higher in the model group than in the control group and the treatment group(both P<0.01).Immunohistochemistry results showed that the expression levels of matrix metalloproteinase 13 and TGF-β1 in the model group were significantly higher than those in the control group and the treatment group(all P<0.01).No significant differences in the above-mentioned indexes were found between the control group and the treatment group(all P>0.05).Overall,local injection of halofuginon attenuates anterior cruciate ligament transection-induced osteoarthritis by inhibiting abnormally elevated TGF-β1 in the subchondral bone and blocking abnormal bone remodeling.Therefore,local injection of halofuginon may be a new therapeutic alternative for osteoarthritis.