基于OPG/RANKL/RANK通路研究回医烙灸对膝骨性关节炎软骨下骨重建的作用机制

Imbalance of the proliferation and apoptosis of osteoclasts and osteoblasts is the cytological basis for the abnormality remodeling of the subchondral bone in the knee osteoarthritis (KOA). Abnormal bone remodeling in subchondral bone and cartilage degeneration aggravates KOA.How to maintain the balance of bone remodeling is one of the key problems that need to solve . In Hui medicine,cautery moxibustion is one of the most effective treaments to KOA and can reduce degeneration of articular cartilage and maintain balance of subchondral bone remodeling ,but the mechanism of subchondral bone remodeling is unknown.In our previous studies on rabbits,we have found that cautery moxibustion therapy can increase the apoptosis of osteoclast and decrease the expression of IL-1 and MMP-3 in the extracellular matrix of subchondral bone.While IL-1 and MMP-3 are key factors of the OPG/RANKL/RANK signal pathway,and activating this pathway can regulate the proliferation and apoptosis of osteoclasts and osteoblasts and the synthesis and metabolism of extracellular matrix.Therefore,the hypothesis that the cautery moxibustion throughs mediating the OPG/RANKL/RANK signaling pathway to treat KOA is proposed.In this study, the model of rabbits KOA is adopted to be treated by cautery moxibustion.Reseach contents:1.The effects of the cautery moxibustion on the proliferation and apoptosis of osteoclasts and osteoblasts in subchondral bone through mediating the OPG/RANKL/RANK signaling pathway.2.The effects of the cautery moxibustion on the extracellular matrix environment of subchondral bone through mediating the OPG/RANKL/RANK signaling pathway.The purpose of the study is to testify the hypothesis that the cautery moxibustion may treat KOA through mediating OPG/RANKL/RANK signaling pathway.The results of this study provide theoretical and experimental basis for the treatment of cautery moxibustion to KOA.

破骨细胞和成骨细胞增殖、凋亡失衡是膝骨性关节炎（KOA）软骨下骨异常骨重建的细胞学基础。软骨下骨异常骨重建与软骨退变相互影响加重KOA，特色回医烙灸治疗KOA疗效确切，减少软骨退变，调控软骨下骨异常骨重建，但调控骨重建作用机制不明。前期研究发现，回医烙灸促进兔KOA模型软骨下骨破骨细胞凋亡及降低胞外基质IL-1和MMP-3表达；而IL-1和MMP-3是OPG/RANKL/RANK通路中的关键作用因子，激活该通路可调控破骨、成骨细胞增殖、凋亡及胞外基质合成代谢。由此提出回医烙灸可能通过介导该信号通路调控骨重建的假说。本课题采用烙灸干预兔KOA模型，主要研究：1.阐明回医烙灸通过OPG/RANKL/RANK通路对软骨下骨破骨、成骨细胞增殖、凋亡的影响；2.探讨回医烙灸通过OPG/RANKL/RANK通路对软骨下骨细胞外基质环境合成代谢的影响。此研究结果为回医烙灸治疗KOA提供理论和实验依据。

破骨细胞和成骨细胞增殖、凋亡失衡是KOA软骨下骨异常骨重建的细胞学基础。OPG/RANKL/RANK信号通路是KOA软骨下骨破坏病程中最重要的信号通路之一。通过维持OPG/RANKL/RANK信号在一定水平，减少软骨下骨破骨、成骨细胞增殖、凋亡及胞外基质环境的稳定，成为治疗KOA软骨下骨骨重建的关键作用环节。因此，干预此信号通路成为防治软骨下骨异常骨重建的理想选择。本研究采用临床疗效确切的回医烙灸干预KOA 兔模型，分析OPG/RANKL/RANK通路对破骨、和成骨细胞的增殖、凋亡及细胞外基质环境的调控，探讨回医烙灸治疗KOA的作用机制。本研究内容：采用Hulth造模方法制备兔KOA模型；造模成功后随机分为模型组、回医烙灸组、拮抗剂组和空白组。回医烙灸组在兔左后膝关节进行每日1次的回医烙灸治疗，每次10分钟；拮抗剂组的烙灸干预方法同回医烙灸组，每周烙灸干预结束后经实验兔腹腔内注射重组人骨保护素FC融合蛋白（5μg/kg）。空白组和模型组均不予处理。干预前后对实验兔进行一般行为学观察；干预6周后取材；采用TB和HE染色观察软骨下骨组织形态学变化； 分别采用双重免疫荧光染色、RT-PCR、Western Blot等方法对软骨下骨组织破骨、成骨细胞的增殖和凋亡、OPG、RANK、RANKL、MMP-3、MMP-9、IL-1、COL I和OPN、羟脯氨酸，己糖胺，蛋白聚糖和钙进行检测。本研究发现：回医烙灸干预兔KOA模型后，形态学观察发现烙灸组表现为纤维化范围的减少以及关节软骨下骨的浅层和中部区域的裂纹，说明回医烙灸疗法具有良好的软骨下骨修复作用。根据双重免疫荧光染色、RT-PCR、Western Blot定量检测等结果，分别提示回医烙灸可以通过减少软骨下骨细胞的凋亡，对软骨下骨中OPG、RANK、RANKL、MMP-3、MMP-9、IL-1、COL I和OPN蛋白以及主要成分表达有调节作用。 回医烙灸可以通过调节OPG/RANKL/RANK信号通路上相关蛋白及基因表达而减少软骨下骨细胞的凋亡，并且可通过增加软骨下骨主要成分的表达，从而抑制了软骨下骨区域的破坏和降解。课题的科学意义是验证回医烙灸是通过介导OPG/RANKL/RANK信号通路治疗KOA软骨下骨骨重建的假说，阐明其部分作用机制，为回医烙灸治疗KOA提供理论和实验依据。