回医烙灸通过Wnt/β-catenin通路治疗膝骨性关节炎软骨退变的机制研究

The wear and tear of articular cartilage is the major cause of Knee Osteoarthritis（KOA）.How to delay the retrogression of articular cartilage is one of the key problems that need to be solved to cure KOA.In Hui medicine, cautery moxibustion is one of the most effective treatments to KOA,but the mechanism of cautery moxibustion to treat KOA can be expound.In our previous studies on rabbits, we have found that cautery moxibustion therapy can reduce the apoptosis of cartilage cells , MMP-3 and the IL-1 in the knee joint.While IL-1 and MMP-3 are key factors of the Wnt/β-catenin signal pathways,and activating this pathway can decrease the apoptosis of cartilage cells. In the present study，through analyzing the regulation of Wnt/β-catenin signal pathway to the apoptosis of cartilage cells and the extracellular matrix of cartilage cells in the knee joint. Therefore the hypothesis that the cautery moxibustion possibly treats KOA through mediating the Wnt/β-catenin signaling pathway is proposed.In this study the model of cautery moxibustion intervention of rabbits' KOA is adopted. Research contents:1. The mechanism of the cautery moxibustion influence multiplication and apoptosis of cartilage cells through mediating the Wnt/β-catenin signaling pathway;2. The mechanism of the cautery moxibustion regulate the extracellular matrix of cartilage cells through mediating the Wnt/β-catenin signaling pathway.The purpose of the study is to testify the hypothesis that the cautery moxibustion may treat KOA through mediating Wnt/β-catenin signaling pathway.The study also helps to find material basis of Four liquids theory and to further develop the Four liquid theory in Hui medicine.

延缓膝关节软骨退变是防治膝骨性关节炎(KOA)亟待解决的问题。特色的回医烙灸治疗KOA临床疗效确切，并能减少关节软骨退变，但作用机制不明。我们前期研究发现回医烙灸可减少KOA模型软骨细胞的凋亡及细胞外基质中IL-1和 MMP-3，而IL-1和MMP-3是Wnt/β-catenin通路上下游关键作用因子，激活该通路也可减少膝关节软骨细胞的凋亡。在此基础之上提出回医烙灸可能是通过Wnt/β-catenin通路治疗KOA软骨退变的假说。本课题采用烙灸干预兔KOA模型，主要研究：1.烙灸通过Wnt/β-catenin通路对软骨细胞增殖及凋亡的调节机制；2.烙灸通过Wnt/β-catenin通路对软骨细胞外基质合成代谢的调控机制。从而验证回医烙灸介导Wnt/β-catenin通路治疗KOA软骨退变的假说，阐明其部分作用机制。本课题还有助于寻找回医药学四液病理学说的物质基础，深化回医药学四液学说理论。

Wnt/β-catenin信号通路是膝骨性关节炎（Knee Osteoarthritis,KOA）软骨破坏病程中最重要的信号通路之一。通过维持Wnt/β-catenin信号在一定水平，减少软骨细胞的凋亡以及维持软骨基质环境的稳定，成为治疗KOA软骨退变的关键作用环节。因此，干预此信号通路成为防治软骨退变的理想选择。回医烙灸是中医化脓灸的发展与深化，具有刺激面积大、施灸量可控及铁制灸具传递热量迅速和均匀等优点，具有浓郁的民族特色，值得进一步挖掘和推广。. 通过分析回医烙灸干预后实验兔步态评分、影像学评分和软骨形态学评分的变化与Wnt蛋白和磷酸化β-catenin蛋白表达的相关性，探讨回医烙灸通过Wnt/β-catenin通路对软骨细胞凋亡的调节机制，以及对软骨细胞外基质环境的调控机制，验证回医烙灸通过介导Wnt/β-catenin信号通路治疗KOA。.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软骨表面稍粗糙，裂隙表浅，软骨细胞排列整齐，说明回医烙灸疗法具有良好的软骨修复作用。根据TUNEL、qRT-PCR、Western-Blot定量、分光光度法检测等结果，分别提示回医烙灸可以通过减少软骨细胞的凋亡，对软骨蛋白中Bcl-2、 Bax、Wnt3a、Wnt4、Wnt5a、Wnt10b、磷酸化β-catenin蛋白以及软骨细胞外基质金属蛋白酶MMP2、MMP3、ADAMTS-4、ADAMTS-5及FZD8蛋白表达有调节作用。. 回医烙灸可以通过降低β-catenin信号通路上相关蛋白及基因表达而减少软细胞的凋亡，并且可通过减少软骨外细胞基质金属蛋白酶、提高软骨组织中蛋白聚糖含量减少软骨细胞凋亡。. 课题的科学意义是验证回医烙灸是通过介导Wnt/β-catenin信号通路治疗KOA软骨退变的假说，阐明回医烙灸治疗KOA软骨退变的部分作用机制，有助于寻找回医药四液病理学说的物质基础。