基于Wnt/β-catenin信号通路探讨按压手法延缓大鼠椎间盘软骨终板退变的机制研究

Mechanism of Improving Degeneration Of Intervertebral Disc Cartilage Endplates in Rats on Wnt/β-catenin Signaling Pathway by Pressing Manipulation

目的基于Wnt/β-catenin信号转导通路,探讨按压手法治疗对退变大鼠腰椎间盘软骨终板的作用机制,为腰椎间盘退变相关疾病的治疗与研究提供新的方向。方法采用大鼠去前肢双足直立的造模方法建立大鼠腰椎间盘退变模型,正常组为对照组,对造模后大鼠分别给予按压手法、假按压连续干预60 d。干预结束后采用HE染色和番红固绿染色观察各组椎间盘组织病理变化,评估按压手法对腰椎间盘退变大鼠的治疗效果,通过Western Blot和实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)技术检测大鼠退变椎间盘软骨终板中Wnt/β-catenin信号通路中关键分子Wnt3a、β-catenin、糖原合酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)及其下游因子基质金属蛋白酶13(matrix metalloproteinase 13,MMP-13)的蛋白和mRNA表达水平变化。结果病理结果表明,与正常组相比,模型组椎间盘软骨终板结构与界限不清,软骨细胞出现明显肥大,分布广泛,番红染色变浅,周围固绿染色范围明显增加,颜色加深退变明显。经按压手法干预后,与模型组相比,软骨终板结构清晰完整,肥大软骨细胞明显减少,番红染色较深且均匀,而假按压组较模型组无明显改善。Western blot和qRT-PCR结果表明,与正常组相比,模型组软骨终板中可见Wnt3a、β-catenin和MMP-13蛋白及mRNA表达水平显著上调(P<0.01),GSK-3β表达下降(P<0.01);按压手法干预后,按压组中Wnt3a、β-catenin、MMP-13蛋白及mRNA表达明显降低(P<0.01),GSK-3β蛋白及mRNA表达增多(P<0.05)。假按压组各蛋白及mRNA表达水平较模型组无明显变化(P>0.05)。结论按压手法可能通过影响Wnt/β-catenin信号通路的激活,延缓大鼠腰椎间盘软骨终板退变程度。

Objective To explore the mechanism of pressing manipulation therapy on the cartilage endplates of lumbar discs in degenerated rats based on the Wnt/β-catenin signaling pathway and provide a new treatment strategy for the treatments and researches of lumbar disc degeneration-related diseases.Methods The lumbar intervertebral disc degeneration model of rats was established by using the forelimb removal and bipedal upright modeling method,and the normal group was used as the control group.The model rats were given pressing manipulation and sham pressing manipulation for 60 days after the 5-month modeling of the lumbar intervertebral disc degeneration.After the interventions,the histopathological changes of the intervertebral discs in each group were observed by HE staining and Safranin O-Fast Green staining,and the therapeutic effects of pressing manipulation on lumbar intervertebral disc degeneration rats were evaluated.Changes in protein and mRNA expression of Wnt-3 a,β-catenin,GSK-3βand the downstream factor matrix metalloproteinase-13(MMP-13)of the Wnt/β-catenin signaling pathway in the cartilage endplates of different groups were detected by Western Blot and qRT-PCR.Results The pathological results showed that compared with those of the control group,the cartilage endplate structure and boundary of the intervertebral disc in the model group were unclear,with the Saffron O staining becoming lighter,and the chondrocytes appeared significantly hypertrophic and widely distributed,while the range of Fast Green staining was significantly increased and deepened in color.After the pressing manipulation intervention,compared with those of the model group,the cartilage endplate structure was clear and intact.And the hypertrophic chondrocytes were significantly reduced,and the Saffron O staining was darker and more uniform.While the sham pressing group showed no significant improvement compared with the model group.Western Blot and qRT-PCR showed that Wnt3 a,β-catenin and MMP-13 proteins and mRNAs expression increased significantly in the cartilage endplates(P<0.01),but the GSK-3βexpression decreased(P<0.01)in the model group compared with that of the control group.After the pressing manipulation intervention,however,the expressions of Wnt3 a,β-catenin,MMP-13 protein and mRNA downgraded significantly(P<0.01)and the GSK-3βexpression increased(P<0.05).There was no significant change in the expressions of proteins or mRNAs in the sham pressing manipulation group compared with those of the model group(P>0.05).Conclusion The pressing manipulation may delay the degeneration of cartilage endplates in lumbar intervertebral disc degenration rats by inhibiting the Wnt/β-catenin signaling pathway.