自拟益气化瘀补肾方对膝骨关节炎小鼠β-catenin信号通路的影响

Effect of self-made Yiqi Huayu Bushen Recipe onβ-catenin signaling pathway in mice with knee osteoarthritis

目的研究自拟益气化瘀补肾方对β-catenin信号通路的影响及其治疗膝骨关节炎的作用机制。方法通过改良Hulth法切开小鼠右膝部内侧皮肤,切断内侧副韧带,切除内侧半月板,并切断前交叉韧带,造成小鼠膝骨关节炎模型,分为模型组和自拟益气化瘀补肾方组。假手术组单纯切开小鼠右膝部内侧皮肤。造模2个月后给予自拟益气化瘀补肾方灌胃,治疗2个月。采用Micro-CT扫描小鼠膝关节,ABH/OG染色观察小鼠膝关节软骨细胞变化,免疫组化染色观察小鼠膝关节β-catenin、MMP-13、Col X表达,RT-PCR检测Col X、MMP-9、MMP-13和OC基因表达。结果(1)Micro-CT检测发现,与假手术组比较,模型组膝关节周围骨赘形成增加,自拟益气化瘀补肾方组膝关节周围骨赘形成少于模型组。(2)ABH/OG染色发现,假手术组关节软骨表面光滑,表浅层、移行层、放射层和钙化层4层结构清晰可辨,软骨细胞呈柱状排列,无软骨细胞巢聚;模型组关节软骨组织表浅层细胞数量减少,移行层和放射层见到大量软骨细胞巢聚,潮线前移,出现双潮线;自拟益气化瘀补肾方组关节软骨组织形态较模型组明显改善。(3)β-catenin免疫组化染色结果显示,与假手术组比较,模型组关节软骨细胞β-catenin蛋白表达显著增加,自拟益气化瘀补肾方组关节软骨细胞β-catenin表达比模型组显著减少(P<0.05);免疫组化染色结果还发现,与假手术组比较,模型组关节软骨组织Col X、MMP-13蛋白表达显著增加(P<0.05)。与模型组比较,自拟益气化瘀补肾方组关节软骨组织Col X、MMP-13蛋白表达显著减少(P<0.05)。(4)RT-PCR反应检测结果显示,与假手术组比较,模型组关节软骨组织Col X、MMP-9、MMP-13和OC基因表达显著增加(P<0.05)。与模型组比较,自拟益气化瘀补肾方组关节软骨组织中Col X、MMP-9、MMP-13和OC基因表达显著减少(P<0.05)。结论自拟益气化瘀补肾方可以治疗膝骨关节炎,其作用机制可能是通过降低β-catenin的表达而实现。

Objective To study the effect of self-made Yiqi Huayu Bushen Recipe onβ-catenin signaling pathway and its mechanism for knee osteoarthritis.Methods The mouse model of knee osteoarthritis was established by modified Hulth method,which is used to cut the medial skin of the right knee,clip the medial collateral ligament,cut off the medial meniscus and clip the anterior cruciate ligament.Then the mice were divided into model group and self-made Yiqi Huayu Bushen Recipe group.In the sham operation group,the medial skin of the right knee was cut.Two months after the establishment of the model,the mice in the self-made Yiqi Huayu Bushen Recipe group were given the self-made Yiqi Huayu Bushen Recipe by gavage for 2 months.The mice were killed after 2 months of treatment,and the knee joints were taken for detection of related indexes,such as Micro-CT scan images,the changes of chondrocytes in knee joints by ABH/OG staining,protein expressions ofβ-catenin,MMP-13,Col X by immunohistochemical staining,gene expressions of Col X,MMP-9,MMP-13 and OC by RT-PCR reaction.Results(1)Micro-CT detection found that compared with the sham operation group,the formation of osteophytes around the knee joint in the model group was increased,and the formation of osteophytes in the self-made Yiqi Huayu Bushen Recipe group was less than that in the model group.(2)ABH/OG staining found that in the sham operation group,the surface of articular cartilage was smooth,and the superficial layer,transitional layer,radioactive layer and calcified layer were clear and recognizable.The chondrocytes were arranged in columnar shape,and no chondrocytes nested.In the model group,the number of cells in the superficial layer of articular cartilage tissue decreased,and a large number of cartilage cells nested in the transitional layer and radioactive layer.The tidal line moved forward,and a double tidal line appeared.The articular cartilage morphology in the self-made Yiqi Huayu Bushen Recipe group was significantly improved compared with the model group.(3)β-catenin immunohistochemical staining showed that compared with the sham operation group,the expression ofβ-catenin protein in articular chondrocytes of the model group was significantly increased,but in self-made Yiqi Huayu Bushen Recipe group the expression was significantly reduced compared with the model group(P<0.05);immunohistochemical staining also found that the expressions of Col X and MMP-13 protein in the articular cartilage tissue of the model group was significantly increased compared with the sham operation group(P<0.05).Compared with the model group,the protein expressions of Col X and MMP-13 in the articular cartilage tissue of self-made Yiqi Huayu Bushen Recipe group was significantly decreased(P<0.05).(4)The results of RT-PCR showed that compared with the sham operation group,the gene expressions of Col X,MMP-9,MMP-13 and OC in articular cartilage tissue of the model group were significantly increased(P<0.05).Compared with the model group,the gene expressions of Col X,MMP-9,MMP-13 and OC in self-made Yiqi Huayu Bushen Recipe group were significantly decreased(P<0.05).Conclusion The self-made Yiqi Huayu Bushen Recipe can treat knee osteoarthritis maybe by decreasing the expression ofβ-catenin.