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目的观察补肾强督方对自发性强直性脊柱炎模型DBA/1小鼠关节韧带及附着点骨化程度和DKK1/Wnt通路的影响,探讨补肾强督方防治强直性脊柱炎的作用机制。方法将30只12周龄的雄性DBA/1小鼠随机分为模型组、阳性药物组、补肾强督方低、中、高剂量组,每组6只,另设6只同周龄C57BL/6小鼠作为空白组。补肾强督方低、中及高剂量组分别灌胃低、中、高浓度的补肾强督方,所含生药重量分别为11.25、22.5、45 g/(kg·d),0.2 m L/只,每日1次;阳性药物组灌胃塞来昔布胶囊0.8 mg/只,0.2 m L/只,每日1次;模型组及空白组灌胃等量的生理盐水。连续饲养、灌胃12周。定期观察小鼠体重、饮食、大便、毛发等情况。每两周评价1次小鼠关节炎体征。处死后取小鼠跟腱部位进行大体观察,对跟腱组织行HE染色,用免疫组化法检测小鼠跟腱中碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(bone gamma-carboxyglutamicacid-containing proteins,BGP)、DKK1、Wnt5a的蛋白表达。结果与空白对照组比较,模型组关节炎体征评分明显升高,而补肾强督方各剂量组及阳性药物组评分均低于模型组(P<0.05)。跟腱组织病理观察显示,正常组无炎性细胞及成纤维细胞浸润,组织形态结构正常;模型组出现不同程度的软骨及骨形成、炎性细胞及附着点成纤维样细胞浸润;各给药组小鼠跟腱组织多见散在淋巴细胞浸润,软骨及骨形成较少见。与空白组比较,模型组组织标本骨化评分升高,补肾强督方各剂量组及阳性药物组评分均低于模型组,差异有统计学意义(P<0.05)。与空白组比较,模型组DKK1蛋白表达降低,Wnt5a蛋白表达升高(P<0.05);与模型组比较,补肾强督方高、中剂量组DKK1蛋白表达升高,Wnt5a蛋白降低,差异有统计学意义(P<0.05)。结论补肾强督方可能通过抑制经典Wnt通路来延缓自发性强直性脊柱炎模型DBA/1小鼠关节炎及骨化程度的发生与发展。
Objective To observe the effects of Bushenchaoyu Decoction on the degree of ossification and the DKK1 / Wnt pathway in the ligaments and attachment points of DBA / 1 mice and explore the mechanism of Bushenjiangdu Decoction in preventing and treating ankylosing spondylitis. Methods Thirty male DBA / 1 mice (12 weeks old) were randomly divided into model group, positive drug group, Bushenqiangdu low, medium and high dose group, with 6 rats in each group. 6 mice as a blank group. Bushenqiangdu low, medium and high dose groups were given low, medium and high concentration of Bushenqiangdu prescription, the weight of crude drugs contained were 11.25,22.5,45 g / (kg · d), 0.2 m L / , Once a day. The positive drug group was given celecoxib capsules 0.8 mg / only, 0.2 m L / day once a day. The model group and the blank group were given the same amount of normal saline. Continuous feeding, gavage for 12 weeks. Regular observation of mice weight, diet, stool, hair and so on. Mouse arthritis signs were evaluated every two weeks. The mice were sacrificed and the Achilles tendon was removed for observation. The Achilles tendon tissues were stained with HE, and the alkaline phosphatase (ALP), bone gamma-carboxyglutamic acid -containing proteins, BGP), DKK1, Wnt5a protein expression. Results Compared with the blank control group, the score of arthritis in the model group was significantly higher than that of the model group (P <0.05). Achilles tendon tissue histopathology showed that the normal group infiltration of inflammatory cells and fibroblasts, normal tissue morphology structure; the model group showed varying degrees of cartilage and bone formation, inflammatory cells and attachment points fibroblast-like cell infiltration; each administration A group of mice more common tendon tissue scattered lymphocyte infiltration, cartilage and bone formation is rare. Compared with the blank group, the ossification score of the model group was significantly higher than that of the model group (P <0.05). Compared with the blank group, DKK1 protein expression and Wnt5a protein expression increased (P <0.05). Compared with the model group, the expression of DKK1 protein and Wnt5a protein were decreased Significance (P <0.05). Conclusion Bushenchaoshu prescription may delay the occurrence and development of arthritis and ossification in spontaneously ankylosing spondylitis model DBA / 1 mice by inhibiting the canonical Wnt pathway.