补肾强督方对DBA/1小鼠组织形态学及Wnt通路的影响

Effect of Bushen Qiangdu Recipe on Histomorphology and Wnt Pathway of DBA/1 Mice

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 effect of Bushen Qiangdu Recipe （BQR） on the entheses ossification histomorphology of articular ligament of DBA/1 mice with spontaneous ankylosing spondylitis （AS）, and to study its mechanism for prevention and treatment of AS. Methods Thirty 12-week old male DBA/1 mice were randomly divided into the model group, the positive drug group, low, medium, high dose BQR groups, 6 in each group. Another 6 C57BL/6 mice of the same age were recruited as a blank control group. BQR containing 11.25, 22.50, 45.00 g/kg crude drugs was respectively adminis- tered to mice in low, medium, high dose BQR groups by gastrogavage, 0.2 mL for each mouse, once per day. Celecoxib Capsule （0.2 mL/0.8 mg for each mouse, once per day） was administered to mice inthe positive drug group by gastrogavage. Equal volume of normal saline was administered to mice in the model group and the blank control group by gastrogavage. All mice were fed and intragastically adminis- tered for 12 successive weeks. Body weight, diet, stools, and hair were routinely observed. Signs of ar- thritis were evaluated once per two weeks. Mice were sacrifice, and then general observation of achilles tendon was performed. The achilles tendon tissue was lie stained. Protein expressions of alkaline phos- phatase （ALP）, bone gamma-carboxyglutamic-acid-containing proteins （ BGP）, Dickkopfl （ DKK1 ）, and Wnt5a in the achilles tendon were detected using immunohistochemical method. Results Compared with the blank control group, the scoring of arthritis obviously increased in the model group （P 〈0.05）. But the scoring of arthritis was obviously lower in the 3 BQR groups and the positive drug group than in the model group （P 〈0.05）. Histopathological results of achilles tendon tissue showed that no infiltration of inflammatory cells or fibroblasts occurred in the normal group. Their histomorphological structures were normal. Cartilage formation and bone formation at various degrees occurred in the model group. Filtration of fibroblast-like cells occurred in inflammatory cells and attachment points. Scattered lymphocyte infiltra- tion was often seen in the achilles tendon tissue of each medicated group. Cartilage formation and bone formation were rarely seen. Compared with the blank control group, the scoring of arthritis increased in the model group （P 〈0.05）. Compared with the model group, the scoring of arthritis was decreased in the 3 BQR groups and the positive drug group （P 〈0.05）. Compared with the blank control group, protein expression of DKK1 decreased and protein expression of Wnt5a increased in the model group （P 〈0.05）. Compared with the model group, protein expression of DKK1 increased and protein expression of Wnt5a decreased in middle and high dose BQR groups （P 〈0.05）. Conclusion BQR could delay the occur- rence and development of arthritis and ossification in DBA/1 mice of spontaneous AS model possibly by inhibiting classical Wnt pathway.