益肾调髓方对膝骨关节炎小鼠软骨下骨骨代谢及疼痛的影响

Effects of Yishen Tiaosui Formula on subchondral bone metabolism and pain in mice with knee osteoarthritis

目的:探究益肾调髓方对膝骨关节炎(KOA)小鼠软骨下骨骨代谢及疼痛症状的干预作用及其可能机制,为临床通过调节骨代谢水平缓解KOA疼痛提供依据。方法:将18只雄性C57BL6/J小鼠随机分为假手术组、模型组及益肾调髓组,每组6只。采用内侧半月板失稳术造模,术后益肾调髓组予益肾调髓方灌胃(每天0.2 mL/10 g),假手术及模型组小鼠给予0.9%氯化钠溶液灌胃,术后4周检测小鼠步态变化、免疫组织荧光染色检测软骨下骨神经生长因子(NGF)表达情况及L4脊背神经节中降钙素基因相关肽(CGRP)表达情况评估小鼠疼痛程度,免疫组织化学染色检测软骨下骨碱性磷酸酶(ALP)表达评估骨形成情况,同时借助Micro-CT扫描、ABH/OG染色、TRAP染色等手段检测小鼠膝关节软骨退变及软骨下骨异常骨重塑情况。结果:步态分析、软骨下骨NGF及脊背神经节CGRP检测结果均表明益肾调髓方可减轻KOA小鼠疼痛症状;ABH/OG染色及OARSI评分提示益肾调髓方干预可延缓关节软骨退变;Micro-CT分析、TRAP染色及免疫组化结果证实,益肾调髓方可改善KOA小鼠软骨下骨异常骨重塑。结论:益肾调髓方可缓解KOA小鼠疼痛症状,同时延缓关节软骨退变及软骨下骨异常骨形成,其作用机制可能是通过调节软骨下骨骨代谢,减少致痛因子分泌来实现的。

Objective: To investigate the effects of Yishen Tiaosui Formula on subchondral bone metabolism and pain symptoms of knee osteoarthritis(KOA) in mice and explore its underlying mechanism. Methods: Eighteen male C57BL6/J mice were randomly divided into sham operation group, model group and Yishen Tiaosui group, with 6 rats in each group. The model was made by medial meniscus instability. After operation, the Yishen Tiaosui group was given Yishen Tiaosui Formula(0.2 mL/10 g per day), mice in sham operation group and model group were given 0.9% sodium chloride solution by gavage,and the gait changes of mice and subchondral tissues were detected by immunohistochemical staining at 4 weeks after operation.The expression of nerve growth factor(NGF) and calcitonin gene-related peptide(CGRP) in L4 dorsal ganglion were used to evaluate the pain degree of mice. Immunohistochemical staining was used to detect the expression of alkaline phosphatase(ALP)in subchondral bone to evaluate bone formation. Micro-CT scanning and ABH/OG staining were used at the same time. Color and TRAP staining were used to detect cartilage degeneration and abnormal bone remodeling of subchondral bone in mice.Results: Gait analysis, NGF and spine of subchondral bone CGRP test results of dorsal ganglion showed that tonifying kidney and regulating marrow could alleviate the pain symptoms of KOA mice. ABH/OG staining and OARSI score suggested Yishen Tiaosui Formula. Intervention can delay articular cartilage degeneration;Micro-CT analysis, TRAP staining and immunohistochemical results confirmed that tonifying kidney and regulating marrow can improve the subchondral bone of KOA mice. Abnormal bone remodeling. Conclusion: Yishen Tiaosui Formula can relieve pain symptoms in KOA mice, and at the same time delay articular cartilage degeneration and abnormal subchondral bone formation. Its mechanism of action may be achieved by regulating subchondral bone metabolism and reducing the secretion of pain-causing factors.