单碘乙酸钠注射液诱导膝骨关节炎大鼠模型的推拿干预
Summary
本方案描述了单碘乙酸钠注射液诱导的膝骨关节炎（KOA）大鼠模型中的推拿干预方法，为推拿在KOA动物模型中的应用提供了参考。该方案还研究了推拿治疗KOA的有效机制，其结果将有助于促进其应用。
Abstract
膝骨关节炎 （KOA） 是一种常见的退行性关节疾病，其特征是慢性疼痛和残疾，可发展为无法修复的关节结构损伤。研究关节软骨、肌肉、滑膜和 KOA 膝关节周围其他组织之间的联系非常重要。目前，管理 KOA 包括改变生活方式、运动、药物治疗和手术干预;然而，仍然缺乏对KOA相关疼痛的复杂机制的阐明。因此，KOA疼痛仍然是一个关键的临床挑战和治疗重点。推拿已被发现对运动、免疫和内分泌系统有调节作用，促使人们探索推拿是否可以缓解由炎症因子上调引起的 KOA 症状，以及骨骼肌中的炎症因子是否可以增强 KOA 的进展。
我们将 32 只雄性 Sprague Dawley （SD） 大鼠 （180-220 g） 随机分为四组，每组 8 只动物：抗 PD-L1+推拿（A 组）、模型（B 组）、推拿（C 组）和假手术（D 组）。对于A组、B组和C组，我们将25 μL单碘乙酸钠（MIA）溶液（4 mg MIA稀释在25 μL无菌盐水溶液中稀释）注射到右膝关节腔中，对于D组，注射等量的无菌生理盐水。所有组均在注射前和注射后 2、9 和 16 天使用压力最小到最大的测试（爪子机械戒断阈值、爪子撤出热潜伏期、右膝关节肿胀、Lequesne MG 评分、皮肤温度）进行评估。
Introduction
膝骨关节炎（KOA）是一种常见的退行性骨关节炎，以慢性疼痛和残疾为特征，相对严重的KOA会导致关节不可逆的结构性损伤1。KOA 的全球高患病率已成为全球公共卫生的重大挑战2,3，严重损害患者的生活质量 4,5。研究表明，KOA 影响着全球超过 2.6 亿人6。随着人口老龄化，我国40岁以上KOA总患病率高达17.0%，给受影响者的家庭带来了沉重的负担7,8。
根据中医的说法，KOA属于“麻痹”的范畴9，《黄帝内科经》将肌腱、骨骼和肉与麻痹联系在一起。因此，我们需要注意 KOA 中软骨、肌肉、滑膜以及膝关节周围其他组织之间的联系。在现代医学中，肌肉和骨骼炎症是关节炎免疫稳态的主要组成部分，它们推动了 KOA 疼痛的进展。然而，大多数研究集中在骨关节的软骨炎症和滑膜炎症上，并且缺乏研究骨骼肌炎症现象的文章。因此，需要对骨骼肌在炎症中的作用进行更多研究，以更全面地了解KOA，并为更有效的治疗方式提供思路。在文献综述中，我们发现抑制免疫相关因子程序性死亡配体 1 （PD-L1） 加剧了 KOA 10,11 的进展，并且 IL-15 和 FOXO1 在 KOA12 患者的骨骼肌中存在差异表达，以及终末期 KOA 患者骨骼肌炎症明显， 这也与步态的变化有关。研究表明，骨骼肌还可以分泌与炎症免疫密切相关的因子，如IL-6，TNF-α也与PD-L1通路和KOA13,14,15密切相关。
目前，KOA 的可用治疗方法包括体重控制、运动、药物治疗和手术，通常包括关节内注射、镇痛药物、关节镜手术和膝关节假体周围截骨术16。目前，KOA疾病的进展无法完全停止或逆转，如果不清楚地了解KOA疼痛的复杂机制，KOA患者有过度使用镇痛药物和高副作用发生率的风险8。大多数治疗策略仅在短期内有效，并且仅关注症状的暂时缓解，而不是疾病的发病机制。因此，KOA疼痛仍然是治疗的重点和挑战，构成了严峻的临床挑战。然而，在KOA治疗中，推拿可以直接刺激患处关节周围的骨骼肌，并能提供一些好处。结合前文所述，是研究骨骼肌组织在KOA中的作用的理想疗法，阐明骨骼肌组织在炎症中的作用也可以为KOA推拿提供更好的理论支持。
研究表明，按摩疗法、整骨手法疗法和脊柱推拿可以帮助缓解疼痛和恢复身体功能17,18。这些操作可以作用于局部炎症，并通过外部机械作用缓解痉挛和镇痛。它们可以缓解全膝关节置换术后的疼痛，减少镇痛药物的使用，促进术后恢复，改善生物力学平衡19。瑞典式按摩在治疗骨关节炎方面是可行的，可以减轻压力和提高生活质量20.推拿对KOA的治疗效果已经有了一些研究。
在本次实验中，我们团队将基于MIA诱导的大鼠KOA模型，探讨骨骼肌中PD-L1及相关炎症因子的表达是否能诱导KOA并促进其发展。推拿也将实施，看看它是否可以缓解由炎症因子表达增加引起的KOA症状。推拿将与PD-L1抑制相结合，证明推拿介导的PD-1通路在骨骼肌炎症中影响KOA发展和疼痛的关联，为进一步多层次研究推拿干预KOA的治疗机制奠定了基础。
综上所述，本文从免疫-炎症因子表达的角度，结合行为相关指标，阐述了骨骼肌炎症在KOA及其疼痛发展中的机制，以及推拿对KOA的治疗效果，以期为中医“筋骨失衡”理论提供现代研究证据。
Protocol
Representative Results
Discussion
本研究旨在通过使用标准化的行为指标评估推拿干预后 KOA 的改善情况，并探讨推拿治疗 KOA 的机制以及骨骼肌与 KOA 的关联。与药物和手术治疗不同，推拿对运动、免疫和内分泌系统有积极的调节作用。推拿可以通过作用于不同的靶点来缓解疾病引起的炎症和疼痛。例如，通过调节TLR4通路和miRNA，它可以抑制神经胶质细胞的活化，调节脑功能改变，调节下游炎性细胞因子，抑制外周炎症<sup class="x…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金（NO.82105042,82205302）资助;上海市博士后卓越计划（NO.2020371）;中国博士后科学基金（NO.2021M692156）;上海帆船计划（NO.20YF1450900）;岳阳市中西医结合医院科学基金（NO.2021YYGQ03）.资助者在研究的设计、执行或撰写中没有作用。
Materials
| Anti-PD-L1 | Abcam, Cambridge, MA, USA | ab80276 | |
| electric von Frey esthesiometer | IITC/Life Science, Woodland Hills, CA, USA | ALMEMO 2450 | |
| GraphPad Prism 9.0 | GraphPad Software | Software for stastistical analysis | |
| monosodium iodoacetate | Sigma-Aldrich Inc | I9148 | Resolved into normal saline for injection |
| pentasorbital sodium | Sigma-Aldrich Inc | P3761 | |
| Sprague Dawley (SD) male rats | Shanghai Jihui Experimental Animal Breeding Co., Ltd | No. SCXK (Hu) 2017-0012 | |
| thermal analgesia tester | IITC/Life Science | Model 390 |
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