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Medicine

推拿操作以减少膝关节骨关节炎大鼠的炎症和软骨损失

Published: June 30, 2023
doi:
10.3791/65451
, , , , ,
1Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine

Summary

我们提出了一种对石膏固定诱导的膝关节骨关节炎大鼠进行推拿操作的方案。根据初步结果，它表明该方法的功效依赖于减少炎症和软骨损失。

Abstract

临床试验表明，推拿手法可有效治疗膝关节骨关节炎（KOA），同时需要进一步研究以发现其机制。因此，膝关节骨关节炎动物模型的操作至关重要。该协议为推拿对KOA大鼠的操作提供了标准过程，并对推拿对KOA的机制进行了初步探索。将按压揉捏操作法（一种推拿操作，是指按压和揉捏体表的特定区域）应用于大鼠膝关节周围的5个穴位。通过手指压力记录标准化操作的力和频率，并且在协议中详细描述了操作过程中大鼠的位置。手法的效果可以通过疼痛行为测试和滑膜和软骨的显微镜检查来测量。KOA大鼠的疼痛行为有显著改善。推拿组滑膜组织炎症浸润减少，肿瘤坏死因子（TNF）-α表达明显降低。与对照组相比，推拿组软骨细胞凋亡较少。本研究为推拿对KOA大鼠的操作提供了标准化的方案，并初步证明推拿的治疗效果可能与减少滑膜炎症和延迟软骨细胞凋亡有关。

Introduction

膝关节骨关节炎（KOA）是一种退行性疾病，主要表现为关节疼痛。纤维化、开裂、溃疡和关节软骨脱落是本病的主要原因1。KOA患病率高，可能对患者的日常生活造成深远影响，严重者致残。在 45-84 岁的人群中，KOA 的患病率随着年龄的增长而增加，85 岁及以上人群的患病率为 15%，其中女性占优势 2,3。此外，KOA可能会给个人和社会带来严重的经济负担。一项调查显示，人均KOA的直接医疗保健费用高达8,858美元±每年5,120美元4。随着社会老龄化，KOA已成为一个全球性的健康问题和重大的社会问题，也是科学研究的热点问题。

循证研究表明，推拿手法在治疗KOA5方面的有效性。推拿手法可以缓解KOA患者的疼痛并改善功能障碍，其机制与抗炎作用有关6,7。学者发现，推拿操作有效抑制了炎症因子白细胞介素（IL）-β和5-羟色胺的表达，减缓了兔KOA模型8中关节软骨的退化。结果表明，推拿可促进病灶部位血液循环和代谢，有助于清除IL-1、IL-6和肿瘤坏死因子（TNF）-α等炎症因子，从而缓解KOA9的临床症状。此外，通过推拿操作使关节被动运动，可以促进滑液渗透和扩散到关节软骨中，改善组织营养代谢10。其他研究表明，推拿操作可以有效改善KOA患者的生物力学指标11。对软组织进行手法可以改善四肢的压力分布并增强平衡功能12,13。同时，通过一些关节调整操作，也可以调整下肢的排列以纠正异常步态14,15。

推拿操作治疗KOA的作用机制仍有待探索，因此有必要进行实验研究。推拿在实验动物中的应用关键是建模、动物固定和干预方法的标准化16。建模方法决定了实验动物是否可以表现出疾病的特征。同时，适当的固定方法可以方便推拿操作的干预，更好地体现推拿的效果。干预方法的标准化是推拿操纵中最难的部分。2010年，中国针灸标准基本体系提到了实验动物穴位标准，为动物实验中的针灸和推拿手术提供了可能17.但是，在标准化推拿操作方面仍然存在困难。推拿操纵有多种类型18.具体操作的选择主要取决于要治疗的疾病和表演者喜欢的治疗理论。在推拿对KOA的研究中，更多的关注是按点操作（用拇指或肘部按压特定的穴位）、Yizhichan推手法（通过摆动拇指进行推力操作）和按压揉捏操作（指用手指或手掌按压和揉捏体表的特定区域）19.按压和揉捏操作是应用最广泛的推拿操作之一，它结合了按压和揉捏来移动皮下组织20。穴位按压揉捏可促进血液循环，缓解疼痛，代表推拿对KOA19的治疗效果。

在本协议中，将详细描述按压和揉捏操作对KOA大鼠的操作，包括所选穴位，操作的强度和频率以及大鼠的身体位置，以便为未来的研究提供参考。

Protocol

本研究通过了上海中医药大学附属岳阳市中西医结合医院实验动物伦理委员会（YYLAC-2022-166）的动物伦理评审。 1. 实验动物的制备和分组 动物制备在室温（18-21°C），湿度40%-50%，12小时：12小时昼夜节律交替下饲养10只健康的SPF SD雌性大鼠200-220克。严格按照动物伦理准则和准则的有关规定进行与疼痛有关的动物实验。 动物分组?…

Representative Results

疼痛行为测试MWT结果显示，建模后右后肢MWT明显低于前（p<0.05）。与对照组相比，推拿后大鼠MWT显著升高（p<0.05; 图 5 和 表 2）。 图5.机械抽出阈值检验的结果 （<img alt="Equation 1" src="/files/ftp_upl…

Discussion

本研究为KOA大鼠的推拿操作提供了方案。通过疼痛行为试验和组织形态学发现，将这一系列推拿手法应用于KOA大鼠可以减少滑膜炎症和软骨凋亡，可为KOA动物模型的推拿手法提供参考。

协议期间有几个关键程序。首先，选择适当的方法来诱导KOA模型很重要。诱导KOA模型的方法多种多样，包括关节腔内注射25、关节内手术26、27、关节制动法<s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了上海市重点临床专科建设项目（批准号：Shslczdzk04001）的支持;上海市科委帆船项目（批准号：22YF1444300）;上海中医药大学预算范围内的项目（批准号：2021LK091）。

Materials

absolute ethanol Supelco PHR1070 For making specimen
ALMEMO admeasuring apparatus ahlborn 2450-1 For Mechanical Withdrawal Threshold test
Anti-Digoxin antibody Sigma-Aldrich SAB4200669 For HE stain IHC or TUNEL
Anti-IL-1 beta abcam ab283818 For HE stain IHC or TUNEL
DAB Substrate kit Solarbio DA1010 For HE stain IHC or TUNEL
Denture base materials Shanghai New Century 20000356 For model making
eosin bioswamp  PAB180016  For HE stain IHC or TUNEL
Finger pressure recordings Suzhou Changxian Optoelectronic Technology CX1003w For Tuina manipulation
formic acid solution Sigma-Aldrich 695076 For decalcification
H2O2 Sigma-Aldrich 386790-M For HE stain IHC or TUNEL
hematoxylin bioswamp  PAB180015 For HE stain IHC or TUNEL
Isoflurane Shanghai Yuyan Scientific Instrument Company S10010533 For gas anesthesia
neutral resins bioswamp  PAB180017 For HE stain IHC or TUNEL
Paraformaldehyde Fix Solution Sigma-Aldrich 100496 For histology
PBS Sigma-Aldrich P3813 For HE stain IHC or TUNEL
Plantar Test Apparatus IITC Life Science / For Paw Withdrawal Latency test
plaster of Paris bandage WANDE 20150023 For model making
Proteinase K Sigma-Aldrich 124568 For HE stain IHC or TUNEL
TNF Alpha Monoclonal antibody Proteintech 60291-1-Ig For HE stain IHC or TUNEL
TUNEL  Servicebio GDP1042 For HE stain IHC or TUNEL
Wax Sigma-Aldrich 327204 For making specimen
xylene Shanghai Sinopharm Group 100092 For making specimen
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Tags

Keywords: Knee OsteoarthritisTuina ManipulationInflammationCartilage LossAnimal ModelStandardizationSynovial InflammationChondrocyte ApoptosisPain BehaviorTNF-α
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Su, X., Song, P., Meng, F., Xu, W., Xing, H., Zhang, H. Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats. J. Vis. Exp. (196), e65451, doi:10.3791/65451 (2023).
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