模仿丁氏滚动法治疗大鼠肌损伤
Summary
该协议描述了一种模仿Ding滚动方法的简单装置，建立了骨骼肌损伤的大鼠模型，并使用苏木精-伊红染色观察受损组织的病理学和酶联免疫吸附测定法来检测血清损伤标志物的变化。
Abstract
丁氏翻滚法是中国传统按摩诊所中最常用的推拿法之一，也是中国当代推拿法中最有影响力的推拿法之一。它以单指禅宗流派中常用的传统卷法为基础，命名为丁氏卷法。由于其抗炎和促进血液循环的作用，丁氏滚动法对肌病有很好的治疗效果。由于施加在人体皮肤上的力面积很大，丁氏滚动法在皮肤面积小的实验动物（如大鼠和兔子）上具有挑战性。此外，推拿应用于人体的强度与应用于实验动物的强度不同，因此在实验过程中可能会发生强度过高或过低而无法达到推拿的治疗效果。该实验旨在根据 Ding 的滚动操作参数（强度、频率、推拿持续时间）创建一种适合大鼠的简单按摩器。该装置可以标准化动物实验中的操作，并减少由于主观因素而施加到不同动物身上的推拿力的变化。建立大鼠骨骼肌损伤模型，采用血浆损伤标志物肌酸激酶（CK）和脂肪酸结合蛋白3（FABP3）评价推拿对骨骼肌损伤的治疗效果。结果表明，该推拿按摩器可降低CK和FABP3的表达水平，减缓骨骼肌损伤的程度。因此，本文介绍的推拿按摩器，模仿丁氏滚动法，有助于实验研究中推拿操作的标准化，对后续推拿治疗肌病分子机制的研究有很大帮助。
Introduction
肌肉损伤是临床和日常生活中常见的创伤性损伤，由外部打击（挫伤）或肌纤维慢性过度拉伤（拉伤）等引起，导致肌肉功能障碍和疼痛，甚至严重影响患者的生活质量1。急性劳损后尽早开始康复是缩短恢复运动时间的关键2 和减轻疼痛 3,4。在现代西医中，肌肉损伤的临床急救遵循休息、冰敷、加压和抬高 （RICE） 原则，以阻止肌肉组织有害出血5 和非甾体抗炎药缓解疼痛6。外泌体7 和组织工程8 等新疗法的发现成为骨骼肌疾病的潜在治疗策略，弥补了先前药物治疗的不足。然而，它也会增加患者的治疗成本，使他们承受巨大的经济压力9.因此，建议使用替代疗法和补充疗法来治疗肌肉骨骼问题10.推拿作为传统医疗方法在中国临床上被广泛使用，因其疗效好、副作用少而受到患者的欢迎。推拿治疗肌肉骨骼疾病可以减轻疼痛并改善功能11,12,13。上海著名推拿法师丁继峰先生创立了丁氏卷法14。它是一种独特的轧碎技术，受力面积大，力均匀柔和，穿透力强。
不同的动物模型基于不同的病因。它们各有利弊，选择正确、合适的动物模型对基础实验具有重要意义，有助于了解骨骼肌损伤后再生和修复的细胞和分子信号通路，为开发治疗骨骼肌疾病的新疗法奠定基础意义。化学诱导的肌肉损伤模型被广泛使用，骨骼肌注射引起肌纤维坏死并产生可以在 2 周内有效再生的再生区域15。notexin 和布比卡因都会导致肌肉损伤。然而，与布比卡因相比，notexin 对骨骼肌的肌毒性损伤更严重，并且自然功能恢复相对较慢16.药物肌内注射成型不仅时间更短，而且对骨骼肌损伤的效果和程度也可控。这种可量化的控制使成功的成型难度降低15,17。
炎症反应是一种重要的生物学反应，已在肌病的背景下进行了广泛研究18,19。在骨骼肌损伤的早期阶段，肌纤维坏死会破坏局部肌肉稳态，许多炎症细胞浸润损伤部位，分泌许多促炎细胞因子19。肌酸激酶 （CK） 是用于评估骨骼肌损伤的传统血清生物标志物。然而，它缺乏组织特异性20 和敏感性21，这限制了其评估药物诱导的肌肉损伤程度和间接报告损伤后肌肉恢复程度的能力。包括脂肪酸结合蛋白 3 （FABP3） 在内的新型生物标志物最近在骨骼肌损伤的啮齿动物模型中显示出相对较高的组织特异性和敏感性。FABP3 是一个结合蛋白家族，主要在心脏和骨骼肌细胞中表达，与脂肪酸代谢、转运和信号传导有关22。因此，我们选择了两种生物标志物CK和FABP3的组合，以评估notexin诱导的骨骼肌损伤和治疗后的恢复程度。
在啮齿类动物中，肌肉较浅，皮肤面积较小，这也决定了啮齿类动物按摩的各种参数不会与人类相同，例如在动物治疗中，按摩治疗师应使用丁氏滚动法以较小的力气对待它们，并且可能由于受伤部位的尺寸较小，不利于该技术的操作， 这最终会导致按摩效果的降低。因此，本实验利用自制的符合丁氏滚动法特点的滚动按摩器，对notexin诱导的大鼠骨骼肌损伤模型的治疗效果进行干预和评价，有助于在实验动物研究中标准化推拿参数，以深入研究推拿的分子作用机制。 一种中医治疗方法，用于肌肉骨骼疾病。
Protocol
Representative Results
Discussion
在这里，我们描述了推拿治疗大鼠骨骼肌损伤的方案，然后分析了治疗后骨骼肌损伤的程度，以验证该方法的有效性。值得注意的是，大鼠骨骼肌损伤模型，包括但不限于药物诱导（notexin、布比卡因）16、钝挫伤26、挤压 27 和缺血再灌注28，可以用推拿进行干预。通过HE染色观察组织病理学变化和ELISA检测确定骨骼肌损伤标志?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金（批准号：82174521），湖南中医药大学研究生创新项目（2022CX109）资助
Materials
| 1 mL syringe | JIANGXI FENGLIN | 20220521 | |
| 1.5 microtubes | Servicebio | EP-150X-J | |
| 15 mL centrifuge tube | Servicebio | EP-1501-J | |
| 30G needle | CONPUVON | 220318 | |
| 5 mL blood collection tube | Servicebio | QX0023 | |
| Acrylic handle | Guangdong Guangxingwang Plastic Materials Co., Ltd | 65643645 | |
| Adjustment splint | CREROMEM | 20220729 | |
| Cotton Swab | INOHV | 22080215 | |
| Enzyme-labeled Instrument | Rayto | RT-6100 | |
| Ethanol | INOHV | 211106 | |
| Fork holder | Yongkang Kangzhe Health Technology Co., Ltd | JL001 | |
| Hair removal cream | Veet, France | LOTC190922002 | |
| Hematoxylin dyeing solution set | Wuhan Google Biotech | G1005 | |
| Imaging system | Nikon, Japan | Nikon DS-U3 | |
| IODOPHOR disfecting solution | Hale&Hearty | 20221205 | |
| Light microscope | Nikon, Japan | Nikon Eclipse E100 | |
| Limit baffle | CREROMEM | 20220724 | |
| Notexin | Latoxan S.A.S. | L8104-100UG | |
| Pentobarbital sodium | Merck KGaA | P3761 | |
| Rat creatine kinase (CK) ELISA kit | LunChangShuoBiotech | YD-35237 | |
| Rat fatty acid-binding protein 3 (FABP3) ELISA kit | LunChangShuoBiotech | YD-35730 | |
| Rubber roller | Hebei Mgkui Chemical Technology Co.,Ltd | 202207 | |
| Screw | Weiyan Hardware | B05Z122 | |
| Sprague Dawley rats | Hunan Slake Kingda Laboratory Animal Co. | SYXK2019-0009 | |
| Spring | Bingzhang Hardware | TH001 | |
| Surgical blade | Covetrus | #23 | |
| Weigh controller | Iyoys | HY-XSQ |
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