强直性脊柱炎小鼠模型中的杜艾灸
Summary
本文详细介绍了杜艾灸治疗实验小鼠强直性脊柱炎的操作规程和注意事项。
Abstract
强直性脊柱炎 （AS） 是一种进行性恶化和致残的关节炎，主要影响中轴骨骼。这种疾病主要累及脊柱和骶髂关节。脊柱与骶髂关节融合可能发生在疾病后期，导致脊柱僵硬和脊柱后凸，以及行走困难，严重影响工作质量和日常生活活动，给患者、家庭和社会带来沉重的负担。非药物治疗作为 AS 的替代疗法越来越受到关注。艾灸是中医（TCM）中使用的一种古老的治疗技术。度艾灸疗法是在普通艾灸的基础上发展起来的一种独特而创新的外用疗法，对AS有一定的治疗效果。度艾灸巧妙地结合了中医的相容技术，将经络、穴位、中草药、艾灸融为一体。本文详细介绍了实验小鼠度灸的操作规程和注意事项，为研究度艾灸治疗AS的机理提供了实验依据。
Introduction
强直性脊柱炎 （AS） 是一种进行性恶化和致残的关节炎，主要影响中轴骨骼。根据最新的流行病学调查，世界上~0.01%-1.8%的人患有这种疾病1，其中大多数是年轻男性（男女比例为~2-3：1）2。AS在中国人群中的发病率为0.2%-0.4%。这种疾病主要累及脊柱和骶髂关节。脊柱与骶髂关节融合可能发生在疾病后期，导致脊柱僵硬和脊柱后凸，以及行走困难，严重影响工作和日常生活活动的质量，给患者、家庭和社会带来沉重的负担1。目前，AS尚无明确的解决方案，所提供的药物和非药物治疗主要集中在减轻疼痛、减缓疾病进展和提高生活质量上。近年来，由于药物治疗选择非常有限3，非药物治疗越来越受到关注，非药物治疗已成为AS的替代疗法。在中国，患者通常更喜欢由中医（TCM）组成的外部治疗，这种治疗副作用小，治疗过程非常方便。
艾灸是中医中使用的一种古老的治疗技术。度艾灸是一种外部治疗技术，由杜经脊柱段的“药泡艾灸”组成。它主要用于AS的治疗，并显示出安全性和有效性。与普通艾灸相比，杜艾灸具有艾灸面积广、艾灸锥大、火力强、耐高温4等特点。一项系统评价和荟萃分析表明，艾灸是治疗 AS5 的有效补充疗法。最近的一项研究证实，艾灸改善了AS患者的临床症状和一些炎症因素6。研究表明，度艾灸可增加脑内β内啡肽含量并发挥中枢镇痛作用7，下调HLA-B27基因8的表达，延缓AS的复发率。度艾灸还通过降低炎症指数 ESR （ESR） 和 C 反应蛋白 （CRP）、I 型胶原 C 末端肽 （CTX-I） 和 Dickkopf1 蛋白 （DKK1） 的水平来改善 AS 的进展9,10。
另一项临床研究表明，度艾灸可以进一步调节无序的T细胞亚群Ig和补体C3，以平衡免疫机制11。在骨代谢方面，艾灸可抑制血清碱性磷酸酶（ALP）和血清磷（S-P）的升高，增加血清钙（S-Ca）、骨密度（BMD）和骨强度12。此外，度艾灸可以在多个点修复脊柱功能，以缓解AS患者的疲劳症状13。一些动物研究揭示了艾灸治疗AS的潜在机制。研究表明，艾灸显著改善了整体健康状况，降低了脊柱韧带组织样本中爪子厚度的水平，并降低了IL-1β、PGE2、IL-6和TNF-α的水平。代谢途径分析将鉴定的代谢物与 TCA 循环以及脂质、氨基酸、肠道菌群和嘌呤代谢联系起来14。另一项研究表明，艾灸抑制促炎细胞因子IL-1β、TNF-α、IL-17和IL-6的表达; 降低 RANKL、RANK、ALP 和 OCN 的 mRNA 水平;并改善了AS小鼠的组织病理学特征15。
虽然这些研究表明艾灸可能有效治疗 AS，但需要更多的研究来证实这些发现并确定 AS 患者艾灸的最佳治疗方案。我们的团队基于中国传统艾灸的基本理论和实际操作，创建并改进了艾灸疗法，在中国临床实践中广泛开展和应用了35年以上。虽然关于独艾灸治疗AS的高质量研究很少，但其治疗AS的适应症已得到证据支持。然而，用度灸治疗AS的机制值得进一步研究。动物研究是探究杜艾灸治疗AS机制的重要方法。杜艾灸的临床操作比较成熟，但很少用于动物实验机理研究。本文详细介绍了杜艾灸治疗AS实验小鼠的操作及注意事项。
Protocol
所有动物实验均经山东中医药大学实验动物福利伦理审查委员会批准。SDUTCM20230831301）。 1.动物准备 提前确定实验组和每组动物数量。 选择雄性BALb/c小鼠，将其饲养在22±1°C的恒温，60±5%的恒湿度和12小时的昼夜循环光照环境中。 2. AS小鼠模型的建立 注：AS模型是用软骨蛋白多糖（PG）诱导?…
Representative Results
PG诱导的模型小鼠出现外周关节炎症状，以四肢和脚趾发红肿胀为特征，早在第14周就逐渐发展为中轴关节炎，与AS的表现非常相似。因此，如果最后一次注射后 14 周的关节炎指数 （AI） 超过 3 分，则 AS 小鼠模型被认为是成功的22。当小鼠的爪子出现肿胀时，每周进行一次度艾灸治疗，共计3次。治疗后，小鼠在异氟烷深度麻醉下通过颈椎脱位实施安乐死，采集其血液，切除脚踝?…
Discussion
AS临床表现为背部、腰椎和关节肿胀疼痛，严重者可出现脊柱畸形和关节僵硬26。早期诊断和治疗可以改善AS患者的生活质量，降低残疾率，并改善预后。根据中医（TCM），AS被认为是由肾脏和度经的阳气不足以及内部冷恶的积累引起的。中医认为，艾灸基于经络的概念，可以产生使身体表面温暖的气。然后，这股气沿着经络和穴位向动。在 《黄帝内经 》（一部非常经典和…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了国家自然科学基金项目（No.82174491）和齐鲁中医学院学术学校传承项目（No.陆维涵[2020]132）。感谢山东中医药大学实验动物中心为我们提供的实验条件。
Materials
| 10% EDTA | Shanghai Macklin Biochemical Co., Ltd.,Shanghai, China | ||
| alcohol pads | HYNAUT, Qingdao Hainuo Biological Engineering Co., LTD, Qingdao, China | ||
| anesthesia machine | Medical Supplies & Services INT. LTD, Keighley, UK | ||
| centrifuge tubes | Axygen, Corning, NewYork, UAS | ||
| complete Frech's adjuvant (CFA) | aladdin,Shanghai, China | F393378 | |
| cotton ball | Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China | ||
| cotton gauze | Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China | ||
| cutting board | self-preparation | ||
| decorin from bovine articular cartilage | Sigma-Aldrich, MO, USA | D8428 | |
| depilatory cream | Veet, Reckitt Benckiser (China) Investment Co. LTD, Shanghai, China | ||
| electronic scale | Shanghai Yajin Electronic Technology Co., Ltd.,Shanghai, China | ||
| Eppendorf tube | Axygen, Corning, NewYork, UAS | ||
| eye lubricant | Beijing Shuangji Pharmaceutical Co., LTD., Beijing, China | ||
| ginger | self-preparation | ||
| GraphPad Prism 7 software | GraphPad Software,Boston, USA | ||
| hair clipper | Super human Group CO LTD, Jinhua, China | ||
| heating pads | Shenzhen Leshuo Tech Co., Ltd.,Shenzhen, China | ||
| incomplete Freund’s adjuvant (IFA) | aladdin,Shanghai, China | F393371 | |
| injection syringe | Shandong Xinhua Ande Medical Supplies Co., LTD, Zibo, China | ||
| isoflurane | Shenzhen Rayward Life Technology Co., LTD, Shenzhen, China | R510-22-10 | |
| joss stick | Shijiazhuang Lidu Fragrant Industry Co., LTD.,Shijiazhuang, China | ||
| juicer | Braun (Shanghai) Co., Ltd.,Shanghai, China | ||
| knife | self-preparation | ||
| lighter | Zhejiang tiger-lighter Co. LTD | ||
| mortar | Luoyang Yinai Ceramic Technology Co., LTD.,Luoyang, China | ||
| Mouse IL-17 ELISA Kit | absin, Shanghai, China | abs520009-96T | |
| Mouse TNF-α ELISA Kit | Wuhan Sanying, Wuhan, China | KE10002 | |
| mulberry paper | Yishui County Mulinsang paper Co., LTD, Linyi, China | ||
| OFT | Xinruan,Shanghai, China | XR-XZ301 | |
| paper cup | self-preparation | ||
| pipettes | OXFORD BIO INSTRUMENTS INC.,Oxford, UK | ||
| refined moxa velvet | self-preparation | ||
| rotarod | Xinruan,Shanghai, China | XR-6C | |
| scientz-48L cryogenic high throughput tissue grinder | Ningbo Xinzhi Biotechnology Co., LTD | ||
| scissors | Shandong Jiaren Medical Supplies Co., Ltd., Zibo, China | ||
| semi-automatic paraffin sectioning machine | Leica Camera AG, Watznach, Germany | ||
| SPSS 25.0 software | International Business Machines Corporation, NewYork, UAS | ||
| TCM powder | self-preparation | ||
| tips | Biosharp, Labgic, Beijing, China | ||
| writing brush | Yishui County Mulinsang paper Co., LTD, Linyi, China |
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Cite This Article
Yu, W., Li, X., Jia, Z., Hou, Y., Dong, T., Wang, X., Liu, Y., Yang, J. Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis. J. Vis. Exp. (200), e65586, doi:10.3791/65586 (2023).