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。

所有实验均经上海中医药大学动物护理与使用专业委员会（批准文号：SYXK2018-0040）批准和监督，符合世界医学会赫尔辛基宣言的规定。
1. 动物准备
<ol><li>House 32 Sprague Dawley （SD） 雄性大鼠，年龄为 8 周，体重 180-220 g，在特定的无病原体饲养装置中，在 24 ± 2 °C 和 60% 湿度下，在 12 小时光照/12 小时黑暗循环下，标准啮齿动物颗粒饮食。所有动物实验操作均符合实验动物福利伦理和动物实验安全规定。</li>
	<li>适应性喂养1周后，将大鼠随机分成四组，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）。</li>
</ol>2.膝关节内注射碘乙酸钠（MIA）
注：除D组外，KOA大鼠模型将通过膝关节腔注射MIA在所有组中制备。对于 D 组，将 25 μL 无菌生理盐水注射到右膝关节腔中。
<ol><li>通过将大鼠置于麻醉笼中，然后引入2.5%异氟醚直至完全麻醉来麻醉大鼠。等待幼鼠仰卧、四肢停止移动、脚趾捏合（踏板反射）、睑反射和肌肉松弛无反应时，确认动物已完全麻醉。</li>
	<li>在麻醉的幼鼠中加入宠物眼膏和眼药水，以防止眼睛干涩。</li>
	<li>剃掉大鼠的右后肢，并用三轮碘伏和75%乙醇交替对膝关节进行消毒。</li>
	<li>为了保持标准化的实验条件，小心地将膝关节屈曲固定在90°的精确角度，确保髌韧带朝上。随后，将25μLMIA溶液注射到大鼠的右膝关节腔中，该溶液由在25μL无菌盐水溶液中稀释的4mg MIA组成。 注：该注射是用专门的显微注射器进行的，确保程序21,22,23的准确性和一致性。将老鼠放回笼子里，直到它们醒来。接受手术的动物在完全康复之前不会回到其他动物的陪伴下。</li>
	<li>在研究结束时，通过注射1%戊巴比妥钠作为100mg / kg剂量对大鼠实施安乐死。</li>
</ol>3. 推拿的实施
注：推拿的实施将以“肌腱骨失衡患者，需先治疗肌腱”的理论为指导。A组和B组均在模型评估成功后的第一天开始干预，两组每天手术一次，持续14天。其余两组仅观察14天，没有任何干预。操作操作人员在实验前必须经过严格的培训，以确保力度、频率和节奏的一致性。
<ol><li>准确定位大鼠穴位，包括EX-LE4、ST35、SP10、ST34、SP9和GB34（图 1和 表1），其中手指揉捏方法将使用实验针灸24进行。</li>
	<li>将大鼠固定在大鼠固定设备上的俯卧位。</li>
	<li>静下肢放松后，用拇指和食指以120-140次/分钟同时揉捏患肢EX-LE4和ST35,4分钟。</li>
	<li>用拇指和食指以120-140次/分钟揉捏患肢前外侧肌肉3分钟，重点放在SP10、ST34和膝关节附近的肌肉上。强调对那些僵硬肌肉的强度的操纵，这样老鼠就不会挣扎。</li>
	<li>用拇指、食指和中指从上到下以120-140次/分钟揉捏患肢小腿后肌3分钟。要充分暴露治疗区域，请用无名指和小指握住患肢的踝关节，轻轻拉扯患肢。重点关注SP9、GB34和膝关节附近的肌肉，强调刚性肌肉和强度，使大鼠不会挣扎。</li>
</ol>4.行为指标的测量
<ol><li>足底机械疼痛阈值的测量 注意：测量 MIA 注射前 1 天以及注射后 2、9 和 16 天的足底机械疼痛阈值。<ol><li>将大鼠带到具有不锈钢网格底部和塑料笼体的足底试验室，在室温（22±2）°C的安静环境中适应30分钟。</li>
		<li>使用带有直径为0.8mm的塑料测试针的电子机械疼痛测试仪垂直刺激大鼠右后足的中心区域。均匀地逐渐增加刺激强度，直到大鼠表现出爪子缩回和回避反应。</li>
		<li>将电子屏幕的读数记录为大鼠右足底的机械疼痛阈值。</li>
		<li>以5分钟的间隔测量每只大鼠5次。</li>
		<li>从五个值中删除最大值和最小值。通过修剪均值法取中间三个值的平均值为结果（表2）。</li>
	</ol></li>
	<li>爪子机械退出阈值的测量 注意：在 MIA 注射前 1 天、注射后 2、9 和 16 天测量爪子机械退出阈值。<ol><li>将大鼠带到具有30°C玻璃底和塑料笼体的加热足底试验室，在室温（22±2）°C的安静环境中适应30分钟。</li>
		<li>在65°C的刺激温度下，用光源加热装置刺激右后足底中心区域，直到大鼠表现出爪子缩回和回避反应。</li>
		<li>记录电子屏幕的时间读数作为大鼠的右足底热痛阈值。</li>
		<li>以5分钟的间隔连续测量每只大鼠5次。</li>
		<li>从五个值中删除最大值和最小值。通过修剪均值法取中间三个值的平均值为结果（表3）。</li>
	</ol></li>
</ol>5.右膝关节肿胀的测量
注：在MIA注射前1天，注射后2,9和16天测量大鼠右膝关节的肿胀。
<ol><li>通过将大鼠置于麻醉笼中并将其暴露于2.5%异氟醚直至完全麻醉来麻醉大鼠。</li>
	<li>将大鼠放在手术台上的平坦位置。</li>
	<li>用游标卡尺测量右膝关节的宽度（图2 和 表4）。</li>
</ol>6. Lequesne MG 评分的测量
注：在MIA注射后2,9和16天测量大鼠的Lequesne MG评分。Lequesne 等人开发了髋关节骨关节炎 （ISH） 严重程度指数，可用于评估治疗干预的有效性。我们考虑四个参数：疼痛刺激、步态改变、关节活动度和关节肿胀。
<ol><li>将大鼠带到室温（22±2）°C的安静环境中的操作平台。</li>
	<li>让两名操作员相互隔离，测量局部疼痛刺激反应、步态变化、关节运动和关节肿胀。<ol><li>用塑料试针刺激右膝关节外侧，根据反应评分0-3分。0 无响应;3、反应良好;1患肢收缩;2患肢收缩痉挛，伴有轻度全身反应，如颤抖、舔舐、吸吮等。</li>
		<li>将大鼠放在手术台上，观察其右后肢的步态。根据回答以 0-3 的等级评分。0 患肢活动无干扰，跑步正常，步法有力;3 患肢不能参与行走、接触地面或踩踏地面;跑步时有 1 次轻度徒步旅行，有强烈的跺脚;2患肢参与行走，但大鼠运动（跋涉）的干扰明显。</li>
		<li>用手弯曲和伸展大鼠的右膝关节，观察关节活动度。根据回答以 0-3 的等级评分。0关节活动角大于90°;3.关节活动角小于15°;1个关节活动角或45°-90°;2.关节活动角为15°-45°。</li>
		<li>触摸大鼠的右膝关节，并将反应与正常大鼠的反应进行比较。根据回答以 0-2 的等级评分。0：无明显肿胀和可见骨斑纹;2 明显肿胀，无骨斑纹;1 轻度肿胀和浅表骨斑纹。</li>
		<li>将这些分数相加以产生每只大鼠的Lequesne MG分数，并通过修剪方法计算平均值（表5）。</li>
	</ol></li>
</ol>7. 皮肤温度的测量
注意：在注射 MIA 前 1 天以及注射后 9 天和 16 天测量皮肤温度。
<ol><li>通过将大鼠置于麻醉笼中，然后引入异氟醚直至完全麻醉，麻醉大鼠。</li>
	<li>将大鼠在室温（22±2）°C下置于手术台上的侧向位置。</li>
	<li>用手轻轻抓住脚，伸直右膝关节。使用 Filr 红外热像仪拍照以确定皮肤温度。</li>
	<li>使用配套的编辑软件读取大鼠膝关节和膝关节周围的皮肤温度。在温度逐渐降低的踝关节和膝关节处设置控制点。</li>
</ol>8. 统计分析
<ol><li>使用统计软件以四分位数表示实验数据。</li>
	<li>使用修剪均值方法处理异常值。行为测试的结果被记录为平均值的平均值±标准误差。</li>
	<li>进行独立样本 t检验以进行组间比较。使用双向重复测量方差分析 （ANOVA） 分析行为数据，然后进行 Bonferroni 的多重比较检验。如果数据不表现出正态性或同方差性，则执行非参数检验（Kruskal-Wallis k 样本）。 注： P < 0.05 表示差异具有统计学意义。所有数据都符合所应用的统计检验的假设。</li>
</ol>

推拿按摩治疗膝骨关节炎的治疗潜力

<ol>
	<li>Katz, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+hip+and+knee+osteoarthritis:+A+review.">Diagnosis and treatment of hip and knee osteoarthritis: A review.</a> JAMA. 325 (6), 568-578 (2021).</li><li>Culvenor, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+knee+osteoarthritis+features+on+magnetic+resonance+imaging+in+asymptomatic+uninjured+adults:+a+systematic+review+and+meta-analysis.">Prevalence of knee osteoarthritis features on magnetic resonance imaging in asymptomatic uninjured adults: a systematic review and meta-analysis.</a> Br J Sports Med. 53 (20), 1268-1278 (2019).</li><li>Safiri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regional+and+national+burden+of+osteoarthritis+1990-2017:+a+systematic+analysis+of+the+Global+Burden+of+Disease+Study+2017.">regional and national burden of osteoarthritis 1990-2017: a systematic analysis of the Global Burden of Disease Study 2017.</a> Ann Rheum Dis. 79 (6), 819-828 (2020).</li><li>Kong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+Chinese+exercises+on+pain+and+disability+in+middle-aged+and+elderly+patients+with+neck+pain:+a+systematic+review+and+meta-analysis+of+randomized+controlled+trials.">Traditional Chinese exercises on pain and disability in middle-aged and elderly patients with neck pain: a systematic review and meta-analysis of randomized controlled trials.</a> Front Aging Neurosci. 14, 912-945 (2022).</li><li>Nevitt, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symptoms+of+knee+instability+as+risk+factors+for+recurrent+falls.">Symptoms of knee instability as risk factors for recurrent falls.</a> Arthritis Care Res (Hoboken). 68 (8), 1089-1097 (2016).</li><li>Hunter, D. J., March, L., Chew, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+in+2020+and+beyond:+a+Lancet+Commission).">Osteoarthritis in 2020 and beyond: a Lancet Commission).</a> Lancet. 396 (10264), 1711-1712 (2020).</li><li>Fan, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+and+evaluation+of+the+Chinese+Osteoarthritis+Treatment+Guidelines+(2021+Edition).">Interpretation and evaluation of the Chinese Osteoarthritis Treatment Guidelines (2021 Edition).</a> Chinese Journal of Evidence-Based Medicine. 22 (06), 621-627 (2022).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+an+epidemiological+survey+on+the+prevalence+of+osteoarthritis+of+the+knee+in+a+middle-aged+and+elderly+population+in+China.">Design of an epidemiological survey on the prevalence of osteoarthritis of the knee in a middle-aged and elderly population in China.</a> Journal of Hebei Medical University. 36 (04), 487-490 (2015).</li><li>Shao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+mechanism+of+regulating+the+&amp;#34;balance+of+tendon+and+bone+force&amp;#34;+in+the+treatment+of+knee+osteoarthritis+by+Tui+Na+manipulation.">Study on the mechanism of regulating the &amp;#34;balance of tendon and bone force&amp;#34; in the treatment of knee osteoarthritis by Tui Na manipulation.</a> Shanghai University of Traditional Chinese Medicine. , 107-108 (2020).</li><li>Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+and+mechanism+of+PD-1/PD-L1+pathway+in+osteoarthritis+in+mice.">Role and mechanism of PD-1/PD-L1 pathway in osteoarthritis in mice.</a> Shandong University. , 130-134 (2020).</li><li>Shoujian, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+promotes+the+repair+and+regeneration+of+contused+skeletal+muscle+by+regulating+Treg+cells+and+macrophages.">PD-1 promotes the repair and regeneration of contused skeletal muscle by regulating Treg cells and macrophages.</a> Shanghai Institute of Physical Education. , 54-58 (2020).</li><li>Levinger, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+level+of+FoxO1+and+IL-15+in+skeletal+muscle,+serum+and+synovial+fluid+in+people+with+knee+osteoarthritis:+a+case+control+study.">The level of FoxO1 and IL-15 in skeletal muscle, serum and synovial fluid in people with knee osteoarthritis: a case control study.</a> Osteoporos Int. 27 (6), 2137-2143 (2016).</li><li>Niu, Q., Xie, C., Jiang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Chinese+medicine+based+on+PI3K/AKT+signaling+pathway+for+the+treatment+of+knee+osteoarthritis.">Advances in Chinese medicine based on PI3K/AKT signaling pathway for the treatment of knee osteoarthritis.</a> Massage and Rehabilitation Medicine. , 1-5 (2022).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+of+checkpoint+receptor+PD-L1+aggravates+osteoarthritis+in+macrophage-dependent+manner+in+the+mice+model.">Blocking of checkpoint receptor PD-L1 aggravates osteoarthritis in macrophage-dependent manner in the mice model.</a> Int J Immunopathol Pharmacol. 33, 2058-2073 (2019).</li><li>Molanouri, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effect+of+aerobic+interval+training+and+selenium+nanoparticles+on+expression+of+IL-15+and+IL-10/TNF-alpha+ratio+in+skeletal+muscle+of+4T1+breast+cancer+mice+with+cachexia.">Combined effect of aerobic interval training and selenium nanoparticles on expression of IL-15 and IL-10/TNF-alpha ratio in skeletal muscle of 4T1 breast cancer mice with cachexia.</a> Cytokine. 90, 100-108 (2017).</li><li>Mahmoudian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early-stage+symptomatic+osteoarthritis+of+the+knee+-+time+for+action.">Early-stage symptomatic osteoarthritis of the knee - time for action.</a> Nat Rev Rheumatol. 17 (10), 621-632 (2021).</li><li>Nahin, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+evaluation+of+complementary+health+approaches+for+pain+management+in+the+United+States.">Evidence-based evaluation of complementary health approaches for pain management in the United States.</a> Mayo Clin Proc. 91 (9), 1292-1306 (2016).</li><li>Bervoets, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+therapy+has+short-term+benefits+for+people+with+common+musculoskeletal+disorders+compared+to+no+treatment:+a+systematic+review.">Massage therapy has short-term benefits for people with common musculoskeletal disorders compared to no treatment: a systematic review.</a> J Physiother. 61 (3), 106-116 (2015).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+tui+na+in+treating+pain+after+total+knee+replacement+for+knee+osteoarthritis.">Effectiveness of tui na in treating pain after total knee replacement for knee osteoarthritis.</a> Chinese Tissue Engineering Research. 25 (18), 2840-2845 (2021).</li><li>Ali, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+therapy+and+quality+of+life+in+osteoarthritis+of+the+knee:+a+qualitative+study.">Massage therapy and quality of life in osteoarthritis of the knee: a qualitative study.</a> Pain Med. 18 (6), 1168-1175 (2017).</li><li>Yoh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-articular+injection+of+monoiodoacetate+induces+diverse+hip+osteoarthritis+in+rats,+depending+on+its+dose.">Intra-articular injection of monoiodoacetate induces diverse hip osteoarthritis in rats, depending on its dose.</a> BMC Musculoskelet Disord. 23 (1), 494-487 (2022).</li><li>Lockwood, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+peripheral+and+central+components+of+the+rat+monoiodoacetate+model+of+osteoarthritis.">Characterisation of peripheral and central components of the rat monoiodoacetate model of osteoarthritis.</a> Osteoarthritis Cartilage. 27 (4), 712-722 (2019).</li><li>McCoy, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+osteoarthritis:+Comparisons+and+key+considerations.">Animal models of osteoarthritis: Comparisons and key considerations.</a> Vet Pathol. 52 (5), 803-818 (2015).</li><li>Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+effect+of+pushing+on+cartilage+in+rats+with+knee+osteoarthritis+based+on+ROCK/LIMK1/Cofilin+signaling+pathway.">Exploring the effect of pushing on cartilage in rats with knee osteoarthritis based on ROCK/LIMK1/Cofilin signaling pathway.</a> Chongqing Medical University. , 42-44 (2022).</li><li>Liu, Z. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuina+for+peripherally-induced+neuropathic+pain:+A+review+of+analgesic+mechanism.">Tuina for peripherally-induced neuropathic pain: A review of analgesic mechanism.</a> Front Neurosci. 16, 1096-1104 (2022).</li><li>Gebremariam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subacromial+impingement+syndrome--effectiveness+of+physiotherapy+and+manual+therapy.">Subacromial impingement syndrome--effectiveness of physiotherapy and manual therapy.</a> Br J Sports Med. 48 (16), 1202-1208 (2014).</li><li>Yao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+microRNAs+reveals+potential+mechanisms+of+manual+therapy+alleviating+neuropathic+pain+through+microRNA-547-3p-mediated+Map4k4/NF-kappab+signaling+pathway.">Transcriptome profiling of microRNAs reveals potential mechanisms of manual therapy alleviating neuropathic pain through microRNA-547-3p-mediated Map4k4/NF-kappab signaling pathway.</a> J Neuroinflammation. 19 (1), 211-221 (2022).</li><li>Valera-Calero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+response+after+cervical+manipulation+and+mobilization+in+people+with+chronic+mechanical+neck+pain:+a+randomized+controlled+trial.">Endocrine response after cervical manipulation and mobilization in people with chronic mechanical neck pain: a randomized controlled trial.</a> Eur J Phys Rehabil Med. 55 (6), 792-805 (2019).</li><li>Kovanur, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measureable+changes+in+the+neuro-endocrinal+mechanism+following+spinal+manipulation.">Measureable changes in the neuro-endocrinal mechanism following spinal manipulation.</a> Med Hypotheses. 85 (6), 819-824 (2015).</li><li>Colombi, A., Testa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+induced+by+spinal+manipulative+therapy+on+the+immune+and+endocrine+systems.">The effects induced by spinal manipulative therapy on the immune and endocrine systems.</a> Medicina (Kaunas). 55 (8), 312-314 (2019).</li><li>Afify, A., Mark, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+assessment+of+chromosome+8+copy+number+in+stage+I+and+stage+II+infiltrating+ductal+carcinoma+of+the+breast.">Fluorescence in situ hybridization assessment of chromosome 8 copy number in stage I and stage II infiltrating ductal carcinoma of the breast.</a> Cancer Genet Cytogenet. 97 (2), 101-105 (1997).</li><li>Kirby, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+influences+on+skeletal+muscle+vascular+tone+in+humans:+insight+into+contraction-induced+rapid+vasodilatation.">Mechanical influences on skeletal muscle vascular tone in humans: insight into contraction-induced rapid vasodilatation.</a> J Physiol. 583, 861-874 (2007).</li><li>Sefton, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+massage+of+the+neck+and+shoulders+produces+changes+in+peripheral+blood+flow+when+assessed+with+dynamic+infrared+thermography.">Therapeutic massage of the neck and shoulders produces changes in peripheral blood flow when assessed with dynamic infrared thermography.</a> J Altern Complement Med. 16 (7), 723-732 (2010).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerobic+exercise+combined+with+glucosamine+hydrochloride+capsules+inhibited+the+apoptosis+of+chondrocytes+in+rabbit+knee+osteoarthritis+by+affecting+TRPV5+expression.">Aerobic exercise combined with glucosamine hydrochloride capsules inhibited the apoptosis of chondrocytes in rabbit knee osteoarthritis by affecting TRPV5 expression.</a> Gene. 830, 146465 (2022).</li><li>Fang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+aerobic+exercise+combined+with+glucosamine+therapy+and+circUNK+in+improving+knee+osteoarthritis+in+rabbits.">The mechanism of aerobic exercise combined with glucosamine therapy and circUNK in improving knee osteoarthritis in rabbits.</a> Life Sci. 275, 119375 (2021).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Casticin+suppresses+monoiodoacetic+acid-induced+knee+osteoarthritis+through+inhibiting+HIF-1alpha/NLRP3+inflammasome+signaling.">Casticin suppresses monoiodoacetic acid-induced knee osteoarthritis through inhibiting HIF-1alpha/NLRP3 inflammasome signaling.</a> Int Immunopharmacol. 86, 106745 (2020).</li><li>Ma, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vanillic+acid+reduces+pain-related+behavior+in+knee+osteoarthritis+rats+Through+the+inhibition+of+NLRP3+inflammasome-related+synovitis.">Vanillic acid reduces pain-related behavior in knee osteoarthritis rats Through the inhibition of NLRP3 inflammasome-related synovitis.</a> Front Pharmacol. 11, 599022 (2020).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroacupuncture+ameliorates+knee+osteoarthritis+in+rats+via+inhibiting+NLRP3+inflammasome+and+reducing+pyroptosis.">Electroacupuncture ameliorates knee osteoarthritis in rats via inhibiting NLRP3 inflammasome and reducing pyroptosis.</a> Mol Pain. 19, 17448069221147792 (2023).</li></ol>

作者声明不存在利益冲突。

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-PD-L1</td>
			<td>Abcam, Cambridge, MA, USA</td>
			<td>ab80276</td>
			<td></td>
		</tr>
		<tr>
			<td>electric von Frey esthesiometer&#160;</td>
			<td>IITC/Life Science, Woodland Hills, CA, USA</td>
			<td>ALMEMO 2450</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 9.0</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Software for stastistical analysis</td>
		</tr>
		<tr>
			<td>monosodium iodoacetate</td>
			<td>Sigma-Aldrich Inc</td>
			<td>I9148</td>
			<td>Resolved into normal saline for injection</td>
		</tr>
		<tr>
			<td>pentasorbital sodium</td>
			<td>Sigma-Aldrich Inc</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley (SD) male rats</td>
			<td>Shanghai Jihui Experimental Animal Breeding Co., Ltd</td>
			<td>No. SCXK (Hu) 2017-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>thermal analgesia tester</td>
			<td>IITC/Life Science</td>
			<td>Model 390</td>
			<td></td>
		</tr>
	</tbody>
</table>

tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis

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。

Knee osteoarthritis (KOA) is a common degenerative osteoarthritis, characterized by chronic pain and disability, and relatively severe KOA will lead to irreversible structural damage to the joint1. The high global prevalence of KOA has become a major global public health challenge2,3, which seriously impairs the quality of life of patients4,5. Studies have shown that KOA affects more than 260 million people worldwide6. With the aging of the population, the total prevalence rate of KOA in China is as high as 17.0% over the age of 40, which puts a heavy burden on the families of those affected7,8.
According to Chinese medicine, KOA belongs to the category of &#34;paralysis&#34;9, and the Yellow Emperor&#39;s Classic of Internal Medicine associates tendons, bones, and flesh with paralysis. Hence, we need to pay attention to the connection between the cartilage, muscle, synovium, as well as other tissues around the knee joint in KOA. In modern medicine, both muscle and bone inflammation, which are major components of the immune homeostasis of arthritis, drive the progression of KOA pain. However, most studies focus on cartilage inflammation and synovial inflammation in bone joints, and there is a paucity of articles examining inflammatory phenomena in skeletal muscles. Therefore, more research on the role of skeletal muscle in inflammation is needed to gain a more comprehensive understanding of KOA and provide ideas for more effective treatment modalities. In a review of the literature, we found that inhibition of the immune-related factor programmed death ligand 1 (PD-L1) exacerbated the progression of KOA10,11 and that there was differential expression of IL-15 and FOXO1 in the skeletal muscle of patients with KOA12, as well as significant skeletal muscle inflammation in patients with end-stage KOA, which was also associated with changes in gait. It has been shown that the skeletal muscle can also secrete factors closely related to inflammatory immunity such as IL-6, and that TNF-&#945; is also closely related to the PD-L1 pathway and KOA13,14,15.
Currently, available treatments for KOA include weight control, exercise, medication, and surgery, which usually include intra-articular injections, analgesic medication, arthroscopic surgery, and periprosthetic osteotomy of the knee16. Currently, KOA disease progression cannot be completely stopped or reversed, and without a clear understanding of the complex mechanisms underlying KOA pain, KOA patients are at risk for overuse of analgesic medications and a high incidence of side effects8. Most treatment strategies are effective only for short periods and focus only on the temporary relief of symptoms rather than on the pathogenesis of the disease. Therefore, KOA pain remains a priority and a challenge for treatment, posing a serious clinical challenge. However, Tuina can directly stimulate the skeletal muscles around the affected joint in KOA treatment and can provide some benefits. Combined with the previous description, it is the ideal therapy to study the role of skeletal muscle tissue in KOA, and clarifying the role of skeletal muscle tissue in inflammation can also provide better theoretical support for Tuina of KOA.
Studies have shown that massage therapy, osteopathic manipulative therapy, and spinal manipulation can help relieve pain and restore body function17,18. These manipulations can act on local inflammation and relieve spasms and analgesia through external mechanical action. They can relieve pain after total knee arthroplasty, reduce the use of analgesic drugs, promote postoperative recovery, and improve biomechanical balance19. Swedish massage is feasible in the treatment of osteoarthritis and can reduce stress and improve quality of life20. The therapeutic effect of Tuina on KOA has been preceded by certain studies.
In this experiment, our team will explore whether the expression of PD-L1 and related inflammatory factors in skeletal muscle can induce KOA and promote its development based on the MIA-induced KOA model in rats. Tuina will also be implemented to see whether it can alleviate KOA symptoms caused by increased inflammatory factor expression. Tuina will be combined with PD-L1 inhibition to demonstrate the association between Tuina-mediated PD-1 pathway in skeletal muscle inflammation affecting KOA development and pain, laying the foundation for further multi-level studies on the therapeutic mechanisms of Tuina intervention in KOA.
In summary, this paper describes the investigation and elaboration of the mechanisms of skeletal muscle inflammation in the development of KOA and its pain, as well as the therapeutic effects of Tuina on KOA, from the perspective of immune-inflammatory factor expression, combined with behavioral related indicators, to provide modern research evidence for the &#34;imbalance of tendon and bone&#34; theory in traditional Chinese medicine.

作者聚焦：推拿治疗膝骨关节炎 - 新视角 

单碘乙酸钠注射液诱导膝骨关节炎大鼠模型的推拿干预

We are trying to broaden the views on the research of knee osteoarthritis by introducing the perspective of refocusing on muscle inflammation during the pathological process of the KOA. There is a recent trend in non-pharmacological therapy treating the knee osteoarthritis. Therapy measures of traditional Chinese medicine, such as TNA and acupuncture, are receiving more and more recognition.There is a calling for detailed mechanism in the treatment. Our experiments indicates the possibility of chronic muscle inflammation in the process of knee osteoarthritis. It also points out the possibility of non-pharmacological therapy such as TNA to treat and control it.

所述方案在岳阳市中西医结合医院的临床环境中实施。 图 1 显示了在大鼠体内推动穴位的确切位置， 表 1 说明了刺激这些穴位的共同好处。表 2 和 表 3 提供了令人信服的证据，表明即使在没有 PD-1 通路抑制的情况下，推拿疗法也能显着缓解疼痛。表 4、 表 5 和 图 2 中提供的研究结果还?...

Watch this Scientific Journal Video about Treating Knee Osteoarthritis with Tuina - A New Perspective at JoVE.com

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。

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...

我们试图通过引入在KOA病理过程中重新关注肌肉炎症的视角来拓宽膝骨关节炎研究的视野。最近有一种非药物疗法治疗膝骨关节炎的趋势。TNA和针灸等传统中医的治疗方法越来越受到认可。在治疗中需要详细的机制。我们的实验表明，在膝骨关节炎的过程中，慢性肌肉炎症的可能性。它还指出了非药物疗法（如TNA）治疗和控制它的可能性。

tuina-intervention-sodium-monoiodoacetate-injection-induced-rat-model

Research

JoVE Journal

Medicine

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

1.1K 次观看.  Shanghai University of Traditional Chinese Medicine. 我们试图通过引入在KOA病理过程中重新关注肌肉炎症的视角来拓宽膝骨关节炎研究的视野。最近有一种非药物疗法治疗膝骨关节炎的趋势。TNA和针灸等传统中医的治疗方法越来越受到认可。在治疗中需要详细的机制。我们的实验表明，在膝骨关节炎的过程中，慢性肌肉炎症的可能性。它还指出了非药物疗法（如TNA）治疗和控制它的可能性。