Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China.
Clinical Medical School, Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China.
J Tradit Chin Med. 2023 Oct;43(5):991-1000. doi: 10.19852/j.cnki.jtcm.2023.05.005.
The aim of this study was to investigate the protective effects of Tuina (a traditional Chinese massage therapy) on intervertebral disc (IVD) degeneration and the regulatory mechanisms of the transforming growth factor-β1 (TGF-β1)/small mothers against decapentaplegic (Smad) signaling pathway.
Thirty New Zealand white rabbits were randomized into five groups: the control group, model group, model + Tuina group (Tuina group), model + TGF-β1 group (TGF-β1 group), and model + TGF-β1 inhibitor SB431542 group (SB431542 group). The model was established by posterolateral annulus fibrosus puncturing (AFP). Recombinant TGF-β1 and inhibitor SB431542 was injected into the TGF-β1 group and SB431542 group with a microsyringe, respectively. The rabbits in the Tuina group received Tuina treatment along the bladder meridian for 4 weeks. Magnetic resonance imaging (MRI) was performed on rabbits before AFP and after 4 weeks of intervention. Lumbar IVDs (L2-L3 to L4-L5) were harvested after intervention. Histopathological changes in the IVDs were measured by hematoxylin and eosin (HE) staining. Type I collagen was analyzed by immunohistochemistry detection. The expression level of matrix metalloproteinase-3 (MMP3) was determined by enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling and Western blotting. Real-time polymerase chain reaction and Western blotting were used to analyze the expression of TGF-β1 and Smad2/3/4 and a disintegrin and metalloproteinase with thrombospondin motifs 5.
Posterolateral AFP induced IVD degeneration in rabbits with histopathological damage and noticeable changes in MRI images. Tuina alleviated histo-pathological changes and reversed the expression of extracellular matrix degeneration-related molecules and apoptosis-related proteins. Furthermore, AFP induced the activation of TGF-β1 and Smad2/3/4, whereas Tuina therapy markedly reduced the protein expression of Smad2/3 and the gene expression of TGF-β1 and Smad2/3/4. Additionally, the TGF-β1/Smad signaling pathway was activated in the TGF-β1 group, while the TGF-β1/Smad signaling pathway was inhibited in the SB431542 group.
Posterolateral AFP induced disc degeneration as determined by MRI assessment and histological analysis. Tuina alleviated disc degeneration, possibly by inhibiting the fibrotic response mediated by the TGF-β1/Smad pathway, thus alleviating extracellular matrix degeneration and reducing cell apoptosis.
本研究旨在探讨推拿（一种传统的中医按摩疗法）对椎间盘（IVD）退变的保护作用及转化生长因子-β1（TGF-β1）/小母亲对抗 Decapentaplegic（Smad）信号通路的调节机制。
将 30 只新西兰白兔随机分为五组：对照组、模型组、模型+推拿组（推拿组）、模型+TGF-β1 组（TGF-β1 组）和 TGF-β1 抑制剂 SB431542 组（SB431542 组）。采用后外侧纤维环穿刺（AFP）建立模型。用微量注射器分别向 TGF-β1 组和 SB431542 组注射重组 TGF-β1 和抑制剂 SB431542。推拿组沿膀胱经推拿治疗 4 周。在 AFP 前和干预 4 周后对兔进行磁共振成像（MRI）检查。干预后采集腰椎（L2-L3 至 L4-L5）IVD。苏木精和伊红（HE）染色测量 IVD 的组织病理学变化。免疫组织化学检测 I 型胶原。酶联免疫吸附试验测定基质金属蛋白酶-3（MMP3）的表达水平。末端脱氧核苷酸转移酶介导的缺口末端标记和 Western blot 法评估细胞凋亡。实时聚合酶链反应和 Western blot 法分析转化生长因子-β1 和 Smad2/3/4 及解整合素和金属蛋白酶与凝血酶 5 的表达。
后外侧 AFP 诱导兔 IVD 退变，组织病理学损伤明显，MRI 图像变化明显。推拿减轻了组织病理学变化，并逆转了细胞外基质退变相关分子和凋亡相关蛋白的表达。此外，AFP 诱导 TGF-β1 和 Smad2/3/4 的激活，而推拿治疗明显降低 Smad2/3 的蛋白表达和 TGF-β1 和 Smad2/3/4 的基因表达。此外，TGF-β1 组 TGF-β1/Smad 信号通路被激活，而 SB431542 组 TGF-β1/Smad 信号通路被抑制。
MRI 评估和组织学分析表明，后外侧 AFP 诱导椎间盘退变。推拿减轻椎间盘退变，可能通过抑制 TGF-β1/Smad 通路介导的纤维化反应，从而减轻细胞外基质退变，减少细胞凋亡。
