转化生长因子β激活激酶1（TAK1）在腰椎后路术后多裂肌损伤性萎缩中的作用和机制

Traumatic atrophy of multifidus muscle after posterior lumbar surgery is a major cause of refractory low back pain, and there is no treatment-related progress. Foreign researches and ours found that early postoperative inflammatory injury and late fibrosis are two key cause of multifidus muscle atrophy. Previous studies have shown ~24 h post skeletal muscle injury is the major period of neutrophil infiltration and the muscle fibers destroyed. Literature reported transforming growth factor-β activated kinase 1 (TAK1) is a pivotal kinase in inflammation signal pathways. And we found the neutrophil infiltration and multifidus muscle injury were obviously reduced by inhibiting TAK1, however, the mechanism is still unknown. And we also found that there were a large number of new-born muscle fibers in multifidus muscle injury area at 1 week postoperatively, however, the new-born muscle fibers were gradually reduced as fibrotic tissue grew in, and that ultimately caused fatty change of multifidus muscle. And it is well-known that the transforming growth factor -β1 which is upstream of TAK1 is a core molecule in skeletal muscle fibrosis. The effects of TAK1 in the fibrosis of kidney, heart and other organs have also been confirmed, however its role in skeletal muscle fibrosis is still unknown. So, this project will analyze the effects of TAK1 in the multifidus muscle injury and fibrosis, using a rat model in vivo, and the mechanisms of TAK1 on neutrophil apoptosis and muscle fibrosis, by cell cultrue in vitro. The object is to provide a theoretical basis for developing new treatments of postoperative multifidus traumatic atrophy.

腰椎后路术后多裂肌损伤性萎缩是顽固性腰背痛的重要原因，目前尚无相关治疗进展。国外研究和我们发现术后早期炎性损伤和后期纤维化改变是多裂肌发生萎缩的两个关键原因。研究表明骨骼肌损伤后24h内是中性粒细胞浸润、破坏肌纤维的主要时期。转化生长因子β激活激酶1（TAK1）是炎症信号通路中的关键激酶，我们观察到通过抑制TAK1能明显减少中性粒细胞浸润、减轻多裂肌损伤，但其机制尚不明确。我们发现，术后1周多裂肌损伤区可见大量肌纤维再生修复，但随后大量纤维疤痕组织长入肌修复区，再生肌纤维逐渐减少，多裂肌发生脂肪化改变。国外研究证实TAK1的上游分子TGF-β1是调控骨骼肌纤维化的核心分子，TAK1在肾、心等脏器中的纤维化作用也已被证实，但其在骨骼肌纤维化中的作用尚不明确。因此本项目拟通过体内手术模型、体外细胞培养，探讨TAK1在多裂肌炎性损伤和纤维化中的作用及机制，为多裂肌损伤性萎缩的治疗提供分子靶标。

腰椎后路术后多裂肌损伤性萎缩是顽固性腰背痛的重要原因，研究表明术后早期炎性损伤和后期纤维化改变是多裂肌发生萎缩的两个关键原因，但确切的分子机制尚不明确。转化生长因子β激活激酶1（TAK1）是炎症信号通路中的关键激酶以及促纤维化的关键分子，但在多裂肌炎症以及纤维化中的具体作用和机制目前尚无相关研究。我们通过构建大鼠腰椎后路术后多裂肌损伤模型，术前12h、4h，术后4h、12h多次多点予以TAK1抑制剂5Z-7-oxozeaenol（15mg/kg）局部注射大鼠局部多裂肌，组织学评估显示5Z-7-oxozeaenol注射组中性粒细胞浸润、肌纤维水肿和肌纤维坏死程度明显降低。体外实验，我们探讨了TAK1在肌细胞炎症反应中作用。用含2%马血清的培养基培养c2c12细胞7天，通过qpcr和免疫荧光证明c2c12细胞分化成肌细胞。通过TNF-α、IL1-β、RNAKL刺激肌细胞，qpcr和免疫荧光表明，刺激可以明显提高炎症因子（ccl2，cxcl1，cxcl5）在肌肉细胞中的表达，同时该促炎效果会被TAK1抑制剂5Z-7-Oxozeanol明显抑制。我们的研究结果明确了TAK1在多裂肌炎症和纤维化中的作用和机制。.纤维性椎间盘退变在临床上与慢性下腰痛有关，我们探讨了TAK1在髓核纤维化中的作用与机制。实验结果显示，TAK1和纤维化标记物（如胶原蛋白I和纤连蛋白）在退变的人的髓核标本中上调。在体外大鼠髓核细胞中，TAK1抑制剂5Z-7-Oxozeanol可抑制氧化应激诱导的髓核细胞纤维化表型。进一步的研究表明，5Z-7-oxozeaenol降低了双氧水诱导的TAK1，细胞外调节蛋白激酶（ERK），c-Jun N端激酶（JNK）和p38 MAPK的磷酸化。通过特异性抑制剂对p38和JNK的抑制部分减轻了双氧水诱导的髓核细胞的纤维化表型。在体内实验中，我们显示了TAK1抑制剂在穿刺诱导的大鼠椎间盘退变中具有出色的抗纤维化作用。这些发现表明TAK1在调节髓核纤维化中起关键作用，提示TAK1可能是纤维化椎间盘退变的潜在治疗靶标。.本课题明确了TAK1在多裂肌损伤与髓核纤维化中的关键作用，为开发治疗下腰痛关键靶点药物提供指导。