基于NF-κB信号通路探讨益肾活血通络方对静水压下髓核细胞凋亡及基质代谢调控机制的研究

Hydrostatic pressure is the main form of mechanics load in human intervertebral disc, which has an important regulatory role in growth and development and matrix metabolism in the intervertebral disc cells. Studies have shown that excessive load will cause matrix decomposition and cell apoptosis, eventually leading to intervertebral disc degeneration. However, the exact biochemical mechanism of the effect of static pressure on nucleus pulposus cell apoptosis and matrix metabolism is not clear. NF-κB signal pathway is closely related to intervertebral disc degeneration, it can be activated and leads to intervertebral disc degeneration when cells are subjected to abnormal stress stimulation. This study intends to put hydrostatic pressure on nucleus pulposus cells using hydrostatic pressure loading system in vitro, and the Yishen Huoxue Tongluo recipe medicated serum of rabbit will be used for nucleus pulposus cells under hydrostatic pressure. The structure, activity, apoptosis rate of the nucleus pulposus cells and the gene and protein expression level of the NF-κB signal pathway were detected in all the experimental group. The main purpose of this study is to explore the molecular mechanism of the apoptosis and matrix metabolism regulation in nucleus pulposus cells induced by hydrostatic pressure, and to reveal the regulation mechanism and target of Yishen Huoxue Tongluo recipe which delay intervertebral disc degeneration caused by hydrostatic pressure, also to povide new micro biological evidence for the treatment of intervertebral disc degenerative diseases with traditional Chinese medicine.

静水压是人体椎间盘承受载荷的主要力学形式，其对椎间盘髓核细胞的生长发育和基质代谢具有重要的调节作用。前期研究表明，过度载荷会引起髓核细胞凋亡和基质分解，最终导致椎间盘退变。但静水压影响髓核细胞凋亡及基质代谢的确切生化机制尚不明确。NF-κB 信号通路与椎间盘退变密切相关，当细胞受到异常应力刺激时NF-κB 信号通路可被激活进而引发椎间盘退变。本研究拟在体外使用静水压加载系统模拟椎间盘内静水压环境，并利用益肾活血通络方含药血清对兔髓核细胞的体外静水压加载过程进行干预，对实验各组髓核细胞的结构、活性、凋亡率以及NF-κB信号通路中相关基因、蛋白的表达水平进行检测，以探明静水压影响髓核细胞凋亡及基质代谢的相关分子生物学机制，揭示益肾活血通络方对静水压下髓核细胞凋亡及基质代谢的调控机制及作用靶点，为中医药治疗椎间盘退行性疾病提供新的微观生物学证据。

静水压是人体椎间盘髓核组织在日常生活中承载负荷的最主要力学形式，其对髓核细胞的生长增殖及功能状态具有重要的调节作用，长期异常压力会导致髓核细胞凋亡率增加，胞外基质代谢紊乱，从而导致椎间盘退变。本课题在体外构建的智能静水压加载装置中模拟人体不同体位时椎间盘的静水压环境，研究益肾活血通络中药对静水压下髓核细胞的保护作用，并通过NF-κB信号通路研究其作用机制。实验结果：（1）光镜观察结果：相对于单纯静压干预组，静压+药物干预组细胞形态更好，胞浆更丰富，生长状况更好；扫描电镜观察结果：跟 0.3MPa和1MPa静水压相比，3MPa静水压下髓核细胞体积明显缩小，细胞呈长梭形，胞浆减少，细胞主级及次级突起出现明显断裂。压力+中药血清干预组较单纯压力干预组髓核细胞的超微结构更完整。（2）细胞存活率检测：在0.3MPa、1MPa、3MPa静水压下，细胞存活率随时间延长分别为逐渐增加、小幅下降、明显下降，与空白组对照，含药血清组髓核细胞活性明显增加；与单纯压力干预组对照，0.3MPa静水压下中药血清干预组髓核细胞活性显著增加，1MPa、3MPa静水压下两者未见明显差异（p>0.05）。（3）使用 Annexin V-FITC/Propidium Iodide 检测各组细胞凋亡情况：在0.3MPa静水压下，随时间增加，椎间盘髓核细胞凋亡率无明显差异，在1MPa、3MPa静水压下增加，所有压力+中药血清干预组髓核细胞凋亡率均低于单纯压力干预组（p<0.05）。（4）Western Blot及RT-PCR检测结果，随时间增加压力+中药血清干预组较单纯压力干预组Collagen Ⅱ、Sox9的蛋白及基因表达增加明显（p<0.05），而NF-κB p65、Caspase-3、ADAMTS-4、MMP-13蛋白及基因表达较对照组明显减少（p<0.05）。本研究部分揭示了益肾活血通络方改善静水压下髓核细胞退变的作用及机制，中药可能通过抑制髓核细胞NF-κB信号通路，使Collagen Ⅱ、Sox9表达增加、Caspase-3、ADAMTS-4、MMP-13表达减少改善静水压下髓核细胞的活性及功能状态。益肾活血通络方治疗椎间盘退行性疾病具有较好临床效果，本课题初步研究了其作用机制，深入研究值得在今后进一步开展。