六味地黄丸对椎间盘髓核细胞凋亡MAPK信号通路的影响

Studies examining the problem from different directions have produced evidence implicating the intervertebral disc in a significant proportion of cases of chronic back pain, leading to the use of the term 'discogenic back pain'.With aging and degeneration, disc cells undergo substantially biologic changes, including alternation of cell type in the nucleus pulposus, increased cell density but decreased number of viable cells as a result of increased cell death and increased cell proliferation, and altered cell phenotype. These changes are involved in the process of disc degeneration through the complicated interactions among them. Disc cell numbers and viability decrease in degenerate IVD. Apoptosis may play a role in the process of IVD degeneration, and that modulation of apoptosis might be a potential therapeutic strategy for IVD degeneration. This has been attributed to apoptosis large body of evidences indicates that mitogen-activated protein kinase (MAPK) signal transduction pathway is associated with the development of IVD disease．p38MAPK is an important subfamily of MAPK, and plays an essential role in the regulation of cell apoptosis of IVD disease．JNK，as a member of the MAPK，is an important signal pathway to perform transduction and adjust the signal of cell death．Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking p38 MAPK and JNK signal could provide a therapeutic approach to slow the course of IVD degeneration.. TNF has been implicated in the catabolic processes leading to matrix degradation in the degenerate IVD. For instance, whilst there is no question that with increasing degrees of degeneration IVD cells exhibit increased TNF-α expression. TNF-α induces the apoptosis of human nucleus pulposus cell via P38MAPK and JNK pathway. The primary goal of this study is to test the hypothesis that, in nucleus pulposus cells apoptosis on the degenerated rabbit IVD mode1 vivo and in vitro induced by TNF-α,invigorating kidney regulate via P38MAPK and JNK pathway,and to define whether invigorating kidney is capable of becoming an alternative therapy for disc degeneration.

椎间盘源性腰痛是临床常见病，多发病，并导致重大的社会负担与经济负担，现代医学治疗手段相对创伤大，副作用多，因而导致其成为临床上的治疗难点。椎间盘退变是导致椎间盘源性腰痛的主要原因，现有研究提示椎间盘髓核细胞的死亡是以凋亡的形式进行的，导致髓核的结构和组成成分发生改变，进而椎间盘组织发生退变。中医认为椎间盘源性腰痛以肾虚为本，补肾法是治疗本病的基本方法。本课题结合前沿科学研究及既往研究基础，拟通过建立椎间盘退变动物及体外培养体系，以六味地黄丸干预，测定髓核细胞凋亡及细胞外基质成分变化，研究该中药复方是否通过调控P38MAPK和JNK信号通路，从而影响TNF-α所导致的髓核细胞过度凋亡及细胞外基质的组分变化，维护椎间盘组织结构的完整性及其力学性能，使通过补肾法调控椎间盘髓核细胞凋亡相关信号通路成为椎间盘源性腰痛新的治疗靶点，为中医药防治椎间盘源性腰痛方面的研究开创新的思路。

背景.椎间盘源性腰痛是临床常见病，多发病，并导致重大的社会负担与经济负担，现代医学治疗手段相对创伤大，副作用多，因而导致其成为临床上的治疗难点。中医认为椎间盘源性腰痛以肾虚为本，补肾法是治疗本病的基本方法。.研究内容.通过建立椎间盘退变动物及体外培养体系，以六味地黄丸干预，测定髓核细胞凋亡及细胞外基质成分变化，研究该中药复方是否通过调控P38MAPK和JNK信号通路，从而影响TNF-α所导致的髓核细胞过度凋亡及细胞外基质的组分变化，维护椎间盘组织结构的完整性及其力学性能。.结果.动物体内实验表明：在8周内兔椎间盘髓核细胞凋亡率伴随椎间盘退变过程中一直上升；六味地黄丸可以调控椎间盘退变模型内髓核细胞凋亡，减缓凋亡速度，上调模型中糖胺多糖含量、硫酸软骨素/硫酸角质素比值、透明质酸含量，适度下调椎间盘退变模型中I型胶原的表达，上调II型胶原的表达，稳定蛋白多糖含量，在一定程度上延缓了椎间盘的退变。这一作用可能是通过部分抑制JNK与p38MAPK通路的全面激活而实现的。.体外实验结果显示：随着培养时间的延长，软骨终板糖胺多糖含量、硫酸软骨素/硫酸角质素比值、透明质酸含量，蛋白多糖及mRNA的表达、Ⅱ型胶原及mRNA的表达逐渐降低，Ⅰ型胶原及mRNA的表达逐渐上升，椎间盘细胞的JNK及P38通路的mRNA含量、蛋白表达量明显逐步升高，六味地黄丸含药血清能延缓这一变化，在培养基中加入TNF-α能明显上调Ⅰ型胶原及mRNA的表达，下调糖胺多糖含量、硫酸软骨素/硫酸角质素比值、透明质酸含量，蛋白多糖及mRNA、II型胶原及mRNA的表达,六味地黄丸含药血清可部分对抗TNF-α所导致的改变。而六味地黄丸含药血清可以延缓这一进程，提示六味地黄丸可以通过一定程度阻滞JNK及P38通路的信号表达，延缓椎间盘退变进程，起到保护椎间盘的作用。.科学意义.为中医补肾法治疗椎间盘源性腰痛提供实验依据，使补肾法干预椎间盘髓核细胞凋亡、调控其相关信号通路成为新的治疗靶点，为中医药防治椎间盘源性腰痛方面的研究开创新的思路。