基于p38MAPK信号通路研究回医烙灸治疗腰椎间盘退变的作用机制

Our pre-clinical studies confirmed the efficacy of cautery moxibustion therapy of Hui medicine treatment of lumbar intervertebral disc degeneration（IDD）.In the present study，through analyzing p38 MAPK signal pathway regulates apoptosis of lumbar endplate cartilage cells，nucleus pulposus cells and extra cellular environment, thus we can expound the mechanism of cautery moxibustion therapy IDD. Our previous studies found that cautery moxibustion therapy could reduce the apoptosis of nucleus pulposus of intervertebral disc and MMP-3 in intervertebral disc，and Moxibustion also could reduce the IL-6 in intervertebral disc.IL-6 and MMP-3 are key factors of p38MAPK signal pathways. At the same time P38MAPK can promote the apoptosis of nucleus pulposus. Therefore we put forward the hypothesis that the cautery moxibustion may be mediated through p38MAPK signaling pathway to treat IDD.p38 MAPK signal pathway regulates apoptosis of lumbar endplate cartilage cells , nucleus pulposus cells and extra cellular environment, such as IL-1、IL-6、MMPs 、TIMPs.This study uses TUNEL method、immunohistochemistry、RT-PCR、Western-Blot and other methods to detect apoptosis of disc cell and P-p38MAPK、p38MAPK、IL-1、IL-6、Bax 、Bcl-2、MMPs 、TIMPs，and we can verify the hypothesis that the cautery moxibustion may be mediated through p38MAPK signaling pathway to treat IDD. The study also helps to find material basis of four liquid and deepens theory of four liquid in Hui medicine.

用临床疗效确切的特色回医烙灸干预腰椎间盘退变(IDD)大鼠模型，分析p38 MAPK信号通路对软骨终板细胞和髓核细胞凋亡及细胞外IL-1、IL-6、MMPs 、TIMPs等基质环境的调控，探讨烙灸治疗IDD的作用机制。我们前期研究发现烙灸可减少髓核细胞的凋亡及椎间盘外基质中IL-6和 MMP-3的水平，IL-6和MMP-3是p38MAPK信号通路下游关键作用因子，同时p38MAPK可促进髓核细胞的凋亡，从而提出烙灸可能是通过介导p38MAPK信号通路治疗IDD的假说。本课题采用TUNEL法、免疫组化、RT-PCR、Western Blot等方法对椎间盘细胞凋亡、Bax、Bcl-2、P-p38MAPK、p38MAPK 、IL-1、IL-6、MMPs 、TIMPs等进行检测。验证烙灸通过介导p38MAPK信号通路治疗IDD的假说，阐明其治疗IDD的作用机制。本课题还有助于深化回医四液学说理论。

用临床疗效确切的特色回医烙灸干预腰椎间盘退变(IDD)大鼠模型，分析p38 MAPK信号通路对软骨终板细胞及细胞外基质环境的调控，探讨烙灸治疗IDD的作用机制。我们前期研究发现烙灸可减少髓核细胞的凋亡及椎间盘外基质中IL-6和 MMP-3的水平，IL-6和MMP-3是p38MAPK信号通路下游关键作用因子，同时p38MAPK可促进髓核细胞的凋亡，从而提出烙灸可能是通过介导p38MAPK信号通路治疗IDD的假说。本项目内容：① 通过观察烙灸疗法治疗前后腰椎正侧位X线片、腰椎MRI及椎间盘形态学的改变，进一步验证回医烙灸疗法的治疗效果。② 研究烙灸疗法干预IDD的作用机制探讨回医烙灸疗法通过p38MAPK信号通路对软骨终板细胞和髓核细胞凋亡的调节作用机制；用TUNEL法、免疫组化、RT-PCR、Western Blot等方法对软骨终板细胞和髓核细胞的凋亡、Bax、Bcl-2、Caspase-3、P-p38MAPK、p38MAPK进行检测。观察回医烙灸疗法通过p38MAPK信号通路对软骨终板细胞和髓核细胞外基质环境的影响；用免疫组化、RT-PCR、Western Blot、分光光度等方法对IL-1、IL-6、MMPs、TIMPs、I型胶原、II型胶原、蛋白多糖进行检测。通过影像学和形态学研究发现回医烙灸疗法可以改变大鼠腰椎间盘的退变。回医烙灸组大鼠间盘终板软骨细胞凋亡指数AI为2.02±0.23低于模型对照组的6.33±1.08（P<0.05），回医烙灸疗法可以减少软骨细胞的凋亡。回医烙灸组椎间盘蛋白多糖含量为2.23±0.03pg/mg、II型胶原纤维152.48±7.45均高于模型对照组的1.03±0.02pg/mg和8.37±9.24（P<0.05），回医烙灸疗法可以改善软骨细胞外基质环境。回医烙灸组椎间盘p-p38MAPK蛋白光密度值为0.34±0.02、Wnt10为3.25±0.16、β-catenin为5.03±0.74均分别低于模型对照组的0.91±0.01、5.81±0.47和7.24±1.26（P<0.05），回医烙灸疗法可以下调p-p38MAPK、Wnt10、β-catenin蛋白的表达，验证回医烙灸通过抑制p38MAPK和WNT10B/β-catenin信号通路治疗IDD的假说，阐明回医烙灸疗法治疗IDD的作用机制。