六味地黄丸对椎间盘体外退变模型髓核细胞MAPK信号级联的影响

Liuwei Dihuang Pill inhibits mitogen-activated protein kinase signaling cascade of nucleus pulposus cells in a model of intervertebral disc degeneration

背景:JNKs和p38作为丝裂原活化蛋白激酶(MAPKs)家族中重要的成员,对细胞增殖、分化、转化及凋亡起着至关重要的作用。目的:观察六味地黄丸对兔椎间盘体外退变模型髓核细胞MAPK信号级联的影响。方法:将100个带终板软骨的椎间盘随机分为空白对照组、肿瘤坏死因子α组、p38-JNK/SAPK阻断组、六味地黄丸组、肿瘤坏死因子α+含六味地黄丸组,每组20个。肿瘤坏死因子α组培养液中含10 mg/L肿瘤坏死因子α2μL;p38-JNK/SAPK阻断组培养液中含p38MAPK特异性阻断剂SB203580,JNK特异性阻断剂SP600125各20μmol/L 10μL,10 mg/L肿瘤坏死因子α2μL;六味地黄丸组培养液中含体积分数为10%六味地黄丸血清;肿瘤坏死因子α+六味地黄丸组培养液中含10 mg/L肿瘤坏死因子α2μL,体积分数为10%六味地黄丸血清,分别于培养的第2,4,8,14天收集标本待测。结果与结论:RT-PCR、Western Blot检测显示,随着培养时间的延长,髓核细胞JNK及P38通路的mR NA含量、蛋白表达量明显逐步升高,而含六味地黄丸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。

BACKGROUND：JNKs and p38 MAPK, as important members of mitogen-activated protein kinase （MAPK）, play critical roles in the cell proliferation, differentiation, transformation and apoptosis. OBJECTIVE：To observe the effects of Liuwei Dihuang Pill on the MAPK signaling cascade of nucleus pulposus cells in a rabbit model of intervertebral disc degeneration. METHODS：100 discs samples with endplate cartilages were equivalently randomized into five groups and cultured in the medium containing 10 mg/L tumor necrosis factor-α（TNF-α, 2μL）, p38-JNK/SAPK blockers （10μL of 20μmol/L SB203580 and SP600125, 2μL of 10μmol/L TNF-α）, 10%Liuwei Dihuang Pill serum, 10 mg/L TNF-α（2μL） plus 10%Liuwei Dihuang Pill serum, respectively. Those cells received no intervention as controls. Samples were collected and detected at 2, 4, 8 and 14 days of culture. RESULTS AND CONCLUSION：Real time-PCR and western blot assay showed that mRNA and protein expressions of JNK and P38 pathways by the nucleus pulposus cells were upregulated with time, while all above indicators were significantly decreased by the Liuwei Dihuang Pill serum. These findings reveal that Liuwei Dihuang Pill can delay the process of disc degeneration by blocking JNK and P38 signaling pathways, thus exerting protective effect on the intervertebral discs.