香叶木素通过抑制髓核细胞凋亡和炎症反应缓解椎间盘退变大鼠神经根性疼痛

Diosmetin alleviates IL-1beta-induced apoptosis and inflammation of nucleus pulposus cells and relieves nerve root pain in rats with intervertebral disc degeneration

目的探究香叶木素(diosmetin,DM)对椎间盘退变的治疗作用及机制,为临床治疗椎间盘退变疾病提供理论依据和药物研发靶点。方法通过CCK-8试剂盒检测DM对大鼠髓核细胞(nucleus pulposus cells,NPCs)活力的影响,选取最适试验浓度;通过RT-PCR、Western blot检测各组髓核细胞中Caspase-3、COX-2、Ki67、MMPs表达变化以及NF-κB p65的活化水平;采用Hoechst 33258染色进一步检测各组细胞的凋亡情况,并通过免疫荧光技术检测各组细胞中NF-κB p65的核转位情况;最后使用von Frey细丝检测IL-1β诱导的椎间盘退变大鼠模型的机械痛阈。结果与IL-1β组相比,DM可从mRNA、蛋白水平显著下调IL-1β诱导的MMPs、COX-2的高表达(P<0.05),并显著上调TIMP-1的表达(P<0.05)。与IL-1β组相比,DM显著抑制了IL-1β诱导的髓核细胞凋亡(P<0.05),并显著下调了Caspase-3的表达(P<0.05),显著上调Ki67的表达(P<0.05)。其次,与IL-1β组相比,DM显著降低了IL-1β诱导的NF-κB p65高度磷酸化以及入核量(P<0.05)。最后,与对照组相比,IL-1β诱导的椎间盘退变大鼠模型机械痛阈明显降低(P<0.05);而与IL-1β组相比,DM显著降低了IL-1β诱导的动物模型的神经根疼痛(P<0.05)。结论 DM可以抑制IL-1β诱导的大鼠髓核细胞凋亡、炎症反应以及NF-κB p65核转位,具有缓解椎间盘退变引起的神经根性疼痛的作用。

This study aims to explore the therapeutic effect of diosmetin(DM) on intervertebral disc degeneration, and to provide theoretical basis for the clinical treatment of intervertebral disc diseases. For this purpose, the effect of DM on the activity of rat nucleus pulposus cells(NPCs) was detected by CCK-8 assay, and the optimal concentration was selected. RT-PCR and Western blot were employed to detect the expression of Caspase-3, COX-2, Ki67, MMPs and the activation level of NF-κB p65 in nucleus pulposus cells of each group. The nuclear translocation of NF-κB p65 was detected by immunofluorescence technique, and the tolerance to nerve root pain was tested by mechanical foot withdrawal test. Data showed that DM significantly decreased the expression of MMPs and COX-2 induced by IL-1 beta(P<0.05), and significantly increased the expression of TIMP-1(P<0.05) in DM group, as compared with IL-1 beta group. DM also significantly inhibited IL-1 beta-induced apoptosis of nucleus pulposus cells, significantly decreased Caspase-3 expression(P<0.05), and significantly increased Ki67 expression(P<0.05). Secondly,compared with IL-1 beta group, DM significantly decreased the phosphorylations of NF-κ B p65 induced by IL-1 beta and the number of nuclear translocation(P<0.05). Finally, compared with the control group, IL-1 beta significantly reduced the nerve root pain tolerance of the animal model, while DM significantly reduced the nerve root pain of the animal model induced by IL-1 beta(P<0.05). All the results suggest that DM can inhibit IL-1 beta-induced apoptosis, inflammation and nuclear translocation of NF-κB p65 in rat nucleus pulposus and alleviate radicular pain caused by intervertebral disc degeneration.