多西环素在纤维连接蛋白诱导髓核细胞退变过程中的作用

Role of doxycycline in the fibronectin-induced degeneration of nucleus pulposus cells

背景:多西环素在阻止间盘退变方面有一定的作用,但是其在椎间盘中抑制基质金属蛋白酶的机制并未完全阐明。目的:从分子内信号传导角度采用细胞实验来阐明多西环素抑制间盘内基质金属蛋白酶的作用机制。方法:体外培养人间盘髓核细胞,在培养液中加入相对分子质量为45 000纤维连接蛋白片段制造退变模型。在退变模型中按照不同作用时间及不同作用浓度分组,均通过Western blot方法检测各组Ⅱ型胶原和基质金属蛋白酶13蛋白的表达情况。然后在10 mg/L多西环素作用24 h后,Western blot方法测定各组ERK1/2、p-ERK1/2、P38、p-P38、JNK、p-JNK蛋白表达水平,并根据以上实验结果选择ERK及JNK通路抑制后,Western blot检测多西环素对MAPK通路蛋白表达的影响。结果与结论:(1)纤维连接蛋白可以诱导髓核细胞退变,表现为基质金属蛋白酶13表达增加、Ⅱ型胶原表达降低。多西环素可以抑制纤维连接蛋白诱导的退变髓核细胞基质金属蛋白酶13的表达,但并不呈时间依赖性和剂量依赖性;(2)多西环素作用于经纤维连接蛋白诱导的髓核细胞后可以抑制其ERK1/2及JNK磷酸化,双重抑制有效地阻断了ERK1/2及JNK在髓核细胞中的激活,与多西环素或MAPK抑制剂单独作用相比,这种联合作用协同地增强了对ERK及JNK磷酸化的抑制作用。因此多西环素可能通过阻断ERK1/2及JNK在髓核细胞中的激活来抑制金属基质蛋白酶表达,进而减少细胞外基质降解,具有预防和治疗间盘退变的潜在作用。

BACKGROUND： Doxycycline has a certain effect on disc degeneration progression, but the mechanism by which doxycycline inhibits the expression of matrix metalloproteinases（MMPs） in intervertebral disc remains unclear.OBJECTIVE： To clarify the mechanism by which doxycycline inhibits MMPs expression in intervertebral disc by cytologic experiment in views of intramolecular signal transduction. METHODS： The human nucleus pulposus cells were cultured in vitro, and 45 000 fibronectin fragments were added into the culture medium to make the degeneration model. The degenerative models were divided according to different action time and different concentrations. The expression levels of type II collagen and MMP13 in each group were detected by western blot assay. After treatment with 10 mg/L doxycycline for 24 hours, the expression levels of ERK1/2, p-ERK1/2, P38, p-P38, JNK, and p-JNK proteins in each group were determined by western blot assay. Based on the above experimental results, the inhibition of ERK and JNK pathways was selected, and the effect of doxycycline on the expression of MAPK pathway protein was detected by western blot assay. RESULTS AND CONCLUSION： Fibronectin could induce the degeneration of nucleus pulposus cells, showing an increase in expression level of MMP13 and a decrease in expression of type II collagen. Doxycycline could inhibit the expression of MMP13 in degenerative nucleus pulposus cells induced by fibronectin, but not in a time-or dose-dependent manner. After degenerative nucleus pulposus cells induced by fibronectin were treated with doxycycline, the phosphorylation of ERK1/2 and JNK was inhibited, and dual inhibition effectively blocked the activation of ERK1/2 and JNK compared with MAPK inhibitors or doxycycline alone. So doxycycline may block ERK1/2 and JNK activation in the nucleus pulposus cells to inhibit MMP expression, and thus reduce the extracellular matrix degradation. Thereafter, doxycycline exerts a potential role in preventing and treating disc degeneration.