中药胡椒碱在终板软骨细胞退变中的保护作用的研究

Protective effects of traditional Chinese medicine piperine on the degeneration of endplate chondrocyte

目的:探讨中药胡椒碱对椎间盘终板软骨细胞退变的保护作用。方法:分离获取大鼠终板软骨细胞,体外培养建立终板软骨细胞加力退变模型。设空白对照组(未加力细胞)、加力组(10%间歇性循环牵张力,10%ICMT,0.5 Hz,8 h/d,加力3 d)及加力加药物组(加力组中加入中药胡椒碱40μmol/L)。倒置相差显微镜观察细胞形态学变化,甲苯胺蓝染色鉴定细胞表型。实时聚合酶链反应检测各组细胞中SOX-9基因,II型胶原,蛋白聚糖,MMP13的表达情况,核质分离及免疫印迹检测胞核与胞质中NF-κB信号通路相关蛋白P65、p-P65表达变化。结果:细胞体外加力后表型丢失,甲苯胺蓝染色可见细胞外基质减少,加力加药组细胞外基质未见明显减少。加力组细胞(ICMT)较空白对照组细胞(NC组)SOX-9(ICMT/NC=0.22,P<0.01)、蛋白聚糖(ICMT/NC=0.15,P<0.01)、II型胶原(ICMT/NC=0.18,P<0.01)表达明显降低,NF-κB信号通路相关基因P65入核增加,NF-κB信号通路相关基因MMP13(ICMT/NC=1.68,P<0.05)表达明显上升。40μmol/L胡椒碱处理的加力加药组较加力组细胞核内p-P65减少,NF-κB信号通路相关基因MMP13(40μmol/L胡椒碱/ICMT=0.70,P<0.05)表达降低,40μmol/L胡椒碱处理的加药加力组中II型胶原(40μmol/L胡椒碱/ICMT=1.93,P<0.05)、蛋白聚糖(40μmol/L胡椒碱/ICMT=1.78,P<0.05)、SOX-9(40μmol/L胡椒碱/ICMT=1.70,P<0.05)等基因表达明显回升。结论:中药胡椒碱可以抑制椎间盘终板软骨细胞的退变发生,从而起到对椎间盘及终板软骨的保护作用。

AIM： To investigate the protective effects of the traditional Chinese medicine piperine on the intervertebral disc degeneration and the end plate cartilage degeneration. METHODS： Rats endplate chondrocytes were isolated and cultured in vitro. Rebulated vitro loading induce degeneration model and cells were divided into control group （chondrocyte without loading）, loading group（ 10% ICMT,0.5 Hz, 8 h/d,loading 3 days） and loading group add medicine （added piperine before the cells accepted loading）. The changes of cellular morphology were observed by inverted phase contrast microscope, the phenotype of endplate chondrocyte were identified by toluidine blue staining. The SOX-9, type II collagen, aggrecan genes and MMP13 were detected by real time-polymerase chain reaction （RTPCR） to verify the degeneration mode. The changes of P65,p-P65 levels were detected by Western blot. RESULTS：Chondrocytes lost the original morphology after loading in vitro. Decrease of extracellular matrix was observed after toluidine blue staining in loading group, but no significant decrease was observed when adding piperine in loading group. The levels of SOX-9 （ ICMT/NC = 0.22, P 〈 0.01 ）, aggrecan （ICMT/NC = 0.15, P 〈 0.01 ） and Type II collagen （ICMT/NC =0.18, P 〈0.01 ） were significantly decreased, while MMP13 （ICMT/NC = 1.68, P 〈 0.05 ） was significantly increased. Western blot analysis showed that in loading cells nuclear phosphorylation p65 was increased and piperine treatment attenuated increase in nuclear phosphorylation p65. Moreover, The levels of Type II collagen （40 p, mol/L piperine/ ICMT = 1.93, P 〈 0.05 ）,aggrecan （40 μmol/L piperine/ICMT = 1.78, P 〈 0.05） and SOX-9 （40 μmol/L piperine/ ICMT = 1.70, P 〈 0.05 ） were significantly increased,MMP13 （40 μmol/L piperine/ ICMT =0.70, P 〈 0.05 ） significantly reduced in treatment group. CONCLUSION： Traditional Chinese medicine piperine can inhibit the degeneration of ehondrocyte of intervertebral disc, so it can play a protective role for the intervertebral disc and the endplate cartilage.