身痛逐瘀汤方对椎间盘退变中PI3K/AKT通路影响的研究

Biomechanical Study on the Expression of PI3K/AKT in the Degeneration of Intervertebral Disc with Shentong Zhuyu Decoction

目的:探明静水压下髓核细胞凋亡及基质代谢的相关分子生物学机制,揭示身痛逐瘀汤方对静水压下髓核细胞凋亡及基质代谢的调控机制及作用靶点。方法:将8只新西兰兔处死后无菌条件下取出髓核,分为8组,将8组髓核细胞分离培养、鉴定及传代后,加入身痛逐瘀汤方含药血清中,在体外静水压加载系统中进行干预,在0.3MPa,1MPa及3MPa压力下作用4h,24h后,使用倒置相差显微镜观察髓核细胞加压前后的形态及生长状况;采用透射电镜观察各组髓核细胞超微结构的变化及差异;使用Cell Counting Kit-8法检测各组髓核细胞的增殖活性;使用Annexin V-FITC/Propidium Iodide双染法检验各组髓核细胞的凋亡状况;使用凝胶电泳迁移法(EMSA)检验各组髓核细胞中PI3K/AKT(磷脂酰肌醇3-激酶)/(丝氨酸/苏氨酸激酶)的活跃情况;Western Blot法检验Sox9,CollagenⅡ,BAD,Caspase-9及GSK-3在髓核细胞中含量的变化。结果:在同一压力与作用时间下倒置相差显微镜及透射电镜示中药干预组较单纯压力组髓核细胞形态及超微结构保存更完整,生长状况更好;CCK-8法示中药干预组髓核细胞增殖活性更高;Annexin V-FIT检验结果示中药干预组髓核细胞凋亡百分比更低;凝胶电泳迁移法及Western Blot法示PI3K/AKT通路相关蛋白表达水平明显升高。结论:身痛逐瘀汤能明显激活PI3K/AKT信号通路的表达,延缓椎间盘的退变。

Objective：To explore the molecular biological mechanism of apoptosis and matrix metabolism of nucleus pulposus cells under hydrostatic pressure,and to reveal the regulation mechanism and action target of Shentong Zhuyu Decoction on apoptosis and matrix metabolism of nucleus pulposus cells under hydrostatic pressure.Methods：The nucleus was removed from 8 New Zealand rabbits after being executed in sterile condition,and divided into 8 groups.After 8 groups of nucleus pulposus cells were isolated,cultured,identified and passaged,they were added to the serum of Shentong Zhuyu Decoction and intervened in hydrostatic loading system in vitro.The morphology and growth of nucleus pulposus cells were observed by inverted phase contrast microscope before and after 4 hand 24 hexposure to 0.3 MPa,1 MPa and 3 MPa pressure.The ultrastructural changes and differences of nucleus pulposus cells were observed by transmission electron microscope.The Cell Counting Kit-8 assay was used to detect the proliferative activity of nucleus pulposus cells.The apoptosis of nucleus pulposus cells was examined by Annexin V-FITC/Propidium Iodide double staining method,and the EMSA was used to test the PI3 K/AKT of nucleus pulposus cells in each group.The changes of content of Sox9,CollagenⅡ,Bad,Caspase-9 and GSK-3 in nucleus pulposus cells were examined by Western blot method.Results：Under the same pressure and action time,the inverted phase contrast microscope and transmission electron microscopy showed that the morphology and ultrastructure of the nucleus pulposus cells were more complete and the growth condition was better than that of the simple pressure group.CCK-8 method showed that the proliferative activity of nucleus pulposus cells was higher in Chinese herbal intervention group.Annexin V-FIT test showed that the percentage of apoptosis was lower in the Chinese herbal intervention group and the EMSA and Western Blot showed that the expression of PI3 K/AKT pathway related genes and proteins were increased significantly.Conclusion：Shentong Zhuyu Decoction can obviously activate the expression of PI3 K/AKT signaling pathway and delay the degeneration of intervertebral disc.