骨形态发生蛋白7通过PI3K/Akt通路可抑制人髓核细胞的凋亡

Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathway

背景:骨形态发生蛋白7可促进人髓核细胞的细胞外基质合成,减缓椎间盘退变。近年来,有学者提出其可能通过对抗髓核细胞凋亡从而发挥上述作用,但其进一步的分子机制一直未被详细阐明。目的:观察骨形态发生蛋白7对无血清诱导下发生凋亡的人髓核细胞产生的作用及其对PI3K/Akt通路的影响,分析并探讨骨形态发生蛋白7抑制人髓核细胞凋亡的分子机制。方法:通过改良Pfirrmann分级及相关条件选取12例患者获取椎间盘组织,采用酶消化法获取人髓核细胞后分组实验,以含体积分数15%胎牛血清的培养基正常培养的髓核细胞设为空白组;使用无血清培养基培养48 h诱导凋亡作为阳性对照组;在无血清条件下,通过加入不同剂量的骨形态发生蛋白7以及同时添加PI3K/Akt通路拮抗剂LY294002形成处理组和拮抗组。使用流式细胞术检测各组细胞凋亡率;免疫荧光观察p-Akt表达;蛋白印迹法检测Akt,p-Akt,BAD和Caspase 9等通路蛋白的表达变化。结果与结论:无血清凋亡诱导下,流式细胞术结果显示,随着骨形态发生蛋白7处理浓度上升,髓核细胞凋亡率明显下降,加入LY294002共同作用后细胞凋亡率再次升高(P<0.05)。p-Akt免疫荧光和蛋白印迹法检测结果进一步表明,与凋亡阳性对照组相比,加入骨形态发生蛋白7的实验组中,p-Akt表达明显增加,其下游凋亡相关蛋白BAD、Caspase 9蛋白表达减少(P<0.05),而在同时加入Akt通路拮抗剂LY294002后,p-Akt蛋白表达下降而凋亡相关蛋白的表达又恢复到相对较高的水平(P<0.05)。结果证明,骨形态发生蛋白7在无血清诱导的人类髓核细胞凋亡中通过激活PI3K/Akt通路,拮抗了BAD-Caspase 9相关的细胞凋亡过程,抑制了髓核细胞的退变。

BACKGROUND： Bone morphogenetic protein 7 （BMP-7） can promote extracellular matrix synthesis of human nucleus pulposus cells, and delay intervertebral disk degeneration. Recently scholars proposed that the above effects can be achieved through anti-apoaptosis, but further molecular mechanism remains poorly understood. OBJECTIVE： To observe the effects of BMP-7 on the apoptotic human nucleus pulposus cells under serum-free induction and the PI3K/Akt pathway, investigate the molecular mechanism underlying the anti-apoptosis effect of BMP-7. METHODS： Human nucleus pulposus tissue were harvested from 12 patients according to the modified Pfirrmann classification, and human nucleus pulposus cells were obtained from digestion of herniated nucleus pulposus tissue. The collected nucleus pulposus cells were divided into four groups. Blank group： cells werecultured in normal culture medium containing 15% feral bovine serum; positive control group： coils were induced to apoptosis in the serum-free culture medium for 48 hours; treated group： cells were induced to apoptosis in the serum-free culture medium containing different dosages of BMP-7; antagonism group： coils were induced to apoptosis in the serum-free culture medium containing different dosages of BMP-7 and LY294002, an PI3K/Akt pathway inhibitor. Cellular apoptosis rate was quantified by flow cytometry. Immunoflurorescenco was performed to observe p-Akt expressions. Changes of Akt, p-Akt, BAD, and Caspase 9 expression were detected with western blot analysis. RESULTS AND CONCLUSION： Results from flow cytometry showed that, the apoptosis rate of nucleus pulposus cells was significantly decreased as the increase of BMP-7 concentration, under the serum-free induction; after the ceils were induced with LY294002, the apoptosis rate was increased again （P 〈 0.05）. The p-Akt immunoflurorescence and western blot analysis demonstrated that, compared with positive control group, the p-Akt expression was significantly increased, while the expression of downstream apoptosis-related proteins such as BAD and Caspase 9, was significantly decreased in the cells treated with BMP-7 （P 〈 0.05）. Furthermore, in the group treated with BMP-7 and LY294002, the p-Akt expression was decreased, while the expression of apoptosis-related proteins was recovered to high levels （P 〈 0.05）. BMP-7 activates the PI3K/Akt pathway, suppresses the downstream BAD and Caspase 9 signaling, and inhibits the apoptosis of human nucleus pulposus cells induced by serum-free situation.