柚皮苷通过Wnt/β-连环蛋白通路调控人髓核细胞凋亡的机制

The mechanism of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc by Wnt/β-catenin signaling pathway

目的观察柚皮苷通过Wnt／β-连环蛋白（β-catenin）通路对人退变椎间盘髓核细胞凋亡的影响。方法对体外培养的人退变椎间盘髓核细胞染色鉴定后，分别用柚皮苷0μg／ml（对照组）、10、20、40、80、100μg／ml的培养基培养48h，细胞计数试剂盒（CCK-8）及流式细胞法选择其抑制人退变椎间盘髓核细胞凋亡的最佳浓度。实时定量反转录聚合酶链反应（RT-qPCR）检测β-catenin、细胞周期蛋白D1（Cyclin D1）、c-Mye癌基因（c-Myc）表达。酶联免疫吸附试验（ELISA）检测B淋巴细胞瘤-2（bcl-2）、bcl-2相关X蛋白（bax）、半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、Ⅱ型胶原及蛋白聚糖表达。Western blot检测Ⅱ型胶原蛋白及蛋白聚糖表达。结果细胞染色结果均为阳性。CCK-8及流式细胞法测定20 μg/ml柚皮苷为抑制人髓核细胞凋亡的最佳浓度。liT-qPCR检测显示，在人退变椎间盘髓核细胞（对照组）中，与正常髓核细胞比较，β-catenin基因过表达，CyclinD1、c-Myc基因表达下调。加入20 μg/ml柚皮苷后，可以明显抑制β-catenin基因的表达（P=0．000），促进Cyclin D1、c-Myc基因表达（P：0．000、0．001）。ELISA显示，20 μg/ml柚皮苷可以明显上调bcl-2、蛋白聚糖及Ⅱ型胶原蛋白表达（P=0．001、0．000、0．023），下调bax、Caspase-3蛋白表达（P=0．001、0．001），并可以促进Ⅱ型胶原蛋白及蛋白聚糖表达。结论柚皮苷可能通过调控Wnt／β-catenin信号通路和线粒体凋亡通路抑制人退变椎间盘髓核细胞凋亡。

Objective To study the effects of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc. Methods Nucleus pulposus cells were identified via the toluidine blue and safranin O. Next, P3 cells were cultured with naringin 0 μg/ml （control group）, naringin group （10, 20, 40, 80, 100 μg/ml）. After 48 hours, the best concentration of naringin was selected by cell counting kit-8 （ CCK-8） and flow cytometry. β-catenin, Cyclin D1, c-Myc gene expression was detected by real-time quantitative reverse transcriptase-polymerase chain reaction （ RT-qPCR）. The expression of B cell lymphoma/leukemia-2 （ bcl-2 ）, bcl-2 associated X protein （ bax）, Caspase-3 protein, type Ⅱ collagen and proteoglycan was determined by enzyme linked immunosorbent assay （ELISA）. Western blotting detected of type Ⅱ collagen and proteoglycan expression. Results The results of cells staining were positive. CCK-8 and flow cytometry were used to determine the optimum concentration of naringin （20 μg/ml） to inhibit the apoptosis of nucleus pulposus ceils. In the human degeneration nucleus pulposus cells, the expression of β-catenin geue was overexpressed and the expression of Cyclin D1 and c-Myc gene was down regulated. 20g/ml naringin could down regulate the expression of β-catenin gene （ P = 0. 000 ）, and up regulate the expression of Cyclin DI and c-Myc gene （ P=0. 000, 0. 001 ）. The naringin （20 μg/ml） could obviously regulate up the expression of bcl-2, type Ⅱ collagen and proteoglycan protein （P = 0. 00l, 0. 000, 0. 023）, and regulate down the expression of bax and Caspase-3 protein, （P =0. 001, 0. 001 ）. Western blotting showed that 20 μg/ml naringin could promote the expression of type Ⅱ collagen and proteoglycan. Conclusion Naringin may inhibit the apoptosis of nucle- us pulposus ceils by regulating the Wnt/β-catenin pathway and the mitochondrial apoptosis pathway.