人参皂苷Rb1通过AKT/GSK-3β/NFAT途径抑制硫化砷诱导的神经细胞PC12毒性

Ginsenoside Rb1 inhibits arsenic sulfide-induced toxicity in PC12 nerve cells via AKT/GSK-3β/NFAT pathway

目的探索人参皂苷Rb1抑制硫化砷诱导的神经细胞--嗜铬细胞瘤细胞(PC12)毒性的作用机制。方法以PC12细胞为研究对象,加入0、2.5、5、10、15、20、50μmol/L硫化砷后观察细胞的损伤情况,采用噻唑蓝法检测细胞活性。在硫化砷损伤的PC12细胞培养基内加入5、10、20μmol/L人参皂苷Rb1,采用磺酰罗丹明B法检测其对细胞增殖能力的影响;用流式细胞术检测其对细胞凋亡的影响;ATP试剂盒荧光素酶法测定其对细胞内ATP含量的影响;2′,7′-二氯四氟乙烷双乙酸盐探针染色法检测其对细胞内活性氧(ROS)含量的影响。Western blot检测PC12细胞中蛋白激酶B(AKT)、糖原合成酶激酶3(GSK-3β)、活化T细胞核因子(NFAT)、细胞色素C(Cyt C)、半胱氨酸蛋白酶3(Caspase-3)、B淋巴细胞瘤2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)的蛋白表达。结果不同浓度硫化砷对PC12细胞有损伤,且随着浓度升高及作用时间延长,细胞损伤越严重;硫化砷可抑制PC12细胞的增殖。人参皂苷Rb1对硫化砷损伤的PC12细胞有保护作用,可有效抑制硫化砷诱导的PC12细胞的凋亡。硫化砷可降低PC12细胞内的ATP含量,增加ROS的含量;人参皂苷Rb1可增加ATP的含量,降低ROS的含量,加强细胞代谢。硫化砷刺激PC12细胞后细胞内p-AKT、p-GSK-3β、NFAT-c3、Bcl-2、Cleaved Caspase-3蛋白的表达显著降低,Caspase-3与Bax蛋白的表达明显提高,线粒体内Cyt C表达明显提高(P<0.01);人参皂苷Rb1干预后,细胞内p-AKT、p-GSK-3β、NFAT-c3、Bcl-2、Cleaved Caspase-3蛋白表达明显升高,Caspase-3与Bax蛋白的表达明显降低,线粒体内Cyt C表达明显降低(P<0.01)。结论人参皂苷Rb1可抑制硫化砷诱导的PC12细胞凋亡,从而保护神经细胞,其可能是通过调节AKT/GSK-3β/NFAT信号通路来实现的。

Aim To explore the mechanism of ginsenoside Rb1 inhibiting arsenic sulfide-induced toxicity of neurocyte pheochromocytoma cells( PC12). Methods PC12 cells were treated with 0,2. 5,5,10,15,20,50 μmol/L arsenic sulfide. Cell damage was observed,and cell viability was measured by thiazole blue method. 5,10,20 μmol/L ginsenoside Rb1 was added into PC12 cell culture medium damaged by arsenic sulfide,and its effect on cell proliferation was detected by sulforhodamine B assay,cell apoptosis was detected by flow cytometry,ATP content in cells was detected by ATP kit luciferase assay,and intracellular reactive oxygen species( ROS) content was detected by 2’,7’-dichlorotetrafluoroethane diacetate probe dyeing. Western blot was used to detect the protein expressions of protein kinase B( AKT),glycogen synthase kinase 3β( GSK-3β),nuclear factor of activated T cell( NFAT),cytochrome C( Cyt C),cysteine protease 3( Caspase-3),B-cell lymphoma-2( Bcl-2),Bcl-2 associated X protein( Bax) in PC12 cells. Results The PC12 cells were damaged by different concentrations of arsenic sulfide,with the increase of the concentration and the prolongation of the action time,the cell damage became more serious;Arsenic sulfide could inhibit the proliferation of PC12 cells. Ginsenoside Rb1 had protective effect on PC12 cells damaged by arsenic sulfide and could effectively in-hibit apoptosis of PC12 cells induced by arsenic sulfide. Arsenic sulfide could decrease ATP content and increase ROS content in PC12 cells,and ginsenoside Rb1 could increase ATP content,reduce ROS content and enhance cell metabolism.After arsenic sulfide stimulation,the protein expressions of p-AKT,p-GSK-3β,NFAT-c3,Bcl-2 and Cleaved Caspase-3 in PC12 cells was significantly decreased,the protein expressions of Caspase-3 and Bax was significantly increased,and the expression of Cyt C in mitochondria was significantly increased( P< 0. 01). After ginsenoside Rb1 intervention,the protein expressions of p-AKT,p-GSK-3β,NFAT-c3,Bcl-2 and Cleaved Caspase-3 increased significantly,the protein expressions of Caspase-3 and Bax decreased significantly,and the expression of Cyt C in mitochondria decreased significantly( P<0. 01). Conclusion Ginsenoside Rb1 can inhibit arsenic sulfide-induced apoptosis of PC12 cells and protect neurons,which may be achieved by regulating AKT/GSK-3β/NFAT signaling pathway.