白桦脂醇对Aβ25-35诱导的PC12细胞氧化应激和凋亡的影响

Effects of Betula alcohol on oxidative stress and apoptosis induced by Aβ25-35 in PC12 cells

目的探讨白桦脂醇对Aβ25-35诱导的PC12细胞氧化应激和凋亡的影响。方法以40μmol/L Aβ25-35作用于PC12细胞,实验随机分对照组、Aβ25-35组、N-乙酰半胱氨酸组(NAC组)和白桦脂醇5、10、20μmol/L组,采用MTT法检测细胞的存活率,Annexin V-FITC/PI双染流式细胞术检测凋亡情况,JC-1试剂盒检测线粒体膜电位,LDH、MDA、SOD试剂盒分别检测LDH、MDA和SOD水平,比色法检测caspase3、caspase8、caspase9和Cyt c的酶活性,Western blot法检测凋亡相关蛋白Bcl2、Bax、caspase3、caspase8、caspase9和Cyt c的活性和表达。结果与Aβ25-35组比较,白桦脂醇和NAC均对Aβ25-35诱导的PC12细胞可提高细胞存活率和SOD活性(P<0. 01),并降低LDH释放率和凋亡率(P <0. 01),抑制caspase3、caspase8、caspase9和Cyt c酶活性(P <0. 01),同时下调Bax、caspase3、caspase8、caspase9和Cyt c表达水平(P<0. 01),并上调Bcl2的活性和表达(P<0. 01)。结论通过预防给予有效剂量的白桦脂醇能够抑制Aβ25-35诱导产生的细胞凋亡,其机制可能与减少ROS生成从而降低氧化应激水平,继而抑制细胞凋亡途径有关。

Objective To investigate the effect of betulin on Aβ25-35-induced oxidative stress and apoptosis of PC12 cells.Methods PC12 cells were treated with 40μmol/L of Aβ25-35.The cells were randomly divided into control group,Aβ25-35 group,NAC group,and betulin 5,10 and 20μmol/L groups.Apoptosis was detected by V-FITC/PI double staining flow cytometry;mitochondrial membrane potential was detected by JC-1 kit;LDH,MDA and SOD levels were detected by LDH,MDA and SOD kits,respectively;the enzyme activity of caspase-3,caspase-8,caspase-9 and Cyt c was detected by colorimetry;the activity and expression of Bcl 2,Bax,caspase-3,caspase-8,caspase-9 and Cyt C were detected by western blotting.Results Compared with the Aβ25-35 group,betulin and NAC improved the cell survival rate and SOD activity(P<0.01),reduced the LDH release rate and apoptosis rate(P<0.01),inhibited caspase-3,caspase-8,caspase-9 and Cyt c enzyme activity(P<0.01),and downregulated Bax,caspase-3,caspase-8,caspase-9 and Cyt c enzyme activity(P<0.01)and expression level(P<0.01).In contrast,these treatments upregulated the activity and expression of Bcl2(P<0.01).Conclusions Betulin inhibited the Aβ25-35-induced apoptosis.The mechanism may be related to the reduction in ROS production,thus reducing the level of oxidative stress,and thereby inhibiting apoptosis.