摘要
目的观察银杏叶提取物对顺铂诱导小鼠耳蜗毛细胞(HEI-OC1)中AMPK及SIRT1蛋白表达水平的影响,探讨银杏叶提取物抗损伤和凋亡作用的机制。方法取对数生长期的HEI-OC1细胞,添加高糖DMEM培养基调整细胞密度到1×10^4/ml,将其加入6孔板中,每孔2 ml。将HEI-OC1细胞分为对照组、顺铂组、顺铂+银杏叶组,对照组HEI-OC1细胞仅采用DMEM培养基培养,顺铂组细胞中加入8μg/ml顺铂进行干预24小时,顺铂+银杏叶组预先给予HEI-OC1细胞加入50μg/ml银杏叶提取物作用24小时,再给予含有8μg/mL顺铂的DMEM培养基作用24小时;采用CCK-8试剂盒检测各组培养24、48、72和96 h时的增殖情况;采用Western blot检测三组细胞凋亡相关蛋白Caspase-3、BCL-2和Bax以及AMPK和SIRT1表达;采用流式细胞术检测三组细胞不同干预后的凋亡情况。结果CCK-8结果显示,与对照组相比,顺铂组和顺铂+银杏叶提取物组HEI-OC1细胞的增殖能力均明显下降,在各个时间点均低于对照组(P<0.05),而顺铂+银杏叶组的HEI-OC1细胞增殖能力在48、72和96 h时均显著高于顺铂组(均为P<0.05),且在第72 h和96 h与对照组比较差异无统计学意义(P>0.05)。流式细胞术检测显示对照组细胞凋亡率为2.3%±0.2%,显著低于顺铂组(26.8%±4.3%)及顺铂+银杏叶组(7.2%±1.5%)(P<0.05)。顺铂+银杏叶组HEI-OC1细胞的Bax及Caspase-3蛋白表达水平较顺铂组显著下降,而AMPK、SIRT1及Bcl-2蛋白表达水平较顺铂组显著升高。结论银杏叶提取物能够通过AMPK/SIRT1信号通路途径抑制顺铂诱导的HEI-OC1细胞的损伤与凋亡。
Objective To investigate the effects of ginkgo biloba extract on the expression of AMPK and SIRT1 in cisplatin-induced mouse cochlear hair cells(HEI-OC1),and to explore its mechanism of anti-injury and apoptosis.Methods HEI-OC1 cells in logarithmic growth phase were cultured,high glucose DMEM medium was added to adjust the cell density to 1×10^4/ml,and it was added to a 6-well plate at 2 ml per well.HEI-OC1 cells were divided into three groups normal group,cisplatin intervention group(8μg/mL cisplatin),cisplatin+ginkgo extract(ginkgo biloba extract 50 mg/L).And cultured for 7 days after intervention.The normal group of HEI-OC1 cells were cultured only in DMEM medium.The cells in the cisplatin-injured group were treated with 8μg/ml cisplatin for 24 hours.The ginkgo biloba+cisplatin group was pre-administered to HEI-OC1 cells by adding 50μg/ml ginkgo biloba extract for 24 hours.The proliferation of each group was detected at 24,48,72 and 96 h using CCK-8 kit.Western blot was used to detect the expression of apoptosis-related proteins caspase-3,BCL-2 and Bax,and the expression of AMPK and SIRT1.Flow cytometry was used to detect apoptosis in three groups of cells after different interventions.Results The results of CCK-8 showed that compared with the normal group,the proliferation of HEI-OC1 cells decreased significantly after cisplatin intervention,which was lower than that of the control group at each time point(P<0.05),while HEI-after ginkgo biloba extract intervention.The proliferation of OC1 cells was significantly higher than that of cisplatin intervention group at 48,72 and 96 h(P>0.05),and there was no significant difference between the 72 h and 96 h groups(P>0.05).The results of flow cytometry showed that the apoptosis rate of the normal group was 2.3%±0.2%,which was significantly lower than that of the cisplatin group 26.8%±4.3%and the cisplatin+ginkgo biloba extract group7.2%±1.5%(P<0.05).The expression levels of Bax and caspase-3 in HEI-OC1 cells of cisplatin+ginkgo biloba extract group were significantly lower than those in cisplatin group,while the expression levels of AMPK,SIRT1 and Bcl-2 protein were significantly higher than those in cisplatin group.Conclusion Ginkgo biloba extract can inhibit the damage and apoptosis of cisplatin-induced HEI-OC1 in mouse cochlear hair cells through AMPK/SIRT1 signaling pathway.
作者
雷文静
陆菲
米文娟
查定军
Lei Wenjing;Lu Fei;Mi Wenjuan;Zha Dingjun(Otolaryngology Head and Neck Surgery,First Affiliated Hospital of Air Force Military Medical University,Xi'an,710032,China)
出处
《听力学及言语疾病杂志》
CAS
CSCD
北大核心
2019年第5期519-523,共5页
Journal of Audiology and Speech Pathology
基金
国家自然科学基金项目(81870732)
陕西省科技重点项目(2018PT-01)
陕西省科技重点项目(2017ZDXM-SF-061)
西京助推—先进学科建设项目(XJZT14X07)
西京助推—卫勤专项研究项目(XJZT15D02)联合资助
关键词
银杏叶提取物
顺铂
耳蜗毛细胞
凋亡
Ginkgo biloba extract
Cisplatin
Cochlear hair cells
Apoptosis
