自噬在葡萄籽原花青素改善氟致SH-SY5Y细胞毒性中的作用  

Role of autophagy in improving fluoride-induced SH-SY5Y cytotoxicity by grape seed proanthocyanidins

作　　者：龚泽林 王本杰 赵文欣 杨露 GONG Zelin;WANG Benjie;ZHAO Wenxin;YANG Lu(Department of Preventive Medicine,Medical College of Shihezi University,Shihezi,Xinjiang 832002,China)

机构地区：[1]石河子大学医学院预防医学系,新疆石河子832002

出　　处：《现代医药卫生》2024年第20期3440-3446,共7页Journal of Modern Medicine & Health

基　　金：石河子大学自主支持立项项目(ZZZC202015A);石河子大学高层次人才项目(RCZK201954);石河子大学创新发展项目(CXFZ20218)。

摘　　要：目的探究自噬过程在葡萄籽原花青素提取物(GSPE)缓解氟致人神经母细胞瘤SH-SY5Y细胞毒性中的作用。方法用氟化钠(60 mg/L)处理SH-SY5Y细胞24 h构建氟染毒细胞模型,随机分为对照组、氟化钠组、氟化钠+低剂量GSPE组、氟化钠+中剂量GSPE组、氟化钠+高剂量GSPE组;采用细胞计数试剂-8法(CCK-8法)检测各组细胞活力值变化;采用Hoechst33342染色分析各组细胞凋亡水平变化;采用免疫荧光方法分析各组细胞LC3表达情况;采用蛋白免疫印迹法(Western blot)检测cleaved-caspase 3、cleaved-PARP、LC3、p62等蛋白表达情况。随后纳入雷帕霉素重新分组,检测各组cleaved-PARP、LC3、p62等蛋白表达情况,并采用Hoechst33342染色分析各组细胞凋亡水平变化。结果与对照组比较,氟化钠组SH-SY5Y细胞凋亡水平显著提高(P<0.05),经GSPE干预后细胞活力上升且细胞自噬水平上升(P<0.05)。与氟化钠组比较,雷帕霉素干预可以有效抑制细胞凋亡,且雷帕霉素与原花青素联合后细胞自噬水平进一步提升(P<0.05)。结论GSPE能够抑制氟化钠诱导的SH-SY5Y细胞凋亡,其作用可能与激活自噬有关。Objective To explore the role of autophagy in improving fluoride-induced SH-SY5Y cytotoxicity by grape seed proanthocyanidins(GSPE)in human neuroblastoma.Methods SH-SY5Y cells were treated with sodium fluoride(60 mg/L)for 24 h to establish a fluoride-infected cell model,and then were randomly divided into the control group,sodium fluoride-infected group,sodium fluoride+low dose GSPE group,sodium fluoride+middle dose GSPE group and sodium fluoride+high dose GSPE group.The cell viability values of each group were detected by CCK-8 assay.Hoechst33342 staining was used to analyze the apoptosis level of each group.Immunofluorescence method was used to analyze the expression of LC3 in each group.The expressions of cleaved-caspase 3,cleaved-PARP,LC3,and p62 were detected by Western blot.Subsequently,Rapamycin was included in the regrouping,and the expressions of cleaved-PARP,LC3,p62 and other proteins in each group were detected,and the changes of apoptosis level in each group were analyzed by Hoechst33342 staining.Results Compared with the control group,the apoptosis level of SH-SY5Y cells in the sodium fluoride group was significantly increased(P<0.05),while the cell viability and autophagy level of SH-SY5Y cells were increased after GSPE intervention(P<0.05).Compared with the sodium fluoride group,Rapamycin intervention could effectively inhibit apoptosis,and the autophagy level was further enhanced in the combination of rapamycin and GSPE together(P<0.05).Conclusion GSPE can inhibit SH-SY5Y cell apoptosis induced by NaF,and this role may be related to the activation of autophagy.

关 键 词：葡萄籽原花青素 氟化钠 人神经母细胞瘤细胞 自噬 

分 类 号：R739.9[医药卫生—肿瘤]