摘要
目的探讨藏红花素对缺氧诱导的视网膜神经节细胞凋亡的作用及其可能机制。方法于2021年1月至2022年1月采用不同浓度藏红花素处理视网膜神经节细胞RGC-5,四甲基噻唑蓝(MTT)法检测细胞存活情况并筛选合适浓度。培养RGC-5细胞并用氯化钴(CoCl_(2))处理建立缺氧模型,分为缺氧组、藏红花素组、阳性对照(抗坏血酸)组和藏红花素+跨膜受体蛋白信号通路抑制剂(DAPT)组,另设对照组。Cell counting kit-8法检测细胞存活情况;采用流式细胞术检测细胞凋亡率;钙荧光探针(Flou-4)实验检测各组细胞钙离子水平;实时定量PCR法检测跨膜受体蛋白(Notch1)、发状分裂相关增强子1(Hes-1)mRNA表达情况;蛋白质印迹法检测凋亡蛋白B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关蛋白(Bax)、钙依赖性蛋白酶家族1(Cal⁃pain1)蛋白表达情况。结果藏红花素组细胞活力0.83±0.08高于缺氧组0.45±0.04,细胞凋亡率(17.92±1.21)%低于缺氧组(51.82±5.36)%,钙离子水平0.27±0.04低于缺氧组0.76±0.05,差异有统计学意义(P<0.05);藏红花素+DAPT组细胞活力0.50±0.06低于藏红花素组0.83±0.08,细胞凋亡率(36.50±3.50)%高于藏红花素组(17.92±1.21)%,钙离子水平0.65±0.05高于藏红花素组0.27±0.04,差异有统计学意义(P<0.05)。与缺氧组比较,藏红花素组Bcl-2蛋白表达水平升高,Notch1、Hes-1mRNA表达、Bax和Calpain1蛋白表达水平降低(P<0.05)。与藏红花素组比较,藏红花素+DAPT组Bcl-2蛋白表达水平降低,Notch1、Hes-1mRNA表达、Bax和Calpain1蛋白表达水平升高(P<0.05)。结论藏红花素对体外培养的缺氧RGC-5细胞凋亡有一定的抑制作用,可能是通过抑制钙离子内流,阻滞Notch1/Hes-1通路,提高细胞内抑凋亡蛋白Bcl-2表达水平发挥作用。
Objective To investigate the effect of crocin on hypoxia-induced retinal ganglion cell apoptosis and its possible mecha⁃nism.Methods In this study,RGC-5 retinal ganglion cells were treated with different concentrations of crocin from January 2021 to January 2022,and cell survival was detected by tetramethylthiazole blue(MTT)method and suitable concentrations were screened.RGC-5 cells were cultured and treated with cobalt chloride(CoCl_(2))to establish a hypoxia model.They were assigned into hypoxia group,crocin group,positive control(ascorbic acid)group and crocin+transmembrane receptor protein signal pathway inhibitor(DAPT)group,another control group was set up.Cell survival was detected by Cell counting kit-8 method;the apoptosis rate was detect⁃ed by flow cytometry;the level of Ca2+was detected by Flou-4 experiment;the mRNA expressions of transmembrane receptor protein(Notch1)and hairy division related enhancer 1(Hes-1)were detected by real-time quantitative PCR;the expressions of apoptotic pro⁃tein B-cell lymphoma factor 2(Bcl-2),Bcl-2 related protein(Bax)and calcium dependent protease family 1(Calpain1)were detected by Western blotting.Results The cell viability 0.83±0.08 in crocin group was higher than that in hypoxia group 0.45±0.04,the apoptosis rate(17.92±1.21)%was lower than that in hypoxia group(51.82±5.36)%,and the level of calcium ion 0.27±0.04 was lower than that in hypoxia group 0.76±0.05.The differences were statistically significant(P<0.05).The cell viability 0.50±0.06 in crocetin+DAPT group was lower than that in crocetin group 0.83±0.08,the apoptosis rate(36.50±3.50)%was higher than that in crocetin group(17.92±1.21)%,and the level of calcium ion 0.65±0.05 was higher than that in crocetin group 0.27±0.04,the differences were statistically signif⁃icant(P<0.05).Compared with hypoxia group,the expression level of Bcl-2 protein in crocetin group was increased,and the expression levels of Notch1,Hes-1 mRNA,Bax and Calpain1 protein were decreased(P<0.05).Compared with crocin group
作者
王玉风
付珂
王洪亮
WANG Yufeng;FU Ke;WANG Hongliang(Ophthalmology,The First Affiliated Hospital of Nanyang Medical College,Nanyang,He'nan 473000,China)
出处
《安徽医药》
CAS
2024年第1期193-197,I0005,共6页
Anhui Medical and Pharmaceutical Journal
基金
南阳市2021年科技发展计划项目(KJGG155)。
