目的 探讨红景天苷对体外培养的人晶状体上皮细胞(HLEC)氧化损伤的影响.方法 实验研究.应用H2O2诱导HLEC建立氧化损伤模型,观察红景天苷对其产生的作用.对数生长期的HLEC分别加入不同浓度(0、10、30、50、100、200 μmol/L)的红景天苷干预24 h后,运用细胞计数试剂盒法检测各组HLEC增殖活力.实验分为5组:空白对照组、H2O2损伤组、红景天苷低剂量组(30 μmol/L红景天苷+H2O2组)、红景天苷中剂量组(50 μmol/L红景大苷+H2O2组)、红景天苷高剂量组(100 μmol/L红景天苷+H2O2组).采用Hoechst33258染色和流式细胞仪检测细胞凋亡;RT-PCR法检测细胞内B细胞淋巴瘤-2相关x蛋白(Bax)、B细胞淋巴瘤/白血病-2基因(Bcl-2)及半胱氨酸蛋白酶3(Caspase-3)基因的表达.单因素方差分析用于组间均数的多重比较,进一步组间两两比较采用LSD-t检验.结果 细胞计数试剂盒法结果显示,当H2O2的浓度为200 μmol/L时,HLEC的生存抑制率为49.56％±7.07％,接近于细胞生存的半数抑制率(IC50).因此,选择200 μmol/L作为H2O2后续实验浓度.不同浓度红景天甘对细胞活性均无抑制作用,红景天苷(10 μmol/L、30 μmol/L、50 μmol/L、100 μmol/L、200 μmol/L)干预细胞24 h后,细胞存活率分别为100.24％±2.07％、101.18％±2.14％、101.32％±2.48％、101.76％±1.93％、99.28％±1.74％,与空白对照组99.84％ ±2.21％相比,差异均无统计学意义(组间比较F=1.044,P=0.415;两两比较P＞0.05).Hoechst 33258染色可见H2O2损伤组有较多HLEC胞核呈致密浓染(凋亡小体),红景天苷干预组HLEC胞核浓染状况明显减轻.流式细胞仪结果显示空白对照组HLEC凋亡率为2.26％±0.29％,H2O2损伤组HLEC凋亡率为44.56％±4.28％,经30、50、100μmol/L红景天苷处理后,HLEC凋亡率分别降为31.52％±3.05％、24.06％±4.25％和17.16％±2.75％,5个组间细胞凋亡率的差异具有统计学意义,且红景天苷浓度越高,HLEC凋亡率越低(F=117.082,P＜0.01,两两比较P值均＜ 0.01).与空白对照组相比,H2O2损伤组Bax和Caspase-3的表达明显增高,Bcl-2的表达明显降低,差异有统计学意义(P值均＜ 0.01).与H2O2损伤组相比,低、中、高剂量红景天苷组Bcl-2基因表达均上调,Bax和Caspase-3基因表达均下调,其中,高剂量组Bcl-2上调及Bax、Caspase-3下调的更为明显.各组之间差异有统计学意义(Bax:F=493.554,P＜0.01; Bcl-2:F=827.820,P＜0.01;Caspase-3:F =537.237,P＜0.01).结论 红景天苷对发生氧化损伤的HLEC具有保护作用,能够抑制HLEC凋亡.%Objective The aim of the experiment was to investigate the effects of salidroside (Sal) on oxidative damage to human lens epithelial cells (HLEC).Methods Experimental study.The cultured HLECwas intervened with hydrogen peroxide (H2O2) which created oxidative damage model to observe the effect of Sal on HLECs.The cultured cells during the logarithmic phase were interposed by different concentrations Sal(0 μmol/L,10 μmol/L,30 μmol/L,50 μmol/L,100 μmol/L,200 μmol/L)for 24 h.Then the viability of cells was detected by cell counting Kit-8 (CCK-8) assay.The cells were divided into 5 groups:control group,H2O2 group,Sal low dose group (30 μmol/L Sal + H2O2 group),Sal middle dose group (50 μmol/L Sal + H2O2 group),Sal high dose group (100 μmol/L Sal + H2O2 group).The effects of Sal on the apoptosis of the HLEC were determined by Hoechst 33258 staining and flow cytometry assay.Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect B cell lymphoma-2 associated X protein (Bax),B-cell lymphoma-2 (Bcl-2) and Cysteinyl aspartate specific proteinase 3 (Caspase-3) expression.Data between groups were analyzed using one-way analysis of variance (ANOVA),while LSD-t test was used for further comparison between every two groups.Results CCK-8 result showed that when the concentration of H2O2 was 200 μmol/L,the survival of HLEC inhibition rate was 49.56％ ± 7.07％,which was close to the half of the cell survival inhibition rate (IC50).So 200 μmol/L was chosen as the concentration of H2O2 in follow-up experiments.Different concentrations of Sal had no inhibitive influence on HLEC viability.After 24 hours cultivated with Sal (10 μmol/L,30 μmol/L,50 μmol/L,100μmol/L,200 μmol/L),the survival rate of HLEC were 100.24％ ± 2.07％ 、101.18％ ± 2.14％,101.32％ ± 2.48％,101.76％ ± 1.93％ and 99.28％ ± 1.74％ correspondingly.There was no significant difference comparing with that of the control group 99.84％ ± 2.21％ (F =1.044,P =0.415 ; all P ＞ 0.05).Hoechst 33258 staining showed that the chromatin of H2O2 group aggregated and concentrated obviously.And Sal could reduce the aggregation of chromatin of HLEC obviously.FCM results indicated that the apoptosis rate of HLEC was 2.26％ ±0.29％ in control group and 44.56％ ±4.28％ in H2O2 group.After interposal with Sal (30 μmol/L,50 μmol/L,100 μmoL/L),the apoptosis rate of HLEC reduced to 31.52％ ± 3.05％,24.06％ ±4.25％ and 17.16％ ± 2.75％.The differences of apoptosis rates had statistical significance between the five groups (F =117.082,P ＜ 0.001).The HLEC apoptosis rate decreased with higher Sal concentreations (F =117.082,P ＜0.01).The expression of Bax and Caspase-3 in H2O2 group were higher and the expression of Bcl-2 were lower than that in the control group (P ＜ 0.01).Compared with the control group,the expression of Bcl-2 in three Sal dose groups was higher and the expression of Bax,Caspase-3 was lower,especially the high dose Sal group (Bax:F =493.554,P ＜ 0.01 ; Bcl-2:F =827.820,P ＜ 0.01 ;Caspase-3:F =537.237,P ＜ 0.Ol).Conclusions The Sal takes the protective effect on the oxidative damage to HLEC.It could decrease the apoptosis of HLEC.
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