虾青素对晶状体上皮细胞氧化应激损伤的抑制作用及其机制

Inhibitory effect of astaxanthin on oxidative stress damage in lens epithelial cells and its mechanism

目的研究虾青素对过氧化氢(H_(2)O_(2))诱导晶状体上皮细胞HLEB-3氧化应激损伤的调控作用及其可能的作用机制。方法将HLEB-3细胞于不同浓度H_(2)O_(2)(0、50、100、200、500、750μmol/L)下培养,噻唑蓝(MTT)法检测细胞抑制率,计算半数抑制浓度(IC50)。将HLEB-3细胞使用不同浓度(0、5、10、20、50μmol/L)虾青素培养,MTT法检测细胞存活率。将HLEB-3细胞分为4个组,其中正常对照组用完全培养基培养、氧化应激组于250μmol/L H_(2)O_(2)培养基中培养,10μmol/L虾青素组和20μmol/L虾青素组分别于相应浓度虾青素+250μmol/L H_(2)O_(2)培养基中培养,各组均培养24 h。采用流式细胞仪检测细胞凋亡率;采用ELISA法检测细胞一氧化氮(NO)浓度、超氧化物歧化酶(SOD)活性、还原型谷胱甘肽(GSH)活性和丙二醛(MDA)含量;采用Western bolt法检测细胞核核因子E2相关因子2(Nrf2)、细胞质Nrf2和血红素加氧酶-1(HO-1)、醌氧化还原酶1(NQO1)蛋白表达。另将细胞分为正常对照-小干扰RNA(NC-siRNA)组、Nrf2-siRNA组、NC-siRNA+虾青素组和Nrf2-siRNA+虾青素组,分别转染NC-siRNA或Nrf2-siRNA,转染后24 h分别于含0或10μmol/L虾青素和250μmol/L H_(2)O_(2)培养基中培养24 h。采用流式细胞仪检测细胞凋亡率,采用ELISA法检测细胞NO浓度、SOD活性、GSH活性和MDA含量。结果随着H_(2)O_(2)浓度的增加,HLEB-3细胞抑制率升高,不同浓度H_(2)O_(2)处理细胞的抑制率总体比较差异有统计学意义(F=12.358,P<0.05)。H_(2)O_(2)对HLEB-3细胞的IC50为264.20μmol/L。0、5、10、20、50μmol/L虾青素处理HLEB-3细胞的存活率分别为(100.00±0.00)%,(102.20±1.34)%、(109.50±3.60)%、(115.40±4.13)%和(93.60±2.59)%,后续选取10μmol/L和20μmol/L作为实验剂量。氧化应激组细胞凋亡率为(38.50±2.38)%,高于正常对照组的(9.20±0.24)%,差异有统计学意义(P<0.05);10μmol/L虾青素组细胞凋亡率为(27.60±4.33)%,低于氧化应激组,高于20μmol/L虾青素组的(14.90±1.23)%和正常对照组,差异均有统计学意义(均P<0.05)。氧化应激组细胞中NO浓度、MDA含量高于正常对照组、10μmol/L虾青素组和20μmol/L虾青素组,SOD、GSH活性低于正常对照组、10μmol/L虾青素组和20μmol/L虾青素组,差异均有统计学意义(均P<0.05);10μmol/L虾青素组NO浓度、MDA含量高于20μmol/L虾青素组和正常对照组,SOD、GSH活性低于20μmol/L虾青素组和正常对照组,差异均有统计学意义(均P<0.05)。各组间细胞核Nrf2、细胞质Nrf2、HO-1和NQO1蛋白相对表达量总体比较,差异均有统计学意义(F=43.512、20.381、31.014、23.435,均P<0.001);正常对照组、氧化应激组、10μmol/L虾青素组和20μmol/L虾青素组细胞质Nrf2蛋白相对表达量逐渐下降,细胞核Nrf2、HO-1和NQO1蛋白相对表达量逐渐升高,组间两两比较,差异均有统计学意义(均P<0.05)。Nrf2-siRNA组和Nrf2-siRNA+虾青素组细胞凋亡率高于NC-siRNA组和NC-siRNA+虾青素组,NC-siRNA组细胞凋亡率高于NC-siRNA+虾青素组,差异均有统计学意义(均P<0.05);Nrf2-siRNA+虾青素组和Nrf2-siRNA组细胞凋亡率差异无统计学意义(P>0.05)。Nrf2-siRNA组细胞NO浓度、MDA含量高于NC-siRNA组,SOD、GSH活性低于NC-siRNA组,差异均有统计学意义(均P<0.05);NC-siRNA+虾青素组NO浓度、MDA含量低于NC-siRNA组和Nrf2-siRNA+虾青素组,SOD、GSH活性高于NC-siRNA组和Nrf2-siRNA+虾青素组,差异均有统计学意义(均P<0.05);Nrf2-siRNA+虾青素组细胞NO浓度、SOD和GSH活性及MDA含量与Nrf2-siRNA组比较,差异均无统计学意义(均P>0.05)。结论虾青素能提高晶状体上皮细胞抗H_(2)O_(2)诱导的氧化应激损伤能力,其作用可能是通过活化Nrf2相关信号通路而实现的。

Objective To investigate the regulatory effect of astaxanthin on oxidative stress injury induced by hydrogen peroxide(H_(2)O_(2))in lens epithelial cells and its possible mechanism.Methods The HLEB-3 cells were cultured with different concentrations(0,50,100,200,500,750μmol/L)of H_(2)O_(2).The cell inhibition rate was detected by the methyl thiazolyl tetrazolium(MTT)method,and the 50%inhibiting concentration(IC50)was calculated.HLEB-3 cells were cultured with different concentrations(0,5,10,20,50μmol/L)of astaxanthin.The cell survival rate was detected by the MTT method.HLEB-3 cells were divided into four groups for 24-hour culture,namely normal control group cultured with complete medium,oxidative stress group cultured with 250μmol/L H_(2)O_(2),10μmol/L astaxanthin group cultured with 10μmol/L astaxanthin and 250μmol/L H_(2)O_(2),and 20μmol/L astaxanthin group cultured with 20μmol/L astaxanthin and 250μmol/L H_(2)O_(2).The cell apoptosis rate was determined by flow cytometry.The nitric oxide(NO)concentration,superoxide dismutase(SOD)activity,glutathione(GSH)activity and malondialdehyde(MDA)content were detected by ELISA.The protein expressions of nuclear factor erythroid-2 related factor 2(Nrf2)in nuclei,cytoplasmic Nrf2,heme oxygenase-1(HO-1)and NAD(P)H,quinine oxidoreductase 1(NQO1)were detected by Western bolt.The cells were divided into four groups,namely normal control-small interfering RNA(NC-siRNA)group,Nrf2-siRNA group,NC-siRNA+astaxanthin group and Nrf2-siRNA+astaxanthin group.The cells were transfected with NC-siRNA or Nrf2-siRNA accordingly.The cells were co-cultured for 24 hours with 0/10μmol/L astaxanthin and 250μmol/L H_(2)O_(2)24 hours after transfection,respectively.The cell apoptosis rate was determined by flow cytometry.The NO concentration,SOD activity,GSH activity and MDA content were detected by ELISA.Results With the increase of H_(2)O_(2)concentration,the inhibition rate of HLEB-3 cells increased.There were significant differences in the inhibition rate of HLEB-3 cells treated with different concentrations of H 2O 2(F=12.358,P<0.05).The IC50 value of H_(2)O_(2)on HLEB-3 cells was 264.20μmol/L.The survival rates of HLEB-3 cells treated with 0,5,10,20 and 50μmol/L astaxanthin were(100.00±0.00)%,(102.20±1.34)%,(109.50±3.60)%,(115.40±4.13)%,(93.60±2.59)%,respectively.Then 10μmol/L and 20μmol/L were chosen as the experimental dose.The cell apoptosis rate of oxidative stress group was(38.50±2.38)%,which was higher than(9.20±0.24)%of normal control group,with a statistically significant difference(P<0.05).The cell apoptosis rate of 10μmol/L astaxanthin group was(27.60±4.33)%,which was lower than(38.50±2.38)%of oxidative stress group,but higher than(14.90±1.23)%of 20μmol/L astaxanthin group and(9.20±0.24)%of normal control group,showing statistically significant differences(all at P<0.05).The NO and MDA contents were higher and the SOD and GSH concentrations were lower in oxidative stress group than in normal control group,10μmol/L astaxanthin group and 20μmol/L astaxanthin group,and the differences were statistically significant(all at P<0.05).The NO and MDA contents were higher and the SOD and GSH concentrations were lower in 10μmol/L astaxanthin group than in normal control group and 20μmol/L astaxanthin groups,and the differences were statistically significant(all at P<0.05).There were significant differences in the relative expression levels of nuclear Nrf2,cytoplasmic Nrf2,HO-1 and NQO1 proteins among normal control group,oxidative stress group,10μmol/L astaxanthin group and 20μmol/L astaxanthin group(F=43.512,20.381,31.014,23.435;all at P<0.001).The relative expression of nuclear Nrf2 protein gradually decreased,and the relative expression of nuclear Nrf2,HO-1 and NQO1 proteins increased gradually in normal control group,oxidative stress group,10μmol/L astaxanthin group and 20μmol/L astaxanthin group,and there were significant differences when compared in pairs(all at P<0.05).The apoptosis rates of Nrf2-siRNA group and Nrf2-siRNA+astaxanthin group were higher than those of NC-siRNA group and NC-siRNA+astaxanthin group,and the differences were statistically significant(all at P<0.05).The cell apoptosis rate was higher in NC-siRNA group than in NC-siRNA+astaxanthin group,showing a statistically significant difference(P<0.05).There was no significant difference in the apoptosis rate between Nrf2-siRNA+astaxanthin group and Nrf2-siRNA group(P>0.05).The NO and MDA concentrations were higher and the SOD and GSH activities were lower in Nrf2-siRNA group than in the NC-siRNA group,with statistically significant differences(all at P<0.05).The NO and MDA concentrations were lower and the SOD and GSH activities were higher in NC-siRNA+astaxanthin group than in NC-siRNA group and Nrf2-siRNA+astaxanthin group,and the differences were statistically significant(all at P<0.05).There was no significant difference in NO and MDA concentrations or the SOD and GSH activities between Nrf2-siRNA+astaxanthin group and Nrf2-siRNA group(all at P>0.05).Conclusions Astaxanthin enhances the resistance of lens epithelial cells to H_(2)O_(2)-induced oxidative stress damage,which may be achieved by activating the Nrf2-related signaling pathway.