The Protective Effect Of Beta-Casomorphin-7 Via Promoting Foxo1 Activity And Nuclear Translocation In Human Lens Epithelial Cells

ObjectiveTo investigate the protective effect of beta-casomorphin-7(?-CM-7)in o-xidative stressed human lens epithelial cells(HLECs)and to explore the possible mechanism for oxidative stress in human lens epithelial cells induced by high glucose.Methods1.we use MTT assay to determine the effect of different concentrations of glucose in HLECs.The most statistically significant glucose concentration was screened out from the normal glucose concentration as the high glucose group(We abbreviate mmol/L as m M).The effects of different concentrations of?-CM-7 on the activity of HLECs were determined by MTT assay and the most statistically significant concentration group with high glucose group was screened as ?-CM-7 group.The effects of 4 concentrations of 5m M,10 m M,20 m M and 40 m M glucose on the expression of Foxo1 and Sp1 in HLECs were detected by Western blot.2.According to different treatment methods,the cells were divided into 5groups: normal control group(5m M control),high glucose group(40m M HG),?-CM-7 group(10-5mol/L,10-6mol/L,10-7mol /L).We measured oxidative stress-induced reactive oxygen species(ROS)by flow cytometry;we used bioassay kit to assay the content of oxidized malondialdehyde(MDA)and antioxidant enzyme superoxide dismutase(SOD);we used immunoblot andImmunofluorescence assay to determine the molecular biology of Foxo1,SP1 and the downstream related protein GSH-px,as well as the localization of Foxo1 and Sp1 in HLECs.Results1.When cells were treated with different concentrations of glucose,the viability of HLECs decreased with the increase of glucose concentration(p<0.01).Compared with the normal glucose concentration,the viability of HLECs decreased with the increase of glucose concentration when the glucose concentration was 40 m M(P<0.01);At 50 m M,cells irreversibly die;Western blot results showed that the expression of Foxo1 and SP1 in HLECs decreased(p<0.01)when the glucose concentration increased from 5m M to 40 m M;2.Compared with the normal control group,when cells were treated with high glucose(40m M)after pretreatment with ?-CM-7(10-5mol / L,10-6mol /L,10-7mol / L),the viability of HLECs increased(P <0.01).Compared with high glucose group,ROS level in ?-CM-7 group was lower(p <0.01),MDA content was lower(p<0.01)and SOD expression was higher(p <0.01)Compared with high glucose group,the expression of total Foxo1 in ?-CM-7 group(10-5mol / L)increased(p<0.01),Foxo1 transferred to nucleus and increased in nucleus(p<0.01),The expression of Foxo1 in the cytoplasm decreased(p<0.01),the expression of GSH-px increased(p<0.01).It was observed by immunofluorescence that Foxo1 and Sp1 expression in the high glucose group was very weak compared to the normal control group.After pretreatment with?-CM-7(10-5mol/L),the total amount of Foxo1 expression was higher than high glucose group and transferred into the nucleus.The expression of Sp1 in the nucleus became stronger with the transfer of Foxo1 into the nucleus.Conclusions1.High concentration of glucose can reduce the activity of HLECs,increase the content of oxidase,decrease the content of antioxidant enzyme anddecrease the relative expression of Foxo1 and Sp1.2.?-CM-7 protects HLECs against oxidative damage by up-regulating the relative expression of Foxo1 and promoting Foxo1 nuclear translocation.