| Objective:In order to simulate the mechanism of diabetic cataract,human lens epithelial cells(HLE-B3)were cultured in vitro with appropriate concentration of glucose to induce apoptosis and oxidative stress.To explore effect of baicalin to the apoptosis and oxidative stress of human lens epithelial cells(HLE-B3)induced by high glucose and whether the effect cat through the signal pathways Keap1-Nrf2-ARE.To provide a new idea for delaying the occurrence and development of diabetic cataract.Methods:1.Different concentrations of glucose(25 mmol/L,50mmol/L,100mmol/L,200mmol/L)were used to treat HLE-B3 which were cultured in vitro for different times(0h,24 h,48h).The cell proliferation of each group was detected by cell counting kit-8(CCK8)method,so as to determine the optimal time and concentration of high glucose.2.Different concentrations of baicalin(0μmol/L、5μmol/L、10μmol/L、25μmol/L、50μmol/L、100 μmol/L)were used to treat HLE-B3 which were cultured in vitro for different times(0h,24 h,48h).The cell proliferation of each group was detected by cell counting kit-8(CCK8)method,so as to determine the optimal time and concentration of baicalin.3.Human lens epithelial cells(HLE-B3)cultured in vitro were randomly divided into 3 groups including control group,high glucose group(200mmol/L glucose)and baicalin group(5μmol/LBai+200mmol/L glucose).CCK8 was used to detect cell proliferation in each group to determine the optimal concentration of baicalin.3.Divided HLE-B3 into three groups randomly including control group,high glucose group(200mmol/L glucose)and baicalin group(5μmol/L Bai+200mmol/L glucose).CCK8 and Flow cytometry was used to detect the proliferation and apoptosis rate of every group.RT-PCR was used to detect the expression of mRNA of Keap-1、Nrf2、NQO1、HO-1、Bax、Bcl-2.Western-blot was used to detect the expression of protein of Keap-1、Nrf2、NQO1、HO-1、Bax、Bcl-2.Also the expression of SOD、MDA、GSH-Px were detected.Statistical Analysis:SPSS 22.0 was used for statistical analysis,and all measurement data were expressed as mean ± standard deviation.One-way analysis of variance(ANOVA)was used for comparison between the means of multiple groups of samples,and LSD-t test was used for pair comparison between groups.P<0.05 indicated statistically significant difference.Results:1.According to the results of CCK8,200mmol/L glucose was selected as the optimum concentration and time for inducing the damage of apoptosis and oxidative stress of HLE-B3 as well as 5μmol/L Bai was selected as the optimum concentration of HLE-B3.2.The proliferation of high glucose group detected by CCK8 was lower than control group and the baicalin group(5μmol/L)was higher than the high glucose group(P<0.05).3.Flow cytometry showed that apoptosis rate of high glucose group was higher than control group and the baicalin group(5μmol/L)was lower than the high glucose group(P<0.05).4.RT-PCR showed that the expression of mRNA of Nrf2、NQO1、HO-1and Bcl-2 of high glucose group were lower than control group while Keap1、Bax were lower compared to high glucose group.and the expression of mRNA of Nrf2、NQO1、HO-1and Bcl-2 in baicalin group was higher than the high glucose group while Keap1、Bax were higher(P<0.05).5.Western-blot showed the expression of protein in high glucose group of Keap-1、Bax were higher than control group and Nrf2、NQO1、HO-1、Bcl-2were lower than control group,and about the high glucose group,the results were reverse(P<0.05).6.The expression of SOD、GSH-Px were lower in high glucose group compared to control group(P<0.05),and baicalin group is higher than high glucose group(P<0.05).The expression of MDA was higher in high glucose group than control group,and baicalin group is lower than high glucose group(P<0.05).Conclusion:1.The apoptosis and oxidative stress of human lens epithelial cells(HLE-B3)induced by high glucose,and low concentration of baicalin can alleviate the injury2.The protective effect of baicalin may be related to the regulation the signal pathways Keap1-Nrf2-ARE. |
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