| Objective: To expore the impact of tBHQ on the expression of Nrf2,HO-1,HIF-1α and VEGF in SD rats retina Müller cells which are under high glucose condition,and discuss the protective mechanism of tBHQ on the SD rats retinal Müller cells under high glucose condition.Methods: SD rat retinal Müller cells are cultured in vitro.CCK-8 experiments screening high glucose concentration and tBHQ intervention concentration in high glucose environment;The experiments are divided into normal control group,high glucose group,high glucose + tBHQ group,and the apoptosis of each group is detected by flow cytometry.Western Blot is used to detect the expression of Nrf2,HO-1,HIF-1α and VEGF in each group.The mRNA expression of Nrf2,HO-1,HIF-1α and VEGF in each group is detected by q RT-PCR.Then,the results are analyzed statistically.Results: 1.Through the screening of CCK-8 experiment,the high glucose concentration is 45mmol/L and the tBHQ intervention concentration is 20umol/L.So the experimental groups are divided into three groups: normal control group(5.5mmol/L DMEM),high glucose group(45mmol/L DMEM),high glucose + t BHQ group(45mmol/L DMEM + 20μmol/L tBHQ).2.The result of flow cytometry showed that the apoptotic rate(%)in control group was 3.95 ± 0.68,in high glucose group was 39.77 ± 1.76,and in high glucose + t BHQ group was 20.84 ± 0.23.Compared with the normal control group,apoptosis rate of the high glucose group increased significantly(P=0.000,P<0.05),The apoptosis rate in high glucose + tBHQ group was significantly lower than that in high glucose group(P=0.000,P<0.05),but significantly higher than that in normal control group(P=0.000,P<0.05),and the difference was statistically significant.3.The results of the protein expression of Nrf2,HO-1,HIF-1alpha and VEGF which were detected by Western Blot in each group were: The relative expression of Nrf2 was 0.06 ± 0.01 in normal control group,0.17 ± 0.02 in high glucose group and 0.4 ±0.06 in high glucose + tBHQ group.The expression of Nrf2 protein in high glucose group was significantly higher than that in normal control group(P=0.006,P<0.05),high glucose+ tBHQ group expressed further(P=0.000,P<0.05),and the difference was statistically significant.The relative expression of HO-1 was 0.19 ± 0.03 in normal control group,0.47 ± 0.02 in high glucose group and 0.72 ± 0.05 in high glucose + tBHQ group.The expression of HO-1 protein in high glucose group was significantly higher than that in normal control group(P=0.000,P<0.05),high glucose+ tBHQ group expressed further(P=0.000,P<0.05),and the difference was statistically significant.The relative expression of HIF-1α was 0.06 ± 0 in normal control group,0.67 ± 0.07 in high glucose group,0.18 ± 0.04 in high glucose + tBHQ group;The relative expression of HIF-1α in high glucose group was significantly higher than that in normal control group(P=0.000,P<0.05),and the expression in high glucose + tBHQ group was significantly lower than that in the high glucose group(P=0.000,P<0.05),but significantly higher than the normal control group(P=0.017,P<0.05),the difference was statistically significant.The relative expression of VEGF was 0.07 ± 0.02 in normal control group,0.6 ± 0.05 in high glucose group and 0.26 ± 0.07 in high glucose + tBHQ group.The relative expression of VEGF in high glucose group was significantly higher than that in normal control group(P=0.000,P<0.05),and the expression in high glucose + tBHQ group was significantly lower than that in the high glucose group(P=0.000,P<0.05),but significantly higher than the normal control group(P=0.003,P<0.05),the difference was statistically significant.4.The results of mRNA expression of Nrf2,HO-1,HIF-1α and VEGF which were detected by qRT-PCR in each group were: The mRNA relative expression of Nrf2 was 1.07 ± 0.07 in normal control group,1.53 ± 0.06 in high glucose group and 2.68± 0.09 in high glucose + tBHQ group;The mRNA relative expression of Nrf2 in high glucose group was significantly higher than that in the normal control group(P=0.000,P<0.05),which was further increased in high glucose + tBHQ group(P=0.000,P<0.05),the difference was statistically significant.The mRNA relative expression of HO-1 was 0.95 ± 0.05 in normal control group,1.5 ± 0.04 in high glucose group and 2.94 ± 0.05 in high glucose + tBHQ group;The mRNA relative expression of HO-1 in high glucose group was significantly higher than that in the normal control group(P=0.000,P<0.05),which was further increased in high glucose + tBHQ group(P=0.000,P<0.05),the difference was statistically significant.The mRNA relative expression of HIF-1α in normal control group was 0.99 ± 0.02,in high glucose group was 2.56 ± 0.09,in high glucose + t BHQ group was 1.48 ± 0.05.The mRNA relative expression of HIF-1α in high glucose group was significantly higher than that in normal control group(P=0.000,P<0.05),while the expression of HIF-1α in high glucose + t BHQ group was significantly lower than that in high glucose group(P=0.000,P<0.05),but significantly higher than that in the normal control group(P=0.000,P<0.05),the difference was statistically significant.The mRNA relative expression of VEGF in normal control group was 1.09 ± 0.08,in high glucose group was 3.04 ± 0.19 and in high glucose + t BHQ group was 1.6 ± 0.08.The mRNA relative expression of VEGF in high glucose group was significantly higher than that in normal control group(P=0.000,P<0.05),while the expression of VEGF in high glucose + t BHQ group was significantly lower than that in high glucose group(P=0.000,P<0.05),but significantly higher than that in the normal control group(P=0.003,P<0.05),the difference was statistically significant.Conclusion: SD rat Müller cells activate oxidative stress response and increase the rate of apoptosis and the production of VEGF and HIF-1α in high glucose environment.TBHQ upregulates Nrf2 and HO-1 by activating endogenous antioxidant Nrf2 / ARE signaling pathway to inhibit the expression of VEGF and HIF-1α and reduce the rate of apoptosis in order to achieve the protective effect on Müller cells;the anti-oxidative effect of tBHQ is expected to become a new treatment ideas for DR. |
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