| Objective: By culturing human retinal pigment epithelium(ARPE-19)cells in vitro,a cellular hyperglucose model was established,and whether high sugar affected retinal pigment epithelium(RPE)cells through oxygen-sensing pathways was explored,and the effect of Danhuang Mingmu decoction-containing serum on RPE cells damaged by high glucose through oxygen-sensing pathway was explored.Methods:1.Preparation of medicinal serum: according to the random method,40 male SD rats were divided into blank group and Danhuang Mingmu decoction group: Danhuang Mingmu decoction group used distilled water to configure Danhuang Mingmu decoction liquid and then gavage.Blank group gavage with equal amounts of distilled water.Serum was extracted 7 days after irrigation.2.Set different concentrations of Danhuang Mingmu decoction containing medicinal serum: 1/50,1/30,1/20,1/10,1/4,1/2,1/1,0(for complete medium).After 24 hours of incubation of different drugs,the maximum non-toxic concentration(TC0)of RPE cells cultured with drug-containing serum was screened for Danhuang Mingmu decoction.3.Intervention study of Danhuang Mingmu decoction on RPE cells damaged by high glucose: the cells were divided into 4 groups,namely:blank group,high-glucose model group,HIF inhibitor group,and Danhuang Mingmu decoction group.After 24 hours of drug action,the cell morphology of each group was observed under a microscope,and the viability of each group of cells was detected by CCK-8 method.4.Flow cytometry to detect the apoptosis rate and reactive oxygen radical(ROS)fluorescence intensity of each group of cells;ELISA method detected intracellular prolyl hydroxylase(PHD3),M2 pyruvate kinase(PKM2)and lactic acid concentration,RT-q PCR gene expressionlevel,WB(Western Blotting)to detect ERK2,Raf1,HIF2α,NDRG3 protein expression.Results:1.The TC0 of the medicinal serum of Danhuang Mingmu decoction was detected by CCK-8 method as 2%.2.The cell morphology of each group after 48 h of culture was obversed by optical inverted light microscope: the cell morphology of the blank group was normal and grew well;Compared with the blank group,the cells in the high-glucose model group had poor growth status,irregular morphology,and cells showed cells such as shiny cells and round contraction.Compared with the high-glucose model group,the HIF inhibitor group and the Danhuang Mingmu Tang group had a significant improvement in the higher cell morphology of the sugar model group.3.The cell viability of each group was detected by CCK-8 method:(100±0.000)%,(40.83±0.52)%,(67.28±0.81)%,(73.19±0.73)%(P<0.01)in blank group,high glucose model group,HIF inhibitor group and Danhuang Mingmu Tang group,respectively,which were statistically significant.Compared with the blank group,cell viability was significantly reduced in the high-glucose model group(P<0.01).Compared with the high-glucose model group,cell viability was improved in the HIF inhibitor group and Danhuang Mingmu Tang group(P<0.01).4.Flow cytometry was used to detect apoptosis rate: compared with the blank group,the early apoptosis rate,late cell apoptosis rate and total apoptosis rate of high glucose model group were significantly increased(P<0.01).Compared with the high-glucose model group,the early,late and total apoptosis rates were significantly reduced in the HIF inhibitor group and the Danhuang Mingmu decoction group(P<0.01).Compared with the HIF inhibitor group,the early,late and total apoptosis rates were significantly reduced in the Danhuang Mingmu decoction group(P<0.01).5.Flow cytometry was used to detect fluorescence intensity: compared with the blank group,the fluorescence intensity of ROS in cells in the high-glucose model group was significantly increased(P<0.01);Compared with the high-glucose model group,the intracellular ROS fluorescence intensity was significantly reduced in the HIF inhibitor group and Danhuang Mingmu decoction group(P<0.01).Compared with the HIF inhibitor group,the intracellular ROS fluorescence intensity was significantly reduced in the Danhuang Mingmu decoction group(P<0.01).6.PHD3,PKM2 and lactic acid concentrations were detected by ELISA:compared with the blank group,the intracellular PKM2 and lactate concentrations in the high-glucose model group increased significantly(P<0.01)and PHD3 concentrations decreased significantly(P<0.01).Compared with the high-glucose model group,the concentrations of intracellular PKM2 and lactic acid in the HIF inhibitor group and Danhuang Mingmu decoction group decreased significantly(P<0.01),and PHD3 concentration increased significantly(P<0.01).Compared with the HIF inhibitor group,the concentration of PKM2 and lactic acid in the cells was high(P<0.01)and the concentration of PHD3 was low(P<0.01)in the Danhuang Mingmu decoction group.7.PCR detected ERK2,Raf1,HIF2α and NDRG3 gene expression levels:Compared with the blank group,the expression levels of ERK2,Raf1,HIF2α and NDRG3 genes were significantly increased in the high-glucose model group,which was statistically significant(P<0.01).The expression of Raf1 and NDRG3 genes in the HIF inhibitor group and the Danhuang Mingmu decoction group were significantly lower than those in the high-glucose model group(P<0.01).Compared with Danhuang Mingmu decoction group,ERK2 and HIF2α gene expression in Danhuang Mingmu decoction group was slightly higher than that in HIF inhibitor group,which was statistically significant(P<0.01),while the expression of Raf1 and NDRG3 genes in Danhuang Mingmu decoction group was slightly lower than that in HIF inhibitor group(P<0.01).8.WB detected the expression levels of ERK2,Raf1,HIF2α and NDRG3proteins: compared with the blank group,the expression levels of ERK2,HIF2α and NDRG3 proteins in the high-glucose model group were significantly increased and had significant statistical significance(P<0.01),and the expression of Raf1 protein in the HIF inhibitor group and Danhuang Mingmu Tang group were significantly higher than those in the high-glucose model group(P<0.01).Compared with the Danhuang Mingmu Tang group,the expression of ERK2 and HIF2αproteins in the Danhuang Mingmu Tang group was slightly higher than that in the HIF inhibitor group(P<0.01),while the expression of Raf1 and NDRG3 proteins in the Danhuang Mingmu Tang group was lower than that in the HIF inhibitor group(P<0.01).Conclusion:1.In a high sugar environment,Danhuang Mingmu decoction can inhibit the expression of ROS in RPE cells and inhibit the level of reactive oxygen species in cells.2.Danhuang Mingmu decoction has antioxidant damage protection effect on RPE cells.3.Danhuang Mingmu soup contains medicinal serum by improving the hypoxia state,inhibiting the secretion and accumulation of hypoxia factors and inflammatory factors. |
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