| Objective: Diabetic Retinopathy(DR)is one of the most common complications of diabetes.Inflammation and oxidative stress are important approaches to the early onset of diabetic retinopathy.Retinal vascular endothelial cells are important targets of hyperglycemia in the early stage of the disease.With the help of transcriptional sequencing technology,the genes closely related to inflammation and oxidative stress in the retinal vascular endothelial cells of Macaca rhesus are screened and verified,thus providing a molecular basis for the early prevention and early treatment of DR in clinical work..Methods: 25mmol/L high glucose was used to stimulate RF/6A cells to establish high glucose model of vascular endothelial cells.The expression of IL-6,TNF-alpha and MCP-1 in RF/6A under high glucose model was measured by ELISA and qRT-PCR.The level of nitric oxide was detected in the cells under high glucose model.ROS+ER double staining and flow cytometry were used to observe ROS production under high glucose stimulation.The expression level of ROS in RF/6A cells,so as to identify the successful establishment of high glucose model.On this basis,25mmol/L high glucose RF/6A cells were detected by RNA-Seq technology,and the normal cultured RF/6A cells were used as the control,through the gene ontology analysis,we found the differential genes related to the early inflammation and oxidative stress process of diabetic retinopathy.The genes differential expression was verified by qRT-PCR Results: the results of ELISA showed that the expression of IL-6 in the high glucose stimulation group(137.7 + 18.18pg/ml)was significantly higher than that of the normal group(51.89 + 2.33pg/ml),and the difference was statistically significant(P<0.01),and the expression of TNF-a(97.25 + 6.17pg/ml)in the high glucose stimulation group was significantly higher than that in the normal group(56.52 + 4.25pg/ml),and the difference was significantly higher than that in the normal group.The difference was statistically significant(P<0.05);the expression of MCP-1 in the high glucose stimulation group(164.60 + 25.89pg/ml)was significantly higher than that in the normal group(81.73 + 6.66pg/ml),and the difference was statistically significant(P<0.01).RT-PCR results showed that compared with the normal control group,the expression level of IL-6 and TNF-alpha mRNA increased in varying degrees,but there was no significant difference in MCP-1 mRNA expression between high glucose group and normal control group.The results of nitrate reductase method: the activity of NO in the high glucose cell group was 0.071 + 0.015 umol/h/mg,which was significantly higher than that of the normal group(0.017 + 0.011)(P<0.05).Flow cytometry was used to detect the level of ROS expression in RF/6A cells stimulated by high glucose: compared with the normal cell group(33.8 + 5.4%),the output of ROS increased in the high glucose group(68.7 + 6.3%),and the two had statistical difference(P<0.01).Through RNA-seq technology,we found 449 differential genes,including 297 up regulated genes and 152 down regulated genes.These differentially expressed genes are involved in the formation and regulation of multicellular organisms.Using blast software to compare genes to KEGG database,we get the pathway annotation information of genes.The results showed that the TGF-beta signaling pathway and complement pathway were more obvious.In addition,many pathways related to amino acid metabolism have also been affected,such as tryptophan,serine,and other metabolic pathways.The most significant genes were CTSW,TGM5 and TXNIP.The genes related to inflammation include FOXP3,LTF,GGT5,CITED1,LOC704750,IL23 A and CARD9.The genes related to oxidative stress include TXNIP,DHCR24,PDILT,PDIA2 and PPARGC1 B.Through the validation of RT-PCR,we screened 3 genes related to inflammation and oxidative stress as research objects.DHCR24 regulates cell physiological changes through oxidative stress mechanism.IL-23 A and CARD9 regulate the early RF/6A lesions induced by high glucose through inflammatory mechanism.Conclusion: high glucose stimulation can promote early inflammation and oxidative stress in RF/6A.The effect of high glucose on retinal vascular endothelial cells is multi-level and multifaceted.DHCR24,IL-23 A and CARD9 have the opportunity to be the new target for the early treatment of diabetic retinopathy. |