| Objective:Based on literature mining technology,analyze and summarize the relevant information and biological significance of metabolomics differential metabolites of diabetes,in order to discover key differential metabolites that may have potentially important influence;use molecular biology,network pharmacology Various methods such as science and literature mining analyze the changes of key differential metabolites acting on high glucose-induced rat retinal microvascular endothelial cell damage,explore the mechanism of action of differential metabolites,and provide new targets for the diagnosis and treatment of diabetic retinopathy and Multi-angle clinical monitoring indicators.Method:1.Retrieve the literature related to diabetes metabolomics in recent years,screen and frequency analyze the metabolites collected,select the metabolites with frequency? 2 as the follow-up research objects,and conduct pathway enrichment analysis to obtain The metabolic pathway with the highest proportion,and the key differential metabolites are screened from the pathway.2.Through cell viability experiments,we found that both dihydrosphingosine and sphingosine 1-phosphate can aggravate the damage of rat retinal microvascular endothelial cells induced by high glucose,and after giving a larger dose(concentration of 1 mM)serine,Did not cause significant damage to rat retinal microvascular endothelial cells induced by high glucose;we also conducted an experimental study on the effect of dihydrosphingosine and sphingosine 1-phosphate on the migration ability of microvascular endothelial cells,and found two kinds of metabolism The substance will inhibit the migration of retinal microvascular endothelial cells.The above experiments have shown that dihydrosphingosine and sphingosine 1-phosphate will exacerbate the high glucose-induced damage to the rat retinal microvascular endothelial cell model.3.By detecting the biochemical indicators such as cell SOD and MDA,it was found that the dihydrosphingosine and sphingosine 1-phosphate induced SOD activity value in the rat retinal microvascular endothelial cell high glucose model was significantly down compared with the high glucose group.MDA levels increased significantly,indicating that both can induce oxidative damage to rat retinal microvascular endothelial cells.In order to further verify the oxidative damage of dihydrosphingosine and sphingosine 1-phosphate,cell apoptosis and ROS levels were also measured.In the experiment,the rate of apoptosis stimulated by metabolites increased,and the level of reactive oxygen species also increased significantly,which further confirmed that dihydrosphingosine and sphingosine 1-phosphate did cause high glucose model of rat retinal microvascular endothelial cells.Oxidative damage will also indicate the direction of subsequent mechanism research.4.Utilize network pharmacology technology for "component-target-pathway-disease"interaction,analyze two metabolites of dihydrosphingosine and sphingosine 1-phosphate,and finally obtain two metabolites and diabetic retinopathy Related metabolic pathways,among which the top three are PI3K-AKT signaling pathway,MAPK signaling pathway and AGE-RAGE signaling pathway,combined with KEGG database information,found that AGE-RAGE signaling pathway is a signaling pathway closely related to diabetes complications,In the network pharmacology research,we found that among the top ten pathways enriched in pathways,the JAK-STAT pathway serves as a downstream signal of the AGE-RAGE pathway,which is closely related to the signal transduction of cells,and studies have found that high glucose Inducing human umbilical vein endothelial cell damage is closely related to the JAK2-STAT3 signaling pathway.Based on this,we think whether the two differential metabolites of dihydrosphingosine and sphingosine 1-phosphate can also be activated by the RAGE-JAK2/STAT3 signaling pathway.Induce the damage of rat retinal microvascular endothelial cells,and verify these three proteins by Western Blot.Result:1.Use literature mining technology to screen out the literatures related to metabolomics of diabetes,and further screen out 32 metabolites with metabolite frequency? 2 in metabolomics articles on rats.Enrichment analysis of 32 differential metabolites revealed that 15 metabolites involved in lipid metabolism pathways accounted for the most,accounting for 48.39%;10 differential metabolites involved in sugar metabolism pathways,accounting for 32.26%;participated The differential metabolites of amino acid metabolism pathway accounted for 35.48%.According to MetPA analysis results,sphingolipid metabolism is the first influential metabolic pathway.The metabolites involved in lipid metabolism include sphinganine,serine,sphingosine 1-phosphate,sphinganine,and sphingosine.There are five kinds,among which,the metabolite level of metabolomics research is only sphinganine,serine and sphingosine 1-phosphate,so these three differential metabolites are the research objects of subsequent experiments.2.Through cell viability experiments,we found that both sphinganine and sphingosine 1-phosphate can aggravate the damage of rat retinal microvascular endothelial cells induced by high glucose,and after giving a larger dose(concentration of 1 mM)serine,Did not cause significant damage to rat retinal microvascular endothelial cells induced by high glucose;we also conducted an experimental study on the effect.of sphinganine and sphingosine 1-phosphate on the migration ability of microvascular endothelial cells,and found two kinds of metabolism The substance will inhibit the migration of retinal microvascular endothelial cells.The above experiments have shown that sphinganine and sphingosine 1-phosphate will exacerbate the high glucose-induced damage to the rat retinal microvascular endothelial cell model.3.By detecting the biochemical indicators such as cell SOD and MDA,it was found that the sphinganine and sphingosine 1-phosphate induced SOD activity value in the rat retinal microvascular endothelial cell high glucose model was significantly down compared with the high glucose group.MDA levels increased significantly,indicating that both can induce oxidative damage to rat retinal microvascular endothelial cells.In order to further verify the oxidative damage of sphinganine and sphingosine 1-phosphate,cell apoptosis and ROS levels were also measured.In the experiment,the rate of apoptosis stimulated by metabolites increased,and the level of reactive oxygen species also increased significantly,which further confirmed that sphinganine and sphingosine 1-phosphate did cause high glucose model of rat retinal microvascular endothelial cells.Oxidative damage will also indicate the direction of subsequent mechanism research.4.Utilize network pharmacology technology for "component-disease-pathway"interaction,the analysis of two metabolites of sphinganine and sphingosine 1-phosphate,and finally to obtain the metabolic pathways of the two metabolites and diabetic retinopathy Among them,the strongest correlation is the AGE-RAGE signaling pathway.Combined with the KEGG database information,it is found that the AGE-RAGE signaling pathway is a related signaling pathway related to diabetic complications.In network pharmacology research,we found that the pathway is enriched Among the top ten pathways,the JAK-STAT pathway is used as a downstream signal of the AGE-RAGE pathway,which is closely related to the signal transduction of cells,and studies have found that high glucose-induced damage to human umbilical vein cells and the JAK2-STAT3 signaling pathway Closely related,based on this,we used Western Blot technology to verify the protein expression of the RAGE-JAK2/STAT3 signaling pathway in the rat retinal microvascular endothelial cell model induced by high sucrose induced by two sphingosine metabolites.The results showed that both sphingosine metabolites can up-regulate the expression of RAGE,JAK2 and STAT3 proteins.We believe that the two differential sphingosine metabolites are very likely to induce damage to the retinal microvascular endothelial cells by activating RAGE-JAK2/STAT3 signaling.Conclusion:1.Sphinganine and sphingosine 1-phosphate as two differential metabolites on the sphingolipid metabolism pathway can exacerbate the high glucose-induced injury of rat retinal microvascular endothelial cells,reduce cell viability and inhibit cellular Migration ability,and will cause oxidative damage to retinal endothelial cells.2.Sphinganine and sphingosine 1-phosphate may induce a series of oxidative stress damages such as increased ROS level and apoptosis in rat retinal microvascular endothelial cells by activating RAGE-JAK2/STAT3 signaling pathway.The molecular biological mechanism of the differential metabolites of diabetes and diabetic retinopathy metabolomics provides new targets for the diagnosis and treatment of diabetic retinopathy and provides laboratory data for a more comprehensive and complete clinical testing index. |
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