| Part I Gene Biomarkers Related to Th17 Cells in Macular Edema of Diabetic Retinopathy: Cutting-Edge Comprehensive Bioinformatics Analysis and In Vivo ValidationBackground: Previous studies have shown that in proliferative diabetic retinopathy(PDR),there is a significant increase in T helper 17(Th17)-related cytokines in the vitreous,suggesting that Th17 cells play an important role in the inflammatory response of diabetic retinopathy(DR).However,there have been no reports on the cellular infiltration and gene-related studies of Th17 cells in DR,particularly in diabetic macular edema(DME)retinas.The primary objective of this study is to comprehensively explore and identify the genes associated with Th17 cells in immune cells from individuals with diabetic retinopathy(DR)accompanied by macular edema.Methods: The dataset GSE160306 was downloaded from the Gene Expression Omnibus(GEO)database,which contains 9 NPDR samples and 10 DME samples.Immu Cell AI algorithm was used to estimate the abundance of Th17 cells in 24 kinds of infiltrating immune cells.The differentially expressed Th17 related genes(DETh17RGs)between NPDR and DME were documented by difference analysis and correlation analysis.Through aggregate analyses such as gene ontology(GO)and Kyoto Encyclopedia of Gene and Genome(KEGG)pathway enrichment analysis,a protein-protein interaction(PPI)network was constructed to analyze the potential function of DETh17 RGs.Cyto Hubba plug-in algorithm,Lasso regression analysis and support vector machine recursive feature elimination(SVM-RFE)were implemented to comprehensively identify Hub DETh17 RGs.The expression archetypes of Hub DETh17 RGs were further verified in several other independent datasets related to DR.The Th17 RG score was defined as the genetic characterization of six Hub DETh17 RGs using the GSVA sample score method,which was used to distinguish early and advanced diabetic nephropathy(DN)as well as normal and diabetic nephropathy.Finally,real-time quantitative PCR(q PCR)was implemented to verify the transcription levels of Hub DETh17 RGs in the STZ-induced DR model mice(C57BL/6J).Results: For the bioinformatics analysis,a total of 238 DETh17 RGs were identified,with 212 genes showing a positive correlation and only 26 genes displaying a negative correlation.Six genes(CD44,CDC42,TIMP1,BMP7,RHOC,FLT1)were identified as Hub DETh17 RGs.Because DR and DN have a strong correlation in clinical practice,the verification of multiple independent datasets related to DR and DN proved that Hub DETh17 RGs can not only distinguish PDR patients from normal people,but also distinguish DN patients from normal people.It can also identify the initial and advanced stages of the two diseases(NPDR vs DME,Early DN vs Advanced DN).In this study,wet assay validation was conducted to assess the expression levels and trends of Hub DETh17 RGs in a STZ-induced DR mouse model using q PCR.The results showed that the transcript levels and trends of these genes were generally consistent with the human transcriptome levels,with the exception of CDC42 and TIMP1.Conclusion: This study uncovered that hub genes,namely CD44,CDC42,TIMP1,BMP7,RHOC,and FLT1,which are closely associated with Th17 cells,likely play a role in the intricate molecular mechanisms responsible for the development and progression of both diabetic retinopathy(DR)and diabetic macular edema(DME).Moreover,through the utilization of bioinformatics analysis,it was suggested that these pivotal genes might also contribute to the perturbing mechanisms involved in ocular and renal complications arising from diabetes.Part II Expression and function study of TIMP-1,a pivotal gene related to Th17 cells,in macular edema of diabetic retinopathyObjective: To quantitatively analyze the expression of TIMP-1 in the vitreous and fibrovascular membranes of proliferative diabetic retinopathy patients who meet the inclusion criteria,investigate the correlation between TIMP-1 expression levels in the vitreous and fibrovascular membranes,as well as the correlation between TIMP-1 and clinical baseline characteristics.Subsequently,further study the changes in TIMP-1protein expression levels and phenotypes in human retinal microvascular endothelial cells(HRMECs)before and after TIMP-1 silencing under high glucose conditions,in order to provide new insights into the pathogenesis of DR microvascular lesions.Methods: For the in vivo experiments,a total of 24 patients with proliferative diabetic retinopathy(PDR)and 16 patients with idiopathic macular epiretinal membrane(ERM)were included in the study.All patients underwent standard pars plana 25 G vitrectomy,and the vitreous and membrane tissues were collected immediately after surgery for examination.Enzyme-linked Immunosorbent Assay(ELISA)and protein immunoblot(Western Blot,WB)were performed on the vitreous and membrane tissues to assess the expression levels of TIMP-1.The correlation between the expression levels of TIMP-1 in the vitreous and membrane tissues was investigated,as well as the correlation between TIMP-1 expression levels in the vitreous and fibrovascular proliferative membranes of the PDR group with the clinical baseline characteristics.The aim was to identify any correlation between TIMP-1expression in the two tissues and to determine the relationship between TIMP-1expression in PDR and clinical characteristics.For the in vitro experiments,the study mapped TIMP-1 expression in human retinal,RPE,and choroidal tissues using singlecell RNA sequencing data.After confirming the expression of TIMP-1 in HRMECs,HRMECs were divided into four groups according to different treatments: normal control group,high glucose group,high glucose + si NC group,and high glucose +si TIMP-1 group.Various assays,including CCK-8,Ed U,Ki-67,Transwell,scratch,and tubuloplasty assays,were performed to assess the impact of the different treatments on the cells.The expression levels of Wnt pathway-related proteins,apoptosis-related proteins,and tubuloplasty-related proteins were also examined.Results: The expression of TIMP-1 was significantly higher in both vitreous and membrane tissues of the PDR group compared to the ERM group.In the ERM group,there was a low positive correlation between TIMP-1 expression in the vitreous and macular pre-membrane,whereas in the PDR group,there was a significant moderate positive correlation between TIMP-1 expression in the vitreous and fibrovascular proliferative membranes.Furthermore,there was a significant moderate positive correlation between TIMP-1 expression in both vitreous and fibrovascular proliferative membranes of the PDR group and serum total cholesterol and triglycerides.In the PDR group,TIMP-1 expression in the vitreous and fibrovascular proliferative membranes was significantly and moderately correlated with serum total cholesterol and triglycerides,and TIMP-1 expression in the vitreous was significantly and positively correlated with body mass index and diastolic blood pressure.In the CCK-8 assay,Ed U assay,Ki-67 assay,Transwell assay,scratch assay,tubuloplasty assay,immunoblotting assay for Wnt pathway-related proteins,Cleaved caspase-3 and Bax in apoptosisrelated proteins,and tubuloplasty-related proteins,the quantitative detection of HRMECs was significantly higher in the high glucose group,high glucose + si NC group,high glucose + si TIMP-1 groups,compared to the control group.The quantitative detection values of HRMECs were not significantly different in the high glucose group and high glucose + si NC group.However,the quantitative detection values of HRMECs were significantly higher in the high glucose + si TIMP-1 group compared to the high glucose and high glucose + si NC groups,respectively.In contrast,the results of immunoblotting experiments for apoptosis-related protein Bcl-2 showed the opposite trend to the quantitative detection values of the above experiments.Conclusion: The mechanism of vitreous-fibrovascular proliferative membrane formation in PDR may involve the molecule TIMP-1,which has been found to positively correlate with serum lipid-related indicators.In a high-glucose state,silencing TIMP-1 may lead to the promotion of HRMECs proliferation,migration,apoptosis,and lumen formation,as well as activation of the Wnt pathway.These findings suggest that targeting TIMP-1 could be a promising therapeutic strategy for the treatment of PDR.Part III CD8+T Cell-Related Gene Biomarkers in Macular Edema of Diabetic RetinopathyBackground: CD8+T lymphocytes have a strong pro-inflammatory effect in all parts of the tissue,and some studies have demonstrated that its concentration in the vitreous increased significantly,suggesting that CD8+T cells play a pivotal role in the inflammatory response of diabetic retinopathy(DR).However,the infiltration of CD8+T cells in the DR retina,especially in diabetic macular edema(DME),and its related genes are still unclear.The primary objective of this study is to explore and identify the genes associated with CD8+ T cells in immune cells from individuals with diabetic retinopathy(DR)accompanied by macular edema.Methods: Download the GSE16036 dataset from the Gene Expression Omnibus(GEO)database.The Immu Cell AI program was performed to evaluate the abundance of 24 immune cells including CD8+T cells.The CD8+T cell-related genes(DECD8+TRGs)between non-proliferative diabetic retinopathy(NPDR)and DME were detected via difference analysis and correlation analysis.Enrichment analysis and protein-protein interaction(PPI)network mapping were implemented to explore the potential function of DECD8+TRGs.Lasso regression,support vector machine recursive feature elimination(SVM-RFE),Cyto Hubba plug-in and MCODE plug-in in Cytoscape software,and Weighted Gene Co-Expression Network Analysis(WGCNA)were performed to comprehensively analyze and obtain Hub DECD8+TRGs.Hub DECD8+TRGs expression patterns were further validated in other two DR-related independent datasets.The CD8+TRG score was defined as the genetic characterization of Hub DECD8+TRGs using the GSVA sample scoring method,which can be administered to distinguish early and advanced diabetic nephropathy(DN)as well as normal and DN.Finally,the transcription level of DECD8+TRGs in DR model mouse were verified by quantitative real-time PCR(q PCR).Results: A total of 371 DECD8+TRGs were identified via bioinformatics analysis,consisting of 294 positively associated genes and only 77 negatively associated genes.Eight genes(IKZF1,PTPRC,ITGB2,ITGAX,TLR7,LYN,CD74,SPI1)were recognized as Hub DECD8+TRGs.DR and DN,which have strong clinical correlation,have been proved to be associated with CD8+T cell-related hub genes by multiple independent data sets.Hub DECD8+TRGs can not only distinguish PDR from normal and DN from normal,but also play a role in the early and progressive stages of the two diseases(NPDR vs DME,Early DN vs Advanced DN).The q PCR transcript levels and trends of Hub DECD8+TRGs in the STZ-induced DR mouse model were validated through wet assays and found to be consistent with the corresponding human transcriptome levels.Conclusion: This study revealed that a set of hub genes,including IKZF1,PTPRC,ITGB2,ITGAX,TLR7,LYN,CD74,and SPI1,which are closely associated with CD8+ T cells,are likely to play a role in the intricate molecular mechanisms underlying the development and progression of diabetic retinopathy(DR)and diabetic macular edema(DME).Furthermore,the application of bioinformatics analysis suggests that these pivotal genes may also be involved in the disruptive mechanisms contributing to ocular and renal complications in diabetes. |
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