Flavonoid Intervention In Diabetic Microangiopathy And Early Screening Markers For Diabetic Retinopathy

Objective:Diabetes mellitus(DM)is a metabolic diseases characterized by chronic hyperglycemia,which easily induces Diabetic microvascular complications(DMVC),among which Diabetic kidney disease(DKD)and Diabetic retinopathy(DR)are the most common and serious.The natural flavonoids such as Scutellarin(SCU)and Luteolin(LTD)have good cardiovascular protective effects.Our previous study found that SCU,LTD and its new derivative LTD2 significantly improved vascular microcirculation lesions in Zucker Diabetic Fatty rats,but the molecular mechanism needs to be further revealed.In this study,db/db mice were used to establish the animal model of DMVC,and the protective effect and molecular mechanism of natural flavonoids SCU/LTD and their derivatives LTD2 on DKD and DR were investigated,providing scientific experimental basis for the clinical application of natural flavonoids SCU/LTD and their derivatives LTD2 in DMVC.In addition,through the study of clinical diabetes patients’ specimens,miRNA biomarkers associated with the early screening of DR were found to improve the accuracy of early screening.Methods:1.The test compounds ameliorate renal lesions in db/db mice and the underlying molecular mechanismsModel building,administration and treatment methods:Normal mice and db/db mouse were fed for 16 weeks and divided into Normal group,Model group,Metformin(Met)group(200 mg/kg/d),SCU group(45 mg/kg/d),LTD group(45mg/kg/d),LTD2 group(45mg/kg/d),Met group(200mg/kg/d)+SCU group(45mg/kg/d),Met group(200mg/kg/d)+ LTD group(45mg/kg/d),Met group(200mg/kg/d)+ LTD2group(45mg/kg/d),The purpose of drugs combination is to observe the synergistic effect and therapeutic effect of drugs.Detection evaluation indicators:1)The changes of Fasting blood glucose(FBG),Urine albumin(UALB),Urine albumin creatinine ratio(UACR)were monitored during administration.The mouse were sacrificed after administration,and serum was tested Fasting insulin,Blood urea nitrogen(BUN),Serum creatine(SCr),Glycosylate hemoglobin type A1C(Hb A1c),Total cholesterol(TC),Triglyceride(TG)and other indexes,and the kidney tissues were tested by histopathology.All of the above were used to evaluate the protective effect of the test compounds on DKD.2)The levels of serum cytokines Interleukin-6(IL-6),Tumor necrosis factor-α(TNF-α)and Vascular endothelial growth factor(VEGF)were detected by Bio-Plex suspension chip technology;Serum endothelin(ET-1)levels were determined by ELISA.3)Renal tissue was used to perform transcriptomic detection analysis and experimental validation to explore the underlying molecular mechanism of the test compounds antagonizing DKD.4)Westem Blotting and immunofluorescence assays were used to determine the protein expression of Adenosine monophosphate-activated protein kinase(AMPKa),p-AMPKa(thr172),mammalian target of rapamycin(mTOR),p-mTOR(ser2448),Endothelial nitric-oxide synthase(eNOS),p-eNOS(ser1177),and their phosphorylation levels in the kidney of db / db mice,respectively.5)The differential genes Arhgef10 l,Ckb,Muc4,and Pvalb were assessed for regulation by the test compounds,and all of them were determined by qRT-PCR.2.The test compounds ameliorate retinopathy in db / db mice and the underlying molecular mechanismsModel building,administration and treatment methods:Normal mice and db/db mouse were fed for 16 weeks and divided into normal group,model group,Met group(200 mg/kg/d),SCU group(45 mg/kg/d),LTD group(45 mg/kg/d),LTD2 group(45mg/kg/d).Detection evaluation indicators:1)During the administration period,blood glucose and body weight were monitored.Electroretinogram(ERG),Optical coherence tomography(OCT),Fundus fluorescein angiography(FFA)and retinal angiography were performed at the end of administration;The mouse were sacrificed after the administration,and the serum was tested the liver function(alanine aminotransferase,aspartate aminotransferase),BUN,SCr,TC,TG.All of the above were used to evaluate the protective effect of the tested compounds on retina.2)Transcriptomic analysis was performed on retinal tissues to explore the potential mechanism of the test compounds to antagonize DR.3)The differential genes Nrm1,S1c17a6,Esmb,Nos3 were assessed for regulation by the test compounds,and all of them were determined by qRT-PCR.3.Targeting clinical specimens for early screening of miRNA biomarkers in diabetic retinopathyThe miRNA and m RNA datasets of DR and neovascularization(NEO)were downloaded from the Gene Expression Omnibus(GEO)database by using the raw signal analysis method.Differential gene analysis was followed by miRNA-m RNA network construction using miRNet and miRWalk.Enrichment analysis was performed in Metascape.We constructed the PPI network by combining 29 genes screened by Gene Cards and calculated the hub genes using Cytoscape.TransmiR was used to predict transcription factors(TF),and Drug Bank was used to predict TF for targeting drugs.Finally,whole blood and clinical data were collected from 58 patients with T2 DM,and qRT-PCR,logistic,and Receiver operating characteristic curve(ROC)were used to verify the value of key miRNAs.Results:1.The test compounds ameliorate renal lesions db/db mice and the underlying molecular mechanisms1.1 Pharmacological effects of tested compounds to ameliorate renal lesions in db / db miceCompared with normal group,FBG,UALB,UACR,SCr and BUN were significantly increased in Model group,while fasting insulin level was decreased.Compared with the model group,the test compounds could reduce FBG level,improve fasting insulin level and reduce UALB,UACR,SCr and BUN levels,indicating that the test compounds had a significant protective effect on DN.Compared with the positive Met group,There were no significant differences among the experimental groups after the intervention treatments.In addition,FBG decreased significantly in Met+LTD2 group,and UALB decreased significantly in Met+SCU and Met+LTD2 groups.1.2 The test compounds reduced the level of serum inflammatory cytokines in db/db miceCompared with the normal group,the level of serum ET-1 in Model group was significantly increased.Compared with Model group,the test compounds can reduce the serum ET-1 level.Especially Met group and Met+SCU group.Compared with the positive Met group,there were no significant differences among the experimental groups after the intervention treatments.1.3 Transcriptomic profiling of the test compounds antagonizes molecular mechanisms underlying DKDTranscriptomic analysis of kidney tissues from db / db mice revealed that the Differentially expressed genes(DEGS)of SCU were enriched in these important pathways of kidney,vascular,and visual system development,and the DEGS of LTD were involved in the development and formation of renal tubules,which provided insights into the molecular mechanisms of their involvement in attenuating kidney injury in DM.1.4 The tested compounds regulated eNOS and AMPK / mTOR signaling pathways in the kidney of diabetic miceCompared with the normal control group,the protein expression levels of AMPKa,p-AMPKa(Thr172),eNOS and p-eNOS(Ser1177)in the Model group were down-regulated,while the protein expression levels of mTOR and p-mTOR were up-regulated.Compared with Model group,the protein expression levels of AMPKa,p-AMPKa(Thr172),eNOS and p-eNOS(Ser1177)in kidney tissue of db/db mice were up-regulated,while the protein expression levels of mTOR and p-mTOR were down-regulated.Compared with the positive Met group,there were no significant differences among the experimental groups after the intervention treatments.1.5 Differential gene regulation by the test compoundsCompared with the normal group,the Model group gene Arhgef10 l and Muc4 were down-regulated significantly.However,Ckb gene was up-regulated significantly,while Pvalb gene was up-regulated significantly.Compared with the Model group,the test compounds up-regulated gene Arhgef10 l and Muc4.Compared with the positive Met group,there were no significant differences among the experimental groups after the intervention treatments.2.The test compounds ameliorate retinopathy in db / db mice and the underlying molecular mechanisms2.1 Pharmacological effects of tested compounds to ameliorate retinopathy in db / db miceCompared with the normal group,TC and TG levels were significantly increased in the Model group,and BUN and Cr levels were increased;compared with the Model group,the test compounds significantly reduced TG,BUN and SCr levels.Compared with the positive Met group,there were no significant differences among the experimental groups after the intervention treatments.Compared with the normal group,the A-wave amplitude of retinal ERG in db/db mice in model group was significantly decreased,the latency of B-wave was significantly prolonged,the number of retinal hemangioma was significantly increased,the proliferation of endothelial cells in the retinal capillary network and peripheral cell apoptosis were significantly increased.Compared with the model group,the test compounds can reverse the above changes,reduce the number of retinal microhemangioma,reduce the proliferation of endothelial cells in the retinal capillary network and peripheral cell apoptosis.Compared with the positive Met group,there were no significant differences among the experimental groups after the intervention treatments.The experimental results showed that the test drugs had obvious protective effect on DR.2.2 Transcriptomic profiling of the test compounds antagonizes molecular mechanisms underlying DRTranscriptomic analysis of retinal tissue from db/db mice revealed that the DEGS of SCU are enriched in the developing eye;As blood vessels are an important part of the circulatory system,DEGS may be involved in a key loop of the overall circulatory system.The DEGS of LTD2 are involved in the regulation of angiogenesis,which is closely related to the injury process of angiogenesis in DR,suggesting that LTD2 therapy has the potential to target diabetic retinal injury.2.3 The differential retinal gene expression in db/db mice was regulated by the test compoundsCompared with Normal group,the Model set of genes Nm1,Slc17a6,Esrb and Nos3 increased,compared with Model group,the test compounds up-regulated gene Nos3 and significantly down-regulated gene Nm1.3.Targeting clinical specimens to study miRNA biomarkers for early screening of diabetesDifferential analysis indicated the presence of genes and miRNAs that coregulate DR and NEO.Enrichment analysis showed that key genes are inextricably linked to neovascularization.PPI combined with hub gene results identified four candidate miRNAs,namely hsa-mir-4328,hsa-mir-4422,hsa-mir-548 z,and hsa-mir-628-5p.qRT-PCR,logistic,and ROC results showed that the expression of these miRNAs was downregulated with the onset and progression of DR.At the same time,the reduced levels predicted the risk of evolving DR in T2 DM patients.Conclusion:1.Natural flavonoids SCU/LTD and their derivatives LTD2 can significantly improve the FBG level and fasting insulin level of db/db mice,reduce the levels of UALB,UACR,SCr,BUN,and have a protective effect on DKD.Its mechanism is related to the reduction of the level of serum ET-1 and up-regulation of eNOS protein expression in renal tissue,also correlated with the protein expression levels of AMPK activation and the inhibition of mTOR;It may protect the kidney by regulating the expression of differential genes Arhgef10 l,Ckb,Muc4 and Pvalb.The renal protective effect of combined drug on diabetes db/db mice was better than that of single drug.2.Natural flavonoids SCU/LTD and their derivatives LTD2 can protect DR by improving the amplitude of a wave and latency of b wave of DR in diabetes db/db mice,reducing the number of retinal microangiomas.The test compounds may protect the retina and delay the progress of DR by regulating the expression of differential genes Nrn1,Slc17a6,Esrrb and Nos3.3.Studies through clinical specimens from patients with diabetes have shown that hsa-mir-(4328,4422,548 z and-628-5p)in whole blood are protective factors for DR and can be used as novel biomarkers for diagnosis and prediction.