Study On The Regulation Mechanism Of Long Non-Coding RNA In The Treatment Of Diabetic Macrovascular Disease By Yiqiyangyinhuoxue Therapy Based On Transcriptome Screening

ObjectiveTo investigate the therapeutic effect and mechanism of Danzhi Jiangtang Capsule（DJC）on diabetic macrovascular disease in Goto-Kakizaki（GK）rats.Transcriptome sequencing（RNA-seq）was used to study the differences of mRNA and lncRNA expression in diabetic macrovascular disease model rats and normal rats as well as in the aortic tissues after DJC intervention.To screen the key lncRNAs in the pathogenesis of diabetic macrovascular disease in rats,and to investigate the possible pathogenesis and the intervention effect of DJC.To investigate the mechanism of lncRNA-Kantr targeting miR-296 to competitively regulate IL-1R1/MyD88/p38 signal axis affecting HUVECs inflammatory injury,and to explore the possible mechanism of intervention of DJC in diabetic macrovascular diseases.MethodsGK rats were fed with high lipid and glucose in combination with endothelial nitric oxide synthase（ENOS）inhibitor n-nitrol-l-arginine methyl ester（L-NAME）(0.1g·L^-1·d^-1)to replicate diabetic macrovascular disease.The model rats were randomly divided into model group,DJC high、medium、low dose group(1 260 mg·kg^-1,630mg·kg^-1,320 mg·kg^-1),the atorvastatin group(105 mg·kg^-1)and metformin group(10mg·kg^-1),each group of 12,12 in Wistar rats as normal control group,only continuous irrigation to eight weeks.Various indexes of rats were observed.Randomly selected from the normal control group（Con）,diabetes,vascular lesions in the model group（Mod）,DJC medium-dose group（DJC）in the aorta tissue,4 rats using Illumina Nova Seq 6000 sequencing platform for the RNA-seq mRNA in rat aorta tissue and the expression of lncRNA situation,in view of the sequencing results sequencing quality evaluation,group relations between samples analysis,quantitative analysis,the differences between RNA and RNA analysis,enrichment of mRNA gene annotation and analysis,prediction and functional analysis of LncRNA target genes.Based on the mRNA-lncRNA expression profile of rats with diabetic macroangiopathy,bioinformatics database and analysis method were used to screen the conserved lncRNAs with different lncRNAs in human,rats and mice.For highly conserved lncRNA-kantr,|r|>0.5 was the standard for screening,and the difference mrnas with positive correlation were calculated according to Pearson correlation coefficient.RNA hybrid and Miranda respectively were used to predict the potential interaction of miRNAs with kantr and to conduct conservative analysis.miRNAs with potential Kantr interactions were used to predict highly conserved targeted mRNAs in humans and rats through the mirwalk database,intersections of differentially expressed mRNAs were obtained,and the Kantr-miRNA-mRNA regulatory relationship data were determined and imported into Cytoscape software 3.7 for visualization.Rt-PCR was used to detect lncRNA-Kantr/miR-296/IL-1R1mRNA levels in aortic tissues for validation.The high glucose-induced cell injury model of HUVECs was established in vitro.MTT method was used to screen the intervention concentration of serum containing DJC.MTT method was used to detect the effect of serum contained in DJC on the survival rate of model cells.The levels of IL-1β、IL-6 and TNF-αin HUVECs supernatant were determined by ELISA.Hoechst 33258 staining was used to observe the apoptotic morphology of HUVECs cells,and the apoptotic rate of HUVECs cells was detected by flow cytometry.RNA fluorescence in situ hybridization（RNA-FISH）was used to detect the localization of Kantr in subcells of HUVECs.qRT-PCR was used to detect the content of Kantr in the subcells of HUVECs.Kantr overexpression plasmid and Kantr-siRNA interference model were constructed for cell transfection and efficiency detection.The dual luciferase reporter gene was used to detect the potential binding sites of miR-296 to Kantr and IL-1R1.LncRNA/miRNA/mRNA levels in HUVECs were detected by Rt-PCR,and protein expression levels in HUVECs were detected by Western blot.Results1 Effect of DJC on the treatment of diabetic rats with macrovascular diseasesDJC could reduce the amount of drinking water and random blood glucose in model rats,increase body mass,reduce the degree of aortic tissue damage,reduce the levels of plasma TC,TG,LDL-C,LDL-C,ET-1,IL-1β,IL-6,TNF-αincrease the levels of plasma HDL-C,NO,and decrease the expression of aortic IL-1β,IL-6 and TNF-αmRNA.2 Effect of DJC on mRNA-lncRNA expression profile in diabetic rats（1）AfterRNA-seq,32,883 mRNAs expression profile data were obtained.Compared with Con group,4,593 mRNAs expression profile data were up-regulated and 1,676mRNAs expression profile data were down-regulated in the Mod group.Compared with the model group,1728 mRNAs expression profile data were up-regulated and 2490 mRNAs expression profile data were down-regulated in the DJC group.The expression profile data of 18048 lncRNAs were obtained.Compared with the Con group,743 lncRNAs expression profile data were up-regulated and 1230 lncRNAs expression profile data were down-regulated in the Mod group.Compared with the Mod group,918 lncRNAs expression profile data were up-regulated and 282 lncRNAs expression profile data were down-regulated in the DJC group.（2）Differences in the functional enrichment of mRNA and lncRNAs in diabetic rats with macrovascular disease,GO enrichment of 101 and 150 on the biological process（BP）,43 and 50 on the cell component（CC）,and 40 and 40 on the molecular function（MF）.There are 44 pathways in KEGG pathway in the functional enrichment of mRNA and lncRNAs.（3）Function enrichment of mRNA and lncRNAs was observed in animals with DJC intervention model of diabetic macroangiopathy,and GO enrichment was observed in 94 and 118 biological processes（BP）,46 and 59 cell components（CC）,and 42 and 44 molecular functions（MF）.The KEGG pathway enriched 37 and 51 signaling pathways,respectively.3 The ceRNA network construction analysis of the key lncRNA in diabetic macrovascular disease and the intervention effect of DJC were verified.In the model rat differential lncRNA,ENSRNOT00000086786,namely Kantr,had a common difference and was well conserved in human,rat and mouse sources.Co-expression analysis of Kantr and differentially expressed mRNAs revealed that there were 16 differentially expressed genes,15 of which had statistical significance（P<0.05）,and 1 of which had no statistical significance（P=0.1）.RNAhybrid and miRanda software predicted that Kantr could potentially co-interact with 10 miRNAs,of which 5 were of good conservability,namely:miR-666-3p,miR-383-3p,miR-296-3p,miR-145-5p,miR-23a-5p.Fifteen m RANs were obtained by intersections of 5 miRNAs targeting m RANs and significantly co-expressed differentially expressed genes.The kantr-miRNA-mRNA network relationship was obtained.The Kantr/miR-296/IL-1R1 regulatory relationship was selected for RT-PCR verification.The results showed that the mRNA expressions of Kantr and IL-1R1 were increased in the model group compared with the normal control group（P<0.01）the expression of mir-296 decreased（P<0.01）,compared with the model group,the mRNA expressions of Kantr and il-1r1 were decreased in the DJC group（P<0.01）the expression of miR-296 increased（P<0.05）.4 LncRNA-kantr adsorption of miR-296 competitively regulates the signal axis of IL-1R1/MyD88/p38 which affects the inflammatory injury of HUVECs and the intervention effect of DJC（1）The effect of hyperglycemia on the survival rate of HUVECs and the intervention effect of DJC.Compared with the control group,the survival rate of the model group decreased to 51.5%（P<0.01）.Compared with the model group,the cell survival rate of DJC drug-containing serum group（5%,10%,15%）was significantly increased（P<0.01,P<0.05）.The results showed that DJC drug-containing serum promoted cell growth and the most obvious effect was 15%.（2）The effect of hyperglycemia on inflammatory cytokines of HUVECs and the intervention effect of DJC.In the high-glucose environment,the supernatant IL-1β,IL-6 and TNF-αlevel were increased in the model group compared with the control group（P<0.01）;Compared with the model group,the DJC containing drug serum group（P<0.01）supernatant IL-1β,IL-6,TNF-αserum decreased（P<0.01）.（3）The effect of hyperglycemia on HUVECs cell apoptosis and the intervention effect of DJC.Compared with the control group,the model group showed obvious characteristics of cell apoptosis and increased apoptosis rate（P<0.01）.Compared with the model group,the morphology changes and apoptosis rate of apoptotic cells in serum containing DJC decreased（P<0.01）.（4）The effect of high glucose on the signal axis of kantr-miRNA-296-IL-1R1/MyD88/p38 in HUVECs and the intervention effect of DJC.Compared with the control group,Kantr expression in the model group increased（P<0.01）,miR-296expression decreased（P<0.01）,IL-1R1,MyD88,p38 mRNA expressions increased（P<0.01,P<0.05）,and IL-1R1,MyD88 and p-p38 expressions increased（P<0.01,P<0.05）.Compared with the model group,Kantr expression was decreased（P<0.01）,mir-296 expression was increased（P<0.05）,IL-1R1,MyD88,p38 mRNA expression was decreased（P<0.01,P<0.05）,and IL-1R1,MyD88 and p-p38 protein expression was decreased（P<0.01,P<0.05）.（5）Interaction between lncRNA-kantr and miR-296,a competitive endogenousRNA（ceRNA）network molecule,mediates high glucose-induced HUVECs inflammatory injury.（1）Subcellular localization and expression of kantr in HUVECs.RNA-FISH showed that Kantr was expressed in both cytoplasm and nucleus of HUVECs,and the expression level of Kantr in cytoplasm was significantly higher than that of lncRNA in nucleus,which provided the necessary conditions for the regulation mechanism of ceRNA network.（2）kantr and miR-296 overexpression and silencing of the realization and efficiency.Potential binding sites of miR-296 and kantr existed,and miR-296 minics inhibited luciferase activity of kantr-WT（P<0.01）,but it had no significant effect on kantr-MUT,indicating that kantr has the effect of competitive regulation of mir-296.（3）Effects of kantr overexpression and silencing on high glucose-induced apoptosis of HUVECs cells and inflammatory factors.Compared with the Mock group,the apoptosis rate of kantr（+）group increased（P<0.01）,increased IL-1β,IL-6,TNF-in supernatant（P<0.01）.Apoptosis rate decreased in si Kantr group（P<0.05）,the levels of IL-1β,IL-6,TNF-αin supernatant decreased（P<0.01,P<0.05）.（4）Effects of over-expression and silencing of miR-296 on high glucose-induced apoptosis of HUVECs cells and inflammatory factors.In the high-glucose environment,compared with the Mock group,kantr expression in the mir-296 mimics group was decreased（P<0.01）,apoptosis was decreased（P<0.01）,and secretion of supernatant IL-1β,IL-6 and TNF-αwas decreased（P<0.05,P<0.01）.In the mir-296 inhibitor group,kantr expression increased（P<0.01）,apoptosis increased（P<0.05）,and secretion of supernatant IL-1β,IL-6 and TNF-αincreased（P<0.01）.（6）mir-296 targets the IL-1R1/MyD88/p38 pathway to mediate hyperglycemia induced inflammatory injury of HUVECs.（1）The dual luciferase reporter gene was used to detect the regulatory targets of IL-1R1 and miR-296.IL-1R1 and miR-296 had binding sites,and mir-296 mimics inhibited the luciferase activity of IL-1R1-WT（P<0.01）,but had no significant effect on IL-1R1-MUT,indicating that IL-1R1 was a direct target molecule of miR-296.（2）The effect of over-expression and silencing of miR-296 on the IL-1R1/MyD88/p38 pathway induced by high glucose in HUVECs.In the high-glucose environment,Compared with the Mock group,the expression of IL-1R1,MyD88,p38 mRNA and IL-1R1,MyD88,p-p38 proteins in the miR-296mimics group decreased（P<0.05,P<0.01）,and the expression of IL-1R1,MyD88,p38 mRNA and IL-1R1,MyD88,p-p38 proteins in the mir-296 inhibitor group increased（P<0.05,P<0.01）.（7）LncRNA-Kantr mediated high glucose-induced HUVECs inflammatory damage through the signal axis of miR-296/IL-1R1/MyD88/p38 and the intervention effect of DJC.In a high-glucose environment,compared with the Mock group,the supernatant IL-1β,IL-6 and TNF-αwere decreased in the sikantr and miR-296 mimics groups（P<0.01）,the apoptosis was reduced（P<0.01,P<0.05）,the expressions of IL-1R1,MyD88 and p-p38 were decreased（P<0.01,P<0.05）,and the effect of DJC intervention was similar to that of sikantr and miR-296 mimics.The results showed that DJC could reduce the kantr,increase the expression of miR-296,inhibit the activation of the IL-1R1/MyD88/p38 pathway,and reduce the inflammatory damage induced by high glucose in HUVECs.Conclusion（1）DJC has therapeutic effect on diabetic macrovascular disease in model rats,and the mechanism of action may be related to reducing inflammatory factors and reducing vascular inflammation.（2）Diabetic rats with macrovascular disease and after the intervention of and DJC,mRNA and lncRNA expression profiles were different at the vascular transcriptome level,and the biological functions of these differentially expressed profiles were concentrated in the biological processes and signal pathways of cellular nutrient metabolism,energy metabolism,immune inflammation and so on.（3）LncRNA-kantr（ENSRNOT00000086786）plays an important regulatory role in the pathogenesis of diabetic macrovascular disease in rats,and the regulatory mechanism may be related to the regulatory network of kantr/miR-296/IL-1R1 ceRNA,which may be the therapeutic target for the intervention of DJC.（4）Kantr can target miR-296 and competitively regulate IL-1R1/MyD88/p38 signal axis to affect HUVECs inflammatory injury under high glucose environment.DJC can reduce the hyperglycose-induced HUVECs inflammatory injury,which may be related to kantr to the competitive inhibition of IL-1R1/MyD88/p38 signal axis by targeting miR-296.