| Type 2 diabetes mellitus(T2DM)is a multifactorial disease,and its prevalence has been increasing year by year since the last century.The pathogenesis of this disease is very complicated.The most significant pathological feature is the blockage of the insulin signaling pathway leading to insulin resistance,which is mainly manifested in the reduced sensitivity of insulin target organs to insulin signals.Resveratrol(RSV)is a polyphenolic compound found in a variety of plants.It is a natural phytoalexin that exists mainly in the trans structure in nature.Resveratrol was first isolated in the roots of resveratrol in 1940,and it was detected in more than 70 plantations,including eucalyptus,banyan,etc.The dietary sources mainly include red grapes,mulberries,red wine,etc.Polygonum cuspidatum is the highest.A large number of studies have shown that resveratrol can play a beneficial role in improving insulin resistance,treating type 2 diabetes and complications through a variety of biological effects,including anti-inflammatory,antioxidant stress,cardiovascular and neuroprotective effects,etc.Long non-coding RNA(long non-coding RNA,lncRNA)belongs to a class of RNA with a length of about 200 nucleotides that does not encode proteins.lncRNA were originally thought to be "noise" in genome transcription and had no biological function.However,recent studies have shown that lncRNA also participate in many important regulatory processes such as genomic imprinting and chromatin modification,and have played a role even in the treatment of T2 DM.However,the functional mechanism of most lncRNA is unclear so far.The purpose of this study is to explore the role and mechanism of lncRNA in improving resveratrol's insulin resistance by establishing in vivo(animal)and in vitro(cellular)insulin resistance models,and to provide new ideas and new targets for the treatment of T2 DM.Part one Effect of resveratrol on lipid deposition in liver of high-fat-fed insulin-resistant miceObjective: To observe the effects of resveratrol on blood glucose,insulin,QUICKI,blood lipid and liver lipid deposition in high-fat diet-induced insulin resistance mice.Methods:C57BL / 6J mice were randomly divided into control group(CON)and high-fat group(HFD).After 8 weeks,an intraperitoneal glucose tolerance test(IPGTT)was performed to assess whether an insulin resistance model was established.Twelve mice from the HFD group were randomly selected as the resveratrol intervention group(HFD + RSV),and the HFD + RSV group was given resveratrol 100.mg / kg / d was administered orally once a day.The CON group and the HFD group were administered an equal volume of 0.9% sodium chloride solution containing 0.1% dimethyl sulfoxide(DMSO).After 6 weeks of intervention,the IPGTT test was performed again to measure the blood glucose values at 0,15,30,60,and 120 minutes and calculate the area under the glucose curve(AUC).Collect serum to measure fasting blood glucose(FPG),total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C).Method(ELLSA)measured fasting insulin levels and calculated the insulin sensitivity index(QUICKI).The liver tissue samples were retained for H & E staining and oil red O staining,and the pathological changes and lipid content of liver tissue were observed.Results:1.Establishment of a high-fat diet-induced insulin resistance model in miceFrom the second week of high-fat diet intervention,the weight of mice in HFD group was significantly higher than that in CON group.The IPGTT test was performed on the 8th weekend.Compared with the CON group,the HFD group had significantly higher blood glucose levels at 0,30,60 and 120 minutes.Compared with the CON group,the area under the glucose curve in the HFD group increased significantly,proving that the insulin resistance model was successfully established.2.Changes in body weight and insulin resistance parameters of mice in each group after resveratrol intervention for 6 weeksThe body weight of mice in HFD group increased significantly compared with that in CON group,and after 4 weeks of resveratrol intervention,the body weight of mice in HFD + RSV group changed significantly compared with HFD.The IPGTT experiment was performed at the end of the 6th week.The blood glucose level of the HFD group was significantly higher than that of the CON group at 0 min,15 min,30 min,60 min,and 120 min.Blood glucose levels decreased at min,60 min,and 120 min.Compared with the CON group,the area under the AUC curve in the HFD group increased significantly,and the area under the AUC curve in the HFD + RSV group decreased significantly compared with the HFD group.Compared with the CON group,the fasting blood glucose and insulin levels in the HFD group were significantly increased,while the fasting blood glucose and insulin levels in the HFD + RSV group were significantly lower than those in the HFD group.The QUICKI value in the HFD group was significantly lower than that in the CON group.After resveratrol intervention,the QUICKI value was significantly increased.3.Comparison of blood lipid levels of mice in each groupCompared with the CON group,the HFD group had significantly higher levels of TG,TC,and LDL-C.Compared with the HFD group,the levels of TG and LDL-C in the HFD + RSV group were significantly lower,the levels of HDL-C were significantly increased,and the levels of TC did not change significantly.4.Morphological comparison of liver tissue of mice in each groupH&E staining showed clear and complete liver cell structure in the CON group,uniform cytoplasmic red staining,fewer lipid droplet vacuoles,and no significant steatosis.In the HFD group,the cell structure of the liver tissue was disordered and fuzzy,and vacuoles of varying sizes were seen in the cytoplasm.The lipid droplets squeezed the nucleus to the edge of the cell,showing obvious steatosis.HFD + RSV group significantly reduced the fatty degeneration of liver cells,significantly reduced lipid droplet vacuoles,and morphological changes between the CON group and the HFD group.Oil red O staining,blue nuclei and fewer orange red lipid droplets were seen in the CON group.A large amount of orange-red lipid droplets were seen in the HFD group.Lipid droplets in the HFD + RSV group were significantly reduced,and the number was between the CON and HFD groups.Summary: High-fat diet can cause insulin resistance and liver steatosis in mice.Resveratrol can significantly improve insulin resistance and liver steatosis in mice induced by high-fat diet.Part two Analysis of resveratrol on expression profiles of lncRNA and mRNA in liver of high-fat-fed miceObjective: The effects of resveratrol intervention on liver lncRNA and mRNA expression profiles in high-fat-fed mice were analyzed by transcriptome sequencing analysis.Methods:Total RNA from mouse liver tissue was extracted,a sequencing library was constructed,and transcriptome sequencing was performed using high-throughput sequencing technology.Use Stringtie software to count fragments within each gene segment,and calculate the FPKM(Fragments Per Kilobase Million)value of each gene.The differential expression foldchange was calculated according to the FPKM value,and its logarithmic value log2(foldchange)was used for screening differentially expressed genes.Then real-time polymerase chain reaction(RT-PCR)was used to verify the relative expression levels of the differentially expressed genes in each group of liver tissue.Results:1.Expression profile of lncRNA and mRNA in mouse liverTranscriptome sequencing results showed that a total of 51024 lncRNA and 31055 mRNA were detected in mouse liver tissues.There were 503 differentially expressed lncRNA(344 up-regulated and 159 down-regulated)and 655 differentially expressed mRNAs(418 up-regulated and 237 down-regulated).Compared with the HFD group,the HFD + RSV group had 95 differentially expressed lncRNA(46 up-regulated and 49 down-regulated)and 102 differentially-expressed mRNA(34 up-regulated and 68 down-regulated).Volcano map analysis results showed that the lncRNA and mRNA were significantly different between the HFD group and the CON group and between the HFD + RSV group and the HFD group.2.Expression profile of lncRNA and mRNA in mouse liver reversed by resveratrolBy comparing the differentially expressed genes of the CON group with the HFD group and between the HFD and HFD + RSV groups,it was found that compared with the CON group,among the lncRNA and mRNA up-regulated in the HFD group,25 lncRNA and 50 mRNA were restored and down-regulated in the HFD + RSV group.Among the lncRNA and mRNA down-regulated in the HFD group,25 lncRNA and 23 mRNA were restored and up-regulated in the HFD + RSV group.The heat map shows the expression values of lncRNAs and mRNAs in the CON group,HFD group,and HFD + RSV group,and suggests that there are significant differences in the expression patterns among the three groups.3.RT-q PCR verified that seven lncRNA had significant expression differences in the three groups.Quantitative RT-PCR detection of the above screening results confirmed that there were significant differences in expression of the 7 lncRNAs in the three groups,which may be related to the mechanism of action of resveratrol.Summary: The high-fat diet caused significant changes in the expression profiles of mouse lncRNA and mRNA,and resveratrol intervention could reverse the expression levels of some lncRNA and mRNA.It is suggested that lncRNA may play an important role in resveratrol in improving insulin resistance induced by high fat in mice.Part three GO and KEGG analysis of resveratrol intervention in liver mRNA expression of high-fat-fed mice and verification of mRNA expression related to insulin signaling pathwayObjective: To find out the main biological functions and signal transduction pathways of differential mRNA in resveratrol to improve insulin resistance through GO and KEGG enrichment analysis,and to explore the relationship between lncRNA.Methods:Annotate each gene based on the Gene Ontology and KEGG databases.Calculate the number of each differential gene,screen the GO and KEGG results significantly enriched in the differential genes,and find out the signal transduction pathways related to this study.Real-time polymerase chain reaction(RT-PCR)and Western blot were used to detect the expression levels of SOCS3,Akt,p-Akt,FOXO1,p-FOXO1 and G6 PC in insulin signaling pathway related molecules.Results:1.GO and KEGG analysis of resveratrol reversed mRNAThe GO enrichment of differentially expressed mRNA mainly includes negative regulation of signals,negative regulation of molecular functions,enzyme-linked receptor protein signaling pathway,enzyme inhibitor activity,and endopeptidase activity.The KEGG pathway analysis of differentially expressed mRNA mainly includes metabolic pathways,MAPK signaling pathways,Jak-STAT signaling pathways,insulin signaling pathways,cell cycle and adipokine signaling pathways.Among them,the insulin signaling pathway is related to our research.We selected this pathway and predicted the closely related target gene SOCS3 through pathway analysis.2.Comparison of mRNA levels of insulin signaling pathway related genesFluorescent quantitative RT-PCR test results showed that compared with the CON group,the mRNA expression levels of FOXO1,SOCS3,and G6 PC in the liver of the HFD group mice were significantly increased,while the mRNA levels of the HFD + RSV group were significantly decreased.There was no significant difference in Akt mRNA expression between the three groups.3.Comparison of insulin signaling pathway related protein expressionsWestern blot results showed that compared with the CON group,the liver FOXO1,SOCS3,and G6 PC expression levels of the mice in the HFD group increased significantly,while the expression levels of the three proteins decreased in the HFD + RSV group.There was no significant difference in the total Akt protein levels among the three groups,but the phosphorylated Akt levels in the HFD group were down-regulated compared with the CON group,and expression increased in the HFD + RSV group.At the same time,compared with the CON group,the liver FOXO1 phosphorylation level in the HFD group was decreased,while the expression was increased in the HFD + RSV group.4.Effect of resveratrol on SOCS3 immunofluorescence results in mouse liverThe results of immunofluorescence staining showed that compared with the CON group,the liver SOCS3 expression in the HFD group was significantly increased,and the HFD + RSV group reduced the SOCS3 expression in the HFD group.This result indicates that the expression level of SOCS3 in the liver of mice fed a high-fat diet increased,and the expression of SOCS3 decreased under the intervention of resveratrol,that is,resveratrol inhibited the expression of SOCS3 in the liver.Summary: Resveratrol altered the expression profiles of Lnc RNA and mRNA in livers of high-fat-fed mice.GO and KEGG enrichment analysis showed a significant correlation between insulin signaling pathways,suggesting that resveratrol may affect insulin signaling pathways and related lncRNAs to improve insulin resistance.Part four Role of NONMMUT008655.2 in resveratrol's effect on insulin signaling pathwayObjective: Pathway analysis was used to select lncRNA related to insulin signaling pathways,and explore the role of lncRNA in resveratrol to improve liver insulin resistance at the cellular level.Methods:Based on the lncRNA-mi RNA-mRNA coexpression network and pathway analysis prediction,combined with our sequencing results,we screened out lncRNA NONMMUT008655.2,which is closely related to the insulin signaling pathway.Design and synthesize its sequence-specific si RNA,transfect Hepa cells,and detect the mRNA level of NONMMUT008655.2 after transfection using RT-PCR to evaluate the transfection efficiency.Hepa cells were divided into: control group(CON),PA intervention group(PA),PA + RSV 30 ?M group(PA + RSV),PA + RSV 30 ?M + si RNA control sequence group(si RNA-NC),PA + RSV 30 ?M + NONMMUT008655.2-knockdown group(si RNA-NONMMUT008655.2).After 24 h of transfection,the corresponding groups were given PA and resveratrol,and the protein was extracted after intervention for 24 h.The target gene expression was detected by western blot.Results:1.NONMMUT008655.2 lncRNA-mi RNA-mRNA co-expression networkAmong the 7 lncRNA screened,NONMMUT008655.2 had the highest expression of FPKM and was closely related to the target gene mRNA SOCS3.The trend of SOCS3 was consistent with NONMMUT008655.2.2.Transfection efficiencyCompared with the negative control group,the expression level of NONMMUT008655.2 in the si RNA transfected group was significantly reduced.3.Expression of indexes related to insulin signaling pathway after knocking down NONMMUT008655.2Compared with the CON group,the expressions of FOXO1,G6 PC,and SOCS3 in the PA group were significantly increased,and the expressions of p-Akt and p-FOXO1 were significantly decreased.After knocking down NONMMUT008655.2,compared with the PA group,the expressions of p-Akt and p-FOXO1 increased significantly,while the expressions of FOXO1,G6 PC,and SOCS3 decreased significantly.After resveratrol intervention,the expressions of FOXO1,G6 PC,and SOCS3 were significantly lower than those in the PA group,and the expressions of p-Akt and p-FOXO1 were significantly higher than those in the PA group.The differences were statistically significant.Compared with the NONMMUT008655.2 knock-down group,p-Akt and p-FOXO1 increased significantly and FOXO1 decreased significantly.There was no significant change in G6 PC and SOCS3.Summary: Resveratrol attenuates insulin resistance in PA-induced HEPA cells,affecting expression of insulin signaling pathway molecules.And knocking down lncRNA NONMMUT008655.2 can partially show the effect of resveratrol.It is suggested that resveratrol may exert its effect of improving insulin resistance by down-regulating NONMMUT008655.2.Conclusions:1.High-fat diet can cause insulin resistance in mice.Resveratrol intervention can affect the insulin signaling pathway in the liver and improve insulin resistance induced by high-fat in mice.2.The expression profiles of liver lncRNAs and mRNAs in resveratrol-treated high-fat mice have changed significantly,and have significant correlation with insulin signaling pathway.3.Resveratrol can down-regulate liver lncRNA NONMMUT008655.2 level,which may play an important role in resveratrol improving insulin resistance induced by high fat in mice. |
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