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OBJECTIVE To construct an insulin-resistant(IR)small intestinal organoid model of mice and study the protective effect of flavanomarein(FM)on the intestinal mucosal barrier in the model.METHODS ①Small intestinal organoid models of C57BL/6J and db/db of mice were constructed.The expressions of Ki-67,E-cadherin(E-cad),lysozyme(Lyz)and mucin-2(Muc-2)in small intestinal organ-oids were detected by 3D immunofluorescence.RT-qPCR was used to detect the expressions of fibro-nectin(Fn),glucagon-like peptide-1(GLP-1)and peotide YY(PYY)mRNA while Western blotting was used to detect the expressions of Fn,GLP-1 and PYY protein.The Lyz secretion level was detected by ELISA.② Small intestinal organoids were divided into five groups:C57BL/6J mice 'small intestinal organ-oids as the normal control group,db/db mice' intestinal organoids as the IR model group,db/db mice small intestinal organoids with flavanomarein 25,50 and 100 μmol·L-1 intervention for 48 h as IR model+ FM groups.RT-qPCR was used to detect the expression of Lyz mRNA while Western blotting was used to detect the expression of Lyz protein.RESULTS ① On the 6th day of small intestinal organoid culture,a ring structure with a clear luminal structure was formed and an IR mouse small intestinal organoid model was established.3D Immunofluorescence detection showed that the established small intestinal organoids all expressed Ki-67,E-cad,Lyz and MUC-2.Compared with the normal control group,the expres-sion of Fn mRNA in the IR model group was significantly increased(P<0.05)while the expressions of GLP-1 and PYY mRNA were significantly decreased(P<0.05).Compared with the normal control group,the expression of Fn protein in the IR model group was significantly decreased(P<0.05)while the expressions of GLP-1 and PYY protein were significantly increased(P<0.05).ELISA results showed that compared with the normal control group,the secretion levels of Lyz in the IR model group were signifi-cantly decreased(P<0.01).② RT-qPCR results showed that compared with the normal control group,the expression of Lyz mRNA in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz mRNA in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).Western blotting results showed that compared with the normal control group,the expression of Lyz protein in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz protein in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).CONCLUSION The constructed IR mouse small intestinal organoid model provides a more complete in vitro research model for exploring the pathophysiological mechanism by which drug interventions help repair the intestinal mucosal barrier.FM may maintain the intestinal mucosal barrier by reversing the decrease in Lyz expression levels in IR mice,thereby improving IR.
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ObjectiveTo investigate the effects of flavanomarein on the transcriptome of small intestinal organoids in insulin-resistant mice. MethodFirstly， small intestinal organoids of C57BL/6J and db/db mice were established. Ki-67 and E-cadherin expression was determined by immunofluorescence. Small intestinal organoids were divided into the following three groups： C57BL/6J mouse small intestinal organoids as the normal control group， db/db mouse small intestinal organoids as the model group （IR group）， and db/db mouse small intestinal organoids treated with flavanomarein as the administration group （FM group）. Western blot was used to detect the expression of glucagon-like peptide-1（GLP-1） protein on the small intestinal organoids of the three groups. Finally， transcriptome sequencing was performed on samples from the three groups. ResultOn the 6th day of small intestine organoids culture， a cyclic structure was formed around the lumen， and a small intestine organoids culture model was preliminarily established. Immunofluorescence detection showed that ki-67 and E-cadherin were expressed in small intestinal organoids. Western blot results showed that the expression of GLP-1 protein was increased by flavanomarein. In the results of differential expressed gene （DEG） screening， there were 1 862 DEGs in the IR group as compared with the normal control group， and 2 282 DEGs in the FM group as compared with the IR group. Through protein-protein interaction（PPI） network analysis of the DEGs of the two groups， 10 Hub genes， including Nr1i3， Cyp2c44， Ugt2b1， Gsta1， Gstm2， Ptgs1， Gstm4， Cyp2c38， Cyp4a32， and Gpx3， were obtained. These genes were highly expressed in the normal control group， and their expression was reduced in the IR group. After the intervention of flavanomarein， the expression of the above genes was reversed. ConclusionFlavanomarein may play its role in improving insulin resistance by reversing the expression levels of 10 Hub genes， including Nr1i3， Cyp2c44， Ugt2b1， Gsta1， Gstm2， Ptgs1， Gstm4， Cyp2c38， Cyp4a32， and Gpx3.
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Background: Diabetic kidney disease (DKD), one of the main complications of diabetes mellitus (DM), has become a frequent cause of end-stage renal disease. A clinically convenient, non-invasive approach for monitoring the development of DKD would benefit the overall life quality of patients with DM and contribute to lower medical burdens through promoting preventive interventions. Methods: We utilized 5hmC-Seal to profile genome-wide 5-hydroxymethylcytosines in plasma cell-free DNA (cfDNA). Candidate genes were identified by intersecting the differentially hydroxymethylated genes and differentially expressed genes from the GSE30528 and GSE30529. Then, a protein interaction network was constructed for the candidate genes, and the hub genes were identified by the MCODE and cytoHubba algorithm. The correlation analysis between the hydroxymethylation level of the hub genes and estimated glomerular filtration rate (eGFR) was carried out. Finally, we demonstrated differences in expression levels of the protein was verified by constructing a mouse model of DKD. In addition, we constructed a network of interactions between drugs and hub genes using the Comparative Toxicogenomics Database. Results: This study found that there were significant differences in the overall distribution of 5hmC in plasma of patients with DKD, and an alteration of hydroxymethylation levels in genomic regions involved in inflammatory pathways which participate in the immune response. The final 5 hub genes, including (CTNNB1, MYD88, CD28, VCAM1, CD44) were confirmed. Further analysis indicated that this 5-gene signature showed a good capacity to distinguish between DKD and DM, and was found that protein levels were increased in renal tissue of DKD mice. Correlation analysis indicated that the hydroxymethylation level of 5 hub genes were nagatively correlated with eGFR. Toxicogenomics analysis showed that a variety of drugs for the treatment of DKD can reduce the expression levels of 4 hub genes (CD44, MYD88, VCAM1, CTNNB1). Conclusions: The 5hmC-Seal assay was successfully applied to the plasma cfDNA samples from a cohort of DM patients with or without DKD. Altered 5hmC signatures indicate that 5hmC-Seal has the potential to be a non-invasive epigenetic tool for monitoring the development of DKD and it provides new insight for the future molecularly targeted anti-inflammation therapeutic strategies of DKD.
