Protective Effect Of Combined Intervention Of FoxO1 Inhibitor And IL-10 Gene Transduction On Islets β Cells Of T1DM Mice

Purpose: To understand the clinical characteristics and influencing factors of type 1diabetes mellitus(type 1 diabetes mellitus,T1DM)and Diabetic ketoacidosis(DKA).Exploring the mechanism of IL-10 gene exerts hypoglycemic effect through PI3K/ AKT / Fox O1(Forkhead transceiption factor of class O1,Fox O1)signaling pathway and protect islet β cell function,to verify whether Fox O1 inhibitor combined with IL-10 gene transduction intervention can produce synergistic hypoglycemic effect,providing new ideas and therapeutic targets for in-depth research on the prevention and treatment strategies of T1 DM and related complications.Method: Part I: Analyze the medical records of T1 DM patients admitted to the Children’s Endocrinology Department of the First Affiliated Hospital of Xinjiang Medical University from January 2015 to December2020,investigate the influencing factors of DKA in first-time T1 DM patients,and compare the clinical characteristics and laboratory examinations between the DKA group and the non DKA group.Collect peripheral blood samples from T1 DM patients,detect the levels of IL-10 and Fox O1 in the DKA group,non DKA group,and normal children,and compare their differences;Peripheral blood transcriptome sequencing was performed,differentially expressed genes were screened using bioinformatics methods,and GO functional annotation,KEGG pathway analysis,and GSEA enrichment analysis were performed.A protein interaction network of differentially expressed genes was constructed using a String database.Part II: Detect the expression levels of IL-10 and Fox O1 genes in NOD mice,insulin marker genes insulin gene-2(INS2),neurogenin 3(Ngn3),and pancreatic duodenal homeobox factor-1(Pdx-1)in the pancreatic tissue of NOD mice.Western blot was used to detect the expression levels of phosphatidylinositol3-kinase(phosphatidylinositol-3-kinase,PI3K),Protein kinase B(protein kinase B,Akt)and the Fox O 1 phosphorylation levels,detection of the cyclin-dependent kinase inhibitor1A(cyclindependent kinase inhibitor 1A,Cdkn1a),S-kinase-related protein 2(S-phase kinase associated protein 2,Skp 2)protein expression level in the pancreatic tissuepathway of NOD mice to explore their role in the pathogenesis of T1 DM.Part III:Construct an IL-10 gene overexpression adeno-associated virus(AAV)and transfect it into mice,and they were randomly divided into T1 DM group,AAV-IL-10 group,AAV-GFP group,Fox O1 inhibitor group,Fox O1 inhibition+AAV-IL-10 group,with 10 mice in each group.Monitor the weight,vital signs and insulin content of mice in each group;HE staining was used to evaluate the status of pancreatic islet inflammation in each group of mice;Detect the expression levels of INS2,Ngn3,Pdx-1,IL-10,Fox O1 genes,pathway proteins PI3K/Akt,and cell cycle regulatory proteins Cdkn1 a and Skp2 in pancreatic tissue of mice in each group.Results:Part I:(1)In this study,155 males(48.3%)of 316 children with T1 DM.The main clinical symptoms were polydipsia,polyphagia,polyuria,weight loss,infection,deep breathing,fatigue,abdominal pain,vomiting,etc.The positive rates of T1 DM antibody ICA,IAA and GADA were 3.2%,3.5%,and 6.3%,≥ 1 type of DM antibody positive 8.7%,combined with autoimmune thyroid disease12.65%,combined with vitamin D deficiency 29.4%.(2)Among T1 DM patients,those who reside in rural areas,randomly have high blood sugar levels,high glycated hemoglobin levels,and low C-peptide levels are more likely to develop DKA.(3)ELISA results showed that the serum IL-10 content in DKA and non-DKA children was significantly reduced compared with the normal control group(P<0.001,P<0.001).IL-10 content increased in DKA group compared with non-DKA group(P=0.0004).Serum Fox O1 content in the DKA and non-DKA children was significantly higher compared with the normal control group(P<0.001,P=0.0002).The Fox O1 content was higher in the DKA group compared with the non-DKA group(P<0.001).Q-PCR results showed that IL-10 m RNA expression decreased and Fox O1 m RNA expression increased in DKA group compared with non-DKA group,and the differences were statistically significant.(4)Through transcriptome sequencing and biological analysis,IL-10 RA was increased as a differential gene in children with T1 DM.GO analysis showed that the differentially expressed genes were mainly concentrated in energy metabolism and immune process.The KEGG pathways were enriched to both the T1 DM and the PI3K-Akt signaling pathways.The GSEA regulated by the localization of Fox O transcription factors suggests that its function is related to AKT,and the protein interaction network map constructed using the String database suggests the interaction of IL10 RA,IL-10,Fox O 1,and Akt1.Part II:(1)NOD mice diagnosed with DM developed typical DM symptoms,with a progressive increase in blood glucose,a progressive decrease in body weight,and decreased insulin secretion.HE staining of pancreatic tissue showed significant changes in the islet morphology of NOD mice and infiltration of lymphocytes and monocytes around or within the islets.(2)ELISA results showed that serum IL-10 expression in NOD mice decreased compared with normal control group(P=0.0241),and Fox O1 expression was enhanced than normal group(P=0.0114);The immunohistochemical results showed that the expression of IL-10 in NOD mice decreased(P=0.002)and the expression of p-Fox O1increased(P=0.0235)compared to the control group.Q-PCR results showed that compared with the normal group of mice,the expression of IL-10,INS2,Ngn-3,and Pdx-1 m RNA in the pancreatic tissue of NOD mice decreased,while the expression of Fox O1 increased.Western Blot test results showed increased he experssion of p-PI3 K,p-Akt,while Cdkn1 a,Skp2 protein in the pancreatic tissue of NOD mice decreased compared with normal controls.Part III: After administration of Fox O 1 inhibitor combined with IL-10 gene transduction intervention in NOD mice,the AAV-IL-10 group,Fox O1 inhibitor group,and Fox O1 inhibition + AAV-IL-10 group significantly reduced pancreatitis compared with the T1 DM group.AAV-IL-10 group,Fox O1 inhibitor +AAV-IL-10 group had more insulin secretion(P<0.01)compared with T1 DM group,and INS 2,Pdx-1,Ngn 3 and Pdx-1 m RNA expression was higher compared with T1 DM group.Western Blot test results showed that PI3 K and Akt phosphorylation levels were reduced in the pancreatic tissues of AAV-IL-10 group,and the protein expression of Cdkn1 a and Skp 2 was increased compared with T1 DM group;There was no significant difference in PI3 K and Akt phosphorylation levels between the Fox O1 inhibitor group and the T1 DM group;Meanwhile,PI3 K and Akt phosphorylation of The Fox O 1 inhibition +AAV-IL-10 group,and the AAV-IL-10 group decreased compared with Fox O 1 inhibitor group alone,with a statistically significant difference(P<0.05).Conclusion:(1)Among children with primary T1 DM,children living in rural areas with high glycaemia,high Hb A1 c and low C peptide were more likely to develop DKA.More attention should be paid to children with DM with the above characteristics.(2)The serum IL-10 expression is reduced and Fox O1 expression is increased in T1 DM children.(3)Fox O1 inhibitor combined with IL-10 gene transduction intervention in NOD mice can alleviate pancreatic inflammation to varying degrees and promote pancreatic islets β Cells secrete insulin.By upregulating the expression of insulin marker genes INS2,Ngn3,Pdx-1,and the expression of PI3K-Akt signaling pathway and pathway proteins Cdkn1 a,Skp2,the protection of pancreatic islets is achieved β The role of cells.(4)The effect of IL-10 gene transduction therapy is better than that of Fox O1 inhibitors in improving islet inflammatory infiltration.Gene transduction with IL-10 protein alone reduced the phosphorylation of pathway proteins PI3 K,while the combination of IL-10 and Fox O1 inhibitors can further upregulate the expression of Pdx-1 compared to monotherapy.The exact effect of the combination of the two needs further research.