| Objective: Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia caused by multiple etiologies.Epidemiological data show that the number of people suffering from diabetes in China ranks first in the world.The damage of hyperglycemia to various organs and tissues of the human body is becoming more and more obvious,and the complications gradually increase with the progress of the disease,posing a serious threat to health.Among them,patients with type 2 diabetes account for more than 90% of the total number of diabetic patients.The basic pathophysiological changes are insulin secretion defects and insulin resistance.The function of pancreatic β cells will gradually fail with the development of the disease.Since insulin is essential to maintain blood sugar levels,protecting the function of pancreatic beta cells is the key to the treatment of diabetes.However,the mechanism of islet β-cell damage is not yet fully understood.Pyroptosis is a programmed death initiated by pathogen-associated molecular patterns/damage-associated molecular patterns(PAMPs/DAMPs)and accompanied by the secretion of a large number of inflammatory substances.Many studies have shown that there are a large number of inflammatory factors in the islet β cells of diabetic patients,and the destruction of islet β cells caused by inflammatory factors is closely related to the pathophysiological process of diabetes.The etiology of diabetes has not been elucidated,and pyroptosis may be one of the pathogenesis of diabetes.At the same time,the metabolic disorders of sugar,fat,protein and other substances in diabetic patients will aggravate pyroptosis and cause severe inflammatory reactions.Inflammatory factors further affect the activity and function of pancreatic β cells,resulting in decreased insulin secretion and resistance to insulin,which play a non-negligible role in the prognosis of diabetes and the occurrence and development of its complications.Therefore,it is of great significance for the research,diagnosis and treatment of diabetes to explore the inflammatory response of islet β cells induced by pyroptosis in diabetic patients and its regulatory mechanism.Micro RNA(miRNA)is an endogenous RNA with a length of about 20-24 nucleotides.miRNA plays an important role in many life activities such as transcriptional regulation and epigenetic regulation.Many recent studies have shown that miRNAs play an important role in regulating the pyroptosis process of cells.These small RNAs can bind to downstream m RNAs to prevent the expression of protein-coding genes and prevent them from being converted into proteins.It has been reported that miRNA is involved in the regulation of diabetes,so studying the regulation mechanism of miRNA on islet β-cell pyroptosis in diabetes may become a potential target for the treatment of diabetes.At present,a few literatures show that miR-322-5p can mediate apoptosis,cell proliferation and migration,and relieve inflammation,but there is a lack of research on its effect on pyroptosis.Therefore,we infer that miR-322-5p may be involved in the regulation of islet β-cell pyroptosis in diabetic patients.Programmed cell death 4(PDCD4)is a new tumor suppressor gene discovered in recent years,which is widely expressed in various tissues and organs,and is absent or lowly expressed in various malignant tumors.It is highly expressed in chronic inflammatory diseases such as type 1 diabetes,obesity and atherosclerosis.According to the search of Targetscan,there is a targeting relationship between miR-322-5p and PDCD4 m RNA.It is speculated that miR-322-5p may target PDCD4 expression and participate in the regulation of islet β-cell pyroptosis in diabetic patients.The composition of PI3K/AKT includes phosphatidylinositol 3-kinase(PI3K)and its downstream molecular protein kinase B(AKT),which is a classical pathway that mediates cell proliferation and inhibits apoptosis through downstream substrates,and is closely related to a variety of human diseases.It has been pointed out that the PI3K/AKT signaling pathway is involved in the occurrence of cardiomyocyte pyroptosis.However,the effect and specific mechanism of PI3K/AKT signaling pathway on pyroptosis in pancreatic islet β cells are not fully understood.This study intends to culture islet β cells in vitro to prove that miR-322-5p can alleviate islet β cell pyroptosis and improve insulin secretion level by targeting PDCD4 to activate PI3K/AKT pathway.The aim is to explore the pathological mechanism of diabetes and provide potential therapeutic targets for the treatment of type 2 diabetes.Methods: 1.Culture islet β-cells(Mouse insulinoma 6,Min6)in vitro,starve for 24 hours in serumfree medium,and then use different glucose concentration gradient medium 5.5mmol/L(1g/L),16.7mmol/L(3g/L),25mmol/L(4.5g/L),and 33.3mmol/L(6g/L)were treated for48 hours respectively,and Western blot(WB)was used to detect the NOD-like receptor thermal protein domain-associated protein 3(NOD-Like receptor thermal protein domain associated protein 3,NLRP3)and caspase-1 expression.To investigate the induction of pyroptosis in islet β cells by the above four glucose concentrations,and to determine the optimum glucose concentration for inducing pyroptosis.2.Min6 cells were cultured in vitro,and Min6 cells were divided into control group 5.5mmol/L(1g/L)and high glucose group 33.3mmol/L(6g/L)according to the optimal glucose concentration.The cells were starved for 24 hours in serum-free medium,and the cells were treated for 48 hours.Immunofluorescence was used to detect the co-localization expression of pyroptosis-related protein Caspase-1 and sadermin D(GSDMD).Cell Counting Kit-8(CCK-8)detects cell viability and understands the proportion of pyroptotic cells.Enzyme linked immunosorbent assay(ELISA)was used to measure the levels of inflammatory factor IL-1β and insulin secretion in the cell culture supernatant.The expression level of miR-322-5p was detected by real-time fluorescent quantitative PCR(real-time PCR).To investigate the injury of pyroptosis on the activity of pancreatic islet β cells and insulin secretion under the optimal glucose concentration for inducing pyroptosis.3.Min6 cells were cultured in vitro,and Min6 cells were divided into high glucose group,high glucose miR-322-5p mimics NC group,high glucose miR-322-5p mimics group,high glucose miR-322-5p inhibitor NC group,high glucose miR-322-5p inhibitor group.After 6 hours of transfection with Lipo2000,the medium was changed for 48 hours,and the miRNA transfection efficiency was detected by RT-PCR.The expression levels of pyroptosis-related proteins NLRP3 and Caspase-1 were detected by Western blot.The colocalization expression of pyroptosis-related protein Caspase-1 and GSDMD was detected by immunofluorescence technique.CCK-8 was used to detect cell viability,and ELISA was used to measure the levels of inflammatory factor IL-1β and insulin secretion in the cell culture supernatant.To explore the protective effect of miR-322-5p on islet β-cell pyroptosis.4.Human embryonic kidney cells(293T)cells were cultured in vitro,and the 293 T cells were divided into miR-322-5p mimics NC PDCD4-WT group,miR-322-5p mimics PDCD4-WT group,miR-322-5p mimics NC and PDCD4-MUT group,miR-322-5p mimics PDCD4-MUT group.After 6 hours of transfection with Lipo2000,the medium was changed for 48 hours,and the dual luciferase reporter assay was used to verify the combination of miR-322-5p and the downstream target protein PDCD4.5.Culture Min6 cells in vitro,divide Min6 cells into miR-322-5p mimics NC group,miR-322-5p mimics group,miR-322-5p inhibitor NC group,miR-322-5p inhibitor group,and divide into groups according to Lipo2000 rpm After 6 hours of transfection,the medium was changed for 48 hours,and the expression level of PDCD4 was detected by Western blot technique.To explore the direct regulation of miR-322-5p on the expression of downstream target protein PDCD4.6.Culture Min6 cells in vitro,divide Min6 cells into high glucose group,high glucose miR-322-5p mimics NC group,high glucose miR-322-5p mimics group,high glucose miR-322-5p inhibitor NC group,high glucose miR-322-5p inhibitor NC group,high glucose miR-322-5p inhibitor group.According to group Lipo2000 transfection 6h,change medium and treat 48 h.The expression levels of pathway proteins AKT,P-AKT,PI3 K,and P-PI3 K were detected by Western blot technology.To explore the relationship between miR-322-5p regulation of islet β cell pyroptosis and PI3K/AKT signaling pathway.Results: 1.High glucose can induce islet β-cell pyroptosis,and up-regulate the protein expression levels of pyroptosis-related proteins NLRP3 and Caspase-1 in islet β-cells.When the glucose concentration was 33.3mmol/L(6g/L),it was statistically significant.2.Compared with the control group,the high glucose group(33.3mmol/L)decreased the activity of islet β cells with decreased insulin secretion function,increased secretion of inflammatory factor IL-1β,and fluorescent colocalization expression of pyroptosis-related proteins Caspase-1 and GSDMD increased.The expression level of miR-322-5p in the high glucose group was significantly lower than that in the control group.3.miR-322-5p mimics increased the expression level of miR-322-5p in cells,while miR-322-5p inhibitor significantly down-regulated the expression level of miR-322-5p in islet β cells.Overexpression of miR-322-5p inhibited the protein expression levels of pyroptosis-related proteins NLRP3 and Caspase-1,increased cell viability,increased insulin secretion,decreased secretion of inflammatory factor IL-1β,and fluorescent coexpression of pyroptosis-related proteins Caspase-1 and GSDMD.Positioning expression increased.In contrast,inhibiting miR-322-5p showed the opposite effect.There was no significant statistical significance in miR-322-5p mimics NC group and miR-322-5p inhibitor NC group.4.Cells co-transfected with miR-322-5p mimics and PDCD4-WT,compared with cells cotransfected with miR-322-5p mimics NC and PDCD4-WT,co-transfected with miR-322-5p mimics and PDCD4-WT significantly decreased luciferase activity.However,cotransfection of miR-322-5p mimics and PDCD4-MUT had no significant effect on luciferase activity in 293 T cells.5.Overexpression of miR-322-5p inhibited the content of PDCD4,while inhibiting miR-322-5p significantly up-regulated the protein content of PDCD4.6.miR-322-5p inhibitor down-regulates the phosphorylation of P-PI3 K and P-AKT,while miR-322-5p mimics up-regulates the phosphorylation of P-PI3 K and P-AKT.Conclusion: 1.High glucose can induce pyroptosis in islet β cells and impair the insulin secretion function of islet β cells.2.miR-322-5p activates the PI3K/AKT pathway by targeting PDCD4 in pancreatic islet β cells to alleviate pyroptosis and improve insulin secretion levels. |
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