Study Of IL-1β Expression Induced By High Glucose And Its Injury Mechanism In INS-1 Cells

[Background]With the rapid development of the global economy, the aging tendency of the population and the modernized lifestyles, the prevalence of type 2 diabetes mellitus is increasing. Nowadays, diabetes has become the third most important chronic non-communicable disease, which is subsequent to cardiovascular and cancer. Diabetes can lead to high rate of disability and mortality in patients. Type 2 diabetes has brought heavy economic burden to the country, society and family. So the prevention and treatment of diabetes have become important social health problems.Now it is widely accepted that insulin resistance andβ-cell dysfunction are the two main components in the pathogenesis of type 2 diabetes. The United Kingdom Prospective Diabetes Study showed that when a patient was diagnosed diabetes, hisβ-cell function was only 50% of that of a normal person, and then theβ-cell function declined at the rate of 4%-5% every year. On this basis, about ten to fifteen years before being diagnosed with diabetes, the patient'sβ-cell function had begun to fail.Many previous studies have suggested that glucose toxicity and lipid toxicity are the main pathological mechanisms ofβ-cell dysfunction.β-cell dysfunction and apoptosis caused by high glucose are the important reasons for the onset and development of diabetes. Recent studies have showed that cytokine-mediated inflammation injury also plays an important role in the onset and development of diabetes. In the past, it was generally considered that inflammatory mediators or cytokines are the key factors in the pathogenesis of type 1 diabetes, but recently studies also revealed that inflammation is closely related to type 2 diabetes. Studies indicated that fibrosis could be observed in the pancreatic islets of patients with type 2 diabetes, and fibrosis was the final stage of the chronic inflammatory disease. This is a strong evidence supporting the existence of local inflammation in islets. In addition, some scholars have pointed out that the expression up-regulation of inflammatory cytokines and chemical factors in the blood circulation could be the predictor of type 2 diabetes. Therefore, the harmful role of inflammatory cytokines in the onset and development has been a hot topic in the field of the study on the pathogenesis of the diabetes.Some studies suggested that pancreaticβcells are more sensitive to IL-1βthan to other inflammatory cytokines. IL-1βis a pro-inflammatory cytokine mainly secreted by monocyte-macrophages, fat cells and endothelial cells, and it has wide biological effects. In the onset and development of type 1 diabetes, such factors as IL-1βproduced by macrophages, tumor necrosis factor (TNF)-α, interferon (IFN)-γand so on constitute a regulatory network of cytokines causing the apoptosis of pancreaticβcells. In 2002, Maedler first reported that high glucose could induce the secretion of pro-inflammatory cytokine interleukin-1β(IL-1β) of human pancreaticβcells and using IL-1 receptor antagonist (IL-1Ra) the natural inhibitor of IL-1βcould block the harmful effects caused by high glucose on the islets, thus offer protection to 0 cells. In vivo, Psammomys were fed by high-energy diet and finally suffered from type 2 diabetes, and researchers found that IL-1βcould be expressed in theβcells of this type 2 diabetes animal model, but IL-1βcould not be expressed in theβcells of non-diabetic Psammomys. From then on, in other two kinds of type 2 diabetes animal model-Goto-Kakizaki (GK) rat and human islet amyloid polypeptid transgenic rat (HIP Rat), also found that in vivo,chronic high glucose could induceβcells to synthesize and secret IL-1β. However, some other studies reported that long-time treatment of high glucose could not induceβcells to secret pro-inflammatory cytokine IL-1β.In summary, the harmful role of cytokines in the onset and development of type 2-diabetes has gained more and more attention. Whether high glucose could induceβ-cell to express pro-inflammatory cytokine IL-1β? Whether this kind of IL-1βparticipates in the damage process of P-cell? These topics are controversial and related studies are rare. Therefore, in this study, the rat insulinoma-derived cell line INS-1 is used as theβ-cell model, our purpose is to investigate whether high concentrations of glucose could induce INS-1 cells to express IL-1βand whether this kind of IL-1βdoes damage to cells. Based on the above study, we will further explore the protective effect of NF-κB inhibitors and rosiglitazone on the pancreaticβ-cell and related machnisms, thus offer new ideas to studies related to the protection ofβ-cell.Chapter 1 Study on IL-1βexpression induced by High Glucose and its Injury effects in INS-1 cells[Objective]1.To investigate whether high glucose induce INS-1 cells express cytokine IL-1βand effects of different glucose concentrations on the expression of IL-1βin INS-1 cells.2.To study the effects of the IL-1βthat express by INS-1 cells on cells insulin secretion function and apoptosis.[Methods]1.Cell culture:The rat insulinoma-derived INS-1 cells, a widely usedβ-cell surrogate, were cultured in 5% CO2-95% air at 37℃in RPMI-1640 complete medium containing 10% fetal bovine serum(FBS),11.1 mM D-glucose.2.Experimental groupA:Effect of different glucose concentrations on the expression of IL-1βand Cell viability in INS-1 cells.(1):11.1mmol/l glucose group (11.1mM Group:glucose concentration was 11.1mM/l);(2):16.7mmol/l glucose group (16.7mM Group:glucose concentration was 16.7 mM/l);(3):27.8mmol/l glucose group (27.8mM Group:glucose concentration was 27.8 mM/l);(4):33.3mmol/l glucose group (33.3mM Group:glucose concentration was 33.3 mM/l)B:effects of the IL-1βthat express by INS-1 cells on cells insulin secretion function and apoptosis.(1):The control group (Conctol group, glucose concentration was 11.1mM/l);(2):The high glucose group (HG group, glucose concentration was 33.3mM/l)(3):High glucose+IL-1Ra (500ng/ml) group (HG+IL-1Ra group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+IL-1Ra 500ng/ml for 48 hours);3.experimental methods(1) the level of IL-1βmRNA was measured by real-time PCR.(2) INS-1 cell viability was detected by MTT assay (3) The apoptosis rate of INS-1 cell were measured by Annexin V-PI apoptosis kit.(4)basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) in INS-1 cells were measured by insulin radioimmunoassay kit.[Results]1. the levels of IL-1βmRNA:The RQ values of 11.1mM group,16.7 mM group,27.8 mM group,33.3 mM group were 1±0,2.73±0.46,8.57±0.72,114.48±9.75, respectively. The results are standardied by housekeeping gene (GAPDH) amplification efficiency and analysis by one-way ANOVA. In compared with 11.1mM group, the expression levels of IL-1βmRNA in 27.8mM group,33.3mM group were increased significantly, up to 8 fold,114 fold, respectively (P=0.01,P=0.009)2. INS-1 cell viability:The OD values of 11.1mM group,16.7 mM group, 27.8 mM group,33.3 mM group were 0.98±0.02,0.95±0.007,0.89±0.003,0.76±0.01,respectively. Data analysis by one-way ANOVA. Compared with the 11.1mM group, cell viability in 27.8mM group,33.3mM group were decreased significantly (P=0.05,P=0.002)3. correlation analysis:IL-1βmRNA expression level (RQ value) and INS-1 cell viability (OD value) showed significant negative.correlation.(r=-0.862,P<0.01).4. INS-1 cell insulin secretion function(1)the basal insulin secretion (BIS) in Control group, HG group, HG+IL-1RA group were 16.39±1.78,8.89±1.90,20.85±1.30,respectively. In comparison with Control group, HG group of basal insulin secretion was significantly lower (P=0.009). after IL-1Ra treatment, basal insulin secretion increased significantly (P =0.001).(2) the glucose-stimulated insulin secretion (GSIS) in Control group, HG group, HG+IL-1Ra group were 29.12±1.78,19.91±1.45,31.81±3.85,respectively. In comparison with Control group, HG group glucose-stimulated insulin secretion was significantly lower (P=0.002). In comparison with HG group, in IL-1Ra group glucose-stimulated insulin secretion were significantly increased (P=0.029).5.the apoptosis rate of INS-1 cell:apoptosis rates in Control group, HG group, HG+IL-1Ra group were 3.24±0.78,6.84±0.15,5.76±0.10,respectively. In comparison with Control group, the apoptosis rate of INS-1 cell in HG group was significantly higher (P=0.000). In comparison with HG group, after IL-1Ra treatment, the apoptosis rate of INS-1 cells was significantly lower (P=0.000).[Conclutions]1. High glucose induced INS-1 cells express IL-1β, the levels of IL-1βexpression dependent on glucose concentrations.2. IL-1βexpress by INS-1 cells under high glucose environment can injuryβcell function and induceβcell apoptosis.Chapter2 The damage mechanism of IL-1βexpression induced by High glucose in INS-1 cells[Objective]To investigate the mechanism of damage effects by IL-1βthat expressed by INS-1 cells under high glucose environment.[Methods]1.Cell culture and experimental grouping were just the same as those of the chapter1. (1):The control group (Conctol group, glucose concentration was 11.1mM/l);(2):The high glucose group (HG group, glucose concentration was 33.3mM/l)(3):High glucose + IL-1Ra (500ng/ml) group (HG+IL-1Ra group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+IL-1Ra 500ng/ml for 48 hours);(4):High glucose+NF-κB inhibitors (5umol/l) group (HG+NF-κB inhibitor group, cells were pretreated by NF-κB inhibitor 5umol/l for 1 hours and then exposed in 33.3 mM glucose+NF-κB inhibitor 5umol/l for 48 hours);(5):High glucose+ Rosiglitazone (5umol/l) group (HG+ rosiglitazone_group, cells were cultured in RPMI-1640 complete medium containing 33.3 mM glucose+ Rosiglitazone 5umol/l for 48 hours);2.experimental methods(1) NF-κB protein nuclear translocation of INS-1 cell was analysis by Immunofluorescence.(2) The expressions of NF-κB protein in cell nucleus were detected by western blot.(3) The level of IKKβmRNA and IL-1βmRNA was measured by realtime fluorescence quantitative RT-PCR(4) The apoptosis rate of INS-1 cell were determined by Annexin V-FITC apoptosis kit.(5) Basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS) in INS-1 cells were determined by insulin radioimmunoassay kit.[Results]1. Immunofluorescence:Cells of control group showed a normal attachment state, red fluorescence is mainly distributed in the cytoplasm, suggesting that NF-κB subunit P65 protein mainly expressed in the cytoplasm; in HG group, strong red fluorescencwere seen within the cytoplasm and nucleus of, suggesting that P65 protein happened nuclear translocation; HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group red fluorescence mainly in the cytoplasm, some weak red fluorescence distribution of cell nucleus, indicating P65 protein expression was still mainly in the cytoplasm but individual cells happened P65 protein nuclear translocation.2. The expression of P65 protein:The expression levels of P65 protein in Control group, HG groups, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 343.47±16.30,835.17±29.63,518.15±15.79,486.9±23.77,386.04±11.10,respectively. In comparison with C group, HG group nucleus P65 protein was significantly increased (P=0.000). In comparison with HG group,The IL-1RA, NF-KB inhibitor and rosiglitazone group nucleus P65 protein was significantly decreased (P=0.000, respectively). In comparison with C group, the intervention group nuclear P65 protein expression were still significantly increased (P=0.000,0.000,0.029)3. the expression levels of IKKβmRNA:The RQ values of Control group, HG group, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+rosiglitazone group were 1±0,3.10±0.17,1.21±0.15,1.12±0.15,1.32±0.12,respectively. In compared with Control group, the expression levels of IKKβmRNA in HG group were increased 3 times (P=0.000). In comparison with the HG group, the expression levels of IKKβmRNA in HG+IL-1Ra group, HG+NF-κB inhibitor group, HG + rosiglitazone group were significantly decreased (P=0.000, respectively)4.INS-1 cell insulin release function(1)the basal insulin secretion (BIS) in Control group, HG group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 16.39±1.78,8.89±1.90,21.54± 3.42,20.82±4.54,respectively. In comparison with Control group, HG group of basal insulin secretion was significantly lower (P=0.009). after NF-κB inhibitor and rosiglitazone treatment, basal insulin secretion increased significantly (P=0.011,0.049)(2) the glucose-stimulated insulin secretion (GSIS) in Control group, HG groups, HG+NF-κB inhibitor group, HG+ rosiglitazone group glucose-stimulated insulin secretion were 29.12±1.78,19.91±1.45,37.63±3.67,42.01±5.66,respectively. In comparison with Control group, HG group glucose-stimulated insulin secretion was-significantly.lower (P=0.002). In comparison-with HG group, in NF-κB inhibitor and rosiglitazone group glucose-stimulated insulin secretion were significantly increased P=0.006,0.017)5.the apoptosis rate of INS-1 cell:apoptosis rates in Control group, HG group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 3.24±0.78,6.84±0.15, 5.18±0.13,2.71±0.12,respectively. In comparison with Control group, the apoptosis rate of INS-1 cell in HG group was significantly higher (P=0.000). In comparison with HG group, the apoptosis rate after NF-κB inhibitor and rosiglitazone treatment, was significantly lower (P=0.000)6.the expression levels of IL-1βmRNA:The RQ values of Control group, HG groups, HG+IL-1Ra group, HG+NF-κB inhibitor group, HG+ rosiglitazone group were 1±0,124.70±7.22,36.91±5.89,19.86±1.58,17.81±3.11,respectively. In compared with Control group, the expression levels of IL-1βmRNA in HG group were increased significantly, up to 124 fold (P=0.004). In comparison with the HG group, the expression levels of IL-1βmRNA in HG+IL-1Ra group, HG+NF-κB inhibitor group, HG + rosiglitazone group were significantly decreased (P=0.001,0.004,0.002, respectively).[Conclusions] 1.High glucose induced INS-1 cells express IL-1βand its cause cell damage by activated NF-κB pathway.2.rosiglitazone protect pancreaticβcells by inhibits NF-κB activation, reducedβcell expression of IL-1β.