| Objectives In stress hyperglycemia,cortisol is the main glucose-increasing hormone.First,collect patients with stress hyperglycemia in the Department of Critical Care Medicine,and analyze the functional status of islet β-cells in patients with hyperglycemia.To explore the relationship between changes in islet β-cell function and cortisol under stressful hyperglycemia.Secondly,glucocorticoid-induced rat insulinoma cells(INS-1)were used to construct a cell model to explore the molecular mechanism of changes in the functional state of islet β-cells during stress hyperglycemia.Experiments can provide experimental basis for further elucidating the mechanism of stress-induced hyperglycemia islet β-cell dysfunction.Methods Analysis of β-cell function and cortisol levels in critically ill patients with stress hyperglycemia: 42 patients admitted to the Department of Critical Care Medicine were selected,and the patients were divided into stress hyperglycemia groups(n =30)and nonhyperglycemia groups(n =12)according to inclusion and exclusion criteria.The portable blood glucose meter detects fasting blood glucose(FBG),and the glycosylated hemoglobin automatic meter detects glycosylated hemoglobin(Hb A1c).Blood samples were collected from two groups of patients,and fasting insulin(FINS)and Cortisol were detected by ELISA method.HOMA-β evaluates pancreatic β cell function,HOMA-IR evaluates insulin resistance.Two independent sample t-tests were used for comparison between groups,and Pearson correlation coefficient was used for correlation analysis.P<0.05 is considered statistically significant.The rat insulinoma INS-1 cells was divided into 4 groups,normal group,low-dose group(0.01μM),medium-dose group(0.1μM),and a high-dose group(1μM)according to the addition of different stimulating concentrations of dexamethasone(DXMS).MTT colorimetry was used to detect the OD values of 24,48,and 72 hours after stimulation with different concentrations of DXMS,and the cell survival rate was calculated according to the formula.AnnexinⅤ-PI fluorescent probe was used to detect the total apoptosis rate of INS-1 cells by flow cytometry.Detect the proteins expression of GRP78 CHOP Neur3 and OCT4 by Western blotting and Real-time q PCR methods to detect GRP78 CHOP Neur3 and OCT4 m RNA expression.DCFH-DA,DHE fluorescent probe was used to detect the ROS levels in cells.Mitochondria ROS levels was detected by Mito SOX fluorescent probe.TMRE fluorescent probe detects intracellular mitochondrial membrane potential levels by flow cytometry method.Oroboros high-resolution cellular energy metabolism analysis system(O2K)is used to detect intracellular mitochondrial respiratory chain oxidative phosphorylation levels.Measurement data were described as mean ± standard deviation,and t-test of two independent samples was used for comparison between two groups.Pearson correlation coefficient was used for correlation analysis.The analysis of variance of completely random design data was used for comparison between groups,and Dunnett’s ttest was used for pairwise comparison.The calculated result P<0.05 was considered statistically significant.Results From September 2019 to February 2021,a total of 42 critically ill patients were collected,including 30 cases in the stress hyperglycemia group and 12 cases in the control group.The HOMA-β index of the hyperglycemia group was lower than the control groups,and the cortisol level was higher.Statistically compared with the normal group(P<0.05),indicating that there is pancreatic islet cell dysfunction in critically ill patients with stress hyperglycemia.Pearson correlation analysis showed that stress hyperglycemia correlated with cortisol concentration.Glucocorticoid stimulation of pancreatic islet cells(INS-1)showed that the results of MTT colorimetry were compared with the normal group.The cell survival rate of the three groups of low medium and high doses all decreased.As the concentration of glucocorticoid increases,the stimulation time becomes longer,the lower the survival rate of cells(P<0.05).AnnexinⅤ-PI detected the apoptosis rate of INS-1 cells.The average total apoptosis rate of cells in each group was 0.62% 3.67% 5.51% and 9.94%respectively(P<0.05).Real-time PCR data showed that compared with the normal group,CHOP levels increased and GRP78 decreased.Western blotting further confirmed the results of q PCR,indicating that the endoplasmic reticulum stress response in cells.Dedifferentiation related indicators Oct4 and Neurin3 was normal.DCFH-DA DHE and Mito SOX detected reactive oxygen species(ROS),compared with the normal group,the intracellular ROS level was increased(P<0.05),and it was correlated with the concentrations of glucocorticoid(P<0.05).Mitochondrial membrane potential by TMRE,it was found that compared with the normal group,the membrane potential level of each group added with glucocorticoid decreased(P<0.05).The results of the analysis of mitochondrial respiratory chain complexes I II and IV by O2 K energy metabolism meter showed that the mitochondrial oxidative phosphorylation ability of each group added with glucocorticoid was decreased(P<0.05),indicating that mitochondria were damaged.Conclusions The Critically ill patients with stress-induced hyperglycemia have islet β-cell dysfunction,which is related to cortisol levels.Glucocorticoids caused a decrease in the survival rate of INS-1 cells and induced cells apoptosis.Glucocorticoids induce severe endoplasmic reticulum stress and oxidative stress in INS-1 cells,which may be one of the reasons of islet β-cell apoptosis.The mitochondrial oxidative phosphorylation function of INS-1 cells is decreased,and mitochondria may be damaged,which is another reason for induced cells apoptosis.Figure14;Table13;Reference 86... |
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