| Diabetes has become the third major chronic disease threatening human health after tumor and cardiovascular disease.The prevalence of diabetes in China is increasing year by year and rising rapidly.In type 2 diabetic patients,the secretion function of pancreatic?-cell is decreasing,and insulin resistance is present.Therefore,it is of great significance to protect the secretion function of pancreatic islet cells for the prevention and cure of diabetes.The high-salt diet is an important global topic,and the epidemiology has shown that the high-salt diet is associated with human diabetes.Pancreas islet Min6 cell line from mice is a commonly used cell model in the study of insulin secretion.This experiment mainly investigated the effects of high-salt environment on secretion of pancreas islet Min6 cells,and explored its possible mechanism.ObjectiveTo investigate the effects of high-salt environment on secretion of pancreas islet Min6 cells,and its possible mechanism.MethodsPancreas islet Min6 cell line from mice bought before were used.Three groups weredesigned,thatis,thecontrol,Min6cellstreatedwith high-salt(50mmol·L-1NaCl),and Min6 cells treated with mannitol(100 mmol·L-1)to produce the same osmotic pressure as high-salt.Results were detected 24h,48h and72h after drug administration.First,the effect of high-salt stimulation on the insulin secretion of Min6 cells was investigated.The amount of insulin secretion was measured by enzyme-linked immuno sorbent assay?ELISA?.Secondly,the effect of high-salt stimulation on the proliferation activity of Min6cells was investigated.In order to identify the survival state of Min6 cells before using,Trypan Blue staining was used to detect the number of living cells.After confirming the activity of Min6 cells,Cell Counting Kit-8?CCK8?was used to detect the proliferation inhibition rate of Min6 cells.Finally,the effect of high-salt stimulation on the apoptosis of Min6 cells was investigated.The number of apoptotic cells was observed by Hoechst 33258 staining method,and the percentage of apoptosis of Min6 cells was detected by Annexin V FITC/PI double staining by flow cytometry.The expression of bcl-2 protein in Min6cells was detected by Western blot 48h after high-salt administration.ResultsFirst,at the above three time points,compared with the control group,the amount of insulin secretion of Min6 cells was decreased significantly after high-salt stimulation,with a dose-dependent manner.But there was no obvious difference in insulin secretion between the mannitol group and the control group.Secondly,Min6 cells used in this experiment were in good survival state with dead ones less than 2%.At each time point,compared with the control group,the proliferation inhibition rate of Min6 cells after high-salt stimulation was increased significantly,with a dose-dependent manner.After 72h stimulation by the mannitol,the proliferation inhibition rate was significantly increased,while there was no obvious difference after 24h and 48h stimulation compared with the control group.However,the cell proliferation inhibition rate in the high-salt group was significantly increased compared with that in the mannitol group after 72h stimulation.Finally,the Hoechst 33258 staining showed that,compared with either control or mannitol group,the number of apoptotic cells in Min6 cells after high-salt stimulation was significantly increased at the three time points.Annexin V FITC/PI double staining by flow cytometry confirmed that the proportion of apoptotic Min6 cells after high-salt stimulation was significantly increased.However,the mannitol group showed no obvious changes in the proportion of apoptosis.In the meanwhile,compared with the control group,the bcl-2 protein expression in Min6 cells was significantly inhibited after 48h high-salt stimulation.Conclusion1.High-salt treatment could dose-dependently reduce insulin secretion in Min6 cells, while osmotic pressure itself did not influence this process.2.High-salt treatment could dose-dependently reduce the proliferation activity of Min6 cells.This partly explained the reduction of insulin secretion in Min6 cells caused by high-salt.3.High-salt treatment could dose-dependently promote the apoptosis of Min6 cells by inhibiting the bcl-2 protein expression.This may be one of the mechanisms by which high-salt reducing the insulin secretion in Min6 cells. |
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