The Effect Of Continuous And Fluctuation High-glucose Cultivation And Exenatide Intervention On The Growth Status And Bcl-2 Of HGMC

Objective:Diabetes patients often have large fluctuations in blood glucose when blood sugar control is unstable.In the past,a large number of studies have found that blood glucose fluctuations are closely related to the occurrence of chronic complications of diabetes.However,few studies reported the exact relationship between diabetes blood glucose concentration fluctuation and diabetic complications of kidney disease and its mechanism.Exenatide has been widely used clinically as a hypoglycemic agent of glucagon-like peptide 1(GLP-1)analogues,and it plays a role in preventing the further progress of diabetic nephropathy by controlling blood glucose smoothly.It is unclear whether there are other effects that can improve diabetic nephropathy.Therefore,this study aims to use human glomus mesangial cells(HGMC)cultured in vitro to be cultured with different concentrations of sugar(Normal glucose concentration,sustained high glucose and fluctuating high glucose relative to the human body)and treated with exenatide to observe the growth status of various HGMC cells and the expression of anti-apoptosis factor bcl-2 mRNA and protein,and to study the influence of glucose fluctuation on HGMC and explore the effects other than hypoglycemic action and possible mechanism of exenatide to clarify the relationship between fluctuating blood glucose and chronic renal complications from the point of view of cell biology.It will also provide theoretical support for new applications of GLP-1 drugs in clinical practice and wider application in diabetic nephropathy,and contribute to the development of new target drugs for diabetic chronic complications.Methods:1.Human glomus mesangial cells(HGMC)were cultured in vitro under suitable conditions to observe the morphological changes under an inverted microscope.2.Logarithmic growth phase HGMC were randomly divided into groups in a fetal bovine serum-free medium and grown in different mediums for 24 hours.They were:Group N: normal glucose concentration(complete medium of sugar concentration 5.6 mmol/L);Group H: continuous high glucose concentration(complete medium of sugar concentration 25 mmol/L);Group F: fluctuating glucose concentration(simulated human body glucose fluctuations after eating three meals a day,first cultured in high-glycemic complete medium for 3 hours,then cultured in normal sugar-concentration complete medium for 2 hours,alternating with 3 cycles,then overnight.);In group NG,group HG and group FG,a proper amount of exenatide was added to the medium of N,H and F,so that the concentration of exenatide reached 100nmol/L(final concentration).3.After the intervention is completed,the cells are processed or collected according to the needs of different experimental sections:(1)Observe and record the morphology and growth status of each group of cells under an inverted microscope,and photograph;(2)RNA was collected from cells and the expression of Bcl-2 mRNA in six groups was measured by real-time quantitative PCR(RT-PCR).(3)The cells were collected and the expression of bcl-2 protein was detected by Western Blot.Results:Compared with group N,the number of cells in group H and group HG was similar,but the morphology was more rounded and plump;Compared with the other four groups,the number of cells in the group F and the FG group decreased significantly,the growth state was poor,the morphologic changes,and the number of apoptotic cells increased.In the relative expression of bcl-2 mRNA,the H group was comparable to the N group(P>0.05,the difference was not statistically significant);the NG group and the HG group had comparable expression levels compared to the corresponding non-dosed group(P>0.05).The expression in F group decreased compared with that in the N group(P<0.01,the difference was statistically significant);the expression of the FG group increased relative to the F group(P<0.05,the difference was statistically significant).The relative expression of bcl-2 protein was higher in H group than in N group;F group was lower than N group;NG group had no significant difference compared with N group(P>0.05);HG group was lower than H group(P< 0.05);FG group was higher than F group(P <0.05).Conclusion:Sustained high glucose and fluctuating high glucose can affect the survival state of human mesangial cells cultured in vitro.Sustained high glucose may promote cell proliferation by inhibiting the degradation of bcl-2.Fluctuant high glucose inhibits the expression of bcl-2 mRNA and protein to promote apoptosis,and exenatide can inhibit the excessive proliferation and apoptosis of cells to a certain extent by affecting the degradation or expression of bcl-2,and can promote the cells to better adapt to changes in the environment.