| Objective: 1.To Culture human renal mesangial cells(HRMC)in vitro.2.To observe the effect of persistent high glucose and fluctuating hyperglycemia on HRMC growth,and the changes of the rate of proliferation and the rate of apoptosis were observed.3.To investigate the role of CHOP signaling pathway in the apoptosis of HMRC and Exenatide reducing the degree of apoptosis.4.To Confirme that GLP-1 receptor agonist Exenatide has a protective effect on HRMC,and to explore its possible mechanism.To provide a theoretical basis for the protective effect of GLP-1 receptor agonists on diabetic nephropathy,so that it can be better applied in clinical practice.Methods: 1.The human glomerular mesangial cell line was cultured in vitro and the morphological changes were observed by inverted microscope.2.HMRCs were randomly divided into 6 groups : N group: normal glucose concentration(5.6mmol / L)medium culture group,H Group: high glucose concentration(30mmol / L)medium culture group,F group: fluctuating hyperglycemia culture group(first cultured with high glucose medium for 3h,cultured with normal sugar medium for 2h,repeated 3 times a day,then low sugar medium overnight),N+G group: normal glucose concentration medium culture based on the final concentration of 100 nmol / L of Exenatide,H+G group:high glucose concentration medium culture based on the final concentration of 100 nmol / L of Exenatide,F+G group: fluctuating glicose concentration medium culture based on the final concentration of 100 nmol / L of Exenatide.The 6 groups were intervened separately for 24 h.The morphological changes of the cells were observed by inverted microscope and photographs were taken for recording.The proliferation rate of the 6 groups was determined by MTT assay;Flow cytometry was used to determine the apoptotic rate of the 6 groups;The relative expression of CHOP protein in each group was determined by Western blot;The relative expression of CHOPm RNA was determined by RT-PCR.Results: 1.Human glomerular mesangial cells cultured in vitro were in good growth conditions.2.The morphology of the cells was observed by inverted microscope.The results showed that the cell morphology changed significantly after 24 hours of fluctuating blood glucose cells,the cells were shorter.and the cell boundary was unclear and the number of cells was significantly decreased.3.MTT assay was used to detect the cell proliferation rate.The results showed that the cell proliferation rate of group H was greater than that of N group(p <0.05).The cell rate of group F was less than that of N group,the difference was statistically significant(p <0.05).After treatment with Exenatide,the cell proliferation rate of H + G group was lower than that of H group,the difference was statistically significant(p <0.05),the cell proliferation rate of F + G group was higher than that of F group,the difference was also statistically significant(p <0.05).4.Flow cytometry was used to determine the apoptotic rate of each group.The results showed that the apoptosis rate of F group> H group> N group,the difference was statistically significant(p <0.05);after treatment with Exenatide,the apoptotic rate of F + G group was lower than that of F group(p <0.05),the apoptosis rate of H + G group was lower than that of H group(p <0.05);differences were statistically significant.5.The relative expression of chop m RNA was determined by RT-PCR.The results showed that the relative expression level was F group> H group> N group,the difference was statistically significant(p <0.05),after treatment with Exenatide,the relative expression of chop m RNA in F + G group was less than that in group F(p <0.05),the relative expression of chop m RNA in H + G group was lower than that in H group(p <0.05),the difference was statistically significant.6.The relative expression of CHOP protein was determined by Western blot.The results were consistent with chop m RNA expression.The difference was statistically significant(p <0.05).After treatment with Exenatide The relative expression of CHOP protein in F + G group was lower than that in group F(p <0.05).The relative expression of CHOP protein in H + G group was lower than that in group H(p <0.05);the difference was statistically significant.Conclusion: Persistent high glucose culture stimulates glomerular mesangial cell proliferation,and volatility of hyperglycemia can inhibit mesangial cell proliferation.Persistent high glucose culture and fluctuating hyperglycemia can cause apoptosis in mesangial cells,and fluctuations in blood glucose induced apoptosis of a greater degree.Continuous high glucose culture and fluctuating high glucose culture may induce apoptosis of mesangial cells by inducing endoplasmic reticulum stress apoptotic pathway mediated by CHOP signaling pathway.The GLP-1 receptor agonist,Exenatide,may reduce the level of apoptosis by downregulating CHOP expression,which can protect the renal function,so it can prevent and control the occurrence and development of diabetic nephropathy. |
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