To Explore The Mechanism Of PERK/CHOP Pathway On Apoptosis Induced By Fluctuating Glucose In Human Renal Mesangial Cells

Objective: Diabetes mellitus has become a global epidemic disease that threatens human health and life,because of the acute and chronic complications caused by diabetes,which seriously affect patients' quality of life and even endanger their lives.Compared with continuous hyperglycemia,blood glucose fluctuation is a more serious harmful factor in the occurrence and development of diabetic complications.Diabetic nephropathy is the main microvascular complication of diabetes mellitus,it is confirmed that blood glucose fluctuation is closely related to the increase of urinary albumin excretion rate in diabetic patients.In the past decade,under the influence of genetic or various harmful environmental factors,apoptosis induced by endoplasmic reticulum stress(ERS)is closely related to the pathogenesis of diabetes and its complications,which has become a research hotspot in the field of life.Therefore,at the cellular level of this study,we studied the effects of continuous low glucose or high glucose,especially fluctuating glucose on the apoptosis of human renal mesangial cells;We preliminarily explore continuous low glucose or high glucose,especially fluctuating glucose can cause endoplasmic reticulum stress induced apoptosis in human renal mesangial cells by activating the pathway of PERK/CHOP,which provides a theoretical basis for the clinician realizing glucose control stably,reducing blood glucose fluctuations and delaying the occurrence and development of diabetic nephropathy.Method: Part ?: Human renal mesangial cells were cultured in vitro,and then the cultured mesangial cells were treated in duplicate for 48 hours consistently with 11.11 mM glucose as the control(NC),consistently with 11.11 mM glucose plus 18.89 mM D-Mannitol as high osmotic control(HOS),consistently with 3.3mM glucose as LG,consistently with 30 mM glucose as HG,with 3.3mM and 30 mM glucose altering every 12 hours as FG.Apoptosis of the cells was confirmed by TUNEL assay and Annexin V-FITC/PI method.Part ?: Human renal mesangial cells were cultured in vitro,and then the cultured mesangial cells were treated in duplicate for 48 hours consistently with 11.11 mM glucose as the control(NC),consistently with 3.3mM glucose as LG,consistently with 30 mM glucose as HG,with 3.3mM and 30 mM glucose altering every 12 hours as FG.Western Blotting assay was used to detect the relative protein expression of PERK,phospho-PERK,eIF-2?,phospho-eIF-2?,CHOP,BAX.Results: Part?: There was no significant difference in the percentage of apoptotic cells between HOS group and NC group.Compared with the NC group,the percentage of apoptotic cells were increased in groups of LG,HG and FG(p < 0.05),and the number of FG group was higher than that of LG group and HG group(p < 0.05).Likely,TUNEL assay showed that there was no significant difference in the number of FITC-labeled nuclei between HOS group and the NC group.Compared with the NC group,the number of FITC-labeled nuclei in the groups of LG,HG and FG increased,and that of FG group is higher than LG group and HG group.Part II: There was no significant difference in the protein expression level of PERK and eIF-2? in NC,LG,HG and FG groups.Compared with NC group,the protein expressions of phospho-PERK and phospho-eIF-2? is higher than the groups of LG,HG and FG(p < 0.05),and further increased in FG group(p < 0.05).Compared with the groups of NC,LG and FG,the protein expression of CHOP showed an increase in FG group(p < 0.05).In the level of BAX protein expression,it is greatly higher in HG and FG groups than NC group(p < 0.05),compared with LG group,that of FG group increased a lot(p < 0.05).conclusions: In our vitro study,continuous low glucose or high glucose,and especially the fluctuating glucose may stimulate human renal mesangial cells to undergo ERS and then induce apoptosis.Compared with continuous high glucose or low glucose,the fluctuating glucose causes more severe damage to mesangial cells.Therefore,we hypothesized that hypoglycemia,hyperglycemia,and especially blood glucose fluctuations may induce renal mesangial cells to develop ERS,activate PERK/CHOP pathway,thus induce apoptosis of mesangial cells,causing increased glomerular injury and sclerosis,which leads to the aggravation and progression of diabetic nephropathy.