| Objective:The prevalence of diabetes is increasing,and the prevalence of diabetes in China ranks the first in the world.Pancreaticβcell dysfunction and death are direct central reason to the pathogenesis of diabetes.Understanding the molecular mechanism of pancreaticβcell failure is of great significance for the development of pancreaticβcell protection methods.Studies have shown that pancreaticβcells in patients with type 1 and type 2 diabetes are under oxidative stress and endoplasmic reticulum stress,in which endoplasmic reticulum stimulation can cause pancreaticβcells damage and induce impaired insulin secretion.Nrf2 is involved in the occurrence and development of diabetes by regulating the antioxidant stress pathway.However,how is Nrf2 involved in the ER stress signaling pathway?What is the specific mechanism?The answers are still not fully understood.Therefore,this study focused on the role and mechanism of Nrf2 in endoplasmic reticulum stress induced isletβcell injury and diabetes mellitus.In this study,Nrf2 knockout mice and Akita diabetic mice were crossbred to investigate the role of Nrf2 in pancreaticβcell dysfunction and cell damage under endoplasmic reticulum stress.Methods:1.Monitoring of basic indicators:Four groups of Nrf2-WT,Ins2+/+、Nrf2-KO,Ins2+/+、Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitamice were obtained by crossing Nrf2-KO mice and Akita diabetic mice.The basic indexes of mice aged from 3 weeks to 22 weeks were detected,including body weight,fasting blood glucose,postprandial blood glucose,Glucose tolerance test（GTT）,body composition,organ coefficients and other basic indexes.2.Morphological observation:H&E staining,immunohistochemical staining of insulin and glucagon and immunofluorescence staining were performed on the pancreatic tissue of four groups of mice,which included insulin and glucagon fluorescence staining,TUNEL staining and insulin fluorescence staining.3.Cell toxicity assay:Nrf2-KD MIN6 cell line with stable and low expression of Nrf2was established by sh RNA technique.Endoplasmic reticulum stressor TG and TUN were treated with different doses of time,and the cells were divided into Scramble,Nrf2-KD,Scramble+TUN,Nrf2-KD+TUN,Scramble+TG,Nrf2-KD+TG.Cell viability was detected by MTT,and ATP content was determined by ATP detection kit.4.Detection of cell protein expression:Western Blot was used to analyze the differences in the expression of ER stress-related proteins such as BIP,e IF2α,XBP1,CHOP,PDI,ATF6α,TXNIP,as well as Insulin,proinsulin,Cleaved caspase 3,Cleaved PARP protein in the cells treated with TG and TUN.5.Data analysis:All data will be plotted and analyzed using the statistical software Graphpad Prism5.Results:1.Weight monitoring results:Nrf2-KO,Ins2+/Akitaand Nrf2-WT,Ins2+/Akitagroup had no significant difference in body weight.2.Blood glucose monitoring results:Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitadiabetic groups had significantly higher fasting blood glucose and postprandial blood glucose than Nrf2-WT,Ins2+/+and Nrf2-KO,Ins2+/+non-diabetes mellitus group.There was no significant difference in fasting blood glucose and postprandial blood glucose between Nrf2-KO,Ins2+/Akitaand Nrf2-WT,Ins2+/Akitagroups.3.GTT test results:Nrf2-WT,Ins2+/+、Nrf2-KO,Ins2+/+、Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitawere detected by GTT test at the age of 19 weeks.GTT test showed that the glucose intolerance in the diabetic group was higher than that in the non-diabetic group（P<0.05）.Moreover,Nrf2-KO,Ins2+/Akitagroup showed more severe glucose intolerance than Nrf2-WT,Ins2+/Akitagroup,and the area under the AUC curve was larger.It was suggested that the function of isletβcells in Nrf2-KO,Ins2+/Akitamice was more severely impaired.4.Basic metabolic chamber analysis:Compared with Nrf2-WT,Ins2+/+and Nrf2-KO,Ins2+/+non-diabetic groups,the food intake and water intake in Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitadiabetic groups were significantly increased,while the voluntary motor ability and respiratory ququent were significantly decreased.There were no significant differences in oxygen consumption and carbon dioxide production among all groups,and the respiratory quotient ratio of Nrf2-KO,Ins2+/Akitamice was significantly lower than that of Nrf2-WT,Ins2+/Akitagroup.This result showed that the whole metabolism of Nrf2-KO,Ins2+/Akitamice was abnormal.5.Serum insulin determination:Serum insulin levels of Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitadiabetic group were lower than those of Nrf2-WT,Ins2+/+and Nrf2-KO,Ins2+/+non-diabetic group.The levels of basal insulin and GSIS in the Nrf2-KO,Ins2+/Akitagroup were lower than those in the Nrf2-WT,Ins2+/Akitagroup（P<0.05）,which indicated that insulin secretion and pancreaticβ-cell dysfunction were decreased in the islet of mice in the Nrf2-KO,Ins2+/Akitagroup,suggesting that the damage of pancreaticβ-cells was more serious after Nrf2 gene deletion.6.Pathological morphological observation:H&E staining of pathological sections showed that Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitadiabetic mice were smaller and the number of islets in the same cross section was decreased than Nrf2-WT,Ins2+/+and Nrf2-KO,Ins2+/+non-diabetic mice,suggesting pancreatic islet injury.The quantitative results after immunohistochemical staining showed that the insulin expression in the Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitadiabetic group was lower than that in the Nrf2-WT,Ins2+/+and Nrf2-KO,Ins2+/+non-diabetic mice,and the insulin expression in the Nrf2-KO,Ins2+/Akitamice was the lowest genotype in all the four groups.7.Immunofluorescence results of insulin and glucagon:Immunofluorescence results of insulin and glucagon in pancreatic tissue showed changes in the islet structure of Nrf2-WT,Ins2+/Akitaand Nrf2-KO,Ins2+/Akitadiabetic mice.Quantitative results showed that the intensity of insulin fluorescence and the number of pancreaticβcells decreased in the diabetic group compared with the non-diabetic mice.The intensity of insulin fluorescence was the weakest and the number of pancreaticβcells was the lowest in the Nrf2-KO,Ins2+/Akitamice compared with the other three groups.8.TUNEL staining and insulin fluorescence co-staining:TUNEL staining and insulin fluorescence staining showed apoptosis of pancreaticβcells increasing in the Nrf2-KO,Ins2+/Akitagroup than that in the other three groups.The results showed that Nrf2-KO,Ins2+/Akitagroup had more severe pancreaticβcells injury and even apoptosis.9.Detection of cell toxicity:Cell viability of Scramble,Nrf2-KD,Scramble+TUN,Nrf2-KD+TUN,Scramble+TG and Nrf2-KD+TG cells showed that Nrf2-KD was more sensitive to ER stressors TG and TUN than Scramble.After the determination of ATP content in each group,ATP content in Nrf2-KD+TUN and Nrf2-KD+TG group was lower than that in Scramble+TUN and Scramble+TG groups.To further investigate the possible mechanism of pancreaticβcell injury induced by acute endoplasmic reticulum stress,the types of cell damage/death were examined.Acute exposure to TG and TUN can induce the appearance of Cleaved Caspase-3 and Cleaved PARP in a dose and time dependent manner.The levels of these markers were higher in Nrf2-KD+TG and Nrf2-KD+TUN cells than Scramble+TG,Scramble+TUN.The results showed that Nrf2-KD MIN6 cells were more sensitive to endoplasmic reticulum stress induced cytotoxicity and increased apoptosis.10.Unfolded protein response determination:The protein of the treated Scramble and Nrf2-KD cells was extracted,and the levels of ER stress related proteins BIP,ATF6a,XBP1 and CHOP in Scramble+TUN,Nrf2-KD+TUN,Scramble+TG,Nrf2-KD+TG cells group were significantly up-regulated,while the expression of ER stress related proteins BIP,ATF6a and XBP1 were significantly decreased in Nrf2-KD+TUN Nrf2-KD+TG group compared with that in Scramble+TUN and Scramble+TG group,while the expression of TXNIP was significantly increased.Compared with Scramble+TUN and Scramble+TG group,the expression of insulin protein in Nrf2-KD+TUN Nrf2-KD+TG groups was significantly decreased,while the expression of proinsulin protein was not significantly different.These results indicated that NRF2 responded to endoplasmic reticulum stress induced by TG and TUN by increasing BIP and ATF6α,which are markers of the adaptive response of unfolded proteins,and decreasing TXNIP,which are pro-apoptotic molecules,thus up-regulating the expression level of insulin protein.Conclusion:Nrf2-KO,Ins2+/Akitamice had more serious pancreaticβ-cells damage.Nrf2 can reduce the accumulation of proinsulinin in ER by increasing the expression of BIP and ATF6α,the molecular chaperone of the adaptive response of unfolded proteins,and reduce the apoptosis of pancreaticβcells and the progression of diabetes.Nrf2 plays an important role in preventing the damage of pancreatic beta cells induced by endoplasmic reticulum stress. |
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