Study On The Change Mechanism Of Wnt/β-catenin Signaling Pathway In Pancreatic Islet Cells Of Severely Scalded Rats

ObjectiveTo explore the changes of the Wnt / β-catenin signaling pathway in pancreatic islet cells of severely scalded rats and Exendin-4 treated rats by detecting the Wnt/β-catenin signaling pathway-related proteins(non-phospho β-catenin,TCF7L2,Cyclin D1)and m RNAs in pancreatic islet cells.Methods1.Preparation of the scald model: Rats were randomized into sham group(C),scald group(S),sham + DKK1 group(CD),scald + DKK1 group(SD),sham + Exendin-4 group(CE),and scald + Exendin-4 group(SE).Rats in groups S,SD and SE were immersed in a water bath kettle at 94 °C to the back for 12 s and the abdomen for 6s to produce full-thickness scald injuries involving 50% total body surface area(TBSA),and all rats were injected intraperitoneally with saline(4 ml/100 g)after sterilization,while rats in groups C,CD and CE were immersed in 37 °C warm water for the same time as controls.Rats in groups SD and CD were injected intraperitoneally with DKK1(1 μg/kg,once/d)for 3 d;rats in groups SE and CE were injected intraperitoneally with Exendin-4(4 μg/kg,twice/d)for 3 d;and rats in groups S and C were injected intraperitoneally with the same amount of sterile water for injection(once/d)for 3 d.The following experiments were conducted at day 3(72 h)post injury.2.Tail blood was sampled to measure fasting blood glucose(FBG).3.Abdominal aortic serum was collected,and Elisa was used to measure the insulin levels of serum.4.The intraperitoneal glucose tolerance test(IPGTT)was performed.The FBG and blood glucose values at 5 min,15 min,30 min,60 min,90 min and 120 min after glucose injection were recorded,and the blood glucose-time curves were drawn to calculate the area under the curve(AUC).5.The wet weight of the pancreas tissue was measured.After baking in a 60 °C oven for 72 h,the dry weight was measured,and the dry-wet weight ratio and edema index(EI)were calculated.6.Immunofluorescence staining was performed on pancreatic tissue to observe the distribution of insulin,non-phospho β-catenin and TCF7L2.7.The islets were extracted by digestive enzyme,and the Wnt/β-catenin signaling pathway-related proteins(non-phospho β-catenin,TCF7L2,Cyclin D1)were detected by Western blot.8.The transcription levels of preproinsulin,β-catenin and TCF7L2 m RNAs in the extracted islets were examined by real-time fluorescent quantitative polymerase chain reaction(RT-q PCR).Results1.FBG changes before and after scald in rats: Compared with group C,the FBG and blood glucose differences before and after scald of groups S,SD and SE increased significantly;compared with group CD,the FBG and blood glucose differences before and after scald increased significantly;compared with group CD,the FBG and blood glucose differences before and after scald of group SD increased significantly;compared with S,the FBG and blood glucose differences before and after scald of group SD increased significantly;compared with group CE,the FBG increased significantly and blood glucose differences before and after scald increased significantly in group SE;compared with group S,the FBG and blood glucose differences before and after scald decreased in group SE.2.Serum insulin concentration in rats: Compared with group C,the serum insulin concentrations of groups S,SD and SE increased;compared with group CD,the serum insulin concentration of group SD increased;compared with group S,the serum insulin concentration in group SD increased;compared with group CE,the serum insulin concentration in group SE increased;compared with group S,and the serum insulin concentration in group SE increased significantly.3.The area under the time-glucose curve in IPGTT: Compared with group C,the area under the time-glucose curve of groups S,SD,C,CD,CE and SE was significantly larger.4.EI of pancreatic tissue: Compared with group C,the EI of groups S,SD and SE increased significantly;compared with CD,the EI of group SD increased significantly;compared with group S,the EI of group SE increased significantly;compared with group CE,the EI of group SE increased significantly;compared with group S,the EI of group SE decreased.5.Immunofluorescence distribution of insulin,non-phospho β-catenin and TCF7L2 in rat pancreas tissue: Insulin was mainly located in islet cells;non-phospho β-catenin was mostly located in cell membrane;and TCF7L2 was located in nucleus.6.Content changes of the Wnt/β-catenin signaling pathway-related proteins: Non-phospho β-catenin and TCF7L2: compared with group C,the expressions in groups S,SD and SE decreased;the expression of group SD decreased compared with that of group CD;the expression of group SE decreased with that of group CE;the expression of group SE increased compared with that of group S.Cyclin: compared with C,the expression of group SD;the expression of group SD decreased compared with that of group S;the expression of group SD decreased compared with that of group CD.7.Content changes of the Wnt/β-catenin signaling pathway-related m RNAs: β-catenin,TCF7L2 and preproinsulin: compared with group C,the expressions in groups S and SD decreased;the expression of group SD decreased compared with that of group S;the expression of group SD decreased compared with that of CD.TCF7L2 and TCF7L2: the expressions in group SE increased compared with those in group S;the expressions in group SE decreased compared with those in group CE.Conclusions1.The decreased activity of the Wnt signaling pathway may be the molecular mechanism of pancreatic islet β-cell damage,reduced insulin transcription and secretion ability and glucose metabolism disorders in rats after severe scald injury.2.Exendin-4 may up-regulate the expression of TCF7L2 and increase the secretion and transcription of downstream insulin by enhancing the activity of islet Wnt signaling pathway,thereby ameliorating glucose metabolism disorders in rats after severe scald.