| Objective:As the main gland that secretes insulin,pancreas is an important digestive organ in the human body.It can participate in the metabolism of many essential substances such as sugar,fat,protein and so on.It is the core component of the body's material metabolism system.When the pancreas has pathological changes,it can cause diabetes,pancreatic cancer and other related diseases.This article divides the experiment into two parts around diabetes and pancreatic cancer.Diabetes is a chronic metabolic disease characterized by hyperglycemia caused by decreased insulin secretion.According to the statistics of the International Diabetes Federation at the end of 2019,there are about 463 million diabetics worldwide,including about 116.4 million people with diabetes in China,ranking first in the world.As the hyperglycemia and high free fatty acids caused by diabetes can strengthen the occurrence of oxidative stress,oxidative stress can directly damage?cells and reduce insulin secretion,thereby promoting the occurrence of diabetes.Therefore,diabetes and oxidative stress are interrelated and mutually affected.SP is a kind of neuropeptide substance,which is closely related to pancreatic diseases.It can alleviate the inflammation of islets and increase insulin sensitivity.NK-1 receptor is the most important receptor of SP.It has been found that NK-1 receptor can be expressed on pancreatic islet?cells,but not on pancreatic islet?cells.GLP-1 is a peptide substance that can be secreted by pancreatic islet?cells,and it can play an important role in the synthesis and secretion of insulin by binding to the GLP-1 receptor on pancreatic islet?cells.The first part of this study is to construct a model of islet cell injury induced by oxidative stress,combining with experiments of isolated pancreatic perfusion,to study the protective effect of SP on the pancreas and its mechanism.It will open a new path for the prevention and treatment of diabetes.Pancreatic cancer is a highly deteriorating cancer.Pancreatic ductal adenocarcinoma?PDAC?,as a pathological type of pancreatic cancer,accounts for more than 85%of pancreatic cancer.Because PDAC is more insidious and highly invasive,its treatment is still facing great challenges.Thus understanding the molecular pathological mechanism of PDAC,identifing key signal transduction pathways and screening new therapeutic targets were urgently needed.In recent years,the emergence of high-throughput sequencing technology make it more convenient to screen tumor biomarkers.The second part of this study is to explore the potential biomarkers and therapeutic targets of PDAC through bioinformatics analysis.Method:Part I:1.Effect of SP on H2O2-induced oxidative damage in rat isletsIn healthy male SD rats,collagenase??1mg/mL?was extracted from the bile duct to extract rat islets and cultured.We divided the experiment into three groups:control group,H2O2 treatment group,and H2O2 treatment group after SP pretreatment.The activity of rat islets was measured by FDA/PI staining.The cultured islets were prepared into frozen sections and used.TUNEL kit was used to detect apoptosis,and immunofluorescence staining was used to detect insulin expression.The cultured cell supernatant was collected,and the insulin secretion was detected by an ELISA kit.2.The protective mechanism of SP on H2O2-induced oxidative damage in rat isletsTo determine whether the protective effect of SP on H2O2-induced oxidative damage in rat islets was caused by binding to the NK-1 receptor,we used NK-1 receptor blockers for further research.We divided the experiment into five groups:control group,H2O2treated group,H2O2 treated group after SP pretreatment,H2O2 treated group after SP with NK-1 receptor blocker pretreatment,and H2O2 treated group after NK-1 receptor blocker pretreatment.FDA/PI staining was used to detect rat islet mass activity after culture;ELISA kits were used to detect insulin and GLP-1 secretion.Since NK-1 receptor is expressed on islet?cells,it is not expressed on islet?cells,while GLP-1 receptor is expressed on pancreatic?cells and GLP-1 can be secreted by? cells.In order to further study whether the effect of SP on H2O2-induced rat islets is related to GLP-1,we use GLP-1 receptor blockers for further research.We divided the experiment into five groups:control group,H2O2 treatment group,H2O2 treatment group after SP pretreatment,H2O2 treated group after SP with GLP-1 receptor blocker pretreatment,and H2O2 treated group after GLP-1 receptor blocker pretreatment.FDA/PI staining was used to detect rat islet mass activity after culture;ELISA kits were used to detect insulin secretion.3.Effect of SP on isolated pancreas in ratsThe pancreas was isolated,the blood vessels around the pancreas were ligated,and a pancreas perfusion model was constructed.The 5.6 mmol/L glucose solution and the SP solution with different concentrations?10-99 mol/L,10-66 mol/L?were sequentially perfused through the abdominal aorta,and portal vein effluent was collected.The ELISA kit was used to determine the content of GLP-1 in the effluent.After the isolated pancreas was perfused with a 5.6 mmol/L glucose solution or a10-6 mol/L SP solution,pancreatic tissue homogenate was extracted.The concentration of GLP-1 was detected by ELISA kit.Part?:1.Screening of differentially expressed genesThree mRNA expression profiles,GSE15471,GSE62165,and GSE28735,were selected from the GEO database as research objects.GEO2R was used to analyze differentially expressed genes?DEGs?between pancreatic ductal adenocarcinoma tissue and normal pancreas tissue.The adjusted P value was<0.05.|log2?fold-change?|?1 as the selection criteria.The Venn diagram is used to intersect the DEGs that are up or down in the three data sets,respectively,to screen out the DEGs that are up or down in common.2.Functional analysis of DEGsThe DAVID online database was used to perform gene ontology?GO?and KEGG enrichment analysis of DEGs to study the biological processes,molecular functions,cellular components,and molecular signaling pathways.3.Screening of core genes and analysis of prognosisThe PPI network of DEGs was constructed by using string database,and the connectivity of genes in PPI network was analyzed by using Cytoscape to screen core genes.Kaplan Meier-plotter online software was used to further analyze the survival of core genes to explore the prognostic significance of core genes in patients with PDAC.Results:Part I:1.Effect of SP on H2O2-induced oxidative damage in rat isletsThe results showed that compared with the control group,the activity of pancreatic islet cells decreased significantly after treatment with H2O2?P<0.01?;the apoptosis rate increased significantly?P<0.01?,and the insulin expression in pancreatic islet cells decreased significantly after treatment with H2O2?P<0.01?;the amount of insulin release was significantly reduced after treatment with H2O2?P<0.01?.Compared with the H2O2group,SP treatment can increase islet mass activity?P<0.01?,significantly reduce the number of apoptotic cells?P<0.01?,increase insulin expression in the islet mass cells?P<0.01?,and increase insulin release?P<0.05?.The above results indicate that SP has a protective effect on H2O2-induced oxidative damage of rat islets.2.The protective mechanism of SP on H2O2-induced oxidative damage in rat isletsCombined administration of NK-1 receptor blockers found that the protective effect of SP on oxidative stress islet cell activity was inhibited after administration of NK-1receptor blockers?P<0.05?;The effect of SP on insulin release of oxidative stress islet cells was also inhibited by NK-1 receptor blocker?P<0.05?.In addition,using an ELISA kit to detect GLP-1 content,compared with the control group,H2O2 treatment can reduce GLP-1 secretion by islets?P<0.01?;SP can increase GLP-1 secretion by H2O2stimulation?P<0.05?;combined treatment with NK-1 receptor blockers can cause GLP-1secretion to decrease in the islet cells of the SP group?P<0.05?.The above results indicate that the protective effect of SP on islet oxidative stress injury is achieved by activating the NK-1 receptor,and that SP can regulate the release of GLP-1 by activating the NK-1 receptor.The combined administration of GLP-1 receptor blockers found that the protective effect of SP on oxidative stress islet mass activity was inhibited after administration of GLP-1 receptor blockers?P<0.01?;The effect of SP on insulin release of oxidative stress islet cells was also inhibited by GLP-1 receptor blocker?P<0.01?.The above results indicate that the protective effect of SP on H2O2-induced oxidative damage in rat islets is related to the effect of GLP-1.3.Effect of SP on isolated pancreas in ratsCompared with the control group infused with a 5.6 mmol/L glucose solution,GLP-1 secretion was increased by infusion of SP solutions at different concentrations?10-99 mol/L,10-66 mol/L?,and the differences were statistically significant?P<0.05;P<0.01?.Compared with the control group perfused with only 5.6 mmol/L glucose solution,the content of GLP-1 in pancreatic tissue increased after perfusion of 10-6 mol/L SP solution,the difference was statistically significant?P<0.01?.Part?:1.Screening of differentially expressed genesIn the three GEO datasets GSE15471,GSE62165,and GSE28735,we obtained1552,3750,and 412 DEGs,respectively,including 1374,2507,256 up-regulated genes and 178,1243,156 down-regulated genes.Using the Venn diagram to intersect the three groups of differential genes,the results showed that 170 DEGs that were co-upregulated and 54 DEGs that were co-downregulated were identified.2.Functional analysis of DEGsThrough GO enrichment analysis,it was found that biological processes mainly include collagen catabolism,extracellular matrix decomposition,proteolysis,etc.;cell components are mainly concentrated in extracellular matrix,protein extracellular matrix,etc.;molecular functions are mainly concentrated in serine Type endopeptidase activity,calcium ion binding,etc.KEGG analysis found that DEGs are mainly concentrated in pathways such as ECM-receptor interaction,adhesion,and protein digestion and absorption.3.Screening of core genes and analysis of prognosisTwelve core genes were identified based on the connectivity of genes in the PPI network?COL1A1,COL3A1,COL5A2,COL6A3,FN1,ITGA2,MMP1,MMP9,TOP2A,ALB,EGF,POSTN?.Kaplan Meier-plotter analysis of the relationship between the expression of 12 core genes and the overall survival rate of patients with PDAC.The results showed that the high expression of the five core genes MMP1,MMP9,ITGA2,TOP2A,and POSTN was related to the poor prognosis of patients with PDAC?P<0.05?.Conclusion:1.SP has a protective effect on H2O2-induced oxidative damage of rat islets.The mechanism of this protective effect is that SP binds to NK-1 receptors on?cells to promote GLP-1 secretion by?cells.GLP-1 then binds to GLP-1 receptors on islet?cells,thereby improving islet cell function.2.In this study,we found that five core genes MMP1,MMP9,ITGA2,TOP2A and POSTN may be the new prognostic markers of PDAC and the feasible therapeutic targets for PDAC. |
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