Effect Of Taurine On The Proliferation Of Pancreatic Stem Cells In STZ-Induced Diabetic Rats

Diabetes mellitus(DM)is a metabolic disease with hyperglycemia as the main pathophysiological process caused by progressive functional failure of pancreatic beta cells.The available method of treatment is to stimulate esidual pancreatic islet function so as to achieve normal blood glucose level in short-term.However,for the long-term perspective,the classical clinical symptoms and complications of diabetes occurred as the aggravation of the residual pancreatic islet function.Recent studies have confirmed the existence of stem cells in the pancreas.To study the regulation on the proliferation of pancreatic stem cells and the differentiation into functional pancreatic beta cells in pathological conditions of diabetes have been expected to contribute the treatment of diabetes at cellular and molecular level.A number of animal experiments have shown that taurine can reduce the blood glucose level,attenuate the symptoms,and prevent complications in diabetic animals.Moreover,early results from our research group showed that taurine could increase the pancreatic stem cells and up regulate the expression of stem cell markers in diabetic rats,suggesting that taurine could promote the proliferation of pancreatic stem cells and differentiation into mature beta cells so as to lower down the blood glucose level.However,little information is available regarding the detailed mechanism.This study aims to investigate the effects of taurine on the proliferation of pancreatic stem cells and its regulatory mechanism in diabetic rats at the cellular and molecular level,so as to provide theoretical basis for the clinical application of taurine to treat human and animal diabetes.This study is divided into the following three parts:(1)Diabetes model in rats was established by single intraperitoneal injection of STZ.The pancreatic stem cells of diabetic rat were extracted by pancreatic duct separation,purified and then cultured in vitro.The expression of the pancreatic stem cell surface markers(Nestin,CK-19 and PDX-1)was identified by immunocytochemical method.(2)The pancreatic stem cells of diabetic rats were divided into control group(basic medium)and taurine treatment group(10 mmol/L taurine was added to the basic medium).The expression of surface markers(Nestin,CK-19 and PDX-1)of pancreatic stem cells in each group was detected by immunofluorescence.The effect of taurine on the proliferation and cell cycle of the pancreatic stem cells was detected by MTT assay and flow cytometry.The effect of taurine on PCNA and Ki67 mRNA and protein expressions of the pancreatic stem cells were analyzed by quantitative real-time PCR and Western-blotting.(3)The differences of the gene expression profiles of the pancreatic stem cells betweentaurine treated and untreated control were analyzed by gene chip screening.The screening standard variance analysis was based on the fold change value of more than 2 for up-regulation or down-regulation and the p value less than 0.05.The biological function and signaling pathway of the differentially expressed genes involved were analyzed by GO and KEGG enrichment analyses.Differentially expressed genes were selected for validation of the microarray data by quantitative real-time PCR.The results were as follows:(1)The diabetic model in rat was successfully established by single intraperitoneal injection of STZ(50 mg/kg).The blood glucose level was increased significantly in 72 hr after administration of STZ.Seven days later,the rats showed typical symptoms of diabetes such as polydipsia,polyphagia,polyuria,and emaciation.The final blood glucose concentration of at least 16.7 mmol/L was applied as the criteria.As a result,the diabetes was successfully induced in 70% of the rats,which can be used for in vitro cultivation of the pancreatic stem cells of experimental diabetic rats.(2)The epithelial cells of the pancreatic duct were successfully extracted using mechanical cutting method and collagenase digestion.The pancreatic stem cells were effectively purified by adherent culture,washing and passage.More than 85% of the stem cell surface markers(Nestin,CK-19 and PDX-1)positive cells were detected by immunocytochemical method.(3)The expressions of the pancreatic stem cell surface markers(Nestin,CK-19 and PDX-1)in the taurine treated and untreated control were positive by immunofluorescent staining in the cell cytoplasm,whereas,the staining for Glucagon and Insulin was negative.The semiquantitative analysis of the immunofluorescent positive samples showed that the expressions of Nestin,CK-19 and PDX-1 were slightly increased by taurine treatment,but there was no significant difference(p>0.05).The results showed that there were no significant changes in morphological and stem cell surface markers after taurine treatment,and the pancreatic stem cells extracted from diabetic rats could be passaged stably.(4)The proliferation activity of the pancreatic stem cells in diabetic rats detected by MTT was not changed significantly(p>0.05)after 24 hr treated with taurine compared with the untreated control.However,the proliferation activity was significantly increased(p<0.01)after 48,72 and 96 hr treated with taurine.(5)The results of flow cytometry revealed that the percentage of S phase and proliferation index of the pancreatic stem cells was increased from 15.73% and 50.77% to28.57% and 61.02%,respectively,by taurine treatment,indicating that taurine enhanced the cell proliferation potential of the pancreatic stem cells in diabetic rats(p<0.01).(6)The expressions of cell proliferation associated antigen(PCNA and Ki67)were detected by quantitative real-time PCR and Western Blot in mRNA and protein levels.The result showed that compared with the control,the mRNA expression of PCNA and Ki67 was increased 1.49 and 2.12 times,respectively,by taurine treatment,whereas,the PCNA and Ki67 protein level was increased from 0.26 and 0.34 to 0.56 and 0.53,respectively(p<0.01).(7)Gene expression profiles technology of pancreatic stem cells in STZ-induced diabetic rats was applied to screen differentially expressed gene treated by taurine.As a result,among3028 of differentially expressed m RNAs,2268 mRNAs were up-regulated(Fold change≥2,p<0.05),whereas,760 m RNAs were down-regulated(Fold change≤-2,p<0.05).The results of gene chip analysis were in consistent with the m RNA levels of differentially expressed genes detected by quantitative real-time PCR,indicating that the chip data was highly reliable.(8)The GO enrichment analysis includes the biological process categories,cellular component categories and molecular function categories.Of the biological process categories,17184 genes were available on the entire microarray,1806 differentially expressed genes were having at least one biological process annotation,and the most highly enriched GO terms were volved in negative regulation of apoptotic process,aging,positive regulation of transcription from RNA polymerase II promoter,response to hypoxia,and wound healing.Of the cellular component categories,17746 genes were available on the entire microarray,1863 differentially expressed genes were having at least one cellular component annotation,and the most highly enriched GO terms were volved in nucleus,cytoplasm,extracellular exosome,membrane and focal adhesion.Of the molecular function categories,16594 genes were available on the entire microarray,1757 differentially expressed genes were having at least one molecular function annotation,and the most highly enriched GO terms were volved in protein binding,poly(A)RNA binding,ATP binding,protein complex binding,and protein heterodimerization activity(FDR_bh<0.05).(9)The KEGG enrichment analysis showed that 7808 genes were available on the entire microarray,including 702 up-regulated genes and 216 down-regulated genes.The signal pathways regulated by up-regulated genes were TNF signaling pathway,protein processing in endoplasmic reticulum,pathways in cancer,NOD-like receptor signaling pathway,and proteoglycans in cancer.Whereas,the down-regulated genes were involved in the signaling pathways for alcoholism and systemic lupus erythematosus(FDR_bh<0.05).(10)Ten signaling pathways including TNF signaling pathway,NF-κB signaling pathway,PI3K-AKt signaling pathway,FoxO signaling pathway,TGF-β signaling pathway,Ras signaling pathway,MAPK signaling pathway,Jak-STAT signaling pathway,Notch signaling pathway,and Wnt signaling pathway were screened and identified using bioinformaticsmethod.Furthermore,the analysis of the gene expression profiles showed that NF-κB and Ras/ERK signal transduction pathways have closely related to regulation on the proliferation of pancreatic stem cells by taurine.The results of quantitative real-time PCR revealed that tautine significantly increased the m RNA of the regulated target genes including Chuk,Egfr,Il1r1,Mapk1,Mki67,Nfkb1,Nras,Pcna,Pdgfra,Rela,Ripk1,and Tlr4 in NF-κB and Ras/ERK signaling pathways(p<0.01).These results suggest taurine regulated the proliferation of pancreatic stem cells in diabetic rats through NF-κB and Ras/ERK signaling pathways.In summary,the STZ-induced diabetic rat model was successfully established by single intraperitoneal injection of STZ.The pancreatic stem cells derived from the pancreatic duct in diabetic rats were successfully extracted,separated,purified and stably cultured in vitro by passaging.Taurine significantly increased the viability,DNA synthesis and proliferation index of the pancreatic stem cells in diabetic rats.Moreover,taurine up-regulated the expressions of PCNA and Ki67 at mRNA and protein levels.Furthermore,taurine regulated the proliferation of pancreatic stem cells in diabetic rats through NF-κB and Ras/ERK signaling pathways.