The Role And Mechanism Of Pancreatic Stellate Cells In Pancreatogenic Diabetes Secondary To Chronic Pancreatitis

Part 1 New-onset diabetes mellitus after chronic pancreatitis: a systematic review and meta-analysis Background: Diabetes mellitus(DM)is the major late sequelae of chronic pancreatitis(CP)and increases the mortality,but the exact occurrence of this complication has never been assessed systematically.The aim of this study was to assess the risk of new-onset DM after CP.Methods: Pubmed,Embase,and the Cochrane library were searched for English articles published from August 1978 until September 2018.The main outcome measures studied were newly diagnosed DM,and DM treated with insulin.The methodological quality of all studies was assessed by the Newcastle-Ottawa scale.For meta-analysis,studies were pooled using the random-effects model.Pooled prevalence and 95% confidence interval(CI)were calculated for all outcomes.Results: A total of 15 prospective clinical studies were eligible.The incidence of new-onset DM after CP was 30%(95% CI 27% to 33%).Among all patients,17%(95% CI 13% to 22%)developed insulin-dependent new-onset DM.Nine studies had a follow-up duration longer than 60 months,and the prevalence was 33%(95% CI,29%–37%).The proportion of alcohol CP,sex,age and body mass index had minimal effect on the studied outcomes.Conclusions: This systematic review identified a clinically relevant risk of new-onset DM after CP of which patients should be informed preoperatively.Part 2 Function changes of endocrine cells in mice with chronic pancreatitis Background: Though the intraperitoneal injection of cearulein and pancreatic duct ligation to induce chronic pancreatitis(CP)in mice,we observed the changes of exocrine and endocrine cells morphology and function.Methods: In this study,120 male C57BL/6(8 weeks old)were used as experimental animals.CP model(n=10)was established by combined injection of cearulein and lipopolysaccharide(LPS),and the control group(n=10)was peritoneal injection with the same amount of saline.In the pancreatic duct ligation group(n=50)and the control group(n=50),the pancreatic tail tissue was taken for histological treatment at 3 days,1 week,4 week,12 week and 24 week after surgery.Body weight and fasting glucose were measured weekly.HE staining and Mason staining were used to evaluate histopathological changes.Collagen type I(Col-I),fibronectin(FN)and transforming growth factorβ(TGF-β)protein were detected by immunohistochemistry.mRNA and protein expressions related to pancreatic fibrosis were detected by real-time quantitative PCR and Western blot.Activation of pancreatic stellate cells(PSCs)in pancreatic endocrine and exocrine of CP mice and changes of islet cells and α cells were evaluated by immunofluorescence staining.Results: Compared with the control group,the body weight and pancreatic quality significantly reduced in mice after cearulein plus LPS treatment,and fasting blood glucose did not change.In comparison with the control group,the mice treated with cearulein plus LPS displayed extensive extracellular matrix deposition and activated PSCs in the islet and peri-islet exocrine pancreas.The size of pancreatic islets decreased significantly in mice treated with cearulein plus LPS.In both duct ligation groupand the control group,the mice weight drop to the minimum value in 1 week postoperation,the body weight gradually increased at 3 week and with a similar level of preoperative body weight.Compare to the control group,the quality of pancreas tail is decreased significantly until 4 weeks after the operation,and the quality of the pancreatic tail basically recovered to the preoperative level at 12 weeks after ligation.The level of blood glucose in both groups had no significant difference and changes were not observed before and after surgery.After pancreatic duct ligation 3d,the pancreas tail showed severe acinar architectural damage,and edema.After 1 week,the acini is necrotic.After 4 weeks,significant pancreatic atrophy,acinar cells decreased and different degrees of fibrosis between the acini.After 12 weeks,fatty tissude and fibrous connective tissue replacement.In the early stage of pancreatic duct ligaiton,total beta cell volume has increased compared to control group,but decreased significantly at 12w after ligaiton.One week after PDL the expression of α-SMA is significantly increased in PDL tailas compared to nonligated pancreas and in parallel with the Col-Ⅰ mRNA increased levels.PDL mice displayed activated PSCs in the islet and peri-islet exocrine pancreas.Conclusions: This study was the first to compare the CP model established by injection of cearuein plus LPS and pancreatic duct ligation,and we found that the pancreatitis model established by pancreatic duct ligation was more serious and more suitable for observing the changes of pancreatic islets.Islet hyperplasia occurred in the early stage of pancreatic duct ligation mice,the area and the number of islet decreased significantly in the late stage.In mice with CP,activated PSCs increased both inside and outside the islets,which may be one of the reasons for CP to cause pancreatic endocrine disruption.This provides a basis for further studies on the effects of PSCs activation on the biological characteristics of islet and related mechanisms.Part 3 Exosomal miR-140-3p and miR-143-3p from TGF-β1 treated PSCs targets BCL2 mRNA to increase β cell apoptosis Background: Emerging evidences suggest that exosomal miRNAs are importment factors of the pathogenesis of diabetes mellitus.This study aimed to explore the effect of exosomal miRNA from TGF-β1 activated pancreatic stellate cells on β cells.Methods: Exosomes in the supernatants of mouse PSC lines(mPSCs)were extracted via ultracentrifugation and identified via transmission electron microscopy,nanoparticle tracking analysis,and Western blot.Fluorescence-labeled exosomes were incubated with MIN6 cells for 48 h to detect whether exosomes internalize into MIN6 cells.The supernatant of PSCs was collected with and without TGF-β1(2 ng/ml)treatment.The exosomes were extracted by ultracentrifugation.MIN6 cells were treated with PBS,C-Exo(exosomes extracted from non-stimulated PSCs),or T-Exo(exosomes extracted from TGF-β1-pretreated PSCs).CCK8 and glucose-stimulated insulin secretion assays were conducted to examine the cell viability and insulin secretion of MIN6 cells after incubation with exosomes.The proliferation and apoptosis rates of MIN6 cells in different groups were measured by Ed U,TUNEL,and flow cytometry.The protein expression of caspase 3 in MIN6 cells was determined by Western blot.High-throughput sequencing was performed to compare the differential expression of exosome miRNAs between C-Exo and T-Exo;then,we further validated the results of miRNAs in β cell treated with exosomes.MIN6 cell and islets were transfected with miR-140-3p and miR-143-3p minics and Nc.The ecpression of miR-140-3p and miR-143-3p levels were measured by q RT-PCR,and the cell viability and insulin secretion of MIN6 cells and islets were examined.Mi Randa,Target Scan and star Base were used to predict target genes of differential miRNAs,and the expression of B cell lymphoma 2(BCL2)mRNA and protein was detected after transfection of mimics to verify its targeting relationship with miR-140-3p and miR-143-3p.Results: Transmission microscopy showed that the exosomes were elliptical and heterogeneously distributed with a diameter ranging from 30nm to 100nm.The volume kurtosis was concentrated between 30nm and 150nm,and the exosome marker proteins CD63,CD81,and TSG101 were positive.The co-incubation results of fluorescence-labeled exosomes and MIN6 cells confirmed that MIN6 cells could ingest large amounts of exosomes secreted by PSCs.The level of high glucose used to stimulate the insulin secretion of MIN6 cells in the two exosome treatment groups did not change significantly compared with the control group(p<0.01).However,the insulin content decreased significantly after T-Exo intervention.The MIN6 cells incubated with T-Exo had lower viability and proliferation rate compared with those incubated with PBS or C-Exo.TUNEL and flow cytometry results showed that the rate of apoptosis was significantly increased in MIN6 cells treated with T-Exo compared with those treated with PBS or C-Exo.In addition,T-Exo could increase the protein levels of cleaved caspase3.Exosome second-generation sequencing screening showed that TGF-β1 could alter the miRNA profile in exosomes sereted fromPSCs,36 miRNAs were upregulated.Mi R-140-3p and miR-143-3p increased in exosome derived from TGF-β1 treated PSCs compared to control group.QRT-PCR confirmed that miR-140-3p and miR-143-3p were significantly increased mPSCs treated with TGF-β1 compare to control group.Compared with C-Exo,miR-140-3p and miR143-3p were highly expressed in MIN6 and islets after T-Exo treatment.Compared with the Nc group,the expression level of miR-140-3p and miR143-3p was significantly upregulated in minics gmup(p<0.05),and the cell vability and proliferation rate were remarkably decreased,and the cell apoptosis rate were significantly increased.Compared with Nc group,the mRNA and protein expression of BCL2 in MIN6 cells was significantly downregulated by minics incubation(p<0.05).Conclusions: Exosome from TGF-β1-activated mPSCs could reduce the survival rate and insulin synthesis of β cells,and the mechanism might be that exosomes miR-140-3p and miR-143-3p increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation.