| Alcoholic chronic pancreatitis(ACP)is the most common chronic pancreatitis(CP),which is mainly characterized by pancreatic fibrosis.The incidence and prevalence of ACP are rising but there is a lack of effective treatment strategy.Transforming growth factor(TGF-β1)is the most important fibrogenic factor,which can activate pancreatic stellate cells(PSCs)to secrete extracellular matrix(ECM),resulting in pancreatic fibrosis.Vitamin D(VD)is a multipotent steroid hormone that has been shown to be a multipotent regulator of cell proliferation,apoptosis,differentiation,and autophagy.Studies have shown that VD deficiency is prevalent in patients with CP.Vitamin D receptor(VDR)is the nuclear receptor of VD.Studies have preliminarily shown that VD and its analogues can inhibit pancreatic fibrosis by inhibiting PSC activation and ECM production,suggesting that VD may be a potential drug for the treatment of pancreatic fibrosis.However,the expression characteristics of VDR in PSC and its correlation with TGF-β1 content or the degree of pancreatic fibrosis in ACP are still unclear.Additionally,the inhibitory effect of VD/VDR pathway on the protein expression profile of PSC-mediated pancreatic fibrosis and its regulatory mechanism have not been fully elucidated.This paper will be elaborated in the following three parts on the experimental study of VD and its receptor pathway in inhibiting PSC-mediated pancreatic fibrosis.Part one:The expression characteristics of VDR in PSC of ACP patients and the antifibrotic potential of VD targeting PSCObjects:To clarify the expression characteristics of VDR in PSC and its correlation with the content of TGF-β1 and the degree of pancreatic fibrosis in ACP and explore the antifibrotic potential of VD targeting PSC and evaluate the rationality of using VD in ACP patients.Methods:(1)Immunohistochemical staining was used to detect the expression of VDR in the pancreatic tissue in ACP.(2)Double-labeling immunofluorescence,q RT-PCR and Van Gieson staining were used to compare the differences of VDR~+PSCs,COL1A1m RNA and collagen deposition areas in the pancreas of ACP versus HC and the correlation between VDR~+PSCs and COL1A1 m RNA or collagen deposition areas.(3)Rat PSC lines(RP-2 cells)were used to build the cell models in vitro.q RT-PCR,Western Blot and immunofluorescence were used to detect the influence of alcohol(ALC)on Cal-induced VDR expression.(4)Levels of TGF-β1 in serum and in supernatant of RP-2 cells were detected by ELISA to evaluate the role of TGF-β1 in pancreatic fibrosis of ACP and in the activation of PSCs.(5)The serum 25(OH)D3levels in ACP and HC were detected by mass spectrometry and serum COL1A1 levels were detected by ELISA to evaluate the effect of VD deficiency on COL1A1production in ACP.Results:(1)VDR was expressed in pancreatic islet cells,acinar cells and interstitial cells in ACP patients,while VDR was only expressed in the pancreatic islet in HC.VDR was expressed in most activated PSCs in the pancreas of ACP.(2)The number of VDR~+PSC,the content of COL1A1 m RNA and the deposition areas of collagen in the pancreas of ACP were significantly higher than that in HC and there were positive correlations between the number of VDR~+PSC and the content of COL1A1 m RNA or the collagen deposition areas(r=0.82,P<0.01;r=0.70,P<0.01).(3)ALC could induce the production and secretion of TGF-β1 in RP-2 cells and ALC could enhance the expression of VDR in RP-2 cells induced by Cal(P<0.001).(4)TGF-β1 levels in the serum or pancreas of ACP patients were significantly higher than those of HC(both P<0.001).(5)The serum 25(OH)D3 level in ACP patients was significantly lower than that in HC(P<0.001)and the serum COL1A1 level in ACP patients was significantly higher than that in HC(P<0.001).Conclusions:Deficiency of VD in ACP patients may be a predisposing factor for triggering TGF-β1-induced COL1 synthesis and the activated PSCs are a target of VD/VDR pathway.Therefore,PSC may be a potential target for VD to treat pancreatic fibrosis in ACP.Part two:Inhibitory effect of VD analog calcipotriol on profibrotic protein profile of TGF-β1-induced PSCsObjects:To detect the profibrotic protein profile of Cal antagonizing TGF-β1-induced PSC mediated fibrosis and the antagonistic effect of Cal on fibrosisMethods:(1)RP-2 cells were cultured in 2.5%FBS DMEM in 10 cm culture dish for 24 h,and then were cultured in 1%FBS DMEM for 12 h.The cells were then divided into three groups,(i)control group(vehicle buffer),(ii)TGF-β1(10 ng/ml)group,and(iii)TGF-β1(10 ng/ml)+Cal(100 n M)group.After 48 h,RP-2 cells from each group were collected and protein extraction and pancreatic enzymolysis were carried out.Liquid chromatography-mass spectrometry was used for protein separation and analysis.Quality control,annotation,functional analysis,differential expressed protein screening of mass spectrum data were carried out by bioinformatics analysis methods.(2)Western Blot was used to verify six randomly selected differentially expressed proteins.Small interfering RNA(si-RNA)was used to knock down the VDR gene in RP-2 cells and q RT-PCR was used to detect six selected m RNAs and clarify whether Cal antagonize the expression of the above proteins via VDR pathway.Results:(1)A total of 5712 shared proteins were detected in three groups of samples.Fold change(FC)>|±1.5|,p<0.05 were used as the screening criterion for differentially expressed proteins,there were 253 up-regulated proteins and 267 down-regulated proteins in TGF-β1 versus Ctrl group,and 60 up-regulated proteins and 97down-regulated proteins in TGF-β1+Cal versus TGF-β1 group.(2)Cal significantly down-regulated 16 key profibrotic proteins in TGF-β1-induced RP-2 cells,including7 ECM components(COL4A1,COL5A2,COL1A1,COL1A2,COL5A1,COL3A1and EMILIN1),2 cytoskeletal proteins(VIM andα-SMA),2 fibrosis-associated factors(RUNX1 and TRAF2),TIMP1,CCN1,ITGA11,a scaffold adhesion protein(TGFB1I1),and an enzyme mediating TGF-β1-induced fibrogenesis(ENPP1).(3)Cal antagonized TGF-β1-induced PSC activation and fibrotic protein synthesis through VDR pathway.Conclusions:Cal antagonize many important profibrotic proteins in TGF-β1-induced PSCs through VDR pathway.Some of these proteins belong to ECM and others are involved in PSC activation,differentiation,adhesion and other functions.Therefore,exogenous VD supplementation may be a potential method to treat pancreatic fibrosis.Part three:Calcipotriol down-regulate RUNX1 to inhibit TGF-β1/SMAD3 signaling pathway-mediated collagen 1 synthesis in rat PSCObjects:The aims of this part were to clarify the role of RUNX1 on regulating TGF-β1/SMAD3 signaling pathway-mediated COL1 synthesis in PSCs and clarify the potential mechanism of Cal on antagonizing COL1 synthesis in PSC in order to provide experimental basis and theoretical basis for VD and its analogues in the treatment of pancreatic fibrosis.Methods:RP-2 cells were treated with 10 ng/ml TGF-β1 as the cell model.Immunofluorescence was used to detect the cell localization of RUNX1;q RT-PCR was used to detect RUNX1,COL1A1 and COL1A2 m RNA;Western Blot was used to detect RUNX1,SMAD2/SMAD3/p SMAD3,COL1A1,COL1A2 andβ-Tubulin.Co-IP was used to detect p SMAD3/RUNX1/CBFβcomplex.si-RNA was used to knock down RUNX1,SMAD2 and SMAD3 gene expression;p CMV-Smad3,p CMV-Runx1overexpressing plasmids,p Runx1-luc,p Col1a1-luc and p Col1a2-luc luciferase reporter plasmids were constructed to evaluate the transcription of COL1 regulated by RUNX1.Results:(1)TGF-β1 could enhance RUNX1 expression and intranuclear aggregation in RP-2 cells.(2)TGF-β1 induced an early synthesis of RUNX1 and a delayed synthesis of COL1A1 and COL1A2 in RP-2 cells.(3)Knockdown of SMAD3 or overexpression of SMAD3 could down-regulate or up-regulate RUNX1,COL1A1and COL1A2 synthesis respectively,suggesting that TGF-β1/SMAD3 pathway mediates RUNX1 and COL1 synthesis in RP-2 cells.(4)The results of Co-IP showed that TGF-β1 could increase p SMAD3/RUNX1/CBFβformation in RP-2 cells,knockdown RUNX1 m RNA and inhibition of CBFβbinding with RUNX1 or overexpression of RUNX1 could down-regulate or up-regulate the synthesis of COL1A1 and COL1A2 respectively,suggesting RUNX1 as a coactivator participating in TGF-β1/SMAD3-mediated transcriptional regulation of COL1.(5)Cal did not affect TGF-β1-induced p SMAD3 levels,but significantly inhibited TGF-β1-induced RUNX1 levels and p SMAD3/RUNX1/CBFβcomplex formation.Conclusions:RUNX1 is an early response product mediated by TGF-β1/SMAD3 in PSC.As a co-activator,RUNX1 is involved in the transcription of TGF-β1-induced COL1m RNA in PSC in the form of p SMAD3/Runx1/CBFβ.Cal inhibits TGF-β1/SMAD3-mediated COL1 synthesis by down-regulating RUNX1.This study suggests that RUNX1 may be a potential target of Cal in the treatment of pancreatic fibrosis. |
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