The Role Of MAPK/fibromodulin Pathway In Pancreatic Fibrosis Induced By Oxidative Stress

Chronic pancreatitis?CP?is a progression inflammatory disease,and the main the pathological processes of CP include acinar cell damage,acinar stress,catheter dysfunction and persistent inflammation,atrophy and/or fibrosis,endocrine and exocrine function gradually lost^[1].The main characteristics of pancreas tissue of CP patients are pancreatic atrophy,fibrosis,catheter stricture and calcification.And CP patients usually undergo long course of disease,poor prognosis and quality of life.The incidence of CP in China has been increasing gradually in recent years^[2].At present,the treatment of CP is not satisfactory,and mainly to relieve symptoms and control concurrent symptoms.Therefore,the understanding of the molecular mechanism under the development of CP can provide a new therapeutic direction for the treatment of CP.The main pathological feature of CP is that the activation of pancreatic stellate cells?PSCs?,which will cause the deposition of collagen fibers.In addition,activation PSCs can secrete chemokines and cytokines to further promote the activation of PSCs,causing inflammatory reactions and fibrosis to continue^[3].A variety of factors can cause activation of PSCs,including oxidative stress?OS?.Studies have shown that OS can induce pancreatic fibrosis by activating PSCs in vivo and in vitro^[5-12].Futhermore,antioxidants treatment can reduce abdominal pain in CP patients^[13,14].OS can promote the promotion of PSCs expression of?-SMA,ECM synthesis and ehance proliferation and metastasis by activating the MAPK signaling pathway^[15].However,there is no evidence which explain what the mechanism causes PSCs activation after OS induce the MAPK signaling pathway activation.In addition,AP-1 is the downstream effector of the MAPK signaling pathway.Therefore,FMOD is a small molecular protein and rich in leucine,participating in the ECM regulation and tissue repair process^[16].In the process of liver fibrosis,the expression of FMOD is increased,and it can induce the activation of hepatic stellate cells,thereby increasing the proliferation and invasion ability of hepatic stellate cells^[17].Therefore,FMOD is the promoter of liver fibrosis.Due to there are many similarities between the liver and the pancreatic fibrosis,and the high homologous of HSCs and PSCs in gene,morphology and function^[18],we hypothesized that in CP,FMOD can induce PSCs activation.FMOD is a kind of OS sensitive protein,and in human fibroblast cells,OS can induce FMOD expression.It was found that the FMOD promoter region contained AP-1binding sites through the analysis of biological information software.In addition,AP-1 is the downstream effector of the MAPK signaling pathway.Therefore,in this study,we hypothesized that OS can induce the expression of FMOD through MAPK/AP-1 signaling pathway,further activate PSCs and induce collagen fiber deposition,and then cause pancreatic fibrosis.Part I:Fibromodulin is up-regulated in chronic pancreatitis patients and induce pancreatic stellate cells activationObjective:To investigate the expression of fibromodulin?FMOD?in serum of patients with CP,and whether abnormal expression of FMOD can induce and promote PSCs activation,and its effect on PSCs's biological function.Methods:ELISA was used to detect the expression of FMOD in CP patients?n=10?and normal people serum samples.FMOD overexpression plasmid?FMOD-O/E?was synthesized and transfected into PSCs to construct a FMOD-up-regulated PSCs cell line.PCR and WB were used to detect the changes of PSCs activation markers,?-SMA,after overexpression of FMOD.CCK-8 and migration assay were used to detect the effect of FMOD overexpression to PSCs's biological function.Results:ELISA result showed that compared with normal people?10.10±1.47?,the expression of FMOD?56.52±6.32?in CP patients was significantly higher?t=22.64,p<0.01?.PCR results showed that the relative mRNA expression of PSCs activation marker?-SMA?1.81±0.08,t=5.3,p<0.01?increased as compared with the control group after up regulation of FMOD expression.WB showed that matching results:the protein expression of?-SMA in PSCs could be induced by up-regulation of the expression of FMOD.CCK-8 results showed that,compared with the control group,the proliferation of PSCs increased after up-regulation of the expression of FMOD.The migration assay results showed that compared with the control group?9.2±2?,the number of PSCs invasion cells significantly increased?45.53±2.77?after up-regulation of the expression of FMOD?t=18.43,p<0.01?.Conclusion:the expression of FMOD is abnormal up-regulated in CP patients,could induce PSCs activation and maintain the fibrosis phenotype of PSCs.Therefore,understand the molecular mechanism of abnormal up-regulated FMOD during CP fibrosis can provide new therapeutic targets for inhibiting the activation of PSCs and terminating the process of CP fibrosis.Part II:Oxidative stress induced overexpression of FMOD to promote the activation of PSCsObjective:To investigate whether oxidative stress can induce the activation of human PSCs in vitro,and does this effect depend on the abnormal up regulated expression of FMOD.Methods:the quiescent PSCs cells were treated with menadione?MND?and the same amount of DMSO was used as a control.PCR and WB were used to detect the changes of PSCs activation markers?-SMA after MND treatment.FMOD shRNA was synthesized and transfected into PSCs to construct a FMOD knockdown PSCs cell line.the effect of MND to PSCs activation after FMOD knockdown was detected by PCR WB,immunofluorescence.Results:PCR results showed that compared with DMSO group,the relative mRNA expression level of FMOD and?-SMA was significantly increased?3.39±1.17,P=0.036;2.20±0.52,P=0.023?.WB results also showed that the protein expression of FMOD and?-SMA also significantly increased.PCR results showed that compared with MND group,knockdown the expression FMOD cloud attenuate the MND induce?-SMA mRNA relative expression?2.07±0.11 vs.3.64±0.78,P=0.026;?.In addition,WB and immunofluorescence results also showed that the protein expression of?-SMA also significantly were attenuated.Conclusion:OS induce and promotes the activation of human PSCs by inducing the expression of FMOD in vitro.Part III:OS induces PSCs activation by up regulation of the expression of FMOD through the MAPK/AP-1 signaling pathwayObjective:To explore the mechanism of OS to induce the activation of PSCsMethods:the PSCs cells were treated with menadione?MND?and the same amount of DMSO was used as a control.WB was used to detect the protein expression of three classic MAPK signal pathways:P-38/p-P-38,ERK/p-ERK and JNK/p-JNK.ERK,JNK,P38 specific inhibitor?U0126,SP600125,SB2021906?were used,WB was used to measure the protein expression of FMOD,?-SMA,P-38/p-P-38,ERK/p-ERK and JNK/p-JNK.AP-1 overexpression plasmid was synthesized and transfected into PSCs to up-regulated AP-1.After up regulated AP-1,WB were used to detect the expression of FMOD in PSCs.We used dual luciferase reporter assay and chromatin immunoprecipitation?ChIP?to explore whether AP-1 could directly induce FMOD expression by directly combining with FMOD promoter region.Results:WB results showed compared with control group,the expression of p-P-38,p-ERK and p-JNK in the MND treatment group were significantly increased.And,WB results also showed there was no difference in FMOD expression between control group and MND+SB2021906 group.However,in MND+U0126 group and MND+SP600125group,FMOD expression significantly decreased.Bioinformatics analysis software?rVISTA?was used to analyze the transcription factor database TRANSFAC Professional.And results showed that there is a binding site of AP-1,which located in the exon region of FMOD.In addition,WB results showed that the mRNA and protein expression of FMOD were significantly increased after overexpression of AP-1.The fluorescence reporter assay result showed that compared with control group,the fluorescence of FMOD promoter region was significantly increased in AP-1 overexpression group?6.25±1.52 vs.1±0.23,P<0.001?.ChIP results also showed that in AP-1 overexpression group,the FMOD promoter region fragment was detected,and the enrichment degree was significantly higher than that in negative control group?34.51±2.26 vs.1.06±0.11,P<0.001?.Conclusion:OS induces PSCs activation by up regulation of the expression of FMOD through the MAPK/AP-1 signaling pathway.Part IV:Detect the changes of FMOD,OS,MAPK signaling pathway and the relationship between FMOD expression and pancreatic fibrosis in animal modeObjective:To verify whether if FMOD and OS is up regulated in CP pancreas tiusse and if there is a relation between FMOD and pancreatic fibrosis in the CP mouse model induced by DBTC.Methods:CP mouse model was constructed by DBTC tail intravenous injection.HE was used to evaluate whether the CP mouse model was successfully constructed,and Sirius-Red staining was used to detect the severity of pancreatic fibrosis.Immunohistochemical staining was used to detect the expression of FMOD in CP and normal pancreas tissue sample,and the MDA expression was detected by MDA detection kit.Protein was extracted from CP and normal pancreas tissue sample,and WB was used to detect the protein expression FMOD??-SMA?P-38?p-P-38?ERK?p-ERK?JNK?p-JNK.Results:4 weeks after DBTC injection,CP group survival rate was 70%,100%in control group.Starting from the 2 weeks later,the weight of CP group was significantly lower than the control group,the weight of 2,3,4 weeks was?222.46±3.40 vs.232.10±5.96,P=0.02?,?231.46±6.94 vs.251.74±6.01,P<0.01?,?233.14±6.69 vs.263.74±7.55,P<0.01?.HE results showed that there was many fibrous tissue hyperplasia,vacuolar degeneration and necrosis of alveolar cavity,infiltration of inflammatory cells in lobules and expansion of pancreatic duct in CP group,while cell edema in control group.Sirius-Red staining result showed that in CP group,except for the blood vessel and the pancreatic duct around,there are a large number of fresh red cords and reticular collagen deposition in the acinar lobule,while the VC group only blood vessels and pancreatic duct with fiber deposition around the staining,and there was significant difference between group?CP:control,0.46±0.07 vs.0.08±0.03,P<0.01?.Immunohistochemical results showed that compared with the control group,the expression of FMOD increased.Compared with CP group,the expression of MDA was significantly higher than that in the control group?2.63±1.42 vs.1.50±0.64,P=0.046?.In addition,WB results showed that compared with the control group,the expression of FMOD,?-SMA,P-38,p-P-38,ERK,p-ERK,JNK,p-JNK was significantly increased in the CP group.Conclusion:FMOD is up regulated in CP pancreas tiusse.OS and MAPK pathyway is activation in CP.