Therapeutic Effects And Molecular Mechanisms Of Pirfenidone And Fraxinellone On Chronic Pancreatitis In Mice

Chronic pancreatitis?CP?is defined as a pathological fibro-inflammatory syndrome of the pancreas in individuals with various risk factors.CP is characterized by acinar atrophy,acinar ductal metaplasia,inflammatory cell infiltration,fibrosis,dilatation/distortion/stricture of pancreatic ducts and calcification of the pancreatic parenchyma.The main clinical manifestations of CP patients are recurrent or constant abdominal pain with backache,steatorrhoea,diabetes mellitus and malnutrition.A small number of CP patients even progress to pancreatic cancer with shorter life expectancy.Clinical treatment options comprise pancreatic enzyme replacement,extracorporeal shock wave lithotripsy,endoscopic and surgical interventions,but there is no effective drug to reverse the chronic inflammation and fibrosis of pancreatic tissue.So there is an urgent need to study the pathogenesis of CP and to find effective therapeutic agents.Pancreatic fibrosis is one of the major pathological features of CP.Pancreatic stellate cells?PSCs?play a key role in the process of tissue fibrosis.PSCs are activated by inflammatory response induced by pancreatic injury and secrete extensive extracellular matrix?ECM?.The deposition of ECM in interstitial tissue induces fibrosis,which eventually leads to the decrease of pancreatic endocrine and exocrine functions.Chronic inflammatory response ensures the sustained activation of PSCs and accelerates pancreatic fibrosis.Macrophages play an important role in chronic inflammation.Macrophages of different functional types promote the development of CP.Pirfenidone?PFD?is an antifibrotic agent which has been approved by FDA for treating idiopathic pulmonary fibrosis.Fraxinellone?Frx?is a natural product extracted from Chinese herbal medicine,which exerts anti-inflammatory and anti-tumor activities.Our study will explore the therapeutic effects and molecular mechanisms of PFD and Frx on CP in vivo and in vitro,aiming to provide the experimental support for clinical drug treatment of CP.Experimental CP was induced by repeated intra-peritoneal injections of caerulein in C57BL/6 male mice for a total of 6 weeks.Mice in the control group received repeated intra-peritoneal injections of equal saline.Mice in the treatment group were orally administered with PFD or Frx from the 4^th week of caerulein to the day before sacrifice.The weight changes of mice were monitored per week.The weight of mice in the Cae group was significantly lower than that in the control group,and the weight of mice in PFD or Frx treatment group was higher than that in the Cae group,thus indicating that PFD and Frx could improve the weight changes and nutrition of CP mice.When mice were sacrificed,the pancreas tissues were paraffin embedded.The severity of CP was evaluated by H&E staining,Sirius red staining,Masson's trichrome staining and IHC staining.The staining results of pancreas sections showed acinar atrophy,inflammatory cell infiltration,acinar ductal metaplasia and fibrosis in the Cae group,while the normal histological structure was presented in the control group.The pancreatic tissue damage was significantly reduced and the pathological score was improved in the PFD or Frx treatment group.Moreover,the RNA of pancreas tissues was extracted.RT-PCR and q PCR were performed to analyze the transcription expression of target genes.Compared with the control group,the expression of fibrosis-associated genes??SMA,FN,Col1?1,TGF??and inflammation-associated genes?F4/80,CD68,TNF?,IL-6,CCL2?significantly increased,while PFD or Frx treatment downregulated the expression of fibrosis-associated and inflammation-associated genes,thus providing an explanation for the efficacy of PFD or Frx in vivo.To further investigate the molecular mechanisms of PFD and Frx,a large number of experiments in vitro were carried out,focusing on the PSCs and macrophages that played a key role in the development of CP.Firstly,the effects of PFD or Frx on PSCs activation and ECM synthesis were analyzed.The q PCR,WB and IF were used to detect the expression of PSCs activation marker??SMA?,ECM?FN,Col1?1?,fibrosis-associated cytokine CTGF,MMP2/9,and TIMP1/2.The above experiment results indicated that PFD and Frx could inhibit the PSCs activation and ECM synthesis.To further study the mechanism of PFD on inhibiting PSCs activation,RNA-seq analysis was performed in PSCs to find out the differentially expressed genes between the PFD group and the control group.It was speculated that four signaling pathways were closely related to the mechanism of PFD.The q PCR,WB and IF experiments were conducted to detect the key components of TGF-?/Smad,Wnt/?-catenin,JAK/STAT and Hippo signaling pathways.The above experiment results showed that PFD reduced the phosphorylation of Smad2/3,LRP6 and GSK3?.PFD decreased the expression of active?-catenin,CCND1,c-Myc and LEF1.Besides,PFD downregulated the p-STAT3 without affecting the phosphorylation of YAP.These results proved that PFD inhibited the PSCs activation and ECM synthesis through TGF-?/Smad,Wnt/?-catenin and JAK/STAT signaling pathways.In addition,PFD inhibited the PSCs migration via wound healing assay and Transwell migration assay,and promoted the apoptosis of PSCs by flow cytometry.To investigate the mechanism of Frx on PSCs activation,MAPK and GSK3?/?-catenin signaling pathways were analyzed.WB results showed that Frx reduced the phosphorylation of p38 MAPK and GSK3?,and downregulated the protein expression of active?-catenin,CCND1,c-Myc and LEF1,thus suggesting that Frx inhibited the PSCs activation and ECM synthesis through the p38 MAPK/GSK3?/?-catenin signaling pathway.Macrophages played a key role in the chronic inflammation of pancreas.Our study performed various experiments in vitro to analyze the effects of PFD and Frx on macrophage functions.The M1 phenotypic polarization of macrophages was stimulated by LPS,and the M2 phenotypic polarization of macrophages was induced by IL-4/IL-13.Different functional types of macrophages were treated with PFD or Frx respectively.The results of q PCR indicated that PFD downregulated the transcription of genes i NOS,TNF?and IL-1?without affecting the genes CD206,CD301 and Arg1.The results of WB showed that PFD decreased the expression of CD86,i NOS,TNF?and IL-1?without affecting CD206,IL-4R,Arg1 and TGF?.Moreover,the results of WB and IF showed that PFD reduced the p-STAT3 without affecting the phosphorylation of STAT1,I?B?and p65.Therefore,PFD inhibited the M1 phenotypic polarization of macrophages,and ameliorated the inflammatory response via STAT3 signaling,without affecting the M2phenotypic polarization of macrophages.To further investigate the effects of Frx on macrophage functions,RNA-seq analysis was performed in macrophages to find out the differentially expressed genes between the Frx group and the control group.The RNA-seq analysis suggested that Frx affected both the M1 and M2 phenotypic polarization of macrophages.The results of q PCR and WB indicated that Frx decreased the markers of M1 and M2 phenotypic polarization.WB results also showed that Frx inhibited the phosphorylation of I?B?and p65 stimulated by LPS,and inhibited the expression of IL-4R induced by IL-4/IL-13.Therefore,Frx could inhibit the pro-inflammatory function of macrophages through the NF-?B signaling pathway,and could inhibit the pro-fibrotic function of macrophages by reducing the expression of IL-4R.Moreover,the M2 phenotypic polarization of macrophages could be induced by the PSCs supernatant.The q PCR results showed that Frx reduced the expression of CCL2 secreted by PSCs,and downregulated the expression of TGF?and PDGF secreted by macrophages.In addition,Frx could also antagonize the effect of PSCs supernatant.Consequently,Frx affected the crosstalk between PSCs and macrophages.In conclusion,PFD and Frx alleviated the caerulein-induced CP in mice,and inhibited the PSCs activation and ECM synthesis.Besides,PFD and Frx ameliorated the effects of macrophages on promoting chronic inflammation and fibration.Therefore,PFD and Frx were proposed to be the clinical agents for CP.