Therapeutic Effect And Molecular Mechanism Of Dasatinib On Chronic Pancreatitis Mice

Chronic pancreatitis（CP）is a persistent and progressive pancreatic inflammatory disease,which often leads to destruction of the pancreatic parenchyma,infiltration of inflammatory cells,pancreatic fibrosis and calcification,causing pancreatic exocrine and/or endocrine dysfunction.There is still no specific and effective drug to reverse the process of pancreatic inflammation and fibrosis.Pancreatic fibrosis,one of the pathological characteristics of CP,is the result of the imbalance between the production and degradation of extracellular matrix（ECM）in pancreatic tissue,which is related to multiple signaling pathways in multiple cells.And the activation of pancreatic stellate cells（PSCs）and macrophage polarization（especially M2）a play a key role.Classical Wnt/β-catenin and Hippo/YAP signaling pathways are important pathways related to the growth and development of organisms.In recent years,these two pathways have been found to be related to the occurrence and development of fibrosis,and they participate in the activation of myofibroblasts.Dasatinib is an oral inhibitor of multiple tyrosine kinases with a wide range of effects,and it is currently mainly used in adult patients with imatinib mesylate-resistant or intolerable chronic myelogenous leukemia.Lots of recent basic studies shown that dasatinib could alleviate the fibrosis of multiple organs including lung,heart,liver and kidney.But the research of dasatinib on pancreatic fibrosis is rare.The purpose of this study is to investigate the effect and mechanism of dasatinib on pancreatic inflammation and fibrosis through in vivo and in vitro experiments,focusing on the effects of dasatinib on Wnt/β-catenin and Hippo/YAP pathways in PSCs,and preliminarily explore effects of dasatinib on macrophage polarization.Part I:Dasatinib Alleviates Cerulein-induced Chronic Pancreatitis Induced in MiceOBJECTIVE:To investigate the therapeutic effect of dasatinib on cerulein-induced CP in mice,then analyze the effect of dasatinib on pancreatic inflammation and fibrosis through animal experiments in vivo.METHODS:Eighteen C57BL/6 male mice were randomly divided into Control group,Cae group and Cae+Dasa group.The CP model was established by repeated intraperitoneal injection of caerulein for 6 weeks in Cae group and Cae+Dasa group.Mice in Cae+Dasa group were given dasatinib（20mg/kg/d）orally from 4^th to 6^th week after modeling.The changes of body weight and relative weight of pancreas were observed.Pancreatic tissues were retained for H&E staining,Sirius Red staining,IF staining（α-SMA and Collagen 1）,qRT-PCR of inflammation and fibrosis related genes（includingα-SMA,Col1α1,TGF-β,TNF-α,IL-6 and F4/80）.Serum levels of TGF-β1,biochemical indicators（ALT,Glu,TG,T-CHO）and H&E staining of other vital organs（liver,spleen,lung and kidney）were compared among all groups.RESULTS:Compared with Control group,CP characteristic changes such as pancreatic atrophy,infiltration of inflammatory cells and tissue fibrosis were observed in pancreas of mice in Cae group and Cae+Dasa group,suggesting that cerulein model was successful.Compared with Cae group,the growth rate of body weight and the relative weight of pancreas in Cae+Dasa group were significantly higher,and serum TGF-β1 level,pancreatic pathological score,positive area of Sirius Red staining,positive area of IF staining ofα-SMA and Collagen 1,IHC staining score of CD68 and the levels of inflammation and fibrosis related genes significantly lower（P<0.05）.Result of H&E staining showed that there were no significant differences in the histological structure of other important organs including liver,spleen,lung and kidney among three groups.And there were no significant differences in serum levels of ALT,Glu,TG and T-CHO among three groups（P>0.05）.CONCLUSION:Dasatinib could significantly alleviate pancreatic inflammation and fibrosis in cerulein-induced CP mice,and has no obvious systemic toxicity.Part II:Dasatinib Alleviates Chronic Pancreatitis by Inhibiting PSCs Activation and Macrophage PolarizationOBJECTIVE:To investigate the effect of dasatinib on the activation of PSCs and macrophage polarization in vitro,then analyze the cellular mechanism of dasatinib in alleviating CP.METHODS:The changes of proliferation and apoptosis level of human pancreatic stellate cells（HPSCs）were evaluated through CCK-8 test and flow cytometry（PI and Annexin V double staining）before and after treatment with dasatinib.WB,qRT-PCR and IF experiment were used to analyze the levels of apoptosis-related proteins（Bax and Bcl-2）,proliferation marker PCNA,activation markers（α-SMA and GFAP）and ECM-related proteins（Fibronectin and Collagen 1）in HPSCs.Scratch test and cell migration test were performed to analyze the migration ability of cells.Finally,qRT-PCR and WB experiments were used to analyze the effect of dasatinib on the expression of M1polarization markers（TNF-α,iNOS,IL-1βand CD86）and M2 polarization markers（CD206,CD301,Arg1,TGF-βand IL-4Rα）in RAW 264.7 cells（a mouse peritoneal macrophage cell line）induced by LPS,IL-4+IL-13 and conditioned medium of HPSCs.The effect of dasatinib on the expression of IL-4,IL-13,IFN-γand chemokine（CXCL1,CCL2 and CCL5）in HPSCs were analyzed by qRT-PCR experiment.RESULTS:CCK-8 experiment showed that the proportion of HPSCs surviving cells gradually decreased with the increase of dasatinib dose（P<0.05）.Flow cytometry showed that high dose（60nM）dasatinib could significantly promote the apoptosis of HPSCs（P<0.05）,while low dose（20nM）did not significantly change（P>0.05）.WB assay showed that dasatinib could significantly up-regulate the expression of Bax,and down-regulate the expression of Bcl-2 and PCNA,which meant that dasatinib inhibit cell proliferation and promote cell apoptosis in HPSCs.WB,qRT-PCR and IF experiments showed that dasatinib could significantly down-regulate the expression ofα-SMA,Fibronectin and Collagen 1,and up-regulate the expression of GFAP（P<0.05）,which meant that dasatinib inhibit cell activation of HPSCs.Scratch test and cell migration test showed that dasatinib could significantly increase the cell-free area at scratch and reduce the number of cells passing through the PET membrane（P<0.05）,which meant that dasatinib inhibit cell migration of HPSCs.WB and qRT-PCR showed that conditioned medium of HPSCs could promote M1polarization and inhibit M2 polarization of RAW 264.7 cells.Dasatinib could inhibit the expression of M1 and M2 polarization markers in RAW 264.7 cells at the same time,and inhibit the expression of IL-4 and IL-13 in HPSCs,but did not affect the expression of chemokines and receptors.CONCLUSION:Dasatinib could significantly inhibit the proliferation,activation and migration of PSCs,promote cell apoptosis,inhibit macrophage polarization and interfere with the communication between the two cells,which may be the cellular mechanism of alleviation for CP.Part III:Dasatinib Inhibits PSCs Activation through Regulating MAPK/GSK3β/β-catenin Signaling PathwayOBJECTIVE:To investigate the effect of classical Wnt/β-catenin signaling pathway,then analyze the mechanism of dasatinib in inhibiting PSCs activation.METHODS:RNA transcriptome sequencing and phosphoproteomic analysis were performed to analyze the transcriptional level of genes and protein phosphorylation of HPSCs after treatment of dasatinib.The expression of tyrosine kinase（PDGFR,Src）,MAPK protein,key protein of Wnt/β-catenin pathway（LRP5/6,GSK3β,activeβ-catenin）and target gene（CCND1,c-Myc）in HPSCs cells were analyzed through WB and qRT-PCR assay after treatment with dasatinib.RESULTS:RNA transcriptome sequencing showed that dasatinib could affect many signaling pathways such as MAPK,Hippo,TGF-βin HPSCs cells.Phosphorylated proteome showed that dasatinib inhibited the kinase levels of Src,p38 MAPK and GSK3families.Whether or not stimulated by TGF-β1 or PDGF-BB,dasatinib could significantly inhibit the phosphorylation of tyrosine kinase（PDGFR,Src）,MAPK（including ERK1/2and p38 MAPK）and downstream GSK3β,and down-regulate the expression of activeβ-catenin and Wnt target genes（CCND1,c-Myc）,but did not affect the transcriptional level of Wnt family andβ-catenin.This study showed that the phosphorylation level of LRP5/6（co-receptor of Wnt protein）increased after dasatinib administration,suggesting that LRP5/6 may not be a direct target of dasatinib.CONCLUSION:Dasatinib inhibits the nuclear entry ofβ-catenin through MAPK/GSK3β/β-catenin signaling pathway,thus affecting the expression of Wnt target genes,and inhibiting the cell activation of PSCs.And the phosphorylation of GSK3βis the key point in upstream and downstream connection of the signaling pathway.PartⅣ:Dasatinib Inhibits PSCs Activation through RegulatingHippo/YAP Signaling PathwayOBJECTIVE:To investigate the relationship between Hippo/YAP signaling pathway and pancreatic fibrosis,and analyze the effect of dasatinib on Hippo/YAP signaling pathway in PSCs.METHODS:Thirty Balb/c female mice were randomly divided into Control group,Cae group,Cae+VP group,Cae+Digi group and Cae+Dasa group.The CP model was established by repeated intraperitoneal injection of caerulein for 6 weeks in Cae group,Cae+VP group,Cae+Digi group and Cae+Dasa group.Mice in Cae+VP group,Cae+Digi group and Cae+Dasa group were given VP（YAP inhibitor,100mg/kg/d）,digitoxin（YAP agonist,1mg/kg/d）and dasatinib（20mg/kg/d）through intraperitoneal injection from 4^th to6^th week after modeling.The changes of body weight,relative weight of pancreas and serum TGF-β1 level were observed.Pancreatic tissues were retained for H&E staining,Sirius Red staining,IHC staining ofα-SMA and qRT-PCR.Then,the expression of activation markers of HPSCs,the key proteins（Mst1,Lats1,YAP）and target genes（CTGF,CYR61）of Hippo/YAP pathway were analyzed by qRT-PCR and WB experiments in HPSCs before and after treatment of VP,digitoxin and dasatinib,and the localization of YAP in HSPCs was analyzed by IF experiments.RESULTS:Compared with Cae group,the growth rate of body weight and the relative weight of pancreas in Cae+VP group and Cae+Dasa group were significantly higher,and serum TGF-β1 level,pancreatic pathological score,positive area of Sirius Red staining,IHC staining score ofα-SMA,the mRNA levels of inflammation and fibrosis related genes（α-SMA,Col1α1,TNF-α,CD68）,target genes of Hippo/YAP pathway（CTGF,CYR61）were significantly lower（P<0.05）,while the above changes in Cae+Digi group were significantly aggravated（P<0.05）.WB and qRT-PCR showed that VP and dasatinib could significantly down-regulate the expression ofα-SMA,Fibronectin,Col1α1,CTGF and CYR61 in HPSCs（P<0.05）,while digitoxin could up-regulate the expression of these genes（P<0.05）.WB showed that dasatinib could significantly inhibit the phosphorylation of PP2A,and promote the phosphorylation of Mst1,Lats1 and YAP.IF experiment confirmed that dasatinib could inhibit the nuclear aggregation of YAP.CONCLUSION:Hippo/YAP pathway is related to pancreatic fibrosis.Dasatinib could regulate the Hippo/YAP pathway by inhibiting the phosphorylation of Src/PP2A in HPSCs cells,then inhibiting the nucleation of YAP and cell activation of PSCs.