Effect Analysis And Molecular Mechanism Research Of Nintedanib On Chronic Pancreatitis In Experimental Mouse

ObjectivesBy establishing chronic pancreatitis（CP）model in mouse with caerulein,evaluate the effects of Nintedanib（BIBF 1120,Ninte）on histological changes of pancreas and expression of genes.At the cellular level,determine the effect of Ninte on the activation,proliferation,migration,and apoptosis of pancreatic stellate cell（PSC）,and investigate its potential molecular mechanisms.MethodsEighteen C57BL/6 mice were divided randomly into control group,CP model group and Ninte treatment group（6 mice in each group）.Mice in control group were intraperitoneally injected with 0.9%Na Cl solution,while mice in CP model group and Ninte treatment group were intraperitoneally injected with caerulein（50μg/kg,6 times a day,3d/w,for 6 weeks）.Body weight was monitored every week.Mice in Ninte treatment group were intragastrically treated with Ninte（100 mg/kg,once a day,for 3 weeks）from the 4^thweek until the end of the experiment.Mice were sacrificed to obtain pancreatic tissue.H&E staining,Sirius Red staining,and Masson’s Trichrome staining were used to evaluate the inflammation and fibrosis of pancreatic tissues.The transcription levels of fibrosis and inflammatory factor genes in pancreatic tissues were detected by PCR.CCK-8 assay,flow cytometry and wound healing assay were used to determine the effects of Ninte on PSC survival rate,viability,apoptosis and migration.With no stimulation,PDGF-BB or TGF-β1stimulation,determine the effects of Ninte on the transcription and expression levels of PSC activation,proliferation and fibrosis related markers.Potential molecular targets and signaling pathways were screened by RNA-Seq,and the key targets and signaling pathways were verified by Western blot.ResultsBody weight of mice in control group increased steadily,and the final weight was24.2±0.66 g.Caerulein treatment reduced body weight of mice in the first half of experiment（1-3 weeks）.In the second half of model（4-6 weeks）,body weight of mice in model group continued to lose,and the final weight was 19.2±0.80 g.However,after Ninte intragastrical treatment,the decreasing trend of body weight of mice was reversed,and the final body weight was 21.5±0.87 g.There were significant differences in final body weights among these three groups（P<0.05）.H&E staining,Sirius Red staining,and Masson’s Trichrome staining of mouse pancreatic tissue showed that,compared with the CP control group,acinar atrophy,inflammatory cell infiltration and fibrosis in the pancreatic tissue of mice in the model group were more obvious（P<0.01）.While Ninte treatment reduced inflammatory cell infiltration and fibrosis（P<0.01）.Compared with control group,transcription levels of fibrotic markers（Fn1,Col1α1 and Ctgf）and inflammatory factors（Ccl2,Ccl5 and Cd68）in the pancreatic tissues of mice in the model group significantly increased,while Ninte treatment significantly reduced transcription levels of these markers（P<0.01）.The results showed that Ninte（<1.0μM）had no significant effect on cell survival,viability and apoptosis of PSC,but significantly inhibited migration（P<0.05）.Ninte significantly inhibited the transcription and protein expression of fibrosis markers（FN,Col1α1,PAI-1,CTGF）and proliferation-related markers（c-Myc and CCND1）in PSC with non-stimulation,PDGF-BB or TGF-β1 stimulation.However,the protein expression levels of apoptosis-related markers（Bax,Bcl-xl and PARP）were not significantly changed（P>0.05）.RNA-Seq results indicated that Ninte mainly affected the proliferation and differentiation related signal pathways.Further research verified Ninte could decrease the level of PDGFRα/βphosphorylation,inhibit activation of JAK/STAT3 signaling pathway and phosphorylation of ERK1/2,JNK,and Akt.ConclusionsNinte could significantly alleviated weight loss,pancreatic tissue fibrosis and inflammatory cell infiltration induced by caerulein in mice.In vitro,Ninte could inhibit PSC activation,migration and proliferation,but had no effect on its apoptosis.Further studies on molecular mechanisms suggested that Ninte may mediate inactivation of JAK/STAT3signaling pathway by inhibiting PDGFRα/βphosphorylation,and it may also decrease the phosphorylation levels of ERK1/2,JNK,and Akt.