| Objective: To study the effect of Hic-5 in pancreatic stellate cells(PSCs)on chronic pancreatitis(CP)from the histological and cytological level,and to explore its expression and regulation of downstream pathways.To explore new therapeutic targets for CP,and to provide new methods and possible laboratory evidence for CP treatment.Methods: 1.H&E staining,immunohistochemistry and Western Blot(WB)experiments was performed on human pancreatic tissue to detect the expression of Hic-5 in CP patients.2.The 8-12 weeks old wild type(WT)and Hic-5knockout(KO)C57BL/6 male mice were randomly divided into control group and model group,5 in each group,a total of 4 groups.The impact of Hic-5 on CP at the animal level was analyzed.The pancreatic fibrosis of mice was analyzed by H&E staining,Masson trichrome staining and Siriu’s Red staining.Enzyme-linked immunosorbent assay(ELISA),immunohistochemistry,WB and real-time quantitative PCR(reverse-transcription and quantitative real-time polymerase chain reaction,q RT-PCR)methods was used to detect inflammatory factors,fibrosis-related indicators and downstream pathways.3.Separated pancreatic acinar cells and PSCs from WT and Hic-5 KO mice by collagenase digestion and density gradient centrifugation.The expression of Hic-5 and its effect was analyzed on the activation of PSCs at the cell level.WB method was used to detect the expression of Hic-5 in the two cells.Cell proliferation experiment,Transwell experiment and wound healing experiment were used to detect the effect of Hic-5 on cell proliferation and migration.4.Primary PSCs was treated with different concentrations of triptolide(NF-κB/p65 inhibitor),and analyzed the effect of NF-κB/p65 on the activation of PSCs at the cell level.Cell proliferation experiment,Transwell experiment and wound healing experiment were used to detect the effect of NF-κB/p65 on cell proliferation and migration.5.The 8-12 weeks old WT mice were randomly divided into control group,model group and treatment group.In the treatment group,two weeks after the cerulein was used to induce chronic pancreatitis,the mice were treated with triptolide on the basis of the cerulein treatment.ELISA,H&E staining,Masson trichrome staining,Siriu’s Red staining,immunohistochemistry,WB and q RT-PCR were used to analyze the effects of inhibiting NF-κB/p65 on CP at the animal level.Results: 1.Hic-5 was up-regulated in pancreatic tissues of CP patients and CP mice model.2.In the CP model,the pancreas shrunk,pancreas weight decreased,fibrosis increased,and serum IL-6 levels increased.Hic-5 knockout alleviated the above changes in CP mice.3.Knockout of Hic-5 reduced the expression levels of α-SMA,pancreatic fibrosis-related factors(Col1a1,Col3a1 and TIMP1),NF-κB/p65 and IL-6.4.PSCs would be gradually activated in vitro.Knockout of Hic-5 inhibited cell proliferation,cell migration,NF-κB/p65,IL-6 expression level and the activation of PSCs induced by TGF-β.5.Triptolide is a NF-κB/p65 inhibitor,which could reduce the activation of PSCs by inhibiting the expression of NF-κB/p65 and reduce cell proliferation and migration.6.Triptolide reduced the expression levels of pancreatic fibrosis,fibrosis-related factors(Col1a1,Col3a1 and TIMP1),NF-κB/p65 and IL-6 in the CP mice model induced by cerulein.Conclusion: 1.Hic-5 is critical for the activation of PSCs.Its expression was up-regulated in activated PSCs and promoted the progress of CP.2.The loss of Hic-5 could slow down the activation process of PSCs by inhibiting the NF-κB(P65)/IL-6 signaling pathway to alleviate CP in mice.3.Triptolide,as an inhibitor of NF-κB/p65,could alleviate pancreatic fibrosis and reduce CP in mice. |
