| Objective: Chronic pancreatitis(CP)is an irreversible chronic inflammatory stimulation caused by various factors(alcohol abuse,smoking,injury and genetic factors,etc.),which is mainly manifested by massive fibroblast proliferation and extra-cellular matrix(ECM)deposition in the pancreas leading to acinar cell loss and progressive fibrosis of the pancreas.The pathologic manifestations of CP are pancreatic atrophy,fibrosis,ductal stenosis and calcification,and the course of disease is prolonged,the prognosis is poor,and the quality of life of patients is seriously affected.In recent years,the incidence of CP in China is increasing.At present,the treatment of CP in clinic mainly focuses on relieving symptoms and controlling complications,and the treatment of the root cause of CP is lacking,so the treatment effect is not satisfactory.Therefore,understanding the molecular mechanism of CP development can provide a new direction for the treatment of CP.ECM overdeposition is a key step in CP,and activation of pancreatic stellate cells(PSCs)is considered to be the core event in CP fibrosis.Nuclear factor kappa-B(NF-?B),as a nuclear protein factor involved in inflammatory response,and p65,as its main functional subunit,play an important role,their involvement in CP has been paid more and more attention in recent years.E3 ubiquitin ligase,speckle-type POZ protein(SPOP)can specifically ubiquitin degrade intracellular proteins,playing an important role in the development and metastasis of tumors,bone development and other diseases,but its biological role in CP is still unclear.Therefore,the purpose of this study was to investigate the changes in SPOP expression in CP and its effect on the activation of PSCs in mice.Methods: 1.Animal level analysis of the expression of SPOP and NF-?B in chronic pancreatitis: a model of chronic pancreatitis in mice induced by cerulein was established.Expression of related fibrosis factors and inflammatory factors in pancreatic tissues was detected by Immunohistochemistry Staining(IHC),Western Blotting(WB)and Real-time quantitative polymerase chain reaction(qRT-PCR),and expression changes of SPOP protein and NF-?B/p65 protein were detected.2.Cell level analysis of the effect of SPOP regulating p65 on PSCs activation: mice pancreatic stellate cells were isolated,and the expression of SPOP was down-regulated by siRNA transfection.The effect of siSPOP on the proliferation and migration of PSCs was detected by Cell Proliferation Assays,Wound Healing Assays,Transwell Migration Assay and WB.3.Correlation between SPOP and FADD: Co-immunoprecipitation(Co-IP)and WB were used to prove their relationship in the activation process of PSCs.4.Whether the effect of SPOP on inhibiting PSCs depends on FADD down-regulation: siRNA transfection down-regulates FADD expression,and analyzes the effect of low FADD expression on SPOP-mediated proliferation and migration of PSCs.Results: 1.In the mice model of chronic pancreatitis,pancreatic weight decreased significantly,tissue fibrosis was obvious,and SPOP expression was down-regulated,while NF-?B/p65,IL-6 and expression of ?-SMA,collagen(Col1a1,Col3a1)and TIMP1 were up-regulated.2.Inhibition of SPOP can promote the expression of more abundant ?-SMA,ECM protein,and EMT protein,and promote the proliferation and migration of PSCs in vitro.3.Down-regulation of SPOP expression can reduce the degradation of FADD during PSCs activation,and low SPOP expression is closely related to high FADD expression.4.The down-regulation of FADD strongly inhibited the promotion of SPOP knockdown on the proliferation and migration of PSCs,and restored the expression levels of ECM proteins(?-SMA,Col1a1),EMT proteins(vimentin),NF-?B /p65,and IL-6.Conclusion: 1.Low SPOP expression in Pancreatic tissues of CP.2.SPOP partially inhibits the NF-?B(p65)/IL-6 signaling pathway via the ubiquitination/degradation of FADD,thereby inhibiting the proliferation and migration of PSCs and the progression of CP. |
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