| Part I A Mouse Model of Chronic Pancreatitis on the PNLIP Gene MutationsBackground and Aims: Variants in PNLIP genetic polymorphisms are a novel genetic risk factor for chronic pancreatitis(CP).The first report of a pathogenic PNLIP variant was of two brothers from a consanguineous marriage who were homozygous for a missense mutation(c.662C>T;p.T221M)in PNLIP.Clinical evaluation showed that both brothers had steatorrhea associated with PNLIP deficiency,suggesting insufficient pancreatic exocrine and may be associated with chronic pancreatitis.O ur previous experiments in vitro demonstrated that PNLIP p.T221 M misfolds and triggers endoplasmic reticulum stress(ERS)and unfolded protein response(UPR),suggesting that protein toxicity is potentially another CP pathogenesis.But this is only in vitro,there is no direct evidence in vivo to support our hypothesis.Since many experiments that illuminate the mechanisms of disease can not or should not be performed on patients,experiments in vivo often rely on replication of animal models.Multiple animal models show their own characteristics in the pathophysiology of chronic pancreatitis,such as hyranin or arginine intraperitoneal injection,sodium bezocholate injection into the pancreatic duct,pancreatic duct ligation,and so on.Recently,a few animal models based on the "protein misfolding" theory have been reported,as an example,carboxypeptidase A1(CPA1)mutation trigger chronic pancreatitis through protein misfolding.However,CPA1 is a protease,this study still cannot exclude the possibility that the abnormal proteolytic function of CPA1 leads to the occurrence and development of CP.Hence,the study we plan to carry out,"whether the mutation of PNLIP leads to CP and whether the pathogenic mechanism is caused by protein toxicity",can solve this confusion well.Therefore,we plan to go through further study in vivo based on the previous experiments in vitro.Our objective was to create a mouse model to determine if PNLIP p.T221 M causes CP and to define the mechanism.Accordingly,the primary purpose of the first part of this study was to create a mouse model of Pnlip p.T221 M using CRISPR/Cas9 genome editing technology.To test whether it can mimic human PNLIP p.T221 M mutations in causing chronic pancreatitis,and whether its characterization marks the clinical CP phenotype,such as pancreat ic atrophy,loss of acinar cells,immune cell infiltration,fibrosis,fat change,decreased exocrine function,etc.Methods: We used multiple methods including,gene-editing,immunohistochemistry,histopathology,transmission electron microscopy,western blot and q PCR,to determine if T221 M animals cause CP.Results:1.The PNLIP p.T221 M cell analysis model was successfully constructed.It was confirmed that the mouse PNLIP p.T221 M mutation caused protein misfolding and activation of ER stress,consistent with we reported in human PNLIP p.T221 M.2.Our experiments in vivo confirmed the results in vitro.It was demonstrated by Western blotting that PNLIP clustered in mouse Pnlip p.T221 M acinar cells and could not be secreted extracellular.3.The decrease in the total activity of amylase besides lipase in the pancreas indicated that T221 M mice had reduced exocrine function.4.Changes in pancreatic tissue structure were observed in Pnlip p.T221 M mice,including progressive pancreatic atrophy,acinar cell loss,fibrosis,immune cell infiltration,fatty infiltration.5.The biochemical indexes in Pnlip p.T221 M mice had been changed,including the elevated levels of serum amylase,macrophages,neutrophils,T cells,and B cells.The upregulation of nuclear factor-B(NF-kB)indicates that the expression of Pnlip p.T221 M activates the NF-kB pathway,induces inflammatory response.Conclusions:1.It was demonstrated that mouse Pnlip p.T221 M can mimic the biochemical properties of human PNLIP p.T221 M,including reduced secretion,intracellular aggregation,reduced bioactivity,etc.2.The detection of pancreatic exocrine function,pancreatic tissue structure and biochemical indicators confirmed the spontaneous development of CP in Pnlip p.T221 M mice.It’s characterized by pancreatic tissue atrophy,acinar cell loss,fibrosis,fat alteration and inflammation cells infiltration.Part II Mechanistic Study of PNLIP Mutations leading to Chronic Pancreatitis in MiceBackground and Aims: In the first part of the experiment,we created a knock-in mouse model of p.T221 M at the murine PNLIP site by gene editing techniques.It was also verified that the Pnlip T221 M mice developed a progressive CP phenotype.But it was unclear about its pathogenesis.Previously,premature activation of trypsinogen in the pancreas had been considered as a key factor in the onset of pancreatitis.However,PNLIP has no trypsin activation domain,and it has no known role in the regulation of trypsin activity.Thus,PN LIP mutations leading to CP may be apart from trypsin-dependent mechanisms.Based on our previous research,we propose the scientific hypothesis that the PNLIP p.T221 M mutation may have no effect on trypsin activity,but it will cause protein misfolding and trigger ER stress,which leads to the development of CP.Hence,the next step of the study is to test the hypothesis and provide another underlying mechanism driving CP through mutation-induced protein misfolding and to uncover novel therapeutic inter vention targeted on mitigating proteotoxicity in CP.Methods: We used various methods including histopathology,immunohistochemistry,transmission electron microscopy,western blot and q PCR.Our aim was to explore whether the mechanism of chronic pancreatitis in Pnlip p.T221 M mice is proteotoxicity caused by misfolded protein accumulation.Results:1.We observed only a much smaller increase in the intrapancreatic trypsin activity in Pnlip T221 M mice,with no obvious age-dependent trend.2.By immunoblot,both WT and p.T221 M were present in the soluble fraction whereas only PNLIP p.T221 M was detectable in the insoluble fraction,indicating that p.T221 M misfolded,resulted in a intracellular accumulation as a detergent-insoluble aggregate and secretion defect.3.Pancreas of T221 M mice showed ultrastructural changes in ER stress(expanded ER)and reduction in the size of zymogen granules,indicating that the amount and structure of the digestive enzymes changed,ER stress was activated.4.It was determined that the expression of Pnlip p.T221 M triggered the UPR in the mouse pancreas,especially the PERK(protein kinase RNA-like ER kinase)arm of the UPR.5.Pnlip p.T221 M misfolding triggered cellular stress reactions a,leading to acinar cell apoptosis and the development of CP.Conclusions:Expression of Pnlip p.T221 M in a pre-clinical mouse model results in CP caused by ER stress and proteotoxicity of misfolded PN LIP p.T221 M. |
