Research On The Role Of CFTR In Age-dependent Nonalcoholic Steatohepatitis

Objective: To investigate the expression change and molecular mechanisms of cystic fibrosis transmembrane conductance regulator(CFTR)in the development of NASH using Cystic fibrosis(CF)animal model and nonalcoholic steatohepatitis(NASH)clinical specimens.To provide a breakthrough for the explanation of the etiology of NASH,the expression change and molecular mechanisms of CFTR in the development of age-dependent NASH were investigated.Method:This project was divided into two parts according to the experimental subjects(CFTR mutation animal model(hereinafter referred to as △-9 rabbits)and clinical patients’ tissue specimens): 1.△-9 rabbit animal model: We collected liver and biliary tissues from Wild Type(WT)rabbits and △-9 rabbits at different ages,and comparison of relative liver weight(liver weight/body weight)at the same age to analyze the effect of CFTR mutation on liver development;AKT/GYS2/GSK3β is a key signal pathway that regulate glucose homeostasis and control glycogen synthesis,therefore,we used Western blot to investigate the molecular mechanism of abnormal glucose homeostasis caused by CFTR mutation;to investigate the changes in the regulation of lipid metabolism in △-9 rabbit liver,we used Western blot and immunohistochemical staining to detect the lipid metabolism-related signaling pathway CREBH/PPARα/FGF21;to find the molecular mechanism of NASH-like symptoms caused by CFTR mutation,we analyzed the endoplasmic reticulum stress(ER stress)in the liver.We collected liver tissues from △-9 rabbits of different ages to detect ER stress markers: IRE-lαand XBP-1 by immunohistochemistry and Western blot.2.Liver tissue specimens from patients with advanced fibrosis or cirrhosis in NASH: Surgical or biopsy liver tissues(liver fibrosis or NASH-derived liver paraneoplastic tissues)were collected.The CFTR protein expression level of liver tissues from patients with NASH was analyzed by immunohistochemistry;the whole genomic DNA of peripheral blood was extracted from patients with NASH,and DNA sequencing was performed to detect whether CFTR gene mutation occurred.Results: Compared with WT rabbits,the relative liver weight of △-9 rabbits at the same age was significantly reduce.Western blot show that AKT,GYS2,and GSK3β expression isdramaticly increase in △-9 rabbits above 50 days;however only GSK level is altered in △-9rabbits less than 50 days,demonstrating that CFTR mutation disrupted glucose homeostasis in an age-dependent manner.The CREBH precursor(CREBH-P),activated CREBH protein(CREBH-A),FGF21 and PPARα expression level in the liver of △-9 rabbits progressively increase with age,and the upregulation of the CREBH/PPARα/FGF21 regulatory axis may represent feedback regulation in liver of △-9 rabbits.IRE-1α overexpression is found by Western blot in liver tissues of △-9 rabbits over 60 days,it is evident in comparison with WT rabbits at the same age,and strong activation of IRE1α and its downstream transcriptional activator XBP1 was detected by immunohistochemistry in liver tissues of △-9 rabbits at 64 and411 days,but not in WT rabbits.Decreased CFTR protein expression was detected by immunohistochemistry in liver tissue specimens from patients with advanced fibrosis or cirrhosis stages of NASH,and no CFTR gene mutation was found by DNA sequencing of patients.Conclusion: 1.CFTR mutation leads to overexpression of AKT/GYS2/GSK3β signaling pathway and thus affects glucose homeostasis.2.CFTR mutation leads to overexpression of CREBH/PPARα/FGF21 signaling pathway,thereby affecting hepatic lipid metabolism.3.CFTR mutation leads to activation of IRE1α and its downstream transcriptional activator XBP1,thereby causing in age-dependent endoplasmic reticulum stress in the liver.4.Decreased CFTR protein expression in patients with advanced fibrosis or cirrhotic stage of NASH.