A Mechanistic Study Of Which CREBH Inhibits Non-alcoholic Steatohepatitis By Regulating The Hepatocyte Autophagic Flux

Objective :We established ahepatocyte lipid overload model induced by palmitic acid(PA)to explore whether regulating the expression of CREBH affects endoplasmic reticulum stress(ERS),the expression of intracellular autophagy-related genes and the dynamic process of autophagy flux.Methods : 1.AML12 and LO2hepatocyte lines were treated with 400 μ M PA for 24hours to induce lipid overload model;LO2hepatocytes were starved with HBSS(Hank’s balanced salt solution)for different time(0,0.5,1,2,4h)to induce autophagy.Western blots and QRT-PCR were used to detect the expression of CREBH and autophagy related genes.2.The lentivirus was used as the vector to overexpress CREBH,the AML12 and LO2 liver cell lines with lentivirus stable overexpressing of CREBH(Lv-CREBH)or Null lentivirus(Lv-Null)were transfected and screened,and were given 400 transfusions of M PA for 24h.QRT-PCR andWestern blot were used to detect the expression of ERS-mediated signaling proteins and autophagy related genes,and acridine orange staining was used to observe the changes of intracellular autophagy by laser confocal microscope.3.The AML12 and LO2hepatocyte lines with Lv-CREBH or Lv-Null were treated with 400 μMPA and autolysosome inhibitor(20μMCQ or 10μMBaf-A1)for 24h,and the expression of autophagy-related genes was detected by q RT-PCR andWestern blot methods.And the changes of autophagy flux were observed by laser confocal microscope after thehepatocyte lines were transfected with LC3-GFP-m Cherry double-labeled plasmid and interfered with PA and autolysosome inhibitor(CQ or Baf-A1)for 24h.Results :1.In the PA-induced lipid overload model of AML12 and LO2hepatocytes,we found that the expression and activation of CREBH increased and autophagy was inhibited;in the HBSS-induced LO2 cell starvation model,autophagy was induced and the expression and activation of CREBH increased: the ratio of LC3II/I reached the maximum at 1h of starvation and then decreased,and the expression and activation of CREBH increased significantly at 1h of starvation and then remained stable.Under different metabolic stress conditions induced by PA and starvation,CREBH was activated and autophagy changed differently.2.The cell lines of AML12 and LO2 stable expressing CREBH were successfully verified.In thehepatocyte lipid overload model induced by PA,it was found that,compared with the Lv-Null group,the expression of ERS signal protein decreased significantly and the LC3II/I ratio increased in the overexpression CREBH group,but there was no significant change in the expression of autophagy-related genes.Acridine orange staining showed that red acidic vacuoles decreased after PA intervention,while red acidic vacuoles in overexpressed CREBH cells(with or without PA)increased compared with Lv-Null cells.The results suggest that overexpression of CREBH can improve ERS and autophagy.3.Western blot showed that the ratio of p62 protein to LC3II/I increased significantly after the intervention of autolysosome inhibitor CQ or Baf-A1 in thehepatocyte lipid overload model induced by PA,suggesting the inhibition of autophagy flux.Compared with Lv-Null group,overexpression of CREBH could inhibit the increase of p62 protein / and LC3II/I ratio induced by the CQ or Baf-A1 intervention,suggesting the improvement of autophagy flux.LC3-GFP-m Cherry double-labeled plasmid was transfected into Lv-Null and Lv-CREBH cell lines,it was observed that in thehepatocyte lipid overload model induced by PA,the yellow spots(autophagosome)increased significantly after the intervention of autolysosome inhibitor(CQ or Baf-A1).While compared with the Lv-Null group,overexpression of CREBH could significantly reduce the yellow spots(autophagosome)and increase the red spots(autolysosomes)after the intervention of CQ or Baf-A1.It is suggested that overexpression of CREBH can improve defective autophagy flux caused by autolysosome inhibition in PA-induced lipid overload model.Conclusions: In the model ofhepatocyte lipid overload induced by PA,ERS and autophagy flow disorder occur inhepatocytes.Overexpression of CREBH may improve ERS and restore autophagy flow.CREBH may regulate autophagy in the late stage by promoting the autophagosome-lysosome fusion.Objective: To investigate the changes of CREBH expression in mouse NASH model induced by HF and MCD diet,and the effects of regulating CREBH expression on liver injury,endoplasmic reticulum stress and autophagy activity in NASH mice.Methods: 1.QRT-PCR andWestern blot were used to detect the expression of CREBH in the mouse NAFLD/NASH model induced by HF diet for 14 weeks(W)or 24W,and MCD diet for 4W.2.CREBH knockout(KO)mice were constructed and bred.With wild-type(WT)mice as control,NASH models induced by HF diet 24W and MCD diet 4W were established.The liver injury was evaluated by the detection of biochemical indexes such as serum lipid and transaminase,the staining of H&E,Oil Red O and MASSON in liver tissue sections,the detection of inflammation and fibrosis by q RT-PCR,and.The expression of autophagy-related proteins was detected by q RT-PCR,Western blot and immunofluorescence,and the morphology and number of lipid droplet,endoplasmic reticulum,autophagosomes and autophagy lysosomes in liver cells were observed by electron microscope.3.Adeno-associated virus overexpressing CREBH(AAV-CREBH)or unloaded control virus(AAV-Null)were injected into the tail vein of mice,and the model of NASH induced by HF diet for 24W was established.The liver injury was evaluated by the detection of biochemical indexes such as serum lipid and transaminase,the staining of H&E and Oil Red O in liver tissue sections,the expression of ERS-related signal proteins by western blot.The expression of autophagy-related proteins was detected by q RT-PCR,westernblot and immunofluorescence,and the morphology and number of lipid droplet,endoplasmic reticulum,autophagosomes and autophagy lysosomes in liver cells were observed by electron microscope.Results: 1.HF diet for 14W induced up-regulation of CREBH expression and activation;HF diet for 24W,the level of CREBH expression and activationhad no significant difference compared with the control diet(CD)group;MCD diet for 4W inhibited the expression and activation of CREBH.2.In the NASH model induced by HF/MCD diet,knockout of CREBH gene aggravates liver tissue injury,endoplasmic reticulum stress and autophagy disturbance.InWT group,compared with CD group,NASH mice fed with HF diet for 24 weeks and MCD diet for 4 weeks showedhigh TG and elevated serum transaminase levels,obvious lipid accumulation,ballooning degeneration and inflammation in liver tissue,and increased expression of ERS signal proteins.In the state of CD diet,there was no significant tissue damage in the liver after knockout of CREBH.In the state of CD diet,there was no significant tissue damage in the liver after knockout of CREBH.In the NASH model induced by HF diet and MCD diet,the levels of serum TG and transaminase in CREBH knockout mice werehigher than those inWT mice.In liver tissue,lipid accumulation decreased but ballooning degeneration,inflammation and fibrosis were more severe,ERS signal protein expression increased,and electron microscopy showed that the endoplasmic reticulum cavity ofhepatocytes was significantly dilated.These results suggest that liver injury is more serious.Compared withWT group,KO mice showed significantly decreased expression of lysosome-associated protein LAMP1,accumulation of LC3 and p62 fluorescence spots,increase of autophagosome and decrease of autophagy lysosome under electron microscope,suggesting autophagy flow disturbance.Under the condition of HF/MCD diet,the disturbance of autophagy in the liver of KO group was more obvious than that ofWT group.Knockout of CREBH genehad no significant effect on autophagy related genes.3.In the NASH model induced by HF diet,overexpression of CREBH can promote autophagy,reduce ERS and liver injury.Under the condition of CD diet,overexpression of CREBHhad no significant effect on the liver of mice.In NASH mice induced by HF diet,overexpression of CREBH decreased elevated blood lipids and serum transaminase levels induced by HF diet,inhibited liver lipid accumulation,ballooning degeneration and inflammation,inhibited the expression of ERS signal proteins,increased the expression of lysosome-associated proteins LAMP1 and LAMP2,increased LC3II/LC3 I and decreased p62 protein accumulation,and autophagy lysosome increased under electron microscope.Overexpression of CREBHhad no significant effect on the expression of genes related to autophagy formation.Conclusion: the expression and activation of CREBH are increased in the early stage of NAFLD and inhibited in the stage of NASH.In the NASH model induced by HF or MCD diet,CREBH can alleviate liver tissue injury,ERS and autophagy in mice.Objective: To explore the mechanism of CREBH regulatinghepatic autophagy flux in vitro and in vivo,and to determine whether CREBHhas direct transcriptional regulation on genes regulating autophagy flux.Methods: 1.In the NASH model of CREBH gene knockout with HF diet,RNA-Seq sequencing was carried out,and the results were analyzed by cluster analysis,GO and KEGG function analysis.Autophagy-related genes with different m RNA expression levels were screened inWT and KO mice,and the m RNA expression levels of these genes were verified by q RT-PCR method.2.In the liver of NASH mice induced by MCD diet with CREBH gene knockout,the liver of CD diet mice with AAV overexpression of CREBH through tail vein,and the corresponding control group,the m RNA expression level of the screened genes was detected by q RT-PCR method,and the autophagy related genes with consistent changes,namely Coro1 a and Atg14,were screened.In NASH model mice induced by HF diet,the protein expression levels of Coro1 a and Atg14 were detected byWesternblot method.3.In LO2hepatocytes with stable overexpression of CREBH(Lv-CREBH)and corresponding no-load control(Lv-Null),the m RNA expression levels of Coro1 a and Atg14 were detected by q RT-PCR.The direct transcriptional regulation of CREBH on CORO1 A and ATG14 was detected by chromatin co-immunoprecipitation(Ch IP)and double luciferase reporter geneResults: 1.In the liver of CREBH knockout NASH mice fed with HF diet,10 autophagy related genes with different m RNA expression were screened by RNA-Seq gene sequencing: Dram1,Dcn,Atg14,Mtcl1,Sh3bp4,Trp53inp2,Ctsk,Pycard,Srpx,Coro1 a.2.After q RT-PCR detection of the above 10 genes,we selected the genes with the same trend of m RNA expression in CREBH knockout NASH mice induced by HF diet and MCD diet,but opposite to those in CD diet mice with overexpression of CREBH,namely Coro1 a and Atg14.Referring to the literature,it was found that Coro1 a inhibited autophagy and Atg14 promoted autophagy.Consistent with the results of CREBH promoting autophagy,knockout CREBH up-regulated the gene and protein expression of Coro1 a,but down-regulated the gene and protein expression of Atg14;overexpressed CREBH inhibited the gene and protein expression of Coro1 a and promoted the gene and protein expression of Atg14.3.In LO2 cell line,overexpression of CREBH down-regulated the m RNA expression of Coro1 a,buthad no significant effect on the m RNA expression of Atg14.The results of CHIP and double luciferase reporter genes suggest that CREBH can bind to the promoter region of Coro1 a,buthas no significant effect on the promoter region of Atg14.Conclusion: In liver,CREBH may promote autophagosome-lysosome fusion by targeting negative regulation of Coro1 a expression and indirectly promoting Atg14 expression,thus promoting autophagy flow.