| ObjectiveCrosstalk between NF-?B and Wnt/?-catenin signal pathways plays a significant role in regulating HSC activation and proliferation&genesis and development of hepatic fibrosis,so discussing about the regulatory mechanism of HSC activation through crosstalk between the two signal pathways will be of great importance to investigating pathogenesis of hepatic fibrosis and therapeutic targets.Inhibitor action upon hepatic fibrosis rats and lentiviral transfection of HSC were adopted firstly in this study to discuss about the molecular regulatory mechanism of HSC activation through crosstalk between Oligo-peptide I-C-F-6 and NF-?B signal pathways.On this basis,the influence of Oligo-peptide I-C-F-6 on HSC and CC14-induced ?-catenin,p65 and NF-?B signal pathways in hepatic fibrosis rats was studied so as to ascertain the action mechanism of Oligo-peptide I-C-F-6 resistance against hepatic fibrosis.Methods1.?-catenin and p65 protein inhibitors were used to act on CC14-induced hepatic fibrosis rats,HE and Masson staining were conducted to observe pathomorphological change of rat livers,and then expression levels of signaling molecules and downstream target genes related to NF-?B and Wnt/?-catenin signal pathways were detected using immunohistochemistry,PCR,western blot and ELISA.2.Lentiviral transection of HSC-T6 cell strains was conducted.PCR,western blot,immunofluorescence and immunoprecipitation were used to detect expression levels of signaling molecules and downstream targets related to NF-?B and Wnt/?-catenin signal pathways so as to study whether interaction existed between p65 and ?-catenin.3.Oligo-peptide I-C-F-6 was used to act upon HSC and CCl4-induced hepatic fibrosis rats.HE and Masson staining were implemented to observe pathomorphological changes of rat livers.Western blot was used to detect expression levels of related signaling molecules of ?-catenin and NF-?B signal pathways.Results1.In the CCl4-induced hepatic fibrosis rat model,?-catenin and p65 proteins presented high expression levels.After ?-catenin protein was inhibited,IKBa protein increased,p65 protein expression was elevated,NF-?B signal pathway was significantly activated and rat hepatic fibrosis was aggravated;after p65 protein was inhibited,?-catenin protein level was elevated,Wnt signal pathway was significantly activated and rat hepatic fibrosis was aggravated.2.p65 and ?-catenin proteins presented high expressions in the activated HSC-T6.After lentiviral transfection of HSC cells was used to knock down p65 protein,?-catenin protein expression was elevated,Wnt/?-catenin activation in HSC was enhanced,and synthetic CTGF and TGF-?1 increased;after p-catenin protein was knocked down,p65 protein expression level was elevated,NF-?B signal pathway activation was enhanced,synthetic MMP-2 and MMP-9 was reduced,and synthetic TIMP-1 increased.Immunoprecipitation results indicated that interaction existed between p65 and p-catenin.p65 and ?-catenin are two key signaling molecules in the crosstalk between NF-?B and Wnt/?-catenin signal pathways.3.Oligo-peptide I-C-F-6 can improve hepatic fibrosis degree of hepatic fibrosis rats,lower HSC expression as well as expressions of ?-catenin and p65 proteins in livers of hepatic fibrosis rats,inhibit HSC activation and proliferation and inhibit activation of NF-?B signal pathway.Conclusions1.Under physiological status,p65 and(3-catenin proteins present low expression levels and NF-?B and Wnt/?-catenin signal pathways are under nonactivated states.During the hepatic fibrosis process,p65 and ?-catenin proteins present high expressions,and NF-?B and Wnt/?-catenin signal pathways are under activated status.Single inhibition of NF-?B signal pathway or Wnt/?-catenin signal pathway can't relieve hepatic fibrosis,but instead,it will aggravate hepatic fibrosis.2.p65 interacts with ?-catenin in HSC.p65 regulates activation of Wnt signal pathway through ?-catenin so as to influence expression of downstream target genes.?-catenin regulates activation of NF-?B signal pathway through p65 so as to influence expression of downstream target genes.The two pathways jointly regulate HSC activation.3.Oligo-peptide I-C-F-6 can significantly mitigate hepatic fibrosis degree of hepatic fibrosis rats while lowering expressions of ?-catenin and p65 expressions,HSC activation and proliferation and activation of NF-?B signal pathway.In summary,during the genesis and development process of hepatic fibrosis,independent obstruction of Wnt/?-catenin or NF-?B signal pathway may not exert the anti-hepatic fibrosis effect.In the hepatic fibrosis model and activated HSC,?-catenin interacts with p65 in regulating activation of Wnt/?-catenin and NF-?B signal pathways and influencing expression of downstream target genes so as to affect HSC activation.Oligo-peptide I-C-F-6 can relieve hepatic fibrosis symptoms of hepatic fibrosis rats,which is related to the fact that it can reduce expressions of ?-catenin and p65 proteins,inhibit activation of two signal pathways and reduce expression of downstream target genes so as to inhibit HSC activation in the meantime. |
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