NF-?B P65 Inhibits Hepatic Stellate Cell Activation Through Wnt Signaling And Effects Of Oligo-peptide I-C-F-6 On Liver Fibrosis

ObjectiveIn this study,the expression of NF-?B P65 was silenced after the hepatic stellate cells(HSC-T6)were infected by lentivirus and the mechanism by which NF-?B P65 regulates the activation of hematopoietic stem cells through the Wnt/?-catenin signaling pathway was explored.On this basis,HSC-T6 and carbon tetrachloride-induced rat models of hepatic fibrosis were used to study the effect of Oligo-peptide I-C-F-6 on the expression levels of signaling molecules of the Wnt/p-catenin signaling pathway and downstream target genes through NF-?B NF-?B P65.The molecular mechanism by which Oligo-peptide I-C-F-6 mediates the activation of HSCs and anti-hepatic fibrosis activity by regulating NF-?B P65 and the Wnt/?-catenin signaling pathway was determined.Methods1.HSC-T6 was infected by lentivirus,and the expression levels of NF-?B P65,signaling molecules of the Wnt/?-catenin signal pathway and downstream target genes were detected with Western blotting and polymerase chain reaction(PCR).2.Flow cytometry and Western blotting were used to detect the proliferation and apoptosis of HSC-T6,and the expression levels of NF-?B P65,signaling molecules of the Wnt/?-catenin signal pathway and downstream target genes.3.The collagen deposition,inflammation and the levels of hyaluronic acid(HA),laminin(LN),procollagen ?(PC?)and collagen type ?(COL4)in carbon tetrachloride-induced rat models of hepatic fibrosis were determined using hematoxylin and eosin(HE)staining,Masson staining and enzyme-linked immunosorbent assay(ELISA).4.ELISA,immunohistochemistry and Western blotting were used to detect the expression levels of NF-?B P65,signaling molecules of the Wnt/?-catenin signal pathway and downstream target genes in the carbon tetrachloride-induced rat model of hepatic fibrosis.Results1.When the lentivirus infection did not silence the expression of NF-?B P65,there were high levels of ?-catenin,P-GSK-3?,TGF-?1 and ?-SMA protein expressions and low levels of MMP-2 and 9 mRNA expressions in HSC-T6.2.After lentivirus infection silenced the expression of NF-?B P65 in HSC-T6,the expressions of ?-catenin and P-GSK-3? were decreased,and the expression of downstream target genes was also decreased.3.Oligo-peptide I-C-F-6 could inhibit the proliferation of HSC-T6,promote its apoptosis,suppress the levels of NF-?B P65 and ?-catenin,P-GSK-3p and P-AKT protein expressions,and reduce the expressions of TGF-?1 and a-SMA.4.Oligo-peptide I-C-F-6 could alleviate the hepatic injury and promote collagen degradation in the rats with hepatic fibrosis,and could inhibit the NF-?B P65,?-catenin,P-GSK-3? and P-AKT protein expressions and regulate the expressions of related downstream target genes.Conclusions1.When the expression of NF-?B P65 in HSC-T6 was not silenced,the expression levels of ?-catenin and P-GSK-3? were high,indicating that the Wnt signaling pathway was activated.The increased expression levels of TGF-?1 and a-SMA suggested the activation of HSC-T6.2.After expression of NF-?B P65 in HSC-T6 was silenced,the expressions of P-catenin and P-GSK-3? were decreased,indicating that the activation of the Wnt/?-catenin signaling pathway was inhibited and the activation of HSCs was further mediated.The mechanism may be related to the direct inhibition of the?-catenin expression after silencing of NF-?B P65,or the promotion of the phosphorylation of GSK-3P to cause the degradation of ?-catenin,further mediating the expression of its downstream target genes.3.Oligo-peptide I-C-F-6 could inhibit the proliferation of HSC-T6 and promote its apoptosis,while inhibiting the expressions of TGF-?1 and a-SMA.This finding suggested that Oligo-peptide I-C-F-6 could regulate the activation of the Wnt/?-catenin signaling pathway by reducing the expression of NF-?B P65 in HSC-T6,and further inhibit the activation of HSCs.4.Oligo-peptide I-C-F-6 could significantly alleviate hepatic injury and reduce the extracellular matrix(ECM)deposition in rats with hepatic fibrosis.This may be related to the fact that Oligo-peptide I-C-F-6 could inhibit the activations of NF-?B P65 and the Wnt/?-catenin signaling pathway and thus regulate the expression levels of CTGF and TGF-?1,TNF-?,VEGF and various other downstream cytokines.In summary,NF-?B P65 and the Wnt signaling pathway play an important role in the progression of hepatic fibrosis.Silencing of NF-kB P65 expression in HSC-T6 could inhibit the activation of the Wnt/?-catenin signaling pathway,indicating that NF-?B P65 can regulate the activation of HSCs through the Wnt/?-catenin signaling pathway and affect the development and progression of hepatic fibrosis.Oligo-peptide I-C-F-6 could reduce the NF-?B P65 expression in the rats with hepatic fibrosis and HSC-T6,and regulate the expression of signaling molecules in the Wnt/?-catenin signaling pathway,thereby inhibiting the activation of the Wnt/?-catenin signaling pathway and affecting the transcription and expression of downstream target genes.As a result,Oligo-peptide I-C-F-6 could further inhibit the activation of HSCs,alleviate hepatic injury in rats with hepatic fibrosis,reduce the deposition of ECM,and achieve the anti-hepatic fibrosis effect.This may be one of the mechanisms underlying anti-hepatic fibrosis activity of Oligo-peptide I-C-F-6.