TWEAK/Fn14 Promotes Pro-inflammatory Cytokine Secretion In Hepatic Stellate Cells Via NF-κB/STAT3 Pathways

Background and objectiveLiver fibrosis is an outcome of most types chronic liver diseases,which is characterized by extensive deposition of extracellular matrix(ECM)proteins following liver injury.Hepatic stellate cells(HSCs)are the major ECM-producing cells,and play a critical role in the occurrence and development of liver fibrosis.Previous studies have suggested that the decrease of the number of activated HSCs is a major factor in regression of liver fibrosis.Most importantly,recent studies have shown that tumor necrosis factor-like weak inducer of apoptosis(TWEAK)and its receptor fibroblast growth factor-inducible 14(Fnl4)have a critical role in the occurrence of the liver fibrosis,and there is little information available on the role of the TWEAK/Fnl4 pathway in human HSCs.Therefore,in the present study,we investigate the relationship between activated HSCs and TWEAK,and explore the possible mechanism.MethodsTo explore the role of TWEAK/Fn14 in activated human HSCs,the LX-2 cells were treated with TWEAK or small interfering RNA(siRNA),the expression of pro-inflammatory cytokines was assayed by enzyme linked immunosorbent assay(ELISA)and real-time PCR(RT-PCR);the expression of Fn14.nuclear factor kappa B(NF-kB,p65)and signal transducers and activators of transcription 3(STAT3)was evaluated by western blotting or RT-PCR;and the total and phosphorylated NF-κB(p65)and STAT3 from the nuclear and cytoplasmic protein of LX-2 cells were examined by western blotting;the cell viability was analyzed by CCK-8 and then the percentage of apoptosis cells was analyzed by flow cytometry.Besides,we also examined the role of miR-19b in TWEAK/Fn14 pathway.The LX-2 cells were treated with TWEAK;the expression of miR-19b was assayed by RT-PCR.The miR-19b mimic and miR-19b inhibitor was transfected into the LX-2 cells to overexpress or knockdown miR-19b,respectively;the expression of Fn14 was evaluated by RT-PCR.The LX-2 cells were co-transfected with the wild-type Fn143’UTR reporter plasmid and mutated Fn14 3’ untranslated region(UTR)reporter plasmid and miR-19b mimic,the luciferase activity was detected by a luciferase assay kit,ResultsThe result showed that the expression of Fn14 had a time-and dose-dependent manner to TWEAK in LX-2 cells.And we found that TWEAK can significantly upregulated the expression of Fn14 by downregulating the expression of miR-19b.Furthermore,TWEAK played an important role in modulating pro-inflammatory cytokines interleukin-8(IL-8),interleukin-6(IL-6),regulated upon activation normal T cell expressed and secreted(RANTES)and monocyte chemotactic protein-1(MCP-1)secretion in LX-2 cells by upregulating the expression of Fn14,and the pro-inflammatory cytokines secretion was closely related to the activation of NF-κB(p65)and STAT3 pathway.Furthermore,our research showed that STAT3 and NF-κB(p65)could interact with each other,which resulted in a significant increase of pro-inflammatory cytokines secretion.Conclusions In summary,our study demonstrated that miR-19b was identified as posttranscriptional negative regulators for Fn14,and TWEAK down regulated the expression of miR-19b to up-regulate the Fn14 protein production.And TWEAK/Fn14 activated NF-κB(p65)and STAT3 signalling pathways in LX-2 cells,which interacted with each other,leading to pro-inflammatory cytokine secretion.The research will reveal the effects of TWEAK on the expression of pro-inflammatory cytokines in activated human HSCs,and provide us a novel principle for prevention and treatment of liver fibrosis.