Cellular mechanism of Tβ4 intervention in liver fibrosis by regulating NF-κB signaling pathway.

Abstract

To investigate the inhibitory effect of thymosin-β4 (Tβ4) on the activation of the human hepatic stellate cell line (HSC-LX2) induced by interleukin (IL)-1β. There were 5 groups in this study, i.e., blank control group, negative control group (SI-NC, empty plasmid), model group (20 ng/ml of IL-1β), siRNA-Tβ4 knockdown group (IL-1β and si-Tβ4) and Tβ4 treatment group (IL-1β and 1000 ng/ml of Tβ4). Cell proliferation rate was measured using the Cell Counting Kit-8 (CCK-8) method. The cell cycle change and percentage of apoptotic cells were determined by Propidium Iodide (PI) DNA staining and Annexin V-fluorescein isothiocyanate (FITC) double staining. Cellular nucleic acid levels of p-IKB and nuclear factor-kappa B (NF-κB)/p65 proteins were measured by fluorescent quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Double immunofluorescence staining and Western blot were used to detect nuclear translocation of NF-κB and p65 and levels of cytoplasmic p-IKB protein and nuclear p65 protein. Due to the G0/G1 phase arrest, the number of cells in the Tβ4 treatment group increased, compared with the model group and the siRNA-Tβ4 knockdown group (p<0.01). In the same between-group comparison, apoptotic rate in the Tβ4 treatment group increased significantly (p<0.05). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were markedly higher in the model group and the siRNA-Tβ4 knockdown group than in the blank control group (p<0.01). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were remarkably lower in the Tβ4 treatment group than in the siRNA-Tβ4 knockdown group (p<0.01). The expression levels of NF-κB/p65 and NF-κB/p50 were significantly lower in the Tβ4 treatment group. The expression levels of cytoplasmic p-IKB and nuclear NF-κB/p65 were lower in the Tβ4 treatment group than in the model group (p<0.01). Tβ4 significantly inhibited IL-1β-induced HSC-LX2 cell proliferation. The mechanism may involve decreased activation of the NF-κB pathway, decreased expression of p-IKB and nuclear translocation of p65. Therefore, Tβ4 had the effect of reversing liver fibrosis.