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

OBJECTIVE: To investigate the inhibitory effect of thymosin-beta 4 (T beta 4) on the activation of the human hepatic stellate cell line (HSC-LX2) induced by interleukin (IL)-1 beta. MATERIALS AND METHODS: 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-1 beta), siRNA-T beta 4 knockdown group (IL-1 beta and si-T beta 4) and T beta 4 treatment group (IL-1 beta and 1000 ng/ml of T beta 4). 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-kappa B)/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-kappa B and p65 and levels of cytoplasmic p-IKB protein and nuclear p65 protein. RESULTS: Due to the G0/G1 phase arrest, the number of cells in the T beta 4 treatment group increased, compared with the model group and the siRNA-T beta 4 knockdown group (p<0.01). In the same between-group comparison, apoptotic rate in the T beta 4 treatment group increased significantly (p<0.05). The cellular nucleic acid levels of p-IKB and NF-kappa B/p65 were markedly higher in the model group and the siRNA-T beta 4 knockdown group than in the blank control group (p<0.01). The cellular nucleic acid levels of p-IKB and NF-kappa B/p65 were remarkably lower in the T beta 4 treatment group than in the siRNA-T beta 4 knockdown group (p<0.01). The expression levels of NF-kappa B/ p65 and NF-kappa B/p50 were significantly lower in the T beta 4 treatment group. The expression levels of cytoplasmic p-IKB and nuclear NF-kappa B/p65 were lower in the T beta 4 treatment group than in the model group (p<0.01). CONCLUSIONS: T beta 4 significantly inhibited IL-1 beta-induced HSC-LX2 cell proliferation. The mechanism may involve decreased activation of the NF-kappa B pathway, decreased expression of p-IKB and nuclear translocation of p65. Therefore, T beta 4 had the effect of reversing liver fibrosis.