Department of Clinical Medicine, Guizhou Medical University, Guiyang, P. R. China.
Eur Rev Med Pharmacol Sci. 2019 Feb;23(3):1279-1290. doi: 10.26355/eurrev_201902_17023.
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.
研究胸腺肽β4（Tβ4）对白细胞介素（IL）-1β诱导的人肝星状细胞系（HSC-LX2）激活的抑制作用。
本研究共设 5 组，即空白对照组、阴性对照组（SI-NC，空载质粒）、模型组（20ng/mlIL-1β）、Tβ4 处理组（20ng/mlIL-1β+1000ng/mlTβ4）和 Tβ4 敲低组（20ng/mlIL-1β+si-Tβ4）。采用细胞计数试剂盒（CCK-8）法检测细胞增殖率。碘化丙啶（PI）DNA 染色和 Annexin V-荧光素异硫氰酸酯（FITC）双重染色检测细胞周期变化和细胞凋亡率。荧光定量实时聚合酶链反应（RT-PCR）检测细胞内 p-IKB 和核因子-κB（NF-κB）/p65 蛋白的核酸水平。双免疫荧光染色和 Western blot 检测 NF-κB 和 p65 的核转位以及细胞质 p-IKB 蛋白和核 p65 蛋白的水平。
与模型组和 siRNA-Tβ4 敲低组相比，Tβ4 处理组由于 G0/G1 期阻滞，细胞数量增加（p<0.01）。在相同的组间比较中，Tβ4 处理组的细胞凋亡率显著增加（p<0.05）。与空白对照组相比，模型组和 siRNA-Tβ4 敲低组的细胞内 p-IKB 和 NF-κB/p65 核酸水平显著升高（p<0.01）。与 siRNA-Tβ4 敲低组相比，Tβ4 处理组的细胞内 p-IKB 和 NF-κB/p65 核酸水平显著降低（p<0.01）。Tβ4 处理组的 NF-κB/p65 和 NF-κB/p50 表达水平显著降低。与模型组相比，Tβ4 处理组的细胞质 p-IKB 和核 NF-κB/p65 表达水平较低（p<0.01）。
Tβ4 可显著抑制 IL-1β诱导的 HSC-LX2 细胞增殖。其机制可能涉及 NF-κB 通路的激活减少、p-IKB 的表达减少和 p65 的核转位减少。因此，Tβ4 具有逆转肝纤维化的作用。
