Department of Epidemiology, Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China.
Int J Mol Med. 2018 Dec;42(6):3602-3612. doi: 10.3892/ijmm.2018.3900. Epub 2018 Sep 26.
The therapeutic management of liver fibrosis remains an unresolved clinical problem. The activation of hepatic stellate cells (HSCs) serves a pivotal role in the formation of liver fibrosis. In our previous study, matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry (MALDI‑TOF MS) was employed to identify potential serum markers for liver cirrhosis, such as eukaryotic peptide chain releasing factor 3b polypeptide (eRF3b‑37), which was initially confirmed by our group to serve a protective role in liver tissues in a C‑C motif chemokine ligand 4‑induced liver cirrhosis mouse model. Therefore, eRF3b‑37 was hypothesized to affect the activation state of HSCs, which was determined by the expression of pro‑fibrogenic associated factors in HSCs. In the present study, peptide synthesis technology was employed to elucidate the role of eRF3b‑37 in the expression of pro‑fibrogenic factors induced by transforming growth factor‑β1 (TGF‑β1) in LX‑2 cells that were treated with either control, TGF‑β1 and TGF‑β1+eRF3b‑37. 3‑(4,5‑Dimethyl‑2‑thiazolyl)‑2,5‑diphenyltetrazolium bromide and flow cytometric assays, and fluorescent microscope examinations were performed to evaluate the effects of eRF3b‑37 on proliferation viability, G0/G1 arrest, apoptosis and cell migration. The results of the present study indicated that eRF3b‑37 inhibited the activation of HSCs. The increased mRNA and protein expression of the pro‑fibrogenic factors collagen I, connective tissue growth factor and α‑smooth muscle actin (SMA) stimulated by TGF‑β1 were reduced by eRF3b‑37 via the following mechanisms: i) Inhibiting LX‑2 cell proliferation, leading to G0/G1 cell cycle arrest and inhibition of DNA synthesis by downregulating the mRNA expressions of Cyclin D1 and cyclin dependent kinase‑4, and upregulating the levels of P21; ii) increasing cell apoptosis by upregulating the mRNA level of B‑cell lymphoma-2 (Bcl‑2)‑associated X protein (Bax) and Fas, and downregulating the expression of Bcl‑2; and iii) reducing cell migration by downregulating the mRNA and protein expression of α‑SMA. In addition, eRF3b‑37 is thought to serve a role in HSCs by inhibiting TGF‑β signaling. Therefore, eRF3b‑37 may be a novel therapeutic agent for targeting HSCs for hepatic fibrosis.
肝纤维化的治疗管理仍然是一个未解决的临床问题。肝星状细胞（HSCs）的激活在肝纤维化的形成中起着关键作用。在我们之前的研究中，基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）被用于鉴定肝硬化的潜在血清标志物，如真核肽链释放因子 3b 多肽（eRF3b-37），该标志物最初被我们小组证实，在 C-C 基序趋化因子配体 4 诱导的肝硬化小鼠模型中，在肝组织中发挥保护作用。因此，eRF3b-37 被假设影响 HSCs 的激活状态，这由 HSCs 中促纤维化相关因子的表达决定。在本研究中，采用肽合成技术来阐明 eRF3b-37 在 TGF-β1 处理的 LX-2 细胞中诱导的促纤维化因子表达中的作用，TGF-β1 处理的细胞分别为对照组、TGF-β1 组和 TGF-β1+eRF3b-37 组。采用 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基四氮唑溴盐和流式细胞术以及荧光显微镜检查评估 eRF3b-37 对增殖活力、G0/G1 期阻滞、凋亡和细胞迁移的影响。本研究结果表明，eRF3b-37 抑制 HSCs 的激活。通过以下机制，抑制 TGF-β1 刺激的促纤维化因子胶原 I、结缔组织生长因子和α-平滑肌肌动蛋白（SMA）的 mRNA 和蛋白表达：i）抑制 LX-2 细胞增殖，通过下调细胞周期蛋白 D1 和细胞周期蛋白依赖性激酶-4 的 mRNA 表达，上调 P21 水平，导致 G0/G1 细胞周期阻滞和 DNA 合成抑制；ii）通过上调 B 细胞淋巴瘤-2（Bcl-2）相关 X 蛋白（Bax）和 Fas 的 mRNA 水平，下调 Bcl-2 的表达，增加细胞凋亡；iii）通过下调 α-SMA 的 mRNA 和蛋白表达，减少细胞迁移。此外，eRF3b-37 被认为通过抑制 TGF-β 信号通路在 HSCs 中发挥作用。因此，eRF3b-37 可能成为肝纤维化靶向 HSCs 的新型治疗药物。
