Guizhi Fuling pill attenuates liver fibrosis <i>in vitro</i> and <i>in vivo</i> via inhibiting TGF-β1/Smad2/3 and activating IFN-γ/Smad7 signaling pathways

Liver fibrosis resulting from chronic liver injuries (CLI) is a common health problem globally. Guizhi Fuling pill (GZFL), a modern preparation from traditional Chinese medicine, exhibited anti-dysmenorrhea, anti-inflammatory, and immune-regulative effects. However, the effect of GZFL on liver fibrosis remains unknown. In this research, LX-2 cells were stimulated with acetaldehyde for mimicking liver fibrosis progression in vitro. In addition, carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established as well. The data revealed GZFL obviously suppressed the proliferation and triggered the apoptosis of acetaldehyde-stimulated LX-2 cells. In addition, GZFL prevented acetaldehyde-induced activation of LX-2 cells via downregulation of TGF-β1, p-Smad2, p-Smad3, CUGBP1, and upregulation of p-STAT1 and Smad7. Meanwhile, GZFL significantly alleviated CCl4‑induced liver fibrosis, as evidenced by the decrease of ALT and AST levels. Moreover, GZFL downregulated the expressions of TGF-β1, p-Smad2, p-Smad3, and CUGBP1 in CCl4-treated mice. Furthermore, GZFL remarkably elevated the levels of IFN-γ, p-STAT1, and Smad7 in CCl4-treated mice. To sum up, GZFL was able to inhibit liver fibrosis in vitro and in vivo through suppressing TGF-β1/Smad2/3-CUGBP1 signaling and activating IFN-γ/STAT1/Smad7 signaling. Thus, GZFL might have a potential to act as a therapeutic agent for anti-fibrotic therapy.慢性肝损伤（chronic liver injuries, CLI）所致肝纤维化是全球范围内的常见健康问题。桂枝茯苓丸（Guizhi Fuling pill, GZFL）是一款现代中药制剂，既往研究已证实其具备抗痛经、抗炎及免疫调节活性，但GZFL对肝纤维化的作用效果尚未明确。本研究采用乙醛刺激LX-2细胞，以体外模拟肝纤维化的进展过程；同时构建四氯化碳（carbon tetrachloride, CCl4）诱导的小鼠肝纤维化模型。实验数据表明，GZFL可显著抑制乙醛刺激的LX-2细胞增殖，并诱导其凋亡。此外，GZFL可通过下调转化生长因子-β1（transforming growth factor-β1, TGF-β1）、磷酸化Smad2（p-Smad2）、磷酸化Smad3（p-Smad3）及CUG结合蛋白1（CUGBP1）的表达，同时上调磷酸化STAT1（p-STAT1）与Smad7的水平，从而阻断乙醛诱导的LX-2细胞活化。同时，GZFL可显著减轻CCl4诱导的小鼠肝纤维化，表现为血清谷丙转氨酶（ALT）、谷草转氨酶（AST）水平降低。进一步研究发现，GZFL可下调CCl4造模小鼠肝脏组织中TGF-β1、p-Smad2、p-Smad3及CUGBP1的表达，并显著升高干扰素-γ（interferon-γ, IFN-γ）、p-STAT1及Smad7的表达水平。综上，GZFL可通过抑制TGF-β1/Smad2/3-CUGBP1信号通路、激活IFN-γ/STAT1/Smad7信号通路，同时在体外及体内模型中发挥抗肝纤维化作用。因此，GZFL有望成为抗肝纤维化治疗的潜在候选药物。