Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai, China; Institutes of Integrative Medicine, Fudan University, Shanghai, China.
Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai, China.
J Ethnopharmacol. 2024 May 23;326:117963. doi: 10.1016/j.jep.2024.117963. Epub 2024 Feb 20.
Liver fibrosis is a serious complication of liver disease characterized by excessive collagen deposition, without effective therapeutic agents in the clinic. Fu-Gan-Wan (FGW) is an empirical formula used for the clinical treatment of hepatitis and cirrhosis. It has been shown to reverse experimental liver fibrosis. However, its corresponding mechanisms remain unclear.
This study aimed to elucidate the key pathways and target genes of FGW in attenuating liver fibrosis.
The therapeutic effects of different doses of FGW on liver fibrosis were investigated using a 2 mL/kg 15% CCl-induced mouse model. Then, RNA-seq combined with network pharmacology was used to analyze the key biological processes and signaling pathways underlying the anti-liver fibrosis exertion of FGW. These findings were validated in a TGF-β1-induced model of activation and proliferation of mouse hepatic stellate cell line JS-1. Finally, the key signaling pathways and molecular targets were validated using animal tissues, and the effect of FGW on tissue lipid peroxidation was additionally observed.
We found that 19.5 g/kg FGW significantly down-regulated CCl-induced elevation of hepatic ALT and AST, decreased collagen deposition, and inhibited the expression of pro-fibrotic factors α-SMA, COL1α1, CTGF, TIMP-1, as well as pro-inflammatory factor TGF-β1. Additionally, FGW at doses of 62.5, 125, and 250 μg/mL dose-dependently blocked JS-1 proliferation, migration, and activation. Furthermore, RNA-seq identified the NF-κB signaling pathway as a key target molecular pathway for FGW against liver fibrosis, and network pharmacology combined with RNA-seq focused on 11 key genes. Significant changes were identified in CCL2 and HMOX1 by tissue RT-PCR, Western blot, and immunohistochemistry. We further demonstrated that FGW significantly attenuated CCl-induced increases in p-p65, CCL2, CCR2, and HMOX1, while significantly elevating Nrf2. Finally, FGW significantly suppressed the accumulation of lipid peroxidation products MDA and 4-HNE and reconfigured the oxidation-reduction balance, including promoting the increase of antioxidants GPx, GSH, and SOD, and the decrease of peroxidation products ROS and GSSG.
This study demonstrated that FGW exhibits potential in mitigating CCl-induced hepatic fibrosis, lipid peroxidation, and iron metabolism disorders in mice. This effect may be mediated through the NF-κB/CCL2/CCR2 and Nrf2/HMOX1 pathways.
肝纤维化是一种严重的肝病并发症，其特征是胶原过度沉积，临床上尚无有效的治疗药物。复肝丸（FGW）是一种用于治疗肝炎和肝硬化的经验方，已被证明可逆转实验性肝纤维化。然而，其相应的机制尚不清楚。
本研究旨在阐明 FGW 减轻肝纤维化的关键途径和靶基因。
采用 2mL/kg15%CCl 诱导的小鼠模型研究不同剂量 FGW 对肝纤维化的治疗作用。然后，采用 RNA-seq 结合网络药理学分析 FGW 抗肝纤维化作用的关键生物学过程和信号通路。在 TGF-β1 诱导的小鼠肝星状细胞系 JS-1 激活和增殖模型中验证这些发现。最后，使用动物组织验证关键信号通路和分子靶标，并观察 FGW 对组织脂质过氧化的影响。
我们发现，19.5g/kg FGW 可显著下调 CCl 诱导的肝 ALT 和 AST 升高，减少胶原沉积，并抑制促纤维化因子α-SMA、COL1α1、CTGF、TIMP-1 和促炎因子 TGF-β1 的表达。此外，FGW 在 62.5、125 和 250μg/mL 剂量下可剂量依赖性地阻断 JS-1 的增殖、迁移和激活。此外，RNA-seq 鉴定出 NF-κB 信号通路是 FGW 抗肝纤维化的关键靶标分子途径，网络药理学结合 RNA-seq 集中于 11 个关键基因。组织 RT-PCR、Western blot 和免疫组化显示 CCL2 和 HMOX1 发生显著变化。我们进一步证明，FGW 可显著降低 CCl 诱导的 p-p65、CCL2、CCR2 和 HMOX1 的增加，同时显著增加 Nrf2。最后，FGW 可显著抑制 CCl 诱导的脂质过氧化产物 MDA 和 4-HNE 的积累，并重新构建氧化还原平衡，包括促进抗氧化剂 GPx、GSH 和 SOD 的增加，以及减少过氧化产物 ROS 和 GSSG。
本研究表明，FGW 具有减轻 CCl 诱导的肝纤维化、脂质过氧化和铁代谢紊乱的潜力。这种作用可能是通过 NF-κB/CCL2/CCR2 和 Nrf2/HMOX1 途径介导的。
