Beijing Youan Hospital, Capital Medical University, Beijing, China.
Department of Gastroenterology, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, Shandong, China.
PLoS One. 2024 Jun 10;19(6):e0304185. doi: 10.1371/journal.pone.0304185. eCollection 2024.
The present study aims to investigate the specific protective effects and underlying mechanisms of Ganshuang granule (GSG) on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rat models.
Hepatic fibrosis was experimentally evoked in rats by DMN administration, and varying dosages of GSG were employed as an intervention. Hepatocellular damage was assessed by measuring serum levels of aminotransferase and bilirubin, accompanied by histopathological examinations of hepatic tissue. The hepatic concentrations of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) were quantitated via enzyme-linked immunosorbent assay (ELISA). The expression of α-smooth muscle actin (α-SMA) within hepatic tissue was evaluated using immunohistochemical techniques. The levels of hepatic interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and a spectrum of interleukins (IL-2, IL-4, IL-6, IL-10) were quantified by quantitative real-time PCR (qRT-PCR). Additionally, hepatic stellate cells (HSCs) were cultured in vitro and exposed to TNF-α in the presence of naringin, a principal component of GSG. The gene expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase-1 (MMP-1) in these cells were also quantified by qRT-PCR. Proliferative activity of HSCs was evaluated by the Cell Counting Kit-8 assay. Finally, alterations in Smad protein expression were analyzed through Western blotting.
Administration of GSG in rats with fibrosis resulted in reduced levels of serum aminotransferases and bilirubin, along with alleviation of histopathological liver injury. Furthermore, the fibrosis rats treated with GSG exhibited significant downregulation of hepatic TGF-β1, PDGF, and TNF-α levels. Additionally, GSG treatment led to increased mRNA levels of IFN-γ, IL-2, and IL-4, as well as decreased expression of α-SMA in the liver. Furthermore, treatment with naringin, a pivotal extract of GSG, resulted in elevated expression of MMP-1 and decreased levels of TIMP-1 in TNF-α-stimulated HSCs when compared to the control group. Additionally, naringin administration led to a reduction in Smad expression within the HSCs.
GSG has the potential to mitigate fibrosis induced by DMN in rat models through the regulation of inflammatory and fibrosis factors. Notably, naringin, the primary extract of GSG, may exert a pivotal role in modulating the TGF-β-Smad signaling pathway.
本研究旨在探讨肝爽颗粒（GSG）对二甲基亚硝胺（DMN）诱导的大鼠肝纤维化的具体保护作用及其潜在机制。
采用 DMN 给药法诱导大鼠肝纤维化，并以不同剂量的 GSG 进行干预。通过检测血清转氨酶和胆红素水平，结合肝组织的组织学检查来评估肝细胞损伤。采用酶联免疫吸附试验（ELISA）定量测定血小板衍生生长因子（PDGF）和转化生长因子-β1（TGF-β1）在肝组织中的浓度。采用免疫组织化学技术评估α-平滑肌肌动蛋白（α-SMA）在肝组织中的表达。采用实时定量 PCR（qRT-PCR）定量测定肝组织中干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）和一系列白细胞介素（IL-2、IL-4、IL-6、IL-10）的水平。此外，体外培养肝星状细胞（HSCs）并在 TNF-α存在的情况下用 GSG 的主要成分柚皮苷进行孵育。还通过 qRT-PCR 定量测定这些细胞中组织金属蛋白酶抑制剂-1（TIMP-1）和基质金属蛋白酶-1（MMP-1）的基因表达水平。通过细胞计数试剂盒-8 测定 HSCs 的增殖活性。最后，通过 Western blot 分析 Smad 蛋白表达的变化。
纤维化大鼠给予 GSG 治疗后，血清转氨酶和胆红素水平降低，肝组织损伤减轻。此外，纤维化大鼠给予 GSG 治疗后，肝组织 TGF-β1、PDGF 和 TNF-α水平显著下调。此外，GSG 治疗导致 IFN-γ、IL-2 和 IL-4 的 mRNA 水平升高，同时肝组织中 α-SMA 的表达降低。此外，用 GSG 的主要提取物柚皮苷处理 HSCs 可导致 TNF-α刺激的 HSCs 中 MMP-1 表达增加和 TIMP-1 水平降低，与对照组相比。此外，柚皮苷给药可降低 HSCs 中的 Smad 表达。
GSG 通过调节炎症和纤维化因子，具有减轻 DMN 诱导的大鼠模型纤维化的潜力。值得注意的是，GSG 的主要提取物柚皮苷可能在调节 TGF-β-Smad 信号通路方面发挥关键作用。
