目的: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保护作用及其机制。
方法:通过DMN给药实验诱发大鼠肝纤维化,并采用不同剂量的GSG作为干预措施。通过测量血清转氨酶和胆红素水平来评估肝细胞损伤,伴随着肝组织的组织病理学检查。通过酶联免疫吸附测定(ELISA)定量血小板衍生生长因子(PDGF)和转化生长因子-β1(TGF-β1)的肝脏浓度。使用免疫组织化学技术评估肝组织中α-平滑肌肌动蛋白(α-SMA)的表达。肝干扰素-γ(IFN-γ)的水平,肿瘤坏死因子-α(TNF-α),和白介素(IL-2,IL-4,IL-6,IL-10)的光谱通过定量实时PCR(qRT-PCR)进行定量。此外,肝星状细胞(HSC)在体外培养,并暴露于TNF-α在柚皮苷的存在下,GSG的主要组成部分。也通过qRT-PCR定量这些细胞中金属蛋白酶-1(TIMP-1)和基质金属肽酶-1(MMP-1)的组织抑制剂的基因表达水平。通过细胞计数试剂盒-8测定评价HSC的增殖活性。最后,通过Western印迹分析Smad蛋白表达的改变。
结果:在纤维化大鼠中施用GSG导致血清转氨酶和胆红素水平降低,随着组织病理学肝损伤的减轻。此外,用GSG处理的纤维化大鼠表现出肝TGF-β1,PDGF,和TNF-α水平。此外,GSG治疗导致IFN-γ的mRNA水平增加,IL-2和IL-4以及α-SMA在肝脏中的表达降低。此外,用柚皮苷治疗,GSG的关键提取物,与对照组相比,TNF-α刺激的HSCs中MMP-1的表达升高,TIMP-1的水平降低。此外,柚皮苷给药导致HSC内Smad表达减少。
结论:GSG具有通过调节炎症和纤维化因子减轻DMN诱导的大鼠模型纤维化的潜力。值得注意的是,柚皮苷,GSG的主要提取物,可能在调节TGF-β-Smad信号通路中起关键作用。
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.