Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai, China; Institute of Integrative Medicine, Fudan University, Shanghai, China.
J Ethnopharmacol. 2023 Dec 5;317:116810. doi: 10.1016/j.jep.2023.116810. Epub 2023 Jun 16.
Numerous studies have provided evidence supporting the significant roles of icariin, in the prevention of multiple chronic diseases like diabetes, liver fibrosis, cardiac fibrosis, renal fibrosis, and pulmonary fibrosis. In particular, Icariside II (ISE II), a prominent flavonoid glycoside derived from Epimedium brevicornum Maxim, the principal metabolite of icariin, has demonstrated noteworthy anti-inflammatory and anti-oxidant properties, along with its ability to protect against lung remodeling. However, the research exploring ISE Ⅱ's application in treating pulmonary fibrosis remains limited.
The aim of this study was to assess the therapeutic efficacy of ISE II in models of pulmonary fibrosis, while also investigating its potential mechanisms of action in cell signaling pathways.
An in vitro model of pulmonary fibrosis was established by treating NIH-3T3 cells with transforming growth factor-β1 (TGF-β1). Western blot, RT-qPCR, and scratch test were performed to assess the effect of ISE Ⅱ. In addition, a murine model of pulmonary fibrosis was induced by intratracheal instillation of bleomycin, and the therapeutic effect of ISE Ⅱ was tested by orally administering ISE Ⅱ at a dose of 10 mg/kg. Three weeks later, lung function, micro-CT, hydroxyproline content, pathological staining, and cytokines detection of BALF or serum were used to assess the anti-fibrosis effects of ISE Ⅱ. Next, immunofluorescence staining, flow cytometry, and in vivo transcriptomics were used to investigate the underlying mechanisms of action.
Our data revealed a significant inhibitory effect of ISE Ⅱ on the upregulation of α-smooth muscle actin (α-SMA) and collagen production induced by TGF-β1 in fibroblasts. Meanwhile, ISE Ⅱ exerted a therapeutic effect against bleomycin-induced pulmonary fibrosis in mice by improving lung function, decreasing collagen deposition, and reducing the expression of interleukin (IL)-1β, tumor necrosis factor α (TNF-α), TGF-β1 and platelet-derived growth factor (PDGF) in serum and bronchoalveolar lavage fluid (BALF). Additionally, ISE Ⅱ treatment effectively attenuated the infiltration of M2 macrophages, concurrently downregulating the expression level of M2 marker genes, such as CD206, arginase-1(Arg-1), and Chitinase-Like Protein 3 (YM-1). Importantly, we observed a statistically significant reduction in the M2 phenotype of interstitial macrophages (IMs). However, the impact of ISE Ⅱ on the M2 polarization of alveolar macrophages (AMs) did not reach statistical significance. Lastly, transcriptome sequencing results suggested that the anti-pulmonary fibrosis effects of ISE Ⅱ may be mediated by the suppression of the WNT/β-catenin signaling pathway, which modulated M2 polarization in macrophages and contributed to the amelioration of pulmonary fibrosis. By immunohistochemical analysis, it was verified that ISE Ⅱ treatment dramatically inhibited the activation of β-catenin in fibrosis murine.
Our findings indicated that ISE Ⅱ exerted anti-fibrotic effects by inhibiting pro-fibrotic macrophage polarization. The underlying mechanism of action might be mediated by modulating the WNT/β-catenin signaling pathway to inhibit the M2 program in IMs.
ETHNOPHARMACOLOGICAL 相关性：大量研究提供了证据，支持淫羊藿苷在预防糖尿病、肝纤维化、心脏纤维化、肾纤维化和肺纤维化等多种慢性疾病方面的重要作用。特别是，从淫羊藿属植物中提取的主要代谢物淫羊藿苷衍生的黄酮糖苷伊卡利辛二（ISE II）具有显著的抗炎和抗氧化特性，以及防止肺重塑的能力。然而，关于 ISE Ⅱ在治疗肺纤维化方面的应用研究仍然有限。
本研究旨在评估 ISE II 在肺纤维化模型中的治疗效果，并研究其在细胞信号通路中潜在的作用机制。
通过用转化生长因子-β1（TGF-β1）处理 NIH-3T3 细胞建立肺纤维化的体外模型。采用 Western blot、RT-qPCR 和划痕试验评估 ISE Ⅱ的作用。此外，通过气管内滴注博莱霉素诱导小鼠肺纤维化模型，并通过口服 ISE Ⅱ（剂量为 10mg/kg）测试 ISE Ⅱ的治疗效果。3 周后，通过肺功能、微 CT、羟脯氨酸含量、病理染色和 BALF 或血清细胞因子检测评估 ISE Ⅱ的抗纤维化作用。接下来，采用免疫荧光染色、流式细胞术和体内转录组学研究其作用机制。
我们的数据显示，ISE Ⅱ对 TGF-β1 诱导的成纤维细胞中α-平滑肌肌动蛋白（α-SMA）和胶原产生的上调有显著抑制作用。同时，ISE Ⅱ通过改善肺功能、减少胶原沉积以及降低血清和支气管肺泡灌洗液（BALF）中白细胞介素（IL）-1β、肿瘤坏死因子α（TNF-α）、转化生长因子-β1 和血小板衍生生长因子（PDGF）的表达，对博莱霉素诱导的肺纤维化小鼠发挥治疗作用。此外，ISE Ⅱ治疗有效减轻 M2 巨噬细胞的浸润，同时下调 M2 标志物基因的表达水平，如 CD206、精氨酸酶-1（Arg-1）和几丁质酶样蛋白 3（YM-1）。重要的是，我们观察到间质巨噬细胞（IMs）的 M2 表型显著减少。然而，ISE Ⅱ对肺泡巨噬细胞（AMs）M2 极化的影响未达到统计学意义。最后，转录组测序结果表明，ISE Ⅱ的抗肺纤维化作用可能通过抑制 WNT/β-catenin 信号通路来介导，该通路调节巨噬细胞的 M2 极化，从而改善肺纤维化。通过免疫组织化学分析，证实 ISE Ⅱ治疗可显著抑制纤维化小鼠中β-catenin 的激活。
我们的研究结果表明，ISE Ⅱ通过抑制促纤维化巨噬细胞极化发挥抗纤维化作用。其作用机制可能是通过调节 WNT/β-catenin 信号通路抑制 IMs 中的 M2 程序。
