转自：天然活性分子发现与靶点鉴定
Wogonoside attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis through SOCS1/P53/SLC7A11 pathway
关注下方公众号，查影响因子：
肝纤维化是各种慢性肝病的常见病理特征，其特征是细胞外基质（ECM）过度积聚。肝星状细胞（HSC）是负责ECM产生的主要肝细胞类型，其激活导致纤维组织沉积和瘢痕形成。在生理条件下，HSC位于DISE空间，是维生素A的主要储存场所，并呈现静态表型。作为对外部伤害性刺激的响应，静态的HSC经历了复杂的激活过程，包括维生素A的下调、细胞膜蛋白α-平滑肌肌动蛋白（α-SMA）的从头表达和ECM成分的过量生成。
黄芩是唇形科多年生草本植物，广泛用于治疗肝病的中药。现代药理学研究表明，黄芩提取物或活性成分可以改善肝损伤的累积。汉黄芩苷（WG）是一种从黄芩根中分离的生物活性类黄酮，具有多种药理特性，包括抗癌、抗炎和抗纤维化活性。先前的研究表明，在脂多糖/D-半乳糖胺诱导的小鼠急性肝损伤模型中，WG显著降低血清丙氨酸转氨酶（ALT）、天冬氨酸氨基转移酶（AST）和肝丙二醛（MDA）。
在该研究中，作者旨在探讨WG的抗肝纤维化作用，并探讨其潜在的分子机制。作者发现，WG通过SOCS1/P53/SLC7A11途径诱导HSC铁死亡，从而减轻肝纤维化。相反，铁死亡抑制剂铁抑制素-1（Fer-1）则抵消了WG的抗纤维化作用。作者的研究可能为肝纤维化的治疗提供一个潜在的治疗靶点。
WG减轻CCl4诱导的小鼠肝纤维化并抑制HSC-T6细胞活力。如图1a所示，与正常小鼠相比，在CCl4诱导的小鼠中观察到肝细胞变性、炎性细胞浸润、纤维瘢痕形成和胶原沉积，而肝损伤和纤维化现象在WG治疗后显著减轻。此外，在WG处理的小鼠中，α-SMA和COL1α1的免疫染色信号减少（图1b）。Western bolt分析证实，与载体组相比，CCl4诱导小鼠的α-SMA和COL1α1蛋白显著增加，而与CCl4诱发小鼠相比，WG给药降低了这些蛋白的表达（图1c）。为了进一步验证WG对肝纤维化的影响，将大鼠HSC-T6细胞与10ng/ml PDGF-BB和不同浓度的WG一起培养。CCK-8分析表明，与对照组相比，PDGF-BB组HSC-T6细胞的活力显著增加，而WG以剂量依赖性方式抑制PDGF-BB处理的HSC-T6细胞的活力（图1d）。综上所述，上述数据表明，WG可减轻CCl4诱导的小鼠肝纤维化，并抑制HSC-T6细胞活力。
FIGURE 1. WG alleviates CCl4-induced liver fibrosis and inhibits HSC-T6 cell viability. (a) Images of liver sections stained with H&E, Masson’s trichrome and Sirius Red staining demonstrate liver injury in indicated groups (scale bar: 100 μm). (b) Photomicrographs of liver immunohistochemical staining of α-SMA and COL1α1 (scale bar: 100 μm). (c) Expression of fibrosis indicators α-SMA and COL1α1 in liver tissue was detected by western blot. (d) Cell viability of HSC-T6 cells treated with different concentrations of WG for 48 h was detected by CCK-8. (E, F) mRNA and protein expression of α-SMA and COL1α1 was detected by qRT-PCR and western blot in HSC-T6 cells. Data presented as the mean ± SEM (n = 3 in each group). #p < .05, ##p < .01 versus vehicle mice and vehicle-treated group. *p < .05, **p < .01 versus CCl4-treated mice and PDGF-BB-induced group.
WG触发激活的HSC-T6细胞铁死亡。在经过WG处理的HSC-T6细胞中，观察到GSH含量降低，铁水平和脂质过氧化产物的升高（图2A）。WG处理的HSC-T6细胞中的GPX4和SLC7A11蛋白水平也显着降低（图2B）。TEM分析表明，与PDGF-BB处理的细胞相比，WG处理的HSC-T6细胞显示出皱纹和断裂的线粒体（图2C），WG（60μM）显着降低了活化的HSC-T6细胞的细胞活力（图2D）。Fer-1是一种有效的铁死亡抑制剂，可以阻止WG介导的细胞生长抑制作用（图2D）。然而，凋亡抑制剂（Z-VAD-FMK）和坏死抑制剂（Necrosulfonamide，NSA）对WG处理的HSC-T6细胞的细胞活力没有影响（图2D）。这些数据表明，WG可以抑制HSC-T6细胞中的生存能力并诱导铁死亡。
FIGURE 2. WG triggers ferroptosis in activated HSC-T6 cells. HSC-T6 cells were treated with DMSO (0.02%, w/v), WG (40, 60, 80 μM) and PDGF-BB (10 ng/ml) for 48 h. (a) Ferroptotic events in HSC-T6 cells: iron release, MDA content and GSH expression in cell lysates were detected by relevant kits. Intracellular ROS generation was detected with DCFH-DA probe (10 mM). (b) Western blot analysis of SLC7A11 and GPX4 proteins was performed. (c) Mitochondria in control and WG-treated groups were observed by transmission electron microscope (scale bar: 200 nm). (d) HSC-T6 cells were exposed to Fer-1 (1 μM), Z-VAD-FMK (10 μM) or NSA (0.5 μM) with or without WG (60 μM); cell viability was assayed by CCK-8. Data presented as the mean ± SEM (n = 3 in each group). #p < .05, ##p < .01 versus vehicle-treated group. *p < .05, **p < .01 versus PDGF-BB-induced group. N.S., not significant
阻断HSC-T6细胞中的铁死亡会损害WG降低的ECM表达。为了进一步探讨铁死亡在WG抗纤维化中的作用，使用铁死亡特异性抑制剂Fer-1抑制HSC-T6细胞中的铁死亡。如图3a所示，在WG处理的HSC-T6细胞中发现铁水平升高、GSH含量降低和脂质过氧化产物升高，而Fer-1可减轻WG所诱导的相关铁死亡标志物的变化。此外，经WG处理的HSC-T6细胞中GPX4和SLC7A11的表达降低也被Fer-1逆转（图3b）。这些数据证实，Fer-1可以阻断WG诱导的HSC-T6细胞铁死亡。此外，WG降低了α-SMA和COL1α1的荧光强度，而Fer-1抑制了这一过程（图3c）。Western blot分析证实HSC-T6细胞中的铁死亡阻断消除了由WG诱导的α-SMA和COL1α1表达的降低（图3d）。总之，阻断HSC中的铁死亡会损害WG诱导的ECM减少。
FIGURE 3. Blockade of ferroptosis in HSC-T6 cells impairs WG-reduced ECM expression. HSC-T6 cells were exposed to 1 μM Fer-1, followed by DMSO (0.02%, w/v) WG (60 μM) or PDGF-BB (10 ng/ml) treatment for 48 h. (a) Iron, MDA and GSH content in cell lysates was measured by relevant kits. Intracellular ROS generation was detected with DCFH-DA probe (10 mM). (b) GPX4 and SLC7A11 proteins were measured by western blot. (c) Expression of α-SMA and COL1α1 was measured in HSC-T6 cells by immunofluorescence (scale bar: 20 μm). (d) Western blot analysis of α-SMA and COL1α1 proteins was performed. Data presented as the mean ± SEM (n = 3 in each group). #p < .05 versus vehicle-treated group. *p < .05 versus PDGF-BB-induced group and the PDGF-BB and WG (60 μM)-induced group. N.S., not significant
SOCS1/P53参与WG的抗肝纤维化作用。与正常小鼠相比，CCl4诱导的肝脏中SOCS1，p53和p-p53蛋白的水平显着降低，而在WG处理的组中上述指标则表现升高（图4A）。与未处理的细胞相比，在PDGF-BB激活的HSC-T6细胞中，SOCS1，p53和p-p53蛋白水平也降低了，WG（60和80μM）在活化的HSC-T6细胞中增加了SOCS1，p-p53和p53蛋白（图4B）。免疫荧光染色测定表明WG不仅增加了SOCS1和p53的表达，而且还增加了它们在细胞核中的丰度（图4C）。如图4d、e所示，与正常组相比，siRNA-2显著降低了SOCS1的基因和蛋白质水平。此外，SOCS1 siRNA也降低了P53和p-P53的蛋白水平（图4f）。Pifithrin-α（PFT-α），一种P53特异性抑制剂，降低了P53和p-P53蛋白的表达，而SOCS1蛋白的表达保持不变（图4f）。总之，这些发现表明WG的抗肝纤维化作用与SOCS1/P53途径有关。
FIGURE 4. SOCS1/P53 is involved in the anti-liver fibrosis effect of WG. (a, b) Western blot analysis of SOCS1, P53 and p-P53 were conducted in liver tissues and HSC-T6 cells. (c) SOCS1 and P53 were measured by immunofluorescence in HSC-T6 cells (scale bar: 20 μm). (d, e) HSC-T6 cells were exposed to SOCS1 siRNA treatment for 48 h. The level of SOCS1 was measured by western blot and qRT-PCR. (f) HSC-T6 cells were exposed to SOCS1 siRNA or PFT-α (20 μM) for 48 h, with or without PDGF-BB (10 ng/ml) and WG (60 μM) treatment for 48 h. Protein expression of SOCS1, P53 and p-P53 was measured by western blot. Data presented as the mean ± SEM (n = 3 in each group). #p < .05, ##p < .01 versus CCl4-treated mice and vehicle-treated group. *p < .05, **p < .01 versus CCl4-treated mice and PDGF-BB-induced group as well as the PDGF-BB-induced and WG (60 μM)-induced group. N.S., not significant
阻断SOCS1/P53通路可抵消WG诱导的HSC-T6细胞铁死亡和抗纤维化作用。如图5a所示，与PDGF-BB组相比，WG(60 μM)在HSC-T6细胞中诱导了铁死亡（图5a）。与WG处理的细胞相比，在SOCS1 siRNA和PFT-α处理后，GPX4和SLC7A11蛋白显著增加（图5b）。此外，WG抑制活化的HSC-T6细胞活力，这种效应被SOCS1 siRNA和PFT-α消除（图5c）。由WG引发的α-SMA和COL1α1的减少也被SOCS1 siRNA和PFT-α消除（图5d）。总之，上述结果表明，WG通过SOCS1/P53途径诱导HSC-T6细胞铁死亡，并减轻肝纤维化。
FIGURE 5. Blockade of SOCS1/P53 pathway abrogates WG-induced HSC-T6 cell ferroptosis and anti-fibrotic effect. HSC-T6 cells were exposed to SOCS1 siRNA or PFT-α (20 μM) for 48 h, with or without PDGF-BB (10 ng/ml) and WG (60 μM) treatment for 48 h. (a) Major biomarkers of ferroptosis: iron, GSH and MDA in cell lysates were measured by kits. Intracellular ROS generation was detected with DCFH-DA probe (10 mM). (b) Western blot analysis of SLC7A11 and GPX4 proteins was performed. (c) Viability of HSC-T6 cells was detected by CCK-8. (d) Protein expression of α-SMA and COL1α1 was measured by western blot. Data presented as the mean ± SEM (n = 3 in each group). #p < .05 versus vehicle-treated group. *p < .05 versus PDGF-BB-induced group and PDGF-BB and WG (60 μM)-induced group. N.S., not significant
综上所述，本研究证实了WG对肝脏具有的抗纤维化作用，表现为ECM沉积减少和组织病理学特征改善。此外，在WG处理的HSC中发现了铁死亡，而不是巨噬细胞或肝细胞。相反，抑制铁死亡会阻碍WG的抗纤维化作用。最终，发现SOCS1/P53/SLC7A11通路至少部分参与了HSC铁死亡。这些数据表明，诱导HSC铁死亡是治疗肝纤维化的一个潜在靶点。同时，WG具有开发成药物的潜力。
DOI:https://doi.org/10.1002/ptr.7558
转自：“如沐风科研”微信公众号
如有侵权，请联系本站删除！
