背景:肝纤维化是慢性肝病中普遍存在的病理过程,其特征是细胞外基质(ECM)沉积过多和血管生成异常。值得注意的是,肝星状细胞(HSC)是ECM的主要来源。活化的HSC不仅分泌大量促纤维化细胞因子,而且被赋予促血管生成表型以促进病理性血管生成。因此,HSC的靶向调节已成为解决肝纤维化的关键策略。羟基红花黄色素A(HSYA)是一种药食同源色素,具有良好的药理活性。然而,HSYA抗肝纤维化的确切机制尚不清楚.
目的:本研究的目的是阐明HSYA对肝纤维化和病理性血管生成的影响,以及体外和体内研究的潜在机制。
方法:采用MTT法研究HSYA对TGF-β1诱导的HSCs和VEGFA诱导的内皮细胞的作用及机制。EdU细胞增殖试验,细胞划痕试验,Elisa分析,免疫荧光测定,分子对接,细胞转染试验,蛋白质印迹分析和RT-qPCR分析。在CCl4诱导的肝纤维化小鼠模型中,H&E,Masson,采用天狼星红染色进行组织病理学观察。采用生化试剂盒检测血清转氨酶活性和肝脏生化指标。免疫组织化学,荧光原位杂交(FISH),采用westernblot分析和RT-qPCR分析来确定HSYA在体内的作用机制。
结果:这里,我们的发现证实了HSYA抑制增殖,HSC的迁移和激活,细胞活力的降低证明了这一点,相对迁移率,EdU染色强度,以及体外促纤维化mRNA和蛋白质表达。机械上,HSYA通过部分沉默活化的HSC中的PDGFRB发挥抗纤维化和抗血管生成作用,从而破坏PDGFRB/MEK/ERK信号转导,抑制HIF-1α的表达,VEGFA和VEGFR2蛋白。重要的是,PDGFRB是miR-29a-3p的靶基因。在用miR-29a-3p抑制剂转染的TGF-β1诱导的HSC中,用HSYA处理逆转miR-29a-3p的下调并拮抗PDGFRB信号通路。与我们的体外研究一致,HSYA通过降低血清ALT和AST水平在CCl4诱导的肝纤维化小鼠中表现出良好的肝保护作用,降低四个纤维化指标的含量(HA,PIIIP,ColIV和LN)和羟脯氨酸,并抑制TGF-β1/TGFBR信号通路。在机制方面,HSYA通过使PDGFRB信号通路失活并损害CD31的阳性表达,减轻纤维化肝病理性血管生成。随后,FISH结果进一步证实HSYA影响HSC的活化和通过miR-29a-3p的同时上调和α-SMA和VEGFA的下调而实现的血管生成。此外,用HSYA治疗也在HSC和内皮细胞之间建立了联系,通过抑制内皮细胞的异常增殖得到支持。
结论:从根本上说,目前的研究表明,HSYA通过抑制HSC介导的促纤维化和促血管生成过程来改善肝纤维化,这取决于HSYA对miR-29a-3p/PDGFRB轴的调节作用。这些发现提供了令人信服的证据支持HSYA作为肝纤维化治疗剂的潜力。
BACKGROUND: Liver fibrosis is a prevalent pathological process in chronic liver diseases characterized by excessive extracellular matrix (ECM) deposition and abnormal angiogenesis. Notably, hepatic stellate cells (HSCs) are the primary source of ECM. Activated HSCs not only secrete numerous pro-fibrotic cytokines but also are endowed with a pro-angiogenic phenotype to promote pathological angiogenesis. Therefore, targeted modulation of HSCs has emerged as a pivotal strategy for addressing liver fibrosis. Hydroxysafflor yellow A (HSYA) is a homology of medicine and food colourant with good pharmacological activity. However, the precise mechanisms of HSYA against liver fibrosis remain unclear.
OBJECTIVE: The objective of this study was to elucidate the impact of HSYA on liver fibrosis and pathological angiogenesis, as well as the underlying mechanisms in vitro and in vivo studies.
METHODS: The efficacy and mechanisms of HSYA on TGF-β1-induced HSCs and VEGFA-induced endothelial cells were investigated by MTT assay, EdU cell proliferation assay, cell scratch assay, Elisa assay, immunofluorescence assay, molecular docking, cell transfection assay, western blot analysis and RT-qPCR analysis. In CCl4-induced liver fibrosis mice model, H&E, Masson, and Sirius red staining were used to observe histopathology. Serum transaminase activity and liver biochemical indexes were tested by biochemical kit. Immunohistochemical, fluorescence in situ hybridization (FISH), western blot analysis and RT-qPCR analysis were implemented to determine the mechanism of HSYA in vivo.
RESULTS: Herein, our findings confirmed that HSYA inhibited the proliferation, migration and activation of HSCs, as evidenced by a reduction in cell viability, relative migration rate, EdU staining intensity, and pro-fibrotic mRNAs and proteins expression in vitro. Mechanistically, HSYA played an anti-fibrotic and anti-angiogenic role by partially silencing PDGFRB in activated HSCs, thereby disrupting PDGFRB/MEK/ERK signal transduction and inhibiting the expression of HIF-1α, VEGFA and VEGFR2 proteins. Importantly, PDGFRB was a target gene of miR-29a-3p. Treatment with HSYA reversed the down-regulation of miR-29a-3p and antagonized PDGFRB signaling pathway in TGF-β1-induced HSCs transfected with miR-29a-3p inhibitor. Consistent with our in vitro study, HSYA exhibited a good hepatoprotective effect in CCl4-induced liver fibrosis mice by reducing serum ALT and AST levels, decreasing the contents of four fibrosis indicators (HA, PIIIP, ColIV and LN) and hydroxyproline, and inhibiting the TGF-β1/TGFBR signaling pathway. In terms of mechanisms, HSYA alleviated pathological angiogenesis in fibrotic liver by deactivating PDGFRB signaling pathway and impairing the positive expression of CD31. Subsequently, FISH results further corroborated HSYA affected the activation of HSCs and angiogenesis achieved by the concurrent upregulation of miR-29a-3p and downregulation of α-SMA and VEGFA. Additionally, treatment with HSYA also forged a link between HSCs and endothelial cells, as supported by inhibiting the aberrant proliferation of endothelial cells.
CONCLUSIONS: Fundamentally, the current study has illustrated that HSYA ameliorates liver fibrosis by repressing HSCs-mediated pro-fibrotic and pro-angiogenic processes, which is contingent upon the regulatory effect of HSYA on the miR-29a-3p/PDGFRB axis. These findings provide compelling evidence bolstering the potential of HSYA as a therapeutic agent in liver fibrosis.