背景:灵芝多糖(GLP)具有许多生物学特性,然而,GLP的抗纤维化作用目前尚不清楚。
目的:本研究旨在研究GLP在体内和体外的抗纤维化作用及其潜在的分子机制。
方法:建立CCl4诱导的小鼠和TGF-β1诱导的HSC-T6纤维化细胞模型,以检查从除孢子粒的灵芝孢子中提取的水溶性GLP(25kDa)的抗纤维化作用。.
方法:血清肝损伤标志物,肝组织的组织学和纤维化,使用自动生化分析仪检查胶原蛋白的形成,H&E染色,天狼星红染色,免疫组织化学,免疫荧光,ELISA,西方印迹,和qRT-PCR。RNA测序,富集途径分析,西方印迹,qRT-PCR,和流式细胞术用于鉴定负责GLP抗纤维化作用的潜在分子靶标和信号通路。
结果:我们发现GLP(150和300mg/kg)显著抑制由TLR4/NF-κB/MyD88信号通路介导的CCl4处理小鼠的肝纤维化和炎症。我们进一步证明,GLP显着抑制小鼠和TGF-β1诱导的HSC-T6细胞中的肝星状细胞(HSC)活化,表现为胶原蛋白I和a-SMA表达降低。RNA测序发现炎症,凋亡,细胞周期,ECM-受体相互作用,TLR4/NF-κB,和TGF-β/Smad信号作为GLP给药抑制的主要途径。进一步的研究表明,GLP引发抗纤维化作用,与体内(抑制)或体外(促进)对细胞凋亡的新型双重作用有关。抑制体内细胞周期,体外诱导S期阻滞,和ECM-受体相互作用相关分子表达的减弱,包括整合素ITGA6和ITGA8。此外,GLP显著抑制小鼠TGF-β/Smad信号,并减少TGF-β1或其激动剂SRI-011381诱导的Smad2和Smad3磷酸化,但增加了HSC-T6细胞中Samd7的表达。
结论:这项研究提供了第一个证据,表明GLP可能是治疗肝纤维化的有希望的饮食策略,通过靶向炎症保护肝纤维化和HSC激活,凋亡,细胞周期,和由TGF-β/Smad信号介导的ECM-受体相互作用。
BACKGROUND: Ganoderma lucidum polysaccharide (GLP) has many biological properties, however, the anti-fibrosis effect of GLP is unknown at present.
OBJECTIVE: This study aimed to examine the anti-fibrogenic effect of GLP and its underlying molecular mechanisms in vivo and in vitro.
METHODS: Both CCl4-induced mouse and TGF-β1-induced HSC-T6 cellular models of fibrosis were established to examine the anti-fibrogenic effect of a water-soluble GLP (25 kDa) extracted from the sporoderm-removed spores of G. lucidum..
METHODS: Serum markers of liver injury, histology and fibrosis of liver tissues, and collagen formation were examined using an automatic biochemical analyzer, H&E staining, Sirius red staining, immunohistochemistry, immunofluorescence, ELISA, Western blotting, and qRT-PCR. RNA-sequencing, enrichment pathway analysis, Western blotting, qRT-PCR, and flow cytometry were employed to identify the potential molecular targets and signaling pathways that are responsible for the anti-fibrotic effect of GLP.
RESULTS: We showed that GLP (150 and 300 mg/kg) significantly inhibited hepatic fibrogenesis and inflammation in CCl4-treated mice as mediated by the TLR4/NF-κB/MyD88 signaling pathway. We further demonstrated that GLP significantly inhibited hepatic stellate cell (HSCs) activation in mice and in TGF-β1-induced HSC-T6 cells as manifested by reduced collagen I and a-SMA expressions. RNA-sequencing uncovered inflammation, apoptosis, cell cycle, ECM-receptor interaction, TLR4/NF-κB, and TGF-β/Smad signalings as major pathways suppressed by GLP administration. Further studies demonstrated that GLP elicits anti-fibrotic actions that are associated with a novel dual effect on apoptosis in vivo (inhibit) or in vitro (promote), suppression of cell cycle in vivo, induction of S phase arrest in vitro, and attenuation of ECM-receptor interaction-associated molecule expressions including integrins ITGA6 and ITGA8. Furthermore, GLP significantly inhibited the TGF-β/Smad signaling in mice, and reduced TGF-β1 or its agonist SRI-011381-induced Smad2 and Smad3 phosphorylations, but increased Samd7 expression in HSC-T6 cells.
CONCLUSIONS: This study provides the first evidence that GLP could be a promising dietary strategy for treating liver fibrosis, which protects against liver fibrosis and HSC activation through targeting inflammation, apoptosis, cell cycle, and ECM-receptor interactions that are mediated by TGF-β/Smad signaling.