目的:肠道微生物来源的代谢产物三甲胺-N-氧化物(TMAO)与心血管纤维化的发展有关。内质网(ER)应激发生在ER及其结构功能障碍后。三个信号PERK/ATF-4、IRE-1α/XBP-1s和ATF6在ER应激时被激活。最近的报道表明,PERK/ATF-4和IRE-1α/XBP-1s信号的激活有助于心血管纤维化。然而,TMAO是否通过激活PERK/ATF-4和IRE-1α/XBP-1s信号介导主动脉瓣纤维化尚不清楚.
方法:从主动脉瓣小叶中分离人主动脉瓣间质细胞(AVIC)。PERKIRE-1α,ATF-4、XBP-1s和CHOP表达,用TMAO处理后,分析了Ⅰ型胶原和TGF-β1的产生。使用抑制剂和小干扰RNA确定PERK/ATF-4和IRE-1α/XBP-1s信号通路在TMAO诱导的纤维化形成中的作用。
结果:病变瓣膜产生较高水平的ATF-4、XBP-1、Ⅰ型胶原和TGF-β1。有趣的是,TMAO刺激后,患病细胞显示出增强的PERK/ATF-4和IRE-1α/XBP-1s激活。PERK/ATF-4和IRE-1α/XBP-1s的抑制和沉默各自导致对患病细胞中TMAO诱导的纤维化活性的增强抑制。用膳食胆碱补充处理的小鼠具有显著增加的TMAO水平和主动脉瓣纤维化,被3,3-二甲基-1-丁醇(DMB,三甲胺形成的抑制剂)处理。此外,高胆碱和高脂肪的饮食重塑了小鼠的肠道菌群。
结论:TMAO在体内和体外通过激活PERK/ATF-4和IRE-1α/XBP-1s信号通路促进主动脉瓣纤维化。调节饮食,肠道菌群,TMAO,PERK/ATF-4和IRE1-α/XBP-1s可能是预防主动脉瓣纤维化的有希望的方法。
OBJECTIVE: The gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) has been implicated in the development of cardiovascular fibrosis. Endoplasmic reticulum (ER) stress occurs after the dysfunction of ER and its structure. The three signals PERK/ATF-4, IRE-1α/XBP-1s and ATF6 are activated upon ER stress. Recent reports have suggested that the activation of PERK/ATF-4 and IRE-1α/XBP-1s signaling contributes to cardiovascular fibrosis. However, whether TMAO mediates aortic valve fibrosis by activating PERK/ATF-4 and IRE-1α/XBP-1s signaling remains unclear.
METHODS: Human aortic valve interstitial cells (AVICs) were isolated from aortic valve leaflets. PERK IRE-1α, ATF-4, XBP-1s and CHOP expression, and production of collagen Ⅰ and TGF-β1 were analyzed following treatment with TMAO. The role of PERK/ATF-4 and IRE-1α/XBP-1s signaling pathways in TMAO-induced fibrotic formation was determined using inhibitors and small interfering RNA.
RESULTS: Diseased valves produced greater levels of ATF-4, XBP-1, collagen Ⅰ and TGF-β1. Interestingly, diseased cells exhibited augmented PERK/ATF-4 and IRE-1α/XBP-1s activation after TMAO stimulation. Inhibition and silencing of PERK/ATF-4 and IRE-1α/XBP-1s each resulted in enhanced suppression of TMAO-induced fibrogenic activity in diseased cells. Mice treated with dietary choline supplementation had substantially increased TMAO levels and aortic valve fibrosis, which were reduced by 3,3-dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) treatment. Moreover, a high-choline and high-fat diet remodeled the gut microbiota in mice.
CONCLUSIONS: TMAO promoted aortic valve fibrosis through activation of PERK/ATF-4 and IRE-1α/XBP-1s signaling pathways in vitro and in vivo. Modulation of diet, gut microbiota, TMAO, PERK/ATF-4 and IRE1-α/XBP-1s may be a promising approach to prevent aortic valve fibrosis.