■微折叠细胞(M细胞)是特定的肠上皮细胞,用于监测和转胞抗原,微生物,和肠道中的病原体。然而,M细胞发育的机制仍然难以捉摸。
■实时聚合酶链反应,免疫荧光,和蛋白质印迹分析山梨醇调节的M细胞分化的体内和体外的影响,荧光素酶和染色质免疫沉淀用于揭示山梨醇调节M细胞分化的机制。
■这里,与甘露醇组(对照组)相比,我们发现,肠道M细胞的发育在响应山梨糖醇治疗时受到抑制,如受损的肠样物质伴随着早期分化标记物膜联蛋白5,Marcksl1,Spib,SOX8和成熟M细胞标记糖蛋白2表达,这归因于体内和体外核因子κB配体受体激活剂(RANKL)表达的下调。机械上,在M细胞模型中,山梨醇刺激引起磷酸二酯酶4(PDE4)磷酸化的显著上调,导致蛋白激酶A(PKA)/cAMP反应元件结合蛋白(CREB)激活减少,这进一步导致CREB在胞质中的保留和减弱的CREB结合RANKL启动子以抑制RANKL表达。有趣的是,内源性PKA与CREB相互作用,这种相互作用被山梨糖醇刺激破坏。最重要的是,双嘧达莫对PDE4的抑制作用可以挽救山梨糖醇对肠样肠样物质和M细胞分化以及体内和体外成熟的抑制作用。
■这些发现表明山梨糖醇抑制肠类肠样物质和M细胞分化并通过PDE4介导的RANKL表达而成熟;靶向抑制PDE4足以诱导M细胞发育。
UNASSIGNED: Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive.
UNASSIGNED: Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation.
UNASSIGNED: Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro.
UNASSIGNED: These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.