Miao Wei, Wu Xiujuan, Wang Kang, Wang Wenjing, Wang Yumei, Li Zhigang, Liu Jingjing, Li Li, Peng Luying
Key Laboratory of Arrhythmias, Ministry of Education, East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
Int J Mol Sci. 2016 Oct 10;17(10):1696. doi: 10.3390/ijms17101696.
As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection.
作为膳食纤维微生物发酵产生的一种生理性小分子产物，丁酸在维持肠道健康方面发挥着重要作用。我们之前的研究已经证明，丁酸钠（NaB）对肠道屏障功能的影响是通过激活AMP活化蛋白激酶（AMPK）介导的。然而，具体涉及的途径仍不清楚。在Caco-2细胞单层模型中使用钙切换试验，我们在此发现NaB主要通过促进储存-操作性钙内流（SOCE）来增加钙水平而非ATP浓度来激活AMPK。在AMPK激活后，NaB基于降低肌球蛋白II调节轻链（MLC2）在Ser19处的磷酸化以及增加蛋白激酶Cβ2（PKCβ2）在Ser660处的磷酸化来促进紧密连接（TJ）的重新组装。抑制蛋白激酶Cβ（PKCβ）可阻断屏障单层模型中NaB诱导的TJ重新组装。这些结果表明，NaB可激活钙/钙调蛋白依赖性蛋白激酶激酶β（CaMKKβ）途径来介导AMPK磷酸化，进而分别抑制MLC2的磷酸化并促进PKCβ2的磷酸化，从而使AMPK的下游分子协同促进Caco-2屏障模型中TJ的重新组装。这些结果提示了丁酸维持肠道稳态和保护作用的潜在机制。
