Laboratory of Inflammation Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Science, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
Laboratory of Inflammation Pharmacology, Institute of Pharmacology and Morphophysiology, Faculty of Veterinary Science, Universidad Austral de Chile, PO Box 567, Valdivia, Chile.
J Dairy Sci. 2013 Apr;96(4):2507-2520. doi: 10.3168/jds.2012-6111. Epub 2013 Feb 10.
Short-chain fatty acids (SCFA) are produced by bacterial fermentation in the rumen of cattle and are the primary energy source in ruminants. Propionate is one of the main SCFA and it can exert multiple effects on the inflammatory process and neutrophil function via calcium (Ca(2+)) release, reactive oxygen species, and intracellular pH changes. However, currently no evidence has shown whether propionate can induce granule release from bovine neutrophils. The purpose of this study was to analyze the effect of propionate on granule release and to evaluate the expression of two G-protein coupled receptors-GPR41 and GPR43-that are activated by propionate. Neutrophil degranulation was assessed by quantifying the release of the neutrophil enzymes myeloperoxidase (MPO), lactoferrin, and matrix metalloprotease-9 (MMP-9) as markers of azurophil, specific granules, and gelatinase granules, respectively. Isolated bovine neutrophils were treated with millimolar concentrations of propionate (0.3, 3 and 30mM), and the cell-free supernatants were recovered. The stimulation of neutrophils with 0.3mM propionate induced the release of lactoferrin and MMP-9 as revealed by ELISA and gelatin zymography, respectively. Propionate at 30mM induced the release of MPO as demonstrated using an enzymatic assay. The role of intracellular Ca(2+) influx and the signaling pathways that may regulate the propionate effect on granules release were also determined. Reverse transcription (RT)-PCR and real-time PCR were performed to analyze the expression of GPR41 and GPR43 mRNA in bovine neutrophils. Both mRNA were detected, whereas the expression of GPR43 was higher than that of GPR41, and the synthetic agonists for this receptor, phenylacetamides 1 and 2, caused an increase in intracellular Ca(2+), lactoferrin, and MMP-9 release. These results support that propionate-induced granule release is mediated by intracellular Ca(2+) influx and activation of extracellular signal-regulated kinase ERK 1/2. We also propose a potential role of GPR43 in propionate-induced granule release from bovine neutrophils that may be involved in regulatory effects of propionate in the innate immune response in cattle.
短链脂肪酸（SCFA）是在牛瘤胃中通过细菌发酵产生的，是反刍动物的主要能量来源。丙酸是主要的 SCFA 之一，它可以通过钙（Ca(2+))释放、活性氧和细胞内 pH 变化来发挥多种作用，影响炎症过程和中性粒细胞功能。然而，目前尚无证据表明丙酸是否能诱导牛中性粒细胞释放颗粒。本研究旨在分析丙酸对颗粒释放的影响，并评估两种被丙酸激活的 G 蛋白偶联受体-GPR41 和 GPR43 的表达。通过定量测定中性粒细胞酶髓过氧化物酶（MPO）、乳铁蛋白和基质金属蛋白酶-9（MMP-9）的释放来评估脱颗粒作用，分别作为嗜中性粒细胞、特异性颗粒和明胶酶颗粒的标记物。用毫摩尔浓度的丙酸（0.3、3 和 30mM）处理分离的牛中性粒细胞，并回收无细胞上清液。ELISA 和明胶酶谱法分别显示，0.3mM 丙酸刺激中性粒细胞诱导乳铁蛋白和 MMP-9 的释放。用酶法测定显示，30mM 丙酸诱导 MPO 的释放。还确定了细胞内 Ca(2+)内流和可能调节丙酸对颗粒释放影响的信号通路的作用。进行逆转录（RT）-PCR 和实时 PCR 以分析牛中性粒细胞中 GPR41 和 GPR43 mRNA 的表达。均检测到这两种 mRNA，而 GPR43 的表达高于 GPR41，并且该受体的合成激动剂苯乙酰胺 1 和 2 引起细胞内 Ca(2+)、乳铁蛋白和 MMP-9 释放增加。这些结果支持丙酸诱导的颗粒释放是通过细胞内 Ca(2+)内流和细胞外信号调节激酶 ERK 1/2 的激活介导的。我们还提出 GPR43 在丙酸诱导的牛中性粒细胞颗粒释放中的潜在作用，这可能涉及丙酸在牛先天免疫反应中的调节作用。
