Andrani Melania, Borghetti Paolo, Ravanetti Francesca, Cavalli Valeria, Ferrari Luca, De Angelis Elena, Martelli Paolo, Saleri Roberta
Department of Veterinary Science, University of Parma, Strada del Taglio 10, 43126, Parma, Italy.
Porcine Health Manag. 2023 May 23;9(1):23. doi: 10.1186/s40813-023-00316-y.
The interest in acetate and propionate as short chain fatty acids (SCFA) derives from research on alternative strategies to the utilization of antibiotics in pig farms. SCFA have a protective role on the intestinal epithelial barrier and improve intestinal immunity by regulating the inflammatory and immune response. This regulation is associated with an increase in intestinal barrier integrity, mediated by the enhancement of tight junction protein (TJp) functions, which prevent the passage of pathogens through the paracellular space. The purpose of this study was to evaluate the effect of in vitro supplementation with SCFA (5 mM acetate and 1 mM propionate) on viability, nitric oxide (NO) release (oxidative stress), NF-κB gene expression, and gene and protein expression of major TJp (occludin [OCLN], zonula occludens-1 [ZO-1], and claudin-4 [CLDN4]) in a porcine intestinal epithelial cell (IPEC-J2) and peripheral blood mononuclear cell (PBMC) co-culture model upon LPS stimulation, through which an acute inflammatory state was simulated.
Firstly, the inflammatory stimulus induced by LPS evaluated in the IPEC-J2 monoculture was characterized by a reduction of viability, gene expression of TJp and OCLN protein synthesis, and an increase of NO release. The response evaluated in the co-culture showed that acetate positively stimulated the viability of both untreated and LPS-stimulated IPEC-J2 and reduced the release of NO in LPS-stimulated cells. Acetate also promoted an increase of gene expression of CLDN4, ZO-1, and OCLN, and protein synthesis of CLDN4, OCLN and ZO-1 in untreated and LPS-stimulated cells. Propionate induced a reduction of NO release in both untreated and LPS-stimulated IPEC-J2. In untreated cells, propionate induced an increase of TJp gene expression and of CLDN4 and OCLN protein synthesis. Contrarily, propionate in LPS-stimulated cells induced an increase of CLDN4 and OCLN gene expression and protein synthesis. PBMC were influenced by acetate and propionate supplementation, in that NF-κB expression was strongly downregulated in LPS-stimulated cells.
The present study demonstrates the protective effect of acetate and propionate upon acute inflammation by regulating epithelial tight junction expression and protein synthesis in a co-culture model, which simulates the in vivo interaction between epithelial intestinal cells and local immune cells.
对乙酸盐和丙酸盐作为短链脂肪酸（SCFA）的研究兴趣源于对猪场抗生素替代策略的研究。SCFA对肠道上皮屏障具有保护作用，并通过调节炎症和免疫反应来改善肠道免疫力。这种调节与肠道屏障完整性的增加有关，这是由紧密连接蛋白（TJp）功能的增强介导的，紧密连接蛋白可防止病原体通过细胞旁间隙。本研究的目的是评估在猪肠道上皮细胞（IPEC-J2）和外周血单核细胞（PBMC）共培养模型中，体外补充SCFA（5 mM乙酸盐和1 mM丙酸盐）对脂多糖（LPS）刺激下细胞活力、一氧化氮（NO）释放（氧化应激）、NF-κB基因表达以及主要TJp（闭合蛋白[OCLN]、紧密连接蛋白1[ZO-1]和Claudin-4[CLDN4]）的基因和蛋白表达的影响，通过该模型模拟急性炎症状态。
首先，在IPEC-J2单培养中评估的由LPS诱导的炎症刺激的特征是细胞活力降低、TJp基因表达和OCLN蛋白合成减少以及NO释放增加。在共培养中评估的反应表明，乙酸盐对未处理和LPS刺激的IPEC-J2的活力均有正向刺激作用，并降低了LPS刺激细胞中NO的释放。乙酸盐还促进了未处理和LPS刺激细胞中CLDN4、ZO-1和OCLN基因表达的增加以及CLDN4、OCLN和ZO-1蛋白的合成。丙酸盐在未处理和LPS刺激的IPEC-J2中均诱导NO释放减少。在未处理的细胞中，丙酸盐诱导TJp基因表达以及CLDN4和OCLN蛋白合成增加。相反，LPS刺激细胞中的丙酸盐诱导CLDN4和OCLN基因表达及蛋白合成增加。PBMC受到乙酸盐和丙酸盐补充的影响，因为在LPS刺激的细胞中NF-κB表达强烈下调。
本研究证明了乙酸盐和丙酸盐在共培养模型中通过调节上皮紧密连接表达和蛋白合成对急性炎症具有保护作用，该共培养模型模拟了肠道上皮细胞与局部免疫细胞之间的体内相互作用。
