Park Sangwoo, Kovanda Lauren, Sokale Adebayo O, Barri Adriana, Liu Yanhong
Department of Animal Science, University of California, Davis, CA 95616, USA.
BASF Corporation, Florham Park, NJ 07932, USA.
J Anim Sci. 2023 Jan 3;101. doi: 10.1093/jas/skae372.
The objectives of this study were to investigate the in vitro immune-modulatory effects of monoglycerides and zinc glycinate with porcine alveolar macrophages (PAM) and their impact on epithelial barrier integrity using the intestinal porcine enterocyte cell line (IPEC-J2). Cell viability was assessed using a Vybrant MTT assay to determine the appropriate dose range of monoglyceride blend (C4, C8, and C10) and zinc glycinate. In experiment 1, IPEC-J2 cells (5 × 105 cells/mL) were seeded and treated with each compound (monoglycerides: 0, 25, 100, 250, 500, and 1,000 µg/mL; zinc glycinate: 0, 2, 5, 12.5, 25, and 50 µg/mL). Transepithelial electrical resistance (TEER) was measured by Ohm's law method at 0 h (before treatment) and at 24, 48, and 72 h posttreatment. In experiment 2, PAM were collected from 6 clinically healthy piglets (7 wk of age) and seeded at 106 cells/mL. After incubation, the cells were treated with each compound and/or lipopolysaccharide (LPS). The experimental design was a 2 × 6 factorial arrangement with 2 doses of LPS (0 or 1 μg/mL) and 6 doses of each compound (monoglycerides: 0, 50, 100, 250, 500, and 1,000 µg/mL; zinc glycinate: 0, 25, 50, 100, 250, and 500 µg/mL). Cell supernatants were collected to analyze the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) by enzyme-linked immunosorbent assay kits. Data were analyzed by ANOVA using PROC MIXED of SAS with a randomized complete block design. IPEC-J2 cells treated with 250 or 1,000 μg/mL of monoglycerides, or 5 μg/mL of zinc glycinate had increased (P < 0.05) TEER values at 48 or 72 h posttreatment, compared with control. The LPS challenge increased (P < 0.05) the production of TNF-α and IL-1β from PAM. In the non-challenge group, 50 or 100 μg/mL of monoglycerides stimulated (P < 0.05) TNF-α and IL-1β production from PAMs. Treatment with 25 or 100 μg/mL of zinc glycinate also enhanced (P < 0.05) TNF-α production from PAM. In LPS-treated PAM, 1,000 μg/mL of monoglycerides increased (P < 0.05) IL-1β production, while zinc glycinate suppressed (P < 0.0001) the secretion of TNF-α and IL-1β at the doses of 100, 250, and 500 μg/mL. In conclusion, the results of this in vitro study indicate that monoglycerides positively affect the barrier function of the epithelium, while zinc glycinate may have strong immune regulatory benefits. Future animal studies will be required to verify their impacts on animal gut health, systemic immunity, and growth performance.
本研究的目的是利用猪肺泡巨噬细胞（PAM）研究甘油单酯和甘氨酸锌的体外免疫调节作用，以及它们对猪肠上皮细胞系（IPEC-J2）上皮屏障完整性的影响。使用Vybrant MTT法评估细胞活力，以确定甘油单酯混合物（C4、C8和C10）和甘氨酸锌的合适剂量范围。在实验1中，接种IPEC-J2细胞（5×105个细胞/mL）并用每种化合物处理（甘油单酯：0、25、100、250、500和1000μg/mL；甘氨酸锌：0、2、5、12.5、25和50μg/mL）。在处理前0小时以及处理后24、48和72小时，通过欧姆定律方法测量跨上皮电阻（TEER）。在实验2中，从6头临床健康仔猪（7周龄）收集PAM，并以106个细胞/mL接种。孵育后，用每种化合物和/或脂多糖（LPS）处理细胞。实验设计为2×6析因安排，有2种剂量的LPS（0或1μg/mL）和每种化合物的6种剂量（甘油单酯：0、50、100、250、500和1000μg/mL；甘氨酸锌：0、25、50、100、250和500μg/mL）。收集细胞上清液，通过酶联免疫吸附测定试剂盒分析肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的浓度。使用SAS的PROC MIXED程序，采用随机完全区组设计，通过方差分析对数据进行分析。与对照组相比，用250或1000μg/mL甘油单酯或5μg/mL甘氨酸锌处理的IPEC-J2细胞在处理后48或72小时的TEER值增加（P<0.05）。LPS刺激使PAM产生的TNF-α和IL-1β增加（P<0.05）。在非刺激组中，50或100μg/mL甘油单酯刺激（P<0.05）PAM产生TNF-α和IL-1β。用25或100μg/mL甘氨酸锌处理也增强（P<0.05）PAM产生TNF-α。在LPS处理的PAM中，100 μg/mL甘油单酯增加（P<0.05）IL-1β的产生,而甘氨酸锌在100、250和500μg/mL剂量下抑制（P<0.0001）TNF-α和IL-1β的分泌。总之，这项体外研究结果表明，甘油单酯对上皮屏障功能有积极影响，而甘氨酸锌可能具有强大的免疫调节益处。未来需要进行动物研究以验证它们对动物肠道健康、全身免疫和生长性能的影响。
