Niacin Improves Intestinal Health through Up-Regulation of AQPs Expression Induced by GPR109A

Abstract

(1) Background: Changes in the expression of aquaporins (AQPs) in the intestine are proved to be associated with the attenuation of diarrhea. Diarrhea is a severe problem for postweaning piglets. Therefore, this study aimed to investigate whether niacin could alleviate diarrhea in weaned piglets by regulating AQPs expression and the underlying mechanisms; (2) Methods: 72 weaned piglets (Duroc × (Landrace × Yorkshire), 21 d old, 6.60 ± 0.05 kg) were randomly allotted into 3 groups for a 14-day feeding trial. Each treatment group included 6 replicate pens and each pen included 4 barrows (n = 24/treatment). Piglets were fed a basal diet (CON), a basal diet supplemented with 20.4 mg niacin/kg diet (NA) or the basal diet administered an antagonist for the GPR109A receptor (MPN). Additionally, an established porcine intestinal epithelial cell line (IPEC-J2) was used to investigate the protective effects and underlying mechanism of niacin on AQPs expression after Escherichia coli K88 (ETEC K88) treatment; (3) Results: Piglets fed niacin-supplemented diet had significantly decreased diarrhea rate, and increased mRNA and protein level of ZO-1, AQP 1 and AQP 3 in the colon compared with those administered a fed diet supplemented with an antagonist (p < 0.05). In addition, ETEC K88 treatment significantly reduced the cell viability, cell migration, and mRNA and protein expression of AQP1, AQP3, AQP7, AQP9, AQP11, and GPR109A in IPEC-J2 cells (p < 0.05). However, supplementation with niacin significantly prevented the ETEC K88-induced decline in the cell viability, cell migration, and the expression level of AQPs mRNA and protein in IPEC-J2 cells (p < 0.05). Furthermore, siRNA GPR109A knockdown significantly abrogated the protective effect of niacin on ETEC K88-induced cell damage (p < 0.05); (4) Conclusions: Niacin supplementation increased AQPs and ZO-1 expression to reduce diarrhea and intestinal damage through GPR109A pathway in weaned piglets.

Keywords: GPR109A; IPEC-J2 cell; aquaporins; diarrhea; niacin; piglets.

Figure 1

Effect of niacin on the diarrhea rate and mRNA expression of tight junctions in the intestinal mucosa of weaned piglets. (A) Diarrhea rate of weaned piglets. (B–D) QPCR determined the relative mRNA expression of ZO-1 and Occludin in the jejunal, ileal, and colonic mucosa, respectively. CON, control group; NA, niacin group, MPN, GPR109A receptor blocking group. Values are presented as the mean ± SEM (n = 6). ** p < 0.01.

Figure 2

Effect of niacin on the genes and protein expression of AQPs in the intestinal mucosa of weaned piglets. (A–C) QPCR determined the relative mRNA expression of AQP1, AQP3, AQP7, and AQP11 in the jejunal, ileal and colonic mucosa, respectively. (D–F) Western blot determined the relative protein expression of AQP1, AQP3, and AQP7 in the jejunal, ileal and colonic mucosa, respectively. CON, control group, NA, niacin group, MPN, GPR109A receptor blocking group. AQP, aquaporin. Values are presented as the mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Effect of ETEC K88 or niacin on the cell viability of IPEC-J2 cells. The cell viability was determined by Cell Counting Kit-8 (CCK8) assay in our trial. (A) The cell viability after being treated with different concentrations of ETEC K88 for 3.5 h. (B) The cell viability after being treated with different concentrations of niacin for 12 h. (C) The cell viability after being treated with different concentrations of niacin for 12 h, followed by ETEC K88 (1 × 10⁸ CFU/mL) for 3.5 h. Values are presented as the mean ± SEM (n = 3). ** p < 0.01, *** p < 0.001.

Figure 4

Effect of niacin on the cell migration capacity of IPEC-J2 cells. (A) Representative image of the wound-healing assay. The dashed lines indicate wound edges. After scratching, the culture medium was removed, replaced with a fresh medium with or without different concentrations of niacin, and incubated for another 24 h. Scale bar: 50 µm. (B) Quantification of migrated scratch width at different time points. (C) Quantification of the healing rate. The healing or closure rate is expressed as a ratio of the migration distance (after 24 h) compared with the distance immediately after scratching. Values are presented as the mean ± SEM (n = 3). *** p < 0.001.

Figure 5

Effect of niacin on the genes and protein expression of AQPs in the IPEC-J2 cells after treatment with ETEC K88. (A–E) QPCR determined the relative mRNA expression of AQP1, 3, 7, 9, and 11, respectively. (F) Western blot determined the relative protein expression of AQP1, 3, 7, and 9 in IPEC-J2 cells, respectively. NA, niacin. AQP, aquaporin. Values are presented as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Effect of ETEC K88 or niacin on the mRNA and protein expression of GPR109A in IPEC-J2 cells. (A) QPCR determined the relative mRNA expression of GPR109A. (B) Western blot determined the relative protein expression of GPR109A. (C) QPCR determined the relative mRNA expression of GPR109A in IPEC-J2 cells transfected with control siRNA or siRNA targeting GPR109A. There are three siRNA (siGPR109A-1, siGPR109A-2, siGPR109A-3) designed in the present study, and siGPR109A2 was selected for the following trials. (D) Western blot determined the relative protein expression of GPR109A in IPEC-J2 cells transfected with control siRNA or siRNA targeting GPR109A. NA, niacin. GPR109A, G protein-coupled receptor 109A. Values are presented as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Effect of blocking GPR109A on cell viability and the mRNA expression of AQPs in the IPEC-J2 cells. (A) CCK8 determined the cell viability in IPEC-J2 cells transfected with control siRNA or siRNA targeting GPR109A. (B–F) QPCR determined the relative mRNA expression of AQP1, AQP3, AQP7, AQP9, and AQP11 in IPEC-J2 cells transfected with control siRNA or siRNA targeting GPR109A. NA, niacin. AQP, aquaporin. GPR109A, G protein-coupled receptor 109A. Values are presented as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01.