SLC6A14 promotes ulcerative colitis progression by facilitating NLRP3 inflammasome-mediated pyroptosis

Abstract

Background: Ulcerative colitis (UC) is an inflammatory condition with frequent relapse and recurrence. Evidence suggests the involvement of SLC6A14 in UC pathogenesis, but the central regulator remains unknown.

Aim: To explore the role of SLC6A14 in UC-associated pyroptosis.

Methods: Quantitative real-time polymerase chain reaction (qRT-PCR), immunoblotting, and immunohistochemical were used to assess SLC6A14 in human UC tissues. Lipopolysaccharide (LPS) was used to induce inflammation in FHC and NCM460 cells and model enteritis, and SLC6A14 levels were assessed. Pyroptosis markers were quantified using enzyme-linked immunosorbent assay, Western blotting, and qRT-PCR, and EdU incubation, CCK-8 assays and flow cytometry were used to examine proliferation and apoptosis. Mouse models of UC were used for verification.

Results: SLC6A14 was increased and correlated with NLRP3 in UC tissues. LPS-induced FHC and NCM460 cells showed increased SLC6A14 levels. Reducing SLC6A14 increased cell proliferation and suppressed apoptosis. Reducing SLC6A14 decreased pyroptosis-associated proteins (ASC, IL-1β, IL-18, NLRP3). NLRP3 overexpression counteracted the effects of sh-SLC6A14 on LPS-induced FHC and NCM460 cell pyroptosis. SLC6A14 improved the mucosa in mice with dextran sulfate sodium-induced colitis.

Conclusion: SLC6A14 promotes UC pyroptosis by regulating NLRP3, suggesting the therapeutic potential of modulating the SLC6A14/NLRP3 axis.

Keywords: Inflammasome; NLRP3; Pyroptosis; SLC6A1; Ulcerative colitis.

Figure 1

SLC6A14 in ulcerative colitis tissues. A: Hematoxylin and eosin staining and immunohistochemical (IHC) analysis of SLC6A14 expression in normal and ulcerative colitis (UC) tissues (magnification, × 200; scale bar = 100 μm); B: SLC6A14 mRNA levels were assessed by real-time polymerase chain reaction in UC (n = 55) and normal tissues (n = 29); C and D: SLC6A14 protein levels in UC (n = 55) and normal tissue samples (n = 29); E: Quantification of the IHC scores for SLC6A14 expression; F and G: NLRP3 protein levels in UC (n = 55) and normal tissue samples (n = 29); H: Association between SLC6A14 and NLRP3 levels in human colonic tissues. The data represent the means ± SD. ^aP < 0.01 vs the controls. UC: Ulcerative colitis.

Figure 2

Induction of pyroptosis by lipopolysaccharide in FHC and NCM460 intestinal epithelial cells. A: CCK-8 assay showing the impact of lipopolysaccharide (LPS) on FHC and NCM460 cell proliferation; B: EdU assay showing the proliferation of LPS-treated and untreated FHC and NCM460 cells; C: Quantification of EdU-positive cells; D and E: Flow cytometric analysis of apoptosis in cells with and without LPS treatment; F and G: Enzyme-linked immunosorbent assay analysis of the effects of LPS on IL-1β and IL-18 secretion; H: Western blot analysis showing the levels of pyroptosis-associated proteins in LPS-treated and untreated cells; I and J: Western blot analysis showing SLC6A14 levels in LPS-treated and control cells. ^aP < 0.01 vs controls. LPS: Lipopolysaccharide.

Figure 3

Downregulating SLC6A14 promotes proliferation and inhibits apoptosis in lipopolysaccharide-treated intestinal epithelial cells. A and B: The expression of SLC6A14 in lipopolysaccharide (LPS)-, LPS+shCTRL-, and LPS+sh-SLC6A14-treated FHC and NCM460 cells was assessed by quantitative real-time polymerase chain reaction (A) and Western blotting (B); C: Cell viability was determined by CCK-8 assays; D and E: An EdU assay was used to detect cell proliferation, and the scale bar represents 100 μm; F and G: Flow cytometry showing apoptosis in FHC and NCM460 cells, followed by quantitative analyses. ^aP < 0.01 vs controls; ^bP < 0.01 vs LPS+sh-CTRL. LPS: Lipopolysaccharide.

Figure 4

SLC6A14 enhances lipopolysaccharide-induced inflammatory cytokine secretion. A: Enzyme-linked immunosorbent assay analysis of IL-1β and IL-18; B: Western analysis of the levels of pyroptosis-associated proteins. The data are means ± SD (n = 5). ^aP < 0.01 vs controls; ^bP < 0.01 vs LPS+sh-CTRL. LPS: Lipopolysaccharide.

Figure 5

NLRP3 overexpression reverses the suppressive effect of SLC6A14 knockdown on lipopolysaccharide-induced FHC cell pyroptosis. A: CCK-8 assays were performed to determine whether NLRP3 overexpression counteracted the inhibitory effect of SLC6A14 knockdown on lipopolysaccharide (LPS)-induced intestinal epithelial cell (IEC) proliferation; B and C: EdU staining was performed to assess whether NLRP3 overexpression could reverse SLC6A14-mediated promotion of proliferation in LPS-stimulated epithelial cell models; D: Flow cytometry was used to investigate whether NLRP3 overexpression could reverse the proapoptotic effects of SLC6A14 silencing on LPS-treated IECs; E: Quantification of apoptosis. ^aP < 0.01 vs controls; ^bP < 0.01 vs LPS; ^cP < 0.01 vs LPS+sh-SLC6A14. LPS: Lipopolysaccharide; OE: Overexpression.

Figure 6

NLRP3 overexpression reverses the inhibitory effect of SLC6A14 knockdown on lipopolysaccharide-induced FHC cell pyroptosis. A: Effects of NLRP3 overexpression on IL-1β and IL-18 production facilitated by SLC6A14 in lipopolysaccharide (LPS)-treated intestinal epithelial cells (IECs), as shown by enzyme-linked immunosorbent assay; B: Western analysis of the effects of NLRP3 overexpression on pyroptosis-associated protein levels mediated by SLC6A14 in IECs after LPS treatment. ^aP < 0.01 vs controls; ^bP < 0.01 vs LPS; ^cP < 0.01 vs LPS+sh-SLC6A14. LPS: Lipopolysaccharide; OE: Overexpression.

Figure 7

SLC6A14 reduces pyroptosis induced by NLRP3 activation in ulcerative colitis mouse models. A: Measurement of colon length; B: Assessment of body weight changes; C: Disease-activity index; D: Histology (magnification 200 ×, scale bar = 100 μm); E and F: Western blot analysis of SLC6A14; G: Effects of SLC6A14 on IL-1β and IL-18 production in the ulcerative colitis mouse model, as measured by enzyme-linked immunosorbent assay; H: Western blot analysis of the levels of pyroptosis-associated proteins. The data are means ± SD. ^aP < 0.01 vs controls, ^bP < 0.01 vs DSS+LV-sh-CTRL. DSS: Dextran sulfate sodium.