Sulforaphane Ameliorates the Intestinal Injury in Necrotizing Enterocolitis by Regulating the PI3K/Akt/GSK-3 β Signaling Pathway

Abstract

Objective: Necrotizing enterocolitis (NEC) is a serious neonatal disease; this study aims to investigate the role of sulforaphane (SFN) in NEC-induced intestinal injury.

Methods: An animal model of NEC was established in newborn mice and intragastrically administrated with SFN; then, the general status and survival of the mice were observed. H&E staining was used to observe the pathological changes of intestinal tissues. ELISA, immunohistochemical staining, and flow cytometry assays were used to detect the levels of inflammatory factors, including TNF-α, IL-6, and IL-17, the expression of Bax, Bcl-2, TLR4, and NF-κB, and the percentages of the Th17 and Treg cells, respectively. GSK-3β expression levels were measured by immunofluorescence. IEC-6 and FHC cells were induced with LPS to mimic NEC in vitro and coincubated with SFN; then, the inflammatory factor levels and cell apoptosis rate were detected. Finally, Western blot was used to assess the expression of PI3K/Akt/GSK-3β pathway-related proteins in vitro and in vivo.

Results: SFN improved the survival rate of NEC mice during modeling, alleviated the severity of the intestinal injury, and reduced the proportion of Th17/Treg cells. SFN could inhibit TLR4 and NF-κB levels, decrease the release of inflammatory factors TNF-α and IL-6, suppress Bax expression, increase Bcl-2 expression, and inhibit apoptosis both in in vitro and in vivo models of NEC. Meanwhile, SFN regulated the expression of PI3K/Akt/GSK-3β pathway-related proteins in vitro and in vivo.

Conclusion: SFN relieved the inflammatory response and apoptosis by regulating the PI3K/Akt/GSK-3β signaling pathway, thereby alleviating NEC in model mice and cells.

Figure 1

Effects of SFN on survival rate and intestinal histopathological changes in NEC mice. (a) Survival analysis of mice in each group. (b) H&E staining and semiquantitative scoring results of the intestinal tissues of mice in each group, n = 6. NEC, necrotizing enterocolitis; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD.

Figure 2

Changes of TLR4, MD-2, TNF-α, NF-κB, IL-6, CXCL1, and IL-17 contents in the serum of NEC mice. NEC, necrotizing enterocolitis; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD, n = 6.

Figure 3

Changes in the proportion of Th17 and Treg cells in intestinal tissues of mice. NEC, necrotizing enterocolitis; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD, n = 3.

Figure 4

Immunohistochemical staining of TLR4 (a), NF-κB (b), Bax (c), and Bcl-2 (d) and semiquantitative analysis of these proteins expression (e) in intestinal tissues of mice. NEC, necrotizing enterocolitis; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD, n = 6.

Figure 5

Expression levels of PI3K/Akt/GSK-3β pathway-related proteins in intestinal tissue of NEC mice. Immunofluorescence staining results of GSK-3β (a) and fluorescence intensity (b) in intestinal tissues of mice in each group, n = 6, and expression of PI3K/Akt/GSK-3β pathway-related proteins in intestinal tissue of mice, n = 3. NEC, necrotizing enterocolitis; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD.

Figure 6

The mRNA expression of TNF-α, IL-6, and IL-17 in FHC and IEC-6 cells. LPS, lipopolysaccharide; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD, n = 3.

Figure 7

Apoptosis of FHC and IEC-6 cells. LPS, lipopolysaccharide; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD, n = 3.

Figure 8

Expression of PI3K/Akt/GSK-3β pathway-related proteins in FHC (a) and IEC-6 (b) cells. LPS, lipopolysaccharide; SFN, sulforaphane. Compared with the control group, ^▲P < 0.05, ^▲▲P < 0.01; compared with the NEC group, ^★P < 0.05, ^★★P < 0.01. Data represent the mean ± SD, n = 3.