Berberine ameliorates neonatal necrotizing enterocolitis by activating the phosphoinositide 3-kinase/protein kinase B signaling pathway

Abstract

Neonatal necrotizing enterocolitis (NEC) is a severe acquired disease that predominantly affects the small intestine of neonates. NEC is caused by a combination of metabolic products, dysfunctions of the blood vessels, mucus and other unknown factors. Berberine may induce beneficial effects on necrotic and cardiovascular diseases due to its anti-inflammatory and anti-apoptotic effects on epithelial cells. In the present study, the therapeutic effects of berberine were investigated and the potential mechanisms by which it functions within a neonatal NEC mouse model were analyzed. Inflammation and levels of associated factors were measured in the serum of mice with NEC prior to and following treatment with berberine. Apoptotic rates in epithelial cells were analyzed following treatment with berberine. The expression of genes associated with apoptosis and apoptosis signaling were determined in epithelial cells in the small intestines of mice with NEC following treatment with berberine. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was investigated in epithelial cells isolated from mice following treatment with either berberine or PBS. Histology and immunohistochemistry were used to determine the area of infarction and apoptosis. Body weight and food intake were measured to evaluate the physical effects of berberine on mice with NEC. The results indicated that berberine attenuated the inflammation caused by NEC in mice after 10 days of treatment. The apoptosis rate of epithelial cells isolated from experimental mice was decreased following berberine treatment. Western blot analysis indicated that the expression of the anti-apoptotic genes c-Myc and p53 were upregulated by berberine, whereas caspase-3 and -9 levels were downregulated in epithelial cells following treatment with berberine. In addition, the expression and phosphorylation levels of PI3K and AKT were downregulated in epithelial cells following treatment with berberine. An in vitro assay indicated that treatment with PI3K alone increased the expression of AKT and promoted the apoptosis of epithelial cells. Treatment with berberine markedly increased epidermal growth factor (EGF) and Bcl-2 expression levels, the activity of epithelial cells and decreased the infarction area of the small intestine. Accordingly, the body weight and food intake of mice with NEC were increased following berberine treatment. Therefore, the results of the present study demonstrate that berberine inhibits inflammation and apoptosis via the PI3K/AKT signaling pathway and may therefore attenuate the progression of NEC. These results suggest that berberine may be a potential therapeutic agent for the treatment of patients with NEC.

Keywords: apoptosis; berberine; inflammation; neonatal necrotizing enterocolitis; phosphoinositide 3 kinase/protein kinase B signaling pathway.

Figure 1.

Berberine significantly reduces inflammatory cytokine levels in the peripheral blood of mice with neonatal NEC. Mice with neonatal NEC were treated with either berberine or PBS (as a control) and levels of (A) TLR-4, (B) MD-2, (C) TNF-α, (D) NF-κB, (E) IL-6 and (F) Cxcl-1 in the peripheral blood was determined by ELISA. **P<0.01. NEC, necrotizing enterocolitis; TLR, toll like receptor; MD, myeloid differentiation protein; TNF, tumor necrosis factor; NF nuclear factor; IL, interleukin; Cxcl, (C-X-C motif) ligand.

Figure 2.

Berberine reduces the apoptosis of epithelial cells through the mitochondrial apoptosis pathway. (A) The apoptosis rate of epithelial cells in the small intestine was determined by flow cytometry. The protein expression levels of (B) caspase-3 and caspase-9, (C) survivin and Cyto c, (D) caspase-8 and FADD, (E) c-Myc and p53 and (F) IFN-γ in epithelial cells in the small intestine were determined by western blot analysis. **P<0.01. Cyto, cytochrome; FADD, Fas-associated protein with death domain; c-Myc, Myc proto-oncogene protein; p53, tumor suppressor protein p53; IFN, interferon.

Figure 3.

Berberine alleviates the apoptosis of epithelial cells by inhibiting the PI3K/AKT signaling pathway. Western blot analysis was used to determine (A) the expression of pPI3K and PI3K, (B) the phosphorylation levels of pAKT and AKT and (C) the expression of EGF and Bcl-2 in epithelial cells following treatment with either berberine or PBS. (D) The apoptosis rate of epithelial cells following treatment with either PI3K, PI3K+berberine or PBS alone was determined using flow cytometry. (E) The expression and phosphorylation levels of AKT and (F) the expression of EGF and Bcl-2 in epithelial cells following treatment with either PI3K, PI3K+berberine or PBS was determined by western blot analysis. tAKT served as the control. **P<0.01. PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; EGF, epithelial growth factor; Bcl, B-cell lymphoma; p, phosphorylated; tAKT, total AKT.

Figure 4.

Berberine improves the physical activity and physiological functions of mice with neonatal NEC. The (A) body weight and (B) food intake of mice with NEC following treatment with berberine or PBS was determined by physical activity analysis. (C) The area of infarction in the small intestine of mice with neonatal NEC was determined by staining with hematoxylin and eosin. (D) The viability of epithelial cells following treatment with berberine treatment was determined by a viability assay. (E) EGF and Bcl-2 expression in the small intestine were determined by immunohistochemistry. (F) The apoptosis of epithelial cells in the small intestine was determined by a TUNEL assay. The arrows pointed to the positive sites. **P<0.01. NEC, necrotizing enterocolitis; EGF, epithelial growth factor; Bcl, B-cell lymphoma; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.