β-Carotene Attenuates Apoptosis and Autophagy via PI3K/AKT/mTOR Signaling Pathway in Necrotizing Enterocolitis Model Cells IEC-6

Abstract

Background: Necrotizing enterocolitis (NEC) is a devastating disease affecting the gastrointestinal tract in the newborn period. In recent years, the role of apoptosis and autophagy in intestinal mucosal barrier dysfunction has come into prominence in research regarding the pathogenesis of NEC. β-Carotene is a well-known vitamin A precursor, and its content in breast milk is relatively high, especially in the colostrum. In the present study, we investigated the protective effect of β-carotene on necrotizing enterocolitis model cells IEC-6 induced by lipopolysaccharide (LPS).

Methods: CCK-8 assay was performed to evaluate cell viability. The Annexin V-FITC/PI method was used to detect apoptosis. Western blotting was utilized to measure the expression levels of proteins. Immunofluorescence analysis was used to assess the autophagy of IEC-6 cells.

Results: Our findings indicated that β-carotene inhibited the apoptosis of IEC-6 cells by downregulating cleaved caspase-3 levels and Bax levels and upregulating Bcl-2 levels, reducing cell autophagy via downregulating LC3II/I ratio and upregulating p62 levels. In addition, the expression of p-PI3K, p-AKT, and p-mTOR was upregulated after β-carotene treatment. Interestingly, these changes induced by β-carotene were partially reversed by rapamycin and voxtalisib.

Conclusion: In conclusion, our findings indicated that β-carotene can attenuate apoptosis and autophagy of IEC-6 cells induced by LPS via activating the PI3K/AKT/mTOR signaling pathway. Therefore, β-carotene may be a promising drug used in the clinical treatment of NEC.

Figure 1

The CCK-8 assay was used to measure the viability of IEC-6 cells. IEC-6 cells were treated with β-carotene at the indicated doses (0, 1.25, 2.5, 5, 10, 20, 40, 80, and 100 μM) for 24 h.  ^∗ ∗P < 0.01 vs. the 0 μM group.

Figure 2

β-Carotene inhibits LPS-induced apoptosis and autophagy in IEC-6 cells. Cells were treated with LPS (100 μM) for 3 h prior to exposure to β-carotene (10, 20, and 30 μM) for 24 h. (a) Cell viability measured by the CCK-8 assay.  ^∗ ∗P < 0.01 vs. the control group. ^##P < 0.01 vs. the LPS group. (b), (c) Apoptosis was quantified by flow cytometry.  ^∗ ∗P < 0.01 vs. the LPS group. ^##P < 0.01 vs. the LPS + 20 μM βC group. (d)–(g) The expressions of Bax, Bcl-2, cleaved caspase-3, LC3II/I, and p62 detected by Western blot.  ^∗P < 0.05,  ^∗ ∗P < 0.01 vs. the LPS group. ^#P < 0.05, ^##P < 0.01 vs. the LPS group. (h) Representative images of intracellular GFP-LC3 puncta in IEC-6 cells under different conditions. The images were made at × 100 magnification. Each experiment is carried out in triplicate.

Figure 3

Protective effect of β-carotene is related to inhibited autophagy in LPS-treated IEC-6 cells. Cells were exposed to LPS (100 μM) for 3 h followed by incubation with β-carotene (10 μM) with or without RAPA (10 μM) for 24 h. (a) Cell viability measured by the CCK-8 assay. (b), (c) Apoptosis quantified by flow cytometry. (d)–(g) The protein expressions of Bax, Bcl-2, cleaved caspase-3, LC3II/I, and p62 detected by Western blot. (h) The formation of GFP-LC3 puncta detected by immunofluorescence. The images were made at × 100 magnification.  ^∗ ∗P < 0.01 vs. the LPS group. ^##P < 0.01 vs. the LPS + βC group.

Figure 4

β-Carotene induces LPS-induced apoptosis and autophagy via activation of the PI3K/AKT/mTOR signaling pathway in IEC-6 cells. Cells were pretreated with LPS (100 μM) for 3 h following incubation with β-carotene (10 μM) with or without voxtalisib (5 μM) for 24 h. (a) Cell viability measured by the CCK-8 assay. (b), (c) Apoptosis quantified by flow cytometry. (d), (e) The protein expressions of LC3II/I and p62 observed by Western blot. (f), (g) Western blot analysis for the expression of p-PI3K, PI3K, p-AKT, AKT, mTOR, and p-mTOR.  ^∗ ∗P < 0.01 vs. the LPS group. ^#P < 0.05, ^##P < 0.01 vs. the LPS + βC group.