N-Acetyl-L-cysteine Protects the Enterocyte against Oxidative Damage by Modulation of Mitochondrial Function

Abstract

The neonatal small intestine is susceptible to damage caused by oxidative stress. This study aimed to evaluate the protective role of antioxidant N-acetylcysteine (NAC) in intestinal epithelial cells against oxidative damage induced by H₂O₂. IPEC-J2 cells were cultured in DMEM-H with NAC and H₂O₂. After 2-day incubation, IPEC-J2 cells were collected for analysis of DNA synthesis, antioxidation capacity, mitochondrial respiration, and cell apoptosis. The results showed that H₂O₂ significantly decreased (P < 0.05) proliferation rate, mitochondrial respiration, and antioxidation capacity and increased cell apoptosis and the abundance of associated proteins, including cytochrome C, Bcl-XL, cleaved caspase-3, and total caspase-3. NAC supplementation remarkably increased (P < 0.05) proliferation rate, antioxidation capacity, and mitochondrial bioenergetics but decreased cell apoptosis. These findings indicate that NAC might rescue the intestinal injury induced by H₂O₂.

Figure 1

Cell proliferation in IPEC-J2 cells. Cells were treated with 0 (NC) to 1000 μM NAC and 0 or 100 μM H₂O₂, respectively, for a 2-day period. Cell viability was quantified by CCK-8 assay. Data are expressed as means ± SEM of at least three independent experiments. ^a–eValues with different letters are significantly different (P < 0.05).

Figure 2

DNA synthesis in IPEC-J2 cells. DNA synthesis during the proliferation of IPEC-J2 cells was quantified by EdU incorporation (red color) using Cell-Light™ EdU Kit (Rui Bo Biotechnology Limited Company, Guangzhou, China). Nuclei are shown in blue color. Cells were treated with 0 (NC) or 800 μM NAC and 0 or 100 μM H₂O₂, respectively. (a) The percentage of EdU-positive cells (the number of red nuclei versus the number of blue nuclei in at least five different microscopic fields of vision). (b) Representative images of EdU staining (magnification ×200) of cells. Data are expressed as means ± SEM of at least three independent experiments. ^a–cValues with different letters are significantly different (P < 0.05).

Figure 3

Mitochondrial respiration of IPEC-J2 cells measured by the XF-24 Extracellular Flux Analyzer and Cell Mito Stress Test Kit from Seahorse Biosciences (North Billerica, MA, USA). (a) Schematic and (b) oxygen consumption rate (OCR) assessed by extracellular flux analysis. OCR was measured under basal conditions followed by the sequential addition of oligomycin (0.5 μM), FCCP (1 μM), rotenone (1 μM), or antimycin A (1 μM). Each data point represents an OCR measurement. (c) Individual parameters for basal respiration, proton leak, maximal respiration, spare respiratory capacity, nonmitochondrial respiration, and ATP production were determined. Cells were treated with 0 (NC) or 800 μM NAC and 0 or 100 μM H₂O₂, respectively. Data were expressed as means ± SEM of at least three independent experiments. ^a–cValues with different letters are significantly different (P < 0.05).

Figure 4

The TCA cycle intermediates, pyruvic acid, and lactic acid of IPEC-J2 cells measured by an Agilent 7890B-5977A GC-MS equipped with HP-5ms (30 m × 250 μm × 0.25 μm) capillary column (Agilent J&W, Santa Clara, CA, USA). Cells were treated with 0 (NC) or 800 μM NAC and 0 or 100 μM H₂O₂, respectively. Data were expressed as means ± SEM of at least three independent experiments. ^a–cValues with different letters are significantly different (P < 0.05).

Figure 5

The concentrations of T-AOC and LDH in the IPEC-J2 cells. Cells were treated with 0 (NC) or 800 μM NAC and 0 or 100 μM H₂O₂, respectively. Data were expressed as means ± SEM of at least three independent experiments. ^a–bValues with different letters are significantly different (P < 0.05).

Figure 6

Cell apoptosis in the IPEC-J2 cells. (a) Representative flow cytometry diagrams and (b) apoptosis rate. Cells were treated with 0 (NC) or 800 μM NAC and 0 or 100 μM H₂O₂, respectively. Data were expressed as means ± SEM of at least three independent experiments. ^a–cValues with different letters are significantly different (P < 0.05).

Figure 7

Abundances of proteins (cytochrome C, Bax, Bcl-XL, cleaved caspase-3, and caspase-3) in IPEC-J2 cells determined by western blot analysis. Cells were treated with 0 (NC) or 800 μM NAC and 0 or 100 μM H₂O₂, respectively. Data were expressed as means ± SEM of at least three independent experiments. ^a–cValues with different letters are significantly different (P < 0.05).