Protective Mechanism of Leucine and Isoleucine against H2O2-Induced Oxidative Damage in Bovine Mammary Epithelial Cells

Abstract

Leucine and isoleucine possess antioxidative and anti-inflammatory properties. However, their underlying protective mechanisms against oxidative damage remain unknown. Therefore, in this study, the protective mechanism of leucine and isoleucine against H₂O₂-induced oxidative damage in a bovine mammary epithelial cell lines (MAC-T cells) were investigated. Briefly, MAC-T cells exposed or free to H₂O₂ were incubated with different combinations of leucine and isoleucine. The cellular relative proliferation rate and viability, oxidative stress indicators, and inflammatory factors were determined by specific commercial kits. The genes related to barrier functions was measured by real-time quantitative PCR. The protein expression differences were explored by 4D label-free quantitative proteomic analyses and validated by parallel reaction monitoring. The results revealed that leucine and isoleucine increased cell proliferation, total antioxidant status (TAS), and the relative mRNA expression of occludin, as well as decreased malondialdehyde (MDA), total oxidant status (TOS)/TAS, IL-6, IL-1β, and TOS. When leucine and isoleucine were combined, MDA, TOS/TAS, and the relative mRNA expression levels of claudin-1, occludin, and zonula occludens-1 increased when compared to leucine or isoleucine alone. Proteomics analyses revealed that leucine significantly upregulated the propanoate metabolism; valine, leucine, and isoleucine degradation; and thermogenesis pathways, whereas isoleucine significantly upregulated the peroxisome and propanoate metabolism pathways. In conclusion, leucine protected MAC-T cells from H₂O₂-induced oxidative stress by generating more ATP to supplement energy demands, and isoleucine improved the deficit in peroxisome transport and promoted acetyl-CoA production. The findings of this study enhance our understanding of the protective mechanisms of leucine and isoleucine against oxidative damage.

Figure 1

Oxidative stress model. (a) Effects of H₂O₂ on the cellular viability of MAC-T cells. Different letters (A–E) indicate significant differences at the same concentration (p < 0.05). Effects of different H₂O₂ concentrations for 6 h on oxidative stress indicators in MAC-T cells: MDA (b), TOS (c), TAS (d), and TOS/TAS (e). Different letters (A–D) among treatments indicate significant differences (p < 0.05).

Figure 2

Effects of different leucine concentrations for 6 (a), 12 (b), 24 (c), 48 (d), or 72 h (e) on the relative proliferation rate of MAC-T cells. Different letters (A–C) among treatments indicate significant differences (p < 0.05).

Figure 3

Effects of different isoleucine concentrations for 6 (a), 12 (b), 24 (c), 48 (d), or 72 h (e) on the relative proliferation rate of MAC-T cells. Different letters (A–D) among treatments indicate significant differences (p < 0.05).

Figure 4

Effects of different combinations of leucine and isoleucine for 24 h on the relative proliferation rate of MAC-T cells. Different letters (A–E) among treatments indicate significant differences (p < 0.05).

Figure 5

Effects of different combinations of leucine and isoleucine for 24 h on the relative proliferation rate (a) and oxidative stress indicators (TAS (b), TOS (c), MDA (d), and TOS/TAS (e)) in MAC-T cells due to H₂O₂-induced oxidative damage. Different letters (A–D) among treatments indicate significant differences (p < 0.05).

Figure 6

Effects of different combinations of leucine and isoleucine for 24 h on IL-6 (a), IL-1β (b), TNF-α (c), and IL-10 (d) in MAC-T cells due to H₂O₂-induced oxidative damage. Different letters (A, B) among treatments indicate significant differences (p < 0.05).

Figure 7

Effects of different combinations of leucine and isoleucine for 24 h on the mRNA expression levels of claudin-1 (a), occludin (b), and ZO-1 (c) in MAC-T cells due to H₂O₂-induced oxidative damage. Different letters (A–E) among treatments indicate significant differences (p < 0.05).

Figure 8

Distribution of proteins identified in MAC-T cells of the control, H1L1I, H1L2I, H2L1I, and H2L1.5I groups. (a) Volcano plot of differentially expressed (DE) proteins. The horizontal coordinate indicates the fold change (log2 transformation) and the vertical coordinate indicates the p-values (log10 transformation). Red dots indicate upregulation and green dots indicate downregulation. (b) Upregulated and downregulated DE proteins. (c) Venn diagram of DE proteins identified in MAC-T cells due to H₂O₂-induced oxidative damage (fold change ≥ 1.5).

Figure 9

Cluster analysis heat map based on GO enrichment classifications: (a) biological process (BP); (b) cellular component (CC); and (c) molecular function (MF). The DE proteins among the comparison groups were subjected to GO enrichment, and a cluster analysis was performed to determine the correlative relationships among the DE protein functions. The horizontal direction indicates the enrichment test results of different groups, and the vertical direction indicates the description of DE enrichment-related functions. Different sets of DE proteins and color blocks correspond with the functional descriptions and indicate the degree of enrichment, where red indicates stronger enrichment (the deeper the red, the stronger the enrichment) and blue indicates weaker enrichment (the lighter the blue, the weaker the enrichment).

Figure 10

Bubble chart of DE proteins enriched in KEGG pathways. The bubble size represents the number of DE proteins in the enriched pathway terms, and the bubble color represents the p value.

Figure 11

PRM protein expression quantities of the candidate proteins which were ALDH6A1, ACSS2, NDUFAF7, ATP5MF, and PEX16. ∗ indicates p < 0.05 and ∗∗ indicates p < 0.01 vs. control group. ## indicates p < 0.01 vs. H1L1I group.

Figure 12

The protective mechanism of leucine (a) and isoleucine (b) against H₂O₂-induced oxidative damage in bovine mammary epithelial cells. Genes in black was validated in PRM and 4D label-free quantitative proteomic analysis, while genes in white was only validated in 4D label-free quantitative proteomic analysis.