Lactobacillus rhamnosus GG Modulates Mitochondrial Function and Antioxidant Responses in an Ethanol-Exposed In Vivo Model: Evidence of HIGD2A-Dependent OXPHOS Remodeling in the Liver

Abstract

The gut microbiota plays a central role in host energy metabolism and the development of metabolic disorders, partly through its influence on mitochondrial function. Probiotic supplementation, particularly with Lactobacillus rhamnosus GG, has been proposed as a strategy to modulate the microbiota and improve host metabolic health. Adolescent binge-like alcohol consumption is a critical public health issue known to induce neuroinflammation, oxidative stress, mitochondrial dysfunction, and intestinal dysbiosis, contributing to disorders such as alcoholic liver disease (ALD). This study aimed to evaluate the effects of L. rhamnosus GG supplementation on mitochondrial physiology in Sprague Dawley rats exposed to binge-like ethanol (BEP group) or saline (SP group) during adolescence (postnatal days 30-43). Starting on postnatal day 44, L. rhamnosus GG was administered orally for 28 days. Fecal colonization was confirmed by qPCR, and mitochondrial function was assessed in the liver, heart, and bone marrow through quantification of NADH, ATP, ADP/ATP ratio, total antioxidant capacity, and the expression of mitochondrial genes Higd2a, MnSOD1, and AMPKα1. L. rhamnosus GG supplementation induced tissue-specific mitochondrial adaptations. In the liver, it increased Higd2a expression and restored antioxidant and energy balance in ethanol-exposed rats. In the bone marrow, it reversed ethanol-induced metabolic stress and enhanced AMPKα1 expression. In contrast, in the heart, L. rhamnosus GG had minimal impact on mitochondrial energy markers but increased antioxidant capacity, indicating a more limited, redox-focused effect. These findings suggest that L. rhamnosus GG exerts context-dependent, tissue-specific benefits on mitochondrial physiology, primarily through the modulation of antioxidant defenses, activation of AMPKα1, and remodeling of respiratory complexes. This probiotic may represent a promising therapeutic strategy to mitigate mitochondrial dysfunction associated with early-life alcohol exposure.

Keywords: ADP/ATP ratio; AMPKα1; Higd2a; Lactobacillus rhamnosus GG; MnSOD; NADH; OXPHOS; binge-like ethanol exposure; microbiota; mitochondrial physiology.

Figure 1

Schematic of the experimental design. Abbreviations: BEP, ethanol exposure; LrhGG, Lactobacillus rhamnosus GG; SP, saline exposure.

Figure 2

Relative gene expression of Higd2a, MnSOD1, and AMPKα1 in the liver, heart, and bone marrow of rats exposed to ethanol (BEP) or saline (SP) during adolescence, following treatment with Lactobacillus rhamnosus GG (Lrh) or water (P). Data are presented as mean ± SEM (n = five biological replicates per group, with two technical replicates per sample). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test, with significance set at p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

NADH levels, ADP/ATP ratio, and total antioxidant capacity in the bone marrow of rats exposed to ethanol (BEP) or saline (SP) during adolescence following Lactobacillus rhamnosus GG (Lrh) or water (P) treatment. Data are presented as mean ± SEM (n = five biological replicates per group, with two technical replicates per sample). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test, with significance set at p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

NADH levels, ADP/ATP ratio, and total antioxidant capacity in the liver of rats exposed to ethanol (BEP) or saline (SP) during adolescence following Lactobacillus rhamnosus GG (Lrh) or water (P) treatment. Data are presented as mean ± SEM (n = five biological replicates per group, with two technical replicates per sample). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test, with significance set at p < 0.05. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

NADH levels, ADP/ATP ratio, and total antioxidant capacity in the heart of rats exposed to ethanol (BEP) or saline (SP) during adolescence following Lactobacillus rhamnosus GG (Lrh) or water (P) treatment. Data are presented as mean ± SEM (n = five biological replicates per group, with two technical replicates per sample). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s post hoc test, with significance set at p < 0.05.

Figure 6

Changes in Higd2a expression are associated with remodeling of the OXPHOS complex. (a) Western blot analysis of OXPHOS complexes using the Total OXPHOS Rodent WB Antibody Cocktail (ab110413). (b) Quantification of OXPHOS complexes normalized to total protein loading, as determined by SDS-PAGE Coomassie blue staining. Data are presented as mean ± SEM (n = five biological replicates). Statistical analysis was performed using Two-way ANOVA (or Mixed Model), followed by mixed-effects analysis multiple comparisons, with a significance level set at p < 0.05. (c) Western blot analysis of Higd2a protein in liver mitochondrial extracts from rats exposed to ethanol (BEP) or saline (SP) during adolescence and treated with Lactobacillus rhamnosus GG (Lrh) or water (P). (d) Quantification of Higd2a protein levels normalized to Porin. Data are presented as mean ± SEM (n = five biological replicates). Statistical analysis was performed using one-way ANOVA, followed by Fisher’s LSD test, with a significance level set at p < 0.05. * p < 0.05; *** p < 0.001.

Figure 7

Tissue-specific effects of Lactobacillus rhamnosus GG on mitochondrial function in ethanol-exposed rats. This graphical model illustrates the differential impact of L. rhamnosus GG on mitochondrial indicators in the heart, liver, and bone marrow of rats subjected to adolescent binge-like ethanol exposure. In the heart, L. rhamnosus GG treatment resulted in a reduction in NADH and ATP levels and downregulation of HIGD2A and AMPKα1, with no significant change in MnSOD1, indicating limited mitochondrial recovery and redox-focused adaptation. In the liver, L. rhamnosus GG selectively restored mitochondrial function in ethanol-exposed rats (BEP-Lrh), as evidenced by increased ATP levels, ADP/ATP ratio, antioxidant capacity, and upregulation of HIGD2A, MnSOD1, and AMPKα1, while showing minimal benefit in non-exposed controls (SP-Lrh). In the bone marrow, LrhGG reversed ethanol-induced metabolic imbalance by increasing ATP levels, restoring the ADP/ATP ratio, and enhancing antioxidant defenses via AMPKα1 upregulation, with MnSOD1 and HIGD2A remaining stable. These findings highlight the context-dependent and tissue-specific efficacy of L. rhamnosus GG in restoring mitochondrial bioenergetics and redox homeostasis in metabolically distinct organs. Downward arrows (↓) indicate a decrease, and upward arrows (↑) indicate an increase in the corresponding mitochondrial or molecular parameter.