Hydrogen sulfide (H₂S) is a critical gasotransmitter that plays a dual role in physiological and pathological processes, particularly in the gastrointestinal tract. While physiological levels of H₂S exert cytoprotective effects, excessive concentrations can lead to toxicity, oxidative stress, and inflammation. The aim of this study was to investigate the dose-dependent effects of exogenous H₂S on mitochondrial functions and biogenesis in intestinal epithelial cells under non-stressed conditions. Using a Caco-2 monolayer model, we evaluated the impact of sodium hydrosulfide (NaHS) at concentrations ranging from 1 × 10⁻⁷ M to 5 × 10⁻³ M on mitochondrial metabolism, redox balance, antioxidant defense, inflammatory responses, autophagy/mitophagy, and apoptosis. Our results demonstrated a biphasic response: low-to-moderate H₂S concentrations (1 × 10⁻⁷ M–1.5 × 10⁻³ M) enhance mitochondrial biogenesis through PGC-1α activation, upregulating TFAM and COX-4 expression, and increasing the mtDNA copy number. In contrast, higher concentrations (2 × 10⁻³–5 × 10⁻³ M) impair mitochondrial function, induce oxidative stress, and promote apoptosis. These effects are associated with elevated reactive oxygen species (ROS) production, dysregulation of antioxidant enzymes, and COX-2-mediated inflammation. H₂S-induced autophagy/mitophagy is a protective mechanism at intermediate concentrations but fails to mitigate mitochondrial damage at toxic levels. This study underscores the delicate balance between the cytoprotective and cytotoxic effects of exogenous H₂S in intestinal cells, helping to develop new therapeutic approaches for gastrointestinal disorders.

The intestinal epithelial barrier is critical for maintaining gut homeostasis, yet its integrity can be compromised by inflammation and microbial dysbiosis. Here, we demonstrate that Lactobacillus rhamnosus GG (LGG) and Lactobacillus paracasei IMPC2.1 (L. paracasei) show their effectiveness in enhancing epithelial barrier function and modulating autophagy, counteract the epithelial barrier dysfunction, induced by Lipopolysaccharide (LPS), in Caco-2 cells by modulating tight junction (TJ) protein expression through regulation of inflammation and apoptosis. LPS exposure significantly reduced transepithelial electrical resistance (TEER) and increased paracellular permeability, effects that were partially reversed by both probiotic strains. Western blot analysis revealed that LPS downregulated ZO-1, Occludin, and p-mTOR, while upregulating autophagy markers LC3-II and Beclin1, without affecting p62 levels. The latter finding indicated an impairment of autophagy flux, confirmed by immunofluorescence experiments. Co-treatment with LGG or L. paracasei restored TJ protein expression and alleviated the LPS-induced impairment of autophagic flux. Both probiotics suppressed LPS-induced cyclooxygenase-2 (Cox-2) and Bax upregulation, suggesting anti-inflammatory and anti-apoptotic effects. In the complex interplay between inflammation, autophagy, and apoptosis, these findings highlight a key regulatory mechanism in probiotic-mediated epithelial protection, underscoring the therapeutic potential of LGG and L. paracasei in mitigating gut barrier dysfunction.