ADSCs-Exo Attenuate HIRI in Miniature Pigs: Pharmacological Evidence for a Potential Association Between Copper-Associated Alterations and Ferroptosis-Related Responses

Hepatic ischemia–reperfusion injury (HIRI) involves multiple forms of regulated cell death, but the relationship between copper-associated alterations and ferroptosis-related responses remains incompletely understood. This study investigated whether pharmacological modulation of intracellular copper homeostasis was accompanied by corresponding changes in ferroptosis-associated responses, and vice versa, during HIRI, and evaluated whether adipose-derived mesenchymal stem cell-derived exosomes (ADSCs-Exo) attenuate injury-associated alterations under OGD/R and HIRI conditions. Primary piglet hepatocytes were subjected to oxygen–glucose deprivation/reoxygenation (OGD/R), followed by bidirectional pharmacological modulation of copper homeostasis and ferroptosis-associated responses. The major response patterns were further evaluated in a laparoscopic miniature pig model of HIRI combined with partial hepatectomy. OGD/R induced copper and iron dyshomeostasis, mitochondrial dysfunction, redox imbalance, alterations in cuproptosis-related markers, and ferroptosis-associated changes. Relative to the OGD/R group, ADSC-Exo treatment attenuated these injury-associated abnormalities. Pharmacological reduction in intracellular copper loading was accompanied by decreased Fe2+ accumulation and restoration of GSH, AIFM2, SLC7A11, and GPX4, whereas increased copper loading produced the opposite pattern. Conversely, pharmacological enhancement in ferroptosis-associated responses increased Cu2+ accumulation and aggravated alterations in the cuproptosis-related markers SLC31A1, FDX1, LIAS, and DLAT, whereas pharmacological suppression of ferroptosis-associated responses attenuated these changes. These bidirectional pharmacological response patterns are consistent with a potential reciprocal association between copper-associated alterations and ferroptosis-related responses but do not establish bona fide cuproptosis, direct molecular interaction, or target-specific causality. In miniature pigs, HIRI was accompanied by hepatic injury, mitochondrial dysfunction, hepatic copper accumulation, alterations in cuproptosis-related markers, ferroptosis-associated changes, and reduced GPX4 expression. Relative to untreated IRI animals, ADSC-Exo administration was associated with attenuation of these injury-related abnormalities. The coordinated changes in GPX4, TAX1BP1, and MAP1LC3B are hypothesis-generating and support the working hypothesis that TAX1BP1/MAP1LC3B-associated autophagic processes may contribute to GPX4 homeostasis; however, autophagic flux, GPX4 turnover, and direct TAX1BP1–GPX4 interaction were not assessed. Because a non-injured ADSC-Exo-only control was not included, the present study does not determine the independent effects of ADSCs-Exo under baseline conditions. Thus, the observed ADSC-Exo-associated effects should be interpreted specifically within the OGD/R and HIRI settings examined here.