Both ethical imperatives and scientific limitations increasingly challenge the traditional reliance on animal models for toxicity testing and drug evaluation, particularly in the era of precision medicine. In response, a paradigm shift is underway, marked by the development of advanced in vitro and in silico technologies that can offer human-relevant and mechanistically informed alternatives. This review examines well-established alternatives, such as receptor binding assays, the monocyte activation test, and enzyme-linked immunosorbent assays, highlighting their applications, mechanisms, and limitations. We further explore emerging human-relevant technologies that include organoids, organ-on-a-chip systems, microphysiological systems, and artificial intelligence-powered modeling platforms. Special emphasis is placed on immune-integrated microphysiological systems as next-generation platforms to evaluate immunotherapy, vaccine responses, and immune toxicities. These models recapitulate dynamic human physiological processes, such as hematopoiesis and germinal center reactions, beyond the capabilities of traditional animal systems. Collectively, these technologies represent scientifically superior and ethically progressive trajectories for preclinical testing. Their integration into regulatory and industrial workflows requires continued refinement, cross-sector collaboration, and standardization.

Excessive generation of reactive oxygen species (ROS) and proinflammatory cytokines is a pivotal mechanism causing sensorineural hearing loss (SNHL) through damage to auditory hair cells. In particular, high expression levels of tumor necrosis factor-alpha (TNF-α) were observed in SNHL. Alpha-lipoic acid (ALA) has demonstrated protective effects against cochlear hair cell damage and hearing deterioration. This study investigated the protective effect of ALA against TNF-α-triggered ototoxicity in auditory hair cells and in a noise-induced hearing loss mouse model. Cells were pretreated with ALA prior to TNF-α treatment, after which cell viability, ROS generation, and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant activity were assessed. In addition, we observed the hearing levels, the number of outer hair cells (OHCs), and Nrf2-related protein expression in ALA-pretreated mice before noise exposure. ALA pretreatment significantly enhanced cell viability, suppressed ROS production, and activated Nrf2-mediated antioxidant activity compared with TNF-α treatment alone. In the animal model, noise-exposed mice exhibited elevated TNF-α expression in cochlear tissues. ALA pretreatment decreased the auditory brainstem response threshold and reduced OHCs damage. Furthermore, ALA administration upregulated Nrf2-related antioxidant protein expression in cochlear tissues. Taken together, these findings suggest that ALA pretreatment confers cytoprotective effects against TNF-α-triggered auditory hair cell damage through activation of the Nrf2 signaling-mediated antioxidant activity.

Mitochondria are crucial for energy metabolism and their dysfunction is implicated in the development of various human diseases. Direct mitochondrial transplantation has shown potential in reversing mitochondrial dysfunction in recipient cells. Mesenchymal stem cells (MSCs) present a promising approach as donor cells for such transplantation. We have previously demonstrated that tomatidine, a natural steroidal alkaloid, promotes the differentiation of human embryonic stem cells (hESCs) into mature cardiomyocytes by enhancing mitochondrial quantity and function. In this study, we assessed the capacity of hESC-derived cardiomyocytes (hESC-CMs) and MSCs as donor cells for mitochondrial transplantation. Mitochondria were extracted from MSCs, immature hESC-CMs, and tomatidine-treated mature hESC-CMs. Treating MSCs with mitochondria derived from mature hESC-CMs led to a marked increase in mitochondrial protein levels, such as COX IV and MIC60, in the recipient MSCs, in comparison to those receiving mitochondria from immature hESC-CMs or MSCs. Transplantation of mature hESC-CM-derived mitochondria significantly enhanced the proliferation of recipient MSCs. These findings indicate that mature hESC-CMs are highly effective as donor cells for mitochondrial transplantation in addressing mitochondrial dysfunction.

Aging contributes to hepatic steatosis by increasing de novo lipogenesis. The Forkhead box O6 (FOXO6) transcription factor links insulin signaling to lipid metabolism. Activated FOXO6 induces hyperlipidemia and decreases peroxisome pro-liferator-activated receptor alpha (PPARα), thereby promoting hepatic lipogenesis. In this paper, we describe the role of FOXO6 in hepatic steatosis in aged male rats and liver cells, and examine the relationship between FOXO6 and PPARα, and the functional consequences of their altered interaction. We find that FOXO6 induces lipid accumulation by inhibiting PPARα in aged male rat livers. Our data show that AKT signaling negatively regulates FOXO6-induced hepatic lipid accumulation, and that a key β-oxidation gene, PPARα, is decreased in aged livers. We further demonstrate that FOXO6 activation decreases PPARα expression and increases lipid accumulation. Furthermore, interaction between FOXO6 and PPARα promotes hepatic steatosis in aged males. Also, high glucose upregulates FOXO6, reduces β-oxidation gene expression, and increases cellular TG-mediated lipid accumulation. Transcriptional activation of FOXO6 by aging and high glucose causes lipid accumulation by downregulating PPARα and hyperglycemia-responsive genes in aged male rats and liver cell cultures. We provide evidence that age-related insulin resistance suppresses β-oxidation through interaction between FOXO6 and PPARα, thereby promoting hepatic lipid accumulation in aged male rats.