In Vitro Evaluation of Redox-Associated Responses Induced by Mud Extract in L929 and RAW 264.7 Cells

While natural muds are widely used in traditional balneotherapy and dermatological applications, the cellular basis of their redox-related effects remains insufficiently defined. In this study, we evaluated the effects of a mineral-rich mud extract on L929 fibroblasts and RAW 264.7 macrophages. This study was designed as an initial in vitro exploratory investigation to evaluate the cellular responses induced by a complex mud-derived material containing multiple inorganic components under standardized extract conditions. The mud extract showed no overt cytotoxicity up to 1000 μg/mL under the tested conditions. Intracellular reactive oxygen species (ROS) levels remained near baseline across the measured time points, with limited cell type-dependent variation. In parallel, antioxidant-related responses were observed primarily in RAW 264.7 cells, including a transient early increase in superoxide dismutase (SOD)-associated activity and subsequent increases in catalase (CAT) and glutathione peroxidase (GPx) activities. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis further showed dose-dependent upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) transcripts, particularly in RAW 264.7 cells. Collectively, these findings suggest that the mud extract is associated with coordinated antioxidant-related responses under non-cytotoxic conditions. However, because the present study was conducted in two murine cell lines and relied partly on assay systems potentially susceptible to matrix effects, the results should be interpreted as supportive of redox-associated modulation rather than definitive proof of a therapeutic mechanism. Furthermore, these findings should be interpreted as preliminary evidence obtained from a standardized aqueous extract system and not as definitive proof of component-specific mechanisms or direct applicability.