Single-Cell Transcriptome Reveals the Regulatory Role of STAT3 in Diquat-Induced Oxidative Stress in Piglet Hepatocytes

Oxidative stress (OS) is known to cause severe liver injury in weaning piglets; however, the cellular and molecular mechanisms underlying this process remain poorly understood. In this study, we employed a diquat (DQ)-induced OS model in weanling piglets and performed single-cell transcriptome sequencing of liver tissue to elucidate the key molecular and cellular events involved in OS-induced hepatic damage. First, piglets were treated with 12 mg/kg DQ and the same amount of saline, and the histopathology, biochemical indicators, and single-cell RNA sequencing (scRNA-seq) of piglets were analyzed. Mouse hepatocytes were used to verify the mechanism of differentially expressed genes, including <i>STAT3</i> knockdown/overexpression, reactive oxygen species (ROS) detection and apoptosis assay. DQ exposure caused significant oxidative damage in the liver of piglets, which was manifested as decreased superoxide dismutase (SOD) activity (<i>p</i> < 0.05), glutathione (GSH) consumption (<i>p</i> < 0.05) and increased malondialdehyde (MDA) (<i>p</i> < 0.05). Cell type-specific responses were revealed by scRNA-seq, with hepatocytes showing the most pronounced transcriptomic alterations (752 genes up-regulated and 918 genes down-regulated). The expression of <i>STAT3</i> was up-regulated in hepatocytes (<i>p</i> < 0.05) and down-regulated in B cells. The functional enrichment of macrophages involved FOXO/MAPK signaling and OS pathways. In vitro experiments showed that DQ treatment (IC50 = 125.8 μmol/L) led to an increase in ROS content and apoptosis, <i>STAT3</i> silencing aggravated ROS and apoptosis (<i>p</i> < 0.05), and <i>STAT3</i> overexpression alleviated ROS and apoptosis (<i>p</i> < 0.05). STAT3 activation increases HO-1 and Bcl-2, while inhibiting Bax and shifting the Bax/Bcl-2 ratio toward cell survival. It has been shown that DQ induces OS and apoptosis in a cell type-dependent manner, in which <i>STAT3</i> plays a key regulatory role in antioxidant defense and cell survival. Targeting <i>STAT3</i> may be a therapeutic strategy for DQ-induced hepatotoxicity.