Glutathione contributes to alleviating hepatic injury via host-microbiome interaction and CAR-dependent pathway

Glutathione (GSH) is a potent antioxidant regulating oxidative stress, but whether exogenous GSH supplementation mitigates stress injury through host-microbiome and liver interactions remains unclear. This study aimed to determine the regulatory mechanism of GSH using in vivo (28-day-old weaned piglets) and in vitro (alpha mouse liver 12 (AML12) cells) stress injury models. Thirty-five healthy weaned piglets (mean body weight (9.52±0.20) kg) were fed diets supplemented with 0.01%, 0.03%, or 0.06% GSH for 4 weeks, followed by being injected intraperitoneally with paraquat (PQ) on days 28, 30, and 32. AML12 cells exposed to tert-butyl hydroperoxide (tBHP) were used to evaluate related mechanisms, and CINPA1was used to inhibit constitutive androstane receptor (CAR) activity. Our results showed that PQ challenge induced hepatic morphological and functional impairments, accompanied by suppression of antioxidant capacity and immune function. Notably, exogenous GSH treatment significantly increased CD4⁺/CD8⁺ T lymphocyte ratio, GSH, immunoglobulin A and interleukin-10 levels in serum, reduced the secretion and gene expression of pro-inflammatory factors to alleviate liver injury. Moreover, GSH treatment significantly promoted CAR nuclear translocation and regulated the expression of detoxification genes in response to liver inflammation. Additionally, GSH treatment improved the diversity and relative abundance of colonic probiotic bacteria such as UCG_002, Christensenellaceae_R_7_group, Prevotellaceae_NK3B31_group, Prevotella, Oscillospira, and unclassified_UCG_010. Furthermore, in vitro results showed that 3 mmol L^-1 GSH administration could increase the expression of CAR pathway and antioxidant related genes, and inhibit cellular ROS production in tBHP-induced AML12 cells. However, CAR inhibition by CINPA1 prevented GSH from alleviating tBHP-induced oxidative stress injury in AML12 cells. Our results indicate that exogenous GSH treatment alleviated liver injury in piglets through host-microbiome interaction and a CAR-dependent signaling pathway.