Hydrogen gas (H2) delivered by intraperitoneal injection alleviated methionine- and choline-deficient diet-induced metabolic dysfunction–associated steatotic liver disease in mice via inhibiting GSDMD- and GSDME-mediated pyroptosis

Background Hydrogen gas (H 2 ), which is the lightest and diffusible gas molecule, has strong abilities to alleviate excessive oxidative stress, inflammation, and apoptosis. Inhalation of H 2 is beneficial for preventing the damage of the lung, heart, brain, liver, kidneys, and many other organs. However, the effect of intraperitoneal injection of H 2 on metabolic dysfunction–associated steatotic liver disease (MASLD) is unclear. Objective The aim of this study is to investigate whether intraperitoneal injection of H 2 can improve MASLD, and if so, what are the key innate immune mechanisms involved? Methods The MASLD mouse model was established by feeding a methionine- and choline-deficient (MCD) diet for 3 weeks. H 2 was daily given by intraperitoneal injection since the eighth day of MCD diet feeding, and lasted for 2 weeks. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined to evaluate liver injury. Hematoxylin and eosin (H&E) staining, Oil Red O staining, qPCR analysis of hepatic lipid metabolism genes, and detection of hepatic triglyceride (TG) levels were performed to evaluate hepatic steatosis. Masson trichrome staining and Collagen-I and Collagen-III protein levels were used to evaluate liver fibrosis. The liver 3-nitrotyrosine (3-NT) was detected by immunoblotting and immunofluorescence, and the levels of malondialdehyde (MDA) and reduced glutathione (GSH) were measured using kits to evaluate redox homeostasis. The activation of TLR4-mediated innate immune signaling and pyroptosis were tested by immunoblotting and immunofluorescence. Moreover, hepatic protective effect and anti-pyroptosis effect of H 2 were further confirmed by H 2 -rich DMEM-treated HepG2 cells in vitro . Results Supplementing with H 2 by intraperitoneal injection protected MCD diet-fed mice against hepatic steatosis and fibrosis by down-regulating de novo lipogenesis and fatty acid uptake genes, as well as hepatic Collagen-Ⅰ and Collagen-Ⅲ protein levels, while up-regulating lipid export genes. Mechanistically, H 2 modulated hepatic redox homeostasis by suppressing 3-NT and MDA levels, while increasing the reduced GSH levels. Subsequently, reactive oxygen species (ROS)-related innate immune signaling, including the expression of TLR4, and the activation of NF-κB, ERK1/2, p38 MAPK, and JNK in the liver, were all inhibited by H 2 treatment. These further contributed to inhibiting the expression of TNF-α, IL-1β, and IL-18 in the liver. The maturation of IL-1β and IL-18, the full-length of the classical pyroptosis trigger GSDMD, and the cleavage of GSDMD processed by Caspase-1 in NLRP3 inflammasome (including NLRP3, ASC, Caspase-1) were all blocked by H 2 . In addition, H 2 decreased both the full-length and cleaved forms of Caspase-11, Caspase-8, Caspase-3 and GSDME, and thus inhibiting the non-canonical pyroptosis signaling in the liver of MASLD mice. The anti-pyroptosis effects of H 2 in vitro were further confirmed by the reduced expression of inflammatory cytokines, the decreased full-length and cleaved forms of GSDMD and GSDME, and the reduced number of HepG2 cells with pyroptotic morphology. Conclusion H 2 is an anti-pyroptosis gas molecule, intraperitoneal injection of H 2 is a novel therapeutic strategy for MASLD that deserves further investigation.