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

Yun Chen¹Kangrong Wang¹Wenhai Guo²Chengqin Lu¹Wenting Suo¹Qiuling Li¹Yao Deng¹Xinling Chen¹Min Dai²Xiaodong Zhang¹Jiean Xu¹Wen Su¹Shuangling Yang³Hongzhi Yang²Fuman Yan^1*Haimei Liu^1*Yaxing Zhang^1*

• ¹Department of Physiology, School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China
• ²Sun Yat-sen University, Guangzhou, Guangdong Province, China
• ³Guangzhou Xinhua University, Guangzhou, China

Background: Hydrogen gas (H2), which is the lightest and diffusible gas molecule, has strong abilities to alleviate excessive oxidative stress, inflammation, and apoptosis. Inhalation of H2 is beneficial for preventing the damage of the lung, heart, brain, liver, kidneys, and many other organs. However, the effect of intraperitoneal injection of H2 on MASLD is unclear. Objective: The aim of this study is to investigate whether intraperitoneal injection of H2 can improve MASLD, and if so, what are the key innate immune mechanisms involved? Methods: The MASLD mouse model was established by feeding a MCD diet for three weeks. H2 was daily given by intraperitoneal injection since the 8 th day of MCD diet feeding, and lasted for two weeks. Moreover, hepatic protective effect and anti-pyroptosis effect of H2 were further confirmed by H2-rich DMEM-treated HepG2 cells in vitro.Results: Supplementing with H2 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, H2 modulated hepatic redox homeostasis by suppressing 3-NT and MDA levels, while increasing reduced GSH levels. Subsequently, 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 H2 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, and the fulllength and cleavage of the classical pyroptosis trigger GSDMD processed by Caspase-1 in NLRP3 inflammasome were all blocked by H2. In addition, H2 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 reduced expression of inflammatory cytokines, the full-length and cleaved forms of GSDMD and GSDME, and the reduced number of HepG2 cells with pyroptotic morphology further confirmed the anti-pyroptosis effect of H2.H2 is an anti-pyroptosis gas molecule, intraperitoneal injection of H2 is a novel therapeutic strategy for MASLD that deserves further investigation.