DGKζ in Glycerophospholipid Metabolism Regulates the DAG and PA Balance and Interacts With PTEN to Alleviate Brain Damage in Septic Mice With Hydrogen Inhalation: A Comparative Metabolomic and Phosphoproteomic Analysis.

OBJECTIVES: Therapeutic effects of hydrogen (H(2)) on sepsis-associated encephalopathy (SAE), a severe neuroinflammatory disease, have been reported, but the underlying mechanism remains unclear. Metabolomic and phosphoproteomic analyses were utilized to explore the therapeutic mechanism of H(2). METHODS: Caecal ligation and puncture (CLP) was used to establish an animal model of sepsis, after which the animals were treated with hydrogen. Mouse brains were obtained for analysis via tandem mass tag-based quantitative proteomics with IMAC enrichment of phosphopeptides and LC-MS/MS analysis to provide a broad overview of the metabolites. The metabolic profiles of mice in the SAE and SAE + H(2) groups were compared by multivariate statistical analysis. Different proteins (or enzymes) were verified by western blot (WB) and immunofluorescence (IF) analyses. ELISA was used to measure the level of DAG and PA. The influence of diacylglycerol kinase ζ (DGKζ) on glycerophospholipid metabolism in the mouse hippocampus was analyzed via coimmunoprecipitation (co-IP), and protein‒protein interactions were detected via LC‒MS/MS analysis. RESULTS: A total of 1476 metabolites were identified, including 131 metabolic biomarkers in negative ion mode and 41 metabolic biomarkers in positive ion mode. These values were different from the standard, with variable importance for the projection (VIP) > 1 and p < 0.05. The correlated differential phosphoproteins found in the combined metabolomic and phosphoproteomic analyses participated in 131 pathways, and the differentially abundant metabolites were involved in 10 metabolic pathways, eight of which were related. The roles and interactions of these differentially expressed proteins and metabolites suggest that glycerophospholipid metabolism is activated in septic mice after the inhalation of hydrogen. Additionally, we quantified the downregulation of choline-phosphate cytidylyltransferase A (Pcyt1α)/CTP/CCTα and DGKζ and the upregulation of the metabolite sn-glycero-3-phosphoethanolamine in the glycerophospholipid metabolism pathway in mice in the SAE + H(2) group compared with mice in the SAE group. The WB and IF results revealed that DGKζ expression increased in septic mice but decreased after H(2) treatment. The ELISA showed that the expression of DAG was increased in SAE mice compared with Sham mice, while it decreased in SAE + H(2) mice compared with SAE mice. Correspondingly, the PA level was reduced in SAE group compared with Sham group and was increased after the inhalation of H(2). Furthermore, the regulation of DGKζ in hydrogen treatment in septic mice may be related to the interaction with phosphatase and tensin homolog (PTEN). CONCLUSION: H(2) downregulates the levels of DGKζ and CCTα to alleviate brain damage in septic mice, and changes in DGKζ expression are balancing the transformation between the DAG anf PA, and it might also interact with PTEN. Thus, DGKζ may be a potential target in septic mouse therapy.