Phosphorylation‐mediated PI3K‐Art signalling pathway as a therapeutic mechanism in the hydrogen‐induced alleviation of brain injury in septic mice

Our previous studies illustrated that 2% H(2) inhalation can protect against sepsis‐associated encephalopathy (SAE) which is characterized by high mortality and has no effective treatment. To investigate the underlying role of protein phosphorylation in SAE and H(2) treatment, a mouse model of sepsis was constructed by caecal ligation and puncture (CLP), then treated with H(2) (CLP + H(2)). Brain tissues of the mice were collected to be analysed with tandem mass tag‐based quantitative proteomics coupled with IMAC enrichment of phosphopeptides and LC–MS/MS analysis. In proteomics and phosphoproteomics analysis, 268 differentially phosphorylated proteins (DPPs) showed a change in the phosphorylated form in the CLP + H(2) group (p < 0.05). Gene ontology analysis revealed that these DPPs were enriched in multiple cellular components, biological processes, and molecular functions. KEGG pathway analysis revealed that they were enriched in glutamatergic synapses, tight junctions, the PI3K‐Akt signalling pathway, the HIF‐1 signalling pathway, the cGMP‐PKG signalling pathway, the Rap1 signalling pathway, and the vascular smooth muscle contraction. The phosphorylated forms of six DPPs, including ribosomal protein S6 (Rps6), tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein gamma (Ywhag/14–3‐3), phosphatase and tensin homologue deleted on chromosome ten (Pten), membrane‐associated guanylate kinase 1 (Magi1), mTOR, and protein kinase N2 (Pkn2), were upregulated and participated in the PI3K‐Akt signalling pathway. The WB results showed that the phosphorylation levels of Rps6, Ywhag, Pten, Magi1, mTOR, and Pkn2 were increased. The DPPs and phosphorylation‐mediated molecular network alterations in H(2)‐treated CLP mice may elucidate the biological roles of protein phosphorylation in the therapeutic mechanism of H(2) treatment against SAE.