Proteomics and phosphoproteomics revealed dysregulated kinases and potential therapy for liver fibrosis.

Liver fibrosis is the initial stage of most liver diseases, and it is also a pathological process involving the liver in the late stages of many metabolic diseases. Therefore, it is important to systematically understand the pathological mechanism of liver fibrosis and seek therapeutic approaches for intervention and treatment of liver fibrosis. Disordered proteins and their post-translational modifications, such as phosphorylation, play vital roles in the occurrence and development of liver fibrosis. However, the regulatory mechanisms that govern this process remain poorly understood. In this study, we analyzed and quantified the liver proteome and phosphoproteome of CCl₄-induced early liver fibrosis model in mice. Proteomic analysis revealed that the pathways involved in extracellular matrix (ECM) recombination, collagen formation, metabolism and other related disorders, and protein phosphorylation modification pathways were also significantly enriched. In addition, western blotting and phosphoproteomics demonstrated that phosphorylation levels were elevated in the context of liver fibrosis. A total of 13,152 phosphosites were identified, with 952 sites increased while only 156 ones decreased. Furthermore, the upregulated phosphorylation sites, which exhibited no change at the proteome level mainly shared a common [xxxSPxxx] motif. Consequently, the kinases-substrates analysis ascertained the overactive kinases of these up-regulated substrates, which ultimately led to the identification of 13 significantly altered kinases within this dataset. These kinases were mainly catalogued into the STE, CMGC, and CAMK kinase families. Among them, STK4, GSK3α and CDK11B were subsequently validated though cellular and animal experiments, and the results demonstrated that their inhibitors could effectively reduce the activation of hepatic stellate cells and ECM production. These kinases may represent potential therapeutic targets for liver fibrosis, and their inhibitors may serve as promising anti- hepatic fibrosis drugs.