SHP2 accelerated acute liver injury via modulating TGFβ1/EZH2/BRD4 signaling induced proliferation and fibrosis of HSC

Abstract

To explore the specific molecular mechanisms by which SHP2 promotes liver injury. Single-cell transcriptomics and bioinformatics analyses were employed for predictive screening. C57BL/6J mice were assigned to normal, model, and model+PHPS1 groups. Liver injury and fibrosis were evaluated by Masson’s trichrome staining, H&E staining, serum alanine aminotransferase (ALT) levels, and immunofluorescence. In vitro, LX-2 cells were stimulated with BCG plus LPS or CCl₄, and treated with TGFβ1, SHP2, and EZH2 inhibitors. Protein expression, apoptosis, and proliferation were assessed by Western blotting, immunofluorescence, flow cytometry, and colony formation assays. Single-cell transcriptomic analysis revealed that the HIPPO pathway was highly activated in hepatic stellate cells during liver injury, and these cells were closely associated with the degree of post-injury fibrosis. Bioinformatics analysis demonstrated interactions between the PI3K and Hippo signaling pathways and proteins of interest in the pathological model of liver injury. In mice, the model group displayed elevated hepatic fibrosis and liver injury compared with the normal group, both of which were attenuated by treatment with the SHP2 inhibitor PHPS1. In LX-2 cells, the model group exhibited increased expression of TGFβ1, p-SHP2, p-STAT3, nuclear BRD4, the fibrotic markers α-SMA and Collagen-I, and the proliferation-related proteins c-Myc and p53, accompanied by decreased nuclear EZH2 expression; both cell proliferation and apoptosis were enhanced. Treatment with the TGFβ1 inhibitor TGFβ1-IN-1 or the SHP2 inhibitor PHPS1 reversed these protein alterations, suppressed proliferation, and promoted apoptosis. When EZH2 inhibitor or BRD4-overexpressing lentivirus was sequentially added on top of PHPS1 treatment, the effects of PHPS1 were progressively abrogated. Our findings are consistent with a model in which SHP2 may contribute to the progression of liver fibrosis through a network involving the TGFβ1/EZH2/BRD4 axis.