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. 2023 Jul 9;19(11):3576–3594. doi: 10.7150/ijbs.85133

Figure 5.

Figure 5

P2RY12 orchestrates T cells aerobic glycolysis through HK2 in ConA-induced immune hepatitis. (A-C) GSEA results indicate that genes related to “carbohydrate metabolic process” (A), “glycolysis gluconeogenesis” (B) and “mitochondrion organization” (C) are differentially enriched in hepatic T cells gathered from WT and P2RY12-/- mice after ConA administration 12 hr. NES, normalized enrichment score. FDR, false-discovery rate. (D, E) Seahorse analysis of maximum ECAR (measured after oligomycin injection, Oligo) and baseline OCR and maximum OCR in hepatic T cells from WT mice and P2RY12-/- mice after ConA administration 12 hr. (F) Total intracellular ATP production in hepatic T cells from WT mice and P2RY12-/- mice induced by ConA was measured by ATP-dependent luminescent activity. (G) Heat map depicting changes in the expression of protein involved in “glycolysis” in hepatic T cells. (H) Relative protein expression levels of HK2, GPI, PFK2, MCT1, Tpi and β-actin in hepatic T cells gathered from WT mice and P2RY12-/- mice after ConA administration 12 hr. (I) Flow cytometric analysis of HK2+ hepatic T cells gathered from WT mice and P2RY12-/- mice after ConA administration 12 hr. Pooled data are presented in the right panel. (J) Schematic diagram of HK2 function in glycolysis metabolism. (K) Targeted metabolomics analysis the level of glucose in hepatic T cells. (L) Schematic diagram of cell adoptive transfer. T cells from WT mice splenic lymphocytes were co-culture with PBS or 3-BrPA for 8 hr, then transferred into Rag1-/- mice via tail vein injection, followed by challenging these mice with ConA. (M) The serum levels of AST and ALT were detected. (N) Photomicrographs of representative H&E-stained mouse livers. Massive hepatocyte necrosis (dark arrows) was observed in T cells co-culture with PBS mice. (O) The proportion of IFN-γ in CD4+ T cells, CD8+ T cells from liver lymphocytes. (P) The serum level of IFN-γ was detected. Data are mean ± SEM. *P< 0.05; **P< 0.01; ***P < 0.001 vs indicated group. (two-tailed Student's t-test). Data are representative of three independent experiments with similar results.