Mechanism of Qiling Fuzheng Qingjie granules in alleviating doxorubicin-induced T cell immune dysfunction via mitochondrial energy metabolism

Figures

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  Fig. 1. QFQ and immune response-related targets, PPI network in immune treatment and analysis of GO and KEGG enrichment. A Venn diagram of shared QFQ and colon immune response- related targets. B Visualization of the PPI network by Cytoscape 3.9.1. Using the Centiscape 2.2 plugin in Cytoscape 3.9.1, the lines within the PPI network were visually organized according to their degree values, reflecting the strength of protein interactions. Larger nodes with deeper colors and closer proximity to the center indicated higher degree values, signifying stronger interactions among the targets. Top 10 GO terms in C biological process (BP), D Cellular component (CC) and E Molecular function (MF) categories. F Top 15 KEGG pathways

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  Fig. 2. Effects of QFQ on body weight and immune organs in mice. A Schematic of the doxorubicin-induced mouse model and QFQ treatment regimen. B Effect of QFQ on body weight in mice. C Effect of QFQ on the weight and index of thymus in mice. D Effect of QFQ on the weight and index of spleen in mice. E Thymic tissue sections from each group of mice were analyzed by H&E staining. Representative H&E-stained images are shown (magnifications: 100 × , 400 ×). Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 3. Effects of QFQ on bone marrow hematopoietic function in mice. A Representative bone marrow smears from each group of mice. MB and ProM: Myeloblast and Promyelocyte, My and MM: Myelocyte and Metamyelocyte, NSG: Neutrophil Segmented Granulocyte, NST: Neutrophilic Stab Cell, ProE and BasoE: Proerythroblast and Basophilic Erythroblast, PolyE: Polychromatic Erythroblast, OrthoE: Orthochromatic Erythroblast, Lymph: Lymphocyte. B Effect of QFQ on the proportion of reticulocytes in the peripheral blood of mice. Reticulocytes are indicated by red arrows. RET: Reticulocyte. C Effects of QFQ on hematological parameters in mice. WBC: White Blood Cell, Lymph: Lymphocyte, GR: Granulocyte, MONO: Monocyte, RBC: Red Blood Cell, HGB: Hemoglobin, PCV: Packed Cell Volume, PLT: Platelet, MPV: Mean Platelet Volume. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 4. Effects of QFQ on the proportions of immune cell subsets in mouse splenocytes. A Flow cytometry gating strategy diagram. B The frequency of immune cell subsets among CD45⁺ leukocytes is shown in representative flow cytometry plots and quantified in the accompanying figures. C t-SNE analysis of immune cell subsets in mouse splenocytes. Data are presented as the mean ± SD, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 5. Effects of QFQ on splenic T cell subsets in mice. A Flow cytometry analysis of major immune cell populations in the spleen. B Frequency of Th1, Th2 cells and the Th1/Th2 ratio among CD3⁺T cells. C Frequency of Th17, Treg, KLRG-1⁺ Treg cells and the Th17/Treg ratio among CD3⁺T cells. D Composition of Tem/Teff, Tcm and Tn within CD4+T cells. E Composition of Tem/Teff, Tcm and Tn within CD8+T cells. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 6. Effects of QFQ on T cell activation and viability in mice. A Representative images of cell activation at 36 h and 72 h, with cell clones indicated by red arrows. (magnifications: 100 ×). B Comparison of the number and diameter of cell clones among the groups. C Effects of QFQ on cell viability in mice. D Flow cytometry analysis of the expression of CD25⁺ and CD27⁺ among CD4⁺ or CD8⁺T cells. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 7. Profiling of T cell proliferation and CD8⁺T cell cytotoxic function. A Representative flow cytometry plots and statistical analysis of CD4⁺ and CD8⁺T cell proportions. B Proliferative capacity of CD4⁺ and CD8⁺T cells in vitro. C IFN-γ production and perforin expression in CD8⁺T cells. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 8. Effects of QFQ on mitochondrial membrane potential in mouse T cells. A Representative Mito-Tracker Deep Red 633 staining images and comparisons of red fluorescence expression levels among the groups. Mito-Tracker Deep Red 633 stains the mitochondria of live cells, emitting red fluorescence, while Hoechst 33,342 stains the nuclei, emitting blue fluorescence. MTDR 633: Mito-Tracker Deep Red 633. (magnifications: 1000 ×). B Representative JC-1 flow cytometry plots and quantitative analysis of red versus green fluorescence in each group. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001

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  Fig. 9. Effects of QFQ on cellular mitochondrial and glycolytic metabolism. Metabolic profiles were determined using Seahorse XF technology. A Comparisons of ATP production, basal respiration, OCR, non-mitochondrial oxygen consumption, proton leak, and ECAR among the groups in the mitochondrial stress test. B Comparisons of ECAR, PER, OCR, real-time glycolytic ATP production rate, real-time OXPHOS ATP production rate, and total ATP production rate among the groups in the real-time ATP rate assay. C Comparisons of ECAR, OCR, PER, basal glycolytic capacity, basal proton efflux rate, basal PER percentage from glycolysis, and compensatory glycolytic rate among the groups in the glycolytic function assay, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 10. Anti-tumor activity of QFQ and its impact on CD8⁺T cell exhaustion in A20 tumor-bearing model. The therapeutic effects of QFQ were evaluated by monitoring body weight changes (A), visualizing tumor sizes (B), and quantifying tumor weight (C) and volume (D). The effects of QFQ on CD8⁺T cell exhaustion were investigated by analyzing CD8⁺T cell subsets, including PD-1, CTLA-4 and CD28 molecules (E), *P < 0.05, **P < 0.01, ****P < 0.0001

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  Fig. 11. The potential mechanism by which QFQ regulates T cell immune dysfunction induced by DOX