Fig.1 Controlled release kinetics of hydrogen from Mg. (a) The volume of released H2 and (b) the concentration of dissolved H2 in PBS solution increased with the exposed surface area (mm2 per mL PBS) of Mg plates (PBS: pH = 7.4); (c) the volume of released H2 and (d) the concentration of dissolved H2 in PBS increased as the pH value decreases.
Fig.2 Hydrogen induces tumor cell apoptosis at certain critical concentrations. (a) A series of morphology images of HCT116 cells from a live cell imaging microscope. (b) Schematic illustration of H2-emitting device. (c) The cell viability of HCT116 cells with different treatments. (d) The cell cycle of the HCT116 cells in the Ctrl and M-H2 groups; and (e) flow cytometry of cell apoptosis after different treatments and the corresponding apoptosis rate was statistically quantified. L-H2 and M-H2 groups represent the low and medium concentrations of H2 in the culture medium, respectively. The ctrl group represents no H2-treatment.
Fig.3 Hydrogen regulated gene expressions related to the P53-mediated apoptosis signaling pathway. (a) A volcano plot showing up/down-regulated genes after M-H2 treatment vs. the Ctrl group. Genes with an absolute fold change of >2 and a P value of <0.05 are highlighted in green and red color separately, denoting the down- and up-regulated genes, respectively. (b) Circular visualization of the gene-annotation enrichment analysis after gene set enrichment analysis (GSEA) was conducted using the Molecular Signatures Database (MSigDB). (c) The gene set enrichment analysis of the P53, lysosome and apoptosis pathway. (NES: normalized enrichment score). (d) A heat map of differentially expressed genes associated with three apoptotic pathways after H2 treatment, a fold change of >2 and a p value of <0.05 compared to the Ctrl group was analyzed. (e, f) An analysis of the enriched differentially expressed genes using the Kyoto Encyclopedia of Genes (KEGG) and Genomes (GO) database.
Fig. 4 The up-regulated p53 activates the mitochondrial apoptosis pathway by hydrogen. (a) The p53, cathepsins, Bax, Bcl-2 and LC3B expression after M-H2, L-H2 and Ctrl treatment, respectively. (b) Confocal fluorescence images of mitochondria membrane potential and apoptosis of HCT116 with or without H2 treatment. The mitochondria membrane potential was stained by Mito-Tracker Red CMXRos (red), apoptosis cells were stained by Annexin V-FITC (green) and the nuclei were stained by Hoechst 33342 (blue). The scale bar is 25 μm. (c) The analysis of cytochrome C expression in HCT116 cells using the APC-conjugated cytochrome C antibody. Scale bar: 25 μm. (d) The analysis of the caspase-3 expression in HCT116 cells using FITC-conjugated cleaved caspase-3 antibody. Scale bar: 25 μm.
Fig. 5 Schematic illustration of the anti-tumor effect of H2 from biodegradable Mg
Fig. 6 Hydrogen released from Mg wires inhibits tumor growth in vivo. (a) The anti-tumor evaluation of the H2 therapies through Mg degradation on tumor-bearing mice. (b) Micro-CT images of Mg wires after 0 d, 8 d, 16 d, and 24 d implantation in tumor tissue; (c) Evolution of hydrogen released and wire volumes in tumor-bearing mice after 8 d, 16 d and 24 d implantation. (d) Growth curve of tumor volume after different treatments. (e) The HCT116 tumor weight and the corresponding representative pictures of tumors at the end of the experiment. (f) Bodyweight change after different treatments.
Fig. 7 Hydrogen induces tumor tissue apoptosis in tumor-bearing mice. (a) TEM photos of the tumor tissues at various scales; (b) H&E images of tumor tissues dissected from each group after different treatments on day 24; (c) Apoptosis was analyzed by TUNEL staining in tumor tissue after 24 days of treatment; (d) Confocal laser scanning microscopy of caspase-3 staining in tumor tissues after different treatments.