Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS- stimulated RAW 264.7 macrophages Hong-Guang Chen, Ke-Liang Xie, Huan-Zhi.

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Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS- stimulated RAW 264.7 macrophages Hong-Guang Chen, Ke-Liang Xie, Huan-Zhi Han, Wei-Na Wang, Da-Quan Liu, Guo-Lin Wang, Yong-Hao Yu International Journal of Surgery Volume 11, Issue 10, Pages 1060-1066 (December 2013) DOI: 10.1016/j.ijsu.2013.10.007 Copyright © 2013 Surgical Associates Ltd Terms and Conditions

Fig. 1 Effects of hydrogen treatment on cell viability and injury of RAW 264.7 macrophages. The injury and viability of RAW 264.7 macrophages were measured with LDH release (A) and MTT assay (B). Cells were treated with different concentrations of hydrogen (0, 0.15, 0.30 and 0.60 mM) for 24 h. The relative absorbance is expressed as % of control, and results are presented as mean ± SD (n = 6 each group). International Journal of Surgery 2013 11, 1060-1066DOI: (10.1016/j.ijsu.2013.10.007) Copyright © 2013 Surgical Associates Ltd Terms and Conditions

Fig. 2 Effects of hydrogen treatment on the production of inflammatory cytokines in LPS-activated RAW 264.7 macrophages. Cells were stimulated with 1 μg/mL of LPS or PBS with absence or presence of hydrogen-rich medium (0.6 mM). The levels of TNF-α (A), IL-1β (B), HMGB1 (C) and IL-10 (D) in the culture media were measured at baseline (0 h) as well as 3 h, 6 h, 12 h and 24 h after LPS or PBS administration. Results are presented as mean ± SD (n = 6 each group at every time point). aP < 0.05 compared with the Con group; bP < 0.05 compared with the LPS group. International Journal of Surgery 2013 11, 1060-1066DOI: (10.1016/j.ijsu.2013.10.007) Copyright © 2013 Surgical Associates Ltd Terms and Conditions

Fig. 3 The concentration-dependent effects of hydrogen treatment on inflammatory cytokines production in LPS-stimulated RAW 264.7 macrophages. Cells were challenged with LPS (1 μg/mL) for 24 h in the absence or presence of different concentrations of hydrogen. The culture media were collected for measurement of TNF-α (A), IL-1β (B), HMGB1 (C) and IL-10 (D). Results are presented as mean ± SD (n = 6 each group). aP < 0.05 compared with the cells without hydrogen and LPS; bP < 0.05 compared with the cells with LPS stimulation; cP < 0.05 compared with the cells with 0.15 mM hydrogen and LPS. International Journal of Surgery 2013 11, 1060-1066DOI: (10.1016/j.ijsu.2013.10.007) Copyright © 2013 Surgical Associates Ltd Terms and Conditions

Fig. 4 Effect of hydrogen treatment on HO-1 activity in LPS-stimulated RAW 264.7 macrophages. (A) At the indicated time points, the HO-1 activity was detected. (B) Cells were challenged with 1 μg/mL of LPS or PBS in the absence or presence of different concentrations of hydrogen. After incubation for 24 h, HO-1 activity was detected. Results are presented as mean ± SD (n = 6 each group at each time point). aP < 0.05 compared with the Con group; bP < 0.05 compared with the LPS group. cP < 0.05 compared with the cells without hydrogen and LPS; dP < 0.05 compared with the cells with LPS stimulation. eP < 0.05 compared with the cells with 0.60 mM hydrogen and LPS. International Journal of Surgery 2013 11, 1060-1066DOI: (10.1016/j.ijsu.2013.10.007) Copyright © 2013 Surgical Associates Ltd Terms and Conditions

Fig. 5 Effect of hydrogen treatment on HO-1 expression in LPS-stimulated RAW 264.7 macrophages. Cells were challenged with 1 μg/mL of LPS or PBS for 24 h in the absence or presence of different concentrations of hydrogen. HO-1 expression was assayed by western blot analysis. Results are presented as mean ± SD (n = 6 each group). aP < 0.05 compared with the cells without hydrogen and LPS; bP < 0.05 compared with the cells with LPS stimulation; cP < 0.05 compared with the cells with 0.15 mM hydrogen and LPS stimulation. dP < 0.05 compared with the cells with 0.30 mM hydrogen and LPS. International Journal of Surgery 2013 11, 1060-1066DOI: (10.1016/j.ijsu.2013.10.007) Copyright © 2013 Surgical Associates Ltd Terms and Conditions

Fig. 6 HO-1 inhibitor ZnPP could reverse the regulative effect of hydrogen treatment on inflammatory cytokines production in LPS-stimulated RAW 264.7 macrophages. Cells were cultured with 1 μg/mL LPS or PBS under hydrogen treatment for 24 h in the presence or absence of 20 μM of ZnPP. Culture media were harvested for measurement of TNF-α (A), IL-1β (B), HMGB1 (C) and IL-10 (D). Results are presented as mean ± SD (n = 6 each group). aP < 0.05 compared with the cells without hydrogen and LPS; cP < 0.05 compared with the cells with LPS stimulation. bP < 0.05 compared with the cells with hydrogen and LPS. International Journal of Surgery 2013 11, 1060-1066DOI: (10.1016/j.ijsu.2013.10.007) Copyright © 2013 Surgical Associates Ltd Terms and Conditions