Upregulation of Heme Oxygenase-1 by Hemin Alleviates Sepsis-Induced Muscle Wasting in Mice

Abstract

Hemin, an inducer of heme oxygenase-1 (HO-1), can enhance the activation of HO-1. HO-1 exhibits a variety of activities, such as anti-inflammatory, antioxidative, and antiapoptotic functions. The objective of this study was to investigate the effects of hemin on sepsis-induced skeletal muscle wasting and to explore the mechanisms by which hemin exerts its effects. Cecal ligation and perforation (CLP) was performed to create a sepsis mouse model. Mice were randomly divided into four groups: control, CLP, CLP plus group, and CLP-hemin-ZnPP (a HO-1 inhibitor). The weight of the solei from the mice was measured, and histopathology was examined. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to assess the expression levels of HO-1 and atrogin-1. Furthermore, we investigated the antioxidative effects of HO-1 by detecting malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity. CLP led to dramatic skeletal muscle weakness and atrophy, but pretreatment with hemin protected mice against CLP-mediated muscle atrophy. Hemin also induced high HO-1 expression, which resulted in suppressed proinflammatory cytokine and reactive oxygen species (ROS) production. The expression of MuRF1 and atrogin-1, two ubiquitin ligases of the ubiquitin-proteasome system- (UPS-) mediated proteolysis, was also inhibited by increased HO-1 levels. Hemin-mediated increases in HO-1 expression exert protective effects on sepsis-induced skeletal muscle atrophy at least partly by inhibiting the expression of proinflammatory cytokines, UPS-mediated proteolysis, and ROS activation. Therefore, hemin might be a new treatment target against sepsis-induced skeletal muscle atrophy.

Figure 1

Hemin ameliorated muscle mass loss and mitigated the protein breakdown rates. (a) Gross comparisons of muscle mass (8-week-old mice, 3 days after CLP surgery). (b) The weight of the solei from mice in the hemin group was improved, especially on days 3, 5, and 7 after surgery. ^∗p < 0.001 (hemin vs. CLP), n = 6. (c) Protein breakdown rates as measured by the tyrosine concentration. ^∗p < 0.001 (control vs. CLP), ^∗∗p < 0.001 (hemin vs. CLP), ^#p < 0.001 (ZnPP vs. hemin), n = 6.

Figure 2

Histopathology examination of TA muscles at day 5 after surgery. (a) Skeletal muscle fibers from the CLP group exhibited a smaller cross-sectional area (CSA) than those from the control and hemin groups (H&E staining, 40x magnification, scale bar = 50 μm). (b) Quantification of the CSA of TA muscles from the different groups. ^∗p < 0.001 (CLP vs. the control group), ^∗∗p < 0.01 (hemin vs. the CLP group), ^#p < 0.05 (ZnPP vs. the hemin group), n = 6.

Figure 3

Hemin induces high HO-1 expression and increased HO-1 activation. (a) Hemin pretreatment can promote HO-1 mRNA expression at 1, 4, and 7 days after surgery (^∗p < 0.001 vs. CLP at 1 day, ^∗∗p < 0.001 vs. CLP at 4 days, ^#p < 0.05 vs. CLP at 7 days). (b) Hemin enhanced the enzyme activity of HO-1, but ZnPP treatment reversed this effect (^∗p < 0.01 vs. CLP, ^#p < 0.01 vs. CLP + hemin; n = 6). (c) Western blot analysis of levels of the HO-1 protein. (d) Results of the corresponding semiquantitative analysis of levels of the HO-1 protein based on the optical density measured using the ImageJ software; the data are presented as means ± SEM and are representative of three separate experiments (^∗p < 0.01 vs. control, ^#p < 0.01 vs. CLP).

Figure 4

Sepsis induced high serum levels of TNF-α and IL-6. Hemin pretreatment reduced the levels of TNF-α and IL-6. (a) ^∗p < 0.0001 vs. control, ^#p < 0.05 vs. CLP; n = 6. (b) ^∗p < 0.0001 vs. control, ^#p < 0.01 vs. CLP; n = 6 (1 day after surgery).

Figure 5

Oxidative stress response in TA muscle at 24 hours after CLP. (a) MDA concentration. ^∗p < 0.05 vs. the control group; ^∗∗p < 0.05 vs. the CLP group; n = 6. (b) SOD activity measurement. ^∗p < 0.01 vs. the control group; ^∗∗p < 0.01 vs. the CLP group; n = 6.

Figure 6

The levels of CLP-induced muscle atrophy markers were elevated, and HO-1 suppressed the mRNA and protein expression of atrogin-1 and MuRF1. (a) HO-1 inhibited the mRNA expression of atrogin-1 at 1, 4, and 7 days after surgery (^∗p < 0.01 vs. CLP at 1 day, ^#p < 0.05 vs. CLP at 4 days, ^∗∗p < 0.01 vs. CLP; n = 6). (b) HO-1 suppressed the mRNA expression of MuRF1 during sepsis at 1, 4, and 7 days after surgery (^∗p < 0.01 vs. CLP at 1 day, ^#p < 0.01 vs. CLP at 4 days, ^∗∗p < 0.01 vs. CLP at 7 days; n = 6). (c, d) Western blot analysis of levels of the atrogin-1 and MuRF1 proteins. Results of the corresponding semiquantitative analysis of levels of the HO-1 protein based on the optical density measured using the ImageJ software; the data are presented as means ± SEM and are representative of three separate experiments (^∗p < 0.01 vs. control, ^#p < 0.01 vs. CLP). The protein expression of atrogin-1 and MuRF1 was upregulated in the CLP group. Hemin suppressed the protein expression of these two ligases; however, ZnPP administration abrogated the protective effects of hemin (1 day after surgery).