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. 2014 Nov 12:14:442.
doi: 10.1186/1472-6882-14-442.

Vitamin C treatment attenuates hemorrhagic shock related multi-organ injuries through the induction of heme oxygenase-1

Bing Zhao, Jian Fei, Ying Chen, Yi-Lin Ying, Li Ma, Xiao-Qin Song, Jie Huang, Er-Zhen Chen  1 , En-Qiang Mao
Affiliations

Vitamin C treatment attenuates hemorrhagic shock related multi-organ injuries through the induction of heme oxygenase-1

Bing Zhao et al. BMC Complement Altern Med. .

Abstract

Background: Vitamin C (VitC) has recently been shown to exert beneficial effects, including protecting organ function and inhibiting inflammation, in various critical care conditions, but the specific mechanism remains unclear. Induction of heme oxygenase (HO)-1, a heat shock protein, has been shown to prevent organ injuries in hemorrhagic shock (HS) but the relationship between VitC and HO-1 are still ill-defined so far. Here we conducted a systemic in vivo study to investigate if VitC promoted HO-1 expression in multiple organs, and then tested if the HO-1 induction property of VitC was related to its organ protection and anti-inflammatory effect.

Methods: Firstly, to determine the HO-1 induction property of VitC, the HO-1 level were measured in tissues including kidney, liver and lung of the normal and HS model of Sprague-Dawley (SD) rats after VitC treatment (100 mg/kg body weight). Secondly, to testify if VitC prevented HS related organ injuries via inducing HO-1, the HS model of rats were separately pre- and post-treated with VitC, and some of them also received Zinc protoporphyrin (Znpp), a specific HO-1 inhibitor. The HO-1 activity in tissues was tested; the organ injuries (as judged by histological changes in tissues and the biochemical indicators level in serum) and inflammatory response in tissues (as judged by the level of pro-inflammatory cytokines Tumor necrosis factor-α and Interleukin-6 ) were analyzed.

Results: The HO-1 mRNA and protein level in kidney, liver, and lung were highly induced by VitC treatement under normal and HS conditions. The HO-1 activity in tissues was enhanced by both VitC pre- and post-treatment, which was shown to improve the organ injuries and inhibit the inflammatory response in the HS model of rats. Of note, the beneficial effects of VitC were abolished after HO-1 activity was blocked by Znpp.

Conclusions: VitC led to a profound induction of HO-1 in multiple organs including the kidney, liver and lung, and this property might be responsible for the organ protection and inflammation inhibitory effects of both pre- and post-treatment with VitC in HS.

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Figures

Figure 1
Figure 1
HO-1 mRNA and protein expression in tissues was highly induced under normal condition by VitC. A) HO-1 mRNA in the kidney (a), liver (b), and lung (c) are expressed relative to the level measured in the control animals. B) HO-1 protein expression in the kidney (a), liver (b), and lung (c) were detected using Western-blot. Each gel is representative of six independent experiments. Densitometric analysis was used to calculate the normalized protein ratio (HO-1 to β-actin). C) distribution of HO-1 protein in the kidney (a, d, scale bar: 200 μm), liver (b, e, scale bar: 100 μm), and lung (c, f, scale bar: 200 μm) were demonstrated by immunohistochemistry. The arrowheads indicate the HO-1-positive cells. Each image is representative of six independent experiments (n = 6). Tissue samples were collected at 2, 6, 12 and 24 hours after VitC treatment. Data represent the mean ± SEM. *p < 0.05 vs. the control. Control: normal saline-treated rats. V6h: the rats treated with VitC for 6 hrs.
Figure 2
Figure 2
The increased HO-1 mRNA and protein expression in tissues after HS were enhanced by VitC. A) HO-1 mRNA in the kidney (a), liver (b), and lung (c) are expressed relative to the level measured in Sham group. B) HO-1 protein expression in the kidney (a), liver (b), and lung (c) were detected using Western-blot. Each gel is representative of six independent experiments. Densitometric analysis was used to calculate the normalized protein ratio (HO-1 to β-actin). Tissue samples were collected at 2, 6, 12 and 24 hours after HS operation. Data represents the mean ± SEM. *p < 0.05 vs. the Sham; # p < 0.05 vs. the HS at the same time point. Sham: the rats which underwent sham operation and received normal saline (NS). HS: the rats which underwent HS operation and received NS. HSV-post: the rats which underwent HS operation and then received VitC immediately.
Figure 3
Figure 3
HO-1 activity in tissues was enhanced by both VitC pre- and post-treatment. Tissue samples were separately removed at 0 and 12 hours after sham or HS operation for HO-1 activity assay. *p < 0.05 vs. the Sham at the same time point; **p < 0.05 vs. the ShamV; *** p < 0.05 vs. the HS; **** p < 0.05 vs. the HSV-post. Data represents the mean ± SEM, n = 6. Sham: the rats which received NS and underwent sham operation; ShamV: the rats which received VitC and underwent sham operation; ShamVZ: some ShamV rats further received ZnPP; HS: the rats which received NS and underwent HS operation; HSV-post: the rats which underwent HS operation and then received VitC immediately; HSV-postZ: some HSV-post rats further received ZnPP.
Figure 4
Figure 4
Both VitC pre- and post-treatment relieved histological damages through HO-1 induction. A) representative light microscopy images of the kidney (a, d, g, j, m, p, s, scale bar: 200 μm), liver (b, e, h, k, n, q, t, scale bar: 100 μm), and lung (c, f, I, l, o, r, u, scale bar: 200 μm), which were stained with hematoxylin and eosin. B) histological damage score of the kidney (a), liver (b), and lung (c). Tissue samples were collected at 12 hours after the HS operation. Data represents the mean ± SEM, n = 6. *p < 0.05 vs. the Sham; **p < 0.05 vs. the HS; ***p < 0.05 vs. the HSV-pre; ****p < 0.05 vs. the HSV-post; $ p > 0.05 vs. the Sham. Sham: the rats which received NS and underwent sham operation. ShamV: the rats which received VitC and underwent sham operation. HS: the rats which underwent HS operation and received NS. HSV-pre: the rats which received VitC at 6 hours before HS operation. HSV-preZ: some HSV-pre rats further received ZnPP. HSV-post: the rats which underwent HS operation and received VitC immediately. HSV-postZ: some HSV-post rats further received ZnPP.
Figure 5
Figure 5
Both VitC pre- and post-treatment decreased the serum level of biochemical indicators through HO-1 induction. Serum level of biochemical indicators including ALT (A), AST (B), BUN (C), Cre (D), LDH (E), and lactate (F) were tested. Serum was collected at 12 hours after HS operation. Data represents the mean ± SEM, n = 6. *p < 0.05 vs. the Sham; **p < 0.05 compared to the HS; ***p < 0.05 compared to the HSV; ****p < 0.05 compared to the HSV-post; $ p > 0.05 compared to the Sham. Sham: the rats which received NS and underwent sham operation. ShamV: the rats which received VitC and underwent sham operation. HS: the rats which received NS and underwent HS operation. HSV-pre: the rats which received VitC at 6 hours before HS operation. HSV-preZ: some HSV-pre rats further received ZnPP. HSV-post: the rats which underwent HS operation and received VitC immediately. HSV-postZ: some HSV-post rats further received ZnPP.
Figure 6
Figure 6
Both VitC pre- and post-treatment decreased the tissues level of proinflammatory cytokines through HO-1 induction. The mRNA and protein levels of the proinflammatory cytokines TNF-α (A, B) and IL-6 (C, D) in tissues collected at 12 hours after HS operation were tested. The data represents the mean ± SEM, n = 6. *p < 0.05 vs. the Sham; **p < 0.05 vs. the HS; ***p < 0.05 vs. the HSV-pre; ****p < 0.05 vs. the HSV-post. Sham: the rats which received NS and underwent sham operation. ShamV: the rats which received VitC and underwent sham operation. HS: the rats which received NS and underwent HS operation. HSV-pre: the rats which received VitC at 6 hours before HS operation. HSV-preZ: some HSV-pre rats further received ZnPP. HSV-post: the rats which underwent HS operation and received VitC immediately. HSV-postZ: some HSV-post rats further received ZnPP.
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Pre-publication history
    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/442/prepub

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