Human serum albumin improves arterial dysfunction during early resuscitation in mouse endotoxic model via reduced oxidative and nitrosative stresses

Abstract

Human serum albumin (HSA) is used as a resuscitation fluid in sepsis. This study investigated the potential protective properties of HSA on vascular function in a mouse endotoxic model in terms of oxidative and nitrosative stresses. Swiss mice were treated with either lipopolysaccharide (LPS) (50 mg/kg i.p.) or vehicle. One and five hours later, mice were infused with HSA (4%, 10 ml/kg), normal saline (0.9% NaCl, 30 ml/kg), or no fluid. Six hours after treatment, vascular reactivity was assessed on aortae and small mesenteric arteries. Measurements of NO and superoxide anion (O2(-)) by spin trapping and nuclear factor (NF)-kappaB, inducible NO synthase (iNOS), and peroxynitrite by Western blotting and immunohistochemical studies were conducted. HSA partially prevented the reduction of blood pressure induced by LPS and completely prevented both vascular hyporeactivity to phenylephrine and myogenic tone as well as endothelial dysfunction induced by the endotoxin. This was associated with a decreased up-regulation of NF-kappa B, iNOS, and peroxynitrite in the vascular wall. LPS-induced tissue increases in both NO and O2(-) production was decreased by HSA. These data demonstrate the protective effect of HSA treatment in experimental endotoxic shock by reducing the inflammatory process leading to oxidative and nitrosative stresses and vascular hyporeactivity.

Figure 1

Telemetric blood pressure recordings. Mean arterial blood pressure (MAP) in sham group (•), HSA group (▪), saline group (▴), and no fluid resuscitation group (○) mice recorded during an 8-hour monitoring period. Data are expressed as means ± SE of n = 7 mice for each group. *P < 0.05, significantly different in sham group versus no fluid resuscitation group, HSA group versus no fluid resuscitation group, or saline group versus no fluid resuscitation group.

Figure 2

HSA improves vascular contraction to Pe, myogenic tone, and endothelial function. A: Concentration-effect curves of aortae in sham group (•), HSA group (▪), saline group (▴), and no fluid resuscitation group (○), n = 5 for each group, exposed to Pe (3 nmol/L to 30 μmol/L). **P < 0.01, significantly different between HSA group and no fluid resuscitation group. Please note that there was no difference in contraction for Pe between aortae from HSA group and sham group. B: Concentration-effect curves of aortae harvested from sham group (•), HSA group (▪), saline group (▴), and no fluid resuscitation group (○) mice, n = 5 for each group of mice, exposed to Ach (1 nmol/L to 1 μmol/L). *P < 0.05, significantly different between HSA group and no fluid resuscitation group. Please note that there was no difference in percentage of relaxation to acetylcholine between aortae from HSA group and sham group. C: Diameter changes in response to increasing steps of pressure (myogenic tone) on mesenteric arteries isolated from sham group (•), HSA group (▪), saline group (▴), and no fluid resuscitation group (○) mice, n = 5 or 6 for each group of animals. **P < 0.01, significantly different between HSA group and no fluid resuscitation group. Please note that there was a difference in myogenic tone between aortae from HSA group and sham group.

Figure 3

HSA decreases NO-dependent vasorelaxation, NO release, and iNOS expression in aorta. A: Concentration-effect curves to Pe (3 nmol/L to 30 μmol/L) in absence (○, ▪) or in presence (♦, ⋄) of L-NAME (100 μmol/L) in mice aortae from no fluid resuscitation group (open symbols) and HSA group (filled symbols), respectively. B: Diameter change in response to increasing steps of pressure (myogenic tone) in the absence (○, ▪) or in the presence (♦, ⋄) of L-NAME (100 μmol/L) on mice mesenteric arteries isolated from no fluid resuscitation group (open symbols) and HSA group (filled symbols), respectively, **P < 0.01, significantly different between vessels from no fluid resuscitation group with and without the inhibitor. Please note that the inhibitor does not affect the contraction in vessels from HSA group. C: Quantification of the amplitude of NO-Fe(DETC)₂ signal in unit/weight [mg of the dried sample A/W_(ds), n = 6] in aorta from the four groups of mice. *P < 0.05, significantly different in sham group versus no fluid resuscitation group or in HSA group versus no fluid resuscitation group. D–F: Immunohistochemical staining for inducible NO synthase of aortae from sham group (D), no fluid resuscitation group (E), and HSA group mice (F). Green fluorescence was linked to secondary Alexa 488 anti-mouse-conjugated antibody. Scale bars = 150 μm.

Figure 4

HSA reduces NO overproduction and iNOS expression in heart and lung. A and B: Quantification of the amplitude of NO-Fe(DETC)₂ signal in unit/weight [mg of the dried sample A/W_(ds), n = 6] in heart (A) and lung (B) from the four groups of mice. *P < 0.05, significantly different in sham group versus no fluid resuscitation group and in HSA group versus no fluid resuscitation group. C: Representative Western blots of three experiments, revealing reduced iNOS expression in lung from HSA group mice.

Figure 5

HSA reduces the activation of NF-κB RelA/p65 in aortic wall. A–C: Immunohistochemical staining for NF-κB p65 subunit in aortae from sham group (A), no fluid resuscitation group (B), and HSA group (C) of mice (n = 3). Green fluorescence was linked with secondary Alexa 488 anti-rabbit-conjugated antibody. The corresponding phase contrast pictures are also shown (D–F). Scale bars = 150 μm.

Figure 6

HSA reduces LPS-induced oxidative stress. A–C: Quantification of the amplitude of O₂⁻-CMH signal in unit/weight [mg] of the dried sample A/W_(ds), n = 6] in aorta (A), heart (B), and lung (C) from the four groups of mice. *P < 0.05, significantly different in sham group versus no fluid resuscitation group and in HSA group versus no fluid resuscitation group. D–F: Immunohistochemical staining for nitrotyrosine of aortae from sham group (D), no fluid resuscitation group (E), and HSA group (F) (n = 3). Green fluorescence was linked with secondary Alexa 488 anti-mouse-conjugated antibody. Scale bars = 150 μm.