GLP-1 Analog Liraglutide Improves Vascular Function in Polymicrobial Sepsis by Reduction of Oxidative Stress and Inflammation

Figures

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  Figure 1. Scheme for treatment and cecal ligation and puncture (CLP) procedure. Male C57BL/6J mice were treated with liraglutide (Lira) or the vehicle (saline) twice daily, every 12 h, for 3 consecutive days via intraperitoneal injections (i.p.) at a dose of 2 × 100 µg/kg/d for Lira or an adequate volume of the vehicle. One day after initial administration, a mid-grade polymicrobial sepsis was induced by cecal ligation at the medium position below the ileocecal valve, followed by needle perforation (puncture) of the cecum. Created with BioRender.com.

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  Figure 2. Treatment with the GLP-1 receptor agonist liraglutide prevents CLP-induced endothelial dysfunction in polymicrobial septic mice. (A,B) Endothelium-dependent (acetylcholine, ACh) and independent (nitroglycerine, NTG) relaxation of thoracic aortic rings was measured in isometric tension recordings. (C,D) Vasoconstrictor responses prompted by 2 μM prostaglandin F2α and increasing concentrations of KCl. Data are the mean ± SEM of 22–34 (A), 21-35 (B) or 26–36 (C,D) independent measurements. * p < 0.05 vs. B6J; # p < 0.05 vs. CLP.

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  Figure 3. Treatment with the GLP-1 receptor agonist liraglutide protects against CLP-induced complications of leukopenia and hyperthermia without altering blood glucose levels in polymicrobial septic mice. (A,D) On day 0 and 3, animals were weighed and their temperature taken to determine Δ body weight and Δ body temperature, respectively. (B,C) Non-fasting blood glucose levels and white blood cell counts were evaluated in whole blood 2 days following sepsis induction. Data are the mean ± SEM of 22–26 (A), 19-22 (B) and 16-24 (D) measurements from individual animals or 6 individual blood samples (each pooled from 3 to 4 mice) (C). * p < 0.05 vs. B6J; # p < 0.05 vs. CLP.

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  Figure 4. Treatment with the GLP-1 receptor agonist liraglutide reduces CLP-induced vascular, cardiac and systemic oxidative stress in polymicrobial septic mice. (A) The whole blood oxidative burst was determined by chemiluminescence (L-012) after phorbol ester (PDBu) stimulation. (B) The superoxide-specific DHE product, 2-hydroxyethidium, was quantified in aortic tissue and expressed as a percentage of B6J controls. Representative chromatograms are shown besides the quantification. (C) 3-nitrotyrosine (3-NT)-positive proteins in plasma as a consequence of peroxynitrite formation were measured by dot blot analysis. A representative image is shown below the densitometric quantification. (D,E) Cardiac and aortic cryosections were stained with dihydroethidium to visualize ROS formation as red fluorescence and autofluorescence from aortic laminae as green. The representative photomicrographs are shown next to the densitometric analysis. E, endothelium; M, media. Data are the mean ± SEM of 6 individual blood samples (each pooled from 3 to 4 mice) (A), 6-8 (B) and 4 (D,E) measurements from individual animals or 4 individual plasma samples (each pooled from 3 to 4 mice) (C). * p < 0.05 vs. B6J; #…

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  Figure 5. Treatment with the GLP-1 receptor agonist liraglutide suppresses CLP-induced vascular inflammation in polymicrobial septic mice. Aortic gene expression levels of (A) interleukin 6 (Il6), (B) inducible nitric oxide synthase (iNos), (C) tumor necrosis factor alpha (Tnfα) and (D) intercellular adhesion molecule-1 (Icam1) were measured by quantitative RT-PCR in aortic tissue. (E,F) Abundance of 3-NT-positive proteins and Nox2 protein expression in the aorta was determined by immunohistochemistry. Representative immunohistochemical images are shown next to the densitometric quantification. Magnification of 40× and scale bar of 50 μm. Data are the mean ± SEM of 3 individual samples (each pooled from 3 to 4 mice) (A–D) or 3 individual animals (E,F). * p < 0.05 vs. B6J; # p < 0.05 vs. CLP.

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  Figure 6. Summary scheme of beneficial cardiovascular effects of the GLP-1 receptor agonist liraglutide on CLP-induced polymicrobial sepsis. CLP-induced mid-grade polymicrobial sepsis led to a reduction in body weight and blood sugar in both vehicle and Lira-treated mice. However, in contrast to the vehicle, Lira protected from sepsis-induced systemic and vascular inflammation and oxidative stress from inflammatory cell-derived Nox2 activity, which translated into a normalized endothelial function yet without a net benefit on mortality. These data demonstrate a new role for GLP-1 receptor agonists like liraglutide in reducing the cardiovascular complications of polymicrobial sepsis based on their antioxidant and anti-inflammatory properties. Created with BioRender.com.