Green propolis extract attenuates acute kidney injury and lung injury in a rat model of sepsis

Abstract

Sepsis is the leading cause of acute kidney injury (AKI) and lung injury worldwide. Despite therapeutic advances, sepsis continues to be associated with high mortality. Because Brazilian green propolis (GP) has promising anti-inflammatory, antioxidant, and immunomodulatory properties, we hypothesized that it would protect kidneys and lungs in rats induced to sepsis by cecal ligation and puncture (CLP). Male Wistar rats were divided into groups-control (sham-operated); CLP (CLP only); and CLP + GP (CLP and treatment with GP at 6 h thereafter)-all receiving volume expansion and antibiotic therapy at 6 h after the procedures. By 24 h after the procedures, treatment with GP improved survival, attenuated sepsis-induced AKI, and restored renal tubular function. Whole-blood levels of reduced glutathione were higher in the CLP + GP group. Sepsis upregulated the Toll-like receptor 4/nuclear factor-kappa B axis in lung and renal tissues, as well as increasing inflammatory cytokine levels and macrophage infiltration; all of those effects were attenuated by GP. Treatment with GP decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells in renal and lung tissue, as well as protecting the morphology of the renal mitochondria. Our data open the prospect for clinical trials of the use of GP in sepsis.

Figure 1

Fingerprint analysis of the ethanol extract of propolis. Chromatograms were plotted at 275 nm with reverse-phase high-performance liquid chromatography on a C18 column (4.6 mm × 250 mm; particle diameter, 5 µm; pore diameter, 100 Å) and gradient elution with methanol and acidic water (pH 2.7). The chromatographic profile includes the following compounds: (1) caffeic acid phenethyl ester (in approximately 15 min); (2) p-coumaric acid (in approximately 20 min); (3) trans-Cinnamic acid (in 35–36 min); (4) aromadendrin (in 38 min); and (5) artepillin C (in 61–62 min).

Figure 2

Effects of propolis on survival. *p < 0.05 versus CLP.

Figure 3

Renal function measured by inulin clearance (glomerular filtration rate) in control, cecal ligation and puncture (CLP), and CLP + green propolis (CLP + GP) group rats 24 h after the CLP or sham procedure. *p < 0.05 versus CLP + GP; ***p < 0.001 versus control.

Figure 4

Changes in oxidative stress markers in control, cecal ligation and puncture (CLP), and CLP + green propolis (CLP + GP) group rats 24 h after the CLP or sham procedure. (A) Whole-blood reduced glutathione (GSH) levels; (B) Immunoblotting of protein expression of manganese superoxide dismutase (MnSOD) in renal tissues; *p < 0.05 versus control; ***p < 0.001 versus CLP + GP.

Figure 5

Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway in renal tissue in control, cecal ligation and puncture (CLP), and CLP + green propolis (CLP + GP) group rats 24 h after the CLP or sham procedure. (A) Immunoblotting of TLR4 protein expression in renal tissue; (B) Immunoblotting of NF-κB protein expression in renal tissues; (C) Macrophage infiltration in renal tissues analyzed by immunohistochemistry. We analyzed 30 grid fields (0.087 mm² each) per kidney cortex, counting the number of infiltrating CD68 + cells in each field, on the basis of which we calculated the mean numbers in the renal cortical tubulointerstitium and the mean counts per kidney. *p < 0.05 versus control and CLP + GP; ***p < 0.001 versus control and CLP + GP; **p < 0.01 versus control and CLP + GP.

Figure 6

Effects of green propolis on apoptosis and mitochondrial morphology in renal tissues in control, cecal ligation and puncture (CLP), and CLP + green propolis (CLP + GP) group rats 24 h after the CLP or sham procedure. (A) Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) + cells. We quantified TUNEL + cells in 30 randomly selected fields; (B) Mitochondrial morphology. *p < 0.05 versus CLP + GP; ***p < 0.001 versus control.

Figure 7

Effects of green propolis on Toll-like receptor 4 (TLR4) protein expression, macrophage infiltration, and apoptosis in lung tissue in control, cecal ligation and puncture (CLP), and CLP + green propolis (CLP + GP) group rats 24 h after the CLP or sham procedure. (A) TLR4 expression in macrophage cells. A double-labeling technique was used for CD68 and TLR4 analyzed by immunofluorescence; (B) Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) + cells analyzed by immunofluorescence. *p < 0.05 versus control and CLP + GP.