Salivary Gland Extract from Aedes aegypti Improves Survival in Murine Polymicrobial Sepsis through Oxidative Mechanisms

Abstract

Sepsis is a systemic disease with life-threatening potential and is characterized by a dysregulated immune response from the host to an infection. The organic dysfunction in sepsis is associated with the production of inflammatory cascades and oxidative stress. Previous studies showed that Aedes aegypti saliva has anti-inflammatory, immunomodulatory, and antioxidant properties. Considering inflammation and the role of oxidative stress in sepsis, we investigated the effect of pretreatment with salivary gland extract (SGE) from Ae. aegypti in the induction of inflammatory and oxidative processes in a murine cecum ligation and puncture (CLP) model. Here, we evaluated animal survival for 16 days, as well as bacterial load, leukocyte migration, and oxidative parameters. We found that the SGE pretreatment improved the survival of septic mice, reduced bacterial load and neutrophil influx, and increased nitric oxide (NO) production in the peritoneal cavity. With regard to oxidative status, SGE increased antioxidant defenses as measured by Trolox equivalent antioxidant capacity (TEAC) and glutathione (GSH), while reducing levels of the oxidative stress marker malondialdehyde (MDA). Altogether, these data suggest that SGE plays a protective role in septic animals, contributing to oxidative and inflammatory balance during sepsis. Therefore, Ae. aegypti SGE is a potential source for new therapeutic molecule(s) in polymicrobial sepsis, and this effect seems to be mediated by the control of inflammation and oxidative damage.

Keywords: Aedes aegypti; CLP model; oxidative stress; saliva; salivary gland extract; sepsis.

Figure 1

Experimental protocol of the CLP model and pre-treatments. CLP: cecal ligation and puncture; CEF: ceftriaxone; CFU: colony-forming unit; ROS: reactive oxygen species; SGE: salivary gland extract; TEAC: Trolox equivalent antioxidant capacity.

Figure 2

Effect of SGE on survival rate (%) and body weight (g) of septic mice within 16 days. (A) Survival of CLP animals pretreated with SGE (3.5 µg/animal), CEF (20 mg/kg), or saline. (B) Body weight during time evaluated for survival. Data presented as mean ± SD (n = 5). Kaplan–Meier analysis (# p < 0.005 CLP versus CLP + saline). CEF: ceftriaxone; CLP: cecal ligation and puncture SGE: salivary gland extract.

Figure 3

Effect of SGE on the number of circulating cells and migration to the peritoneal cavity in animals with sepsis. (A) Neutrophils in the blood. (B) neutrophils in the peritoneal cavity. (C) monocytes in the blood. (D) Monocytes in the peritoneal cavity. Total cell counts in the peritoneal cavity and blood were determined 12 h and 24 h after CLP. The results were expressed as the mean ± SD (five animals/group). * p < 0.05 compared to the sham control group; # p < 0.05 compared to the saline control group. CLP: cecal ligation and puncture; SGE: salivary gland extract; CEF: ceftriaxone.

Figure 4

Effect of SGE on NO and ROS production in septic mice. (A) NO levels in serum 12 h and 24 h after CLP; (B) NO levels in the peritoneal cavity 12 h and 24 h after CLP; (C) ROS production by peritoneal macrophages stimulated or not with 40 μM t-BHP. The results were expressed as the mean ± SD (five animals/group). * p < 0.05 compared to sham control group; # p < 0.05 compared to the saline control group. CLP: cecal ligation and puncture; SGE: salivary gland extract; CEF: ceftriaxone; NO: nitric oxide; ROS: reactive oxygen species.

Figure 5

Effect of SGE on antioxidant activity in septic mice. (A) TEAC in serum 12 h and 24 h after CLP; (B) TEAC in the peritoneal cavity 12 h and 24 h after CLP. The results were expressed as the mean ± SD (five animals/group). * p < 0.05 compared to the sham control group; # p < 0.05 compared to the saline control group. CLP: cecal ligation and puncture; SGE: salivary gland extract; CEF: ceftriaxone; TEAC: Trolox equivalent antioxidant capacity.

Figure 6

Effect of SGE on GSH levels in septic mice. (A) GSH levels in serum 12 h and 24 h after CLP. (B) GSH levels in peritoneal cavity 12 h and 24 h after CLP. (C) GSH levels in other tissue after 12 h CLP. The results were expressed as the mean ± SD (five animals/group). * p < 0.05 compared to the sham control group; # p < 0.05 compared to saline-pretreated sepsis group. CLP: cecal ligation and puncture; SGE: salivary gland extract; CEF: ceftriaxone; GSH: glutathione.

Figure 7

Effect of SGE on lipid peroxidation products in septic mice. (A) MDA levels in serum at 12 and 24 h after CLP. (B) MDA levels in the peritoneal cavity 12 and 24 h after CLP. (C) MDA levels in other tissue 12 h after CLP. The results are expressed as the mean ± SD (five animals/group). * p < 0.05 compared to the sham group; # p < 0.05 compared to saline-pretreated septic group. CLP: cecal ligation and puncture; SGE: salivary gland extract; CEF: ceftriaxone; MDA: malondialdehyde.

Figure 8

Probable mechanism of action of Ae. aegypti’s SGE in CLP sepsis model. (A) The D7 protein of SGE binds to LTB4 and reduces TNF-α production, decreasing neutrophil and macrophage recruitment to the inflammatory site. (B) The SGE has lysozyme, crecropin, D7 protein, defensin A1, and gambicin, which interact with bacterial surfaces and result their elimination. (C) By a different mechanism, SGE modulates oxidative stress, and SOD converts the superoxide into oxygen or hydrogen peroxide, which are less reactive molecules. (D) SGE reduce lipid peroxidation by restoring antioxidant system. (E) NADH restores the reduced GSH to its active form, increasing GSH bioavailability and reducing lipid peroxidation. CLP: cecal ligation and puncture; SGE: salivary gland extract; LTB4: Leukotriene B4; TNF-α: tumor necrosis factor - alfa; SOD: superoxide dismutase; GSH: glutathione.