Serratiopeptidase Attenuates Lipopolysaccharide-Induced Vascular Inflammation by Inhibiting the Expression of Monocyte Chemoattractant Protein-1

Abstract

Lipopolysaccharide (LPS) has potent pro-inflammatory properties and acts on many cell types including vascular endothelial cells. The secretion of the cytokines MCP-1 (CCL2), interleukins, and the elevation of oxidative stress by LPS-activated vascular endothelial cells contribute substantially to the pathogenesis of vascular inflammation. However, the mechanism involving LPS-induced MCP-1, interleukins, and oxidative stress together is not well demonstrated. Serratiopeptidase (SRP) has been widely used for its anti-inflammatory effects. In this research study, our intention is to establish a potential drug candidate for vascular inflammation in cardiovascular disorder conditions. We used BALB/c mice because this is the most successful model of vascular inflammation, suggested and validated by previous research findings. Our present investigation examined the involvement of SRP in vascular inflammation caused by lipopolysaccharides (LPSs) in a BALB/c mice model. We analyzed the inflammation and changes in the aorta by H&E staining. SOD, MDA, and GPx levels were determined as per the instructions of the kit protocols. ELISA was used to measure the levels of interleukins, whereas immunohistochemistry was carried out for the evaluation of MCP-1 expression. SRP treatment significantly suppressed vascular inflammation in BALB/c mice. Mechanistic studies demonstrated that SRP significantly inhibited the LPS-induced production of proinflammatory cytokines such as IL-2, IL-1, IL-6, and TNF-α in aortic tissue. Furthermore, it also inhibited LPS-induced oxidative stress in the aortas of mice, whereas the expression and activity of monocyte chemoattractant protein-1 (MCP-1) decreased after SRP treatment. In conclusion, SRP has the ability to reduce LPS-induced vascular inflammation and damage by modulating MCP-1.

Keywords: lipopolysaccharides; monocyte chemoattractant protein-1; oxidative stress; serratiopeptidase; vascular inflammation; vascular smooth muscle cells.

Figure 1

The effect of SRP on oxidative stress biomarkers. Column figure shows the oxidative stress biomarkers measured in aortic homogenates of vehicle control (0.9% NS), LPS-, LPS+SRP-, and LPS+AT-treated animals. (A) Superoxide Dismutase; (B) reactive oxygen species intensity; (C) glutathione activity; (D) malondialdehyde (MDA) represent mean ± SEM. n = 6 for each group. * represents significant values when compared to controls at p < 0.05, # represents significant values when compared to LPS-treated animals at p < 0.05, *** represents significant values when compared to control group at p < 0.05, ** represents significant values when compared to control group p < 0.05, ## represents significant values when compared to LPS-treated group at p < 0.05.

Figure 2

The effect of Serratiopeptidase on inflammatory biomarkers. Column figure shows the inflammatory biomarkers measured in whole aorta homogenates of vehicle control (0.9% NS), LPS-, LPS+SRP-, and LPS+AT-treated animals. (A) Interleukins-2; (B) interleukins-4; (C) interleukins-6; (D) TNF-alpha. Data represent mean ± SEM. n = 6 for each group. * represents significant values when compared to controls at p < 0.05, # represents significant values when compared to SRP-treated animals at p < 0.05, ## represents significant values when compared to LPS-treated group at p < 0.05.

Figure 3

Aortic representative slices (5 μm-thick) stained with H&E. (A) Control group. (B) Endothelial cells in LPS-induced mice had significant damage, including disordered organization, indistinct borderlines, and partial deletion (arrow). (C) LPS + SRP-treated mice, who suffered less damage than LPS-treated mice. (D) LPS+AT-treated mice, who suffered less damage than LPS mice. Scale bars indicate 10 μm. (n = 6). Original magnifications—100×.

Figure 4

Immunohistochemistry analysis. MCP-1 expression of the heart ventricular sections from each mouse (20×, scale bar: 50 μm; n = 6 per group, at least 10 randomly selected heart sections per mouse). Data are presented as mean standard deviation. One-way ANOVA was applied.