Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis

Abstract

Rosmarinic acid (RA) reduced the mortality of mice infected with Japanese encephalitis virus (JEV). Significant decreases in viral loads (P < 0.001) and proinflammatory cytokine levels (P < 0.001) were observed in JEV-infected animals treated with RA compared to levels in infected mice without treatment, at 8 to 9 days postinfection.

FIG. 1.

RA treatment significantly increases the survival of JEV-infected mice. (A) Survival of mice infected with 3 × 10⁵ PFU of JEV was significantly increased in groups that received RA treatment (15 mice for each group). Treatment with RA alone has no significant effect on survival. Observation of animal survival experiments was performed in a masked manner to avoid bias toward any one group of animals. (B to E) Cryostat sections from control (B), JEV-infected (C), and JEV-infected and RA-treated (D) mouse brains were processed for Iba-1. While the control sections exhibited only resting microglia (B; arrow), the infected brains showed the presence of activated microglia (C; arrowheads). The group of JEV-infected and RA-treated mice had mostly resting microglia (D; arrow). Scale bar, 25 μm. (E) Iba-1-positive activated microglia were counted and plotted as a graph. Values represent the means ± standard errors of the means (SEM) from five random fields in three animals in each group (P < 0.001).

FIG. 2.

Antiviral efficacy of RA. C, control mice; I, JEV-infected mice; RA, JEV-infected and RA-treated mice; RA*, JEV-infected and RA-treated mice (dead). RA treatment in JEV-infected mice completely reduced the levels of viral proteins. Proteins isolated from control, JEV-infected, and JEV-infected and RA-treated BALB/c mice were analyzed by immunoblot analysis. A significant decrease in the levels of JEV-specific proteins (84 kDa and 17 kDa) was observed in RA-treated samples compared to levels in infected mice. Data shown are representative of four individual animals from each group.

FIG. 3.

RA abrogates the increased expression of proinflammatory mediators. (A) Expression of IL-12, TNF-α, IFN-γ, MCP-1, and IL-6 was observed by CBA in control animals, JEV-infected animals, JEV-infected and RA-treated animals, animals treated with RA alone, and JEV-infected and RA-treated animals at death. Levels of IL-12, TNF-α, IFN-γ, MCP-1, and IL-6 were significantly reduced in RA-treated samples compared to levels in infected mice. P < 0.001 (mean ± SEM). JEV-infected mice that succumbed even after RA treatment had significantly high levels of proinflammatory cytokines compared to uninfected mice. P < 0.001 (mean ± SEM) (four mice for each group). (B) Protein levels of control, JEV-infected, JEV-infected and RA-treated, RA-treated, and JEV-infected and RA-treated animals at death were analyzed by immunoblotting. Significant reductions in the levels of pNF-κB and Cox-2 in RA-treated samples were observed compared to levels in infected samples (P < 0.05). RA treatment reversed the level of IκB-α (P < 0.05). Mice that died even after RA treatment had increased levels of pNF-κB and Cox-2 and decreased levels of IκB-α compared to the control group. Data shown are for two individual animals from a total of four animals in each group.

FIG. 4.

RA decreases the proinflammatory mediators in vitro. Mouse microglial cell line BV-2 was used to study the in vitro induction of proinflammatory cytokines and a chemokine following JEV infection. There were significant reductions in the levels of IL-6, MCP-1, IL-12, IFN-γ, and TNF-α in JEV-infected and RA-treated samples compared to levels in JEV-infected samples. Values represent the means ± SEM from three independent experiments performed in duplicate (P < 0.05).