Primers used in Real time-PCR assays.

<p>Primers used in Real time-PCR assays.</p

Effect of MEPT leaves and Luteolin on HSV-2-induced MAPK, JNK1/2, NF-kB and IkBα activation.

<p>Expression of MAPK (A), JNK (B), NF-kB (C) and IkBα (D) was determined by Western blot, using GAPDH as the internal control. The HSV-2-infected peritoneal macrophage(s) were treated with MEPT (86.5 μg/ml) or Luteolin (40.2 μg/ml) and after 4 h, equal amounts of protein (40 μg/sample) extract from whole cells were harvested in buffer (200 μl/well) containing 50 nM Tris-Cl, 150 mM NaCl, 1% NP-40, 1% Triton X-100, and 1% protease inhibitor cocktail. The soluble fraction was separated by centrifugation, subjected to SDS-PAGE and blotted to pre-equilibrated PVDF membrane. The membrane was then blocked in 5% NFDM in 1X TBST, rinsed and incubated with specific antibody at 4°C overnight. Immunoblotting was performed with peroxidase-labelled specific antibodies and visualized by ECL Western blot detection kit. The average expression of NF-kB was significantly higher in the HSV-2-induced macrophage, as compared to the control and MEPT or luteolin co-treated group (*, P<0.05; **, P<0.001). Lane 1, cells only control; Lane 2, cells + HSV-2; Lane 3, cells + HSV-2 + MEPT leaves; Lane 4, cells + HSV-2 + luteolin.</p

<i>Pedilanthus tithymaloides</i> Inhibits HSV Infection by Modulating NF-κB Signaling

<div><p><i>Pedilanthus tithymaloides</i> (PT), a widely used ethnomedicinal plant, has been employed to treat a number of skin conditions. To extend its utility and to fully exploit its medicinal potential, we have evaluated the <i>in vitro</i> antiviral activity of a methanolic extract of PT leaves and its isolated compounds against Herpes Simplex Virus type 2 (HSV-2). Bioactivity-guided studies revealed that the extract and one of its constituents, luteolin, had potent antiviral activity against wild-type and clinical isolates of HSV-2 (EC₅₀ 48.5–52.6 and 22.4–27.5 μg/ml, respectively), with nearly complete inhibition at 86.5–101.8 and 40.2–49.6 μg/ml, respectively. The inhibitory effect was significant (p<0.001) when the drug was added 2 h prior to infection, and was effective up to 4 h post-infection. As viral replication requires NF-κB activation, we examined whether the observed extract-induced inhibition of HSV-2 was related to NF-κB inhibition. Interestingly, we observed that treatment of HSV-2-infected cells with extract or luteolin suppressed NF-κB activation. Although NF-κB, JNK and MAPK activation was compromised during HSV replication, neither the extract nor luteolin affected HSV-2-induced JNK1/2 and MAPK activation. Moreover, the PT leaf extract and luteolin potently down-regulated the expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, NO and iNOS and the production of gamma interferon (IFN-γ), which are directly involved in controlling the NF-κB signaling pathway. Thus, our results indicate that both PT leaf extract and luteolin modulate the NF-κB signaling pathway, resulting in the inhibition of HSV-2 replication.</p></div

Effect of MEPT leaves and Luteolin on pro-inflammatory cytokine release in HSV-2-infected peritoneal macrophages.

<p>Peritoneal macrophages were cultured overnight and infected with HSV-2, washed after 1 h and then treated with MEPT leaves (100 μg/ml) or Luteolin (20 μg/ml). The cells were further incubated for 24 h, and the cell-free supernatants were subjected to sandwich ELISA to determine the level of (A) TNF-α, (C) IL-1β, (E) IL-6 and (G) IFN-γ (pg/mL). The ELISA data are expressed as Mean ± SD from triplicate experiments, yielding similar results. Asterisks indicate statistically significant (*, P<0.05; **, P<0.001) induction of TNF-α, IL-1ß, IL-6, and IFN-γ release, compared to the infected macrophages.</p

Effect of MEPT leaves and Luteolin on NO production and iNOS expression in HSV-2 infected murine macrophages.

<p>(A) Macrophages (10⁶ cells/ml) were infected with HSV-2 treated with the MEPT leaves (100 μg/ml) or Luteolin (20 μg/ml), and incubated for 24 h. The supernatant was removed and the concentration of NO was determined by Griess reagent. Data are expressed as Mean ± SD from triplicate experiments, yielding similar results (m moles of nitrite). Asterisks indicate a statistically significant increase (*, P<0.05; **, P<0.001) in nitrite generation, compared to the infected macrophages. (B) HSV-2-infected macrophages were treated with the MEPT leaves or Luteolin, incubated for 12 h, following which RNA was isolated and subjected to RT–PCR analysis for the expression of iNOS2 mRNA. The data are expressed as Mean ± SD from triplicate experiments yielding similar results. The asterisk indicates a statistically significant increase (*, P<0.001) in iNOS2 expression, compared to the infected macrophage.</p

Chemical structure of the isolated compound(s) Luteolin (A) and Tetradecanediol (B) isolated from the antiviral fraction of <i>Pedilanthus tithymaloides</i> leave extracts.

<p>Chemical structure of the isolated compound(s) Luteolin (A) and Tetradecanediol (B) isolated from the antiviral fraction of <i>Pedilanthus tithymaloides</i> leave extracts.</p