Anti-Inflammatory Activity of <i>Odina wodier</i> Roxb, an Indian Folk Remedy, through Inhibition of Toll-Like Receptor 4 Signaling Pathway

<div><p>Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of <i>Odina wodier</i> (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular <i>in vivo</i> antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1β, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.</p></div

Effect of OWB extract and CA on the JNK, MAPK, IkB-α and MyD88 expression.

<p>Expression of (A) JNK, (B) MAPK, (C) IkB-α and (D) MyD88 were determined by the Western blot, using GAPDH as the internal control. The LPS (1 µg/ml) induced peritoneal macrophage(s) were treated with OWB (100 µg/ml) or CA (10 µg/ml), and after 24 h the protein extract from whole cell were harvested in buffer, containing 20 mM Tris (pH 7±0.5), 50 mM NaCl, 5% NP-40 and 0.05% DOC. 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 and MAPK was significantly higher in the LPS-induced macrophage, as compared to the control and OWB or CA co-treated group (** <i>P</i></p

Effect of OWB extract on Carrageenan- and Dextran- induced paw edema, and Cotton pellet-induced granuloma in rats.

<p>(A and C) Inflammation in the right hind paw of Wistar rats was made by subcutaneous injection of carrageenan or dextran respectively, under the sub plantar aponeurosis. The test groups were administered with 200 or 400 mg kg⁻¹ of OWB extract orally, 1 h before carrageenan or dextran injection; while the control group received distilled water or Indomethacin (10 mg/kg). After 6 h, the paw volume was measured and % of inhibition was compared with the control groups. (B) Photographs showing Carrageenan-induced Paw edema (CIPE) in the hind limb of rats, 6 h after carrageenan challenge. Redness and swelling of paw are evident with respect to control and OWB extract treatment. (D) Subcutaneously implemented sterile cotton-pellets (10 mg each) in the axilla regions of the rats, under anesthesia, were treated orally with the extract (200 or 400 mg kg⁻¹) daily for 7 consecutive days, with respect to the normal saline or Indomethacin (10 mg/kg). After scarification, on 8^th day, the cotton-pellets were removed, cleaned and the dry weight of each pellet was taken to calculate the percentage of inhibition with respect to the cotton-pellet weight, compared with the control. Results are expressed as Mean ± SD, (n = 6), *, <i>P</i>P</p

Effect of OWB extract and CA on pro-inflammatory and anti-inflammatory cytokine release in LPS-induced peritoneal macrophages by sandwich ELISA and RT-PCR.

<p>Peritoneal macrophages were cultured overnight and incubated with LPS (1 µg/ml), washed after 4 h and then treated with the OWB (50 or 100 µg/m) or CA (10 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) IL-6 (pg/mL). In a separate set similarly treated cells were cultured for 5 h, and collected in TRI Reagent for mRNA extraction and subsequent RT–PCR analysis (vide Materials and methods) to study the cytokine and β-actin mRNA expression. The data were shown for the expression of (B) TNF-α, (D) IL-1β, (F) IL-6 and (H) IL-12. The ELISA and RT–PCR data are expressed as Means ± SD from triplicate experiments, yielding similar results. Asterisks indicate statistically significant (**P, 0.05) induction of TNF-α, IL-1β, IL-6, and IL-12 release; and increase or decrease (**P, 0.05; *P, 0.001) in cytokine expression, compared to the infected macrophages.</p

Primer used in Real time-PCR assay.

<p>Primer used in Real time-PCR assay.</p

Immunomodulatory activity of OWB extract and CA via nitrite generation and iNOS2 expression in LPS-induced murine macrophages.

<p>(A) Macrophages (10⁶ cells/ml) were incubated with LPS (1 µg/ml), OWB (50 and 100 µg/ml) or CA (10 µg/ml) for 24 h. The cell-free supernatants were collected for nitrite assay, as described in the Materials and methods. Data were expressed as Means ± SD from triplicate experiments, yielding similar results (µ moles of nitrite). Asterisks indicate a statistically significant increase (**P, 0.05) in nitrite generation, compared to the infected macrophages. (B–C) The LPS-stimulated macrophages were treated with the OWB extract and CA, incubated for 24 h, following which RNA was isolated and subjected to the RT–PCR analysis for the expression of iNOS2 mRNA. The data were 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 macrophages.</p

Effect of OWB extract and CA on TLR4 and MyD88 expression.

<p>The LPS-stimulated macrophages were treated with the OWB or CA and incubated for 24 h, following which RNA was isolated for RT–PCR analysis of the expression of MyD88 (A) and TLR4 (B) mRNA. The RT–PCR data are expressed as Means ± SD from triplicate experiments. The expression of TLR4 and MyD88 was significantly higher in the LPS-induced macrophage as compared to the control and OWB or CA co-treated group (** <i>P</i></p

Effect of OWB extract and CA on protein denaturation.

<p>Each value represents the Mean ± SD; n = 6) * p<0.001** p<0.01.</p

<i>In</i><i>vivo</i> efficacy of

<p>HM. BALB/c mice were fed with HM (0.25 or 0.5 mg/kg) or ACV (5 mg/kg) and after 8 h of drug treatment the animals were infected with HSV-2G (9 X 10⁵ pfu per animal) intravaginally. The challenged animals of test groups were fed with HM twice daily for 7 days. Development of lesions and death were observed three times daily, while brain and vaginal tissue were collected after sacrification on days 2, 4, 6 or 8 after infection, homogenized and centrifuged. The supernatant was used for the determination of virus yield by plaque assay. Mean lesion score [A], Mean±S.D. of virus yield at log₁₀ (PFU/organ) in vaginal tissue [B] and brain [C]. </p

Anti-HSV efficacy of HM.

<p>[A] <b>Plaque </b><b>Reduction </b><b>Assay</b>. Infected cells were treated with HM or ACV at 0.5-50 μg/ml, overlaid with methylcellulose and plaques developed after 2-3 days were stained. The % of plaque number reduction was calculated, and the effective concentration of drug that inhibited the number of viral plaques was interpolated from the dose-response curve. [B] <b>Time </b><b>course </b><b>analysis</b>. Inhibitory effects of HM and ACV at various time points prior to infection (-3 to -1 h), at the time of infection (0 h) and post-infection (2-24 h) with HSV-2 were evaluated by plaque reduction assay. Each bar represents the mean ± S.E.M of three independent experiments.</p