Aqueous extract of Vitex trifolia L. (Labiatae) inhibits LPS-dependent regulation of inflammatory mediators in RAW 264.7 macrophages through inhibition of Nuclear Factor kappa B translocation and expression.
BACKGROUND
Vitex trifolia L. (Labiatae), a widespread tree found from the Asia-Pacific to the east Africa regions is used in the traditional medicine of the Pacific islands to treat inflammatory-associated conditions.


OBJECTIVE
We herein evaluated its in vitro regulatory effects on the expression profile of lipopolysaccharide (LPS)-induced inflammatory genes focusing on regulation of chemokines C-X-C motif 10 (CXCL-10) and C-C motif ligand 3 (CCL-3) and cyclo-oxygenase (COX)-2. Furthermore, the plant effect on the LPS-mediated activation of Nuclear Factor kappa B (NF-κB) was also studied.


METHODS
Aqueous extract of Vitex trifolia leaves was prepared and evaluated for its effect on LPS-induced stress and toxicity-related genes in RAW 264.7 macrophage cells using RT(2) Profiler Polymerase Chain Reaction (PCR) Array System. Effects of the extract on LPS-induced chemokines CCL-3 and CXCL-10, COX-2, and NF-κB p50 and p65 mRNA levels were also studied using Reverse Transcription quantitative PCR (RT-qPCR) technique. Translocation of the nuclear factor was further assessed by measuring its nuclear p65 subunit via an ELISA-based TransAM method.


RESULTS
Vitex trifolia extract at 5000μg/ml exerted a significant inhibitory effect on the expression of various LPS-induced inflammatory genes in RAW 264.7 cells after 8h of incubation time. Using RT-qPCR, this anti-inflammatory effect was further confirmed by significant inhibition of CCL-3 and CXCL-10 mRNA production in LPS-stimulated RAW 264.7 cells upon treatment with 2500μg/ml of Vitex trifolia extract. Furthermore, the inhibitory activity of this plant on LPS-induced COX-2 mRNA was also observed at a concentration of 2500μg/ml in a time-dependent manner. TransAM assays showed that LPS-induced NF-κB translocation was also inhibited by Vitex trifolia extract even at a concentration of extract as low as 250μg/ml. RT-qPCR assays showed that aqueous extract of Vitex trifolia leaves had a significant inhibitory activity on LPS-induced p50 mRNA synthesis. Interestingly, however, no effect on p65 subunit mRNA expression was observed. Moreover, PCR array analysis showed that LPS-induced inflammatory and apoptosis genes under NF-κB control are also repressed by the extract.


CONCLUSIONS
The anti-inflammatory properties of Vitex trifolia extract seem associated with inhibition of NF-κB translocation through a reduction in the expression level of NF-κB p50 but interestingly not p65 subunit mRNA. The regulatory effects of Vitex trifolia on NF-κB and consequently on inflammation mediators such as chemokines CCL-3 and CXCL-10, and COX-2 provide new evidence of its efficacy and emphasise its high potential therapeutic value. However, further in vivo experiments are still required to validate its utilization as a remedy against inflammatory diseases.
Institut Pasteur de Nouvelle-Calédonie, BP61, 98845 Noumea, New Caledonia.
Mariko
M
Matsui
Minou
M
Adib-Conquy
Agnès
A
Coste
Shilpa
S
Kumar-Roiné
Bernard
B
Pipy
Dominique
D
Laurent
Serge
S
Pauillac
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology / Q000627:therapeutic use*; D001692:Biological Transport / Q000187:drug effects; D054405:Chemokine CCL3 / Q000235:genetics / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D015870:Gene Expression / Q000187:drug effects; D007249:Inflammation / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008070:Lipopolysaccharides; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics; D052002:NF-kappa B p50 Subunit / Q000037:antagonists & inhibitors / Q000235:genetics; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000235:genetics; D020950:Vitex
D000893:Anti-Inflammatory Agents; C496534:Ccl3 protein, mouse; D054405:Chemokine CCL3; D054357:Chemokine CXCL10; D018925:Chemokines; C485433:Cxcl10 protein, mouse; D052246:Cyclooxygenase 2 Inhibitors; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D010936:Plant Extracts; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D051546:Cyclooxygenase 2