Protium javanicum Burm. Methanol Extract Attenuates LPS-Induced Inflammatory Activities in Macrophage-Like RAW264.7 Cells

Abstract

Protium javanicum Burm. f. is a medicinal plant used in traditional medicine. Gum and oleoresins from this plant have been used as anti-inflammatory agents for treating ulcers, headaches, eyelid inflammation, and rheumatic pain. However, its anti-inflammatory mechanism of action is still unknown. To better understand the mechanism, we used lipopolysaccharide- (LPS-) treated RAW264.7 cells to measure inflammatory mediators with the Griess assay and to identify target signaling molecules by immunoblot analysis. In this study, we report that the Protium javanicum methanol extract (Pj-ME) plays an important role in suppressing nitric oxide (NO) levels without cytotoxicity. The effect of Pj-ME in LPS-induced expression leads to reduced inflammatory cytokine expression, specifically inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and tumor necrosis factor (TNF-α). Pj-ME significantly inhibited LPS-induced protein expression of the nuclear factor-kappa B (NF-κB) signaling pathway in a time-dependent manner. Syk and Src were identified as putative signaling molecules of Pj-ME-mediated anti-inflammatory activity, which were inhibited by Pj-ME. We demonstrated that Pj-ME controls the STAT3 signaling pathway by suppressing STAT3 and JAK phosphorylation and also downregulates the gene expression of IL-6. Therefore, these results elucidate Pj-ME as a novel anti-inflammatory naturally derived drug with anti-inflammatory and antioxidant properties which may be subject to therapeutic and prognostic relevance.

Figure 1

Effects of Pj-ME on NO production in LPS-activated macrophages. ((a) and (d)) Murine macrophage-like RAW264.7 cells or peritoneal macrophages pretreated with Pj-ME (0-200 μg/ml) or L-NAME (0-1 mM) for 30 min and then treated with LPS (1 μg/ml) for 24 h. LPS-induced NO production levels were determined by the Griess assay. ((b) and (e)) To evaluate the cytotoxic activity of Pj-ME or L-NAME, RAW264.7, and HEK293 cells, and peritoneal macrophages were treated with Pj-ME (0-200 μg/ml) and L-NAME (0-1.5 mM) for 24 h. Cell viability was then determined by the MTT assay. (c) Phytochemical fingerprinting was performed by LC/MS spectrophotometric analysis. Putative components were included in each peak. Data ((a), (b), (d), and (e)) expressed as mean ± SD are representative of 3 independent experiments. ^##: p< 0.01 with respect to the untreated group;  ^∗p< 0.05 and  ^∗∗p< 0.01 with respect to the LPS-treated group.

Figure 2

Effect of Pj-ME on inflammatory gene expression. ((a) and (b)) Semiquantitative RT-and real-time PCR analysis was carried out to detect mRNA expression levels of inflammatory genes iNOS, COX-2, and TNF-α in RAW264.7 cells pretreated with Pj-ME (50 to 200 μg/ml) for 30 min followed by LPS exposure for 6 h. Data (b) expressed as mean ± SD are representative of 3 independent experiments.  ^∗∗p< 0.01 with respect to the LPS-treated group.

Figure 3

Effect of Pj-ME on the NF-κB and its upstream signaling cascade in LPS-stimulated RAW264.7 cells. ((a) left panel, (a) right panel and (c)) Western blot analysis was performed to detect protein expression levels in whole cell lysates or nuclear extracts from RAW264.7 cells treated with Pj-ME (200 μg/ml) for 30 min followed by LPS exposure (1 μg/ml) over various lengths of incubation times. Levels of phosphorylated and total p85, IKKα/β, IκBα, p50, and p65 at 5, 15, 30, and 60 min, and Syk and Src levels at 2, 3, and 5 min were determined. β-Actin was used as a loading control. (b) HEK293 cells cotransfected with NF-κB-Luc (1 μg/ml) and β-gal (as transfection control) plasmid constructs were treated with Pj-ME in the presence or absence of the adaptor molecule MyD88 (1 μg/ml). Luciferase activity was measured by using luminescence. ((d) and (e)) Inhibitory activity of Pj-ME (100 and 200 μg/ml) on autophosphorylation of Syk and Src overexpressed in HEK293 cells was determined by Western blot analysis with antibodies specific to phospho-Src or phospho-Syk. Data (b) expressed as mean± SD are representative of 3 independent experiments. ^##p< 0.01 with respect to untreated group and  ^∗∗p< 0.01 with respect to treated group.

Figure 4

Effect of Pj-ME on the upstream JAK/STAT3 signaling cascade in LPS-stimulated RAW264.7 cells. (a) Western blot analysis was performed to determine protein expression levels in whole cell lysates of RAW264.7 cells treated with Pj-ME (200 μg/ml) for 30 min followed by LPS treatment (1 μg/ml) over different amounts of time. Levels of phosphorylated and total STAT3 and JAK2 at 6, 9, 12, and 24 h were determined with their specific antibodies. β-Actin was used as a loading control. (b) STAT3-specific expression of IL-6 was determined by real-time PCR from LPS-treated RAW264.7 cells. Data (b) expressed as mean± SD are representative of 3 independent experiments. ^##p< 0.01 with respect to untreated group and  ^∗∗p< 0.01 with respect to treated group.

Figure 5

Putative suppressive pathway of Pj-ME in displaying its anti-inflammatory response. It is considered that Pj-ME targets the activation of protein tyrosine kinases such as JAK and Src and Syk linked to the activation of intracellular signaling pathway for the nuclear translocation of NF-κBand STAT3. Suppression of this pathway leads to the downregulation of iNOS-mediated NO production and the expression of other cytokines such as NO and IL-6.