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. 2018 Aug 1:2018:3430684.
doi: 10.1155/2018/3430684. eCollection 2018.

Suppression of Proinflammatory Cytokines and Mediators in LPS-Induced RAW 264.7 Macrophages by Stem Extract of Alternanthera sessilis via the Inhibition of the NF- κ B Pathway

Katyakyini Muniandy  1 , Sivapragasam Gothai  1 , Khaleel M H Badran  1 , S Suresh Kumar  2 , Norhaizan Mohd Esa  3 , Palanisamy Arulselvan  1   4   5
Affiliations

Suppression of Proinflammatory Cytokines and Mediators in LPS-Induced RAW 264.7 Macrophages by Stem Extract of Alternanthera sessilis via the Inhibition of the NF- κ B Pathway

Katyakyini Muniandy et al. J Immunol Res. .

Abstract

Alternanthera sessilis, an edible succulent herb, has been widely used as herbal drug in many regions around the globe. Inflammation is a natural process of the innate immune system, accompanied with the increase in the level of proinflammatory mediators, for example, nitric oxide (NO) and prostaglandin (PGE2); cytokines such as interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor alpha (TNFα); and enzymes including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) via the activation and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) subunit p65 due to the phosphorylation of inhibitory protein, IκBα. Inflammation over a short period of time is essential for its therapeutic effect. However, prolonged inflammation can be detrimental as it is related to many chronic diseases such as delayed wound healing, cardiovascular disease, arthritis, and autoimmune disorders. Therefore, ways to curb chronic inflammation have been extensively investigated. In line with that, in this present study, we attempted to study the suppression activity of the proinflammatory cytokines and mediators as a characteristic of anti-inflammatory action, by using stem extract of A. sessilis in the lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophage cell line. The results showed that the extract has significantly inhibited the production of the proinflammatory mediators including NO and PGE2; cytokines comprising IL-6, IL-1β, and TNFα; and enzymes covering the iNOS and COX-2 by preventing the IκBα from being degraded, to inhibit the nuclear translocation of NF-κB subunit p65 in order to hinder the inflammatory pathway activation. These results indicated that the stem extract of A. sessilis could be an effective candidate for ameliorating inflammatory-associated complications.

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Figures

Figure 1
Figure 1
Effect of stem extract of A. sessilis on the cell viability on the RAW macrophage cell line. The cell viability was expressed as the percentage compared with the untreated (control) cell group. The error bars were added into the plot to signify the standard deviation between triplicates.
Figure 2
Figure 2
The effect of stem extract of A. sessilis on NO production in LPS-stimulated RAW 264.7 macrophage cells. The nitric oxide level was studied by observing the nitrite level production by Griess reagents. The values of nitrite production were expressed in μM, and the data presented were the mean values of three experiments ± S.D. ###p < 0.001, the LPS-treated group versus the control group; ∗∗∗p < 0.001, the treated group significantly different from the LPS-treated group.
Figure 3
Figure 3
The effect of stem extract of A. sessilis on prostaglandin E2 (PGE2) (a), interleukin IL-6 (b), IL-1β (c), and tumor necrosis factor-α (TNFα) (d) in LPS-stimulated RAW 264.7 macrophage cells. The values of cytokine production were expressed in pg/mL, and the data presented were the mean values of three experiments ± S.D. ###p < 0.001, the LPS-treated group versus the control group; ∗∗∗p < 0.001 and ∗∗p < 0.01, the treated group significantly different from the LPS-treated group.
Figure 4
Figure 4
Effect stem extract of A. sessilis on the nuclear translocation of the unit of NF-κB in LPS-stimulated RAW264.7 macrophages. The treated cells were fixed and processed for immunostaining with specific antibodies. The nucleus was stained with Hoechst dye (blue color), and NF-κB subunit p65 (red color) was observed with a fluorescent microscope with the magnification of images, ×600.
Figure 5
Figure 5
Effect of stem extract of A. sessilis on the protein expression of inflammatory enzymes, cyclooxygenase- (COX-) 2, inducible nitric oxide synthase (iNOS), inflammatory regulator, nuclear factor NF-κB p65, and inhibitor of κB (IκBα) on the macrophage induced with LPS and treated with various concentrations of A. sessilis stem extract (50, 100, and 200 μg/mL) for 24 h. Protein was extracted from the cells and furthered to electrophoresis, and protein expression was detected by Western blots according to its appropriate antibodies. The experiments were carried out in triplicate, and images shown are representatives of triplicates.
Figure 6
Figure 6
Liquid chromatogram for stem extract of A. sessilis.
Figure 7
Figure 7
Image above shows the NF-κB inflammatory signaling pathway. The stem extract of A. sessilis has inhibited the phosphorylation, ubiquitination, and degradation of the inhibitory protein, IκBα complex. Hence, the NF-κB subunit p65 stayed intact with the inhibitory protein, stopping the cytoplasm-residing NF-κB subunit p65 from being translocated to the nucleus to activate the proinflammatory gene expression.
See this image and copyright information in PMC

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