Targeting MAPK/NF-κB Pathways in Anti-Inflammatory Potential of Rutaecarpine: Impact on Src/FAK-Mediated Macrophage Migration

Studies have discovered that different extracts of Evodia rutaecarpa and its phytochemicals show a variety of biological activities associated with inflammation. Although rutaecarpine, an alkaloid isolated from the unripe fruit of E. rutaecarpa, has been exposed to have anti-inflammatory properties, the mechanism of action has not been well studied. Thus, this study investigated the molecular mechanisms of rutaecarpine (RUT) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. RUT reserved the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL)-1β in the LPS-induced macrophages. RUT showed an inhibitory effect on the mitogen-activated protein kinases (MAPKs), and it also inhibited nuclear transcription factor kappa-B (NF-κB) by hindering IκBα and NF-κB p65 phosphorylation and p65 nuclear translocation. The phospho-PI3K and Akt was concentration-dependently suppressed by RUT. However, RUT not only suggestively reduced the migratory ability of macrophages and their numbers induced by LPS but also inhibited the phospho-Src, and FAK. Taken together, these results indicate that RUT participates a vital role in the inhibition of LPS-induced inflammatory processes in RAW 264.7 macrophages and that the mechanisms involve PI3K/Akt and MAPK-mediated downregulation of NF-κB signaling pathways. Notably, reducing the migration and number of cells induced by LPS via inhibiting of Src/FAK pathway was also included to the anti-inflammatory mechanism of RUT. Therefore, RUT may have potential benefits as a therapeutic agent against chronic inflammatory diseases.

Anti-Inflammatory Effects of Phlebia sp. Extract in Lipopolysaccharide-stimulated RAW 264.7 Macrophages

Lichens are a life form in which algae and fungi have a symbiotic relationship. A lichen has various biological activities, including anti-inflammatory and anti-proliferative activities. Inflammation is a response caused by various factors, such as infection by pathogens or tissue damage; excessive reactions can contribute to the etiology of chronic diseases, such as asthma, brain damage, and serious tissue damage. This study demonstrates the anti-inflammatory effect of ethyl acetate extract from Phlebia sp. on NF-κB and AP-1 pathways in the lipopolysaccharide-treated RAW 264.7 cell. Especially, Phlebia sp. extract inhibits the phosphorylation of AP-1 signaling (c-Fos and c-Jun) and its upstream MKK/MAPKs (MKK4, MKK7 and JNK), which induced a decrease in the production of the inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in downstream of AP-1 signaling. Furthermore, Phlebia sp. extract inhibited the production of final inflammatory effector molecules involved in AP-1 signaling, including nitric oxide (NO) and prostaglandin E2 (PGE2). Here, we report that Phlebia sp. extract has the potential to be developed as an anti-inflammatory agent.

Cyclophilin A Impairs Efferocytosis and Accelerates Atherosclerosis by Overexpressing CD 47 and Down-Regulating Calreticulin

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don’t-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Immunomodulatory and Antioxidant Potential of Biogenic Functionalized Polymeric Nutmeg Oil/Polyurethane/ZnO Bionanocomposite

The current study is focused on the biosynthesis of nutmeg oil/ polyurethane/ZnONPs bionanocomposite film for immunomodulatory and antioxidant activities. The fabricated film was prepared by using naturally extracted nutmeg oil functionalized with ZnONPs in the presence of polyutherane (PU) medium. The bionanocomposite film was obtained by incorporating dropwise 10 % (w/v) of nutmeg oil to the PU solution/ZnONPs blend. The active constituents of nutmeg oil were determined by gas chromatography coupled with mass spectrometry (GC-MS). The morphological characteristics of the resulting bionanocomposite film were confirmed using various microscopic and spectroscopic methods. Immunomodulatory potential of bionanocomposite was evaluated for RAW 264.7 macrophages. The results exhibited an excellent reduction in inflammatory cytokines (IL-6, IL-10, and TNFα) secretions after the treatment with bionanocomposite. The bionanocomposite exerted the highest inhibitory effects on certain cell signaling constituents that influence the initiation of expression of proinflammatory cytokines. The bionanocomposite was also tested for DPPH and ABTS free radicals scavenging assays and showed excellent antioxidant potential with IC50 values (0.28 ± 0.22 and 0.49 ± 0.36), respectively. The outcomes suggested promising immunomodulatory and antioxidant potentials for the biogenic synthesized nutmeg oil/PU/ZnONPs polymeric bionanocomposite.

Dracocephalum moldavica Ethanol Extract Suppresses LPS-Induced Inflammatory Responses through Inhibition of the JNK/ERK/NF-κB Signaling Pathway and IL-6 Production in RAW 264.7 Macrophages and in Endotoxic-Treated Mice

The excessive synthesis of interleukin-6 (IL-6) is related to cytokine storm in COVID-19 patients. Moreover, blocking IL-6 has been suggested as a treatment strategy for inflammatory diseases such as sepsis. Sepsis is a severe systemic inflammatory response syndrome with high mortality. In the present study, we investigated the anti-inflammatory and anti-septic effects and the underlying mechanisms of Dracocephalum moldavica ethanol extract (DMEE) on lipopolysaccharide (LPS)-induced inflammatory stimulation in RAW 264.7 macrophages along with septic mouse models. We found that DMEE suppressed the release of inflammatory mediators NO and PGE2 and inhibited both the mRNA and protein expression levels of iNOS and COX-2, respectively. In addition, DMEE reduced the release of proinflammatory cytokines, mainly IL-6 and IL-1β, in RAW 264.7 cells by inhibiting the phosphorylation of JNK, ERK and p65. Furthermore, treatment with DMEE increased the survival rate and decreased the level of IL-6 in plasma in LPS-induced septic shock mice. Our findings suggest that DMEE elicits an anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophages and an anti-septic effect on septic mouse model through the inhibition of the ERK/JNK/NF-κB signaling cascades and production of IL-6.

Tocotrienol-rich Fraction Modulated Genes Responsible for Inflammation in Lipopolysaccharide-stimulated RAW 264.7 Macrophages

Background: Inflammation plays a vital role in the pathogenesis of chronic non-communicable diseases (NCDs), the leading health issue worldwide. An earlier study reported that tocotrienol-rich fraction (TRF) showed better anti-inflammation effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Aim: This study aimed to investigate the anti-inflammatory effects of tocotrienol-rich fraction at the molecular level by looking at the genes that were differentially regulated and pathways affected in LPS-stimulated macrophages exposed to TRF using the microarray approach. Methods: A microarray study was carried out in LPS-stimulated RAW 264.7 macrophages. Total ribonucleic acid (RNA) was extracted from the RAW 264.7 cells treated with TRF (10µg/mL), alpha-tocopherol (10 µg/mL) or LPS (10 ng/mL). Untreated cells served as control. Enrichment analyses, such as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), were conducted for genes listed in the differentially expressed genes (DEGs). Results: The microarray analysis showed that the expression of five genes [Hamp, Interleukin-1a (IL-1a), IL-b, C-X-C motif chemokine ligand 2 (CXCL2) and colony-stimulating factor 3 (CSF3)] and one gene (SLC1A4), an amino acid transporter, was modulated (fold change 2, P< 0.05) in the TRF-treated cells. With a more stringent analysis (fold change 3, P < 0.05), only one gene (CSF3) was downregulated in the TRF-treated in RAW 264.7 cells. Analysis using the GO and KEGG pathways revealed interactions between pro-inflammatory agents such as tumor necrosis factor-alpha (TNFα) and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-B), as well as signaling pathways of interleukin (IL)-1 and IL-17. Conclusion: TRF modulated the expression of genes responsible for acute and chronic inflammation that were part of the lipoxygenase (LOX) and cyclooxygenase (COX) inflammatory pathways. Further investigation on the effects of TRF in different cell lines and in vivo studies should be conducted in the future.

Triterpenoids From Kadsura coccinea With Their Anti-inflammatory and Inhibited Proliferation of Rheumatoid Arthritis-Fibroblastoid Synovial Cells Activities

One new 3,4-seco-17,13-friedo-lanostane triterpenoid heilaohuacid A (1), one new 3,4-seco-17,14-friedo-lanostane triterpenoid heilaohuacid B (2), five new 3,4-seco-lanostane triterpenoids heilaohuacids C-D (3–4) and heilaohumethylesters A-C (7–9), one new 3,4-seco-cycloartane triterpenoid heilaohuacid E (5), and one new intact-lanostane triterpenoid heilaohuacid F (6), together with twenty-two known analogues (10–31), were isolated from heilaohu. Their structures were determined using HR-ESI-MS data, 1D and 2D NMR spectra, 13C NMR calculations, and electronic circular dichroism (ECD) calculations. Heilaohuacids A and B (1 and 2) contain a 3,4-seco ring A and unprecedented migration of Me-18 from C-13 to C-17 or C-14 to C-18. This type of lanostane triterpenoid derivatives was rarely reported so far. More importantly, all compounds against inflammatory cytokines IL-6 and TNF-α levels on LPS-induced RAW 264.7 macrophages were evaluated, and compounds 4 and 31 significantly inhibited the release level of IL-6 with IC50 values of 8.15 and 9.86 μM, respectively. Meanwhile, compounds 17, 18, and 31 significantly inhibited proliferation of rheumatoid arthritis-fibroblastoid synovial (RA-FLS) cells in vitro with IC50 values of 7.52, 8.85, and 7.97 μM, respectively.