METHODS: The antioxidant and anti-inflammatory activity of DE'RAAQSIN was assessed by measuring the levels of ROS and nitric oxide (NO) produced, using the DCF-DA assay and the Griess reagent assay, respectively. The molecular pathways activated by DE'RAAQSIN were investigated via qPCR.
RESULTS: LPS stimulation of RAW264.7 cells increased the production of nitric oxide (NO) and ROS and resulted in the overexpression of the inducible nitric oxide synthase (iNOS) gene. Furthermore, LPS induced the upregulation of the expression of key proinflammatory genes (IL-6, TNF-α, IL-1β, and CXCL1) and of the antioxidant gene heme oxygenase-1 (HO-1). DE'RAAQSIN demonstrated potent antioxidant and anti-inflammatory activity by significantly reducing the levels of ROS and of secreted NO, simultaneously counteracting the LPS-induced overexpression of iNOS, IL-6, TNF-α, IL-1β, and HO-1. These findings were corroborated by in silico activity prediction and physicochemical analysis of the main agarwood oil components.
CONCLUSIONS: We propose DE'RAAQSIN as a promising alternative managing inflammatory disorders, opening the platform for further studies aimed at understanding the effectiveness of DE'RAAQSIN.
AIM OF THE STUDY: Phytochemical investigation and assessment of pharmacological mechanism(s) involved in anti-ulcer effect of methanolic extract of the seeds of E. conferta.
MATERIALS AND METHODS: Bioactive phytoconstituents were isolated by column chromatography. These were identified by spectroscopic techniques including infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and mass spectrometry. Methanolic extract (MEC) of the seeds was prepared by cold maceration and its anti-ulcerogenic potential was evaluated using indomethacin (50 mg/kg) and water immersion stress models in male rats. The animals were pre-treated with different doses of MEC (400 and 800 mg/kg) and the therapeutic effect was compared with standard drug i.e. ranitidine (RANT; 50 mg/kg). The ameliorative effects of MEC were investigated on gastric juice pH, total acidity, free acidity and ulcer index. The assays of malionaldehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and pro-inflammatory cytokines i.e. interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were carried out to find out the possible mechanism(s) of protection. Further, histopathological changes were also studied.
RESULTS: Chromatography studies and further confirmation by spectroscopic techniques revealed the presence of four different compounds in MEC i.e oleic acid (1), stearic acid (2), ascorbic acid (3) and quercetin (4). MEC exhibited anti-ulcerogenic effect in dose dependent manner which may be attributed to suppression of pro-inflammatory cytokines (IL-6, TNF-α) and MDA (112.7%), and up-regulation of protective factors such as CAT (90.48%), SOD (92.77%) and GSH (90.01%). Ulcer inhibition, reduction in total and free acidity and increase in gastric juice pH were observed in MEC treated rats as compared to disease control animals. Histopathological findings confirmed decreased cell infiltration, less epithelial cell damage and regeneration of gastric mucosa in dose dependent manner.
CONCLUSIONS: The anti-ulcer effect of MEC may be attributed to its ability to scavenge free radicals and anti-inflammatory property via suppression of TNF-α and IL-6, thus offers a complete and holistic approach for management of peptic ulcer.
AIM OF THE STUDY: As allergy could be mediated by both IgE and IgG, we further evaluated the anti-allergy potential of CNAE in both in vitro model of IgG-induced macrophage activation and in vivo anaphylaxis models to further dissect the mechanism of action underlying the anti-allergic properties of CNAE.
MATERIAL & METHODS: The anti-allergy potential of CNAE was evaluated in in vivo anaphylaxis models of ovalbumin-challenged active systemic anaphylaxis (OVA-ASA) and IgE-challenged passive systemic anaphylaxis (PSA) using Sprague Dawley rats as well as IgG-challenged passive systemic anaphylaxis (IgG-PSA) using C57BL/6 mice. Meanwhile, in vitro model of IgG-induced macrophage activation model was performed using IC-21 macrophages. The release of soluble mediators from both IgE and IgG-mediated pathways were measured using enzyme-linked immunosorbent assay (ELISA). The signaling molecules targeted by CNAE were identified by performing Western blot.
RESULTS: IgG, platelet-activating factor (PAF) and IL-6 was suppressed by CNAE in OVA-ASA, but not IgE. In addition, CNAE significantly suppressed PAF and IL-6 in IgG-PSA but did not suppress histamine, IL-4 and leukotrienes C4 (LTC4) in IgE-PSA. CNAE also inhibited IL-6 and TNF-α by inhibiting the phosphorylation of ERK1/2 in the IgG-induced macrophage activation model.
CONCLUSION: Overall, our findings supported that CNAE exerts its anti-allergic properties by suppressing the IgG pathway and its mediators by inhibiting ERK1/2 phosphorylation, thus providing scientific evidence supporting its traditional use in managing allergy.
AIM OF THE STUDY: This study explores the anti-inflammatory effect of TPTQ in silico, in vitro, and in vivo.
MATERIALS AND METHODS: In silico testing used the Gnina application, opened via Google Colab. The TPTQ structure was docked with the nuclear factor kappa B (NF-ĸB) protein (PDB: 2RAM). In vitro testing began with testing the cytotoxicity of TPTQ against Raw 264.7 cells, using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method. A phagocytic activity test was carried out using the neutral red uptake method, and interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) secretion tests were carried out using the enzyme-linked immunosorbent assay (ELISA) method. In vivo, tests were carried out on mice by determining cluster of differentiation 8+ (CD8+), natural killer cell (NK cell), and IL-6 parameters, using the ELISA method.
RESULTS: TPTQ has a lower binding energy than the native ligand and occupies the same active site as the native ligand. TPTQ decreased the phagocytosis index and secretion of IL-6 and TNF-α experimentally in vitro. TPTQ showed significant downregulation of CD8+ and slightly decreased NK cells and IL-6 secretion in vivo.
CONCLUSION: The potent inhibitory effect of TPTQ on the immune response suggests that TPTQ can be developed as an anti-inflammatory agent, especially in the treatment of Covid-19.
METHODS: Interleukin (IL)-6 cytokine production in histamine-induced HaCaT cells were measured using enzyme-linked immunosorbent assay (ELISA) and cytotoxicity effects were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the inhibitory effects of MS65 on nuclear factor-kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways.
RESULTS: Histamine enhanced IL-6 production in HaCaT cells, with the highest production of IL-6 at 97.41 ± 2.33 pg/mL after 24 h of exposure. MS65 demonstrated a promising anti-inflammatory activity by inhibiting IL-6 production with half maximal inhibitory concentration (IC50) value of 4.91 ± 2.50 μM and median lethal concentration (LC50) value of 28.82 ± 7.56 μM. In gene expression level, we found that MS65 inhibits NF-κB and MAPK pathways through suppression of IKK/IκB/NFκB and c-Raf/MEK/ERK inflammatory cascades.
CONCLUSION: Taken together, our results suggest that MS65 could be used as a lead compound on developing new medicinal agent for the treatment of allergic skin diseases.
METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer.
RESULTS: The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts.
CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.
OBJECTIVE: This study aimed to investigate the immuno-modulatory effects of agarwood leaf extract (ALE) derived from Aquilaria malaccensis using RAW264.7 murine macrophages.
METHODS: In this study, RAW264.7 macrophages were incubated with ALE alone for 26 hours or ALE for 2 hours, followed by bacterial lipopolysaccharide for 24 hours. The nitrite and cytokine production (tumour necrosis factor-alpha (TNFα), interleukin (IL)-1β, IL-6, and IL-10), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) expression in the macrophages were assayed.
RESULTS: The study showed that ALE alone was immunostimulatory on the macrophages by increasing the nitrite, TNFα, and IL-6 production and COX2 expression (p<0.05 vs. untreated unstimulated cells). Pre-treatment of ALE suppressed nitrite level and iNOS expression but enhanced TNFα and IL-6 production and COX2 expression (p<0.05 vs. untreated lipopolysaccharides (LPS)-stimulated cells). ALE also increased IL-10 production regardless of LPS stimulation (p<0.05 vs. untreated cells).
CONCLUSION: ALE was able to promote the immune response of macrophages by upregulating pro-inflammatory cytokine levels and COX2 expression. It also regulated the extent of the inflammation by reducing iNOS expression and increasing IL-10 levels. Thus, ALE may have a role in enhancing the innate immune system against infection; however, its validation from in vivo studies is still pending.