Molecular Mechanism Of Anti-inflammatory Activity Research Articles

In vitro, in vivo and in silico evaluation of the anti-inflammatory potential of Hyssopus officinalis L. subsp. aristatus (Godr.) Nyman (Lamiaceae)

Ethnopharmacological relevanceMedicinal properties of hyssop have been used in traditional medicine since ancient times, inter alia, in diseases/conditions with an inherent inflammatory process.Aim of the study: Accordingly, the aim of this study was to investigate the anti-inflammatory properties of hyssop herb preparations (essential oil and methanol extracts) in vivo, in vitro and in silico. Materials and methodsFor in vitro testing of essential oils and extracts of hyssop herb, the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme assays were used. In vivo anti-inflammatory potential of the extracts (at doses of 50, 100 and 200 mg/kg) was assessed using the carrageenan-induced rat paw edema test. Molecular docking and dynamics were used for in silico testing of the inhibitory activity of chlorogenic (CA) and rosmarinic (RA) acids, as the dominant compounds in the tested methanol extracts against COX-1 and COX-2 enzymes. ResultsSignificant inhibitory activity was shown in the COX-2 test regarding extracts (essential oils did not exhibit any significant activity). Namely, all analyzed extracts, at a concentration of 20 μg/mL, showed a percentage of inhibition of COX-2 enzyme (54.04–63.04%), which did not indicate a statistically significant difference from the positive control of celecoxib (61.60%) at a concentration of 8.8 μM. In vivo testing showed that all methanol extracts of hyssop herb, at the highest test dose of 200 mg/kg in the third and fourth hours, after carrageenan administration, exhibited a statistically significant (p < 0.05) inhibitory effect on the increase in rat paw edema in relation to control. This activity is comparable or higher in relation to the reference substance, indomethacin, at a concentration of 8 mg/kg. The preliminary in silico results suggest that investigated compounds (RA and CA) showed better inhibitory activity against COX-1 and COX-2 than standard non-steroidal anti-inflammatory drug (NSAID), ibuprofen, as evident from the free binding energy (ΔGbind in kJ mol−1). The binding energies of the docked compounds to COX-1 and -2 were found to be in the range between −47.4 and −49.2 kJ mol−1. Ibuprofen, as the one NSAID, for the same receptors targets, showed remarkably higher binding energy (ΔGbind = −31.3 kJ mol−1 to COX-1, and ΔGbind = −30.9 kJ mol−1 to COX-2). ConclusionThe results obtained not only support the traditional use of hyssop herb in inflammatory conditions in folk medicine, but also open the door to and the need for further in vivo testing of extracts in order to examine the molecular mechanism of anti-inflammatory activity in living systems and possibly develop a new anti-inflammatory drug or supplement.

Pharmacological and Molecular docking studies of new copper (II) complexes of N2-Phenyl-N4,N6-di(thiazol-2-yl)-1,3,5-triazine-2,4,6-triamine

A new series of copper(II) complexes of tri substituted 1,3,5 triazine based ligand N2-Phenyl-N4,N6-di(thiazol-2-yl)-1,3,5-triazine-2,4,6-triamine have been synthesized. The structural features of the ligand and its metal complexes have been characterized by micro analytical data, magnetic susceptibility measurements, molar conductance, IR, UV-Visible, 1HNMR, Mass and EPR spectral techniques. The non-electrolytic behaviour of the complexes was confirmed from their conductance data. The electrochemical behaviour of the complexes has been studied by cyclic voltammetry. The free ligand and its metal complexes have been screened for their in vitro biological activity against bacteria Staphylococcus aureus and Escherichia coli and the fungi Candida albicans and Aspergillus niger by well diffusion method.The free ligand and the Cu(II) complexes have been screened for in vivo anticonvulsant activity by maximal electroshock method and have shown significant activity. Carrageenan induced rat paw edema is the most widely used primary test to screen inflammation which measures the ability of the compounds to reduce local edema induced in the rat paw by carrageenan. The ligand and Cu[(PDTTA)(ac)2] areidentified as a new lead structure, demonstrated anti-inflammatory properties in preliminary studies in mice. Molecular docking study was conducted to study the molecular mechanism of anti-inflammatory activity. Cu[(PDTTA)(ac)2] is found to be potent inhibitor of PDE4 enzyme which is involved in the inflammation process.

A Novel Curcumin-Mycophenolic Acid Conjugate Inhibited Hyperproliferation of Tumor Necrosis Factor-Alpha-Induced Human Keratinocyte Cells.

Curcumin (CUR) has been used as adjuvant therapy for therapeutic application in the treatment of psoriasis through several mechanisms of action. Due to the poor oral bioavailability of CUR, several approaches have been developed to overcome the limitations of CUR, including the prodrug strategy. In this study, CUR was esterified with mycophenolic acid (MPA) as a novel conjugate prodrug. The MPA-CUR conjugate was structurally elucidated using FT-IR, 1H-NMR, 13C-NMR, and MS techniques. Bioavailable fractions (BFs) across Caco-2 cells of CUR, MPA, and MPA-CUR were collected for further biological activity evaluation representing an in vitro cellular transport model for oral administration. The antipsoriatic effect of the BFs was determined using antiproliferation and anti-inflammation assays against hyperproliferation of tumor necrosis factor-alpha (TNF-α)-induced human keratinocytes (HaCaT). The BF of MPA-CUR provided better antiproliferation than that of CUR (p < 0.001). The enhanced hyperproliferation suppression of the BF of MPA-CUR resulted from the reduction of several inflammatory cytokines, including IL-6, IL-8, and IL-1β. The molecular mechanisms of anti-inflammatory activity were mediated by an attenuated signaling cascade of MAPKs protein, i.e., p38, ERK, and JNK. Our results present evidence for the MPA-CUR conjugate as a promising therapeutic agent for treating psoriasis by antiproliferative and anti-inflammatory actions.

Anti-Inflammatory Effect Fraction of Bletilla striata and Its Protective Effect on LPS-Induced Acute Lung Injury

Bletilla striata is a well-known traditional Chinese herb with anti-inflammatory properties that is widely used in the treatment of lung conditions such as silicosis, tuberculosis, and pneumogastric hemorrhage. However, little information on the anti-inflammatory ingredients and their activities is available. In this study, an effect fraction of Bletilla striata (EFBS) was enriched, and its anti-inflammatory activities and underlying mechanisms were investigated. EFBS was enriched by polyamide column chromatography and characterized by HPLC; an LPS-induced acute lung injury model was used to evaluate the anti-inflammatory activities of EFBS. Meanwhile, the main anti-inflammation-contributing ingredients and possible molecular mechanism of anti-inflammatory activity in EFBS were verified by component-knockout method combined with LPS-induced RAW264.7 cell model. The EFBS mainly consisted of coelonin (15.88%), batatasin III (32.49%), 3′-O-methylbatatasin III (6.96%), and 3-hydroxy-5-methoxy bibenzyl (2.51%). Pretreatment with the EFBS (20 mg/kg and 60 mg/kg) for five days prior to the administration of LPS resulted in decreases in wet-to-dry lung weight ratio, neutrophil number, MPO activity, total protein concentration, NO level, and MDA level, as well as IL-1β, IL-6, MCP-1, and TNF-α concentrations in the bronchoalveolar lavage fluid. Western blot analysis demonstrated the increased expressions of iNOS, COX-2, and NF-κB p65 in the LPS treatment group, all of which were ameliorated by EFBS pretreatment. Histological examination confirmed the protective effect of the EFBS. Additionally, component-knockout assay confirmed that these four quantitative components contributed significantly to the anti-inflammatory effect of EFBS. Coelonin, batatasin III, 3′-O-methylbatatasin III and 3-hydroxy-5-methoxy bibenzyl were the main anti-inflammatory components of EFBS and could regulate the expression of downstream inflammatory cytokines by inhibiting p65 nuclear translocation. These findings uncover, in part, the molecular basis underlying the anti-inflammatory activity of Bletilla striata.

Cinnamaldehyde Inhibits Inflammation of Human Synoviocyte Cells Through Regulation of Jak/Stat Pathway and Ameliorates Collagen-Induced Arthritis in Rats.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit ‎CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1β-induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6-induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-κB pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1β-induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug. SIGNIFICANCE STATEMENT: In this study, we found that cinnamaldehyde (Cin) suppressed proinflammatory cytokines secretion in rheumatology arthritis synoviocyte cells by Janus kinase/signal transducer and activator of transcription pathway. The in vivo results showed that Cin ameliorated collagen-induced arthritis in rats. These findings indicate that Cin is a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug.

Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation

Chloroquine (CQ) and amodiaquine (AQ) have been used for treating or preventing malaria for decades, and their application has expanded into treating inflammatory disease in humans. CQ and AQ are applicable for controlling rheumatoid arthritis, but their molecular mechanisms of anti-inflammatory activity remain to be elucidated. In this study, we examined the effects of CQ and AQ on T cell activation and T cell-mediated immune response. CQ had no significant effect on T cell numbers, but decreased the population of T cells with a high division rate. However, AQ treatment significantly increased the number of cells with low division rates and eliminated cells with high division rates, resulting in the inhibition of T cell proliferation triggered by T cell receptor stimulation, of which inhibition occurred in developing effector T helper and regulatory T cells, regardless of the different exogenous cytokines. Interestingly, the cyclin-dependent kinase inhibitor p21 was significantly and dose-dependently increased by CQ, and more potently by AQ, while other cell cycle regulators were unchanged. Both CQ and AQ elevated the transcription level of p21 though the activation of p53, but also blocked p21 protein degradation in the presence of cycloheximide, causing p21 protein accumulation mainly in the nucleus. Sustained treatment of developing T cells with either CQ or AQ suppressed IFN-γ production in a dose dependent manner and potently inhibited the differentiation of IFN-γ-producing Th1 cells. These results demonstrate that CQ and AQ increase the expression level of p21 and inhibit T cell proliferation and the development of IFN-γ-producing Th1 cells, thereby revealing beneficial roles in treating a wide range of chronic inflammatory diseases mediated by inflammatory T cells.

Piceatannol inhibits phorbol ester-induced expression of COX-2 and iNOS in HR-1 hairless mouse skin by blocking the activation of NF-κB and AP-1.

The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory activity of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) in mouse skin in vivo. Female HR-1 hairless mice were topically treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) with or without piceatannol pretreatment. Epidermal protein expression was assessed by Western blot analysis. The cyclooxygenase-2 (COX-2) expression was detected by immunohistochemistry. The DNA binding of nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) was examined by the electrophoretic mobility gel shift assay. The catalytic activity of IκBα kinase-β (IKKβ) was measured by in vitro kinase assay. Pretreatment with piceatannol attenuated TPA-induced expression of COX-2 and inducible nitric oxide synthase (iNOS) in mouse skin. Piceatannol diminished nuclear translocation and the DNA binding of NF-κB through the blockade of phosphorylation and subsequent degradation of IκBα. Piceatannol attenuated the catalytic activity of IKKβ and inhibited the phosphorylation of mitogen-activated protein (MAP) kinases in TPA-treated mouse skin. In addition, piceatannol decreased TPA-induced expression of c-Fos and the DNA binding of AP-1. Piceatannol inhibits TPA-induced COX-2 and iNOS expression by blocking the activation of NF-κB and AP-1 via suppression of the IKKβ activity and phosphorylation of MAP kinases, which provides a mechanistic basis of its anti-inflammatory effects in mouse skin.

The anti-inflammation effect of baicalin on experimental colitis through inhibiting TLR4/NF-κB pathway activation.

Baicalin holds a protective effect on inflammatory responses in several diseases. However, its molecular mechanism of anti-inflammatory activity on ulcerative colitis (UC) remains unknown. The present study was conducted to verify whether the anti-inflammation effect of baicalin on experimental colitis is via inhibiting TLR4/NF-κB pathway activation. The inflammatory response in RAW264.7 cells was induced by LPS and in rats by intrarectal administration of TNBS. Western blot analysis was carried out to examine toll-like receptor 4 (TLR4), NF-κB, p-NF-κB p65, IκB and p-IκB protein expressions in cells. Furthermore, intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), cyclo-oxygenase-2 (Cox-2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 levels in cell supernatant and rat serum were detected by appropriate kits. An immunohistochemical assay was applied to examine TNF-α and IL-1β protein expression in colon tissues and TLR4 and p-NF-κB p65 protein expressions in RAW264.7 cells. Baicalin ameliorates the considered inflammatory symptoms of induced colitis. It could also down-regulate pro-inflammatory mediators in the colon mucosa. The decline in the production of pro-inflammatory cytokines was correlated with the decrease in mucosal TLR4 protein expression. The expression of p-NF-κB p65 protein was significantly decreased, which correlated with a similar decrease in p-IκB protein. Consistent with the in vivo results, baicalin blocked LPS-stimulated nuclear translocation of p-NF-κB p65 in mouse macrophage RAW264.7 cells. The present study indicates for the first time that the mechanism for baicalin on abrogating experimental colitis was targeted inhibition of the TLR4/NF-κB pathway activation.

Molecular mechanism of anti-inflammatory activity of Pluchea indica leaves in macrophages RAW 264.7 and its action in animal models ofinflammation

Ethnopharmacological relevance: Pluchea indica Less.(Asteraceae) is a Thai medicinal plant used in traditional medicine for the treatment of hemorrhoids, lumbago, leucorrhoea and inflammation. This study investigated the molecular mechanism of anti-inflammatory activity of Pluchea indica leaf extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and also determined its action in acute inflammation animal models. Materials and methodsThe inhibitory effect of Pluchea indica leaf extract on LPS-induced nitric oxide (NO) production was evaluated by Griess reaction. Protein and mRNA expressions were determined by real time RT-PCR and Western blotting analysis, respectively. Inducible nitric oxide synthase (iNOS) promoter activity was evaluated by iNOS promoter based reporter gene assay. In vivo anti-inflammatory effect was examined in ethylphenylpropiolate (EPP)-induced ear edema and carrageenan-induced paw edema in rat models. ResultsEthyl acetate fraction of ethanol extract of Pluchea indica leaves (EFPI) exhibited the potent inhibitory effect on NO production in LPS-induced macrophages and also inhibited PGE2 release. EFPI reduced iNOS mRNA and protein expression through suppressed iNOS promoter activity and nuclear translocation of subunit p65 of nuclear factor-κB, but did not inhibit phosphorylation of the mitogen-activated protein kinases (MAPKs). Moreover, EFPI possessed anti-inflammatory activities on acute phase of inflammation as seen in EPP-induced ear edema and carrageenan-induced paw edema inrats. ConclusionsThese data support the pharmacological basis of Pluchea indica plant as a traditional herbal medicine for treatment of inflammation.

Study of the Molecular Mechanism of Anti-inflammatory Activity of Bee venom in Lipopolysaccharide Stimulated RAW 264.7 Macrophages

Purpose: Bee venom (BV) is traditionally used in many inflammatory chronic conditions but its mechanism of action at molecular level is not fully understood. This study was undertaken to elucidate the mechanism of action of bee venom at the molecular level Methods: We used lipopolysaccharide (LPS) stimulation in Raw 264.7 macrophage (RM) cells and studied the effect of BV on cell proliferation, inflammation related protein expression by western blotting and RNA expression by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Bee venom was toxic to RM cells above10 μg/ml but reduced the production of nitric oxide (NO) at 2–10 μg/ml in LPS stimulated RM cells by inhibiting the expression of inducible nitric oxide synthase (iNOS) and cyclooxigenase (COX)-2 via nuclear factor (NF)-κB. However, bee venom also induced the pro-inflammatory cytokine, interleukin (IL)-1β via p38 mitogen activated protein kinase (MAPK) which is known to stimulate inflammatory activity.Conclusion: It seems that NFκB and p38 MAPK signal pathways are involved in triggering the functional activation of LPS-stimulated macrophage. We suggest that some components of bee venom can cause inflammation by inducing IL-1β via p38 MAPK while others act as anti-inflammatory by suppressing iNOS and COX2 via NFκB.Keywords: Bee venom, Cyclooxygenase-2, Interleukin 1beta, Inducible nitric oxide synthase, Lipopolysaccharide, Macrophage, Mitogen activated protein kinase, Nuclear factor kappa-B.

Molecular Mechanisms of Anti-Inflammatory Activity Mediated by Flavonoids

Flavonoids (or bioflavonoids) are naturally occurring compounds, ubiquitous in all vascular plants. These compounds have been considered to possess anti-inflammatory properties, both in vitro and in vivo. Although not fully understood, these health-promoting effects have been mainly related to their interactions with several key enzymes, signaling cascades involving cytokines and regulatory transcription factors, and antioxidant systems. The biological effects of flavonoids will depend not only on these pharmacodynamic features but also on their pharmacokinetics, which are dependent on their chemical structure, administered dose schedule and route of administration. Thus, the therapeutic outcome mediated by flavonoids will result from a complex and interactive network of effects, whose prediction require a deep and integrated knowledge of those pharmacokinetic and pharmacodynamic factors. The aim of the present review is thus to provide an integrated update on the bioavailability and biotransformation of flavonoids and the mechanisms of activity at the molecular, cellular, organ and organism levels that may contribute to their anti-inflammatory effects.