Engineering a Remedy to Modulate and Optimize Biopharmaceutical Properties of Rebamipide by Synthesizing New Cocrystal: In Silico and Experimental Studies.

PURPOSE: Rebamipide (REB) a potent anti-ulcer agent, has not been exploited to its full potential, owing to it extremely poor solubility, leading to highly diminutive bioavailability (<10%). The purpose is to carry out its solid-state modification. METHOD: Cocrystallisation was done with three GRAS coformers namely citric acid (CA), 3,4-dihydroxybenzoic acid (DHBA) and oxalic acid (OXA) employing the liquid-assisted grinding method. Cocrystal formation was based upon amide-carboxyl and amide-hydroxyl supramolecular synthons. Characterization of novel cocrystals i.e. RCA, RDHBA and ROXA was carried out by DSC, PXRD and additionally by FT-IR spectroscopy. Chemical structures have been determined utilizing the PXRD pattern by Material Studio®. Furthermore, cocrystals were subjected to solubility and intrinsic dissolution rate (IDR) evaluation. Also, pharmacodynamic and pharmacokinetic studies were performed and compared with pure rebamipide. RESULT: The appearances of a single sharp melting endotherm in DSC, along with novel characteristic peaks in PXRD infer the existence of a new crystalline form. Shifting in characteristic vibrations in FT-IR spectroscopy supports the establishment of distinct hydrogen-bonded networks. Structural determination revealed that RCA crystallizes in 'Bb2b' space groups whereas RDHBA in 'P1' and ROXA crystallize out in the 'P-1' space group. All the cocrystals exhibited superior apparent solubility and almost 7-13 folds increase in IDR. Furthermore, 1.6-2.5 folds enhancement in relative bioavailability and remarkable amplification in anti-ulcer, anti-inflammatory and the antioxidant potential of these cocrystals were observed. CONCLUSION: The study ascertains the advantages of cocrystallization, with RCA showing greatest potential and suggests a viable alternative approach for improved formulation of rebamipide.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

Local and Systemic Profiles of Inflammatory Cytokines in Carrageenan-induced Paw Inflammation in Rats.

OBJECTIVE: Carrageenan (CA)-induced edema has been described as highly reproducible model of acute inflammation. However, little is known about the cytokines attributed to the CA-induced inflammation. In this study, we aimed to investigate the local and systemic expression profiles of various inflammatory cytokines following the subplantar injection of CA in rats. METHODOLOGY: Acute inflammation was induced in male Wistar rats by subplantar injection of CA. Serum and paw tissue were examined for the level of 19 specific inflammatory cytokines using antibody array. Further, the CA-elicited level of key inflammatory cytokines, cytokine-induced neutrophil chemoattractant (CINC)-2, CINC-3, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Edema was peaked 3 h postinjection of CA in hind paw. Among 19 specific cytokines profiled using antibody array, CA significantly (p < 0.05) elicited the levels of CINC-2, CINC-3, IL-1ß, IL-6, ß-NGF, TNF-α, and VEGF in paw tissue and that of CINC-2 and CINC-3 in serum. Consistently, levels of CINC-2, CINC-3, IL-1ß, IL-6, and TNF-α in tissue and CINC-2 and CINC-3 in serum were upregulated in CA-treated rats when compared to control, quantified by ELISA. CONCLUSIONS: This study corroborates the distinct pattern of inflammatory cytokines involved during CA-induced acute inflammation. Furthermore, data provide new evidence on elevated expression of rat CXC chemokines: CINC-2 and CINC-3 at the site of inflammation as well as their significant reflection in the circulation, thereby suggesting their frontline role in CA-induced acute inflammation.

P2X4 receptors mediate PGE2 release by tissue-resident macrophages and initiate inflammatory pain.

Prostaglandin E2 (PGE2) is a key mediator of inflammation and contributes to pain hypersensitivity by promoting sensory neurons hyperexcitability. PGE2 synthesis results from activation of a multi-step enzymatic cascade that includes cyclooxygenases (COXs), the main targets of non-steroidal anti-inflammatory drugs (NSAIDs). Although NSAIDs are widely prescribed to reduce inflammatory symptoms such as swelling and pain, associated harmful side effects restrict their long-term use. Therefore, finding new drugs that limit PG production represents an important therapeutic issue. In response to peripheral inflammatory challenges, mice lacking the ATP-gated P2X4 channel (P2X4R) do not develop pain hypersensitivity and show a complete absence of inflammatory PGE2 in tissue exudates. In resting conditions, tissue-resident macrophages constitutively express P2X4R. Stimulating P2X4R in macrophages triggers calcium influx and p38 MAPK phosphorylation, resulting in cytosolic PLA2 (cPLA2) activation and COX-dependent release of PGE2. In naive animals, pain hypersensitivity was elicited by transfer into the paw of ATP-primed macrophages from wild type, but not P2X4R-deficient mice. Thus, P2X4Rs are specifically involved in inflammatory-mediated PGE2 production and might therefore represent useful therapeutic targets.

Shrimp that have received carrageenan via immersion and diet exhibit immunocompetence in phagocytosis despite a post-plateau in immune parameters.

The effect of carrageenan on the immune response of white shrimp Litopenaeus vannamei, was studied in vitro and in vivo. Shrimp haemocytes receiving carrageenan at 1 mg ml⁻¹ experienced change in cell size, reduction in cell viability, increase in PO activity, serine proteinase activity, and RB in vitro. Shrimp received carrageenan via immersion at 200, 400 and 600 mg L⁻¹ after 3 h and orally at 0.5, 1.0 and 2.0 g kg⁻¹ after 3 weeks showed higher proliferation of haematopoietic tissues (HPTs) together with increases in haemocyte count and other immune parameters. Shrimp that fed a diet containing carrageenan at 0.5 g kg⁻¹ after 3 weeks significantly up-regulated gene expressions of several immune-related proteins. The immune parameters of shrimp that received carrageenan via immersion and orally increased to a plateau after 3 h and after 3 weeks, but decreased after 5 h and 6 weeks, respectively. Phagocytosis and clearance of Vibrio alginolyticus remained high in shrimp that had received carrageenan via immersion after 5 h and orally after 6 weeks, respectively. Resistances of shrimp against V. alginolyticus and white spot syndrome virus were higher over 24-144 h and 72-144 h, respectively in shrimp that received carrageenan at 600 mg L⁻¹ via immersion after 3 and 5 h. It was concluded that carrageenan effectively triggers an innate immunity in vitro, and increases mitotic index of HPT, immune parameters, gene expressions and resistance against pathogens in vivo. Shrimp received carrageenan via immersion and orally exhibited immunocompetence in phagocytosis and clearance of V. alginolyticus, and resistance to pathogen despite the trend in immune parameters to recover to background values.

Inhibition of pro-inflammatory mediators: role of Bacopa monniera (L.) Wettst.

Bacopa monniera (L.) Wettst is a renowned plant in the Ayurvedic system of medicine. The present study seeks to identify the anti-inflammatory activity of two fractions from the methanolic extract of Bacopa, viz. the triterpenoid and bacoside-enriched fractions. The ability of these two fractions to inhibit the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 was tested using lipopolysaccharide (LPS)-activated peripheral blood mononuclear cells and peritoneal exudate cells in vitro. We found that triterpenoid and bacoside-enriched fractions significantly inhibited LPS-activated TNF-α, IL-6 and nitrite production in mononuclear cells. Significant antioxidant activity was exhibited by the bacoside enriched fraction compared to the triterpenoid fraction. Carrageenan-induced hind paw oedema assay revealed that triterpenoid and bacoside-enriched fractions exerted anti-oedematogenic effect, while in the arthritis model only the triterpenoid fraction exerted an anti-arthritic potential. The present study provides an insight into the ability of Bacopa monniera to inhibit inflammation through modulation of pro-inflammatory mediator release.

The Role of Carrageenan in Inflammatory Bowel Diseases and Allergic Reactions: Where Do We Stand?

Carrageenan (CGN) is a high molecular weight polysaccharide extracted from red seaweeds, composed of D-galactose residues linked in ß-1,4 and α-1,3 galactose-galactose bond, widely used as a food additive in processed foods for its properties as a thickener, gelling agent, emulsifier, and stabilizer. In recent years, with the spread of the Western diet (WD), its consumption has increased. Nonetheless, there is a debate on its safety. CGN is extensively used as an inflammatory and adjuvant agent in vitro and in animal experimental models for the investigation of immune processes or to assess the activity of anti-inflammatory drugs. CGN can activate the innate immune pathways of inflammation, alter the gut microbiota composition and the thickness of the mucus barrier. Clinical evidence suggests that CGN is involved in the pathogenesis and clinical management of inflammatory bowel diseases (IBD), indeed food-exclusion diets can be an effective therapy for disease remission. Moreover, specific IgE to the oligosaccharide α-Gal has been associated with allergic reactions commonly referred to as the "α-Gal syndrome". This review aims to discuss the role of carrageenan in inflammatory bowel diseases and allergic reactions following the current evidence. Furthermore, as no definitive data are available on the safety and the effects of CGN, we suggest gaps to be filled and advise to limit the human exposure to CGN by reducing the consumption of ultra-processed foods.

Infrared radiation from cage bedding moderates rat inflammatory and autoimmune responses in collagen-induced arthritis.

The development of collagen type II (CII)-induced arthritis (CIA), a model of rheumatoid arthritis, in rats housed in cages with bedding composed of Celliant fibres containing ceramic particles, which absorb body heat and re-emit the energy back to the body in the form of infrared radiation (+IRF rats), and those housed in cages with standard wooden shaving bedding (-IRF control rats) was examined. The appearance of the first signs of CIA was postponed, while the disease was milder (judging by the arthritic score, paw volume, and burrowing behaviour) in +IRF compared with -IRF rats. This correlated with a lower magnitude of serum anti-CII IgG antibody levels in +IRF rats, and lower production level of IL-17, the Th17 signature cytokine, in cultures of their paws. This could be partly ascribed to impaired migration of antigen-loaded CD11b + dendritic cells and their positioning within lymph nodes in +IRF rats reflecting diminished lymph node expression of CCL19 /CCL21. Additionally, as confirmed in rats with carrageenan-induced paw inflammation (CIPI), the infrared radiation from Celliant fibres, independently from immunomodulatory effects, exerted anti-inflammatory effects (judging by a shift in pro-inflammatory mediator to anti-inflammatory/immunoregulatory mediator ratio towards the latter in paw cultures) and ameliorated burrowing behaviour in CIA rats.

Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs.

BACKGROUND: Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as "tomatinho do mato" and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated. RESULTS: In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1ß, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract. CONCLUSION: S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial-binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS-induced TLR4-mediated NF-kappaB activation and to the pathogenesis of Crohn's disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen-binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1-mediated bacterial aggregation via interaction with its bacterial-recognition motif. Competition and ELISA studies identify poly-sulfated and poly-phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose-response studies in Dmbt1(-/-) and Dmbt1(+/+) mice utilizing the DSS-induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands providing a molecular basis for its broad bacterial-binding specificity and its inhibitory effects on LPS-induced TLR4-mediated NF-kappaB activation.

Euryops arabicus displays anti-inflammatory activities in experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Euryops arabicus (Asteraceae) is grown in Arab Peninsula. Its aerial parts possess ethnomedicinal applications against several inflammatory conditions. AIM OF THE STUDY: To evaluate the anti-inflammatory activity of Euryops arabicus (E. arabicus) organic extract as well as its major polymethoxylated flavonoids. MATERIALS AND METHODS: Acute toxicity of the total extract of E. ararbicus was evaluated by assessing LD50. In vivo anti-inflammatory activity was evaluated in rats injected with carrageenan in the plantar area. Paw edema volume, histological changes and rats'stair climbing and motility were assessed. In vitro anti-inflammatory activity of the isolated compounds was evaluated in peripheral blood mononuclear cells (PBMCs) challenged with carrageenan. Inflammation markers were assessed in cellular lysates and collected media. RESULTS: The extract was found safe and considered unclassified with an oral LD50 > 2000 mg/kg in rats. Pretreatment of rats with a total extract of E. arabicus at doses of 100 and 250 mg/kg significantly inhibited carrageenan-induced increase in paw edema volume and histopathological changes. Also, it significantly ameliorated diminution of climbing and motility. Phytochemical studies led to the isolation and identification of five polymethoxylated flavonoids. The anti-inflammatory properties of the isolated compounds were evaluated in carrageenan-challenged peripheral blood mononuclear cells (PBMCs). All compounds exhibited appreciable antioxidant activities. Further, pre-incubation of the cells with the isolated metabolites significantly ameliorated the rise in cyclo-oxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) induced by carrageenan challenge. Further, the compounds inhibited the leakage of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and myeloperoxidase (MPO) in media collected from stimulated cells. CONCLUSION: E. arabicus exhibited in vivo anti-inflammatory effects in the carrageenan model as it ameliorated rat paw edema, histopathological changes and movement dysfunction. in vitro activity of isolated compounds was confirmed in stimulated PBMCs. Thus, the anti-inflammatory activity of E. arabicus can be attributed, at least partly, to its anti-oxidant, anti-inflammatory and anti-chemotactic properties.

Anti-inflammatory and anti-arthritic activity in extract from the leaves of Eriobotrya japonica.

ETHNOPHARMACOLOGICAL RELEVANCE: The Eriobotrya japonica (EJ) is a Chinese medicinal plant that is currently grown in Brazil. E. japonica leaves infusion is traditionally used in the treatment of inflammation; however, there are few scientific studies showing the effects of these properties on joint articular and persistent experimental inflammation. AIM OF THE STUDY: The present research had objective investigation of the effect of infusion obtained from leaves of E. japonica (EJLE) on acute and persistent experimental articular inflammation. MATERIALS AND METHODS: The Swiss mice were treated orally with EJLE and analyzed for acute pleural inflammation (30, 100, and 300 mg/kg), paw edema induced by carrageenan (100 mg/kg), acute knee inflammation induced by zymosan (100 mg/kg), and persistent inflammation induced by Complete Freund's Adjuvant (CFA) (30 and 100 mg/kg). Mechanical hyperalgesia, cold and edema were analyzed. RESULTS: The chromatographic analysis of EJLE revealed the presence of corosolic acid, oleanolic acid, and ursolic acid. EJLE presented anti-inflammatory activity in the pleurisy model, inhibiting leukocyte migration, protein extravasation and nitric oxide production. In the articular inflammation model, EJLE reduced the number of leukocytes in the joint cavity, paw edema and hyperalgesia (4 h after induction). In the persistent inflammation model induced by CFA, the extract reduced paw edema after 11 days of mechanical and cold hyperalgesia on day 6. CONCLUSIONS: The EJLE has anti-inflammatory and antihyperalgesic potential in models of acute and persistent experimental articular inflammation, making this infusion a new possibility for complementary treating acute or chronic articular inflammatory diseases.

Cell-mediated immune response to unrelated proteins and unspecific inflammation blocked by orally tolerated proteins.

Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.

Tumor bearing decreases systemic acute inflammation in rats--role of mast cell degranulation.

OBJECTIVE AND DESIGN: To investigate the effect of experimental tumor bearing on acute inflammation models in rats. METHODS: Four and 7 days after Walker tumor implantation in the right armpit, carrageenan or dextran- induced edema in the contralateral paw, carrageenan induced neutrophil migration into peritoneal cavities, cutaneous vascular permeability induced by bradykinin, histamine, serotonin, substance P, capsaicin or compound 48/80, and mesenteric mast cell degranulation induced by compound 48/80 were evaluated. The control group did not receive tumor implantation. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Bonferroni test. RESULTS: On the 7(th) day after tumor inoculation, there were significant decreases in both carrageenan and dextran- induced paw edema. Tumor bearing did not change the neutrophil infiltration induced by carrageenan. There were decreases in cutaneous vascular permeability induced by compound 48/80, serotonin or bradykinin, but not that induced by histamine, substance P. A significant inhibition of mesenteric mast cell degranulation induced by compound 48/80 was observed, on the 4(th) and 7(th) days after tumor inoculation. CONCLUSION: Tumor bearing can limit mast cell function and vascular events in acute systemic inflammation in rats, without changes in neutrophil migration.

Mefenamic acid-loaded solid SMEDDS: an innovative aspect for dose reduction and improved pharmacodynamic profile.

AIM: The current investigation is focused on solid self-microemulsifying drug-delivery systems (S-SMEDDS) of mefenamic acid (MFA) for improving pharmacodynamic activity. Methodology & results: Solubility assessment in various lipid excipients and optimization of pseudoternary plots were carried out for development of liquid SMEDDS. The optimized liquid SMEDD formulation was spray dried to solid dosage form and observed with enhanced amorphization or molecular dispersion of MFA in S-SMEDDS, as evident from x-ray diffractometry and differential scanning calorimetry studies. Enhanced in vitro dissolution rate of optimized formulation was observed, resulting in multifold enhancement in absorption profile of MFA, as compared with pure drug and marketed product. These studies further substantiate the dose reduction in SMEDDS by gaining equivalent therapeutic profile with marketed product. Enhanced analgesic and anti-inflammatory activity was observed with S-SMEDD formulations in acetic acid-induced writhings and carrageenan-induced paw edema models, respectively. CONCLUSION: The optimized S-SMEDD formulation holds great promise for enhancement of its physiochemical and biological attributes.

Antiinflammatory effects of etoricoxib alone and combined with NSAIDs in LPS-induced reactive arthritis.

OBJECTIVE: Nonsteroidal anti-inflammatory drugs constitute the primary therapeutic approach in reactive arthritis. Here, we compared etoricoxib, a specific COX-2 inhibitor, with other cyclooxygenase inhibitors on articular incapacitation, edema, leukocyte migration, and gastric damage, in a model of LPS-induced reactive arthritis in rats. METHODS: E. coli Lipopolysaccharide was injected into a carrageenan-primed knee-joint of rats. The effects of etoricoxib, piroxicam, indomethacin, as well the combination of etoricoxib either with piroxicam or indomethacin, were evaluated on articular incapacitation and edema. Afterwards, the synovial leukocyte ontent and the stomach bleeding points were counted. RESULTS: Etoricoxib, piroxicam, and indomethacin dose-dependently inhibited incapacitation and edema. However, only etoricoxib inhibited both mononuclear and polymorphonuclear leukocyte migration. Piroxicam inhibited only mononuclear migration, while indomethacin even increased polymorphonuclear content in inflamed synovia. Associating etoricoxib with either subeffective doses of piroxicam or indomethacin did not enhance the hyponociceptive or the antiedematogenic effect, but prevented the anti-leukocyte migration effect and increased gastric damage. CONCLUSION: The present results suggest that the selective COX-2 inhibitor etoricoxib could be a better option than non-selective COX inhibitors, since it presented a potent inhibitory effect on the clinical signals and also a potent inhibition on cell migration.

Inhibitory effect of amygdalin on lipopolysaccharide-inducible TNF-alpha and IL-1beta mRNA expression and carrageenan-induced rat arthritis.

Amygdalin is a cyanogenic glycoside plant compound found in the seeds of rosaceous stone fruits. We evaluated the antiinflammatory and analgesic activities of amygdalin, using an in vitro lipopolysaccharide (LPS)-induced cell line and a rat model with carrageenan-induced ankle arthritis. One mM amygdalin significantly inhibited the expression of TNF-alpha and IL-1beta mRNAs in LPS-treated RAW 264.7 cells. Amygdalin (0.005, 0.05, and 0.1 mg/kg) was intramuscularly injected immediately after the induction of carrageenan-induced arthritic pain in rats, and the anti-arthritic effect of amygdalin was assessed by measuring the weight distribution ratio of the bearing forces of both feet and the ankle circumference, and by analyzing the expression levels of three molecular markers of pain and inflammation (c-Fos, TNF-alpha, and IL-1beta) in the spinal cord. The hyperalgesia of the arthritic ankle was alleviated most significantly by the injection of 0.005 mg/kg amygdalin. At this dosage, the expressions of c-Fos, TNF-alpha, and IL-1beta in the spinal cord were significantly inhibited. However, at dosage greater than 0.005 mg/kg, the painrelieving effect of amygdalin was not observed. Thus, amygdalin treatment effectively alleviated responses to LPStreatment in RAW 264.7 cells and carrageenan-induced arthritis in rats, and may serve as an analgesic for relieving inflammatory pain.

Acute inflammatory and neuropathic pain in Lewis and Fischer rats.

Inbred, histocompatible Lewis and Fischer 344 rats (LEW and FIS) have been used to identify an inverse relationship between hypothalamic-pituitary-adrenal (HPA) axis activity and susceptibility to autoimmune and chronic inflammatory disorders, with LEW showing blunted HPA axis activity and increased susceptibility toward the development of autoimmunity and chronic inflammation, and FIS showing the opposite relationship. In the present study, LEW and FIS were used to determine the relationship between HPA axis function and acute inflammatory pain (carrageenan-induced hindpaw inflammation) and neuropathic pain (partial sciatic nerve ligation; PSNL). The results showed that carrageenan-induced thermal and mechanical allodynia and hyperalgesia were greater in FIS than in LEW. Similarly, FIS showed more carrageenan-induced hindpaw swelling and higher levels of myeloperoxidase (a measure of neutrophils) in the carrageenan-inflamed hindpaw. After PSNL, LEW showed a profound mechanical allodynia and hyperalgesia, whereas mechanical sensitivity in FIS was unaltered. However, FIS, but not LEW, developed thermal allodynia and hyperalgesia after PSNL. These results provide strong evidence for a positive relationship between HPA axis activity and acute inflammatory pain. The results also support a relationship between HPA axis activity and neuropathic pain, but the relationship is complex and may depend on the pain assay.

In vivo potentiation of concanavalin A-bound L1210 vaccine by antimacrophage agents.

Carrageenan potentiated in vivo the primary and secondary responses by concanavalin A (Con A)-bound L1210 murine leukemia vaccine and induced in histocompatible animals enhanced immune resistance to subsequent inoculations of live L1210 cells. This enhancement was critically dependent on the vaccine preparation and the administration timing of carrageenan. Carrageenan potentiated Con A-free vaccine to a much lesser extent if at all, than did Con A-bound vaccine, and it was effective only on the day of or one day after vaccine administration. This enhancement was L1210 specific as evidenced by the lethal growth of P388 leukemia cells in animals inoculated with Con A-bound L1210 vaccine and carrageenan. This is explicable by the increase of spleen but not peritoneal exudate effector cells as measured by suppression of in vitro L1210 cell growth. The following findings suggested that potentiation of Con A-bound vaccine by carrageenan was achieved by affecting suppressor macrophages. Silica and trypan blue, other antimacrophage agents, potentiated Con A-bound vaccine. The enhancement was nullified by the macrophage-stabilizing agent, poly-2-vinylpyridine N-oxide. This hypothesis was further evidenced by the following findings. Phagocytic peritoneal cells induced by Con A-bound vaccine and adhering to the plastic vessel suppressed in vitro spleen cell blastogenesis. Antimacrophage agents abrogated not only production but also activity of these suppressor cells. The combined administration of Con A-bound L1210 vaccine and carrageenan produced an enhanced antitumor immune response and prolonged the life span of tumor-bearing animals marginally but significantly.

Enhanced effect of κ-carrageenan on TNBS-induced inflammation in mice.

BACKGROUND: As a sulfated polysaccharide, carrageenan has been widely used as common food additive. METHODS: In the present study, we investigated the effects of κ-carrageenan on TNBS-induced gut inflammation in mice. BALB/c mice were pretreated with κ-carrageenan for 14days prior to the administration of TNBS. RESULTS: Our results showed that κ-carrageenan pretreatment aggravated the loss of body weight and further increased the mortality rate. Histological and morphological analyses revealed that the TNBS-induced colonic inflammation was deteriorated by the κ-carrageenan administration. The ratio of CD4(+)CD25(+)CD127dim/CD4(+) of the κ-carrageenan+TNBS groups was significantly lower than that of the TNBS group. The expression of IL-2, TNF-α and IL-6 was significantly increased, whereas the expression of IL-10 was significantly decreased in the κ-carrageenan+TNBS groups. In addition, κ-carrageenan, together with TNBS, decreased the enzyme activity of SOD and GSH-px and up-regulated the expression of TLR4, NF-κB, p-ERK, p-JNK, p-Jun., IL-8 and MDA in the colonic mucosa. CONCLUSIONS: κ-Carrageenan aggravated the TNBS-induced intestinal inflammation, and such an effect could be associated with the oxidative stress and activation of TLR4-NF-κB and MAPK/ERK1/2 pathway.