Effects of Amburoside A and Isokaempferide,Polyphenols from Amburana cearensis,on Rodent Inflammatory Processes and Myeloperoxidase Activity in Human Neutrophils

Abstract: The present study evaluated the anti‐inflammatory activity of amburoside A (a phenol glucoside) and isokaempferide (a flavonol) isolated from the trunk bark of Amburana cearensis, a medicinal plant used in northeast Brazil for the treatment of asthma. Animals (male Wistar rats or Swiss mice) pre‐treated with amburoside A (25 and 50 mg/kg) or isokaempferide (12.5, 25 and 50 mg/kg), orally or intraperitoneally, showed a significant inhibition of the paw oedema induced by carrageenan (1%), prostaglandin E₂ (30 nmol/paw), histamine (200 µg/paw) or serotonin (200 µg/paw). Histological and morphometric evaluations of the rat paw oedema induced by carrageenan showed that amburoside A and isokaempferide also inhibited the accumulation of inflammatory cells. Amburoside A reduced significantly the paw oedema and the increase in vascular permeability induced by dextran, as related to the control group. Similar results were observed with the isokaempferide pre‐treatment. Furthermore, amburoside A or isokaempferide inhibited both leucocyte and neutrophil migrations, in mouse peritoneal cavity, after the carrageenan injection. The polyphenols were not cytotoxic and blocked N‐formyl‐methyl‐leucyl‐phenylalanine‐induced myeloperoxidase release and activity in human neutrophils. In addition, amburoside A and isokaempferide at 50 and 100 µg/ml concentrations reduced significantly the lipopolysaccharide‐mediated increase in tumour necrosis factor‐α (TNF‐α) levels. These results provide, for the first time, evidence to support the anti‐inflammatory activity of amburoside A and isokaempferide that seems to be related to an inhibition of inflammatory mediators, such as TNF‐α, as well as histamine, serotonin and prostaglandin E₂, besides leucocyte infiltration in a dose‐ or concentration‐dependent manner. These anti‐inflammatory effects can be explained, at least in part, by the ability of these compounds to reduce neutrophil degranulation, myeloperoxidase activity, mediators as well as TNF‐α secretion.     

The role of Syk/IĸB‐α/NF‐ĸB pathway activation in the reversal effect of BAY 61‐3606, a selective Syk inhibitor,on hypotension and inflammation in a rat model of zymosan‐induced non‐septic shock

Spleen tyrosine kinase (Syk), a non‐receptor tyrosine kinase, plays an important role in allergic diseases and inflammation. Syk triggers several intracellular signalling cascades including Toll‐like receptor signalling to activate inflammatory responses following fungal infection but the role of this enzyme in zymosan (ZYM)‐induced non‐septic shock and its impacts on hypotension and inflammation in rats is not well understood. This study was conducted to determine the effects of Syk inhibition on ZYM‐induced alterations in the expression and/or activities of Syk, inhibitor ?B (I?B)‐α, and nuclear factor‐?B (NF‐?B) p65. We also examined the effect of Syk inhibition on inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)‐2, and tumour necrosis factor (TNF)‐α, and activity of myeloperoxidase (MPO) that contribute to hypotension and inflammation. Administration of ZYM (500 mg/kg, ip) to male Wistar rats decreased blood pressure and increased heart rate. These changes were associated with increased expression and/or activities of Syk, NF‐κB p65, iNOS and COX‐2 and decreased expression of IκB‐α with enhanced levels of nitrite, nitrotyrosine, 6‐keto‐PGF_1α, and TNF‐α and activity of MPO in renal, cardiac and vascular tissues. ZYM administration also elevated serum and tissue nitrite levels. The selective Syk inhibitor BAY 61‐3606 (3 mg/kg, ip) given 1 hour after ZYM injection reversed all of these changes induced by ZYM. These results suggest that Syk/I?B‐α/NF‐?B pathway activation contributes to hypotension and inflammation caused by the production of vasodilator and proinflammatory mediators in the zymosan‐induced non‐septic shock model.     

Bis‐(3‐hydroxyphenyl) diselenide inhibits LPS‐stimulated iNOS and COX‐2 expression in RAW 264.7 macrophage cells through the NF‐kB inactivation

Objectives Previously, we reported that diaryl diselenide compounds have strong inhibitory effects on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production in macrophages. In this study, we investigated the molecular mechanisms underlying NO suppression and prostaglandin E₂ (PGE₂) production by diaryl diselenide compounds, bis‐(2‐hydroxyphenyl) diselenide (DSE‐A), bis‐(3‐hydroxyphenyl) diselenide (DSE‐B), bis‐(4‐hydroxyphenyl) diselenide (DSE‐C), dipyridyl diselenide (DSE‐D) and diphenyl diselenide (DSE‐E). Methods The effect of these compounds on NO suppression and PGE₂ production was investigated in RAW 264.7 macrophages. Key findings Our data indicate that of the above, DSE‐B most potently inhibits NO and PGE₂ production, and that it also significantly reduces the releases of tumour necrosis factor (TNF)‐α, interleukin(IL)‐1β and IL‐6. Consistent with these observations, DSE‐B also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2), and the mRNA levels of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6. Furthermore, DSE‐B inhibited LPS‐induced nuclear factor‐κB (NF‐κB) activation, which was associated with the prevention of the inhibitor κB‐α (IκB‐α) degradation and a subsequent reduction in nuclear p65 protein levels. Conclusions Taken together, our data suggest that the anti‐inflammatory properties of DSE‐B are due to reduction in the expression of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6 through the down‐regulation of NF‐κB binding activity.     

Discovery of Novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole Derivatives as Potential Anti‐Inflammatory Agents

A novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 5 with good anti‐inflammatory activity was identified from our in‐house library. Based on hit compound 5 , two series of 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 6a – g and 7a – h were designed and synthesized as novel anti‐inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF‐α production in LPS‐stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm ) and TNF‐α (IC₅₀ = 1.87 μm ) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti‐inflammatory activity than ibuprofen did on xylene‐induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF‐κB in LPS‐stimulated RAW 264.7 macrophages.     

Myrrh mediates haem oxygenase-1 expression to suppress the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages

Objectives To elucidate a novel anti‐inflammatory mechanism of myrrh against lipopolysaccharide (LPS)‐induced inflammation. Methods RAW264.7 macrophages were cultured in DMEM and then cells were treated with LPS or LPS plus a myrrh methanol extract (MME) for 24 h. The culture medium was collected for determination of nitric oxide (NO), prostaglandin (PG)E₂, interleukin (IL)‐1β, and tumour necrosis factor (TNF)‐α, and cells were harvested by lysis buffer for Western blot analysis. Key findings Our data showed that treatment with the MME (1～100 µg/ml) did not cause cytotoxicity or activate haem oxygenase‐1 (HO‐1) protein synthesis in RAW264.7 macrophages. Furthermore, the MME inhibited LPS‐stimulated NO, PGE₂, IL‐1β and TNF‐α release and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)‐2 protein expression. Zn(II) protoporphyrin IX, a specific inhibitor of HO‐1, blocked the inhibition of iNOS and COX‐2 expression by the MME. Conclusions These results suggest that among mechanisms of the anti‐inflammatory response, the MME inhibited the production of NO, PGE₂, IL‐1β and TNF‐α by downregulating iNOS and COX‐2 gene expression in macrophages and worked through the action of HO‐1.     

Isopropoxy‐Carvacrol,a Derivative Obtained from Carvacrol,Reduces Acute Inflammation and Nociception in Rodents

Monoterpenes, compounds mainly presented in essential oils, have important pharmacological actions. Isopropoxy‐carvacrol (IPC) is a derivative of the monoterpene carvacrol, and its pharmacological properties have not yet been investigated. The aim of this study was to analyse the acute anti‐inflammatory and antinociceptive properties of IPC. Mice (25–30 g) and rats (150–230 g) were pre‐treated (i.p.) with IPC at the doses of 10, 30 or 100 mg/kg or vehicle (Tween 80, 0.5%), 30 min. before injection of the phlogistic agents. Both the first and the second phases of formalin‐induced nociception were significantly reduced by IPC (100 mg/kg). Injection of carrageenan in mice paw reduced the threshold of stimulus intensity, applied with an analgesymeter, necessary to cause paw withdrawal, which was significantly reduced by 100 mg/kg of IPC. The area under curve (0–4 hr) of rat paw oedema induced by injection of carrageenan was also significantly diminished by the administration of IPC (100 mg/kg). Administration of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) markedly increased mice ear oedema and myeloperidase (MPO) activity. Topical co‐administration of IPC (0.3–3 mg/ear) during the induction did not affect TPA‐induced ear oedema, but significantly decreased MPO activity in the ears, when compared with the vehicle. In in vitro experiments, IPC reduced lipoperoxidation induced by different stimuli, showed nitric oxide scavenger activity and did not interfere with murine macrophage viability in concentrations up to 100 μg/mL. These results demonstrate that IPC exerts acute anti‐inflammatory and antinociceptive activities, suggesting that it may represent an alternative in the development of new future therapeutic strategies.     

Design,Synthesis, and Biological Evaluation of 1,5‐Diaryl‐1,2,4‐triazole Derivatives as Selective Cyclooxygenase‐2 Inhibitors

A series of 1,5‐diaryl‐1,2,4‐triazole derivatives were synthesized and evaluated as cyclooxygenase‐2 (COX‐2) inhibitors. The results of the preliminary biological assays in vivo showed that eight compounds 5b , 6b , 6c , 7c , 8b , 8d , 9c , and 9d have potent anti‐inflammatory activity (P < 0.01), while compounds 6b , 6c , and 9c exhibit marked potency. Compound 6c was then selected for further investigation. In the COX inhibition assay in vitro, compound 6c was identified as a potent and selective inhibitor of COX‐2 (COX‐2 IC₅₀ = 0.37 µM; SI = 0.018), being equipotent to celecoxib (COX‐2 IC₅₀ = 0.26 µM; SI = 0.015). In a rat carrageenan‐induced paw edema assay, 6c exhibited moderate anti‐inflammatory activity (35% inhibition of inflammation) at 2 h after administration of 15 mg/kg as an oral dose. A docking study also revealed that compound 6c binds in the active site of COX‐2 in a similar mode to that of the known selective COX‐2 inhibitor SC‐558.     

Synthesis of Novel Substituted Thiourea and Benzimidazole Derivatives Containing a Pyrazolone Ring as Anti‐Inflammatory Agents

Two series of new 1‐(alkyl/aryl)‐3‐{2‐[(5‐oxo‐4,5‐dihydro‐1H‐pyrazol‐3‐yl)amino]phenyl}thioureas 2a – h and 5‐[2‐(substituted amino)‐1H‐benzimidazol‐1‐yl]‐4H‐pyrazol‐3‐ols 3a – i were designed and synthesized as anti‐inflammatory agents. The cyclooxygenase inhibitory activity of the newly synthesized compounds was investigated. All the compounds showed non‐selective inhibition of COX‐1 and COX‐2 enzymes which was consistent with their docking results. Compounds 2c , 2f , 2g , 3b , and 3g that showed the highest COX‐2 inhibitory activity were selected for further in vivo testing as anti‐inflammatory agents using diclofenac as a reference drug. Two of the test compounds ( 2c and 3b ) showed potent anti‐inflammatory activity comparable to that of diclofenac with lower ulcerogenic effect relative to indomethacin. SAR study of the two series as cyclooxygenase inhibitors and anti‐inflammatory agents was also provided.     

Inhibition of key enzymes in the inflammatory pathway by hybrid molecules of terpenes and synthetic drugs: In vitro and in silico studies

The aim of this work was to compare the anti‐inflammatory activity of compounds prepared from terpenes and the synthetic drugs ibuprofen and naproxen. The anti‐inflammatory activity of the hybrid compounds was compared with the activity of the parent compounds. This was accomplished using in vitro inhibition of lipoxygenases (LOX) and COX‐2, and in silico docking studies in 15‐LOX and COX‐2. The synthesized hybrids showed an inhibition of COX‐2 and LOX between 9.8%–57.4% and 0.0%–97.7%, respectively. None of the hybrids showed an improvement in the inhibitory effect toward these pro‐inflammatory enzymes, compared to the parent terpenes and non‐steroidal anti‐inflammatory drugs. The docking studies allowed us to predict the potential binding modes of hybrids 6 – 15 within COX‐2 and 15‐LOX active sites. The relative affinity of the compounds inside the binding sites could be explained by forming non‐covalent interactions with most important and known amino acids reported for those enzymes. A good correlation (r² = 0.745) between docking energies and inhibition percentages against COX‐2 was found. The high inhibition obtained for compound 10 against COX‐2 was explained by hydrogen bond interactions at the enzyme binding site. New synthetic possibilities could be obtained from our in silico models, improving the potency of these hybrid compounds.     

Antihypertensive methyldopa,labetalol, hydralazine,and clonidine reversed tumour necrosis factor‐α inhibited endothelial nitric oxide synthase expression in endothelial‐trophoblast cellular networks

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor‐α (TNF‐α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre‐incubated with (or without) low dose TNF‐α (0.5 ng/mL) or TNF‐α plus soluble fms‐like tyrosine kinase‐1 (sFlt‐1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR‐8/SVneo cells were co‐cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF‐α on eNOS mRNA expression. After pre‐incubating endothelial cells with TNF‐α and sFlt‐1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF‐α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF‐α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF‐α. The anti‐angiogenic molecule sFlt‐1 may antagonise the potential benefit of these medications by interfering with the NOS pathway.     

Meloxicam inhibits fipronil‐induced apoptosis via modulation of the oxidative stress and inflammatory response in SH‐SY5Y cells

Oxidative stress and inflammatory responses have been identified as key elements of neuronal cell apoptosis. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in human neuroblastoma SH‐SY5Y cells treated with fipronil (FPN). Based on the cytotoxic mechanism of FPN, we examined the neuroprotective effects of meloxicam against FPN‐induced neuronal cell death. Treatment of SH‐SY5Y cells with FPN induced apoptosis via activation of caspase‐9 and ‐3, leading to nuclear condensation. In addition, FPN induced oxidative stress and increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Pretreatment of cells with meloxicam enhanced the viability of FPN‐exposed cells through attenuation of oxidative stress and inflammatory response. FPN activated mitogen activated protein kinase (MAPK) and inhibitors of MAPK abolished FPN‐induced COX‐2 expression. Meloxicam also attenuated FPN‐induced cell death by reducing MAPK‐mediated pro‐inflammatory factors. Furthermore, we observed both nuclear accumulation of p53 and enhanced levels of cytosolic p53 in a concentration‐dependent manner after FPN treatment. Pretreatment of cells with meloxicam blocked the translocation of p53 from the cytosol to the nucleus. Together, these data suggest that meloxicam may exert anti‐apoptotic effects against FPN‐induced cytotoxicity by both attenuating oxidative stress and inhibiting the inflammatory cascade via inactivation of MAPK and p53 signaling. Copyright © 2015 John Wiley & Sons, Ltd.     

Screening of curcumin‐derived isoxazole,pyrazoles, and pyrimidines for their anti‐inflammatory,antinociceptive, and cyclooxygenase‐2 inhibition

Curcumin has shown pharmacological properties against different phenotypes of various disease models. Different synthetic routes have been employed to develop its numerous derivatives for diverse and improved therapeutic roles. In this study, we have synthesized curcumin derivatives containing isoxazole, pyrazoles, and pyrimidines and then the synthesized molecules were evaluated for their anti‐inflammatory and antinociceptive activities in experimental animal models. Acute toxicity of synthesized molecules was evaluated in albino mice by oral administration. Any behavioral and neurological changes were observed at dose of 10 mg/kg body weight. Additionally, cyclooxygenase‐2 (COX‐2) enzyme inhibition studies were performed through in vitro assays. In vivo anti‐inflammatory studies showed that curcumin with pyrimidines was the most potent anti‐inflammatory agent which inhibited induced edema from 74.7% to 75.9%. Compounds 7 , 9 , and 12 exhibited relatively higher prevention of writhing episodes than any other compound with antinociceptive activity of 73.2%, 74.9%, and 71.8%, respectively. This was better than diclofenac sodium (reference drug, 67.1% inhibition). Similarly, COX‐2 in vitro inhibition assays results revealed that compound 12 (75.3% inhibition) was the most potent compound. Molecular docking studies of 10 , 11 , and 12 compounds in human COX‐2 binding site revealed the similar binding modes as that of other COX‐2‐selective inhibitors.     

Synthesis,characterization, molecular modelling and biological evaluation of thieno‐pyrimidinone methanesulphonamide thio‐derivatives as non‐steroidal anti‐inflammatory agents

This study reports the synthesis, molecular docking and biological evaluation of eight ( 5 ‐ 8 and 5a ‐ 8a ) newly synthesized thieno‐pyrimidinone methanesulphonamide thio‐derivatives. The synthetic route used to prepare the new isomers thioaryl and thio‐cycloesyl derivatives of the heterocyclic system 6‐phenylthieno[3,2]pyrimidinone was economically and environmentally very advantageous and characterized by the simplicity of procedure, reduction in isolation steps, purification phases, time, costs and waste production. The study in silico for the evaluation of cyclooxygenase (COX)‐1 and COX‐2 selective inhibition was carried out by AutoDock Vina, an open‐source program for doing molecular docking which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. The research in vitro for the biological evaluation was performed by using human cartilage and chondrocytes cultures treated with 10 ng/mL of interleukin‐1beta as inflammation models. The anti‐inflammatory activity of each new compound at the concentration of 10 μmol/L was determined by assaying COX‐2, inducible nitric oxide synthetase (iNOS) and intercellular adhesion molecule 1 (ICAM 1) through Western blot. The examined derivatives showed interesting pharmacological activity, and the compound N‐[2‐[2,4‐difluorophenyl)thio]‐4‐oxo‐6‐phenylthieno[3,2‐d]pyridine‐34H‐yl]methanesulphonamide ( 7 ) was excellent COX‐2 inhibitor. In agreement with the biological data, compound 7 was able to fit into the active site of COX‐2 with highest interaction energy. These results can support the design of novel specific inhibitors of COX‐2 by the comparative modelling of COX‐1 and COX‐2 enzymes with the available pharmacophore.     

Effects of Nimesulide,Acetylsalicylic Acid,Ibuprofen and Nabumetone on Cyclooxygenase‐1‐ and Cyclooxygenase‐2‐Mediated Prostanoid Production in Healthy Volunteers ex vivo

Abstract: The beneficial actions of non‐steroidal anti‐inflammatory drugs (NSAIDs) have been associated with inhibition of cyclooxygenase‐2 (COX‐2), whereas some of their adverse effects are associated mainly with inhibition of COX‐1. Selective COX‐2 inhibitors reduce the risk of gastrointestinal adverse events, but increase the risk of thromboembolic events pointing to importance of optimal COX‐1/COX‐2 inhibition in drug safety. We compared the effects of acetylsalicylic acid, ibuprofen, nabumetone and nimesulide on COX‐1 and COX‐2 pathways in healthy volunteers in an ex vivo set‐up using single oral doses commonly used to treat acute pain. In a randomized, double‐blind four‐phase cross‐over study, 15 healthy volunteers were given orally a single dose of either acetylsalicylic acid 500 mg, ibuprofen 400 mg, nabumetone 1 g or nimesulide 100 mg. Blood samples were drawn before and 1, 3, 6, 24 and 48 hr after the drug for the assessment of COX‐1 and COX‐2 activity. COX‐1 activity was measured as thromboxane₂ production during blood clotting and COX‐2 activity as endotoxin‐induced prostaglandin E₂ synthesis in blood leucocytes. The data show that after a single oral dose these four NSAIDs have different profiles of action on COX‐1 and COX‐2. As expected, acetylsalicylic acid appeared to be COX‐1‐selective and ibuprofen effectively inhibited both COX‐1 and COX‐2. Nabumetone showed only a slight inhibitory effect on COX‐1 and COX‐2. Nimesulide caused almost complete suppression of COX‐2 activity and a partial reduction of COX‐1 activity. This confirms the relative COX‐2 selectivity of nimesulide.     

Beneficial Pharmacological Effects of Levosimendan on Antioxidant Status of Acute Inflammation Induced in Paw of Rat: Involvement in Inflammatory Mediators

Levosimendan (LEVO) is a new calcium sensitizer with positive inotropic and vasodilating properties that represents a new pharmacological class of inotropic drugs that stimulate elevated cardiac output. The purpose of this study was to examine anti‐inflammatory effect and antioxidant activity of LEVO in a carrageenan (CAR)‐induced inflammatory paw oedema rat model. The CAR‐induced rat groups received LEVO 1, 2 and 3 mg/kg by intraperitonally and indomethacin (IND) 25 mg/kg by oral gavage. LEVO inhibited CAR‐induced paw oedema and suppressed the production of TNF‐α, IL‐1 and IL‐6 at doses of 2 and 3 mg/kg. In contrast to CAR‐injected paws, 2 and 3 mg/kg doses of LEVO and IND increased superoxide dismutase (SOD) activity and also both doses of LEVO, and IND decreased the 8‐isoprostaglandin F2α (8‐ISO) level. A 2 mg/kg dose of LEVO produced 39%, 46%, 61% and 64.7% anti‐inflammatory effects (p < 0.0001) for the 1st, 2nd, 3rd and 4th hours, respectively. Other results of our current study have shown that SOD and glutathione for CAR‐injected groups were lower, and 8‐ISO level was higher than those for the healthy rat group. LEVO may be provided as a pharmacological agent in the prevention or treatment of diseases in which acute or chronic inflammation occurs based on a pathogenic factor.     

Antinociceptive and Anti‐Inflammatory Effects of Ketamine and the Relationship to Its Antidepressant Action and GSK3 Inhibition

Ketamine (KET), a NMDA antagonist, exerts an antidepressant effect at subanaesthetic doses and possesses analgesic and anti‐inflammatory activities. We evaluated the involvement of KET antinociceptive and anti‐inflammatory effects with its antidepressant action. Male Swiss mice were subjected to formalin, carrageenan‐induced paw oedema and forced swimming tests, for assessing antinociceptive, anti‐inflammatory and antidepressant effects. The treatment groups were as follows: control, KET (2, 5 and 10 mg/kg), lithium (LI: 5 mg/kg) and KET2 + LI5 combination. Immunohistochemistry analyses (TNF‐α, iNOS, COX‐2 and GSK3) in oedematous paws were performed. KET5 and KET10 reduced licking times in neurogenic (22 and 38%) and inflammatory (67 and 78%) phases of the formalin test, respectively, as related to controls. While LI5 inhibited the second phase by 24%, the licking time was inhibited by 26 and 59% in the KET2 + LI5 group (first and second phases). Furthermore, oedema volumes were reduced by 37 and 45% in the KET5 and KET10 groups, respectively. Oedema reductions were 29% in the LI5 group and 48% in the KET2 + LI5 group. In the forced swimming test, there were 23, 38 and 53% decreases in the immobility time in KET2, KET5 and KET10 groups, respectively. While LI5 caused no significant effect, decreases of 52% were observed with KET2 + LI5. KET also decreased TNF‐α, iNOS, COX‐2 and GSK3 immunostainings in oedematous paws, effects intensified with KET2 + LI5. We showed that KET presents antinociceptive and anti‐inflammatory effects associated with its antidepressant response. Furthermore, our results indicate the close involvement of GSK3 inhibition and blockade of inflammatory responses, in the antidepressant drug effect.     

Gastric acid is the key modulator in the pathogenesis of non‐steroidal anti‐inflammatory drug‐induced ulceration in rats

1. In the present study, we investigated the role of gastric acid (GA) secretion on non‐steroidal anti‐inflammatory drug (NSAID)‐induced ulcerogenesis in vivo. Rats were administered single oral doses of selective cyclo‐oxygenase (COX)‐1 (SC‐560; 2.5 mg/kg), COX‐2 (DFU; 25 mg/kg) or non‐selective COX (indomethacin; 25 mg/kg) inhibitors. Three groups (basal, histamine‐stimulated and histamine with lansoprazole) were pylorus ligated 2 h after inhibitor administration and killed 2 h later. Another group without pylorus ligation received only inhibitors and was killed after 18 h. 2. At 4 h, indomethacin increased the ulcer index (UI) and myeloperoxidase (MPO) activity in basal and histamine‐stimulated states, whereas SC‐560 only increased MPO activity. Histamine‐stimulated, but not basal, GA was further enhanced by indomethacin and SC‐560 via increased proton pump expression. Lansoprazole (10 mg/kg) reduced the UI, MPO activity and GA to basal levels with SC‐560 and DFU and to near basal with indomethacin. Indomethacin and SC‐560 significantly inhibited prostaglandin (PG) E₂, without significantly affecting COX‐1 and COX‐2 expression. Although DFU inhibited PGE₂ by one‐third, it did not affect COX expression. 3. At 18 h, indomethacin significantly increased the UI and MPO activity, whereas PGE₂ synthesis was less inhibited, indicating a return to control levels. In contrast, PGE₂ synthesis was higher than control with SC‐560. Furthermore, COX‐2 expression was significantly elevated with indomethacin and SC‐560, explaining the source of augmented PGE₂ synthesis. Proton pump expression remained elevated, comparable with 4 h levels, with indomethacin and SC‐560. However, DFU had no significant effect on the aforementioned parameters. 4. The data suggest that NSAID‐induced ulcerogenesis is dependent on the amount of GA secretion derived from increased proton pump expression and requires inhibition of both COX‐1 and COX‐2.     

Immunocompatibility and Toxicity Studies of Poly‐L‐Lysine Nanocapsules in Sprague–Dawley Rats for Drug‐Delivery Applications

Poly‐L‐Lysine (PLL) nanocapsules are the emerging drug‐delivery vehicle for the therapeutics of targeted diseases. The study was designed for the synthesis and characterization of PLL nanocapsules and to know its immunocompatibility and toxicity behavior for in vivo drug‐delivery applications. Alteration in hematologic parameters, immunomodulatory gene expression by RT‐PCR studies, toxicity markers status, immunoblotting of major inflammatory marker proteins, and histopathologic studies from major tissues of rat after intravenous administration of PLL nanocapsules after 30 days were assessed. In vivo toxicity markers activity, hematologic parameters alteration, and RT‐PCR analysis of important immunomodulatory genes such as monocyte chemotactic protein‐1(MCP 1), tumor necrosis factor‐alpha (TNF‐α), Intercellular adhesion molecule‐1 (ICAM‐1), and interleukin‐6 (IL‐6) showed least changes when compared with control. The immunoblotting of major inflammatory markers such as cyclooxygenase‐2 (COX‐2), lipo‐oxygenase‐15 (LOX‐15), and nitric oxide synthase (NOS) found have least expression showing the immunocompatibility of PLL nanocapsules. Histopathologic studies of important tissues showed almost normal architecture after treatment using different concentration of PLL nanocapsules after the experimental period. The results showed a promising outcome and further confirmed the immunocompatibility and non‐toxicity of PLL nanocapsules in vivo for drug‐delivery applications.     

NF‐κB/p53‐activated inflammatory response involves in diquat‐induced mitochondrial dysfunction and apoptosis

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N‐acetyl‐cystein attenuated the cytotoxicity of caspase‐3 pathways. These results demonstrate that diquat‐induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF‐κB and p53 proteins. Importantly, an inhibitor of NF‐κB nuclear translocation blocked the increase of p53. Both NF‐κB and p53 inhibitors also blocked the diquat‐induced inflammatory response. Pretreatment of cells with meloxicam, a COX‐2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat‐induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF‐κB‐mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation‐related neurodegenerative disease.     

Effect of inflammatory factor‐induced cyclo‐oxygenase expression on the development of reperfusion‐related no‐reflow phenomenon in acute myocardial infarction

No reflow after reperfusion therapy for myocardial infarction is a strong predictor of clinical outcome. Increased levels of inflammatory factors, including C‐reactive protein (CRP), in patients with acute myocardial infarction (AMI) undergoing primary percutaneous coronary intervention (PCI) may affect myocardial perfusion. However, why the no‐reflow phenomenon increases in inflammation stress after PCI is not clear. The aim of the present study was to determine the effects and molecular mechanisms underlying the effects of CRP on the expression of cyclo‐oxygenase (COX) on the development of the no‐reflow phenomenon. There was a significant increase in plasma levels of CRP and interleukin (IL)‐6 in no‐reflow patients, suggesting that inflammatory factors play an important role in the development of the no‐reflow phenomenon. The mechanisms involved were further evaluated after reperfusion in a rat model mimicking the no‐reflow phenomenon. Compared with normal reflow rats, there were significant increases in both COX‐1 and COX‐2 in cardiac tissue from no‐reflow rats. The COX inhibitor indomethacin (5 mg/kg, i.p.) significantly reduced the no‐reflow area. In another series of experiments, human coronary artery endothelial cells (HCAEC) were treated with CRP at clinically relevant concentrations (5–25 μg/mL). C‐Reactive protein significantly increased COX‐1 and COX‐2 levels in a time‐ and concentration‐dependent manner. In addition, extracellular signal‐regulated kinase (ERK) and Jun N‐terminal kinase (JNK) were activated in CRP (5, 10, 25 μg/mL)‐treated HCAEC cultures. Furthermore, the ERK inhibitor pd98059 (30 μmol/L) and the JNK inhibitor sp600125 (10 μmol/L) blocked CRP‐induced COX‐1 and COX‐2 expression for 12 h. Together, the findings of the present study suggest that CRP can promote the development of the no‐reflow phenomenon by increasing COX‐1 and COX‐2 expression, which is regulated, in part, via ERK and JNK activity.