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.     

LE135, a retinoid acid receptor antagonist,produces pain through direct activation of TRP channels

Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RAR_β, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels.Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours.Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies.Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels.     

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.     

Comparison of cyclooxygenase inhibitory activity and ocular anti-inflammatory effects of ketorolac tromethamine and bromfenac sodium

ABSTRACT

Objective: To compare the cyclooxygenase (COX) activity and anti-inflammatory effects of the nonsteroidal anti-inflammatory drugs (NSAIDs) ketorolac tromethamine (ketorolac) and bromfenac sodium (bromfenac).

Methods: Cyclooxygenase activity and selectivity was determined in vitro by measuring prostaglandin E₂ (PGE₂) production following incubation of varying concentrations of NSAID with human recombinant COX‐1 or COX‐2 and arachidonic acid. Anti-inflammatory effects were evaluated in a rabbit model in which an ocular inflammatory response was induced by intravenous injection of 10?µg/kg lipopolysaccharide (LPS). In study animals, one eye was treated with 50?µL (+/–) ketorolac 0.4% (Acular LS) or bromfenac 0.09% (Xibrom) and the other eye with 50?µL buffered saline. In control animals, both eyes were treated with vehicle. All animals were treated twice: 2 hours and 1 hour before LPS.

Main outcome measures: PGE₂ production in vitro, measured by enzyme immunoassay; fluorescein isothiocyanate (FITC)-dextran leakage into the anterior chamber, measured by fluorophotometry; aqueous PGE₂ levels in vivo, measured by ELISA immunoassay.

Results: Ketorolac was six times more active against COX‐1 (?IC₅₀ = 0.02?µM) than COX‐2 (?IC₅₀ = 0.12?µM) while bromfenac was ≈ 32 times more active against COX‐2 (?IC₅₀ = 0.0066?µM) than COX‐1 (?IC₅₀ = 0.210?µM). In the animal model, both drugs resulted in nearly complete inhibition of FITC-dextran leakage and PGE₂ production in the anterior chamber of treated eyes. There was also a 79% inhibition (?p < 0.001) of FITC-dextran leakage in the contralateral eyes of bromfenac-treated rabbits, and a 22.5% inhibition (not statistically significant) in the contralateral eyes of ketorolac-treated rabbits.

Conclusions: Ketorolac is relatively COX‐1 selective while bromfenac is potently selective for COX‐2 over COX‐1. In the animal model, both ketorolac 0.4% and bromfenac 0.09% demonstrated maximal anti-inflammatory activity in treated eyes. Only bromfenac 0.09% had a significant effect on the contralateral eye, suggesting possible systemic absorption of this drug.     

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.     

Diphlorethohydroxycarmalol,Isolated from Ishige okamurae,Increases Prostaglandin E2 through the Expression of Cyclooxygenase-1 and -2 in HaCaT Human Keratinocytes

Prostaglandin (PG) E₂, the most abundant prostaglandin in the human body, is synthesized from arachidonic acid via the actions of cyclooxygenase (COX) enzymes. PGE₂ exerts homeostatic, cytoprotective, inflammatory, and in some cases anti-inflammatory effects. Also, it has been reported that PGE₂ is involved in hair growth. Diphlorethohydroxycarmalol (DPHC) is a phlorotannin compound isolated from the brown algae Ishige okamurae, with various biological activities in vitro and in vivo. In this study, the biological effect and mechanism of action of DPHC on prostaglandin synthesis in HaCaT human keratinocytes was examined. The results showed that, in these cells, DPHC significantly and dose-dependently induced PGE₂ synthesis by increasing the protein and mRNA levels of COX-1 and COX-2. Interestingly, DPHC-induced COX-1 expression preceded that of COX-2. Also, while both rofecoxib and indomethacin inhibited PGE₂ production, the latter was seems to be the more potent. From above results, we can expect that DPHC has some beneficial effects via increasing of PGE₂ production.     

Activation of transient receptor potential A1 by a non-pungent capsaicin-like compound, capsiate

BACKGROUND AND PURPOSE

Capsiate is produced by ‘CH-19 Sweet’ (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels.

EXPERIMENTAL APPROACH

Using Ca²⁺ imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons.

KEY RESULTS

We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC₅₀ for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.     

Cloning and functional characterization of dog transient receptor potential vanilloid receptor-1 (TRPV1)

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.     

Spirodela polyrhiza extract modulates the activation of atopic dermatitis-related ion channels,Orai1 and TRPV3, and inhibits mast cell degranulation

Context: Spirodela polyrhiza (L.) Schleid. (Lemnaceae), Spirodelae Herba (SH), has been known to relieve inflammation, urticaria and skin symptoms including pruritus, eczema and rash.

Objective: The effects of SH extract on two calcium ion channels, Orai1 and TRPV3, and their potential as novel therapeutics for atopic dermatitis (AD) were investigated. The regulatory role of Orai1 on mast cell degranulation was evaluated.

Materials and methods: The dried leaves of SH were extracted by 70% methanol. Effects of SH extract (100?μg/mL) in an HEK293T cell line overexpressing human Orai1 or TRPV3 were assessed. Ion channel modulation in transfected HEK293T cells was measured using a conventional whole-cell patch-clamp technique. IgE-antigen complex-stimulated mast cell degranulation was measured by β-hexosaminidase assay with morphological observation after treatment with 20, 50 and 100?μg/mL SH extract.

Results: SH extract (100?μg/mL) significantly inhibited Orai1 activity (63.8?±?0.97%) in Orai1-STIM1 co-overexpressed HEK293T cells. SH extract significantly increased TRPV3 activity (81.29?±?0.05% at ?100?mV) compared with the positive control 2-APB (100?μM), which induced full activation. SH extract inhibited degranulation in IgE-antigen complex-stimulated RBL-2H3 mast cells by decreasing β-hexosaminidase activity (3.14?±?0.03, 2.56?±?0.12 and 2.29?±?0.08?mU/mg, respectively).

Conclusion: Our results suggested that SH extract could treat abnormal skin barrier pathologies in AD through modulation of the activities of the calcium ion channels Orai1 and TRPV3 and inhibition of mast cell degranulation. This is the first report of an herbal effect on the modulation of ion channels associated with skin barrier disruption in AD pathogenesis.     

New 1‐Benzyl‐4‐hydroxypiperidine Derivatives as Non‐imidazole Histamine H3 Receptor Antagonists

A series of 1‐benzyl‐4‐(3‐aminopropyloxy)piperidine and 1‐benzyl‐4‐(5‐aminopentyloxy)piperidine derivatives has been prepared. The 1‐benzyl‐4‐hydroxypiperidine derivatives obtained were evaluated for their affinities at recombinant human histamine H₃ receptor, stably expressed in HEK 293T cells. All compounds investigated show moderate to pronounced in‐vitro affinities. The most potent antagonists in this series 9b2 (hH₃R, pK_i = 7.09), 9b1 (hH₃R, pK_i = 6.78), 9b5 (hH₃R, pK_i = 6.99), and 9b6 (hH₃R, pK_i = 6.97) were also tested in vitro as H₃ receptor antagonists – the electrically evoked contraction of the guinea‐pig jejunum. The histaminergic H₁ antagonism of selected compounds 9b1 , 9b2 , and 9b4 – 9b6 was established on the isolated guinea‐pig ileum by conventional methods; the pA₂ values were compared with the potency of pyrilamine. The compounds did not show any H₁ antagonistic activity (pA₂ < 4; for pyrilamine pA₂ = 9.53).     

皮肤角质形成细胞中TRPV1受体激活后的蛋白质组差异分析

目的 研究皮肤角质形成细胞中辣椒素受体TRPV1激活后细胞中蛋白质组学的变化,并鉴定差异蛋白质。方法 皮肤角质形成细胞株HaCaT细胞经辣椒素(CAP)刺激24 h后,提取蛋白样品,采用双向凝胶电泳技术分离HaCaT细胞总蛋白,用PDQuest软件分析对照组和CAP刺激组蛋白组图谱的表达差异点,再运用串联飞行时间质谱(MALDI-TOF-MS)鉴定差异蛋白。结果 CAP刺激HaCaT细胞后,磷酸甘油醛异构酶、烯醇化酶、6-磷酸葡萄糖酸内酯酶和组织蛋白酶D蛋白表达水平增高,角蛋白14和60S酸性核糖体蛋白P2表达量降低,这些蛋白可能在TRPV1激活后影响细胞功能的过程中有重要作用。结论 利用蛋白质组学技术,研究皮肤角质形成细胞株HaCaT细胞中TRPV1受体激活后蛋白表达差异,为研究TRPV1受体的作用机制,以及为研发针对性药物,提供新的线索和方向。     

Scorpion Toxin,BmP01, Induces Pain by Targeting TRPV1 Channel

The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation.     

Capsaicin causes protein synthesis inhibition and microtubule disassembly through TRPV1 activities both on the plasma membrane and intracellular membranes

TRPV1 is a non-selective cationic channel that is activated by capsaicin, acidic pH and thermal stimuli. Sustained TRPV1 channel activation causes severe cytotoxicity that leads to cell death. In this study, we investigated the mechanisms of capsaicin-induced cytotoxicity in HEK293 cells stably expressing TRPV1 with a focus on protein synthesis regulation and cytoskeleton reorganization. Capsaicin inhibited protein synthesis in TRPV1-expressing HEK cells with an IC(50) of 15.6nM and depolymerized microtubules within 10min after exposure. These effects were completely blocked by pretreatment of cells with the TRPV1 antagonist A-425619, both in the presence and absence of extracellular calcium. Protein synthesis inhibition induced by capsaicin was not a result of eIF2alpha hyperphosphorylation, but rather closely correlated with cytosolic calcium elevation caused by calcium flux through cell surface and intracellular TRPV1, and/or ER calcium depletion through intracellular TRPV1. Microtubule dependent cell process shrinkage may serve as a mechanism for rapid alteration of the neurotransmission network upon TRPV1 activation. Taken together, the present studies demonstrate that intracellular pool of TRPV1 plays an important role in regulating cell morphology and viability upon receptor activation.     

The M-channel blocker linopirdine is an agonist of the capsaicin receptor TRPV1

Linopirdine is a well known blocker of voltage-gated potassium channels from the Kv7 (or KCNQ) family that generate the so called M current in mammalian neurons. Kv7 subunits are also expressed in pain-sensing neurons in dorsal root ganglia, in which they modulate neuronal excitability. In this study we demonstrate that linopirdine acts as an agonist of TRPV1 (transient receptor potential vanilloid type 1), another ion channel expressed in nociceptors and involved in pain signaling. Linopirdine induces increases in intracellular calcium concentration in human embryonic kidney 293 (HEK293) cells expressing TRPV1, but not TRPA1 and TRPM8 or in wild-type HEK293 cells. Linopirdine also activates an inward current in TRPV1-expressing HEK293 cells that is almost completely blocked by the selective TRPV1 antagonist capsazepine. At low concentrations linopirdine sensitizes both recombinant and native TRPV1 channels to heat, in a manner that is not prevented by the Kv7-channel opener flupirtine. Taken together, these results indicate that linopirdine exerts an excitatory action on mammalian nociceptors not only through inhibition of the M current but also through activation of the capsaicin receptor TRPV1.     

Nootkatol prevents ultraviolet radiation-induced photoaging via ORAI1 and TRPV1 inhibition in melanocytes and keratinocytes

Skin photoaging occurs due to chronic exposure to solar ultraviolet radiation (UV), the main factor contributing to extrinsic skin aging. Clinical signs of photoaging include the formation of deep, coarse skin wrinkles and hyperpigmentation. Although melanogenesis and skin wrinkling occur in different skin cells and have different underlying mechanisms, their initiation involves intracellular calcium signaling via calcium ion channels. The ORAI1 channel initiates melanogenesis in melanocytes, and the TRPV1 channel initiates MMP-1 production in keratinocytes in response to UV stimulation. We aimed to develop a drug that may simultaneously inhibit ORAI1 and TRPV1 activity to help prevent photoaging. We synthesized nootkatol, a chemical derivative of valencene. TRPV1 and ORAI1 activities were measured using the whole-cell patch-clamp technique. Intracellular calcium concentration [Ca²⁺]_i was measured using calcium-sensitive fluorescent dye (Fura-2 AM). UV-induced melanin formation and MMP-1 production were quantified in B16F10 melanoma cells and HaCaT cells, respectively. Our results indicate that nootkatol (90 μM) reduced TRPV1 current by 94% ± 2% at –60 mV and ORAI1 current by 97% ± 1% at –120 mV. Intracellular calcium signaling was significantly inhibited by nootkatol in response to ORAI1 activation in human primary melanocytes (51.6% ± 0.98% at 100 μM). Additionally, UV-induced melanin synthesis was reduced by 76.38% ± 5.90% in B16F10 melanoma cells, and UV-induced MMP-1 production was reduced by 59.33% ± 1.49% in HaCaT cells. In conclusion, nootkatol inhibits both TRPV1 and ORAI1 to prevent photoaging, and targeting ion channels may be a promising strategy for preventing photoaging.     

Anti-inflammatory potential of Phaseolus calcaratus Roxburgh, a oriental medicine, on LPS-stimulated RAW 264.7 macrophages

Objectives The seed of Phaseolus calcaratus Roxburgh (PHCR) has traditionally been used as a herbal medicine, considered to have anti‐inflammatory potential. Here we examined the ability of PHCR seed extract to inhibit inflammatory responses of macrophages to bacterial toxin and the mechanism involved. Methods In the present study, we prepared four fractions from an ethanol extract of PHCR seed and investigated their effects on the production of nitric oxide and cytokines, and the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Key findings The fractions inhibited LPS‐induced nitric oxide production and cyclooxygenase‐2 (COX‐2) expression in the cells. The ethyl acetate fraction at 100 µg/ml almost completely suppressed NO production, iNOS and COX‐2 expression, and TNF‐α and IL‐6 secretion in cells stimulated with LPS. The fraction also inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) and p38 in LPS‐stimulated cells with the attendant suppression of IκBα nuclear translocation and nuclear factor (NF)‐κB activation. Furthermore, PHCR seed extracts contained a large number of phenolic compounds having antioxidant potentials against 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radicals and hydroxyl radicals. We identified catechin‐7‐O‐β‐d ‐glucopyranoside as one of the active compounds responsible for the biological activity of PHCR seed extract. Conclusions These results suggest for the first time that ethanol extracts from PHCR seed have anti‐inflammatory potential on LPS‐stimulated macrophages through the down‐regulation of ERK/p38‐ and NF‐κB‐mediated signalling pathways.     

Analysing the role of COX-2 in acute oesophagitis and in melatonin-exerted protection against experimental reflux oesophagitis in rats

Objectives Cyclooxygenase(COX)‐2 is implicated in variety of pathophysiological processes, although its role in acute reflux oesophagitis is debatable. This study was designed to evaluate the role of COX‐2 during oesophagitis and in melatonin‐elicited protection in rats. Methods Reflux oesophagitis was induced in rats by ligating the pyloric end and the limiting ridge of the stomach for 5 h. Celecoxib (COX‐2 blocker; 10 mg/kg), 16,16‐dimethyl prostaglandinE₂ (dmPGE₂; a synthetic analogue of PGE₂; 10 µg/kg), melatonin (20 and 40 mg/kg) and omeprazole (10 mg/kg) were given intra‐peritoneally 45 min before induction of oesophagitis in rats. Alterations in COX‐1 and 2 gene expression and protein levels level were analysed via RT‐PCR and Western blotting, respectively. Mucosal PGE₂ level and myeloperoxidase (MPO) activity were measured using an enzyme immunoassay (EIA) kit and spectrophotometrically, respectively. Key findings COX‐2 over‐expression during reflux oesophagitis promotes inflammation of the oesophagus as celecoxib pretreatment significantly reduced tissue damage and MPO activity in rats with reflux oesophagitis (RE‐rats). By contrast, dmPGE₂ pretreatment significantly exacerbated tissue injury and simultaneously increased COX‐2 expression, PGE₂ levels and MPO activity in RE‐rats. Further, melatonin pretreatment significantly reduced the tissue injury, COX‐2 over‐expression, PGE₂ level and MPO activity in RE‐rats. Melatonin offered more potent suppression of COX‐2, PGE₂ and MPO activity than the proton‐pump inhibitor omeprazole; however, both reduced the lesion injury to a similar extent. Melatonin at a dose of 20 mg/kg failed to inhibit significantly the dmPGE₂‐induced tissue damage, COX‐2 expression, PGE₂ level and MPO activity in RE‐rats while at a higher dose of 40 mg/kg it significantly attenuated these changes. Conclusion Our results suggest that COX‐2 plays an important pro‐inflammatory role during acute reflux oesophagitis in rats and its inhibition contributes significantly to melatonin‐exerted protection against reflux oesophagitis.     

Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma (PPARγ)

Objectives Peroxisome proliferator‐activated receptor gamma (PPARγ) plays a critical role in regulation of diverse biological processes, including lipid metabolism and adipogenesis, cell division and apoptosis, and is involved in variety of disease conditions, such as obesity, atherosclerosis, inflammation and tumour. Developing a cell‐based reporter gene model targeting PPARγ would be useful to screen human PPARγ agonists that could be beneficial to patients with these diseases. Methods We stably co‐transfected human embryonic kidney (HEK) cell line 293T cells with phPPARγ‐IRES2‐EGFP vector to express human PPARγ (hPPARγ), a reporter vector pPPRE×3‐TK‐LUC, and control vector pRL‐CMV. The efficiency of the co‐transfection was evaluated with flow cytometry of hPPARγ expressing cells. Specificity of hPPARγ activity was determined by dual luciferase reporter assay of co‐transfected cells exposed to PPARγ agonist rosiglitazone, PPARα agonist WY14643 and retinoic acid receptor alpha (RARα) agonist all‐trans‐retinoic acid (ATRA). Key findings The phPPARγ‐IRES2‐EGFP co‐transfected HEK293T cells showed concentration‐ and time‐dependent luciferase induction upon exposure to the rosiglitazone, while WY14643 and ATRA were unable to activate the co‐transfected HEK293T cells. Conclusions These data indicated that the HEK293T cells could be stably transfected with hPPARγ. This cell‐based drug screening platform could be used targeting specific nuclear receptor of hPPARγ with effectiveness and specificity for hPPARγ agonists discovery.     

Synthesis and Anti‐neuroinflammatory Activity of Lactone Benzoyl Hydrazine and 2‐nitro‐1‐phenyl‐1H‐Indole Derivatives as p38α MAPK Inhibitors

Inhibition of p38 mitogen‐activated protein kinases (MAPKs) would allow significant modulation of the neuroinflammation condition associated with Alzheimer's disease (AD). Inspired from the pharmacophore of natural NF‐κB and p38α MAPK inhibitor 5,6‐dehydrokawain and p38α MAPK inhibitors 1a, 1‐pyrazolyl‐3‐(4‐((2‐anilinopyrimidin‐4‐yl)oxy)napththalen‐1‐yl)ureas, and 1b , a class of indole–pyrimidinyl compounds which were patented respectively, we designed, de novo synthesized, and evaluated two kinds of novel series of lactone benzoyl hydrazine derivatives and 2‐nitro‐1‐phenyl‐1H‐indole derivatives in an effort to develop pharmacologically tractable agents to alleviate the progression of AD. Fourteen of the seventeen synthesized compounds exhibit significant inhibitory effect on the nitric oxide (NO) production induced by lipopolysaccharide (LPS)‐induced microglia activation with IC₅₀ less than the control 5,6‐dehydrokawain. Notably, compound 27 , 6‐methoxy‐2‐nitro‐1‐(1H‐1, 2, 3‐triazol‐1‐yl)‐1H‐indole, with IC₅₀ values of 1.6  μ m can markedly inhibit p38 α MAPK and NO release in BV‐2 microglial cells. The molecular dynamic (MD) simulations demonstrate that compound 27 inhibits p38 α MAPK through binding to the Glu71 and Asp168 residues. Moreover, in vitro study shows that all compounds can easily cross the blood–brain barrier (BBB) and did not exhibit any acute cellular toxicity checked by MTT assay. These investigations provide promising chemical lead candidate as anti‐neuroinflammatory agents for AD.