COX-2 Activity Research Articles

Pyrimidine Derivatives as Selective COX-2 Inhibitors with Anti-Inflammatory and Antioxidant Properties

Pyrimidine derivatives exhibit a wide range of biological activities, including anti-inflammatory properties. The aim of this study was to investigate the effects of tested pyrimidine derivatives on the activity of cyclooxygenase isoenzymes (COX-1 and COX-2), antioxidant properties, and their ability to inhibit the growth of inflammatory cells. In vitro tests were conducted to assess the ability of pyrimidine derivatives L1–L4 to inhibit COX-1 and COX-2 activity using the TMPD oxidation assay (N,N,N′,N′-tetramethyl-p-phenylenediamine). The compounds’ ability to inhibit the growth of lipopolysaccharide (LPS)-stimulated THP-1 (human leukemia monocytic) monocyte cells and their impact on reactive oxygen species (ROS) levels in an inflammatory model were also evaluated. The binding properties of human serum albumin (HSA) were assessed using UV–Vis spectroscopy, circular dichroism (CD), and isothermal titration calorimetry (ITC). Among the tested pyrimidine derivatives, L1 and L2 showed high selectivity towards COX-2, outperforming piroxicam and achieving results comparable to meloxicam. In the sulforhodamine B (SRB) assay, L1 and L2 demonstrated dose-dependent inhibition of LPS-stimulated THP-1 cell growth. Additionally, ROS assays indicated that these compounds reduced free radical levels, confirming their antioxidant properties. Binding studies with albumin revealed that L1 and L2 formed stable complexes with HSA. These results suggest that these compounds could serve as a basis for further research into anti-inflammatory and anticancer drugs with reduced toxicity.

Calcitonin gene-related peptide promotes epithelial reparative and anti-colitic functions of IL-4 educated human macrophages.

Interleukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn's disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function. Thus, human (MIL4)s were treated with CGRP and the cells phagocytotic, epithelial wound repair and anti-colitic functions were assessed. Initial studies confirmed up-regulation of expression of the CGRP receptor, which was localized to the cell surface and was functional as determined by CGRP-evoked increases in cAMP. M(IL4,CGRP)s had increased mannose receptor (CD206) and FcgRIIa (CD32a) mRNA expression, a subtle, but significant increase in phagocytosis, and decreased chemokine production following exposure to E. coli. When delivered systemically (106 cells, ip.) to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same blood donor. Conditioned medium (CM) from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM in the human CaCo2 epithelial cell line in vitro wounding assay. Moreover, M(IL4,CGRP)s displayed increased cyclooxygenase (COX)-1 and prostaglandin D2, and CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers. These data confirm the human M(IL4)s' anti-colitic effect that was enhanced by CGRP, and may be partially dependent on macrophage COX1/PDG2 activity. Thus, input from neurone-derived molecules is a local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer.

In Vitro Evaluation of Pharmacokinetic Properties of Selected Dual COX-2 and 5-LOX Inhibitors.

Evaluation of pharmacokinetic properties is a significant step at the early stages of drug development. In this study, an in vitro evaluation of the pharmacokinetic properties of five newly synthesized compounds was performed. These compounds belong to N-hydroxyurea and hydroxamic acid derivatives and analogs of NSAIDs indomethacin, flurbiprofen, diclofenac, ibuprofen, and naproxen (compounds 1, 2, 3, 11, and 12, respectively) with dual COX-2 and 5-LOX inhibitory activity. Two in vitro methods (biopartitioning micellar chromatography (BMC) and PAMPA) were used to evaluate passive gastrointestinal absorption, while high-performance affinity chromatography (HPAC) and differential pulse voltammetry (DPV) were used to evaluate binding to human serum albumin (HSA). The introduction of N-hydroxyurea and hydroxamic acid groups into the structure of NSAIDs decreases both expected passive gastrointestinal absorption (BMC k values were from 3.02 to 9.50, while for NSAIDs were from 5.29 to 13.36; PAMPA -logPe values were between 3.81 and 4.76, while for NSAIDs were ≤3.46) and HSA binding (HPAC logk values were from 2.03 to 9.54, while for NSAIDs were ≥11.03; DPV peak potential shifts were between 7 and 34, while for NSAIDs were ≥54). Structural modifications of all tested compounds that increase lipophilicity could be considered to enhance their passive gastrointestinal absorption. Considering lower expected HSA binding and higher lipophilicity of tested compounds compared to corresponding NSAIDs, it can be expected that the volume of distribution of compounds 1, 2, 3, 11, and 12 will be higher. Reduced HSA binding may also decrease interactions with other drugs in comparison to corresponding NSAIDs. All tested compounds showed significant microsomal instability (25.07-58.44% decrease in concentration) in comparison to indomethacin (14.47%) and diclofenac (20.99%).

Structure-Activity relationship of 5‑butyl‑4,6-dimethyl-2-{[4-(o-fluorophenyl)-1-piperazinyl]-2-oxoethyl}-pyrrolo[3,4-c]pyrrole-1,3(2H,5H)‑dione: An experimental and theoretical study

Synthesis of 5‑butyl‑4,6-dimethyl-2-{[4-(o-fluorophenyl)-1-piperazinyl]-2-oxoethyl}-pyrrolo[3,4-c]pyrrole-1,3(2H,5H)‑dione 3 is reported. The compound was obtained by direct alkylation of 3,4-pyrrolodicarboximide 1 with (1-chloroacetyl-4-(o-fluorophenyl)piperazine) 2 in DMF. Its physico-chemical features were characterized by 1H NMR, 13C NMR, FT-IR, MS spectra, and elemental analysis. Single-crystal X-ray diffraction was also recorded. A colorimetric inhibitor screening assay was used to determine its inhibitory potency towards COX-1 and COX-2. It was found that the compound exhibits anti-inflammatory activity higher than the reference structure – Meloxicam (MXM). According to the experimental results, the tested compound inhibited the activity of COX-1 and COX-2 respectively. In the next step, molecular docking was performed to estimate the binding affinity of compound 3 and commercially available reference compounds for COX-1/COX-2 enzymes. It was found that the binding affinity of 3 is higher than for the reference compounds. Quantum Theory of Atoms In Molecules (QTAIM) and Reduced Density Gradient (RDG) methods were used to reveal non-covalent interactions present in the COX-1/COX-2 binding pocket. Finally, Born-Oppenheimer molecular dynamics (BOMD) based on semiempirical forces was carried out to gain insight into the dynamic features of the studied complexes and confirmed the fact that the weak hydrogen bonds play the most important role in stabilizing the ligand conformation for the studied COX-2 complexes with compound 3 and Rofecoxib.

Pharmacological assessment of delphinidin in counteracting polystyrene microplastic induced renal dysfunction in rats

Polystyrene microplastics (PSMP) are toxic environmental contaminants which can damage various body organs including kidneys. Delphinidin (DEL) is a potential anthocyanidin flavonoid with significant pharmacological benefits. This research was conducted to analyze the protective effect of DEL to avert PSMP prompted renal dysfunction. Rats (n = 24) were divided into 4 separate groups: Control, PSMP (0.01 mgkg−1), PSMP (0.01 mgkg−1) + DEL (25 mgkg−1) and only DEL (25 mgkg−1). Our results showed that PSMP exposure reduced the expressions of Nrf-2 and antioxidant genes while increasing the expression of Keap1. Besides, PSMP intoxication escalated the level of kidney injury markers (urea, KIM-1, creatinine and NGAL) while inducing substantial reduction in the levels of creatinine clearance. Moreover, PSMP significantly reduced the levels of GSH, GST, SOD, HO-1, CAT, GSR, GPx while escalating MDA and ROS. Conversely, inflammatory biomarkers including IL-1β, TNF-α, NF-kB, IL-6 and COX-2 activity were increased due to PSMP intoxication. Our results showed that PSMP administration increased the expressions of Bax and caspase-3 while decreasing the expression of Bcl-2. However, DEL treatment significantly restored the PSMP-induced renal impairments. Therefore, it is suggested that DEL could be used as a therapeutic compound to alleviate PSMP-induced kidney damage in rats, possibly due to its strong pharmacological properties.

Bis-chalcones obtained via one-pot synthesis as the anti-neurodegenerative agents and their effect on the HT-22 cell line

In the area of research on neurodegenerative diseases, the current challenge is to search for appropriate research methods that would detect these diseases at the earliest possible stage, but also new active structures that would reduce the rate of the disease progression and minimize the intensity of their symptoms experienced by the patient. The chalcones are considered in the context of candidates for new drugs dedicated to the fight against neurodegenerative diseases. The synthesis of bis-chalcone derivatives (3a-3d), as aim molecules was performed. Their structures were established by applying 1H NMR, 13C NMR, MS, FT-IR and UV–Vis spectra. All bis-chalcones were synthesized from terephthalaldehyde and appropriate aromatic ketone as substrates in the Claisen-Schmidt condensation method and evaluated in the biological tests and in silico analysis. Compounds exerted antioxidant activity using the HORAC method (3a-3d) and decreased the activities of GPx, COX-2 (3b-3d), GR (3a-3c) and CAT (3a,3b). The high anti-neurodegenerative potential of all four bis-chalcones was observed by inhibition of acetyl- (AChE) and butyrylcholinesterase (BChE) and a positive effect on the mouse hippocampal neuronal HT-22 cell line (LDH release and PGC-1α, PPARγ and GAPDH protein expression). TD-DFT method (computing a number of descriptors associated with HOMO–LUMO electron transition: electronegativity, chemical hardness and potential, first ionization potential, electron affinity) was employed to study the spectroscopic properties. This method showed that the first excited state of compounds was consistent with their maximum absorption in the computed UV–Vis spectra, which showed good agreement with the experimental spectrum using PBE1PBE functional. Using in silico approach, interactions of bis-chalcones with selected targets (aryl hydrocarbon receptor (AhR) PAS-A Domain, ligand binding domain of human PPAR-γ, soman-aged human BChE-butyrylthiocholine complex, Torpedo californica AChE:N-piperidinopropyl-galanthamine complex and the COX-2-celecoxib complex) were characterized. Results obtained in in silico models were consistent with in vitro experiments.

In-silico based Designing of benzo[d]thiazol-2-amine Derivatives as Anal-gesic and Anti-inflammatory Agents.

Benzo[d]thiazoles represent a significant class of heterocyclic com-pounds renowned for their diverse pharmacological activities, including analgesic and anti-inflammatory properties. This molecular scaffold holds substantial interest among medicinal chemists owing to its structural versatility and therapeutic potential. Incorporating the benzo[d]thiazole moiety into drug molecules has been extensively investigated as a strategy to craft novel therapeutics with heightened efficacy and minimized adverse effects. The aim of the present research work was to design, synthesize and characterize the new benzo[d]thiazol-2-amine derivatives as potent analgesic and anti-inflammatory agents. The synthesis of the presented benzo[d]thiazol-2-amine derivatives was performed by condensing-(4-chlorobenzylidene) benzo[d]thiazol-2-amine with a number of substituted phenols in the presence of potassium iodide and anhydrous potassium carbonate in dry acetone. IR spectroscopy, 1HNMR spectroscopy, 13CNMR spectroscopy and Mass spectroscopy methods were used to characterize the structural properties of all 13 newly syn-thesized derivatives. The molecular properties of these newly synthesized derivatives were estimated to study the attributes of drug-like candidates. Benzo[d]thiazol-2-amine derivatives were molecularly docked with selective enzymes COX-1 and COX-2. Analgesic and anti-inflammatory activities of synthesized compounds were evaluated by us-ing albino rats. Findings of the research suggested that compounds G3, G4, G6, G8 and G11 possess higher binding affinity than diclofenac sodium, when docking was performed with enzyme COX-1. Compounds G1, G3, G6, G8andG10 showed lower binding affinity than Indometha-cin when docking was performed with enzyme COX-2.In vitro evaluation of the COX-1 and COX-2 enzyme inhibitory activities was performed for synthesized compounds. Compounds G10 and G11 exhibited significant COX-1 and COX-2 enzyme in-hibitory action with an IC50 value of 5.0 and 10 μM, respectively. Using the hot plate method and the carrageenan-induced rat paw edema model, the synthesized compounds were screened for their biological activities, including analgesic and anti-inflammatory activities. Highest analgesic action was exhibited by derivative G11 and the compound G10 showed the highest anti-inflammatory response. Inhibition of COX may be considered as a mechanism of action of these compounds. It was concluded that synthesized derivatives G10 and G11 exhibited significant analgesic and anti-inflammatory effect; therefore, the said compounds may be subjected to further clinical investigation for establishing these as future compounds for the treatment of pain and inflammation.

Antinociceptive and Anti-Inflammatory Activities of Acetonic Extract from Bougainvillea x buttiana (var. Rose).

Background:Bougainvillea x buttiana is an ornamental plant with antioxidant, anti-inflammatory, and cytotoxic activities, which has been traditionally used to treat respiratory diseases. This study aimed to investigate whether the acetonic extract of Bougainvillea x buttiana var. Rose (BxbRAE-100%) has analgesic and anti-inflammatory properties and its potential action mechanisms. Methods: Analgesic and anti-inflammatory activities were evaluated using three murine pain models and two acute inflammation models. In vitro, the ability of the extract to inhibit proteolytic activity and the activities of the enzymes phospholipase A2 (PLA2) and cyclooxygenase (COX) were evaluated. In silico analysis was performed to predict the physicochemical and Absorption, distribution, metabolism, and excretion (ADME) profiles of the compounds previously identified in BxbRAE-100%. Results: In vivo BxbRAE-100% decreased the nociceptive behaviors in the writhing model, the tail immersion, and the formalin test, suggesting that the extract has the potential to relieve pain at peripheral and central levels. Additionally, topical or oral BxbRAE-100% treatment reduced dose-dependent 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced ear inflammation and carrageenan-induced paw edema, respectively. In vitro, BxbRAE-100% significantly inhibited proteolytic activity and PLA2, COX-1 and COX-2 activities. In silico, the compounds previously identified in BxbRAE-100% met Lipinski's rule of five and showed adequate ADME properties. Conclusions: These results support the use of B. x buttiana in Traditional Mexican Medicine and highlight its potential for the development of new treatments for pain and inflammation.

Anti-Platelet Activity of Sea Buckthorn Seeds and Its Relationship with Thermal Processing.

Sea buckthorn (Hippophae rhamnoides L.) is a tree or shrub with small, orange berries. Sea buckthorn seeds have shown many properties beneficial to human health, including antioxidant, anti-hypertensive, anti-hyperlipidemic, and retinoprotective activities. Seeds, as a component of food, are often exposed to high temperatures, which can increase or decrease their biological activity. In our previous study, we showed that both raw and roasted sea buckthorn seeds had significant antioxidant activity, which was measured in human plasma in vitro. In this paper, we evaluated the effect of extracts from raw and roasted sea buckthorn seeds on several parameters of hemostasis in vitro, including thrombus formation in full blood (measured by the Total Thrombus formation Analysis System-T-TAS), blood platelet activation (based on the exposition of P-selectin, the active form of GPIIb/IIIa on their surface and platelet-derived microparticles formation), aggregation (measured with impedance aggregometry), adhesion to fibrinogen and collagen, arachidonic acid metabolism in washed platelets stimulated by thrombin, and COX-1 activity. We also measured the levels of free 8-isoprostane in plasma and the total non-enzymatic antioxidant status of plasma. The extract from roasted seeds (50 µg/mL) significantly prolonged the time of occlusion measured by T-TAS-the AUC10 (area under the curve) value was decreased by approximately 18%. Both extracts decreased the exposition of the active form of GPIIb/IIIa on the surface of platelets activated with 10 μM ADP (by 38.4-62.2%) and 20 μM ADP (by 39.7-51.3%). Moreover, the extract from raw seeds decreased the exposition of P-selectin on the surface of platelets stimulated with 20 μM ADP (by 31.2-34.9%). The adhesion of thrombin-stimulated platelets to fibrinogen and collagen was inhibited only by the extract from roasted sea buckthorn seeds (by 20-30%). Moreover, the extract from raw seeds inhibited the level of TBARS (thiobarbituric acid-reactive substances, an indicator of enzymatic peroxidation of arachidonic acid) in washed platelets stimulated with thrombin; the activity of COX-1 was inhibited by both extracts, although the effect of the extract from raw seeds was stronger. These results indicate that sea buckthorn seeds have anti-platelet activity that is not decreased by thermal processing, but more research is needed to determine which exact chemical compounds and mechanisms are responsible for this phenomenon.

In vivo anti-gastric ulcer activity of 7-O-methyl aromadendrin and sakuranetin via mitigating inflammatory and oxidative stress trails

Ethnopharmacological relevanceEucalyptus genus has been used for a very long time in conventional treatment as an anti-ulcer remedy. Aim of the studyThe study aimed to explore the gastroprotective potential of 7-O-methyl aromadendrin (7-OMA), and sakuranetin (SKN) in comparison with omeprazole. The study tackled the contribution of their anti-inflammatory, antioxidant, and antiapoptotic capabilities to their anti-gastric ulcer effects. Materials and methodsAn ethanol-induced gastric ulcer model in rats was adopted and the consequences were confirmed by a molecular docking study. ResultsThe oral pretreatment of rats 1 h before ethanol using omeprazole (20 mg/kg) or 7-OMA (20 or 40 mg/kg) or SKN (20 or 40 mg/kg) exhibited gastroprotective and anti-inflammatory properties to different extents. These amendments witnessed as restorations in the stomach histological architecture in H and E-stained sections, mucus content in periodic acid-Schiff (PAS) stained sections with increased cellular proliferation, as demonstrated by increased immunohistochemical staining of PCNA, and increments in stomach COX-1 activity and eNOS. The highest dose of SKN showed the best corrections to reach 4.8, 1.8, and 2.1 folds increase in PAS, COX-1 and eNOS, respectively as compared to the untreated ethanol-induced gastric ulcer group; effects that were comparable to that of omeprazole. Moreover, reductions in COX-2 activity, and the protein expression of NF-κB, IL-6, TNF-α and NOx, in addition to the gene expression of inducible iNOS were also noted. Moreover, the antioxidant and antiapoptotic capabilities of omeprazole, 7-OMA, and SKN were perceived. SKN (40 mg/kg) succeeded to show the unsurpassed results to reach 293.6%, 237.1%, 274.7%, 248.2%, and 175.4% in total and reduced GSH, catalase, SOD, and Bcl2, respectively, as well as 50.0%, 46.8%, and 52.1 % in oxidized GSSG, TBARS and caspase-3, respectively. The gastroprotective potential of the tested compounds can be assigned to their anti-inflammatory, antioxidant and antiapoptotic properties.7-OMA and SKN were studied using molecular docking into the binding sites of the most significant inflammatory targets, including COX-2, TNF-α, iNOS, and NF-κB. Pharmacokinetic and physicochemical parameters in silico were appropriate. ConclusionThe prophylactic use of 7-OMA and SKN could be considered as an add-on to recurrent gastric ulcers and might influence its therapeutic approaches.

Efficacy and mechanism of the Ermiao San series of formulas for rheumatoid arthritis based on Chinmedomics strategy

BackgroundThe Ermiao San Series of Formulas (ESSF) refers to Ermiao San (TS), Sanmiao Wan (TW), and Simiao Wan (FW), which are widely used traditional Chinese medicine (TCM) formulas for treating rheumatoid arthritis (RA). However, the therapeutic advantages and underlying mechanisms of ESSF treatment are unclear, especially regarding the improper selection of these three formulas when treating RA. PurposeTo explore the efficacy and mechanisms of ESSF treatment for RA. MethodsComplete Freund's adjuvant was used to induce RA in rats. Chinmedomics strategy, which included metabolomics, serum pharmacochemistry of TCM, molecular docking, western blotting and qPCR, was applied to reveal the therapeutic advantages, pathways, and targets of ESSF. ResultsIn the early stages of treatment, TS quickly reduced joint swelling and the arthritis score index and regulated pathways such as arachidonic acid metabolism and purine metabolism. TW increases the regulation of tryptophan metabolism and pyrimidine metabolism pathways, promoting the recovery of the thymus and spleen. FW increases the regulation of linoleic acid metabolism and has the greatest effect on immune organ and bone recovery. In addition, 54, 67, and 86 bioactive compounds were detected in the serum from TS, TW, and FW, respectively. Berberine, phellodendrine, atractylolide III, limonin, 25R-inokosterone, coixol, and stigmasterol were found to act on the key enzymes COX-2, mPGES-1, ALOX5, and XDH in arachidonic acid metabolism and purine metabolism pathways. Western blot and qPCR results showed that ESSF can reduce the activity of these targets, thereby inhibiting the expression of the inflammatory factors IL-1β, IL-6, IL-17, and TNF-α; the tissue injury factors MMP-3 and CRP; and the rheumatoid factors CCP Ab and RF, thereby achieving anti-RA efficacy. ConclusionESSF has a good therapeutic effect on RA. TS focus on rapid swelling reduction in the early stages of RA, TW focus on the recovery of immune organ function, and FW can be used for bone recovery in the later stage of RA treatment. The key mechanism of treating RA is that ESSF reduces the activity of COX-2, mPGES-1, ALOX5, and XDH. These findings provide valuable guidance for targeted therapy for RA and for the clinical application of ESSF.

Cannabidiol and Beta-Caryophyllene Combination Attenuates Diabetic Neuropathy by Inhibiting NLRP3 Inflammasome/NFκB through the AMPK/sirT3/Nrf2 Axis.

In this study, we investigated in detail the role of cannabidiol (CBD), beta-caryophyllene (BC), or their combinations in diabetic peripheral neuropathy (DN). The key factors that contribute to DN include mitochondrial dysfunction, inflammation, and oxidative stress. Briefly, streptozotocin (STZ) (55 mg/kg) was injected intraperitoneally to induce DN in Sprague-Dawley rats, and we performed procedures involving Randall Sellito calipers, a Von Frey aesthesiometer, a hot plate, and cold plate methods to determine mechanical and thermal hyperalgesia in vivo. The blood flow to the nerves was assessed using a laser Doppler device. Schwann cells were exposed to high glucose (HG) at a dose of 30 mM to induce hyperglycemia and DCFDA, and JC1 and Mitosox staining were performed to determine mitochondrial membrane potential, reactive oxygen species, and mitochondrial superoxides in vitro. The rats were administered BC (30 mg/kg), CBD (15 mg/kg), or combination via i.p. injections, while Schwann cells were treated with 3.65 µM CBD, 75 µM BC, or combination to assess their role in DN amelioration. Our results revealed that exposure to BC and CBD diminished HG-induced hyperglycemia in Schwann cells, in part by reducing mitochondrial membrane potential, reactive oxygen species, and mitochondrial superoxides. Furthermore, the BC and CBD combination treatment in vivo could prevent the deterioration of the mitochondrial quality control system by promoting autophagy and mitochondrial biogenesis while improving blood flow. CBD and BC treatments also reduced pain hypersensitivity to hyperalgesia and allodynia, with increased antioxidant and anti-inflammatory action in diabetic rats. These in vivo effects were attributed to significant upregulation of AMPK, sirT3, Nrf2, PINK1, PARKIN, LC3B, Beclin1, and TFAM functions, while downregulation of NLRP3 inflammasome, NFκB, COX2, and p62 activity was noted using Western blotting. the present study demonstrated that STZ and HG-induced oxidative and nitrosative stress play a crucial role in the pathogenesis of diabetic neuropathy. We find, for the first time, that a CBD and BC combination ameliorates DN by modulating the mitochondrial quality control system.

Unveiling Anticancer Potential of COX-2 and 5-LOX Inhibitors: Cytotoxicity, Radiosensitization Potential and Antimigratory Activity against Colorectal and Pancreatic Carcinoma.

Apart from cytotoxicity, inhibitors of the COX-2 enzyme have demonstrated additional effects important for cancer treatment (such as radiosensitization of tumor cells and cell antimigratory effects); however, the relationship between the inhibition of other inflammation-related enzyme 5-LOX inhibitors and anticancer activity is still not well understood. In our study, the cytotoxicity of thirteen COX-2 and 5-LOX inhibitors previously presented by our group (1-13) was tested on three cancer cell lines (HCT 116, HT-29 and BxPC-3) and one healthy cell line (MRC-5). Compounds 3, 5, 6 and 7 showed moderate cytotoxicity, but good selectivity towards cancer cell lines. IC50 values were in the range of 22.99-51.66 µM (HCT 116 cell line), 8.63-41.20 µM (BxPC-3 cell line) and 24.78-81.60 µM (HT-29 cell line; compound 7 > 100 µM). In comparison to tested, commercially available COX-2 and 5-LOX inhibitors, both cytotoxicity and selectivity were increased. The addition of compounds 6 and 7 to irradiation treatment showed the most significant decrease in cell proliferation of the HT-29 cell line (p < 0.001). The antimigratory potential of the best dual COX-2 and 5-LOX inhibitors (compounds 1, 2, 3 and 5) was tested by a wound-healing assay using the SW620 cell line. Compounds 1 and 3 were singled out as compounds with the most potent effect (relative wound closure was 3.20% (24 h), 5,08% (48 h) for compound 1 and 3.86% (24 h), 7.68% (48 h) for compound 3). Considering all these results, compound 3 stood out as the compound with the most optimal biological activity, with the best dual COX-2 and 5-LOX inhibitory activity, good selectivity towards tested cancer cell lines, significant cell antimigratory potential and a lack of toxic effects at therapeutic doses.

Seasonal energy investment and metabolic patterns in a farmed fish

The present research focuses on the seasonal changes in the energy content and metabolic patterns of red porgy (Pagrus pagrus) sampled in a fish farm in North Evoikos Gulf (Greece). The study was designed in an effort to evaluate the influence of seasonality in several physiological feauteres of high commercial importance that may affect feed intake and growth. We determined glycogen, lipids and proteins levels, and cellular energy allocation (CEA) as a valuable marker of exposure to stress, which integrates available energy (Ea) and energy consumption (Ec). Metabolic patterns and aerobic oxidation potential were based on the determination of glucose transporter (GLU), carnitine transporter (CTP), L-lactate dehydrogenase (L-LDH), citrate synthase (CS), cytochrome C oxidase subunit IV isoform 1 (COX1) and 3-hydroxyacyl CoA dehydrogenase (HOAD) relative gene expression. To integrate metabolic patterns and gene expression, L-LDH, CS, COX and HOAD activities were also determined. For further estimation of biological stores oxidized during seasonal acclimatization, we determined the blood levels of glucose, lipids and lactate. The results indicated seasonal changes in energy content, different patterns in gene expression and reorganization of metabolic patterns during cool acclimatization with increased lipid oxidation. During warm acclimatization, however, energy consumption was mostly based on carbohydrates oxidation. The decrease of Ec and COX1 activity in the warm exposed heart seem to be consistent with the OCLTT hypothesis, suggesting that the heart may be one of the first organs to be limited during seasonal warming. Overall, this study has profiled changes in energetics and metabolic patterns occurring at annual temperatures at which P. pagrus is currently farmed, suggesting that this species is living at the upper edge of their thermal window, at least during summer.

Ethnopharmacological study on Adenosma buchneroides Bonati inhibiting inflammation via the regulation of TLR4/MyD88/NF-κB signaling pathway

Adenosma buchneroides Bonati, also known as fleagrass, is an important medicinal plant used by the Akha (Hani) people of China for treating inflammation-related skin swelling, acne, and diarrhoea, among other conditions. In this study, we aimed to evaluate the anti-inflammatory activities and explore the molecular mechanisms of fleagrass on treating skin swelling and acne. The results demonstrated that fleagrass inhibited the enzymatic activities of 5-LOX and COX-2 in vitro, and decreased the release of NO, IL-6, TNF-α, and IL-10 in the LPS-induced RAW264.7 macrophages. The levels of proteins associated with the nuclear factor-kappa B (NF-κB) pathway were examined by western blotting and immunofluorescence, demonstrating that fleagrass downregulated the expression of TLR4, MyD88, NF-κB/p65, and iNOS and blocked the nuclear translocation of NF-κB/p65. Furthermore, fleagrass exhibited acute anti-inflammatory activity in paw oedema models. The results confirm that fleagrass exhibits remarkable anti-inflammatory activity and can be used in alleviating inflammation, suggesting that fleagrass has the potential to be a novel anti-inflammatory agent.Graphical

Naturally occurring benzophenones and xanthones from Garcinia smeathmannii (Planch. & Triana) Oliv. displayed anti-inflammatory effects by modulating the activities of inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

Introduction: There is a growing interest in studying natural products for the identification of novel lead compounds for drug development for treating inflammatory diseases. Although some studies have focused anti-inflammatory activity of benzophenones and xanthones, exploring additional targets such as enzymes and cytokines, involved in their inflammatory response could provide more comprehensive understanding of the compounds' anti-inflammatory effects. In this study, four xanthones ananixanthone (1), smeathxanthone A (2), smeathxanthone B (3), and 1,3,5,8-tetrahydroxy-2-(3-methybut-2-enyl)-4-(3,7-dimethyloct-2,6-dienyl) xanthone (4); and three benzophenones guttiferone O (5), guttiferone M (6), and aristophenone A (7) from Garcinia smeathmannii (Planch. & Triana) Oliv. were investigated for their effect on nitric oxide production, cyclooxygenase, lipoxygenase inhibition, and Th1/Th2 cytokines production in activated RAW 264.7 macrophages. Methods: The Griess reagent method and the ferrous oxidation-xylenol orange assay were used to evaluate the inhibition of NO production and the 15-lipoxygenase activity respectively. Cyclooxygenase activity was assessed using the fluorometric COX activity assay kit and measurement of Th1/Th2 cytokines was performed using a flow cytometer. Results: All the tested compounds exhibited a dose-dependent inhibition of NO production with varying degrees of inhibitory effects on 15-LOX activity. Compound (6), displays the best inhibitory effect on COX-1/COX-2 activity. A general trend of the tested compounds on cytokines profiles revealed that compound (5) showed a pronounced enhancement of anti-inflammatory cytokines (IL-4 and IL-10). Conclusion: This observation supports future exploration of ananixanthone (1), guttiferone O (5), and guttiferone (6) as potential candidates for the development of anti-inflammatory drugs.

Degree of Cyclooxygenase-2 Inhibition Modulates Blood Pressure Response to Celecoxib and Naproxen.

Non-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of adverse cardiovascular events via suppression of cyclooxygenase (COX)-2-derived prostacyclin (PGI2) formation in heart, vasculature, and kidney. The Prospective Randomized Evaluation of Celecoxib Integrated Safety versus Ibuprofen Or Naproxen (PRECISION) trial and other large clinical studies compared the cardiovascular risk of traditional NSAIDs (i.e. naproxen), which inhibit both COX isozymes, with NSAIDs selective for COX-2 (i.e. celecoxib). However, whether pharmacologically equipotent doses were used - that is, whether a similar degree of COX-2 inhibition was achieved - was not considered. We compared drug target inhibition and blood pressure response to celecoxib at the dose used by most patients in PRECISION with the lowest recommended naproxen dose for osteoarthritis, which is lower than the dose used in PRECISION. Sixteen healthy participants (19-61 years) were treated with celecoxib (100 mg every 12h), naproxen (250 mg every 12h), or placebo administered twice daily for seven days in a double-blind, crossover design randomized by order. On Day 7 when drug levels had reached steady state, the degree of COX inhibition was assessed ex vivo and in vivo. Ambulatory blood pressure was measured throughout the final 12h dosing interval. Both NSAIDs inhibited COX-2 activity relative to placebo, but naproxen inhibited COX-2 activity to a greater degree (62.9±21.7%) than celecoxib (35.7±25.2%; p<0.05). Similarly, naproxen treatment inhibited PGI2 formation in vivo (48.0±24.9%) to a greater degree than celecoxib (26.7±24.6%; p<0.05). Naproxen significantly increased blood pressure compared to celecoxib (differences in least-square means of mean arterial pressure: 2.5 mm Hg (95% CI: 1.5, 3.5); systolic blood pressure: 4.0 mm Hg (95% CI: 2.9, 5.1); diastolic blood pressure: 1.8 mm Hg (95% CI: 0.8, 2.8); p<0.05 for all). The difference in systolic blood pressure relative to placebo was associated with the degree of COX-2 inhibition (p<0.05). Celecoxib 200 mg/day inhibited COX-2 activity to a lesser degree than naproxen 500 mg/day, resulting in a less pronounced blood pressure increase. While the PRECISION trial concluded the non-inferiority of celecoxib regarding cardiovascular risk, this is based on a comparison of doses that are not equipotent.ClinicalTrials.gov identifier: NCT02502006 (https://clinicaltrials.gov/study/NCT02502006).

Comparison of Anti-Inflammatory and Antibacterial Properties of Raphanus sativus L. Leaf and Root Kombucha-Fermented Extracts.

In the cosmetics industry, the extract from Raphanus sativus L. is fermented using specific starter cultures. These cosmetic ingredients act as preservatives and skin conditioners. Kombucha is traditionally made by fermenting sweetened tea using symbiotic cultures of bacteria and yeast and is used in cosmetic products. The aim of this study was to evaluate the cosmetic properties of radish leaf and root extract fermented with the SCOBY. Both unfermented water extracts and extracts after 7, 14, and 21 days of fermentation were evaluated. The analysis of secondary plant metabolites by UPLC-MS showed higher values for ferments than for extracts. A similar relationship was noted when examining the antioxidant properties using DPPH and ABTS radicals and the protective effect against H2O2-induced oxidative stress in fibroblasts and keratinocytes using the fluorogenic dye H2DCFDA. The results also showed no cytotoxicity to skin cells using Alamar Blue and Neutral Red tests. The ability of the samples to inhibit IL-1β and COX-2 activity in LPS-treated fibroblasts was also demonstrated using ELISA assays. The influence of extracts and ferments on bacterial strains involved in inflammatory processes of skin diseases was also assessed. Additionally, application tests were carried out, which showed a positive effect of extracts and ferments on TEWL and skin hydration using a TEWAmeter and corneometer probe. The results obtained depended on the concentration used and the fermentation time.

Isolation, identification and quantification of flavonoids from the flowers of &lt;i&gt;Staphylea pinnata&lt;/i&gt; L.

Introduction. Search for new plant species containing biologically active substances (hereinafter – BAS) is one of the leading tasks of pharmacognosy as a science. The search for flavonoid glycosides in plant raw materials is especially relevant, since they have anti-inflammatory, antioxidant, immunostimulating, as well as weak fungicidal and bacteriostatic action. Staphylea pinnata L. is an endemic plant of the Caucasus, cultivated not only in Georgia, but also in the Russian Federation in the Northern and Northwestern Caucasus. In foreign literature there are studies of antioxidant, antiproliferative and cytotoxic activity of leaf extracts of several species of S. pinnata L., as well as inhibitory activity of COX-1, COX-2 and LTB4 formation. Meanwhile, no serious Russian-language scientific studies on either the chemical composition or pharmacological action of generative organs of S. pinnata were found in the literature. This work is part of a comprehensive phytochemical study of S. pinnata. The aim of the work is to study the qualitative and quantitative composition of flavonoids in the studied object.Aim. To isolate, identify and quantify flavonoids in flowers and buds of Staphylea pinnata L.Materials and methods. Alcohol-water extracts from dried generative organs of the studied plant were used as analyzed solutions. Solutions were analyzed on a spectrophotometer SF-2000 (LLC "OKB Spectr", Russia) after sample preparation with aluminum chloride and on an HPLC Nexera-i LC-2040 (Shimadzu Corporation, Japan) equipped with a column and sample thermostat, degasser and autosampler using an individually selected elution gradient of the mobile phase (0.1 % orthophosphoric acid/acetonitrile solution). The primary data were processed using LabSolutions Single LC software (Shimadzu Corporation, Japan). Compounds from the flavonoid group were identified by retention times. Detection was carried out using a UV detector with an absorption wavelength of 365 ± 2 nm.Result and discussion. Alcohol-water extracts from flowers and buds of S. pinnata L. were obtained. Quantitative evaluation by spectrophotometry for flavonoid content was carried out. A gradient elution mode for HPLC was selected for simultaneous determination of 7 flavonoid glycosides. These chromatographic conditions allowed the identification and quantification of astragaline, cynaroside, cosmosiin, narcissin and rutin in flowers and buds of Staphylea pinnata L. Flavonoid glycosides: raponticin and kaempferol were not detected.Conclusion. Flavonoid glycosides were isolated from the generative organs of S. pinnata L., a technique for quantitative determination of flavonoid glycosides in alcohol-water extracts was developed, astragalin, cynaroside, cosmosiin, narcissin and rutin were detected and quantified.

PGE2-PKA Control of Mitochondrial Membrane Potential Dictates Macrophages Proliferation and Efferocytic Function During Lung Injury

In the lungs, phenotypically different alveolar macrophages (AMs) co-exist with interstitial monocyte-derived macrophages (Mo-Macs) to regulate lung homeostasis. We have shown that airspace-recruited Mo-Macs resolve inflammation, restoring lung-fluid homeostasis. Analyzing publicly available datasets (Immgen Consortium), we found that compared to Mo-Macs, AMs are also enriched with pathways regulating mitochondria function, but their significance remain elusive. During development, AMs are generated by differentiation of lung-recruited fetal liver monocytes by granulocyte macrophage-colony stimulating factor (GM-CSF). In contrast, Mo-Macs require macrophage colony-stimulating factor (M-CSF) to differentiate from monocytes. Here, we mimic the developmental program of AM versus Mo-Macs by differentiating bone marrow monocytes towards AM-like cells (G-BMDM) or Mo-Mac-like cells (M-BMDM) to understand the role of mitochondrial activity in regulating macrophage functions. We confirmed that G-BMDMs highly express canonical AM-like genes on the cell surface, such as Itgax, Siglecf, Chil3, Ear2, Egr2, and Car4. M-BMDM mimics Mo-Macs as they show higher C1q, Mmp9, Folr2, and Cx3cr1 expression. G-BMDM and M-BMDM showed similar mitochondrial mass. However, G-BMDM showed higher mitochondrial membrane potential (ΔΨm) than M-BMDM, which was associated with compromised efferocytic capacity. These findings were recapitulated in vivo at homeostasis where ΔΨm was higher in AM than Mo-Macs or monocytes. AM compared to other tissue resident macrophages expresses very low level of Ptgs1 &amp; 2 the rete limiting enzymes for PGE2 production. Prostaglandin E2 (PGE2) is a potent lipid mediator and modulator of immune cell function and activates protein kinase A (PKA) through generating cAMP. We, therefore, assessed if PGE2 was a factor in diversifying ΔΨm between G-BMDM and M-BMDM. Using the COX2 sensor (COXFLuor), we found that M-BMDM generated more PGE2 than G-BMDM. PGE2 reduced ΔΨm in G-BMDM to the level of M-BMDM. However, this response was not seen in G-BMDM upon inhibiting EP2 receptor (TG4-155; 10μm) or PKA (H89; 5μm), indicating PGE2 functioned by ligating its receptor and elevating PKA activity. During infection or injury many cell types upregulate Ptgs2 expression and activity to produce local PGE2. Further studies showed that during endotoxin induced acute lung injury, Mo-Macs have high COX2 activity and are the major source of PGE2 in the alveolar milieu to promote AM proliferation and efferocytic function, and help resolve edema. However, inhibition of PKA (H89; 1mg/Kg, i.t), impaired AM proliferation and efferocytosis, leading to unresolved injury. These findings indicate that the PGE2-PKA axis stabilizes mitochondrial function in Mo-Macs to maintain the AM-proliferative-efferocytotic niche for resolving edema. This study is funded by NIH grant 5R01-HL155941-03 and P01 HL151327-03. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.