Cyclooxygenase Inhibitor Research Articles

Anti-inflammatory and antinociceptive effects of LQFM275 - A new multi-target drug.

Compound (4-(3,5-di-tert-butyl-4-hydroxybenzylamine)benzenesulfonamide) (LQFM275) was designed and synthesized from darbufelone and sulfanilamide as a new multi-target for the treatment of inflammatory diseases. LQFM275 showed a great range of safe cytotoxicity profile (100-400μM) evaluated by MTT assay, preventing damage induced by lipopolysaccharide (LPS) in EA.hy926 cell line. In mice, the acute oral treatment with LQFM275 (57, 114, and 228mg/kg) reduced the number of writhing by 26, 37, and 49%, respectively. LQFM275 (114mg/kg) also presented an antinociceptive effect, reducing by 57% the nociceptive response in the second phase of the formalin test and by 47% the Carrageenan(Carra)-induced hyperalgesia. That effect was dependent on its anti-inflammatory activity. LQFM275 (114mg/kg) also reduced 42% and 31% of the Carra and LPS-induced edema, respectively. The pleurisy test attenuated the leukocyte migration induced by Carra and LPS by reducing the number of polymorphonuclear cells (by 39 and 36%, respectively). The production of reactive oxygen species in the pleural exudate was reduced, which is shown by a decrease in myeloperoxidase (MPO) activity (Carra=35% and LPS=40%) and in levels of pro-inflammatory cytokines TNF-α and IL-1β (Carra=48% and LPS=47 e 36%). On the other hand, it increased the levels of anti-inflammatory cytokines, IL-4, and IL-10 (Carra=50% and LPS=21 and 53%). Moreover, LQFM275 demonstrated to be a dual COX-2 and 5-LOX inhibitor (IC50=81 and 167μM, respectively). Therefore, the promising anti-inflammatory and antinociceptive effects of LQFM275 provide an opportunity for a new multi-target drug development.

Molecular Biomarkers of NSAID-induced Gastritis

Nonsteroidal anti-inflammation drug (NSAID) has been commonly used by people to treat myalgia and arthritis. Long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) can cause chronic gastritis due to its topical and systemic effects. The topical effects of NSAID comprise cytotoxic and apoptotic effects on gastric mucosa which involve many apoptosis biomarkers while systemic effects are associated with cyclooxygenase (cox) inhibition in the reduction of prostaglandins production that plays a role in gastric mucosa’s protectors. Apoptosis biomarkers that are involved in NSAID-induced gastritis comprise tumor necrosis factor-alpha (TNF-α) as well as cytochrome-c and SMAC which act as apoptosis inducers via extrinsic and intrinsic pathways respectively, caspase-8, caspase-9, caspase-3 in apoptosis pathway, Apoptosis Inducing Factor (AIF), longevity of apoptotic inhibitor proteins and NF-kB activity, resulting in apoptosis and cytotoxic effect to develop gastritis. This review will help us to understand the safety of NSAID drugs and to find effective treatments to regulate its mediators and not just suppress gastric acid secretion.

Sonochemical synthesis of 3-arylsufonyl indole derivatives as potential inhibitors of cyclooxygenases

Sonochemical synthesis of 3-arylsufonyl indole derivatives as potential inhibitors of cyclooxygenases

Design, synthesis, and evaluation of benzhydrylpiperazine-based novel dual COX-2/5-LOX inhibitors with anti-inflammatory and anti-cancer activity.

Piperazine derivatives were screened using the ChEMBL database, paving the way for the design, synthesis, and evaluation of a novel series of dual COX-2/5-LOX inhibitors and identifying their role in mitigating cancer cell proliferation. Compound 9d with 4-Cl substitution at the terminal phenyl ring showed promising inhibition of COX-2 (IC50 = 0.25 ± 0.03 μM) and 5-LOX (IC50 = 7.87 ± 0.33 μM), outperforming the standards celecoxib (IC50 = 0.36 ± 0.023 μM) and zileuton (IC50 = 14.29 ± 0.173 μM), respectively. The two most active derivatives 9d and 9g indicated a significant anti-inflammatory response in a paw edema model by inhibiting PGE2, IL-6, and TNF-α and an increase in IL-10 concentrations. Interestingly, 9d effectively reduced pain by 55.78%, closely comparable to the 59.09% exhibited by the standard indomethacin, and was also devoid of GI, liver, kidney, and cardiac toxicity. Furthermore, 9d demonstrated anti-cancer potential against in vitro A549, COLO-205, and MIA-PA-CA-2 human cancer cell lines and an in vivo Drosophila cancer model. The pharmacokinetic investigations revealed that 9d has good oral absorption characteristics.

2-(4-Bromobenzyl) tethered 4-amino aryl/alkyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines: design, synthesis, anticancer assessment via dual topoisomerase-I/II inhibition, and in silico studies.

A series of 2-(4-bromobenzyl) tethered 4-amino aryl/alkyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines (7a-7u) were designed, synthesized, characterized and screened against a panel of cancer cell lines. Compound 7a, in particular, emerged as a potent antiproliferative agent against FaDu cells (HTB-43) with an IC50 value of 1.73 μM. 7a induced morphological alterations in FaDu cells were observed via brightfield microscopy and DAPI staining, confirming cytotoxicity. Autophagy and apoptotic effects of 7a were confirmed by acridine orange staining, Rhodamine 123 staining, and western blot analysis, which revealed dose-dependent increases in LC3A/B and cleaved caspase-3 levels, respectively. Further, 7a impaired cell migration and colony formation, as demonstrated by scratch and clonogenic assays. Additionally, 7a reduced oxidative stress and induced G2/M phase cell cycle arrest in MCF-7 cells. 7a emerged as a dual topoisomerase I and II inhibitor, and results were supported by molecular docking and simulation studies. In anti-inflammatory studies, 7a exhibited selective inhibition of COX-2 over COX-1, supporting its dual anticancer and anti-inflammatory properties.

Microwave assisted synthesis, biological assessment and in-silico studies of some new 5,5′-(phenylmethylene)bisuracil and dihydropyrido[2,3-d:6,5-d']dipyrimidine derivatives as selective COX-2 inhibitors with potential anti-inflammatory activities

Microwave assisted synthesis, biological assessment and in-silico studies of some new 5,5′-(phenylmethylene)bisuracil and dihydropyrido[2,3-d:6,5-d']dipyrimidine derivatives as selective COX-2 inhibitors with potential anti-inflammatory activities

The role of the cyclooxygenase-2 pathway in tissue ischemia and revascularization following skeletal muscle injury induced by bothropic snake venom

Bothrops asper venom (Bav) contains metalloproteinases that disrupt the microvascular system, impairing muscle tissue regeneration after injury. This study investigated the impact of the cyclooxygenase-2 (COX-2) pathway on vascular injury and revascularization in muscle injuries induced by Bav. Mice were injected with Bav into the gastrocnemius muscle and treated with lumiracoxib, a selective COX-2 inhibitor, 30 min, 2 days, and 6 days post-Bav injection. Muscle tissue was analyzed at 24 h, 7 days, and 21 days post-injection. A decrease in COX-2 expression at 24 h post-Bav injection indicated significant necrosis and tissue loss. Both Bav injection and lumiracoxib treatment influenced the decrease of prostaglandin (PG)D2 and PGE2 production. Seven and 21 days post-Bav injections, COX-2 expression increased, along with PGDs levels unaffected by lumiracoxib, indicating that the other isoform COX-1 pathway could contribute to the release of PGs. Bav/lumiracoxib treated animals presented exacerbated limb ischemia, implying that COX-2-derived prostaglandins preserve vessel integrity. CD31, an angiogenesis marker, initially (24 h) decreased post-Bav injection but increased at 7 and 21 days in Bav/lumiracoxib mice, suggesting a down-modulatory role for COX-2-derived prostaglandins in early angiogenesis and tissue regeneration. Vascular endothelial growth factor (VEGF) production rose 7 days post-Bav injection, supporting its role in angiogenesis. Previous treatment with lumiracoxib promoted release of VEGF levels 21 days post-Bav injury showing that the inhibition of COX-2 pathway in the early stage of revascularization stimulates the neovascularization regulated by elevated release of VEGF. Similarly, metalloproteinases (MMPs), such as MMP-9, MMP-10, and MMP-13, crucial for vascular remodeling, were elevated 21 days after Bav/lumiracoxib treatment. In conclusion, the COX-2 pathway is essential to decrease the high grade of ischemia caused by acute injury induced by Bav. However, the decrease of activity in the COX-2 pathway in the first stages of revascularization contributes to the elevated production of key pro-angiogenic mediators that up-regulate the restoration of microvasculature and blood flow in muscle tissue injured by botropic venoms.

Synthesis, in silico and bio-evaluation studies of new isothiocyanate derivatives with respect to COX inhibition and H2S release profiles.

The development of H2S-donating derivatives of non-steroidal anti-inflammatory drugs (NSAIDs) is considered important to reduce or overcome their gastrointestinal side effects. Sulforaphane, one of the most extensively studied isothiocyanates (ITCs), effectively releases H2S at a slow rate. Thus, we rationally designed, synthesized, and characterized new ITC derivatives (I1-3 and I1a-e) inspired by the natural compound sulforaphane. The anti-inflammatory properties of these compounds were evaluated by their inhibitory activities against cyclooxygenase targets COX-1 and COX-2. Additionally, the cytotoxicity of the compounds was tested using the MTT assay on LPS-induced RAW 264.7 cells, revealing no cytotoxic effects at low doses. Notably, compounds I1 and fluorine-containing ester derivative I1c emerged as the most potent and selective COX-2 inhibitors, with selectivity indexes of 2611.5 and 2582.4, respectively. The H2S-releasing capacities of ITC derivatives were investigated and compared with that of sulforaphane, showing that while compounds I1-3 exhibit slow and similar H2S release to sulforaphane, the release from compounds I1a-e was not as pronounced as that of the standard. Physics-based molecular modeling studies including molecular docking and molecular dynamics (MD) simulations, binding free energy calculations and absorption, distribution, metabolism, and excretion (ADME) analyses were also conducted. MD simulations analysis underscored the crucial amino acids such as Tyr385, Trp387, Phe518, Val523, and Ser530 in the interactions between I1c hit compound and COX-2. The combined in silico and in vitro findings suggest that compounds I1 and I1c are promising NSAID candidates against selective COX-2 inhibition.

Bioengineered NanoAid synergistically targets inflammatory pro-tumor processes to advance glioblastoma chemotherapy.

Through transcriptomic analysis of patient-derived glioblastoma tissues, we identify an overactivation of inflammatory pathways that contribute to the development of a tumor-promoting microenvironment and therapeutic resistance. To address this critical mechanism, we present NanoAid, a biomimetic nanoplatform designed to target inflammatory pro-tumor processes to advance glioblastoma chemotherapy. NanoAid employs macrophage-membrane-liposome hybrids to optimize the delivery of COX-2 inhibitor parecoxib and paclitaxel. By inheriting macrophage characteristics, NanoAid not only efficiently traverses the blood-brain barrier and precisely accumulates within tumors but also enhances cancer cell uptake, thereby improving overall anticancer efficacy. Notably, the combination of parecoxib and paclitaxel effectively disrupts inflammatory pro-tumor processes while inducing a synergistic effect that inhibits tumor growth, overcomes therapeutic resistance, and minimizes adverse effects. This results in substantial tumor growth inhibition and extends the median survival of tumor-bearing mice. Thus, our study bridges clinical insights with fundamental research, potentially revolutionizing tumor therapy paradigms.

Constitutive surface expression of the thromboxane A2 receptor is Pim kinase-dependent.

The thromboxane A2 receptor (TPαR) plays an important role in the amplification of platelet responses during thrombosis. Receptor activity is regulated by internalization and receptor desensitization. The mechanism by which constitutive surface expression of the TPαR is regulated is unknown. Recently, it has been demonstrated that proviral insertion in murine lymphoma (Pim) kinase inhibitors reduce platelet functional responses in a TPαR-dependent manner. To investigate whether Pim kinases regulate constitutive TPαR surface expression. TPαR surface expression was measured in platelets, and human embryonic kidney 293T (HEK293T) cells transfected with tagged TPαRs in the presence and absence of Pim kinase inhibitors using flow cytometry and confocal microscopy. TPαR-dependent calcium flux was assessed using Fluo-4 AM. Site prediction modeling and site-directed mutagenesis were used to identify the TPαR PIM kinase phosphorylation site. Surface expression of TPαR and calcium responses to U46619 were reduced in platelets and HEK293T cells following Pim kinase inhibition. Overexpression of kinase-dead Pim-1 also reduced TPαR surface expression on HEK293T cells. Reduced surface expression of the TPαR was found to be mediated by increased receptor internalization in a dynamin and β-arrestin-dependent manner. Four putative Pim kinase phosphorylation sites in the TPαR were mutated, and serine 57 in the first intracellular loop of TPαR was identified to be a novel regulatory site important for maintaining TPαR surface expression and thromboxane A2-dependent functional responses. Pim kinase inhibition may offer a novel therapeutic approach to limit cellular responses to thromboxane A2, independent of cyclooxygenase inhibition and direct antagonism of the receptor.

Novel Celecoxib Derivative, RF26, Blocks Colon Cancer Cell Growth by Inhibiting PDE5, Activating cGMP/PKG Signaling, and Suppressing β-catenin-dependent Transcription.

Previous studies have reported that the cGMP-specific PDE5 isozyme is overexpressed in colon adenomas and adenocarcinomas and essential for colon cancer cell proliferation, while PDE5 selective inhibitors (e.g., sildenafil) have been reported to have cancer chemopreventive activity. This study aimed to determine the anticancer activity of a novel PDE5 inhibitor, RF26, using colorectal cancer (CRC) cells and the role of PDE5 in CRC tumor growth in vivo. The objective of this study was to characterize the anticancer activity of a novel celecoxib derivative, RF26, in CRC cells previously reported to lack COX-2 inhibition but have potent PDE5 inhibitory activity. Anticancer activity of RF26 was studied using human CRC cell lines. Its effects on intracellular cGMP levels, cGMP-dependent protein kinase (PKG) activity, β-catenin levels, TCF/LEF transcriptional activity, cell cycle distribution, and apoptosis were measured. CRISPR/cas9 PDE5 knockout techniques were used to determine if PDE5 mediates the anticancer activity of RF26 and validate PDE5 as a cancer target. RF26 was appreciably more potent than celecoxib and sildenafil to suppress CRC cell growth and was effective at concentrations that increased intracellular cGMP levels and activated PKG signaling. RF26 suppressed β-catenin levels and TCF/LEF transcriptional activity and induced G1 cell cycle arrest and apoptosis within the same concentration range. CRISPR/cas9 PDE5 knockout CRC cells displayed reduced sensitivity to RF26, proliferated slower than parental cells, and failed to establish tumors in mice. Further evaluation of RF26 for the prevention or treatment of cancer and studying the role of PDE5 in tumorigenesis are warranted.

Multifunctional Plumeria rubra nanoparticles: Antimicrobial, antiviral, antiparasitic and anti-inflammatory activities.

Novel natural nanomaterials are extensively investigated nowadays due to their potential biological activities and low toxicity. In the present investigation, polyvinyl alcohol was used as a green reducing agent to synthesize Plumeria rubra nanoparticles. Plumeria rubra flowers were collected and extracted using a Soxhlet apparatus and examined through gas and liquid chromatography which revealed the presence of palmitic acid, linoleic acid, N-(O-Nitrophenylthio)-l-leucine, plumieridin B, lupeol, and rutin. The antimicrobial activity of the flower extract showed MIC values between 15.6 and 250.0μg/mL. The optimum conditions for Plumeria rubra nanoparticle synthesis were 30min stirring time and a 1:1 PVA/Plumeria rubra ratio. The optimized nanoparticles showed antimicrobial activity reached 7.8μg/mL with entrapment and loading efficiencies %, zeta size, zeta potential, and PDI were 89.0%, 40.0%, 18.5nm, -1.65mV, and 0.29, respectively. The cytotoxic effect (IC50) of Plumeria rubra nanoparticles reached 503.2μg/mL. Plumeria rubra nanoparticles showed low antiviral activity against 229E (low-pathogenic coronavirus), with selectivity index 2.90. Moreover, the synthesized Plumeria rubra nanoparticles' anti-inflammatory effect showed effective and selective COX-2 inhibition (IC50=4.2μM, SI=15.57). On top of that, the synthesized nanoparticles reduced the parasitic tachyzoites' count by 37, 43, and 49% in the spleen, brain, and liver, respectively.

Relaxant Effect of Rosuvastatin in Isolated Rat Aorta with Perivascular Adipose Tissue

Objective: Rosuvastatin displays favorable pleiotropic effects on vascular system to reduce the risk of cardiovascular events besides providing an intensive reduction in LDL-C levels. The role of perivascular adipose tissue (PVAT) in modulating the vasorelaxant effect of rosuvastatin is not evaluated so far. The present study aimed to investigate the vascular relaxant effect of rosuvastatin in rat aortic rings with intact PVAT, as well as to evaluate the possible mechanisms underlying this effect in relation to nitric oxide (NO) and prostaglandin pathways. Methods: Thoracic aorta rings with intact PVAT, isolated from male Wistar rats (n=5), were mounted on an isolated organ bath system. Endothelium-dependent responses to acetylcholine (Ach,10-6-10-4M) were obtained in aortic rings precontracted submaximally with phenylephrine (Phe,10-6-3x105M). The concentration-dependent relaxant effect of rosuvastatin (10-7-10-4M) was examined in the absence and presence of NO inhibitor, L-NOARG (10-4M, 30min.) and cyclooxygenase inhibitor, indomethacin (10-5M, 30min.). Vascular relaxation capacity of aortic rings was checked by the nitrovasodilator, sodium nitroprusside (SNP,10-6 M) at the end of the experiments. Results: Rosuvastatin (10-7-10-4M) produced concentration-dependent relaxations in Phe-precontracted rat aortic rings with intact PVAT. Pretreatment with L-NOARG significantly attenuated the relaxant responses to rosuvastatin in isolated rat aortic rings with intact PVAT. However, pretreatment with indomethacin did not modify the relaxations to rosuvastatin. In the aortic rings, maximal relaxation responses to Ach and SNP were determined to be 75.87±2.68% and 102.54±2.92%, respectively. Conclusions: This study will provide a basis for investigating the interaction between PVAT and statins in vascular homeostasis.

In-vitro and In-silico Anti-inflammatory Activity of a Common Bangladeshi Fruit: An Approach to Find Selective COX-2 Inhibitors

Background: From the ancient time, medicinal plants are utilized to mitigate different disease. In Ayurveda and Siddha indigenous medicinal systems, the bark of Syzygium cumini (L.) (Myrtaceae) is used for sore bronchitis, asthma, throat, dysentery, seasickness and ulcers. Objectives: The present experiment was executed to explore the anti-inflammatory property of Syzygium cumini fruit from it’s methanolic extract through phytochemical screening as well as the in-silico approach. Methods: In this experiment, standard method such as human red blood cell (HRBC) membrane stabilizing method was utilized to discover the phytochemical responsible for anti-inflammatory activity present in methanolic crude extract of Syzygium cumini fruits. In-silico ADME/T and molecular docking study was performed to assess the potential of stated phytochemicals against Cyclooxygenase-II enzyme. Findings: The presence of flavonoids, alkaloids, glycosides, tannins and saponins were confirmed by phytochemical screening. In membrane stabilizing method, the extract showed a maximum membrane stabilizing effect on HRBC with 62.381% at 1000 µg/mL in hypotonic solution 37.619% hemolysis were found when compared with standard drug Diclofenac sodium exhibited 87.143% protection. Twenty-three phytochemicals exhibit notable pharmacokinetics properties and passed drug likeness screening test in silico. In molecular docking study, Dihydromyricetin showed docking score (-9.1 kJ/mol) near as high as standard drug Celecoxib (-9.5 kJ/mol) with significant non-bonding interactions with target enzyme. Conclusions: The result of the present study demonstrates a favorable baseline in progression for the possible use of fruit of Syzygium cumini to treat inflammation.

FXR Activation Accelerates Early Phase of Osteoblast Differentiation Through COX-2-PGE2-EP4 Axis in BMP-2-Induced Mouse Mesenchymal Stem Cells.

Farnesoid X receptor (FXR), a nuclear receptor, is expressed in calvaria and bone marrow stromal cells and plays a role in bone homeostasis. However, the mechanism of FXR-activated osteoblast differentiation remains unclear. In this study, we investigated the regulatory mechanism underlying FXR-activated osteoblast differentiation using bone morphogenetic protein-2 (BMP-2)-induced mouse ST-2 mesenchymal stem cells. We also synthesized a novel FXR agonist, FLG390, and compared its biological effects in osteoblast differentiation with a known FXR agonist, chenodeoxycholic acid (CDCA). As an FXR agonist, FLG390 accelerated osteoblast differentiation to a comparable extent with CDCA, enhancing alkaline phosphatase (ALP) activity and the expression of osteoblast differentiated-related genes such as ALP, collagen type 1 α1 chain (COL1A1), and runt-related transcription factor 2 (RUNX2). FXR activation elevated the expression of cyclooxygenase (COX)-2 and the production of prostaglandin (PG) E2 in the early phase of osteoblast differentiation. A selective COX-2 inhibitor and an antagonist of EP4 receptors, one of PGE2 receptors, partially suppressed FXR-activated osteoblast differentiation. Moreover, treatment with either inhibitor during the first 6 h after initiating osteoblast differentiation repressed FXR-activated osteoblast differentiation to the same extent as did the treatment for 6 d. Therefore, a novel FXR agonist, FLG390, exhibited potency comparable to CDCA. FXR activation promoted the early phase of osteoblast differentiation via the COX-2-PGE2-EP4 axis, representing a potential target for control of bone metabolism.

Mechanisms and effects of aspirin to cancer cells

Aspirin, everyone knows it is an anti-inflammatory drug, has great promise mostly for the prevention and treatment of cancer by using a number of methodologies. Its anti-cancer property come from its induction of apoptosis, inhibition of cell growth, and disruption of important signaling pathways. By exploring the mechanisms through which aspirin affects cancer cells, particularly having the impact on signaling pathways like non-coding RNAs (ncRNAs), and controlling the activity of platelets. It is also mentioned that the inhibition of cyclooxygenase (COX) enzymes is involved in aspirin’s primary action, leading to lower rate of inflammatory mediators produced and changing cellular signaling networks. The modulation of ncRNAs, especially in connection to specific cancer pathways, concentrating its role in cancer treatment for many areas. However, although huge potential of the drug exhibits, the use of aspirin is not without risks. Common symptoms such as gastrointestinal discomfort and bleeding are in considering. As all mentioned below, numerous good results in improved treatment approaches for patients will come out, follow the continued investigation into the many ways that aspirin affects cancer cells.

Metabolic kinetics and safety assessment of the antitumor drug meloxicam

Meloxicam is a selective cyclooxygenase-2 (COX-2) inhibitor with anti-inflammatory effects, and its anti-tumour effects have been the subject of considerable attention in recent years. This research provides a comprehensive analysis of the studies on the anti-tumour aspects of meloxicam, including the mechanism of anti-tumour action, basic pharmacokinetic studies, and an evaluation of the safety profile. The anti-tumour mechanism of meloxicam is primarily characterized by the inhibition of the COX-2/PGE2 signaling pathway, the regulation of apoptosis-related proteins, the inhibition of tumour angiogenesis and the regulation of immune function. In terms of pharmacokinetics, meloxicam has rapid oral absorption, high bioavailability and wide distribution. It is mainly metabolized by the liver, undergoing hydroxylation and oxidation to form biologically inactive metabolites. With an elimination half-life of approximately 20 hours, the safety evaluation is primarily concerned with the assessment of potential gastrointestinal, hepatic, renal and cardiovascular adverse reactions that may arise from long-term use. The application of nanotechnology has the potential to mitigate the observed toxicity).

NSAIDs in Sports Medicine: Balancing Quick Relief with Long-Term Health Impacts

Introduction and PurposeNonsteroidal anti-inflammatory drugs (NSAIDs) are regularly used in sports medicine to manage pain and inflammation caused by acute and chronic injuries. Their mechanism involves inhibition of cyclooxygenase (COX) enzymes, reducing the body’s natural inflammatory response. This review aims to analyze the effects of NSAID use in sports contexts, focusing on short- and long-term benefits and risks, specifically concerning tissue healing, athletic performance, and general health outcomes.Materials and MethodsWe reviewed studies from PubMed, Google Scholar and ResearchGate, focusing on key search terms including NSAIDs in sports injuries, NSAID mechanism, short-term and long-term effects of NSAIDs, athletic performance and NSAID, pain management in sports, NSAID side-effects and NSAID alternativesConclusionsShort-term NSAID use can provide effective pain relief and support functional recovery, benefiting professional and recreational athletes in their sports. However, prolonged use may decrease natural tissue healing abilities, increase susceptibility to various side effects, and potentially affect long-term performance. Alternative pain management strategies are recommended to complement NSAID use, providing athletes with safer, long-term options for recovery. This review highlights the need for a balanced approach to NSAID usage, recommending cautious decision making among sportspeople, coaches, and healthcare workers.

Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity.

Endothelial dysfunction is a hallmark of several pathological conditions, including cancer, cardiovascular disease and inflammatory disorders. In these conditions, perturbed TCA cycle and subsequent succinate accumulation have been reported. The role of succinate as a regulator of immunological responses and inflammation is increasingly being recognized. Nevertheless, how endothelial cell function and phenotype are altered by elevated intracellular succinate has not been addressed yet. Thus, we employed numerous in vitro functional assays using primary HUVECs and diethyl succinate (DES), a cell membrane-permeable succinate analogue. An MTS assay 1 h post stimulation with DES suggested reduced metabolic activity in HUVECs. Concurrently, elevated production of ROS, including mitochondrial superoxide, and a reduction in mitochondrial membrane potential were observed. These findings were corroborated by Seahorse mito-stress testing, which revealed that DES acutely lowered the OCR, maximal respiration and ATP production. Given the link between mitochondrial stress and apoptosis, we examined important survival signalling pathways. DES transiently reduced ERK1/2 phosphorylation, a response that was followed by a skewed pro-apoptotic shift in the BAX to BCL2L1 gene expression ratio, which coincided with upregulating VEGF gene expression. This indicated an induction of mixed pro-apoptotic and pro-survival signals in the cell. However, the BAX/BCL-XL protein ratio was unchanged, suggesting that the cells did not commit themselves to apoptosis. An MTS assay, caspase 3/7 activity assay and annexin V/propidium iodide staining confirmed this finding. By contrast, stimulation with DES induced acute endothelial barrier permeability, forming intercellular gaps, altering cell size and associated actin filaments without affecting cell count. Notably, during overnight DES exposure gradual recovery of the endothelial barrier and cell sprouting was observed, alongside mitochondrial membrane potential restoration, albeit with sustained ROS production. COX-2 inhibition and EP4 receptor blockade hindered barrier restoration, implicating a role of COX-2/PGE2/EP4 signalling in this process. Interestingly, ascorbic acid pre-treatment prevented DES-induced acute barrier disruption independently from ROS modulation. In conclusion, succinate acts as a significant regulator of endothelial mitochondrial function and barrier integrity, a response that is counterbalanced by upregulated VEGF and prostaglandin production by the endothelial cells.

Shikonin induces the apoptosis and pyroptosis of EGFR-T790M-mutant drug-resistant non-small cell lung cancer cells via the degradation of cyclooxygenase-2

BackgroundThe T790M mutation in the epidermal growth factor receptor (EGFR) gene is the primary cause of resistance to EGFR-tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients. Previous research demonstrated that certain traditional Chinese medicine (TCM) monomers exhibit anti-tumor effects against various malignancies. This study aims to investigate the potentials of shikonin screened from a TCM monomer library containing 1060 monomers in killing EGFR-T790M drug-resistant NSCLC cells and elucidate the underlying mechanisms.MethodsMTT method was used to screen for the TCM monomers with significant killing effects on H1975 cells carrying the EGFR-T790M mutation. The influences of the identified monomer shikonin on cell growth were determined by the colony formation assay. Annexin-V/PI staining and JC-1 staining were applied to detect the effects of shikonin on cell apoptosis. The influences of shikonin on cell membrane integrity were detected by lactate dehydrogenase (LDH) release assay. Reactive oxygen species (ROS) generation was analyzed using DCFH-DA as probe. The mechanisms of shikonin affecting the stability of cyclooxygenase-2 (COX-2) were evaluated by using specific inhibitors for protein degradation pathways. Western blotting was performed to assess the effects of the alteration of COX-2 expression or enzymatic activity on the related signal pathways as well as the apoptotic and pyroptotic markers.ResultsShikonin was identified as a potent cytotoxic compound against EGFR-T790M-mutant NSCLC cells. Shikonin induced cell apoptosis and pyroptosis by triggering the activation of the caspase cascade and cleavage of poly (ADP-ribose) polymerase and gasdermin E by elevating intracellular ROS levels. Further investigations revealed that shikonin induced the degradation of COX-2 via the proteasome pathway, thereby decreasing COX-2 protein level and enzymatic activity and subsequently inhibiting the downstream PDK1/Akt and Erk1/2 signaling pathways through the induction of ROS production. Notably, COX-2 overexpression attenuated shikonin-induced apoptosis and pyroptosis, whereas COX-2 inhibition with celecoxib enhanced the cytotoxic effects of shikonin.ConclusionsCombination treatment with shikonin and COX-2 inhibitor may be a suitable therapeutic strategy for EGFR-T790M-mutant NSCLC treatment.