Inhibitor NS-398 Research Articles

The selective cyclooxygenase-2 inhibitor NS398 ameliorates cisplatin-induced impairments in mitochondrial and cognitive function.

Chemobrain is a condition that negatively affects cognition in cancer patients undergoing active chemotherapy, as well as following chemotherapy cessation. Chemobrain is also known as chemotherapy-induced cognitive impairment (CICI) and has emerged as a significant medical contingency. There is no therapy to ameliorate this condition, hence identification of novel therapeutic strategies to prevent CICI is of great interest to cancer survivors. Utilizing the platinum-based chemotherapy cisplatin in an investigative approach for CICI, we identified increased expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the adult mouse hippocampus, and in human cortical neuron cultures derived from induced pluripotent stem cells (iPSCs). Notably, administration of NS398, a selective COX-2 inhibitor, prevented CICI in vivo without negatively affecting the antitumor efficacy of cisplatin or potentiating tumor growth. Given that dysfunctional mitochondrial bioenergetics plays a prominent role in CICI, we explored the effects of NS398 in cisplatin-induced defects in human cortical mitochondria. We found that cisplatin significantly reduces mitochondrial membrane potential (MMP), increases matrix swelling, causes loss of cristae membrane integrity, impairs ATP production, as well as decreases cell viability and dendrite outgrowth. Pretreatment with NS398 in human cortical neurons attenuated mitochondrial dysfunction caused by cisplatin, while improving cell survival and neurite morphogenesis. These results suggest that aberrant COX-2 inflammatory pathways may contribute in cisplatin-induced mitochondrial damage and cognitive impairments. Therefore, COX-2 signaling may represent a viable therapeutic approach to improve the quality of life for cancer survivors experiencing CICI.

Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

Acetaminophen and ibuprofen are widely administered to babies due to their presumed safety as over-the-counter drugs. However, no reports exist on the effects of cyclooxygenase inhibitors on undifferentiated spermatogonia and spermatogonial stem cells. Infancy represents a critical period for spermatogonial stem cell formation and disrupting spermatogonial stem cells or their precursors may be associated with infertility and testicular cancer formation. The goal of this study was to examine the molecular and functional impact of cyclooxygenase inhibition and silencing on early steps of undifferentiated spermatogonia (u spg) and spermatogonial stem cell development, to assess the potential reproductive risk of pharmaceutical cyclooxygenase inhibitors. The effects of cyclooxygenase inhibition were assessed using the mouse C18-4 undifferentiated juvenile spermatogonial cell line model, previously shown to include cells with spermatogonial stem cell features, by measuring prostaglandins, cell proliferation, and differentiation, using cyclooxygenase 1- and cyclooxygenase 2-selective inhibitors NS398, celecoxib, and FR122047, acetaminophen, and ibuprofen. Cyclooxygenase 1 gene silencing was achieved using a stable short-hairpin RNA approach and clone selection, then assessing gene and protein expression in RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence studies. Cyclooxygenase 2 inhibitors NS398 and celecoxib, as well as acetaminophen, but not ibuprofen, dose-dependently decreased retinoic acid-induced expression of the spg differentiation gene Stra8, while NS398 decreased the spg differentiation marker Kit, suggesting that cyclooxygenase 2 is positively associated with spg differentiation. In contrast, short-hairpin RNA-based cyclooxygenase 1 silencing in C18-4 cells altered cellular morphology and upregulated Stra8 and Kit, implying that cyclooxygenase 1 prevented spg differentiation. Furthermore, RNA sequencing analysis of cyclooxygenase 1 knockdown cells indicated the activation of several signaling pathways including the TGFb, Wnt, and Notch pathways, compared to control C18-4 cells. Notch pathway genes were upregulated by selective cyclooxygenase inhibitors, acetaminophen and ibuprofen. We report that cyclooxygenase 1 and 2 differentially regulate undifferentiated spermatogonia/spermatogonial stem cell differentiation. Cyclooxygenases regulate Notch3 expression, with the Notch pathway targeted by PGD2. These data suggest an interaction between the eicosanoid and Notch signaling pathways that may be critical for the development of spermatogonial stem cells and subsequent spermatogenesis, cautioning about using cyclooxygenase inhibitors in infants.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

Buxu Tongyu Granule Alleviates Myocardial Ischemia by Activating Vascular Smooth Muscle Cell Soluble Guanylate Cyclase to Inhibit Abnormal Vasomotion

Myocardial ischemia is a serious threat to human health, and vascular dysfunction is its main cause. Buxu Tongyu (BXTY) Granule is an effective traditional Chinese medicine (TCM) for treating myocardial ischemia. However, the underlying mechanism of BXTY is still unclear. In this study, we demonstrate that BXTY ameliorates myocardial ischemia by activating the soluble guanylate cyclase (sGC)–3′,5′-cyclic guanosine monophosphate (cGMP)–protein kinase G (PKG) signaling pathway in vascular smooth muscle cells (VSMCs) to dilate the arteries. BXTY was given by gavage for ten consecutive days before establishing an animal model of acute myocardial ischemia in mice via the intraperitoneal injection of pituitrin. The results showed that BXTY alleviated the symptoms of myocardial ischemia induced by pituitrin in mice, including electrocardiogram abnormalities and changes in plasma enzymes. In addition, BXTY dilated pre-constricted blood vessels and inhibited the vasoconstriction of the superior mesenteric artery in a dose-dependent but endothelial-independent manner. These effects were eliminated by pre-incubating vascular rings with the sGC inhibitors NS 2028 or ODQ, or with the PKG inhibitor KT 5823. Moreover, BXTY increased the protein expression of sGC-β1 and the intracellular second messenger cGMP level in mouse aortic vascular smooth muscle cells (MOVAs). NS 2028 or ODQ reversed these effects of BXTY. The expression level of the cGMP downstream effector protein PKG-1 increased after treating MOVAs with BXTY. NS 2028, ODQ, or KT 5823 also reversed this effect of BXTY. In conclusion, BXTY can improve the symptoms of acute myocardial ischemia in mice, and activating the sGC–cGMP–PKG pathway in VSMCs to induce vasodilation is its key pharmacodynamic mechanism.

Smooth muscle dysfunction in the pre-inflammation site in stenotic Crohn's-like colitis: implication of mechanical stress in bowel dysfunction in gut inflammation.

Background and Aims: Gut smooth muscle dysfunctions contribute to symptoms such as abdominal cramping, diarrhea, and constipation in inflammatory bowel disease (IBD). The mechanisms for muscle dysfunctions are incompletely understood. We tested the hypothesis that mechanical stress plays a role in muscle dysfunction in a rat model of Crohn's-like colitis where inflammatory stenosis leads to mechanical distention in the pre-inflammation site. Methods: Crohn's-like colitis was induced by intracolonic instillation of TNBS (65mg/kg) in Sprague-Dawley rats. Control rats were instilled with saline. The rats were fed with either regular solid food or exclusively liquid diet. Rats were euthanized by day 7. Results: When rats were fed with solid food, TNBS treatment induced localized transmural inflammation with stenosis in the instillation site and marked distention with no inflammation in the pre-inflammation site of the colon. Smooth muscle contractility was suppressed, and expression of cyclo-oxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2) were increased not only in the inflammation site but also in the pre-inflammation site. Liquid diet treatment, mimicking exclusive enteral nutrition, completely released mechanical distention, eliminated COX-2 expression and PGE2 production, and improved smooth muscle contractility especially in the pre-inflammation site. When rats were administered with COX-2 inhibitor NS-398 (5mg/kg, i. p. daily), smooth muscle contractility was restored in the pre-inflammation site and significantly improved in the inflammation site. Conclusion: Colonic smooth muscle contractility is significantly impaired in stenotic Crohn's-like colitis rats not only in the inflammation site, but in the distended pre-inflammation site. Mechanical stress-induced expression of COX-2 plays a critical role in smooth muscle dysfunction in the pre-inflammation site in Crohn's-like colitis rats.

A179 MECHANISMS OF ACTION INVOLVED IN THE WOUND HEALING EFFECT OF THE DIETARY FIBRE RHAMNOGALACTURONAN

Abstract Background Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions associated with impaired intestinal epithelial barrier. Mucosal healing is the primary goal for IBD treatment since it is a good predictor of clinical remission. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal), a polysaccharide isolated from the plant Acmella oleracea, on intestinal epithelial barrier function with participation of TLR4 and PKC activation. RNAseq data and pathway analysis have indicated the involvement of the canonical nuclear factor kB (NF-kB) signaling pathway. Purpose We hypothesize that RGal increases intestinal epithelial wound healing through FAK-Src/PI3K/NF-kB signaling pathways. Method Caco-2 and T84 cells, and human primary cell monolayers grown from ulcerative colitis patient-derived organoids, were wounded and treated with vehicle (media or 0.5% DMSO in media) or RGal (1000 μg/ml) for 48 h. Wound healing was assessed using either the IncuCyte or ImageXpress Pico live cell imaging systems. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Inhibitors were added at the same time (transcription inhibitor Actinomycin D, 5 μg/ml) or 1 h (FAK inhibitor FAK-14, 10 μM, Src inhibitor PP2, 5 μM, PI3K inhibitor LY294002, 20 μM, NF-kB inhibitors Bay 11-7082 and JSH-23, 10 and 20 μM, respectively, or COX-2 inhibitor NS-398, 20 μM) before RGal treatment. Unwounded Caco-2 monolayers treated with RGal (1000 μg/ml) were collected for Western blotting for COX-2 protein. Result(s) In the wound healing assay, RGal (1000 μg/ml) enhanced wound healing by 12.5% at 48 h in Caco-2 cells and by 14.7% at 24 h in T84 cells, compared to control group. RGal (1000 μg/ml) also accelerated the wound closure in colonoid monolayers obtained from ulcerative colitis patient biopsies by 81.3% at 48 h. Neither proliferation nor apoptosis were involved in the RGal effect on wound healing. Treatment of cells with FAK14 (10 μM), PP2 (5 μM), and PI3K (20 μM) significantly prevented the RGal-induced wound healing. Actinomycin D (5 μg/ml), Bay 11-7082 (10 μM) or JSH-23 (20 μM) treatment significantly reversed the effect of RGal on wound healing, showing that the response was transcriptionally dependent and involved NF-kB signaling. Treatment of cells with NS-398 (20 μM) also reversed the effect of RGal on wound healing. COX-2 protein expression was significantly increased at 6 and 12 h after RGal addition to Caco-2 monolayers. Conclusion(s) These data suggest that the plant-based polysaccharide RGal increases intestinal epithelial cell wound healing by increasing cell migration. The RGal effect is dependent on the activation of the FAK-Src/PI3K signaling pathways and subsequently the transcription factor NF-kB and downstream COX-2 protein expression and activity. Our findings show a novel mechanism of action of RGal in wound healing that could help in the resolution of intestinal inflammation and mucosal healing. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; NSERC Disclosure of Interest None Declared

A PRECLINICAL MODEL OF STENOTIC CROHN’S DISEASE FOR THE STUDY OF MECHANISMS OF MOTILITY DYSFUNCTION IN IBD

Abstract BACKGROUND AND AIMS Motility dysfunction is present in patients with stenotic Crohn’s disease (CD) and contributes to cramps, diarrhea and constipation. However, mechanisms for motility dysfunction in CD are incompletely understood. Mechanical stress, i.e. inflammatory infiltration, edema, stenosis, and distention, is commonly encountered in CD. We tested the hypothesis in a rat model of stenotic CD that mechanical stress-induced expression of mechano-sensitive genes such as cyclo-oxygenase-2 (COX-2) may account for motility dysfunction in the inflammation site and the mechanically distended site prior to inflammation. ANIMAL MODEL AND METHODS Rat model of CD-like colitis was induced by intracolonic instillation of TNBS 65 mg/kg in 250 µL of 40% ethanol. Control rats were treated with intracolonic saline instillation. When rats were fed with regular pellet food, instillation of TNBS led to localized transmural inflammation with stenosis in the instillation site (site I) and lumen distention in the colon segment proximal to inflammation (site P). However, when rats were fed exclusively with liquid diet, inflammation-associated stenosis and mechanical distention was eliminated. Rats were euthanized by 7 days. RESULTS 1) Compared to controls, COX-2 mRNA expression was increased by 13.1(± 2.5)-fold in site I and 16.6(±4.3)-fold in the mechanically distended site P, but not in the non-distended site D. This data indicates a mechanosensitive mechanism in COX-2 expression. If mechanical distention in CD was prevented by feeding rats exclusively with clear liquid diet, the increase of COX-2 mRNA expression was attenuated in sites I and P. Further investigation showed that increased expression of COX-2 occurs mainly in the colonic smooth muscle cells (SMC) of the distended colon. 2) We found that COX-2 expression was robustly induced by mechanical stretch (18% static stretch) in vitro in rat colonic smooth muscle cells (SMC). 3) Inflammation led to significant suppression of colon circular muscle contractility not only in site I but also in distended site P of CD-like rats. Muscle contractility in site D was not significantly affected by inflammation. Moreover, clear liquid diet treatment significantly improved muscle contractility in sites P and I. 4) To determine the role of COX-2 in smooth muscle dysfunction in the CD model, COX-2 inhibitor NS-398 (5 mg/kg, i.p. daily) was administered in control and CD rats. We found that NS-398 significantly improved muscle contractility in CD rats in sites P and I. CONCLUSIONS Our study found that up-regulation of COX-2 expression in stenotic CD-like colitis is dependent on mechanical stress. COX-2 inhibitor improved colon smooth muscle contractility in colitis. Thus, mechanosensitive expression of COX-2 may play a critical role in smooth muscle dysfunction and dysmotility in CD.

TRPC5 mediates endothelium-dependent contraction in the carotid artery of diet-induced obese mice.

Little is known about the contribution of the transient receptor potential canonical channel isoform 5 (TRPC5), a Ca2+-sensitive channel, to vasoconstriction in obesity. In this study, we found that the TRPC5 expression and carotid artery contraction of diet-induced obese (DIO) mice were significantly higher than those of wild-type mice. Endothelium-dependent vasocontraction was inhibited by the TRPC5 inhibitor clemizole and the knockout of TRPC5 in DIO mouse carotid arteries, while activation of TRPC5 enhanced contraction in wild-type mice. TRPC5-regulated vasocontraction can be inhibited by the ROS scavenger NAC and the COX-2 inhibitor NS-398. Our study suggested that upregulation of TRPC5 contributes to endothelium-dependent contraction, which is involved in ROS production and COX-2 expression in DIO mouse carotid arteries. From these results, we speculated that TRPC5 mediated endothelium-dependent contraction in the carotid artery of DIO mice, which was achieved by increasing the levels of ROS and COX-2 expression.

Anoxia/reoxygenation enhances spontaneous contractile activity via TRPA1 channel and COX2 activation in isolated rat whole bladder.

Bladder ischemia/reperfusion is an important etiologic factor for overactive bladder disease. The occurrence of this disease is closely associated with enhanced spontaneous contractile activity of the bladder. However, the relationship between bladder ischemia/reperfusion and altered spontaneous bladder contractions (SBC) remains poorly studied. Therefore, the present study investigated whether ischemia/reperfusion affects SBC ex vivo. SBC was measured using isolated whole bladder preparations from rats. The preparations were exposed to anoxia (95% N2 ) for 0.5-6 h, followed by reoxygenation (95% O2 ) in Krebs medium. Anoxia followed by reoxygenation significantly enhanced the amplitude of SBC without affecting its frequency in an anoxic duration-dependent manner. The 5 h anoxia/reoxygenation-induced enhancement of SBC amplitude was completely suppressed by an antioxidant combination of L(+)-ascorbate/D, L-α-tocopherol, or N-acetyl cysteine. Additionally, the enhanced SBC amplitude was inhibited in a concentration-dependent manner by the nonselective TRP antagonist ruthenium red, or selective TRPA1 antagonists HC-030031 or AP-18. A similar inhibitory effect was obtained after repeated treatment with the TRPA1 agonist allyl isothiocyanate, as it induced acute desensitization of TRPA1 channels. Further, the enhanced SBC amplitude was significantly diminished by the nonselective cyclooxygenase (COX) inhibitor indomethacin or selective COX2 inhibitor NS-398, but not by the selective COX1 inhibitor SC-560 and 5-lipoxygenase inhibitor MK-886. The study findings reveal that the spontaneous contractile activity of the bladder is significantly enhanced in response to anoxia/reoxygenation, and that oxidative stress and activation of TRPA1 and COX2 (the resulting production of prostaglandins) are involved in the enhanced SBC activity.

Mesenchymal Stem Cells Attenuate Acute Lung Injury in Mice Partly by Suppressing Alveolar Macrophage Activation in a PGE2-Dependent Manner.

Mesenchymal stem cells (MSCs) have been demonstrated to attenuate acute lung injury (ALI). We also found that they can suppress the activation of alveolar macrophages (AMs), which can partly account for their therapeutic effects. MSCs do not inherently own immunosuppressive effects, when co-cultured with inflammatory immune cells, MSCs can be activated by inflammatory cytokines and meanwhile exert immunosuppressive effects. In order to further research, RNA sequencing (RNA-seq) of MSCs cultured before and after co-culturing with activated macrophages was performed. The data suggested a total of 5268 differentially expressed genes (DEGs) along the process. We used the data of 2754 upregulated DEGs to develop a signaling network of genes and the transcription factors targeting them in order to predict the altered functions of MSCs after exposure to inflammatory stimuli. This constructed network revealed some critical target genes and potential roles of MSCs under inflammatory conditions. According to the network, Ptgs2 was assumed to be an important gene participating in the immunosuppressive effects of MSCs. We also identified significant increases in the expression of COX2 protein and the secretion of PGE2 from MSCs. The use of the COX2 inhibitor NS-398 restrained the secretion of PGE2 and reversed the suppression of macrophage activation by MSCs in vitro. In addition, a selective antagonist of PGE2 binding receptor (EP4 receptor), GW627368X, also reversed the inhibitory effects of MSCs on AMs and the protective effects in ALI mouse. In summary, the therapeutic effects of MSCs on ALI partly occur through suppressing AM activation via PGE2 binding to EP4 receptor.

COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis.

Giardia duodenalis, the causative agent of giardiasis, is among the most important causes of waterborne diarrheal diseases around the world. Giardia infection may persist over extended periods with intestinal inflammation, although minimal. Cyclooxygenase (COX)-2 is well known as an important inducer of inflammatory response, while the role it played in noninvasive Giardia infection remains elusive. Here we investigated the regulatory function of COX-2 in Giardia-induced pro-inflammatory response and defense-related nitric oxide (NO) generation in macrophage-like cell line, and identified the potential regulators. We initially found that Giardia challenge induced up-regulation of IL-1β, IL-6, TNF-α, prostaglandin (PG) E2, and COX-2 in macrophages, and pretreatment of the cells with COX-2 inhibitor NS398 reduced expressions of those pro-inflammatory factors. It was also observed that COX-2 inhibition could attenuate the up-regulated NO release and inducible NO synthase (iNOS) expression induced by Giardia. We further confirmed that Giardia-induced COX-2 up-regulation was mediated by the phosphorylation of p38 and ERK1/2 MAPKs and NF-κB. In addition, inhibition of reactive oxygen species (ROS) by NAC was shown to repress Giardia-induced activation of MAPK/NF-κB signaling, up-regulation of COX-2 and iNOS, increased levels of PGE2 and NO release, and up-expressions of IL-1β, IL-6, and TNF-α. Collectively, in this study, we revealed a critical role of COX-2 in modulating pro-inflammatory response and defense-related NO production in Giardia-macrophage interactions, and this process was evident to be controlled by ROS-dependent activation of MAPK/NF-κB signaling. The results can deepen our knowledge of anti-Giardia inflammatory response and host defense mechanisms.

Inhibition of COX-2 ameliorates murine liver schistosomiasis japonica through splenic cellular immunoregulation

BackgroundWe have reported the positive association of the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) axis with liver fibrosis induced by Schistosoma japonicum (Sj) infection, and TLR4 signaling controlled this axis. However, how COX-2 regulates immune response during Sj infection is still unclear.MethodsHematoxylin and eosin staining was used to evaluate the effect of the COX-2-specific inhibitor NS398 on liver granulomatous inflammation and fibrosis. Flow cytometry was used to explore the frequency and amount of different immune cell infiltration in the spleen during Sj infection.ResultsNS398 significantly reduced the size of liver granuloma, spleen, and mesenteric lymph node (MLN) and alleviated chronic granulomatous inflammation. Mechanically, this might be by decreasing the number of Sj-induced macrophages and T helper type 1 (Th1), Th2, T follicular helper (Tfh), T follicular regulatory (Tfr), and germinal center B (GC B) cells. There were no differences in the number of neutrophils, myeloid-derived suppressor cells, Th17 cells, regulatory T cells (Treg), or total B cells in the spleen of the mice with or without NS398 treatment.ConclusionsCOX-2/PGE2 inhibition may represent a potential therapeutic approach for schistosomiasis japonica through splenic cellular immunoregulation.Graphical

A64 THE DIETARY FIBRE RHAMNOGALACTURONAN PROMOTES INTESTINAL EPITHELIAL CELL MIGRATION THROUGH THE NF-κB SIGNALING PATHWAY

BackgroundDamaged intestinal epithelial barrier is characteristic of inflammatory bowel diseases (IBD) and mucosal healing is the primary goal for IBD treatment. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal), a polysaccharide isolated from the plant Acmella oleracea, on intestinal epithelial barrier function with participation of TLR4 and PKC activation. We also observed that RGal accelerates wound healing in human colonic epithelial Caco-2 cells. RNAseq data and pathway analysis have indicated the involvement of the canonical nuclear factor kB (NF-kB) signaling pathway.AimsWe hypothesize that RGal increases intestinal epithelial wound healing through NF-kB signaling pathway.MethodsCaco-2 cells monolayers were scratched and treated with vehicle (media or 0.5% DMSO in media) or RGal (1000 mg/ml) for 48 h. Wound healing was assessed using the IncuCyte live cell imaging system. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Inhibitors were added at the same time (transcription inhibitor Actinomycin D, 5 mg/ml) or 1 h (NF-kB inhibitors Bay 11–7082 and JSH-23, 10 and 20 mM, respectively, or COX-2 inhibitor NS-398, 20 mM) before RGal treatment. Unwounded Caco-2 monolayers treated with RGal (1000 mg/ml) were collected for Western blotting for COX-2 protein.ResultsIn the wound healing assay, RGal at a concentration of 1000 µg/ml enhanced wound healing by 12.5% at 48 h compared to control group, under 10% serum conditions. Neither proliferation nor apoptosis were involved in the RGal effect on wound healing, suggesting the response was due solely to cell migration. Actinomycin D (5 mg/ml), Bay 11–7082 (10 mM) or JSH-23 (20 mM) treatment significantly reversed the effect of RGal on wound healing, showing that the response was transcriptionally dependent and involved NF-kB signaling. Treatment of cells with NS-398 (20 mM) also reversed the effect of RGal on wound healing. COX-2 protein expression was significantly increased at 6 and 12 h after RGal addition to Caco-2 monolayers.ConclusionsThese data suggest that the plant-based polysaccharide RGal increases intestinal epithelial cell wound healing by increasing cell migration. The RGal effect is dependent on the activation of the transcription factor NF-kB and downstream COX-2 protein expression and activity. Our findings show a novel mechanism of action of RGal in wound healing that could help in the resolution of intestinal inflammation and mucosal healing.Funding AgenciesNSERC

Effects of microsomal prostaglandin E synthase-1 inhibition on resistance artery tone in patients with end stage kidney disease.

The microvasculature is a target organ for the early manifestations of cardiovascular disease. Therefore, a better understanding of the prostaglandin system and characterising the effects of mPGES-1 inhibition and concomitant reduction of PGE2 in vascular beds are of interest. The effects of mPGES-1 inhibition on constriction and relaxation of resistance arteries (diameter: 100-400 μm) from patients with end stage kidney disease (ESKD) and controls (Non-ESKD) were studied using wire-myography in combination with immunological and mass-spectrometry based analyses. Inhibition of mPGES-1 in arteries from ESKD patients and Non-ESKD controls significantly reduced adrenergic vasoconstriction, which was unaffected by the COX-2 inhibitors NS-398 and Etoricoxib, or by the COX-1/COX-2 inhibitor Indomethacin tested in Non-ESKD controls. However, a significant increase of acetylcholine-induced dilatation was observed for mPGES-1 inhibition. In IL-1β treated arteries, inhibition of mPGES-1 significantly reduced PGE2 levels while PGI2 levels remained unchanged. In contrast, COX-2 inhibition blocked the formation of both prostaglandins. Blockade of PGI2 signalling with an IP receptor antagonist did not restore the reduced adrenergic constriction, neither did blocking PGE2 -EP4 or signalling through PPARγ. A biphasic effect was observed for PGE2 , inducing dilatation at nanomolar and constriction at micromolar concentrations. Immunohistochemistry demonstrated expression of mPGES-1, COX-1, PGIS, weak expression for COX-2, as well as receptor expression for PGE2 (EP1-4), thromboxane (TP) and PGI2 (IP) in ESKD and Non-ESKD. Our study demonstrates vasodilating effects following mPGES-1 inhibition in human microvasculature and suggests that several pathways besides shunting to PGI2 are involved.

Effects of different cyclooxygenase inhibitors on prostaglandin E2 production, steroidogenesis and ovulation of bovine preovulatory follicles.

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E2 (PGE2) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17β-estradiol, progesterone, and prostaglandin E2 (PGE2) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixin-meglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE2 concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE2 is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.

Fisetin suppresses cigarette smoke extract-induced epithelial to mesenchymal transition of airway epithelial cells through regulating COX-2/MMPs/β-catenin pathway

Cigarette smoke exposure leads to upregulation of cyclooxygenase‐2 (COX‐2), an inducible enzyme that synthesizes prostaglandin E2 (PGE2) and promotes airway inflammation. COX-2 overexpression is frequently implicated in inflammation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). However, its detailed molecular mechanism in cigarette smoke induced EMT is not clear. Further, fisetin, a bioflavonoid, exhibits antioxidant and anti-inflammatory properties, but its effect in modulating COX-2-mediated inflammation and downstream sequelae remains unexplored. Therefore, we have investigated the mechanism of cigarette smoke-induced COX-2-mediated EMT in airway epithelial cells and examined the role of fisetin in controlling this aberration. MTT, trypan blue staining, gelatin zymography, Western blotting, invasion, wound healing, and tumor sphere formation assays in cigarette smoke extract (CSE) and/or fisetin treated airway epithelial cells, and in-silico molecular docking studies were performed. Results revealed that CSE exposure increased the expression and activity of COX-2, MMP-2/9, and β-catenin and also enhanced expression of EMT markers leading to higher migration and invasion potential of airway epithelial cells. A specific COX-2 inhibitor NS-398 as well as fisetin treatment reversed the expression of EMT biomarkers, reduced the activity of MMP-2/9, and blocked the migration and invasion potential induced by CSE. Further, PGE2 also increased MMPs activity, invasion, and migration potential similar to CSE, which were significantly reversed by fisetin. In-silico studies showed a high binding affinity of fisetin to key EMT associated proteins, validating its anti-EMT potential. Thus, our study firstly unearths the mechanism of CSE-induced EMT in airway epithelial cells via COX-2/MMP/β-catenin pathway, and secondly, it reveals that fisetin could significantly reverse CSE-induced EMT by inhibiting COX-2, indicating that fisetin could be an effective drug candidate for cigarette smoke-induced lung dysfunction.

Ghrelin Regulates Cyclooxygenase-2 Expression and Promotes Gastric Cancer Cell Progression.

Aim To research the molecular mechanism of ghrelin in apoptosis, migratory, and invasion of gastric cancer (GC) cells. Methods After GC AGS cells were handled with ghrelin (10–8 M), cyclooxygenase-2 inhibitor NS398 (100 μM), and Akt inhibitor perifosine (10uM), the rates of apoptosis were detected by TUNEL assay and flow cytometry assay. We assessed the expressions of PI3K, p-Akt, and COX-2 proteins by making use of Western blot analysis. The cell migratory and invasion were detected by using wound-healing and transwell analysis. Results The migratory and invasion were increased in ghrelin-treated cells, while the rates of apoptosis were decreased. GC AGS cells treated with ghrelin showed an increase in protein expression of p-Akt, PI3K, and COX-2. After cells were treated with Akt inhibitor perifosine, the protein expression of p-Akt, PI3K, and COX-2 and the cell migratory, invasion, and apoptosis were partly recovered. After cells were treated with cyclooxygenase-2 inhibitor NS398, the protein expression of COX-2 and the cell migratory and invasion were decreased, while the rates of apoptosis were increased. Conclusion Ghrelin regulates cell migration, invasion, and apoptosis in GC cells through targeting PI3K/Akt/COX-2. Ghrelin increases the expression of COX-2 in GC cells by targeting PI3K/Akt. Ghrelin is suggested to be one of the molecular targets in GC.

COX-2 promotes the osteogenic potential of BMP9 through TGF-β1/p38 signaling in mesenchymal stem cells

This study investigated the effects of transforming growth factor-β1 (TGF-β1) and cyclooxygenase-2 (COX-2) on bone morphogenetic protein 9 (BMP9) in mesenchymal stem cells (MSCs). We found that BMP9 increased mRNA levels of TGF-β1 and COX-2 in C3H10T1/2 cells. BMP9-induced osteogenic markers were enhanced by TGF-β1 and reduced by TGF-βRI-specific inhibitor LY364947. BMP9 increased level of p-Smad2/3, which were either enhanced or reduced by COX-2 and its inhibitor NS398. BMP9-induced osteogenic markers were decreased by NS398 and it was partially reversed by TGF-β1. COX-2 increased BMP9-induced osteogenic marker levels, which almost abolished by LY364947. BMP9-induced bone formation was enhanced by TGF-β1 but reduced by silencing TGF-β1 or COX-2. BMP9’s osteogenic ability was inhibited by silencing COX-2 but partially reversed by TGF-β1. TGF-β1 and COX-2 enhanced activation of p38 signaling, which was induced by BMP9 and reduced by LY364947. The ability of TGF-β1 to increase the BMP9-induced osteogenic markers was reduced by p38-specific inhibitor, while BMP9-induced TGF-β1 expression was reduced by NS398, but enhanced by COX-2. Furthermore, CREB interacted with Smad1/5/8 to regulate TGF-β1 expression in MSCs. These findings suggest that COX-2 overexpression leads to increase BMP9’s osteogenic ability, resulting from TGF-β1 upregulation which then activates p38 signaling in MSCs.

Chronic Low-Level Lead Exposure Increases Mesenteric Vascular Reactivity: Role of Cyclooxygenase-2-Derived Prostanoids.

Lead (Pb) exposure causes hazardous effects as hypertension and other cardiovascular diseases. We evaluated whether chronic Pb exposure alters the peripheral vascular resistance measuring the vascular reactivity of mesenteric resistance arteries in rats to identify the underlying mechanisms that are associated to the development of Pb-induced hypertension. Mesenteric resistance arteries from lead-treated and untreated Wistar rats (1st dose: 10 μg/100 g; subsequent doses: 0.125 μg/100 g, intramuscular, 30 days) were used. Contractile responses to phenylephrine increased, while acetylcholine and sodium nitroprusside-induced relaxation was not affected by lead treatment. Endothelium removal and inhibition of NO synthase by L-NAME similarly enhanced the response to phenylephrine in untreated and lead-treated rats. The antioxidants apocynin and superoxide dismutase (SOD) did not affect vasoconstriction in either group. The vascular expression of cyclooxygenase-2 (COX-2) protein increased after lead exposure. The respective non-specific or specific COX-2 inhibitors indomethacin and NS398 reduced more strongly the response to phenylephrine in treated rats. Antagonists of EP1 (SC19220), TP (SQ29548), IP (CAY10441) and angiotensin II type 1 (losartan) receptors reduced vasoconstriction only in treated rats. These conclusions present further evidence that lead, even in small concentration, produces cardiovascular hazards being an environmental contaminant that account for lead-induced hypertension.

PP2 Ameliorates Renal Fibrosis by Regulating the NF-κB/COX-2 and PPARγ/UCP2 Pathway in Diabetic Mice.

Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis in diabetic nephropathy (DN). We aimed to evaluate the effects of PP2 on renal fibrosis of DN. GSE33744 and GSE86300 were downloaded from the GEO database. Firstly, 839 DEGs were identified between nondiabetic and diabetic mice renal glomerular samples. COX-2 was selected to assess the effects of PP2 on renal glomerulosclerosis. In db/db mice, PP2 decreased the expression of COX-2, phosphorylated p65, and fibrotic proteins, accompanied with attenuated renal glomerulosclerosis. In cultured glomerular mesangial cells, high glucose- (HG-) induced p65 phosphorylation and COX-2 expression were attenuated by PP2 or NF-κB inhibitor PDTC. PP2, PDTC, or COX-2 inhibitor NS-398 ameliorated abnormal proliferation and expression of fibrotic proteins induced by HG. Secondly, 238 DEGs were identified between nondiabetic and diabetic mice renal cortex samples. UCP2 was selected to assess the effects of PP2 on renal tubulointerstitial fibrosis. In db/db mice, PP2 decreased the expression of PPARγ and UCP2, accompanied with attenuated renal tubulointerstitial fibrosis and EMT. In cultured proximal tubular cells, HG-induced PPARγ and UCP2 expression was inhibited by PP2 or PPARγ antagonist GW9662. PP2, GW9662, or UCP2 shRNA ameliorated HG-induced EMT. These results indicated that PP2 ameliorated renal fibrosis in diabetic mice.