Downregulation Of Cyclooxygenase-2 Expression Research Articles

Exploring the Benefits of Herbal Medicine Composite 5 (HRMC5) for Skin Health Enhancement.

The skin, as the body's largest organ, is vital for protecting against environmental stressors, regulating temperature, and preventing water loss. Here, we examined the potential of a mixture of five traditional Korean herbal extracts-Cimicifuga racemosa, Paeonia lactiflora, Phellodendron amurense, Rheum rhaponticum, and Scutellaria baicalensis-referred to as herbal medicine composite 5 (HRMC5) for enhancing skin health and managing menopausal symptoms. High-performance liquid chromatography identified 14 bioactive compounds, including flavonoids, phenolic acids, anthraquinones, and alkaloids. In vitro studies revealed an optimal concentration of 0.625 g/L for cell survival and UV protection, with the mixture demonstrating significant wound-healing properties comparable to epidermal growth factor. HRMC5 exhibited anti-inflammatory effects by downregulating COX2 expression and upregulating the key skin barrier proteins. A 4-week clinical trial involving 20 postmenopausal women showed significant improvements in skin redness, hemoglobin concentration, and skin moisture content. Visual analog scale assessments indicated substantial reductions in facial flushing severity and the associated sweating. The topical application of HRMC5 cream offered potential advantages over ingested phytoestrogens by reducing the systemic side effects. These findings suggest that HRMC5 is a promising non-invasive treatment for vasomotor symptoms in menopausal women and overall skin health, warranting further research on its long-term efficacy and safety in larger populations.

Linoleic Acid Induces Metabolic Reprogramming and Inhibits Oxidative and Inflammatory Effects in Keratinocytes Exposed to UVB Radiation.

Linoleic acid (LA), the primary ω-6 polyunsaturated fatty acid (PUFA) found in the epidermis, plays a crucial role in preserving the integrity of the skin's water permeability barrier. Additionally, vegetable oils rich in LA have been shown to notably mitigate ultraviolet (UV) radiation-induced effects, including the production of reactive oxygen species (ROS), cellular damage, and skin photoaging. These beneficial effects are primarily ascribed to the LA in these oils. Nonetheless, the precise mechanisms through which LA confers protection against damage induced by exposure to UVB radiation remain unclear. This study aimed to examine whether LA can restore redox and metabolic equilibria and to assess its influence on the inflammatory response triggered by UVB radiation in keratinocytes. Flow cytometry analysis unveiled the capacity of LA to diminish UVB-induced ROS levels in HaCaT cells. GC/MS-based metabolomics highlighted significant metabolic changes, especially in carbohydrate, amino acid, and glutathione (GSH) metabolism, with LA restoring depleted GSH levels post-UVB exposure. LA also upregulated PI3K/Akt-dependent GCLC and GSS expression while downregulating COX-2 expression. These results suggest that LA induces metabolic reprogramming, protecting against UVB-induced oxidative damage by enhancing GSH biosynthesis via PI3K/Akt signaling. Moreover, it suppresses UVB-induced COX-2 expression in HaCaT cells, making LA treatment a promising strategy against UVB-induced oxidative and inflammatory damage.

Pyrazoles have a multifaceted anti-inflammatory effect targeting prostaglandin E2, cyclooxygenases and leukocytes’ oxidative burst

Elevated levels of prostaglandin E2 have been implicated in the pathophysiology of various diseases. Anti-inflammatory drugs that act through the inhibition of cyclooxygenase enzymatic activity, thereby leading to the suppression of prostaglandin E2, are often associated with several side effects due to their non-specific inhibition of cyclooxygenase enzymes. Consequently, the targeted suppression of prostaglandin E2 production with innovative molecules and/or mechanisms emerges as a compelling therapeutic strategy for the treatment of inflammatory-related diseases. Therefore, in this study, a systematic analysis of 28 pyrazole derivatives was conducted to explore their potential mechanisms for reducing prostaglandin E2 levels. In this context, the evaluation of these derivatives extended to examining their capacity to reduce prostaglandin E2in vitro in human whole blood, inhibit cyclooxygenase-1 and cyclooxygenase-2 enzymes, modulate cyclooxygenase-2 expression, and suppress oxidative burst in human leukocytes. The results enabled the establishment of significant structure-activity relationships, elucidating key determinants for their activities. In particular, the 4-styryl group on the pyrazole moiety and the presence of chloro substitutions were identified as key determinants. Pyrazole 8 demonstrated the capacity to reduce prostaglandin E2 levels by downregulating cyclooxygenase-2 expression, and pyrazole-1,2,3-triazole 18 emerged as a dual-acting agent, inhibiting human leukocytes' oxidative burst and cyclooxygenase-2 activity. Furthermore, pyrazole 26 demonstrated effective reduction of prostaglandin E2 levels through selective cyclooxygenase-1 inhibition. These results underscore the multifaceted anti-inflammatory potential of pyrazoles, providing new insights into the substitutions and structural frameworks that are beneficial for the studied activity.

The Antiproliferative and Proapoptotic Effects of Cucurbitacin B on BPH-1 Cells via the p53/MDM2 Axis.

Cucurbitacin B (Cu B), a triterpenoid compound, has anti-inflammatory and antioxidant activities. Most studies only focus on the hepatoprotective activity of Cu B, and little effort has been geared toward exploring the effect of Cu B on the prostate. Our study identified that Cu B inhibited the proliferation of the benign prostatic hyperplasia epithelial cell line (BPH-1). At the molecular level, Cu B upregulated MDM2 and thrombospondin 1 (THBS1) mRNA levels. Immunocytochemistry results revealed that the protein expressions of p53 and MDM2 were upregulated in BPH-1 cells. Furthermore, Cu B upregulated THBS1 expression and downregulated COX-2 expression in the BPH-1 cell supernatant. Altogether, Cu B may inhibit prostate cell proliferation by activating the p53/MDM2 signaling cascade and downregulating the COX-2 expression.

Salvia miltiorrhiza and Tanshinone IIA reduce endothelial inflammation and atherosclerotic plaque formation through inhibiting COX-2

The mechanisms of Salvia miltiorrhiza (SM) and Tanshinone IIA (Tan IIA) in the treatment of atherosclerosis was examined by combining network pharmacology and molecular biology experiments. The TCMSP and BATMAN-TCM databases provided 104 SM candidate ingredients and 813 target genes, while GEO and GeneCards databases identified 35 overlapping targets between SM and coronary artery disease (CAD). From these data, we constructed a CAD-target-active ingredient network, and using Gene Ontology (GO) and KEGG pathway analysis, 211 GO terms and 43 pathways were identified, which facilitated the construction of a key active ingredient-target-pathway network. We then constructed a protein-protein interaction (PPI) network and performed molecular docking simulations between Tan IIA and 10 key target proteins to analyze the interactions between the molecule and the protein. SM was found to alleviate CAD by reducing the expression of key pro-inflammatory factors, such as COX-2 (PTGS2), MMP9, ICAM1, TNF-α, and NF-κB. Tan IIA was identified as the primary effective component of SM in treating CAD, with TNF and PTGS2 being its main targets. We further validated these findings using in vitro/in vivo experiments. The results showed that both SM and Tan IIA attenuated the buildup of plaque and the accumulation of lipids in ApoE-/- mice. In addition, SM and Tan IIA reduced vascular inflammatory factors expression in ApoE-/- mice and ox-LDL-cultured HUVECs. Furthermore, our findings showed that Tan IIA reduced vascular endothelial inflammation and prevented plaque formation via COX-2/TNF-a/NF-κB signaling pathway. We have demonstrated for the first time that Tan IIA plays a vital role in attenuating atherosclerosis by downregulating COX-2 expression.

ED Formula, a Complex of Ecklonia cava and Chrysanthemum indicum, Ameliorates Airway Inflammation in Lipopolysaccharide-Stimulated RAW Macrophages and Ovalbumin-Induced Asthma Mouse Model.

Ecklonia cava (E. cava) and Chrysanthemum indicum Linne (C. indicum) are natural raw materials known to have beneficial effects on inflammatory-related diseases, as evidenced by various sources in the literature. This study aimed to investigate the airway-protective effects of a formulation called ED, comprising E. cava and C. indicum, by evaluating its potential anti-inflammatory properties. Methods: The major components of ED were analyzed using high-performance liquid chromatography (HPLC) and its anti-inflammatory activity was assessed in RAW 264.7 cells through measurements of nitric oxide's (NO) inhibitory effect, cyclooxygenase (COX)-2 protein expression, and the mitogen-activated protein kinase (MAPK) signaling pathway. Additionally, the anti-inflammatory effect of ED was evaluated in an ovalbumin-induced asthma model by measuring cytokine levels in serum, bronchoalveolar lavage fluid (BALF), and lung tissue. Through HPLC analysis, the major components of ED, dieckol and luteolin, were identified. ED demonstrated no cytotoxicity and effectively reduced NO production in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Moreover, ED downregulated COX-2 expression through the MAPK signaling pathway in LPS-induced RAW 264.7 cells. In the ovalbumin-induced asthma model, the ED-treated group exhibited reduced levels of inflammatory cytokines in lung tissue. Furthermore, the ED-treated group showed a decrease in the number of inflammatory cells in BALF and lower serum interleukin (IL)-6 levels compared to the ovalbumin-treated group. These results suggest that ED has the potential to be a novel therapeutic agent for improving inflammatory respiratory diseases.

Kinsenoside mitigates myocardial ischemia/reperfusion-induced ferroptosis via activation of the Akt/Nrf2/HO-1 pathway

Ischemia-induced myocardial infarction is regarded as one of the major killers of humans worldwide. Kinsenoside (KD), a primary active ingredient derived from Anoectochilus roxburghii, shows antioxidant and vascular protective properties. Myocardial ischemia/reperfusion (I/R) injury is associated with oxidative damage and could be regulated by KD. However, its targets and the exact mechanism by which it operates remains unclear. The aim of this study was to investigate the role of KD in myocardial I/R injury and to define the mechanism by which it works. We established both myocardial I/R model in vivo and hypoxia/reoxygenation (H/R) cardiomyocyte model in vitro in this study. KD can attenuate I/R-induced myocardial injury in vivo and inhibit H/R-induced injury in vitro in a dose-dependent manner. KD increased mitochondrial membrane potential, SOD activity, and GSH activity in cardiomyocytes, whereas MDA accumulation, iron accumulation, and Mito-ROS production were decreased. We intersected differentially expressed genes (DEGs) from RNA-seq results with ferroptosis-related genes, and found KD significantly downregulated COX2 expression and upregulated GPX4 expression. These findings were further confirmed by Western blot analysis. Additionally, KD increased AKT phosphorylation and Nrf2 translocation into the nucleus, as well as HO-1 expression. When Akt or Nrf2 were inhibited in the KD group, the anti-ferroptosis properties of KD were nullified. Thus, Kinsenoside may exert anti-ferroptosis effect in myocardial I/R injury by decreasing mitochondrial dysfunction and increasing anti-oxidation through the Akt/Nrf2/HO-1 signaling pathway, suggesting it could be used as a potential therapeutic agent for myocardial reperfusion injury.

Natural Antimicrobials Promote the Anti-Oxidative Inhibition of COX-2 Mediated Inflammatory Response in Primary Oral Cells Infected with Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis.

Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis can colonize the tooth root canals, adhere to dentin walls, and frequently cause periodontitis in dogs. Bacterial periodontal diseases are common in domesticated pets, causing severe oral cavity inflammation and a strong immune response. This study investigates the antioxidant effect of a natural antimicrobial mixture (Auraguard-Ag) on the ability of S. aureus, S. pyogenes and E. faecalis to infect primary canine oral epithelial cells as well as its impact on their virulence factors. Our data show that a concentration of 0.25% Ag is sufficient to inhibit the growth of all three pathogens, whereas a concentration of 0.5% will become bactericidal. The sub-inhibitory concentration of 0.125% Ag reveals that the antimicrobial mixture can significantly reduce biofilm formation and exopolysaccharide production. The impact on these virulence factors was further translated into a significantly reduced ability to infect primary canine oral epithelial cells and restore epithelial tight junctions, with no impact on the epithelial cell viability. The post-infection inflammatory cytokines (IL-1β and IL-8) and the COX-2 mediator were also reduced both in mRNA and protein expression levels. The oxidative burst, detected upon infection, was also decreased in the presence of Ag, as our results show a significant decrease in H2O2 released by the infected cells. We show that inhibition of either NADPH or ERK activity will result in a downregulation of COX-2 expression and lower levels of H2O2 in infected cells. Conclusively, our study shows that natural antimicrobials reduce pro-inflammatory events, post infection, through an antioxidative mechanism that involves the downregulation of the COX-2 mediator via the inactivation of ERK in the absence of H2O2. As a result, they significantly reduce the risk of secondary bacterial infections and host oxidative stress caused by Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis accumulation in biofilms in an in vitro canine oral infection model.

Evaluation of in vitro anti-inflammatory effect of PLAY® on UC-MSCs: A COX-2 expression study

Wound healing is an intricate process consisting of multiple phases, each of which is indispensable for requisite tissue repair. Timely initiation and resolution of each overlapping phase is critical in aiding the wound healing cascade. A prolonged inflammatory phase causes overexpression of proteolytic mediators, resulting in extracellular matrix (ECM) degradation and increased oxidative stress. This imbalance in inflammatory mediators, ECM degradation, and upregulated levels of reactive oxygen species retards cell proliferation and migration, resulting in delayed tissue repair and the development of chronic wounds. Cyclooxygenase-2 (COX-2) is a potent inflammatory mediator that plays a pivotal role in the wound healing process at different stages of the tissue repair cascade. Overexpression of COX-2 during the tissue repair process impairs the wound healing cascade and contributes to the formation of chronic wounds. PLAY® is an in-house product of International Stem Cell Services Limited (iCREST) that is a human blood-platelet cocktail of natural growth factors and bioactive modulators. In our study, we hypothesized that PLAY® decreases inflammation in phorbol myristate acetate (PMA)- and 5-fluorouracil (5-FU)- stimulated umbilical cord derived mesenchymal stem cells (UC-MSCs) by downregulating COX-2 expression to its basal levels. We used an experimentally induced inflammatory in vitro model to produce enhanced COX-2 expression in mesenchymal stem cells (MSCs) and PLAY® showed a significant decrease in COX-2 levels in PMA induced UC-MSCs, providing evidence for an innate anti-inflammatory effect of PLAY® and a potential role in accelerated wound healing.

Inhibition of sPLA2 enzyme activity by cell-permeable antioxidant EUK-8 and downregulation of p38, Akt, and p65 signals induced by sPLA2 in inflammatory mouse paw edema model.

The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A2 (PLA2 ). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA2 s. Among these, we found EUK-8 is a potent inhibitor with its IC50 value ranges 0.7-2.0 µM for sPLA2 s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA2 . In vivo effect of EUK-8 was studied in HSF-sPLA2 -induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA2 activity, it also plays an important role in the regulation of sPLA2 -inducedcell signaling cascades. Apart from the sPLA2 inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.

Cross-species comparisons reveal resistance of human skeletal stem cells to inhibition by non-steroidal anti-inflammatory drugs.

Fracture healing is highly dependent on an early inflammatory response in which prostaglandin production by cyclo-oxygenases (COX) plays a crucial role. Current patient analgesia regimens favor opioids over Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) since the latter have been implicated in delayed fracture healing. While animal studies broadly support a deleterious role of NSAID treatment to bone-regenerative processes, data for human fracture healing remains contradictory. In this study, we prospectively isolated mouse and human skeletal stem cells (SSCs) from fractures and compared the effect of various NSAIDs on their function. We found that osteochondrogenic differentiation of COX2-expressing mouse SSCs was impaired by NSAID treatment. In contrast, human SSCs (hSSC) downregulated COX2 expression during differentiation and showed impaired osteogenic capacity if COX2 was lentivirally overexpressed. Accordingly, short- and long-term treatment of hSSCs with non-selective and selective COX2 inhibitors did not affect colony forming ability, chondrogenic, and osteogenic differentiation potential in vitro. When hSSCs were transplanted ectopically into NSG mice treated with Indomethacin, graft mineralization was unaltered compared to vehicle injected mice. Thus, our results might contribute to understanding species-specific differences in NSAID sensitivity during fracture healing and support emerging clinical data which conflicts with other earlier observations that NSAID administration for post-operative analgesia for treatment of bone fractures are unsafe for patients.

MiR-204-5p is sponged by TUG1 to aggravate neuron damage induced by focal cerebral ischemia and reperfusion injury through upregulating COX2

Studies have reported that miR-204-5p is involved in multiple biological processes. However, little is known about the expression and mechanism of miR-204-5p in cerebral ischemia and reperfusion injury. This study found that miR-204-5p expression was significantly downregulated in the blood of patients with ischemic stroke, MCAO/R rat brains, and OGD/R neurons. Overexpression of miR-204-5p markedly reduced infarct volume and neurological impairment and alleviated the inflammatory response in vivo. miR-204-5p promoted neuronal viability and reduced apoptotic cells in vitro. Mechanically, miR-204-5p was negatively regulated by the expression lncRNA TUG1 upstream and down-regulated COX2 expression downstream. Therefore, the TUG1/miR-204-5p/COX2 axis was involved in ischemia and reperfusion-induced neuronal damage. This finding may provide a novel strategy for the treatment of cerebral ischemia and reperfusion injury.

Co-delivery of Dexamethasone and a MicroRNA-155 Inhibitor Using Dendrimer-Entrapped Gold Nanoparticles for Acute Lung Injury Therapy.

Development of nanomedicines for effective therapy of acute lung injury (ALI), a common critical respiratory failure syndrome, remains to be challenging. We report here a unique design of a functional nanoplatform based on generation 5 (G5) poly(amidoamine) dendrimer-entrapped gold nanoparticles (Au DENPs) to co-deliver dexamethasone (Dex) and a microRNA-155 inhibitor (miR-155i) for combination chemotherapy and gene therapy of ALI. In this study, we synthesized Au DENPs with 10 Dex moieties attached per G5 dendrimer and an Au core diameter of 2.1 nm and used them to compress miR-155i. The generated polyplexes own a positive zeta potential (16-26 mV) and a small hydrodynamic diameter (175-230 nm) and display desired cytocompatibility and efficient miR-155i delivery to lipopolysaccharide (LPS)-activated alveolar macrophages, thus upregulating the suppressor of cytokine signaling 1 and IL-10 expression and downregulating the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Likewise, as a synthetic glucocorticoid with a potent anti-inflammatory property, the attached Dex on the surface of Au DENPs could inhibit pro-inflammatory cytokine secretion by down-regulating cyclooxygenase-2 expression in the LPS-activated alveolar macrophages. The integration of Dex and miR-155i within one nanoformulation enables superior downregulation of pro-inflammatory cytokines for successful repair of damaged lung tissues in an ALI model, as demonstrated by histological examinations and pro-inflammatory cytokine downregulation in ALI lesion at the gene and protein levels. Such a combined chemotherapy and gene therapy strategy enabled by dendrimer nanotechnology may hold great promise to treat other types of inflammatory diseases.

Abnormal Pressure Stress Reduces Interleukin-1β-Induced Cyclooxygenase-2 Expression in Cultured Rat Vascular Smooth Muscle Cells.

Elevated mechanical stress on blood vessels associated with hypertension has a direct effect on the function of vascular endothelial cells and vascular smooth muscle cells (VSMCs). In the present study, we have identified the effect of pulsatile pressure stress on cyclooxygenase-2 (COX-2) expression induced by interleukin (IL)-1β in cultured rat VSMCs. VSMCs were isolated from aortic media of Wistar rats and cultured. Pulsatile pressure applied to VSMCs was repeatedly given between either 80 and 160 mmHg, which simulates systolic hypertension, or 80 and 120 mmHg, which simulates normal blood pressure, at a frequency of 4 cycles per min using our original apparatus. Pressure loading that simulates systolic hypertension reduced IL-1β-induced COX-2 expression. The pressure also inhibited the rapid and transient phosphorylation of extracellular signal-regulated kinase (ERK) induced by IL-1β. IL-1β-induced COX-2 expression was significantly inhibited by a specific conventional protein kinase C (PKC) inhibitor. Pressure loading that simulates systolic hypertension also reduced phorbol myristate 13-acetate (PMA) (a PKC activator)-induced COX-2 expression and the rapid and transient phosphorylation of ERK. Pressure loading that simulates normal blood pressure had no effect on IL-1β- and PMA-induced COX-2 expression. The present study shows that pressure stress between 80 and 160 mmHg, which simulates systolic hypertension reduces IL-1β-induced COX-2 expression by affecting a mechanism involving PKC and ERK signaling pathways. Downregulation of COX-2 expression in VSMCs by abnormal pressure stress may further worsen local vascular injury associated with hypertension.

Effect of CO 2 on the Expression of cyclooxigenase in Vascular Smooth Muscle Cells

Objectives Carbon dioxide(CO2) is one of the factors that affect the constriction and dilatation of smooth muscle cells. Cyclooxigenase(COX) catalyzes have important roles in inducing inflammatory responses and prostanoids, generated from arachidonic acid by COX-1 and COX-2, are involved in modulating of vascular tone. This study aimed to clarify the role of CO2 in regulating COX expression in vascular smooth muscle cells and elucidate its effects for vascular endothelial function. Methods A7r5 rat smooth muscle cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37ºC in a 5% CO2 incubator. The cells were exposed to 10% and 20% CO2 for 24 h. The expression of COX-1 and COX-2 was subsequently evaluated by real time PCR, and cell viability was analyzed following exposure to 10% and 20% CO2. Results The expression levels of COX-2 were significantly reduced by concentration dependence in the vascular smooth muscle cells following exposure to 10% and 20% CO2, while no significant difference was found in those of COX-1 before and after exposure. Cell viability also decreased on the depending on the concentration of CO2. Conclusions These results suggested that exposure to CO2 may suppress the vasodilatation via the downregulation of COX-2 expression in vascular smooth muscle cells.

Mitochondrial gene COX2 methylation and downregulation is a biomarker of aging in heart mesenchymal stem cells.

The mitochondria have been proven to be involved in processes of aging; however, the mechansims through which mitoepigenetics affect the cytological behaviors of cardiomyocytes during the aging process are not yet fully understood. In the present study, two senescence models were constructed, replicative senescence (RS) and stress-induced premature senescence (SIPS), using human heart mesenchymal stem cells (HMSCs). First, the differences in age-related gene expression levels and telomere length were compared between the HMSCs in the RS and SIPS models by PCR. Subsequently, protein expression and the mitochondrial DNA (mtDNA) methylation status of cytochrome c oxidase subunit II (COX2) was measured by western blot analysis and bisulfite genomic sequencing (BSP). Finally, the value of the DNA methyltransferase (Dnmt) inhibitor, 5-aza-2′-deoxycytidine (AdC), in delaying the senescence of HMSCs was evaluated. It was found that the p16, p27 and p53 mRNA expression levels increased in the senescent cells, whereas p21 mRNA expression did not. It was also found that telomere shortening only occurred in the RS model, but not in the SIPS model. Along with the senescence of HMSCs, COX2 gene methylation increased and its protein expression level significantly decreased. It was demonstrated that AdC inhibited COX2 methylation and downregulated COX2 expression. The addition of exogenous COX2 or the administration of AdC promoted cell proliferation and delayed cell aging. On the whole, the present study demonstrates that COX2 methylation and downregulation are biomarkers of HMSC senescence. Thus, COX2 may have potential for use as a therapeutic target of cardiovascular diseases and this warrants further investigation.

A new fluorescence method for monitoring PNK activity in vitro, natural compounds screening and intracellular imaging

Polynucleotide kinase (PNK) is critical for nucleic acid metabolism, as its overexpression can increase ROS production and activate the inflammation pathway, leading to chronic inflammation. In this study, we proposed a new fluorescence method for monitoring PNK in vitro, natural compounds screening and intracellular imaging. Quantitative detection depicted that the limit of detection (LOD) of PNK is 0.0021 U/mL in the linear range of 0.01∼10 U/mL. Methodological evaluation showed that both of the selectivity and the inhibitory effect of ADP and EDTA on PNK activity with satisfactory results. Fluorescence imaging of inflammatory cells demonstrated the reliability of platform for capturing PNK change in situ regulated by inhibitor of natural compound NS17 isolated from Kadsura heterolita. Mechanism study showed that the inhibitor NS17 can reduce inflammation by scavenging intracellular ROS, killing inflammatory cells and down-regulating COX-2 expression, the processes of which are related with PNK activity. In summary, above results suggest that this new platform could monitor PNK activity in situ and screen inflammation inhibitors targeted on PNK.

Hejie Zhitong prescription promotes sleep and inhibits nociceptive transmission-associated neurotransmitter activity in a rodent migraine model

BackgroundMigraine is painful disease in which neurotransmitters related to pain transmission play an important role. Hejie Zhitong prescription (HJZT) has been used in the clinic as an effective prescription for the treatment of migraine for many years. Our team aimed to further explore its antimigraine mechanism based on previous research results and to explore the inhibitory effect of HJZT on the transmission of pain related to nitroglycerine (NTG)-induced migraine as well as the synergistic effect of HJZT with pentobarbital sodium on promoting sleep.MethodsSixty mice were randomly assigned to groups and received the corresponding interventions. Sleep latency and sleep time were recorded to calculate the incidence of sleep. Forty-eight Wistar rats were randomly assigned and administered an intervention corresponding to their group. Calcitonin gene-related peptide (CGRP), serotonin (5-HT), substance P (SP), and cholecystokinin (CCK) levels were measured using ELISAs. Levels of the cannabinoid receptor type 1 (CB1R) and cyclooxygenase-2 (COX-2) protein were assessed using immunohistochemistry. The expression of the CGRP and CCK mRNAs in the midbrain and trigeminal ganglion (TG) were measured using real-time quantitative PCR.ResultsHJZT promoted the occurrence of sleep in mice. HJZT downregulated COX-2 expression in the midbrain and TG of rats but upregulated the expression of the CB1R, and decreased the plasma level of the CGRP protein and expression of its mRNA in the midbrain and TG. It also downregulated the expression of the CCK mRNA in the midbrain and TG. The high-dose HJZT treatment increased plasma 5-HT levels, but did not induce changes in the plasma levels of the SP or CCK protein.ConclusionsHJZT exerts a synergistic effect with pentobarbital sodium on promoting sleep. As for anti-migraine, HJZT can inhibits the expression of nociceptive transmission-associated neurotransmitters, including 5-HT, CGRP and CCK, which may be related to its upregulation of CB1R and downregulation of COX-2.

Yokukansan, a Japanese Herbal Medicine, Suppresses Substance PInduced Production of Interleukin-6 and Interleukin-8 by Human U373 MG Glioblastoma Astrocytoma Cells.

Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear. This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP). We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p < 0.05. Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells. Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Lactobacillus acidophilus and Bifidobacterium longum exhibit antiproliferation, anti-angiogenesis of gastric and bladder cancer: Impact of COX2 inhibition

BackgroundProbiotics have gained exponential attention worldwide because they are used as a therapeutic tool for management, prevention and treatment of diseases like cancer. Here, antiproliferation, anti-angiogenesis and antitumor effects of Lactobacillus acidophilus (LA) and Bifidobacterium longum (BL) were examined on gastric (AGS) and bladder (J253) cancer cell lines. MethodsCancer cell viability was monitored with MTT assay. The chick embryo chorioallantoic membrane (CAM) assay was used to screen tumor growth and hemoglobin content of cancer cells alone and with different LA and BL concentrations. Protein expression of COX2 was analyzed with Western blot in cell lines treated with LA, BL, celecoxib at 60 and 100 μM, or their combination. ResultsTreatment with either probiotic showed increased vacuolated gastric cancer (AGS) cells with significant morphological changes at high concentration (2.5 × 107 cfu/ml) of LA and BL bacteria compared to bladder cancer cells (J253). Similarly, AGS cells responded well to LA and BL and there was decreased cancer cell viability, tumor weight and hemoglobin content (p < 0.001). The probiotics were more potent against AGS than J253. High dose (2.5 × 107 cfu/ml) of probiotic bacteria when combined with 60 μM celecoxib downregulated COX2 expression in AGS by 70 % and 95 % (p < 0.001), without affecting COX1 expression. However, J253 cells did not show any changes in the expression of COX2 with probiotics, which is consistent with no effect on cancer cell viability and tumor angiogenesis. ConclusionsThis study is the first to document antiproliferation and anti-angiogenesis of LA and BL against gastric cancer by downregulating COX2 expression. LA and BL might be new, safe agents for targeting COX2 and suppressing growth of some cancers.