Knock-down of long intergenic noncoding RNA cyclooxygenase 2 (lincRNA-COX2) inhibits apoptosis and polarization into M1 in Listeria monocytogenes-infected macrophages / 细胞与分子免疫学杂志

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.

Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats / 中国中药杂志

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

To explore the therapeutic effect of Kangfuxiaomi shuan II on cervicitis in rats

ABSTRACT Purpose: The rat cervicitis model was established with 20% phenol glue to explore the therapeutic effect of Kangfuxiaomi shuan II on rat cervicitis and its mechanism. Methods: After modeling, the rats were treated with Shuangzuotai suppository (37.84 mg/kg), Kangfuxiaoyan shuan (205.6 mg/kg) and Kangfuxiaomi shuan II (40, 80, 160 mg/kg). The histopathological changes and injury degree of cervix in rats were evaluated by vulvar inflammation score and organ index. The therapeutic effect of Kangfuxiaomi shuan II on cervicitis was evaluated by detecting the levels of copper-protein (CP), C-reactive protein (CRP), Rat interleukin 6 (IL-6), superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and epidermal growth factor (EGF), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cervical tissue. Results: Compared with the model group, the vulvar inflammation score and cervical index of rats in other groups decreased significantly (P<0.01). Kangfuxiaomi shuan II could significantly reduce the levels of CP, CRP, and MDA in serum of rats with cervicitis, and significantly increase the activity of SOD in serum of rats with cervicitis (P<0.01). The levels of EGF and iNOS in cervical tissue of rats also increased in different degrees, while the level of COX-2 decreased significantly (P<0.01), which significantly improved the pathological degree of vulvar inflammation in rats with cervicitis. Conclusions: Kangfuxiaomi shuan II has a certain therapeutic effect on cervicitis in rats, and its mechanism may be related to the regulation of inflammatory cytokine network and immunity.

Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

The effect of CA1 administration of orexin-A on hippocampal expression of COX-2 and BDNF in a rat model of orofacial pain / O efeito da administração de CA1 de orexina-A na expressão hipocampal de COX-2 e BDNF em um modelo de dor orofacial em ratos

ABSTRACT The neuropeptide orexin-A and its receptors are widely distributed in both hippocampal circuitry and pain transmission pathways. Objective: Involvement of the CA1 orexin 1 receptor (OX1R) on the modulation of orofacial pain and pain-induced changes in hippocampal expression of cyclooxygenase-2 (COX-2) and brain-derived neurotrophic factor (BDNF) was investigated. Methods: Orofacial pain was induced by an intra-lip injection of capsaicin (100 μg). Reverse transcription polymerase chain reaction and immunoblot analysis were used to indicate changes in hippocampal BDNF and COX-2 expression, respectively. Results: Capsaicin induces a significant pain response, which is not affected by either orexin-A or SB-334867-A, an OX1R antagonist. However, an increased expression of COX-2 and decreased expression of BDNF was observed in the hippocampus of animals that received capsaicin or SB-334867-A (80 nM) plus capsaicin. Meanwhile, orexin-A (40 pM) attenuated the effects of capsaicin on the expression of COX-2 and BDNF. Conclusions: CA1 OX1R activation moderates capsaicin-induced neuronal inflammation and neurotrophic deficiency.

RESUMO O neuropeptídeo orexina-A e seus receptores estão amplamente distribuídos nos circuitos do hipocampo e nas vias de transmissão da dor. Objetivo: O envolvimento do receptor de orexina 1 CA1 (OX1R) na modulação da dor orofacial e alterações induzidas pela dor na expressão do hipocampo de ciclooxigenase-2 (COX-2) e fator neurotrófico derivado do cérebro (BDNF) foi investigado. Métodos: A dor orofacial foi induzida por injeção intra-labial de capsaicina (100 μg). A reação em cadeia da polimerase de transcrição reversa e a análise de imunotransferência foram utilizadas para indicar alterações na expressão de BDNF e COX-2 no hipocampo, respectivamente. Resultados: A capsaicina induz uma resposta significativa à dor, que não é afetada pela orexina-A ou pelo SB-334867-A, um antagonista do OX1R. No entanto, uma expressão aumentada de COX-2 e uma expressão diminuída de BDNF foi observada no hipocampo de animais que receberam capsaicina ou SB-334867-A (80 nM) mais capsaicina. Enquanto isso, a orexina A (40 pM) atenuou os efeitos da capsaicina na expressão de COX-2 e BDNF. Conclusões: A ativação de CA1 OX1R modera a inflamação neuronal induzida por capsaicina e a deficiência neurotrófica.

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

microRNA-142-3p inhibits apoptosis and inflammation induced by bleomycin through down-regulation of Cox-2 in MLE-12 cells

microRNA (miR)-142-3p is implicated in malignancy and has been identified as a biomarker for aggressive and recurrent lung adenocarcinomas. This study aimed to evaluate the inhibitory effect of miR-142-3p on apoptosis and inflammation induced by bleomycin in MLE-12 cells. MLE-12 cells were first transfected either with miR-142-3p mimic or miR-142-3p inhibitor and then the cells were exposed to 50 μg/mL of bleomycin. Thereafter, cell viability, apoptosis and the expression of pro-inflammatory cytokines were assessed using CCK-8, flow cytometry, RT-PCR and western blot analyses. Cox-2, PI3K, AKT and mTOR expressions were detected by western blotting after bleomycin was administered together with NS-398 (an inhibitor of Cox-2). As a result, cell viability was significantly decreased, as well as apoptosis and the expression of IL-1 and TNF-α were remarkably increased after 50 and 100 μg/mL of bleomycin administration. miR-142-3p overexpression alleviated bleomycin-induced apoptosis and overproduction of these two pro-inflammatory cytokines, while miR-142-3p suppression exhibited completely opposite results. Up-regulation of Cox-2 and inactivation of PI3K/AKT/mTOR were found in bleomycin-pretreated cells, while these abnormal regulations were partially abolished by miR-142-3p overexpression and NS-398. In conclusion, this study demonstrated that miR-142-3p overexpression protected bleomycin-induced injury in lung epithelial MLE-12 cells, possibly via regulating Cox-2 expression and PI3K/AKT/mTOR signaling pathway. These findings provide evidence that miR-142-3p may be a therapeutic strategy for idiopathic pulmonary fibrosis (IPF) treatment.

Immunohistochemical angiogenic biomarkers in hepatocellular carcinoma and cirrhosis: correlation with pathological features

OBJECTIVE To investigate immunohistochemical markers of angiogenesis and their association with pathological prognostic features in hepatocellular carcinoma and cirrhotic liver. METHODS Vascular endothelial growth factor, CD105, and cyclooxygenase-2 were immunohistochemically detected in 52 hepatocellular carcinoma tissue samples and 48 cirrhotic liver tissue samples. Semiquantitative measurements of vascular endothelial growth factor and cyclooxygenase-2 were evaluated considering the degree and intensity of immunostaining based on a 7-point final scoring scale. CD105 microvascular density (MVD-CD105) was measured using automated analysis. Morphological aspects evaluated in the hepatocellular carcinoma samples included size (≤2 and >2 cm), differentiation grade, and microvascular invasion. RESULTS The mean vascular endothelial growth factor immunoreactivity score was slightly higher in the hepatocellular carcinoma samples (4.83±1.35) than the cirrhotic liver (4.38±1.28) samples. There was a significant and direct correlation between these mean scores (rs=0.645, p=0.0001). Cyclooxygenase-2 was expressed in all the cirrhotic liver samples but was only found in 78% of the hepatocellular carcinoma samples. The mean cyclooxygenase-2 score was higher in the cirrhotic liver samples (4.85±1.38) than the hepatocellular carcinoma samples (2.58±1.68), but there was no correlation between the scores (rs=0.177, p=0.23). The mean CD105 percentage in the hepatocellular carcinoma samples (11.2%) was lower than that in the cirrhotic samples (16.9%). There was an inverse relationship in MVD-CD105 expression between the hepatocellular carcinoma and cirrhotic samples (rs=-0.78, p=0.67). There were no significant associations between vascular endothelial growth factor expression and morphological characteristics. Cyclooxygenase-2 and CD105 were associated with hepatocellular carcinoma differentiation grade (p=0.003 and p=0.05, respectively). CONCLUSION Vascular endothelial growth factor, cyclooxygenase-2, and MVD-CD105 were highly expressed in cirrhotic liver compared to hepatocellular carcinoma and might be involved in liver carcinogenesis. Additionally, cyclooxygenase-2 and CD105 might be involved in hepatocellular carcinoma differentiation grade.

Cytokine Expression of Microscopic Colitis Including Interleukin-17

BACKGROUND/AIMS: Microscopic colitis is characterized by chronic watery diarrhea with specific pathological changes that can be diagnosed by microscopic examination. We performed immunohistochemical analysis of proinflammatory cytokines to investigate the pathogenic mechanism of microscopic colitis. METHODS: This study consisted of six patients with lymphocytic colitis, six patients with collagenous colitis, and six patients with functional diarrhea but normal pathology. We performed an immunohistochemical analysis of the colonic mucosal biopsies to assess the expression of cyclo-oxygenase-2, interleukin-17, nuclear factor-kappaB, interferon-gamma, inducible nitric oxide synthase, and tumor necrosis factor-alpha. We compared the quantity score of immunohistochemical staining among the groups. RESULTS: The microscopic colitis group showed significantly higher expression of cyclo-oxygenase-2, interleukin-17, nuclear factor-kappaB, and interferon-gamma compared with the control group. Cytokine expression was similar between collagenous colitis and lymphocytic colitis. However, the expression of cyclo-oxygenase-2 was higher in collagenous colitis. CONCLUSIONS: Proinflammatory cytokines, including interleukin-17 and interferon-gamma, are highly expressed in microscopic colitis. The expression of cyclo-oxygenase-2 was higher in collagenous colitis than in lymphocytic colitis. This study is the first on interleukin-17 expression in microscopic colitis patients.

Inflammation and Cancer Development in Pancreatic and Biliary Tract Cancer / 대한소화기학회지

Chronic inflammation has been known to be a risk for many kinds of cancers, including pancreatic and biliary tract cancer. Recently, inflammatory process has emerged as a key mediator of cancer development and progression. Many efforts with experimental results have been given to identify the underlying mechanisms that contribute to inflammation-induced tumorigenesis. Diverse inflammatory pathways have been investigated and inhibitors for inflammation-related signaling pathways have been developed for cancer treatment. This review will summarize recent outcomes about this distinctive process in pancreatic and biliary tract cancer. Taking this evidence into consideration, modulation of inflammatory process will provide useful options for pancreatic and biliary tract cancer treatment.

Expresión de ciclo-oxigenasa 2 en carcinoma de laringe / Cyclooxigenase-2 expression in laryngeal carcinoma

Introducción: La sobrevida de carcinoma escamoso de laringe no ha aumentado significativamente en los últimos 20 años. Ciclo-oxigenasa 2 (COX-2) es una molécula que tiene un rol en la progresión tumoral ya que estimula la proliferación y angiogénesis e inhibe apoptosis celular. Objetivo: Evaluar la expresión de COX-2 en muestras de carcinoma escamoso de laringe. Material y método: Pacientes atendidos en los hospitales Barros Luco Trudeau y San Juan de Dios entre 2008 y 2011 con diagnóstico de carcinoma escamoso de laringe sin tratamiento previo. Se recolectaron tacos de biopsia los cuales fueron estudiados mediante inmunohistoquímica para la expresión de COX-2. Protocolo aprobado por Comité de Ética de la Facultad de Medicina de la Universidad de Chile. Resultados: Se incluyeron 32 casos. En 17 de ellos se describe sobreexpresión de COX-2, lo que equivale al 53% de la muestra. No hubo correlación con edad, sexo ni hábito tabáquico. Conclusión: La sobreexpresión de COX-2 es un fenómeno frecuente en carcinoma escamoso de laringe por lo cual es una molécula interesante para considerar como candidata a terapia adyuvante.

Introduction: Survival of laryngeal squamous cell carcinoma has not improved significantly in the last 20 years. Cyclooxigenase 2 (COX-2) has a role in carcinogenesis since it induces proliferation and angiogenesis, and inhibits apoptosis. Aim: To evaluate the expression of COX-2 in samples of laryngeal carcinoma. Material and methods: Patients attending Hospital San Juan de Dios and Barros Luco-Trudeau with diagnose of laryngeal carcinoma between 2008 and 2011 were included. Formalin fixed paraffin embedded samples were collected and COX-2 expression was assayed with immunohistochemistry. The study was approved by the ethics committee of Facultad de Medicina Universidad de Chile, and all patients consented. Results: 32 cases were included. COX-2 was overexpressed in 17, that is 53%. No correlation was identified with age, sex, or smoking habit. Conclusion: COX-2 overexpression is a frequent phenomenon in laryngeal carcinoma.

Ethyl acetate fraction from Angelica sinensis inhibits IL-1ß-induced rheumatoid synovial fibroblast proliferation and COX-2, PGE2, and MMPs production

BACKGROUND: The root of Angelica sinensis (AS), also known as "Dang-gui," was a popular herbal medicine widely used in the treatment of gynecological diseases in China, Korea, and Japan for a long time. This study aimed to determine the effects of ethyl acetate fraction from Angelica sinensis (EAAS) on the interleukin-1ß (IL-1ß)-induced proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), and production of matrix metalloproteinases (MMPs), cyclooxygenase (COX) 2, and prostaglandin E2 (PGE2), involved in articular bone and cartilage destruction, by RASFs. RESULTS: RASF proliferation was evaluated with cholecystokinin octapeptide (CCK-8) reagent in the presence of IL-1ß with/without EAAS. Expression of MMPs, tissue inhibitor of metalloproteinases-1 (TIMP-1), COXs, PGE2, and intracellular mitogen-activated protein kinase (MAPK) signaling molecules, including p-ERK, p-p38, p-JNK, and NF-κB, were examined using immunoblotting or semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. EAAS inhibited IL-1ß-induced RASF proliferation; MMP-1, MMP-3, and COX-2 mRNA and protein expressions; and PGE2 production. EAAS also inhibits the phosphorylation of ERK-1/2, p38, and JNK, and activation of NF-κB by IL-1ß. CONCLUSION: EAAS might be a new therapeutic modality for rheumatoid arthritis management.

Preventive effect of Ligularia fischeri on inhibition of nitric oxide in lipopolysaccharide-stimulated RAW 264. 7 macrophages depending on cooking method

BACKGROUND: Ligularia fischeri (common name Gomchwi) is known for its pharmaceutical properties and used in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases; however, little is known about its anti-inflammatory effect. In this study the influence of blanching and pan-frying on the anti-inflammatory activity of Ligularia fischeri (LF) was evaluated. RESULTS: Fresh LF and cooked LF showed no significant effect on the viability of macrophages after 24 h incubation. Fresh LF was found to be the most potent inhibitor of nitric oxide (NO) production at 100 µg/ml, while pan-fried LF showed little inhibitory effect on lipoloysaccharide (LPS) stimulated murine machrophage RAW264.7 cells. In contrast with its effect on NO production, pan-fried LF showed significant attenuation of the expression of inducible nitiric oxide synthase (iNOS) compared with fresh LF. In the cooking method of LF, PGE2 production was not affected in the LPS-induced RAW 264.7 cells. In LPS-induced RAW 264.7 cells, pretreatment by fresh and cooked LF increased COX2 mRNA expression. The 3-O-caffeoylquinic acid content of blanching and pan-frying LF increased by 4.92 and 9.7 fold with blanching and pan-frying respectively in comparison with uncooked LF. CONCLUSIONS: Regardless of the cooking method, Ligularia fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells.

Cyclooxygenase-2 and vascular endothelial growth factor in chronic hepatitis, cirrhosis and hepatocellular carcinoma

BACKGROUND/AIMS: Cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) are up-regulated in hepatocellular carcinoma (HCC). To investigate the levels of COX-2 and VEGF expression in chronic hepatitis (CH), cirrhosis, and HCC. METHODS: The immunohistochemical expressions of COX-2 and VEGF were evaluated in tissues from patients with CH (n=95), cirrhosis (n=38), low-grade HCC (LG-HCC; n=6), and high-grade HCC (HG-HCC; n=29). RESULTS: The COX-2 expression scores in CH, cirrhosis, LG-HCC, and HG-HCC were 3.3+/-1.9 (mean+/-SD), 4.2+/-1.7, 5.5+/-1.0, and 3.4+/-2.4, respectively (CH vs. cirrhosis, P=0.016; CH vs. LG-HCC, P=0.008; LG-HCC vs. HG-HCC, P=0.004), and the corresponding VEGF expression scores were 0.9+/-0.8, 1.5+/-0.7, 1.8+/-0.9, and 1.6+/-1.1 (CH vs. cirrhosis, P<0.001; CH vs. LG-HCC, P=0.011; LG-HCC vs. HG-HCC, P=0.075). Both factors were correlated with the fibrosis stage in CH and cirrhosis (COX-2: r=0.427, P<0.001; VEGF: r=0.491, P<0.001). There was a significant correlation between COX-2 and VEGF in all of the tissue samples (r=0.648, P<0.001), and between high COX-2 and VEGF expression scores and survival (COX-2: P=0.001; VEGF: P<0.001). CONCLUSIONS: The expressions of both COX-2 and VEGF are significantly higher in cirrhosis and LG-HCC than in CH. High COX-2 and high VEGF expressions are associated with a high survival rate.

Cyclooxygenase-2 inhibitors modulate skin aging in a catalytic activity-independent manner

It has been proposed that the pro-inflammatory catalytic activity of cyclooxygenase-2 (COX-2) plays a key role in the aging process. However, it remains unclear whether the COX-2 activity is a causal factor for aging and whether COX-2 inhibitors could prevent aging. We here examined the effect of COX-2 inhibitors on aging in the intrinsic skin aging model of hairless mice. We observed that among two selective COX-2 inhibitors and one non-selective COX inhibitor studied, only NS-398 inhibited skin aging, while celecoxib and aspirin accelerated skin aging. In addition, NS-398 reduced the expression of p53 and p16, whereas celecoxib and aspirin enhanced their expression. We also found that the aging-modulating effect of the inhibitors is closely associated with the expression of type I procollagen and caveolin-1. These results suggest that pro-inflammatory catalytic activity of COX-2 is not a causal factor for aging at least in skin and that COX-2 inhibitors might modulate skin aging by regulating the expression of type I procollagen and caveolin-1.

Synergy between adiponectin and interleukin-1beta on the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in fibroblast-like synoviocytes

To determine whether adiponectin may have synergistic effects in combination with the proinflammatory cytokine interleukin (IL)-1beta regarding the production of proinflammatory mediators during arthritic joint inflammation, synovial cells from rheumatoid arthritis (RA) patients were treated with adiponectin, IL-1beta, and their combination for 24 h. Culture supernatant was collected and analyzed by enzyme-linked immunosorbent assay for levels of IL-6, IL-8, prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Adiponectin-mediated intracellular signaling pathways were investigated to elucidate the molecular mechanisms underlying their synergy. The association of proinflammatory mediators with adiponectin was investigated in the synovial fluid of arthritis patients. Adiponectin functioned synergistically with IL-1beta to activate IL-6, IL-8, and PGE2 expression in RA fibroblast-like synoviocytes; Levels of VEGF, MMP-1, and MMP-13 were not synergistically stimulated. Adiponectin and IL-1beta each increased the expression of both adiponectin receptor 1 and IL-1 receptor 1. However, adiponectin and IL-1beta did not synergistically support the degradation of IkappaB-alpha or the nuclear translocation of NF-kappaB. Synergistically increased gene expression was significantly inhibited by MG132, an NF-kappaB inhibitor. Supporting the in vitro results, IL-6 and IL-8 levels were positively associated with adiponectin in synovial joint fluid from patients with RA, but not osteoarthritis (OA). In conclusion, adiponectin and IL-1beta may synergistically stimulate the production of proinflammatory mediators through unknown signaling pathways during arthritic joint inflammation. Adiponectin may be more important to the pathogenesis of RA than previously thought.

A new synthetic chalcone derivative, 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139), suppresses the Toll-like receptor 4-mediated inflammatory response through inhibition of the Akt/NF-kappaB pathway in BV2 microglial cells

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IkappaB and p65/RelA NF-kappaB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-kappaB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-kappaB target genes, such as those for COX-2, iNOS, and IL-1beta, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant-active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IkappaB kinase (IKK)/NF-kappaB signaling pathway.

High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify ‘hit’ molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer anti-inflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.

Study on cow ghee versus soybean oil on 7,12-dimethylbenz(a)-anthracene induced mammary carcinogenesis & expression of cyclooxygenase-2 & peroxisome proliferators activated receptor- γ in rats.

Background & objectives Breast cancer is a leading cause of cancer death in women; dietary fat is the one of the factors that influences its incidence. In the present study we investigated the effect of feeding cow ghee versus soybean oil on 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary cancer in rat and expression of cyclooxygenase-2 and peroxisome proliferators activated receptor- γ (PPAR-γ) in mammary gland. Methods Two groups of 21 day old female rats (30 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). The animals were given DMBA (30mg/kg body weight) through oral intubation after 5 wk feeding. Another two groups (8 each) fed similarly but not given DMBA served as control for the gene expression study. Results In DMBA treated groups, the animal fed soybean oil had higher tumour incidence (65.4%), tumour weight (6.18 g) and tumour volume (6285 mm3) compared to those fed cow ghee (26.6%, 1.67 g, 1925 mm3, respectively). Tumour latency period was 23 wk on soybean oil compared to 27 wk on cow ghee. Histological analysis of tumours showed that the progression of carcinogenesis was more rapid on soybean oil than on cow ghee. The expression of cyclooxygenase-2 was observed only in DMBA treated rats and it was significantly less on cow ghee than on soybean oil. The expression of PPAR-γ was significantly more on cow ghee than on soybean oil. Interpretation & conclusions Our results show that dietary cow ghee opposed to soybean oil attenuates mammary carcinogenesis induced by DMBA; and the effect is mediated by decreased expression of cyclooxygenase-2 and increased expression of PPAR-γ in the former group.