Objective:To investigate the expression of cyclooxygenase-2 and its pathological effect in the experimental nonalcoholic fatty liver of rats, and to explore its possible mechanism. Methods:The rat NAFLD model was es...Objective:To investigate the expression of cyclooxygenase-2 and its pathological effect in the experimental nonalcoholic fatty liver of rats, and to explore its possible mechanism. Methods:The rat NAFLD model was established by giving a fat-enriched diet. The blood samples were obtained form abdominal aorta and the levels of serum ALT, AST and IL-1, changes in the hepatic tissue 6-k-PGF1 α TXB2 were measured. The expression level of COX-2 in rats livers were assayed by immunohistochemistry, RT-PCR and Westernblot. Results: Light microscope analysis revealed that hepatocytes were injured in the model group and slightly in the treatment group. The levels of serum TXB2 and IL-1 in the fatty liver rats were increased. Compared with the model group, the IL-1 and TXB2 increased significantly(P〈 0.05), on the contrary, compared with the normal group, the hepatic tissue 6-Keto-prostagland decreased significantly in the model group(P 〈 0.05), the treatment group also increased but P 〉 0.05. There was no positive expression of COX-2 in hepatic tissue of normal rats. In the model group, there was positive expression of COX-2 antigen and the number of COX positive cells progressively increased at 4, 8, 12 wks. The intensity of expression of COX-2 had significantly increased(P 〈 0.05 ) and the intensity of COX-2 expression in the treated group decreased remarkably compared with the model group(P 〈 0.05). The expression of COX-2 mRNA and the level of COX-2 protein were significantly stronger in the liver of model rats compared with normal rats, and significantly weaker in treated rats, than in 8W and 12W model rats(P 〈 0.05). Conclusion:The increase of COX-2 expression in NAFLD is closely associated with the severity of liver inflammation and damage. COX-2 may play an important role in the progression of rat NAFLD, and the expression of COX-2 mRNA is downregulated by cyclooxygenase-2 inhibitor, which can depress the oxidative stress and control inflammatory response efficiently.展开更多
In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcri...In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.展开更多
Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-...Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott’s method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury.展开更多
Summary: To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expres...Summary: To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a Basis for the chemoprevention of ovarian cancer.展开更多
To investigate the relationship between the expression of cyclooxygenase-2 (COX-2) and menstrual cycle, the regulatory effects of 17-β-estradiol (E2) and medroxyprogesterone acetate (MPA) on the expression of C...To investigate the relationship between the expression of cyclooxygenase-2 (COX-2) and menstrual cycle, the regulatory effects of 17-β-estradiol (E2) and medroxyprogesterone acetate (MPA) on the expression of COX-2 in cervical cancer Hela cells were examined. Cervical cancer specimens were obtained from 47 pre-menopausal patients. The phase of menstrual cycle was determined by case history and HE staining of uterine endometrium. COX-2 was immunohistochemically stained by SABC staining and the staining intensity was determined with computerized image analysis system. Hela cells were incubated with alcohol, E2, E2+MPA, MPA for 12, 24 and 48 h respectively. The expression of COX-2 in Hela cells was detected by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Our results showed that the expression of COX-2 was significantly higher during proliferative phase than secretory phase (P〈0.05), but there was no difference in the positive rate between proliferative phase and secretory phase (P〉0.05). Incubation with E2 could significantly enhance the expression of COX-2 continually. On the contrary, E2+MPA and MPA alone could decrease the expression of COX-2 as compared with the control and E2 group (P〈0.05 and P〈0.01 respectively). It is concluded that the expression of COX-2 in cervical cancer of pre-menopausal patients and Hela cells was regulated by estrogen/progestogen.展开更多
Background: Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-β1) are modulated in variety cancers including Hepatocellular carcinoma (HCC). However, there is a paucity of data concerning their role ...Background: Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-β1) are modulated in variety cancers including Hepatocellular carcinoma (HCC). However, there is a paucity of data concerning their role in the pathologic process of recurrence of HCC following hepatectomy. We herein assessed the role of the hepatic expression of COX-2 and TGF-β as predictors for patients with early recurrence within 2 years of HCC diagnosis. Methods: Sixty patients with HCC who underwent curative hepatectomy between 2000 and 2003 were entered in the present study. The immunoreactivity and distribution patterns of COX-2 and TGF-β1 were examined in both the HCC and the adjacent nonHCC tissues of the liver. Risk factors of tumor recurrence within 2 years, including COX-2 and TGF-β1 expression, were investigated by univariate and multivariate analyses. Results: Among 60 patients, 31 patients had early recurrences within 2 years and 14 patients recurred after 2 years following surgery. Patients with low COX-2 expression in the HCC tissues and adjacent nonHCC tissues had favorable disease-free survival (p = 0.002 and p β1 expression in the nonHCC tissues had also longer disease-free survival (p = 0.045). Based on the expression patterns of COX-2 and TGF-β1, patients with low COX-2 and positive TGF-β1 expression in the nonHCC tissues had favorable overall and disease-free survival (p β1 signaling in nontumor tissues suggested high risk of recurrence and poor survival to the HCC patients following hepatectomy.展开更多
Walnut dreg protein hydrolysates(WDPHs)exhibit a variety of biological activities,however,the cyclooxygenase-2(COX-2)inhibitory peptide of WDPHs remain unclear.The aim of this study was to rapidly screen for such pept...Walnut dreg protein hydrolysates(WDPHs)exhibit a variety of biological activities,however,the cyclooxygenase-2(COX-2)inhibitory peptide of WDPHs remain unclear.The aim of this study was to rapidly screen for such peptides in WDPHs through a combination of in silico and in vitro analysis.In total,1262 peptide sequences were observed by nano liquid chromatography/tandem mass spectrometry(nano LC-MS/MS)and 4 novel COX-2 inhibitory peptides(AGFP,FPGA,LFPD,and VGFP)were identified.Enzyme kinetic data indicated that AGFP,FPGA,and LFPD displayed mixed-type COX-2 inhibition,whereas VGFP was a non-competitive inhibitor.This is mainly because the peptides form hydrogen bonds and hydrophobic interactions with residues in the COX-2 active site.These results demonstrate that computer analysis combined with in vitro evaluation allows for rapid screening of COX-2 inhibitory peptides in walnut protein dregs.展开更多
Objective:To study the inhibitory effect of siRNA mediated by expression vectors on Skp2 expression in human chondrocytes.Method:Three Skp2 sequences siRNA-59,siRNA-318 and siRNA-504 were designed as target sites usin...Objective:To study the inhibitory effect of siRNA mediated by expression vectors on Skp2 expression in human chondrocytes.Method:Three Skp2 sequences siRNA-59,siRNA-318 and siRNA-504 were designed as target sites using online siRNA design tools.Skp2 siRNA expression vectors were successfully constructed in vitro by gene recombination technology,and the influence of recombinant plasmid transfection on Skp2 mRNA expression was detected.DNA electrophoresis was used to verify the results.Results:The sequence of Skp2 interference was correct by sequence analysis.The expression of Skp2 mRNA in siRNA-59,siRNA-318,siRNA-504 transfection group was significantly lower than that in no-load group and NC group(P<0.05),the inhibition rates of Skp2 mRNA in siRNA-59,siRNA-318 and siRNA-504 were respectively 60%,41%and 64%,and the siRNA-504 transfection group had the highest inhibition rate.Conclusion:The siRNA eukaryotic expression vector of Skp2 gene was constructed successfully which effectively inhibit Skp2 mRNA expression in human chondrocytes cell,and can provide strong evidence for the treatment of osteoarthritis.展开更多
Gibberellin 2-oxidases(GA2ox)are important enzymes that maintain the balance of bioactive GAs in plants.GA2ox genes have been identified and characterized in many plants,but these genes were not investigated in Brassi...Gibberellin 2-oxidases(GA2ox)are important enzymes that maintain the balance of bioactive GAs in plants.GA2ox genes have been identified and characterized in many plants,but these genes were not investigated in Brassica napus.Here,we identified 31 GA2ox genes in B.napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes.Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm,and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons.Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups,including two C_(19)-GA2ox and two C_(20)-GA2ox clades.Group 4 is a C_(20)-GA2ox Class discovered recently.Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes.BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome.BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development,and most of them were mainly involved in abiotic responses,regulation of phytohormones and growth and development.Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons,as well as an insight into the biological functions of GA2ox family genes in B.napus.展开更多
Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.M...Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.展开更多
AIM:To examine the expression of cyclooxygenase-2(COX-2)and prostaglandin E2(PGE2)in rat esophageal lesions induced by reflux of duodenal contents.METHODS:Thirty 8-week-old male Wistar rats were exposed to duodenal co...AIM:To examine the expression of cyclooxygenase-2(COX-2)and prostaglandin E2(PGE2)in rat esophageal lesions induced by reflux of duodenal contents.METHODS:Thirty 8-week-old male Wistar rats were exposed to duodenal content esophageal reflux.All animals underwent an esophagoduodenal anastomosis(EDA)with total gastrectomy to elicit chronic esophagitis.In ten rats sham operations with only a midline laparotomy were performed(Control).The rats were sacrificed at the 40th week,their esophagi were taken for hematoxylin and eosin staining and for examination of expression of COX2,PGE2,and proliferating cell nuclear antigen(PCNA),and total bile acids in the esophageal lumen was measured.RESULTS:After 40 wk of reflux,columnar dysplasia,squamous cell carcinoma and adenocarcinoma were observed.Total bile acids in the esophageal lumen were significantly increased in the EDA group compared with the sham operated rats.PCNA labelling index and esophageal tissue PGE2 levels were higher in dysplastic and cancer tissues than in control tissues.Overexpression of COX2 was observed in dysplastic and cancer tissues.CONCLUSION:Reflux of duodenal contents induces COX2 expression and increases prostaglandin synthesis in dysplastic and cancer tissues.This result suggests a possible mechanism by which bile acids promote esophageal cancer.展开更多
Aim: To investigate the expression of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) and the possible mechanism in the development in androgen independent prostate cancer (AIPC). Methods:...Aim: To investigate the expression of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) and the possible mechanism in the development in androgen independent prostate cancer (AIPC). Methods: Immunohistochemistry was performed on paraffin-embedded sections with goat polyclonal against COX-2 and mouse monoclonal antibody against EGFR in 30 AIPC and 18 androgen dependent prostate cancer (ADPC) specimens. The effect of epidermal growth factor (EGF) treatments on the expression of COX-2 and signal pathway in PC-3 and DU-145 cells was studied using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. ELISA was used to measure prostaglandin E2 (PGE2) levels in the media of PC-3 and DU-145 incubated with EGF for 24 h. Results: COX-2 was positively expressed in AIPC and ADPC, which were predominantly in endochylema of prostate cancer (PCa) cells. Intense staining was seen in AIPC (80%) and in ADPC (55.5%), but there was no significant association between the two groups. EGFR expression was also positive in the two groups (61.8% in ADPC and 90% in AIPC, P 〈 0.01). A significant association was found between EGFR expression and a higher Gleason score (P 〈 0.05) or tumor stage (P 〈 0.05). The expression of PGE2 was increased in PC-3 and DU-145 cells after being incubated with EGF. Both p38MAPK and PI-3K pathway were involved in the PC-3 cell COX-2 upregulation course. In DU- 145, only p38MAPK pathway was associated with COX-2 upregulation. Conclusion: EGFR activation induces COX-2 expression through PI-3K and/or p38MAPK pathways. COX-2 and EGFR inhibitors might have a cooperative anti-tumor effect in PCa.展开更多
AIM to determine whether cyclooxygenase-2(COX-2) and prostaglandin E1 receptor(EP1) contribute to disease and whether they help predict prognosis.METHODS We retrospectively reviewed the records of 116 patients with he...AIM to determine whether cyclooxygenase-2(COX-2) and prostaglandin E1 receptor(EP1) contribute to disease and whether they help predict prognosis.METHODS We retrospectively reviewed the records of 116 patients with hepatocellular carcinoma(HCC) who underwent surgery between 2008 and 2011 at our hospital. Expression of COX-2 and EP1 receptor was examined by immunohistochemistry of formalin-fixed, paraffinembedded tissues using polyclonal antibodies. Possible associations between immunohistochemical scores and survival were determined.RESULTS Factors associated with poor overall survival(OS) were alpha-fetoprotein > 400 ng/m L, tumor size ≥ 5 cm, and high EP1 receptor expression, but not high COX-2 expression. Disease-free survival was not significantly different between patients with low or high levels of COX-2 or EP1. COX-2 immunoreactivity was significantly higher in well-differentiated HCC tissues(Edmondson grade Ⅰ-Ⅱ) than in poorly differentiated tissues(Edmondson grade Ⅲ-Ⅳ)(P = 0.003). EP1 receptor immunoreactivity was significantly higher in poorly differentiated tissue than in well-differentiated tissue(P = 0.001).CONCLUSION COX-2 expression appears to be linked to early HCC events(initiation), while EP1 receptor expression may participate in tumor progression and predict survival.展开更多
Background Estrogen receptor (ER)-negative breast cancer cells are more aggressive than ER-positive cells. Elevated levels of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) expression...Background Estrogen receptor (ER)-negative breast cancer cells are more aggressive than ER-positive cells. Elevated levels of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) expression have been detected in cultured human breast cancer cells and are associated with negative hormone receptor status. In this study, we created ERα stable transfectants in MDA-MB-231 cells to explore the effect of ERα on cell growth and COX-2 and VEGF-C expression.Methods The green fluorescent protein (GFP)-ERα plasmids were stably transfected into ER-negative MDA-MB-231 cells. The proliferation and migration of untransfected MDA-MB-231 cells, ERα-transfected MDA-MB-231 cells and ER-positive MCF-7 cells were determined. The expression of COX-2, and the levels of VEGF-C mRNA and the VEGF-C secretion concentration were assayed in these cell lines.Results The proliferation and migration capacities of ERα-tranfected MDA-MB-231 cells were significantly decreased (P 〈0.05). The expression of COX-2 was significantly lower in ERa-tranfected MDA-MB-231 cells than in untranfected MDA-MB-231 cells. The mRNA and protein levels of VEGF-C were lower in ERa-tranfected MDA-MB-231 cells than in untransfected MDA-MB-231 cells (P〈0.05).Conclusions ERα stable transfection inhibits proliferation and migration capacities of MDA-MB-231 cells and decreases expression of COX-2 and VEGF-C. The decreases of proliferation and migration capacities may be related to suppression of COX-2 and VEGF-C expression.展开更多
Objective: To study the relationship between cyclooxygenase-2 (COX-2) expression and tumor angiogenesis in human breast cancer. Methods: Archival primary breast carcinomas (n = 62), adjacent ductal carcinoma in situ (...Objective: To study the relationship between cyclooxygenase-2 (COX-2) expression and tumor angiogenesis in human breast cancer. Methods: Archival primary breast carcinomas (n = 62), adjacent ductal carcinoma in situ (DCIS, n = 13) and DCIS alone (n = 5) were analyzed for COX-2 and VEGF expression by immunohistochemistry using specific monoclonal antibodies. Microvessel density (MVD) was also examined the using CD34 staining. Results: A significant correlation was found between COX-2 and VEGF expression (P<0.01). Both COX-2 and VEGF were significantly correlated with MVD (P<0.05) and P<0.01, respectively). COX-2 and VEGF genes were overexpressed in tumor specimens as compared with normal epithelia. Conclusion: COX-2 is related to tumor angiogenesis in breast cancer. It is likely that VEGF is one of the most important mediators of the COX-2 angiogenic pathway.展开更多
Objective: To evaluate the efficacy and safety of celecoxib treatment of advanced non-small cell lung cancer (NSCLC), and combined therapy by molecular analysis. plus platinum-doublet as first-line chemotherapy in ...Objective: To evaluate the efficacy and safety of celecoxib treatment of advanced non-small cell lung cancer (NSCLC), and combined therapy by molecular analysis. plus platinum-doublet as first-line chemotherapy in to determine the subgroup benefiting from celecoxib Methods: A total of 44 treatment-naive patients of advanced NSCLC with positive cyclooxygenase-2 (COX-2) expression confirmed by immunohistochemical (IHC) staining were designed to receive celecoxib plus platinum-doublet chemotherapy (cisplatin plus gemcitabine, novelbine or docetaxol) from February 2005 to May 2007. On 5-7 day before chemotherapy, 400 mg celecoxib was administered twice a day orally until obvious evidence of disease progression or intolerable toxicity was found. Adverse events were recorded according to NCI-CTC criteria. The primary endpoint was overall survival (OS). The secondary endpoints included progression-free survival (PFS), 1-year survival rate, response rate (RR) and safety. Additionally, we detected epithelial growth factor receptor (EGFR) status including EGFR gene amplification by real-time PCR and gene mutations by DHPLC followed by sequencing. Results: The response rate was 45% (20/44), and the disease control rate (DCR) was 59% (26/44). The median progression-free survival time and median survival time were 6 m and 18 m, respectively. The l-year survival rate was 68%. Chemotherapy cycle numbers and best response were found to be the predictive factors for PFS by COX model analysis (P=0.023 and P=0.000, respectively). No factor was found to affect OS. The most common toxicities included neutropenia and nausea/vomit. EGFR gene amplification was an independent prognostic factor influencing OS (P=0.0002). Patients with EGFR mutations (exon 21) had a tendency of disease progression (P=0.041). Conclusion: Encouraging activities of celecoxib combined with platinum-doublet chemotherapy were demonstrated in treatment-naive patients with advanced NSCLC, with good tolerances. For COX-2 IHC positive patients, positive EGFR amplification and mutation might be related to poor clinical outcomes.展开更多
Migration of dendritic cells (DCs) into tissues and secondary lymphoid organs plays a crucial role in the initiation of innate and adaptive immunity. In this article, we show that cyclosporin A (CsA) impairs the migra...Migration of dendritic cells (DCs) into tissues and secondary lymphoid organs plays a crucial role in the initiation of innate and adaptive immunity. In this article, we show that cyclosporin A (CsA) impairs the migration of DCs both in vitro and in vivo. Exposure of DCs to clinical concentrations of CsA neither induces apoptosis nor alters development but does impair cytokine secretion, chemokine receptor expression, and migration. In vitro, CsA impairs the migration of mouse bone marrow-derived DCs toward macrophage inflammatory protein-3beta (MIP-3beta) and induces them to retain responsiveness to MIP-1alpha after lipopolysaccharide (LPS)-stimulated DC maturation, while in vivo administration of CsA inhibits the migration of DCs out of skin and into the secondary lymphoid organs. CsA impairs chemokine receptor and cyclooxygenase-2 (COX-2) expression normally triggered in LPS-stimulated DCs; administration of exogenous prostaglandin E2 (PGE2) reverses the effects of CsA on chemokine receptor expression and DC migration. Inhibition of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway signaling by CsA may be responsible for the CsA-mediated effects on the regulation of chemokine receptor and cyclooxygenase-2 (COX-2) expression. Impairment of DC migration due to inhibition of PGE2 production and regulation of chemokine receptor expression may contribute, in part, to CsA-mediated immunosuppression.展开更多
文摘Objective:To investigate the expression of cyclooxygenase-2 and its pathological effect in the experimental nonalcoholic fatty liver of rats, and to explore its possible mechanism. Methods:The rat NAFLD model was established by giving a fat-enriched diet. The blood samples were obtained form abdominal aorta and the levels of serum ALT, AST and IL-1, changes in the hepatic tissue 6-k-PGF1 α TXB2 were measured. The expression level of COX-2 in rats livers were assayed by immunohistochemistry, RT-PCR and Westernblot. Results: Light microscope analysis revealed that hepatocytes were injured in the model group and slightly in the treatment group. The levels of serum TXB2 and IL-1 in the fatty liver rats were increased. Compared with the model group, the IL-1 and TXB2 increased significantly(P〈 0.05), on the contrary, compared with the normal group, the hepatic tissue 6-Keto-prostagland decreased significantly in the model group(P 〈 0.05), the treatment group also increased but P 〉 0.05. There was no positive expression of COX-2 in hepatic tissue of normal rats. In the model group, there was positive expression of COX-2 antigen and the number of COX positive cells progressively increased at 4, 8, 12 wks. The intensity of expression of COX-2 had significantly increased(P 〈 0.05 ) and the intensity of COX-2 expression in the treated group decreased remarkably compared with the model group(P 〈 0.05). The expression of COX-2 mRNA and the level of COX-2 protein were significantly stronger in the liver of model rats compared with normal rats, and significantly weaker in treated rats, than in 8W and 12W model rats(P 〈 0.05). Conclusion:The increase of COX-2 expression in NAFLD is closely associated with the severity of liver inflammation and damage. COX-2 may play an important role in the progression of rat NAFLD, and the expression of COX-2 mRNA is downregulated by cyclooxygenase-2 inhibitor, which can depress the oxidative stress and control inflammatory response efficiently.
基金This project was supported by grants from National Natu ral Sciences Foundation of China ( No. 30070929,30271363).
文摘In order to investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splicing variant of COX-2, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of COX-2. The primers were designed and synthesized according to the sequence of rat COX-2 splice variant which was discovered firstly by us. Then the splicing variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. The results showed that the expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than that in labor women and a new band of COX-2 was obtained in myometrium from labor woman. The fragment included an unspliced intron, which pitched between exons 7 and 8. It was suggested that COX-2 gene was not only expressed highly in human myometrium from woman in labor, but also produced splicing variant by alternative splicing.
基金supported by the National Natural Science Foundation of China,No.30471934
文摘Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott’s method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury.
文摘Summary: To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a Basis for the chemoprevention of ovarian cancer.
文摘To investigate the relationship between the expression of cyclooxygenase-2 (COX-2) and menstrual cycle, the regulatory effects of 17-β-estradiol (E2) and medroxyprogesterone acetate (MPA) on the expression of COX-2 in cervical cancer Hela cells were examined. Cervical cancer specimens were obtained from 47 pre-menopausal patients. The phase of menstrual cycle was determined by case history and HE staining of uterine endometrium. COX-2 was immunohistochemically stained by SABC staining and the staining intensity was determined with computerized image analysis system. Hela cells were incubated with alcohol, E2, E2+MPA, MPA for 12, 24 and 48 h respectively. The expression of COX-2 in Hela cells was detected by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Our results showed that the expression of COX-2 was significantly higher during proliferative phase than secretory phase (P〈0.05), but there was no difference in the positive rate between proliferative phase and secretory phase (P〉0.05). Incubation with E2 could significantly enhance the expression of COX-2 continually. On the contrary, E2+MPA and MPA alone could decrease the expression of COX-2 as compared with the control and E2 group (P〈0.05 and P〈0.01 respectively). It is concluded that the expression of COX-2 in cervical cancer of pre-menopausal patients and Hela cells was regulated by estrogen/progestogen.
文摘Background: Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-β1) are modulated in variety cancers including Hepatocellular carcinoma (HCC). However, there is a paucity of data concerning their role in the pathologic process of recurrence of HCC following hepatectomy. We herein assessed the role of the hepatic expression of COX-2 and TGF-β as predictors for patients with early recurrence within 2 years of HCC diagnosis. Methods: Sixty patients with HCC who underwent curative hepatectomy between 2000 and 2003 were entered in the present study. The immunoreactivity and distribution patterns of COX-2 and TGF-β1 were examined in both the HCC and the adjacent nonHCC tissues of the liver. Risk factors of tumor recurrence within 2 years, including COX-2 and TGF-β1 expression, were investigated by univariate and multivariate analyses. Results: Among 60 patients, 31 patients had early recurrences within 2 years and 14 patients recurred after 2 years following surgery. Patients with low COX-2 expression in the HCC tissues and adjacent nonHCC tissues had favorable disease-free survival (p = 0.002 and p β1 expression in the nonHCC tissues had also longer disease-free survival (p = 0.045). Based on the expression patterns of COX-2 and TGF-β1, patients with low COX-2 and positive TGF-β1 expression in the nonHCC tissues had favorable overall and disease-free survival (p β1 signaling in nontumor tissues suggested high risk of recurrence and poor survival to the HCC patients following hepatectomy.
基金supported by the Major Project of Science and Technology Department of Yunnan Province (202002AA100005 and 202102AE090027-2)the Project of Yunnan Province Food and Drug Homologous Resources Functional Food Innovation Team (A3032023057)+2 种基金the YEFICRC project of Yunnan provincial key programs (2019ZG009)Yunnan Province Ten Thousand Plan Industrial Technology Talents project (YNWR-CYJS-2020-010)the Yunnan Provincial Department of Science and Technology Agricultural Joint Special Project (202101BD070001-120)。
文摘Walnut dreg protein hydrolysates(WDPHs)exhibit a variety of biological activities,however,the cyclooxygenase-2(COX-2)inhibitory peptide of WDPHs remain unclear.The aim of this study was to rapidly screen for such peptides in WDPHs through a combination of in silico and in vitro analysis.In total,1262 peptide sequences were observed by nano liquid chromatography/tandem mass spectrometry(nano LC-MS/MS)and 4 novel COX-2 inhibitory peptides(AGFP,FPGA,LFPD,and VGFP)were identified.Enzyme kinetic data indicated that AGFP,FPGA,and LFPD displayed mixed-type COX-2 inhibition,whereas VGFP was a non-competitive inhibitor.This is mainly because the peptides form hydrogen bonds and hydrophobic interactions with residues in the COX-2 active site.These results demonstrate that computer analysis combined with in vitro evaluation allows for rapid screening of COX-2 inhibitory peptides in walnut protein dregs.
基金National Natural Science Foundation of China(82060875)National Natural Science Foundation of China(82160912)Guangxi Natural Science Foundation(2022JJA141229)。
文摘Objective:To study the inhibitory effect of siRNA mediated by expression vectors on Skp2 expression in human chondrocytes.Method:Three Skp2 sequences siRNA-59,siRNA-318 and siRNA-504 were designed as target sites using online siRNA design tools.Skp2 siRNA expression vectors were successfully constructed in vitro by gene recombination technology,and the influence of recombinant plasmid transfection on Skp2 mRNA expression was detected.DNA electrophoresis was used to verify the results.Results:The sequence of Skp2 interference was correct by sequence analysis.The expression of Skp2 mRNA in siRNA-59,siRNA-318,siRNA-504 transfection group was significantly lower than that in no-load group and NC group(P<0.05),the inhibition rates of Skp2 mRNA in siRNA-59,siRNA-318 and siRNA-504 were respectively 60%,41%and 64%,and the siRNA-504 transfection group had the highest inhibition rate.Conclusion:The siRNA eukaryotic expression vector of Skp2 gene was constructed successfully which effectively inhibit Skp2 mRNA expression in human chondrocytes cell,and can provide strong evidence for the treatment of osteoarthritis.
基金supported by the Chongqing Academy of Agricultural Sciences Youth Innovation Team Project(NKY-2018QC01)Chongqing Finance Special Project(NKY-2022AC002)+2 种基金the Natural Science Foundation Project of Yongchuan(2021yc-jckx20013)the Technology Innovation and Application Development(Surface)Project of Yongchuan(2021yc-cxfz30007)the National Oilseed Rape Industrial Technology System Sanxia Comprehensive Experiment Station Project(CARS-13).
文摘Gibberellin 2-oxidases(GA2ox)are important enzymes that maintain the balance of bioactive GAs in plants.GA2ox genes have been identified and characterized in many plants,but these genes were not investigated in Brassica napus.Here,we identified 31 GA2ox genes in B.napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes.Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm,and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons.Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups,including two C_(19)-GA2ox and two C_(20)-GA2ox clades.Group 4 is a C_(20)-GA2ox Class discovered recently.Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes.BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome.BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development,and most of them were mainly involved in abiotic responses,regulation of phytohormones and growth and development.Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons,as well as an insight into the biological functions of GA2ox family genes in B.napus.
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
文摘AIM:To examine the expression of cyclooxygenase-2(COX-2)and prostaglandin E2(PGE2)in rat esophageal lesions induced by reflux of duodenal contents.METHODS:Thirty 8-week-old male Wistar rats were exposed to duodenal content esophageal reflux.All animals underwent an esophagoduodenal anastomosis(EDA)with total gastrectomy to elicit chronic esophagitis.In ten rats sham operations with only a midline laparotomy were performed(Control).The rats were sacrificed at the 40th week,their esophagi were taken for hematoxylin and eosin staining and for examination of expression of COX2,PGE2,and proliferating cell nuclear antigen(PCNA),and total bile acids in the esophageal lumen was measured.RESULTS:After 40 wk of reflux,columnar dysplasia,squamous cell carcinoma and adenocarcinoma were observed.Total bile acids in the esophageal lumen were significantly increased in the EDA group compared with the sham operated rats.PCNA labelling index and esophageal tissue PGE2 levels were higher in dysplastic and cancer tissues than in control tissues.Overexpression of COX2 was observed in dysplastic and cancer tissues.CONCLUSION:Reflux of duodenal contents induces COX2 expression and increases prostaglandin synthesis in dysplastic and cancer tissues.This result suggests a possible mechanism by which bile acids promote esophageal cancer.
文摘Aim: To investigate the expression of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) and the possible mechanism in the development in androgen independent prostate cancer (AIPC). Methods: Immunohistochemistry was performed on paraffin-embedded sections with goat polyclonal against COX-2 and mouse monoclonal antibody against EGFR in 30 AIPC and 18 androgen dependent prostate cancer (ADPC) specimens. The effect of epidermal growth factor (EGF) treatments on the expression of COX-2 and signal pathway in PC-3 and DU-145 cells was studied using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. ELISA was used to measure prostaglandin E2 (PGE2) levels in the media of PC-3 and DU-145 incubated with EGF for 24 h. Results: COX-2 was positively expressed in AIPC and ADPC, which were predominantly in endochylema of prostate cancer (PCa) cells. Intense staining was seen in AIPC (80%) and in ADPC (55.5%), but there was no significant association between the two groups. EGFR expression was also positive in the two groups (61.8% in ADPC and 90% in AIPC, P 〈 0.01). A significant association was found between EGFR expression and a higher Gleason score (P 〈 0.05) or tumor stage (P 〈 0.05). The expression of PGE2 was increased in PC-3 and DU-145 cells after being incubated with EGF. Both p38MAPK and PI-3K pathway were involved in the PC-3 cell COX-2 upregulation course. In DU- 145, only p38MAPK pathway was associated with COX-2 upregulation. Conclusion: EGFR activation induces COX-2 expression through PI-3K and/or p38MAPK pathways. COX-2 and EGFR inhibitors might have a cooperative anti-tumor effect in PCa.
基金Supported by National Natural Science Foundation of China,No.81260331Key Laboratory for High-Incidence Tumor Prevention and Treatment,Ministry of Education,No.GKE2015-ZZ05
文摘AIM to determine whether cyclooxygenase-2(COX-2) and prostaglandin E1 receptor(EP1) contribute to disease and whether they help predict prognosis.METHODS We retrospectively reviewed the records of 116 patients with hepatocellular carcinoma(HCC) who underwent surgery between 2008 and 2011 at our hospital. Expression of COX-2 and EP1 receptor was examined by immunohistochemistry of formalin-fixed, paraffinembedded tissues using polyclonal antibodies. Possible associations between immunohistochemical scores and survival were determined.RESULTS Factors associated with poor overall survival(OS) were alpha-fetoprotein > 400 ng/m L, tumor size ≥ 5 cm, and high EP1 receptor expression, but not high COX-2 expression. Disease-free survival was not significantly different between patients with low or high levels of COX-2 or EP1. COX-2 immunoreactivity was significantly higher in well-differentiated HCC tissues(Edmondson grade Ⅰ-Ⅱ) than in poorly differentiated tissues(Edmondson grade Ⅲ-Ⅳ)(P = 0.003). EP1 receptor immunoreactivity was significantly higher in poorly differentiated tissue than in well-differentiated tissue(P = 0.001).CONCLUSION COX-2 expression appears to be linked to early HCC events(initiation), while EP1 receptor expression may participate in tumor progression and predict survival.
基金This work was supported by a grant from the National Natural Science Foundation of China (No. 30600588).
文摘Background Estrogen receptor (ER)-negative breast cancer cells are more aggressive than ER-positive cells. Elevated levels of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) expression have been detected in cultured human breast cancer cells and are associated with negative hormone receptor status. In this study, we created ERα stable transfectants in MDA-MB-231 cells to explore the effect of ERα on cell growth and COX-2 and VEGF-C expression.Methods The green fluorescent protein (GFP)-ERα plasmids were stably transfected into ER-negative MDA-MB-231 cells. The proliferation and migration of untransfected MDA-MB-231 cells, ERα-transfected MDA-MB-231 cells and ER-positive MCF-7 cells were determined. The expression of COX-2, and the levels of VEGF-C mRNA and the VEGF-C secretion concentration were assayed in these cell lines.Results The proliferation and migration capacities of ERα-tranfected MDA-MB-231 cells were significantly decreased (P 〈0.05). The expression of COX-2 was significantly lower in ERa-tranfected MDA-MB-231 cells than in untranfected MDA-MB-231 cells. The mRNA and protein levels of VEGF-C were lower in ERa-tranfected MDA-MB-231 cells than in untransfected MDA-MB-231 cells (P〈0.05).Conclusions ERα stable transfection inhibits proliferation and migration capacities of MDA-MB-231 cells and decreases expression of COX-2 and VEGF-C. The decreases of proliferation and migration capacities may be related to suppression of COX-2 and VEGF-C expression.
文摘Objective: To study the relationship between cyclooxygenase-2 (COX-2) expression and tumor angiogenesis in human breast cancer. Methods: Archival primary breast carcinomas (n = 62), adjacent ductal carcinoma in situ (DCIS, n = 13) and DCIS alone (n = 5) were analyzed for COX-2 and VEGF expression by immunohistochemistry using specific monoclonal antibodies. Microvessel density (MVD) was also examined the using CD34 staining. Results: A significant correlation was found between COX-2 and VEGF expression (P<0.01). Both COX-2 and VEGF were significantly correlated with MVD (P<0.05) and P<0.01, respectively). COX-2 and VEGF genes were overexpressed in tumor specimens as compared with normal epithelia. Conclusion: COX-2 is related to tumor angiogenesis in breast cancer. It is likely that VEGF is one of the most important mediators of the COX-2 angiogenic pathway.
基金supported by the grants from the National High Technology Research and Development Program of China (863 Program) (No.2006AA02A401)the Capital Development Foundation (No.30772472)Peking University 985 Program (No.2-013-39).
文摘Objective: To evaluate the efficacy and safety of celecoxib treatment of advanced non-small cell lung cancer (NSCLC), and combined therapy by molecular analysis. plus platinum-doublet as first-line chemotherapy in to determine the subgroup benefiting from celecoxib Methods: A total of 44 treatment-naive patients of advanced NSCLC with positive cyclooxygenase-2 (COX-2) expression confirmed by immunohistochemical (IHC) staining were designed to receive celecoxib plus platinum-doublet chemotherapy (cisplatin plus gemcitabine, novelbine or docetaxol) from February 2005 to May 2007. On 5-7 day before chemotherapy, 400 mg celecoxib was administered twice a day orally until obvious evidence of disease progression or intolerable toxicity was found. Adverse events were recorded according to NCI-CTC criteria. The primary endpoint was overall survival (OS). The secondary endpoints included progression-free survival (PFS), 1-year survival rate, response rate (RR) and safety. Additionally, we detected epithelial growth factor receptor (EGFR) status including EGFR gene amplification by real-time PCR and gene mutations by DHPLC followed by sequencing. Results: The response rate was 45% (20/44), and the disease control rate (DCR) was 59% (26/44). The median progression-free survival time and median survival time were 6 m and 18 m, respectively. The l-year survival rate was 68%. Chemotherapy cycle numbers and best response were found to be the predictive factors for PFS by COX model analysis (P=0.023 and P=0.000, respectively). No factor was found to affect OS. The most common toxicities included neutropenia and nausea/vomit. EGFR gene amplification was an independent prognostic factor influencing OS (P=0.0002). Patients with EGFR mutations (exon 21) had a tendency of disease progression (P=0.041). Conclusion: Encouraging activities of celecoxib combined with platinum-doublet chemotherapy were demonstrated in treatment-naive patients with advanced NSCLC, with good tolerances. For COX-2 IHC positive patients, positive EGFR amplification and mutation might be related to poor clinical outcomes.
文摘Migration of dendritic cells (DCs) into tissues and secondary lymphoid organs plays a crucial role in the initiation of innate and adaptive immunity. In this article, we show that cyclosporin A (CsA) impairs the migration of DCs both in vitro and in vivo. Exposure of DCs to clinical concentrations of CsA neither induces apoptosis nor alters development but does impair cytokine secretion, chemokine receptor expression, and migration. In vitro, CsA impairs the migration of mouse bone marrow-derived DCs toward macrophage inflammatory protein-3beta (MIP-3beta) and induces them to retain responsiveness to MIP-1alpha after lipopolysaccharide (LPS)-stimulated DC maturation, while in vivo administration of CsA inhibits the migration of DCs out of skin and into the secondary lymphoid organs. CsA impairs chemokine receptor and cyclooxygenase-2 (COX-2) expression normally triggered in LPS-stimulated DCs; administration of exogenous prostaglandin E2 (PGE2) reverses the effects of CsA on chemokine receptor expression and DC migration. Inhibition of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway signaling by CsA may be responsible for the CsA-mediated effects on the regulation of chemokine receptor and cyclooxygenase-2 (COX-2) expression. Impairment of DC migration due to inhibition of PGE2 production and regulation of chemokine receptor expression may contribute, in part, to CsA-mediated immunosuppression.