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1.
Thorac Cancer ; 2019 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-31496055

RESUMO

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and lethal cancers lacking valid prognostic biomarkers. As an essential component of a large ribonucleoprotein complex, U Three Protein 14a (UTP14a) might play important roles in human tumorigenesis. However, the clinical significance and functions of UTP14a in ESCC still remain unclear. METHODS: From September 2009 to August 2015, 210 patients with ESCC of the thoracic esophagus underwent thoracoscopic esophagectomy in our institute. The corresponding 210 tissue samples and 30 cancer-distant mucosa (CDM) samples were tested for UTP14a expression by immunohistochemical staining. The long-term survival was analyzed by the Kaplan-Meier method and Cox proportional hazards regression analyses. CCK8, cell colony formation, cell cycle, apoptosis, cell invasion, and wound healing assays were carried out with ECA109 cells to evaluate the effects of UTP14a on ESCC in vitro. RESULTS: UTP14a was positively expressed in 88.1% (185/210) of the ESCC samples. UTP14a expression in ESCC was significantly higher than in CDM, as further confirmed by Western blot analysis. High expression of UTP14a in ESCC correlated significantly with tumor invasive depth (pT stage), which predicts poor disease-free survival and disease-specific survival, as indicated by the log-rank test and Cox proportional hazards regression analysis. Additionally, our in vitro experiments further demonstrated that knockdown of UTP14a inhibits cell proliferation and invasion in ECA109 cells. CONCLUSIONS: Our results suggest that UTP14a is aberrantly expressed in ESCC, plays a critical role in cancer progression and could be a potential prognosis predictor of ESCC.

2.
J Neurochem ; 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31314916

RESUMO

Glial glutamate transporter 1 (GLT-1) plays a vital role in the induction of brain ischemic tolerance (BIT) by ischemic preconditioning (IPC). However, the mechanism still needs to be further explained. The aim of this study was to investigate whether peroxisome proliferator-activated receptor gamma (PPARγ) participates in regulating GLT-1 during the acquisition of BIT induced by IPC. Initially, cerebral IPC induced BIT and enhanced PPARγ and GLT-1 expression in the CA1 hippocampus in rats. The ratio of nuclear/cytoplasmic PPARγ was also increased. At the same time, the up-regulation of PPARγ expression in astrocytes in the CA1 hippocampus was revealed by double immunofluorescence for PPARγ and glial fibrillary acidic protein. Then, the mechanism by which PPARγ regulates GLT-1 was studied in rat cortical astrocyte-neuron cocultures. We found that IPC [45 min of oxygen glucose deprivation (OGD)] protected neuronal survival after lethal OGD (4 h of OGD), which usually leads to neuronal death. The activation of PPARγ occurred earlier than the up-regulation of GLT-1 in astrocytes after IPC, as determined by western blot and immunofluorescence. Moreover, the preadministration of the PPARγ antagonist T0070907 or PPARγ siRNA significantly attenuated GLT-1 up-regulation and the neuroprotective effects induced by IPC in vitro. Finally, the effect of the PPARγ antagonist on GLT-1 expression and BIT was verified in vivo. We observed that the preadministration of T0070907 by intracerebroventricular injection dose-dependently attenuated the up-regulation of GLT-1 and BIT induced by cerebral IPC in rats. In conclusion, PPARγ participates in regulating GLT-1 during the acquisition of BIT induced by IPC. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

3.
Brain Res Bull ; 147: 1-13, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30731111

RESUMO

The previous studies have shown that glial glutamate transporter-1 (GLT-1) participates in cerebral ischemic injury in rats. However, the mechanism involved remains to be elucidated. This study was undertaken to investigate whether p38 MAPK was involved in regulating GLT-1 in the process. At first, it was observed that global brain ischemia for 8 min led to obvious delayed neuronal death, GLT-1 down-regulation and p-p38 MAPK up-regulation in CA1 hippocampus in rats. Then, whether p-p38 MAPK was involved in regulating GLT-1 during cerebral ischemic injury was studied in vitro. Astrocyte-neuron co-cultures exposed to oxygen and glucose deprivation (OGD) were used to mimic brain ischemia. It was observed that lethal OGD (4-h OGD) decreased GLT-1 expression and increased p-p38 MAPK expression in astrocytes. The p-p38 MAPK protein rised from 0 min to 48 h that is the end time of the observation, and the peak value was at 12 h, which was 12.45 times of the control group. Moreover, pre-administration of p38 MAPK inhibitor SB203580 or its siRNA dose-dependently increased GLT-1 expression, and meanwhile alleviated the neuronal death induced by lethal OGD. The above results indicated that p38 MAPK signaling pathway participated in regulating GLT-1 during OGD injury in vitro. Finally, back to in vivo experiment, it was found that pre-administration of SB203580 by intracerebroventricular injection dose-dependently reversed the down-regulation of GLT-1 expression and attenuated the delayed neuronal death normally induced by global brain ischemia in CA1 hippocampus in rats. Taken together, it can be concluded that the mechanism of GLT-1 mediating cerebral ischemic injury depends on the activation of p38 MAPK.

4.
Neurochem Res ; 43(10): 2016, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30171421

RESUMO

The order of corresponding author was inadvertently published. Hence, the first and the second corresponding authors should be Min Zhang (hebmuzhangmin@163.com) and Jing-Ge Zhang (zhangjg001@163.com).

5.
Front Mol Neurosci ; 11: 281, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30158854

RESUMO

Sulbactam is an atypical ß-lactam medication and reported to be neuroprotective by up-regulating glial glutamate transporter-1 (GLT-1) in rats. The present study was undertaken to study the role of p38 MAPK signal pathway in sulbactam induced up-regulation of GLT-1 expression in astrocytes and anti-ischemic effect. Neuron-astrocyte co-cultures and astrocyte cultures from neonatal Wistar rats were used. Cerebral ischemia was mimicked by oxygen-glucose deprivation (OGD). Hoechst (HO)/propidium iodide (PI) double fluorescence staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay were used to evaluate neuronal death and cell viability, respectively. Immunocytochemistry and Western blot were used to detect protein expressions. Sulbactam pre-incubation significantly and dose-dependently prevented neuronal death and decline in cell viability induced by OGD in neuron-astrocyte co-cultures, and upregulated GLT-1 expression in astrocyte cultures endured OGD, which suggested that sulbactam might protect neurons against OGD by up-regulating astrocytic GLT-1 expression. It was further shown that the phosphorylated-p38 MAPK expression in astrocytes was up-regulated after the sulbactam pre-incubation and this up-regulation was moderate in amplitude. Especially, the time course of the up-regulation of phosphorylated-p38 MAPK was obviously earlier than that of GLT-1, which suggested possibility that p38 MAPK might be an upstream signal for GLT-1 up-regulation induced by sulbactam. We further found that SB203580, the specific inhibitor of p38 MAPK, dose-dependently inhibited the GLT-1 up-regulation induced by sulbactam either in non- or OGD-treated astrocytes and the protective effect of sulbactam on co-cultured neurons against OGD. Taken together, it might be concluded that sulbactam protects cerebral neurons against OGD by up-regulating astrocytic GLT-1 expression via p38 MAPK signal pathway.

6.
Neurochem Res ; 43(9): 1779-1790, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29995175

RESUMO

Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH + sham group, ischemia group and IH + ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (PB = 404 mmHg, PO2 = 84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, L-NAME + ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of L-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.

7.
Immunol Lett ; 191: 63-72, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28963072

RESUMO

Regulatory T cells (Treg cells) belong to a class of immunosuppressive cells that control the pathological changes of autoimmunity and inflammation. Prostaglandin E2 (PGE2) is a potent lipid mediator of immune inflammation including rheumatoid arthritis (RA) that exerts its effects via four subtypes of G-protein-coupled receptors (EP1-4). The ability of PGE2 to regulate human Treg differentiation has not yet been reported. In the current study, we investigated the effects of PGE2 on the differentiation of naïve T cells from healthy and RA patients into Treg cells and the intracellular signaling involved in this process in vitro. Our data indicate that PGE2 negatively influenced the percentage of Treg cells and Foxp3 mRNA expression. The regulatory effects of PGE2 were associated with increased intracellular cAMP levels and PKA activity. EP2 receptors may mediate the inhibitory role of PGE2, since PGE2 actions were mimicked by EP2 agonist (Butaprost) and cAMP agonist (Sp-8-CPT-cAMPS) but were reversed by an EP2 antagonist (PF-04418948) and a PKA inhibitor (H-89). PGE2 negatively modulated the expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), as well as the production of interleukin (IL)-10 by Treg cells via EP2 receptors and cAMP/PKA signaling. All these findings indicate that PGE2 can inhibit Treg differentiation mediated through the EP2-cAMP/PKA signaling pathway, and suggest novel immune-based therapies for use in RA treatment.


Assuntos
Artrite Reumatoide/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Linfócitos T Reguladores/imunologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Azetidinas/farmacologia , Antígeno CTLA-4/metabolismo , Diferenciação Celular , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Humanos , Interleucina-10/metabolismo , Transdução de Sinais
8.
Int Immunopharmacol ; 20(2): 307-15, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24704498

RESUMO

Cholecystokinin octapeptide (CCK-8), an immunomodulatory peptide, can promote or suppress the development or function of specific CD4(+) T cell subsets by regulating antigen-presenting cell functions. In the current study, we investigated whether CCK-8 exerts a direct effect on T cells through influencing differentiation and cytokine production of distinct CD4(+) T cell subsets in vitro. Our results showed that CCK-8 differentially affects the development and function of CD4(+) T cell populations, with a negative influence on Th1 and Th17 cells and positive regulatory effect on inducible T regulatory cells (iTreg). Notably, CCK-8 suppressed Th1 while slightly enhancing Th2 development and cytokine production. Similarly, CCK-8 inhibited the differentiation of Th17 cells and promoted Foxp3 expression. L-364,718 and LY-288,513, selective antagonists of CCK1R and CCK2R, respectively, suppressed the effects of CCK-8 on CD4(+) T cell subset-specific transcription factors. Our findings strongly indicate that CCK-8 exerts a direct effect on T cells, which is dependent on CCKRs, particularly CCK2R. The collective results aid in further clarifying the mechanism underlying the anti-inflammatory and immunoregulatory effects of CCK-8.


Assuntos
Anti-Inflamatórios/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Sincalida/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Devazepida/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Técnicas In Vitro , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Receptor de Colecistocinina A/antagonistas & inibidores , Receptor de Colecistocinina B/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia
9.
Acta Pharmacol Sin ; 32(11): 1373-80, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21986577

RESUMO

AIM: The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA). METHODS: Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 µmol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions. RESULTS: One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter. CONCLUSION: The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.


Assuntos
Artrite Reumatoide/genética , Ilhas de CpG , Metilação de DNA , Interleucina-10/genética , Regiões Promotoras Genéticas , Adolescente , Adulto , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto Jovem
10.
Int Immunopharmacol ; 11(11): 1685-90, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21664492

RESUMO

Cholecystokinin octapeptide (CCK-8) is a typical brain-gut peptide that exerts a variety of physiological actions in both the peripheral and central nervous systems. Our laboratory has previously reported that CCK-8 produces immunoregulatory action through activating CCK receptor (CCK1R/CCK2R) expression on immune cell surfaces. In the present study, we investigated the effect of CCK-8 on immunoglobulin G1 (IgG1) production in lipopolysaccharide (LPS)-activated B cells in vitro. CCK-8 inhibited the proliferation and IgG1 mRNA expression of LPS-activated B cells and therefore inhibited IgG1 production. The mechanism may be associated with the regulation of CCK-8 on transcription factors Blimp1, Pax5, Xbp1 and Bcl6. CCK-8 inhibited the expression of Blimp1, while the effect on Pax5, Xbp1 and Bcl6 varied with time, suggesting that CCK-8 acted as a complex regulator of LPS-activated B cells. The inhibitory action of CCK-8 was mainly mediated through the CCK2R pathway. These studies indicate that CCK-8 attenuates humoral immune responses and acts as endogenous immune deactivators in autoimmune diseases.


Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Imunoglobulina G/biossíntese , Lipopolissacarídeos/farmacologia , Sincalida/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunoglobulina G/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores da Colecistocinina/antagonistas & inibidores , Receptores da Colecistocinina/fisiologia , Sincalida/fisiologia , Transcrição Genética/efeitos dos fármacos
11.
Immunopharmacol Immunotoxicol ; 33(1): 157-63, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20536341

RESUMO

Cholecystokinin octapeptide (CCK8) can exert the immunoregulatory roles through activating immune cell surface receptors such as T lymphocytes, macrophages, dendritic cells, and so on. In this study, we discussed the effects of CCK8 on lipopolysaccharide (LPS)-activated B cells in terms of the expression of co-stimulatory molecules, and the capacity to activate CD4(+) T cells and cytokines production in vitro. The results revealed that B cells expressed two types of CCK receptors; CCK8 inhibited the expression of co-stimulatory molecules such as CD80 and CD86 on LPS-activated B cells, suppressed the proliferation of allogeneic T cells in a dose-dependent manner, and also reduced the secretion of Th1-type cytokine IFN-γ, whereas enhanced the secretion of Th2-type cytokine IL-4 by LPS-activated B cells. Both CCK1R and CCK2R participated in these effects. Taken together, CCK8 is capable of exerting immunomodulatory functions through B cells.


Assuntos
Antígenos CD/biossíntese , Linfócitos B/efeitos dos fármacos , Citocinas/imunologia , Lipopolissacarídeos/farmacologia , Receptores da Colecistocinina/biossíntese , Sincalida/farmacologia , Animais , Antígenos CD/imunologia , Linfócitos B/imunologia , Antígeno B7-1/biossíntese , Antígeno B7-1/imunologia , Antígeno B7-2/biossíntese , Antígeno B7-2/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Citocinas/biossíntese , Citometria de Fluxo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores da Colecistocinina/imunologia
12.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 38(5): 770-4, 2007 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-17953354

RESUMO

OBJECTIVE: Glutathione(GSH) maintains an optimum cellular redox potential. Elevated levels of GSH render some types of cancer cells resistant against anti-cancer drugs. The aim of this study was to determine the effect of a thiol-depleting agent, diethylmaleate (DEM), on the sensitivity of human breast cancer cells to ADM. METHODS: The ADM-resistant human breast cancer MCF-7/ADM cell lines and ADM-sensitive MCF-7/S cell lines were treated by thiol-depleting agent DEM for 3 h respectively. The changes of sensitivity to ADM were then measured by MTT assay. The intracellular GSH contents were examined by fluorescent-spectrophotometry and the correlation between the changes of sensitivity to ADM and the intracellular GSH content was analyzed. RESULTS: Treatment of MCF-7/ADM and MCF-7/S cells by 0.1 micromol/L DEM for 3 h decreased 37.4% and 29.7% of the intracellular GSH content respectively (P < 0.01). ADM also decreased intracellular GSH content in a ADM-concentration-dependent manner. The combined use of DEM and ADM depleted the intracellular GSH content in both cells significantly more than the sum of single use of ADM and DEM alone. The sensitivity of both cells to ADM increased with the decline of intracellular GSH content. CONCLUSION: The depletion effect of DEM on the intracellular GSH could be enhanced by ADM and such depletion may be involved in the changes of the sensitivity of MCF/7 cells to ADM.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Glutationa/metabolismo , Maleatos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Glutationa/antagonistas & inibidores , Humanos
13.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 38(2): 284-6, 301, 2007 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-17441350

RESUMO

OBJECTIVE: To observe the effect of platelet activating factor (PAF) antagonist, ginkgolide B (GB), on the expression of glial fibrillary acidic protein (GFAP) in astrocytes during focal cerebral ischemia, of which the mechanism will be explored too. METHODS: The focal cerebral ischemia of tree shrews was induced to form via photochemical reaction. Morphological changes were observed by hematoxylin-eosin staining and electron microscopic technology. The GFAP expression in astrocytes was detected by immunohistochemistry. RESULTS: Morphological changes of the brain tissue occurred after focal cerebral ischemia. Astrocytes were more swollen with the prolongation of ischemia. The GFAP expression in astrocytes in the penumbra didn't change obviously at 4 h, but increased significantly at 24 h (P < 0.01), and retained the higher level at 72 h (P < 0.01) after focal cerebral ischemia. Whereas in contralateral cortex, the GFAP expression began to increase at 72 h (P < 0.05) after focal cerebral ischemia. With giving experimental animals GB at 6 h, the GFAP expression decreased until at 24 h after focal cerebral ischemia. CONCLUSION: GFAP expression in astrocytes increases after focal cerebral ischemia. And by inhibiting the astroglial GFAP expression, the ginkgolide B exerts the cerebral protective effects.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Ginkgolídeos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Lactonas/farmacologia , Animais , Astrócitos/patologia , Isquemia Encefálica/prevenção & controle , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ginkgolídeos/administração & dosagem , Injeções , Lactonas/administração & dosagem , Masculino , Fatores de Tempo , Tupaiidae
14.
Fen Zi Xi Bao Sheng Wu Xue Bao ; 40(1): 17-23, 2007 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-17357445

RESUMO

To investigate the pathogenic mechanism of homocysteine-induced endothelial nitric oxide synthase dysfunction and the antagonistic effects by folic acid (FA). Human umbilical vein endothelial cells (HUVEC)were cultured to the third generation. Then HUVEC were cultured with Hcy at different concentrations (0,10,30,100 and 300 micromol/L),with or without FA(100 micromol/L)for 72 hours. The mRNA and protein levels of endothelial nitric oxide synthase (eNOS) were analyzed by RT-PCR and immunohistochemistry respectively. Asymmetric dimethylarginine (ADMA)was measured by reversed-phase high performance liquid chromatography. The dimethylarginine dimethylaminohydrolase(DDAH), activity of eNOS and the production of NO were analyzed simulta- neously. After HUVEC were exposed to Hcy at different concentrations for 72 hours, the level of eNOS mRNA and the content of eNOS protein, the eNOS activity, and the production of nitric oxide (NO) were all significantly and dose-dependently reduced compared with the control group (P< 0.05). The activity of DDAH has a parallel decrease and the ADMA concentration showed a cor- responding increase. The addition of folic acid (100 micromol/L)resulted in partial antagonistic effects against the injury of Hcy on NOS system of endothelial cells, the eNOS protein level and eNOS activity, and NO production increased,and so does the DDAH activity,and the ADMA concentration reduced. But the FA didn't influence the eNOS mRNA expression. The pathogenic mechanism of homocysteine-induced eNOS dysfunction may involve two levels,the level of eNOS protein and eNOS activity,and the level of the expression of eNOS gene. The injury on the level of eNOS protein and eNOS activity may go through the DDAH-ADMA pathway. Folic acid can exert partial protective roles against the Hcy in the level of eNOS protein and eNOS activity,but without impact on the expression of eNOS gene.


Assuntos
Células Endoteliais/efeitos dos fármacos , Ácido Fólico/farmacologia , Homocisteína/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Humanos , Imuno-Histoquímica , Óxido Nítrico Sintase Tipo III/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Chin Med J (Engl) ; 120(23): 2132-7, 2007 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18167189

RESUMO

BACKGROUND: Hyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS). This study was designed to investigate whether the dysfunction of endothelial NO system originates from HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured to the third generation and treated with homocysteine (Hcy) at different concentrations (0, 10, 30, 100, and 300 micromol/L) for 72 hours. The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP). The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR. The activity of DDAH2 and eNOS in cells, and the concentrations of ADMA and NO in culture medium were assayed respectively. RESULTS: Mild increased concentration of Hcy (10 and 30 micromol/L) induced hypomethylation, while high concentration of Hcy (100 and 300 micromol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene. The mRNA expression of DDAH2 increased in mild enhanced concentration of Hcy, and decreased in high concentration of Hcy correspondingly. The inhibition of DDAH2 activity, the increase of ADMA concentration, the reduction of eNOS activity and the decrease of NO production were all consistently relevant to the alteration of Hcy concentration. CONCLUSION: The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway, which showed highly relevant and dose-effect relationship. The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in DDAH2 gene.


Assuntos
Amidoidrolases/genética , Metilação de DNA/efeitos dos fármacos , Homocisteína/farmacologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico/fisiologia , Regiões Promotoras Genéticas , Arginina/análogos & derivados , Arginina/sangue , Células Cultivadas , Humanos , Óxido Nítrico/análise , Óxido Nítrico Sintase Tipo III/genética
16.
Acta Biochim Biophys Sin (Shanghai) ; 38(6): 417-22, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16761100

RESUMO

Recent studies have suggested that antibodies can catalyze the generation of unknown oxidants including hydrogen peroxide (H2O2) and ozone (O3) from singlet oxygen (1O2) and water. This study is aimed to detect the effect of antibody-catalyzed water oxidation on atherosclerosis. Our results showed that both H2O2 and O3 were produced in human leukemia THP-1 monocytes incubated with human immunoglobulin G and phorbol myristate acetate. In the THP-1 monocytes incubated with human immunoglobulin G, phorbol myristate acetate and low density lipoprotein, the intracellular total cholesterol, free cholesterol, cholesteryl ester and lipid peroxides clearly increased, and a larger number of foam cells were observed by oil red O staining. The accumulation of all intracellular lipids was significantly inhibited by vinylbenzoic acid, and only slightly affected by catalase. These findings suggested that the production of O3, rather than H2O2, might be involved in the pathogenesis of atherosclerosis through the antibody-catalyzed water oxidation pathway.


Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Ozônio/farmacologia , Água/química , Compostos Azo/farmacologia , Catálise , Linhagem Celular Tumoral , Colesterol/metabolismo , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Imunoglobulina G/química , Peróxidos Lipídicos/química , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Estirenos/química
17.
Fen Zi Xi Bao Sheng Wu Xue Bao ; 39(6): 509-15, 2006 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-17348203

RESUMO

In the present study, we measured the antibody-catalyzed 03 formation from THP-1 monocytes activated by phorbol myristate acetate (PMA) by indigo carmine bleaching reaction test, and the accumulation of cholesterol in THP-1 monocytes by fluorescence spectrophotometric method, and analyzed the cholesterol ozonation product 5,6-secosterol by high-performance liquid chromatography (HPLC), to explore the potential effect of antibody-catalyzed water oxidation on pathogenesis of atherosclerosis. It was showed that THP-1 monocytes incubated with human IgG and PMA evidently produced an oxidant with the chemical signature of 03 which could bleach indigo carmine, and be intensified or inhibited respectively by catalase and vinylbenzoic acid. In the THP-1 monocytes incubated with human IgG, PMA and LDL, the intracellular accumulated total cholesterol (TC), free cholesterol (FC), cholesteryl ester (CE) and the CE/TC increased evidently, and the cholesterol ozonation product 5,6-secosterol was also produced markly, all of that were inhibited by vinylbenzoic acid. These results demonstrated that the activated THP-1 monocytes possess the ability to produce O3 through antibody-catalyzed water-oxidation pathway, which could be a new mechanism concerned with atheriosclerosis.


Assuntos
Aterosclerose/metabolismo , Imunoglobulina G/farmacologia , Monócitos/efeitos dos fármacos , Ozônio/metabolismo , Linhagem Celular , Colesterol/metabolismo , Ésteres do Colesterol/farmacologia , Cromatografia Líquida de Alta Pressão , Humanos , Monócitos/citologia , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Fluorescência , Acetato de Tetradecanoilforbol/farmacologia
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