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1.
Braz. J. Psychiatry (São Paulo, 1999, Impr.) ; 41(6): 485-493, Nov.-Dec. 2019. tab, graf
Article in English | LILACS | ID: biblio-1055347

ABSTRACT

Objective: Cocaine use disorders (CUDs) represent a major public health problem in many countries. To better understand the interaction between the environmental modulations and phenotype, the aim of the present study was to investigate the DNA methylation pattern of CUD patients, who had concomitant cocaine and crack dependence, and healthy controls. Methods: We studied DNA methylation profiles in the peripheral blood of 23 CUD patients and 24 healthy control subjects using the Illumina Infinium HumanMethylation450 BeadChip arrays. Results: Comparison between CUD patients and controls revealed 186 differentially methylated positions (DMPs; adjusted p-value [adjP] < 10-5) related to 152 genes, with a subset of CpGs confirmed by pyrosequencing. DNA methylation patterns discriminated CUD patients and control groups. A gene network approach showed that the EHMT1, EHMT2, MAPK1, MAPK3, MAP2K1, and HDAC5 genes, which are involved in transcription and chromatin regulation cellular signaling pathways, were also associated with cocaine dependence. Conclusion: The investigation of DNA methylation patterns may contribute to a better understanding of the biological mechanisms involved in CUD.


Subject(s)
Humans , Male , Adult , Young Adult , Crack Cocaine , DNA Methylation , Cocaine-Related Disorders/genetics , Cocaine-Related Disorders/blood , Genome-Wide Association Study/methods , Case-Control Studies , Linear Models , Histone-Lysine N-Methyltransferase/genetics , Statistics, Nonparametric , Mitogen-Activated Protein Kinase 1/genetics , MAP Kinase Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Gene Regulatory Networks , High-Throughput Nucleotide Sequencing , Histocompatibility Antigens/genetics , Histone Deacetylases/genetics
2.
Int. j. morphol ; 37(1): 273-283, 2019. tab, graf
Article in English | LILACS | ID: biblio-990038

ABSTRACT

SUMMARY: In spite of being one of the most powerful anti-cancer drug, the nephrotoxicity of Vincristine (VCR) is not well established in either animals or humans. Hence, this study evaluates the nephrotoxic effect of VCR in rats after sub-chronic long-term administration. Rats were divided into 2 groups (n=10/group) of either control and VCR treated rats (50 mg/kg). Treatments were carried out for 30 consecutive days, after which a series of biochemical and molecular experiments related to kidney function were evaluated. VCR administration significantly decreased the survival rate (69.8 %) and impaired renal function as evidenced by lowered creatinine (Cr) clearance (Ccr), high serum levels of urea and Cr, increased urinary protein levels and resulted in sever cortex pathological alterations, including glomerulus congestion and damage as well as vascular degenerations up to necrosis of both proximal and distal convoluted tubules. Mechanistically, VCR lowered renal antioxidant potential and ATP levels, enhanced lipid peroxidation and induced inflammation. In addition, VCR induced activation of Raf-1-MEK1/2-ERK1/2 signaling pathway leading to downregulation of Bcl2 and upregulation of P53, Bax, and cleaved caspase-3. In conclusion, these findings show a nephrotoxic effect of VCR sulfate in rats after sub-chronic administration and such effect was mediated by activation of ERK1/2 induced apoptosis.


RESUMEN: A pesar de ser uno de los medicamentos de mayor eficacia contra el cáncer, aún no se ha establecido la nefrotoxicidad de la vincristina (VCR) en animales y humanos. Por lo tanto, este estudio evalúa el efecto nefrotóxico de la VCR en ratas después de la administración subcrónica a largo plazo. Las ratas se dividieron en 2 grupos (n = 10 / grupo) de control y ratas tratadas con VCR (50 mg / kg). Los tratamientos se llevaron a cabo durante 30 días consecutivos, después de los cuales se evaluaron una serie de experimentos bioquímicos y moleculares relacionados con la función renal. La administración de VCR disminuyó significativamente la tasa de supervivencia (69,8 %), dificultó la función renal, lo que se observó además en los bajos niveles de creatinina (Cr) (Ccr), los niveles séricos elevados de urea y Cr, un nivel más alto de proteína urinaria, los que dieron lugar a alteraciones patológicas severas de la corteza, incluido el glomérulo congestión y daño, como también degeneraciones vasculares, incluyendo la necrosis de los túbulos contorneados proximales y distales. Mecánicamente, el VCR redujo el potencial antioxidante renal y los niveles de ATP, mejoró la peroxidación lipídica y la inflamación inducida. Además, la VCR indujo la activación de la vía de señalización Raf-1-MEK1 / 2-ERK1 / 2 que conduce a la regulación negativa de Bcl-2 y la regulación positiva de P53, Bax y la caspasa-3. En conclusión, estos hallazgos muestran un efecto nefrotóxico del sulfato de VCR en ratas después de la administración subcrónica. Dicho efecto fue mediado por la activación de la apoptosis inducida por ERK1 / 2.


Subject(s)
Animals , Male , Rats , Vincristine/toxicity , Kidney Diseases/chemically induced , Urea/blood , RNA, Messenger , Blotting, Western , Survival Rate , Rats, Wistar , Apoptosis/drug effects , Oxidative Stress/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Creatinine/blood , MAP Kinase Signaling System , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 3 , Kidney/drug effects , Kidney/pathology , Necrosis
3.
Braz. arch. biol. technol ; 62: e19180699, 2019. graf
Article in English | LILACS | ID: biblio-1055375

ABSTRACT

Abstract Bone morphogenetic protein-4 (BMP4) is a member of the bone morphogenetic protein family which plays an important role in bone formation, inflammation and cardiac hypertrophy. The aim of this study was to investigate the underlying molecular mechanism that BMP4-induced cardiomyocyte hypertrophy. H9c2 cells were used to measure cell surface area and protein synthesis. Western blot was used to examine hypertrophic marker brain natriuretic peptide (BNP) protein expression and phosphorylation of ERK1/2. The results exhibited that cell surface area, protein synthesis and BNP protein expression were increased with BMP4 treatment. While PD98059 inhibited these effects of BMP4. In addition, BMP4 treatment increased phosphorylation of ERK1/2 in a time- and dose-dependent manner. PD98059 treatment decreased phosphorylation of ERK1/2 that was increased by BMP4. These results suggest that BMP4 induces cardiomyocyte hypertrophy through the activation of ERK1/2 cell signaling pathway.


Subject(s)
Cardiomegaly/chemically induced , Bone Morphogenetic Protein 4/administration & dosage , Blotting, Western/instrumentation , Mitogen-Activated Protein Kinase 3 , Wnt Signaling Pathway
4.
National Journal of Andrology ; (12): 199-205, 2018.
Article in Chinese | WPRIM | ID: wpr-689777

ABSTRACT

<p><b>Objective</b>To explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.</p><p><b>METHODS</b>We cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.</p><p><b>RESULTS</b>Compared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).</p><p><b>CONCLUSIONS</b>PPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.</p>


Subject(s)
Apoptosis , Cell Proliferation , DNA (Cytosine-5-)-Methyltransferase 1 , Metabolism , Diosgenin , Pharmacology , Flavonoids , Metabolism , Humans , MAP Kinase Signaling System , Male , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , NF-kappa B , Metabolism , PC-3 Cells , Phosphorylation , Prostatic Neoplasms, Castration-Resistant , Drug Therapy , Metabolism , Pathology , Signal Transduction , Transcription Factor RelA , Metabolism
5.
National Journal of Andrology ; (12): 436-441, 2018.
Article in Chinese | WPRIM | ID: wpr-689737

ABSTRACT

<p><b>Objective</b>To investigate the effects of Qiangjing Tablets (QJT) on sperm quality and the MAPK signaling pathway in the SD rat model of asthenospermia (AS).</p><p><b>METHODS</b>A total of 100 male SD rats were randomly divided into five groups of equal number, blank control, AS model control, high-dose QJT, medium-dose QJT, and low-dose QJT. All the rats were intragastrically administered ORN at 200 mg/kg/d for establishment of the AS model except those in the blank control group, which were given 1% CMC sodium solution at 1 ml/100 g by gavage. Meanwhile the animals of the high-, medium-, and low-dose QJT groups were gavaged with QJT at 6700, 3300 and 1700 mg/kg/d, respectively, qd 6 days a week for 20 days. Then the testis issue and the apoptosis of the testicular cells were observed under the electron microscope, the expression of vimentin in the testis was determined with the immunohistochemical SP method, that of ERK1/2 detected by Western blot, and the concentration of TGF-β1 in the semen measured by ELISA.</p><p><b>RESULTS</b>The AS model controls showed round nuclei of spermatocytes, homogeneously distributed chromatins, broken or lost mitochondria, and expanded rough endoplasmic reticulum in the testis tissue. In comparison, the rats of the high-, medium-, and low-dose QJT groups exhibited round nuclei of spermatocytes, homogeneously distributed chromatins, and well-structured mitochondria, rough endoplasmic reticulum and ribosome, which were all similar those of the blank controls. Compared with the blank controls, the AS model rats manifested significantly increased expressions of ERK1/2 (1.00 ± 0.00 vs 1.26 ± 0.10, P<0.01) and vimentin (0.16 ± 0.01 vs 0.17 ± 0.01, P<0.01) and apoptosis rate of cells in the testis tissue ([9.20 ± 3.07] vs [42.20 ± 9.17] %, P<0.01), but decreased level of TGF-β1 in the semen ([627.67 ± 26.07] vs [566.73 ± 68.44] ng/ml, P<0.05). In comparison with the model controls, the rats of the high- and medium- -dose QJT groups presented remarkably down-regulated expressions of ERK1/2 (1.26 ± 0.10 vs 1.14 ± 0.08, P<0.01; 1.26 ± 0.10 vs 1.18 ± 0.05, P<0.05) and vimentin (0.17 ± 0.01 vs 0.16 ± 0.01, P<0.01; 0.17 ± 0.01 vs 0.17 ± 0.09, P<0.05) and decreased rate of cell apoptosis ([42.20 ± 9.17] vs [21.60 ± 5.94] %, P<0.01; [42.20 ± 9.17] vs [33.95 ± 6.39] %, P<0.05). The concentration of TGF-β1 in the semen was markedly lower in the high-dose QJT than in the AS model control group ([621.78 ± 30.80] vs [566.73 ± 68.44] ng/ml, P < 0.05).</p><p><b>CONCLUSIONS</b>Qiangjing Tablets could improve semen quality in asthenospermia rats by acting against oxidative stress.</p>


Subject(s)
Animals , Apoptosis , Asthenozoospermia , Drugs, Chinese Herbal , Pharmacology , Male , Mitogen-Activated Protein Kinase 3 , Metabolism , Mitogen-Activated Protein Kinases , Metabolism , Random Allocation , Rats , Rats, Sprague-Dawley , Semen , Semen Analysis , Signal Transduction , Spermatozoa , Testis , Metabolism , Transforming Growth Factor beta1 , Metabolism , Vimentin , Metabolism
6.
Article in English | WPRIM | ID: wpr-766056

ABSTRACT

PURPOSE: The aim of this study was to evaluate the capacity of single and combined applications of the bark of the stems and roots of Magnolia officinalis Rehd. et Wils. (Magnoliae Cortex) and Zea mays L. (maize) to modulate inflammation in RAW 264.7 cells stimulated with Porphyromonas gingivalis. METHODS: RAW 264.7 cells were stimulated with P. gingivalis, and Magnoliae Cortex and/or maize was added. Cytotoxicity and the capacity to modulate inflammation were determined with a methylthiazol tetrazolium (MTT) assay, nitrite production, enzyme-linked immunosorbent assay (ELISA), and western blotting. RESULTS: Treatment with Magnoliae Cortex and/or maize inhibited nuclear transcription factor κB (NF-κB) pathway activation and nuclear p44/42 mitogen-activated protein kinase (MAPK) and inducible nitric oxide synthase (iNOS) protein expression in P. gingivalis-stimulated RAW 264.7 cells. Moreover, the treatments suppressed cytokines (prostaglandin E2 [PGE2], interleukin [IL]-1β, and IL-6) and nitrite production. CONCLUSIONS: Both Magnoliae Cortex and maize exerted an anti-inflammatory effect on P. gingivalis-stimulated RAW 264.7 cells, and this effect was more pronounced when the extracts were combined. These findings show that these extracts may be beneficial for slowing the progression of periodontal disease.


Subject(s)
Blotting, Western , Cytokines , Enzyme-Linked Immunosorbent Assay , Inflammation , Interleukins , Magnolia , Mitogen-Activated Protein Kinase 3 , Nitric Oxide Synthase Type II , Periodontal Diseases , Porphyromonas gingivalis , Porphyromonas , Protein Kinases , Transcription Factors , Zea mays
7.
J. appl. oral sci ; 26: e20170231, 2018. graf
Article in English | LILACS | ID: biblio-893679

ABSTRACT

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.


Subject(s)
Animals , Mice , Gene Expression/drug effects , Calcium/pharmacology , Fibroblast Growth Factor 2/drug effects , Cyclic AMP-Dependent Protein Kinases/drug effects , Mitogen-Activated Protein Kinase 1/drug effects , Dental Papilla/drug effects , Mitogen-Activated Protein Kinase 3/drug effects , Time Factors , Calcium Chloride/pharmacology , Enzyme-Linked Immunosorbent Assay , Cells, Cultured , Blotting, Western , Reproducibility of Results , Fibroblast Growth Factor 2/analysis , Fibroblast Growth Factor 2/genetics , Cyclic AMP-Dependent Protein Kinases/analysis , Mitogen-Activated Protein Kinase 1/analysis , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 3/analysis , Real-Time Polymerase Chain Reaction
8.
National Journal of Andrology ; (12): 172-175, 2018.
Article in Chinese | WPRIM | ID: wpr-775199

ABSTRACT

Penile erectile dysfunction (ED) is ascribed to the contraction-relaxation imbalance of smooth muscle cells (SMC), the weakening of their diastolic function and the strengthening of their systolic function. The contraction-related signaling pathways, cell membrane ion channels and SMC phenotypes all participate in the regulation of their contraction and its malfunction may cause a variety of SMC-related diseases. The signaling pathways RhoA/Rock and Raf/MEK/ERK1/2 interact with each other, suppressing the expression of the RhoA protein or reducing the level of Rock2 phosphorylation, which may contribute to the treatment of ED. The poor performance of VDCC or TRPC is reckoned to be an important cause of hypertension- or diabetes-related ED. The expressions of CaV1.2, TRPC1 and TRPC4 can be upregulated by many pathological factors, which may enhance the contraction of SMCs. The pathogenesis of ED may be associated with the differentiation of the phenotypes corpus cavernosal SMCs. This review focuses on the recent progress in the studies of the relationship between SMC contraction and ED.


Subject(s)
Animals , Diabetes Complications , Erectile Dysfunction , Humans , Hypertension , Ion Channels , Metabolism , Male , Mitogen-Activated Protein Kinase 3 , Muscle Contraction , Physiology , Myocytes, Smooth Muscle , Physiology , Penile Erection , Physiology , Phosphorylation , Signal Transduction , Physiology
9.
Article in English | WPRIM | ID: wpr-301012

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the effects of salvianolate on myocardial infarction in a murine in vivo model of ischemia and reperfusion (I/R) injury.</p><p><b>METHODS</b>Myocardial I/R injury model was constructed in mice by 30 min of coronary occlusion followed by 24 h of reperfusion and pretreated with salvianolate 30 min before I/R (SAL group). The SAL group was compared with SHAM (no I/R and no salvianolate), I/R (no salvianolate), and ischemia preconditioning (IPC) groups. Furthermore, an ERK1/2 inhibitor PD98059 (1 mg/kg), and a phosphatidylinositol-3-kinase (PI3-K) inhibitor, LY294002 (7.5 mg/kg), were administered intraperitoneal injection (i.p) for 30 min prior to salvianolate, followed by I/R surgery in LY and PD groups. By using a double staining method, the ratio of the infarct size (IS) to left ventricle (LV) and of risk region (RR) to LV were compared among the groups. Correlations between IS and RR were analyzed. Western-blot was used to detect the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation changes.</p><p><b>RESULTS</b>There were no significant differences between RR to LV ratio among the SHAM, I/R, IPC and SAL groups (P>0.05). The SAL and IPC groups had IS of 26.1%±1.4% and 22.3%±2.9% of RR, respectively, both of which were significantly smaller than the I/R group (38.5%±2.9% of RR, P<0.05, P<0.01, respectively). Moreover, the phosphorylation of ERK1/2 was increased in SAL group (P<0.05), while AKT had no significant change. LY294002 further reduced IS, whereas the protective role of salvianolate could be attenuated by PD98059, which increased the IS. Additionally, the IS was not linearly related to the RR (r=0.23, 0.45, 0.62, 0.17, and 0.52 in the SHAM, I/R, SAL, LY and PD groups, respectively).</p><p><b>CONCLUSION</b>Salvianolate could reduce myocardial I/R injury in mice in vivo, which involves an ERK1/2 pathway, but not a PI3-K signaling pathway.</p>


Subject(s)
Animals , Blotting, Western , Cardiotonic Agents , Pharmacology , Therapeutic Uses , Flavonoids , Pharmacology , Heart Ventricles , Pathology , MAP Kinase Signaling System , Male , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Myocardial Reperfusion Injury , Drug Therapy , Pathology , Organ Size , Phosphorylation , Plant Extracts , Chemistry , Pharmacology , Therapeutic Uses , Protein Kinase Inhibitors , Pharmacology , Staining and Labeling
10.
Article in English | WPRIM | ID: wpr-311389

ABSTRACT

<p><b>OBJECTIVE</b>Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.</p><p><b>METHODS</b>Nuclear and cytoplasmic protein extraction and immuno?uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.</p><p><b>RESULTS</b>Previously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.</p><p><b>CONCLUSION</b>Taken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.</p>


Subject(s)
Apoptosis , Physiology , Cell Line, Tumor , Gene Expression Regulation , Physiology , Heterogeneous-Nuclear Ribonucleoprotein K , Genetics , Metabolism , Humans , Mitogen-Activated Protein Kinase 1 , Genetics , Metabolism , Mitogen-Activated Protein Kinase 3 , Genetics , Metabolism , TNF-Related Apoptosis-Inducing Ligand , Genetics , Metabolism , Up-Regulation , Physiology , X-Linked Inhibitor of Apoptosis Protein , Genetics , Metabolism
11.
National Journal of Andrology ; (12): 406-411, 2017.
Article in Chinese | WPRIM | ID: wpr-812752

ABSTRACT

Objective@#To investigate the expressions of extracellular signal-regulated kinase (ERK) and p-ERK in benign and malignant prostate tissues, and whether it can be used as a marker for the prognosis of advanced prostate cancer (PCa).@*METHODS@#Using immunohistochemical Envision, we detected the expressions of ERK1/2 and p-ERK1/2 in 20 cases of benign prostatic hyperplasia (BPH) and 40 cases of advanced PCa and analyzed their correlation with PCa metastasis, Gleason score, PSA level, and prognosis.@*RESULTS@#The expression of ERK1/2 was remarkably higher in the advanced PCa than in the BPH cases (82.5% vs 55%, P5 yr, and survival ≤ 5 yr groups were 61.9%, 89.5%, 57.9%, and 90.5%, respectively, with statistically significant differences among these groups (P<0.05).@*CONCLUSIONS@#ERK1/2 and p-ERK1/2 proteins are highly expressed in advanced PCa and p-ERK1/2 is associated with the metastasis and prognosis of advanced PCa.


Subject(s)
Biomarkers, Tumor , Metabolism , Extracellular Signal-Regulated MAP Kinases , Metabolism , Humans , Male , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Neoplasm Grading , Neoplasm Metastasis , Prognosis , Prostate , Prostate-Specific Antigen , Metabolism , Prostatic Hyperplasia , Pathology , Prostatic Neoplasms , Mortality , Pathology
12.
Chinese Medical Journal ; (24): 1236-1243, 2017.
Article in English | WPRIM | ID: wpr-330638

ABSTRACT

<p><b>BACKGROUND</b>Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.</p><p><b>METHODS</b>Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.</p><p><b>RESULTS</b>HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.</p><p><b>CONCLUSIONS</b>The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.</p>


Subject(s)
Cell Line , Cell Survival , Physiology , Colorimetry , Humans , Kidney , Cell Biology , Metabolism , Lipopolysaccharides , Toxicity , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , NF-kappa B , Metabolism , Pulmonary Surfactant-Associated Protein A , Metabolism , Sulfonamides , Pharmacology , Tetrazolium Salts , Chemistry , Toll-Like Receptor 4 , Metabolism
13.
Article in Chinese | WPRIM | ID: wpr-360076

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of high concentration insulin on K562 cell proliferation and its underlying mechanism.</p><p><b>METHODS</b>K562 cells were treated by different concentration of insulin and/or anti-IGF-1R antibody (IGF-1R-Ab), MTT assay and flow cytometry were used to detect the K562 cells proliferation and apoptosis, respectivety; Western blot was used to measure the expression and phosphorylation level of IGE-IR, Akt, Erk1/2 in K562 cells under the different concentration of insulin.</p><p><b>RESULTS</b>MTT assay showed that less than 40 mU/ml insulin could promote K562 cell proliferation, while high concentration (> 40 mU/ml) insulin has been shown to inhibit K562 cell proliferation; Flow cytometry showed that 40 mU/ml insulin suppressed K562 cell apoptosis (P < 0.05), while 200 mU/ml insulin could significantly induce K562 cell apoptosis (P < 0.01); 0.01 to 1.0 µg/ml IGF-1R-Ab has significantly enhanced the inhibitory and inducing effects of high concentration (> 40 mU/ml) of insulin on K562 cell proliferation and apoptosis respectively (r = 0.962, P < 0.001); Western blot showed that after K562 cells were treated with different concentrations of insulin ERK, and the p-ERK expression did not change significantly, after K562 cells were treated with 200 mU/ml insulin, the expression of IGF-1R and AKT also not were changed obviously, while the phosphorylation level of IGF-1R and AKT increased.</p><p><b>CONCLUSION</b>High concentration (>40 mU/ml) of insulin inhibits K562 cell proliferation and induces its apoptosis, and its mechanism may be related with the binding IGF-1R by insulin, competitively inhibiting the binding of IGF-1 and IGF-1R, the blocking the transduction of PI3K/AKT signal pathway.</p>


Subject(s)
Antibodies , Pharmacology , Apoptosis , Cell Proliferation , Culture Media , Chemistry , Humans , Insulin , Pharmacology , Insulin-Like Growth Factor I , Metabolism , K562 Cells , Mitogen-Activated Protein Kinase 3 , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt , Metabolism , Receptors, Somatomedin , Allergy and Immunology , Signal Transduction
14.
Acta Physiologica Sinica ; (6): 27-34, 2016.
Article in English | WPRIM | ID: wpr-331686

ABSTRACT

The aim of the present study was to investigate the mechanism of the inhibitory effect of luteolin on the proliferation of breast cancer cells induced by epidermal growth factor (EGF) in vitro. MTT assay was used to detect the inhibitory effect of luteolin on the proliferation of MCF-7 and MDA-MB-231 cells as well as the effect on the proliferation of MCF-7 cells induced by EGF. Western blotting was used to detect the effects of luteolin on the expression of epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (Erk) 1/2 and signal transducers and activators of transcription-3 (STAT3) in MCF-7 cells induced by EGF. The results showed that luteolin could significantly inhibit the proliferation of MCF-7 and MDA-MB-231 cells, and the inhibitory effect on MCF-7 cells was more prominent. Moreover, luteolin could inhibit the proliferation of MCF-7 cells induced by EGF. Western blotting results showed that luteolin and AG1478 (an inhibitor of EGFR signaling) could inhibit the expression of p-EGFR and p-STAT3 in MCF-7 cells induced by EGF. Luteolin, LY294002 (an inhibitor of Akt signaling) and PD98059 (an inhibitor of Erk1/2 signaling) could inhibit the expression of p-Akt and p-Erk1/2 respectively in MCF-7 cells induced by EGF. Our data suggest that luteolin may inhibit EGF-induced activities of EGFR signaling pathway in human breast cancer cell lines, and PI3K/Akt, MAPK/Erk1/2, STAT3 signal pathways may be the major pathways that mediate the inhibitory effect of luteolin on EGFR signaling. Overall, our results may provide a theoretical foundation for the development of luteolin as anti-tumor drug.


Subject(s)
Breast Neoplasms , Cell Line, Tumor , Cell Proliferation , Chromones , Epidermal Growth Factor , Humans , Luteolin , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Morpholines , Phosphatidylinositol 3-Kinases , Quinazolines , ErbB Receptors , Tyrphostins
15.
Yonsei Medical Journal ; : 373-381, 2016.
Article in English | WPRIM | ID: wpr-147352

ABSTRACT

PURPOSE: Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. MATERIALS AND METHODS: Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. RESULTS: We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. CONCLUSION: Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.


Subject(s)
Animals , Caspase 3/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Flavonoids/pharmacology , Glucose/metabolism , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 1 , Male , Mitogen-Activated Protein Kinase 3 , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Inbred OLETF , Rats, Long-Evans , Reactive Oxygen Species/metabolism , Rutin/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
16.
Chinese Journal of Stomatology ; (12): 154-159, 2016.
Article in Chinese | WPRIM | ID: wpr-259425

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).</p><p><b>METHODS</b>Human PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.</p><p><b>RESULTS</b>Phosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).</p><p><b>CONCLUSIONS</b>The endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.</p>


Subject(s)
Antigens, CD , Genetics , Metabolism , Butadienes , Pharmacology , Cadherins , Genetics , Metabolism , Cell Differentiation , Endothelial Cells , Cell Biology , Physiology , Enzyme Inhibitors , Pharmacology , Extracellular Signal-Regulated MAP Kinases , Physiology , Fibroblast Growth Factor 2 , Pharmacology , Humans , Mitogen-Activated Protein Kinase 3 , Metabolism , Nitriles , Pharmacology , Periodontal Ligament , Cell Biology , Metabolism , Phosphorylation , Platelet Endothelial Cell Adhesion Molecule-1 , Genetics , Metabolism , RNA, Messenger , Metabolism , Random Allocation , Signal Transduction , Stem Cells , Cell Biology , Physiology , Time Factors , Vascular Endothelial Growth Factor A , Genetics , Metabolism , Pharmacology
17.
Article in Chinese | WPRIM | ID: wpr-263969

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of silencing ADP-ribosylation factor 6 (Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC-3 and the possible molecular mechanisms.</p><p><b>METHODS</b>Three Arf6-specific small interfering RNA (siRNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by real-time PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC-3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting.</p><p><b>RESULTS</b>Transfection of siRNA-3 resulted in significantly decreased Arf6 mRNA and protein expression with inhibition rates of (91.88±3.13)% and (86.37±0.57)%, respectively. Arf6 silencing by siRNA-3 markedly suppressed the proliferation, migration and invasion of PC-3 cells and reduced the expression levels of p-ERK1/2 and Rac1.</p><p><b>CONCLUSION</b>Silencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC-3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.</p>


Subject(s)
ADP-Ribosylation Factors , Genetics , Metabolism , Cell Line, Tumor , Cell Movement , Down-Regulation , Humans , Male , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Neoplasm Invasiveness , Prostatic Neoplasms , Pathology , RNA Interference , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Real-Time Polymerase Chain Reaction , Transfection , Wound Healing , rac1 GTP-Binding Protein , Metabolism
18.
Article in Chinese | WPRIM | ID: wpr-355330

ABSTRACT

<p><b>OBJECTIVE</b>To explore the effects of hydrogen sulfide (H(2)S) on myocardial fibrosis and expressions of MAPK1/3 and MMP-8 in diabetic rats.</p><p><b>METHODS</b>Forty adult male SD rats were randomized into 4 groups, namely the control group, diabetes mellitus group (STZ group), diabetes mellitus with H(2)S treatment group (STZ+H(2)S group), and normal rats with H(2)S treatment group (H(2)S group). Diabetes was induced by intraperitoneal injections of 40 mg/kg streptozotocin (STZ). The rats in the control group received daily intraperitoneal injections of saline, and those in STZ+H(2)S group and H(2)S group were given NaHS (100 µmol/kg) injections. After 8 weeks, the pathologies of cardiac fibrosis were examined with HE staining, and the expressions of collagen I, MAPK1/3 and MMP-8 were analyzed with Western blotting.</p><p><b>RESULTS</b>Compared with the control group, the diabetic rats showed increased collagen content and obvious interstitial fibrosis in the myocardial tissue with significantly increased expression levels of collagen I, MAPK1/3 and MMP-8 (P<0.05); all these changes were obviously reversed by treatment with H(2)S (P<0.05). Collagen I, MAPK1/3 and MMP-8 expression levels and the degree of myocardial fibrosis were comparable between H(2)S group and control group (P>0.05).</p><p><b>CONCLUSION</b>Hydrogen sulfide can attenuate cardiac fibrosis in diabetic rats, and the mechanism may involve the inhibition of MAPK1/3/MMP-8 signal pathway.</p>


Subject(s)
Animals , Collagen Type I , Metabolism , Diabetes Mellitus, Experimental , Metabolism , Pathology , Fibrosis , Hydrogen Sulfide , Pharmacology , Injections, Intraperitoneal , Male , Matrix Metalloproteinase 8 , Metabolism , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Myocardium , Pathology , Rats , Rats, Sprague-Dawley
19.
Chinese Journal of Cardiology ; (12): 728-731, 2015.
Article in Chinese | WPRIM | ID: wpr-351613

ABSTRACT

<p><b>OBJECTIVE</b>To elucidate the effect of hydroxysafflor yellow A ( HYSA) on the proliferation of vascular smooth muscle cells (VSMCs) and the related mechanism.</p><p><b>METHODS</b>VSMCs derived from SD rats were treated with DMEC culture medium (Control), 10 ng/ml PDGF (PDGF group), pretreatment with HYSA at different doses (1, 5, 10, 20, 40, 60 µmol/L) for 24 h then cotreatment with PDGF. After 24 h, MTT assay, Western blot and immunohistochemical staining were performed to evaluate the inhibitory effects of HYSA on VSMCs proliferation.</p><p><b>RESULTS</b>HYSA inhibited PDGF induced VSMCs proliferation in a dose-dependent manner, dowregulated proliferating cell nuclear antigen (PCNA) expression and blocked PDGF activated PDGFR-MEK-ERK1/2 signaling pathway.</p><p><b>CONCLUSIONS</b>HYSA inhibits VSMCs proliferation possibly via downregulating the expression of PCNA and blocking MEK-ERK1/2 signal transduction in VSMCs.</p>


Subject(s)
Animals , Cell Proliferation , Cells, Cultured , Chalcone , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Proliferating Cell Nuclear Antigen , Quinones , Rats , Rats, Sprague-Dawley
20.
Article in Chinese | WPRIM | ID: wpr-237928

ABSTRACT

<p><b>OBJECTIVE</b>To study the molecular mechanism of Yangjing Zhongyu Decoction (YZD) n-butanol extracts (ZDC) and ethyl acetate extracts (YSYZ) in reducing androgen in porcine granulose cells by mitogen-activated protein kinase (MAPK) pathway.</p><p><b>METHODS</b>Porcine granulose cells were isolated and cultured. They were inoculated by MAPK inhibitor PD98059 at different concentrations, and then they were divided into the blank control group (0), 1, 3, 10, and 25 micromol/L groups. After 24-h culture the cytochrome P450c17a (CYP17) mRNA expression level was detected using Real-time fluorescent quantitative PCR. Contents of androgen (testosterone) in the supernate were detected using RIA and optimal PD98059 concentration screened. After intervened by 10 micromol/L PD98059 for 24 h, the culture solution was intervened by effective ingredients of with or without YZD or YSYZ at various concentrations (0, 1 , 5, 25, 50 mg/mL) at various time points (3, 6, 18, 24 h). Expression levels of p-ERK1/2, c-Fos and CYP17 were detected by Western blot. Testosterone content in the supernate was determined by radioimmunoassay (RIA).</p><p><b>RESULTS</b>Ten pLmol/L PD98059 could obviously decrease p-ERK1/2 protein expression and increase CYP17 mRMA expression, and elevate testosterone content in the supernate (P < 0.05). ZDC and YSYZ at 25 ng/mL could increase p-ERK1/2 protein expression and c-Fos levels, and reduce CYP17 protein expression, and lower testosterone content in the supernate after 6-h intervention (P < 0.01).</p><p><b>CONCLUSION</b>Effective ingredients of YZD could reduce androgen production in porcine granulose cells through increasing activities of MAPK.</p>


Subject(s)
Androgens , Animals , Drugs, Chinese Herbal , Pharmacology , Female , Flavonoids , Granulosa Cells , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Mitogen-Activated Protein Kinases , Metabolism , RNA, Messenger , Swine
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