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Objective:To explore the regulation effects of baicalin on the behavior as well as extracellular regulated protein kinase(ERK)and cAMP-response element binding protein(CREB) in chronic unpredictable mild stimulus(CUMS) model mice.Methods:Thirty ICR mice were randomly assigned to control(CON) group, model(CUMS) group, fluoxetine(FLU) group, baicalin high-dose(BA-H) group and baicalin low-dose(BA-L) group with 6 mice in each group.In addition to the CON group, the mice in the other four groups were modeled by CUMS method.The modeling was carried out for 42 days, and intragastric administration was carried out according to groups from the 21st day to the completion of modeling.After administration, the depression like behavior of mice was measured by sugar water preference test and water maze test.Western blot (WB) and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the protein level and mRNA level of ERK and CREB in mouse hippocampus respectively.SPSS 21.0 was used for statistical analysis.After normal test and variance homogeneity test, one-way ANOVA was used for multi group comparison, and Tukey test was used for pairwise comparison.Results:Results from the sugar preference experiment showed that compared with CON group, the sugar preference rate of CUMS group was decreased ((82.88±2.00)%, (64.49±1.24)%, t=19.11, P<0.05). Compared with CUMS group, sugar preference rate in FLU group ((81.90±1.19) %), BA-H group (77.86±2.51)%) and BA-L group ((67.98±2.56)%) increased ( t=24.83, 11.68, 3.00, all P<0.05). The results of water maze test showed that compared with CON group, the number of crossing platform ((6.33±0.82), (1.83±0.75), t=9.93, P<0.05) and the target quadrant residence time ((46.83±4.78)s, (24.25±6.12)s, t=7.13, P<0.05) of mice in CUMS group were decreased, but the the escape latency was prolonged ((14.88±3.00) s, (70.70±4.77) s, t=24.26, P<0.05). Compared with CUMS group, the number of crossing platform ((5.00±0.89)times, (5.17±0.75)times and (3.33±0.82) times, t=6.64, 7.67, 3.31, all P<0.05), and the residence time in the target quadrant ((36.80±2.66) s, (36.82±5.62) s, (33.28±3.56) s, t=4.61, 3.71, 3.13, all P<0.05) in FLU group, BA-H group and BA-L group increased, but the escape latencies were shortened ((23.37±4.86) s, (34.83±4.72) s, (62.15±5.30) s, t=17.02, 13.10, 2.94, all P<0.05). WB results showed that compared with CON group, the expression of ERK protein ((1.00±0.15), (0.36±0.10), t= 6.26, P<0.05) and CREB protein((1.00±0.12), (0.29±0.03), t=10.32, P<0.05) in hippocampus of mice in CUMS group decreased.Compared with CUMS group, ERK protein in hippocampus of mice in FLU, BA-H and BA-L groups increased ((0.87±0.05), (0.77±0.08), (0.67±0.03), t=8.25, 5.7, 5.39, all P<0.05), and CREB protein in FLU, BA-H and BA-L groups were also increased ((0.90±0.12), (0.84±0.14), (0.62±0.04), t=8.94, 6.59, 12.25, all P<0.05). qPCR results showed that compared with CON group, ERK mRNA ((1.00±0.03), (0.41±0.10), t=9.78, P<0.05) and CREB mRNA ((1.00±0.08), (0.61±0.12), t=4.62, P<0.05) were decreased in CUMS group.Compared with CUMS group, ERK mRNA in hippocampus of mice in FLU, BA-H and BA-L groups were increased ((0.71±0.08), (0.69±0.03), (0.59±0.04), t=4.15, 4.65, 2.84, all P<0.05), CREB mRNA in FLU group and BA-H group were increased ((0.87±0.08), (0.86±0.07), t=3.14, 3.19, all P<0.05). Conclusion:BA can improve the depression-like behavior of CUMS model mice.The mechanism of action may be related to the regulation of ERK and CREB proteins.
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Aim To investigate the effects of morphine preconditioning (MPC) on transient receptor potential vanilloid 1 (TRPV1) channel current in rat dorsal root ganglia (DRG) neurons and the phosphorylation (p) of TRPV1 and extracellular regulated protein kinases (ERK) in PC12 cells that sensitized by nerve growth factor (NGF). Methods DRG neurons isolated from T2-T8 segments of 10 days old SD rat or pheochromo-cytoma (PC12) cells were seeded into 24-well plates or 6-well plates, respectively, and randomly divided into 5 groups:control group (group C), NGF sensiti-zation group (group NGF), and morphine precondi-tioning groups (group MPC 0.3, group MPC 1.0 and group MPC 3.0). DRG neurons were identified by im-munofluorescent method with neuronal specific enolase (NSE). Cells were treated by morphine, NGF and capsaicin to simulate the effects of MPC on DRG neu-rons in T2-T8 segments during myocardial ischemia reperfusion injury (IRI). Afterwards, the inward cur-rent of DRG neurons induced by capsaicin in all groups were detected by whole cell recording; the expression and phosphorylation of TRPV 1 and ERK in PC12 cells were detected by Western blot. Results DRG neu-rons survived and grew nicely,and the staining of neu-ronal specific markers,NSE,was positive. In compar-ison with group C, the inward current of group NGF was enhanced (P <0.05), while MPC inhibited the enhancement (P <0.05). The relative expression of TRPV1,p-TRPV1 and p-ERK in group NGF was up-regulated when compared with group C (P <0.05).Moreover, the up-regulation was also suppressed by MPC (P <0.05). Conclusions MPC inhibits TR-PV1 channel current sensitized by NGF in neurocytes, and the mechanism might be associated with the down-regulation of TRPV1 p-TRPV1 and p-ERK expression.
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OBJECTIVE: To study the effects of scutellarin on the proliferation of cardiac fibroblasts (CFs) in neonate rats induced by angiotensin Ⅱ (ANG Ⅱ) and signal pathway of extracellular regulated protein kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK).METHODS: CFs of neonatal rat were isolated and cultured in vitro, and then divided into blank group (blank culture medium), Ang Ⅱ group (10-7 μmol/L), 50 μmol/L scutellarin group and Ang Ⅱ (10-7 μmol/L)+5, 10, 20, 50 μmol/L scutellarin groups. After cultured for 48 h, CCK-8 assay was used to detect the proliferation ability of cells. Other cells were selected and divided into blank group (blank culture medium), Ang Ⅱ group (10-7 μmol/L) and Ang Ⅱ +5, 10, 20, 50 μmol/L scutellarin groups. After cultured for 48 h, mRNA expression of Col I, Col Ⅲ and α-SMA in cells and hydroxyproline (HYP) content in cell culture fluid were detected; phosphorylation levels of ERK1/2 and p38 MAPK in cells were also detected. RESULTS: 5, 10, 20, 50 μmol/L scutellarin could significantly inhibit the proliferation of CFs induced by Ang Ⅱ (P<0. 05), and decreased mRNA expression of Col I, Col Ⅲ and α-SMA in CFs induced Ang Ⅱ (P<0. 05). 5, 10, 20, 50 μmol/L scutellarin could significantly inhibit the increase of HYP content and the phosphorylation of ERK1/2 and p38 MAPK after induced by Ang Ⅱ (P<0. 05), in dose-dependent manner. CONCLUSIONS: Scutellarin inhibits the proliferation of CFs induced by Ang Ⅱ, the mechanism of which may be associated with reduction of ERK1/2 and p38 MAPK phosphorylation.
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Objective To investigate the correlation between the expression of phospholipase Cβ1 (PLCβ1) and the clinical relevant parameters and prognosis in patients with hepatocellular carcinoma (HCC).Methods By using tissue microarray technique and immunohistochemical method,the expressions of PLCβ1 in tumor and pericancerous tissues were tested in 141 HCC patients.The relationship between the expressions of PLCβ1 and the clinical and pathological characteristics was analyzed.Colony formation assay and apoptosis experiments were used to check the effect of PLCβ1 on proliferation of HCC cells.KaplanMeier analysis and Cox multivariate regression model analysis were adopted to analyze the prognosis of HCC patients.Results The expression level of PLCβ1 in tumor tissues was obviously higher than that in pericancerous tissues,which was closely related to the tumor staging.Kaplan-Meier survival analysis indicated that the survival rate in HCC patients with high expression level of PLCβ1 was lower than that in HCC patients with low expression level of PLCβ1.Cox multivariate regression analysis revealed that high expression of PLCβ1 was an independent prognostic factor for HCC patients.Over expression of PLCβ1 in HCC cells could promote the proliferation of HCC cell and inhibit its apoptosis.Further investigation showed that activation of extracellular regulated protein kinase signaling pathway might be involved in PLCβ1-mediated HCC cell growth.Conclusion PLCβ1 can promote the progression of HCC,and the expression level of PLCβ1 can be regarded as an independent prognostic factor for HCC,and it is expected that PLCβ1 may become an ideal therapeutic target.
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Objective To observe effects of different dosages of moxibustion with ginger-separated moxibustion on expressions of mitogen extracellular kinase (MEK) 1/2 and extracellular regulated protein kinase (ERK) 1/2 of gastric tissue in rats with spleen deficiency; To explore the possible mechanism and the dose-effect relationship. Methods Seventy-five SD rats were randomly divided into blank control group, model group, ginger-separated moxibustion for three zhuang group, six zhuang group and nine zhuang group according to random digits table method, with fifteen rats in each group. The rat model of spleen deficiency was established by intragastric administration with 200% Rhei Radix et Rhizoma infusion at 4 ℃. Ginger-separated moxibustion groups were treated with different dosage of moxibustion at "Zusanli", "Zhongwan" for eight days after the modeling. Pathological changes of gastric tissue by HE staining were observed under light microscope, and immunohistochemistry was used to detect the expressions of MEK1/2 and ERK1/2 protein in gastric tissue of rats. Results Compared with the blank control group rats, the gastric mucosa injury in the model group was obvious, which showed that the damage and abscission was more serious; compared with the model group, the gastric mucosa of rats was partly exfoliated and the damage was improved in three zhuang group, and the surface of gastric mucosa of rats was more complete and damage was improved obviously in six zhuang group and nine zhuang group; compared with the blank control group, the expressions of MEK1/2 and ERK1/2 protein in gastric tissue increased obviously in other groups (P<0.01);compared with three zhuang group, the expressions of MEK1/2 and ERK1/2 protein in gastric tissue increased in six zhuang group and nine zhuang group (P<0.01), but the effects of the two group were similar, without statistical significance (P>0.05). Conclusion Ginger-separated moxibustion can repair gastric mucosa in rats with spleen deficiency, which may be closely associated with its effect in increasing the expressions of MEK1/2 and ERK1/2 protein in gastric tissue and activating the MEK/ERK signal transduction pathway.
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Objective: To investigate the effects of epithelial cell transformingsequence 2 oncogene (ECT 2) gene-silencing on the proliferation and apoptosis of human breast cancer cells, as well as its potential molecular mechanism. Methods: Firstly, the expressions of ECT2 mRNA and protein in normal mammary epithelial cells (MCF-10A) and breast cancer cells (MDA-MB-231, SK-BR-3, MCF-7, and BT474) were detected by real-Time fluorescent quantitative PCR and Western blotting, respectively. After transfection with the specific siRNA targeting ECT 2 gene (ECT2 siRNA) and treatment with extracellular regulated protein kinase (ERK) pathway activator or transfection with microRNA-101 (miR-101) inhibitor, the proliferation and apoptosis of MDA-MB-231 and MCF-7 cells were detected by CCK-8 and FCM assay, respectively. Then the levels of phospho-ERK (p-ERK), Ras-related C3 botulinum toxin substrate 1 (Rac1) and miR-101 in MDA-MB-231 and MCF-7 cells were detected by Western blotting and real-Time fluorescent quantitative PCR. Results: The expression levels of ECT2 mRNA and protein in breast cancer cells were significantly increased as compared with those in normal mammary epithelial cells (both P 0.05). Conclusion: ECT 2 gene-silencing may affect the proliferation and apoptosis of breast cancer cells by ERK-miR-101-Rac1 signaling pathway.
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Objective@#To investigate the impact of transforming growth factor-β1 (TGF-β1) silencing by small interfering RNA (siRNA) on the expression of platelet-derived growth factor-β (PDGF-BB) and its receptor (PDGF-βR) in rats with hepatic fibrosis.@*Methods@#A total of 40 male Sprague-Dawley rats were randomly divided into normal control group, model group, TGF-β1 siRNA treatment group, and negative control group. All rats except those in the normal control group were given subcutaneous injection of 40% carbon tetrachloride to establish a rat model of hepatic fibrosis. The rats in the negative control group and the TGF-β1 siRNA treatment group were given tail vein injection of negative control plasmid or TGF-β1 siRNA plasmid twice a week at a dose of 0.25 mg/kg, and those in the normal control group and the model group were given the injection of sterile isotonic saline twice a week. The rats were sacrificed after 12 weeks and liver tissue samples were collected. Real-time PCR, immunohistochemistry, and Western blot were used to measure the expression of PDGF-BB, PDGF-βR, and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) in liver tissue. A one-way analysis of variance, the q test, and the Kruskal-Wallis test were used for statistical analysis based on data type.@*Results@#Compared with the model group and the negative control group, the TGF-β1 siRNA treatment group had significantly inhibited mRNA and protein expression of PDGF-BB and PDGF-βR (F = 24.785 and 22.92, P < 0.01), as well as significantly inhibited expression of p-ERK1/2 (P < 0.05).@*Conclusion@#Targeted TGF-β1 siRNA can effectively downregulate the expression of PDGF-BB, PDGF-βR, and p-ERK1/2 in liver tissue and thus help to improve hepatic fibrosis.
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ObjectiveTo explore the inductive action of docosapentenoic acid(DPA) on neurite outgrowth in PC12 cells in vitro.MethodsNeurite outgrowth in PC12 cells was examined after the treatment with different concentration of DPA using Motic Zamges Plus software mapping cell image system.Western blot was performed to detect the expression of β Ⅲ-tubulin regulated protein kinase,a neuronal marker as well as ERK and protein kinase B (Akt) phosphorylation.ResultsPC12 cell neurite formation rate was increased in a concentration dependent manner in the induction of DPA,increased by 2.4% (DPA 10 μg/ml,P>0.05),18.6% (DPA 30 μg/ml,P<0.05) and 25.0% (DPA 50 μg/ml,P<0.05) compared with that in the control group.DPA promoted the expression of β Ⅲ-tubulin (P<0.05) and the phosphorylation level of ERK and Akt (P<0.05,P<0.01).ConclusionDPA promotes PC12 cell neurites growth and its mechanism may be related to the activation of ERK and Akt signaling pathways.
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AIM: To investigate the effect of berberine (Ber) on Helicobacter pylori (Hp)-induced human gastric epithelial cells (GES-1) injury and the underlying mechanism .METHODS: Berberine (5, 10 and 20 μmol/L) and PD98059 (20 μmol/L), a selective inhibitor of extracellular regulated protein kinases (ERK)1/2 signaling pathway, were added to Hp-infected GES-1 cells.The cell activity and apoptosis, the levels of interleukin (IL)-1βand IL-8, lactic dehydrogenase ( LDH) activity and the protein levels of Bax , Bcl-2 and p-ERK1/2 in the GES-1 cells were determined by MTT assay, flow cytometry, ELISA, colorimetry and Western blot, respectively.RESULTS: Compared with control group, Hp significantly inhibited the cell activity , increased the apoptotic rate , LDH activity, IL-1βand IL-8 levels, the Bax and p-ERK1/2 protein levels but decreased the Bcl-2 protein level in GES-1 cells (P<0.05).However, these effects of Hp were reversed by berberine at medium-dose and high-dose, as compared with the Hp-infected GES-1 cells ( P<0.05).Moreover, the protective effects of berberine were significantly enhanced by the co-incubation of berberine with PD98059, as compared with the berberine at higher dose (P<0.05).CONCLUSION:Berberine may attenuate Hp-in-duced human gastric epithelial GES-1 cells injury by anti-inflammation, promoting cell growth and anti-apoptosis via the in-hibition of ERK1/2 signaling pathway .
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ABSTRACT:Objective To explore role of U0126,the specific inhibitor of ERK signaling pathway,in early brain injury (EBI)and the autophagy of nerve cells in hippocampus area in subarachnoid hemorrhage (SAH). Methods A total of 48 male adult SD rats were randomly divided into control group,SAH group,DMSO+SAH group,and U0126+SAH group,with 12 in each.We established SAH rat model by the puncture of internal carotid artery.The same amount of saline water,DMSO and U0126 solution of 0.5 mL per rat was injected respectively into the rats of different groups 30 min before modeling.The rats were killed at 24 h.To measure brain water content by Wet and dry method after 24 h,the morphological changes of hippocampus CA1 neural cells were observed by microscopy;the expression levels of ERK,Beclin-1 and LC3 were detected by using immunohistochemical method. Results Compared with that in sham group,brain water content increased obviously in SAH model group.The density of surviving neurons in SAH group was significantly lower than that in control group (P<0 .0 5 ).ERK signaling pathway was activated obviously,the expressions of Beclin 1 and LC3-Ⅱ were significantly higher than those in control group (P<0.05).Compared with SAH model group,in U0126 group brain water content increased obviously.Compared with those in SAH group,the density of surviving neurons was significantly lower (P<0.05), ERK signaling pathway was suppressed,the expressions of Beclin-1 and LC3-Ⅱ were significantly lower (P<0.05). Conclusion The U0126,the ERK signaling pathway inhibitor,can inhibit neuron autophagy and increase EBR of SAH.
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Aim To investigate whether Hcy influenced the intestinal mucosal permeability by regulating MEK-ERK-MLCK pathway. Methods SD rats were divided into 4 groups:normal group, normal+Hcy group, TN-BS/ethanol group, TNBS/ethanol+Hcy group. Experi-mental colitis model with hyperhomocystinemia was es-tablished in rats with intracolonic administration of TN-BS and subcutaneous injection of Hcy. The colonic mucosal tissue was collected for histopathological exam-ination and activity of myeloperoxidase ( MPO ) . The protein expression of MLCK, p-MLCK, MEK, ERK and p-ERK in intestinal mucosal tissues was examined by Western blot method. The mRNA expression of ML-CK was examined by RT-qPCR method. Result Com-pared with the normal group and TNBS group, the DAI and HI scores and the MPO activity were increased in TNBS/ethanol+Hcy group ( P <0. 01 ) . Western blot and RT-qPCR showed that expression of MLCK, p-ML-CK, MEK, ERK and p-ERK increased in small intes-tine in TNBS/ethanol+Hcy group. Conclusion Hcy can increase intestinal permeability in TNBS-induced colitis rats by regulating the expression of MEK-ERK-MLCK signal pathway.
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Objective To observe the changes of the cells of human hepatocellular carcinoma (HepG2)using RNA for silencing the expression of COMMD7 gene,and investigate related mechanism of COMMD7 gene promoting HepG2 proliferation.Methods COMMD7 gene shRNA was designed and constructed into COMMD7-shRNA plasmid.HepG2 cells were divided into the HepG2 group,control-shRNA group (empty vectors were infected) and COMMD7-shRNA group (positive vectors were infected).Cells shapes were observed by fluorescence microscope after infecting.The expression of COMMD7 and expression and phosphosylation of extracellular regulated protein kinase1/2 (ERK1/2) and MEK1/2 protein were measured by Western blot.The cell vitality was measured by cholecystokinin octapeptide (CCK-8),and the apoptosis of cell was detected by flow cytometry.The measurement data with normal distribution were presented as (x) ± s.The comparisons among groups were evaluated with the one-way ANOVA,and pairwise comparison was analyzed by the LSD-t test.Results The cells were oval or spindle shapes and displayed green fluorescent after infected successfully.The results of Western blot showed that the relative quantitative expression of COMMD7 protein in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.90 ±0.18,1.03 ±0.05 and 0.23 ±0.03,respectively,with a significant difference among the 3 groups (F =152.08,P ＜ 0.05),and the expression of COMMD7 protein in the COMMD7-shRNA group was significantly lower than those in the other 2 groups (t =20.74,21.16,P ＜ 0.05).The results of CCK-8 showed that the scores of the HepG2 vitality in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 1.193 ±0.024,1.225 ±0.034 and 1.147 ±0.021,respectively,with a significant difference among the 3 groups (F =6.90,P ＜ 0.05),and the HepG2 vitality in the COMMD7-shRNA group was significantly lower than those in the other 2 groups (t =3.53,3.69,P ＜ 0.05).The results of flow cytometry showed that the apoptosis rate of HepG2 in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 6.1％ ± 0.3％,7.8％ ± 0.5％ and 20.9％ ± 1.4％,showing a significant difference among the 3 groups (F =270.80,P ＜0.05),and the apoptosis rate of HepG2 in the COMMD7-shRNA group was significant higher than those in the other 2 groups (t =21.77,19.36,P ＜0.05).The results of Western blot showed that the relative quantitative expression of phosphorylation (p)-ERK1/2 and p-MEK1/2 in the HepG2 group,control-shRNA group and COMMD7-shRNA group were 0.932 ±0.046,0.945 ±0.017,0.553 ±0.052 and 0.452 ±0.031,0.468±0.027,0.263 ± 0.022,respectively,showing significant differences among the 3 groups (F =93.61,49.16,P ＜ 0.05),and the relative quantitative expression of p-ERK1/2 and p-MEK1/2 in the COMMD7-shRNA group were significantly lower than those in the other 2 groups (t =11.94,12.17,9.33,8.65,P ＜ 0.05).Conclusions COMMD7 gene can promote HepG2 proliferation via activating ERK/mitogen-activated protein kinase (MAPK) signaling pathway,and its mechanism may be promoting the phosphorylation of expression of ERK1/2 and MEK1/2.
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Objective To explore the effects of extracellular regulated protein kinase 1/2 (ERK1/2) signal pathway on cardiomyocyte apoptosis and tumor necrosis factor-α (TNF-α) expression at different glucose-lowing rates,and the influence of glucose-lowing rate on cardiomyocyte injury and inflammatory secretion function,as well as its mechanism.Methods Cardiomyocytes of Wistar neonate rat were maintained in medium supplemented with 25 mmol/L glucose for 72 h.Then the medium was changed to different concentrations of glucose and all cells were divided into five groups.Group A was control group whose medium supplemented with 25 mmol/L glucose.Medium of group B,C,D,E was supplemented with 20,15,10,5 mmol/L glucose (glucose-lowing rate was 5,10,15,20 mmol/L) respectively.Survival rate of cardiomyocyte was measured by CCK8 kit.Cardiomyocyte apoptosis was measured by flow cytometry instrument and laser confocal microscope after Annexin V-PI.TNF-α was measured by ELISA.ERK1/2 protein and phosphorylation were measured by Western blot.Cardiomyocyte apoptosis and TNF-α levels were measured again after U0126 was added.Results At the same time point,along with the glucose-lowing rate increased,survival rate of cardiomyocyte in group A was increased and those in group C,D,E were decreased (P＜ 0.05).TNF-α concentration was increased in group B,C,D and decreased in group E.After 24 h,apoptosis rate decreased in group B and increased in group C,D,E (P＜0.05).ERK1/2 phosphorylation level increased in group B,D,and E(P＜0.05).The ERK1/2 phosphorylation level in group B was the lowest.After U0126 was added,survival rates of cardiomyocyte in all groups were increased (P＜0.01) while TNF-α concentrations were decreased (P＜0.05).In every group,survival rate of eardiomyocyte after 48 h was lower than that after 3 h and 24 h,while TNF-α concentration was higher (P＜0.05).Conclusion Influence of glucose-lowering rate for cardiomyocyte apoptosis and TNF-o is caused by ERK1/2 pathway.In the glucose-lowering course,ERK1/2 pathway promotes cardiomyocytes apoptosis and TNF-α secretion is related with not only osmotic pressure,but also ERK1/2 signal pathway activation as well.
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Objective: To explore the regulative role of extracellular regulated protein kinase-5 (ERK5)/Bcl-2 interacting mediator of cell death (Bim) pathway in hypothermal stimulation induced neonatal rat’s cardiomyocytes (CMs) damage and apoptosis. Methods: CMs were cultured for hypothermal stimulation and the speciifc siRNA was used to down-regulate the ERK5 or Bim in CMs. The cell apoptosis was detected by lfow cytometry, protein expression was examined by Western blot analysis, the intracellular Ca2+, reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were evaluated by lfuorescent labeling and lfow cytometry. Results: In hypothermal stimulated CMs, ERK5 siRNA could promote Bim protein expression, but Bim siRNA could not inlfuence ERK5, while attenuated p-ERK5 expression. ERK5 siRNA induced higher apoptosis rate, while Bim siRNA could decrease such effect. ERK5 siRNA increased the intracellular Ca2+overloading, ROS activation andΔΨm damage, while Bim siRNA played the role to against those effects in hypothermal stimulated CMs. Conclusion: Our study revealed that ERK5/Bim pathway played the important regulative roll in hypothermal stimulation induced neonatal rat’s CMs damage and apoptosis.
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Objective: To investigate the role of extracellular signal-regulated protein kinase (ERK) in estrogen-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7 and the related mechanisms. Methods: Estrogen receptor-positive breast cancer cell line MCF-7 was used in our study. The effects of 17β-E2 on the proliferation of MCF-7 cells was investigated by MTT assay to determine the optimal concentration of 17β-E2 for the following experiment. The effect of PD98059 on 17β-E2-induced proliferation of MCF-7 cells was measured by MTT assay to determine the intermediate concentration of PD98059. The cell cycle was analyzed by flow cytometry and telomerase activity was determined by Telomerase repeat amplification protocol PCR (TRAP-PCR) silver staining. The expression of wild-type p53 and phosphorylated ERK1/2 protein was determined by Western blotting and the expression of wild-type p53 mRNA was detected by RT-PCR. Results: ERK phosphorylation inhibitor PD98059 inhibited the proliferation of MCF-7 cells treated with 17β-E2 in a time- and dose-dependent manner(P<0.01). The intermediate concentrations of PD98059 were 89.28 μmol/L for 24 h, 39.81 μmol/L for 48 h and 21.87 μmol/L for 72 h. Treatment with 20 μmol/L PD98059 for 48 h reversed the promoting effect of 17β-estradiol on the cell cycle transformation of MCF-7, increasing the number of G1 phase cells and decreasing the number of S and M phase cells (P<0.01), inhibited the enhancing effect of 17β-E2 on the telomerase activity of MCF-7 cells(P<0.05), increased the protein expression level and genetic transcription of wild-type p53 (P<0.01), and decreased the expression of p-ERK1/2 protein(P <0.01). Conclusion: ERK plays an important role in 17β-E2-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7, which might be related to the changes of genetic transcription of wild type p53 and telomerase activity.
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Objective:To investigate the role of extracellular signal-regulated protein kinase(ERK) in estrogen-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7 and the related mechanisms.Methods: Estrogen receptor-positive breast cancer cell line MCF-7 was used in our study.The effects of 17?-E2 on the proliferation of MCF-7 cells was investigated by MTT assay to determine the optimal concentration of 17?-E2 for the following experiment.The effect of PD98059 on 17?-E2-induced proliferation of MCF-7 cells was measured by MTT assay to determine the intermediate concentration of PD98059.The cell cycle was analyzed by flow cytometry and telomerase activity was determined by Telomerase repeat amplification protocol PCR(TRAP-PCR) silver staining.The expression of wild-type p53 and phosphorylated ERK1/2 protein was determined by Western blotting and the expression of wild-type p53 mRNA was detected by RT-PCR.Results: ERK phosphorylation inhibitor PD98059 inhibited the proliferation of MCF-7 cells treated with 17?-E2 in a time-and dose-dependent manner(P