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【Objective】 To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. 【Methods】 The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5 (si-Annexin A5) in human glioma cell line (U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. 【Results】 Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively (P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased, and the tumor grade and positive rate were significantly positively correlated (r=1.000, P=0.000). The cell viability of U251 cells in the si-Annexin A5 group after 48 h and 72 h of culture was significantly reduced by 29.46% and 40.43%, respectively, compared with that in the control group (P<0.05). Compared with the control group, in the si-Annexin A5 group the colony formation rate was reduced by 68.58%, while the apoptosis rate was increased by 24.41 times (P<0.05); the cell migration rate and invasion rate were reduced by 65.35% and 68.80% (P<0.05). The protein expression of p-Raf, p-MEK1/2, p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%, 70.37%, 60.26% and 54.95%, respectively, and that of E-Cadherin was increased by 3.58 times (P<0.05). 【Conclusion】 Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.
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Objective: To explore the mechanism of the intervention of Xinkeshu Tablets (XKST) on atherosclerosis (AS) and provide reference for the secondary development and clinical application of XKST. Methods: The integrated pharmacology platform was used to predict the key targets and pathways of the intervention of XKST on AS and its molecular mechanism was also explored. Results: In the integrative analysis of heterogeneous network of “TCM-component-target-pathway”, 80 relevant effective ingredients were found, including B4GALT4, B4GALT2, PRKCD, GCK, GNB1, and other key targets; Endocrine system, thyroid hormone signaling pathway, nervous system, estrogen signaling pathway, and chemokine signaling pathway were key pathways related with its anti-atherosclerosis. Conclusion: According to the analysis and prediction of the enrichment information, the effect of XKST on common regulating PI3K/Akt/eNOS and Raf/MEK/ERK signaling pathway and protecting vascular endothelial cells is first prompted, thus achieving the intervention in AS.
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OBJECTIVE: To explore the effect of amarogentin on the induction of liver cancer cell line Huh-7 apoptosis and the regulation of PKA/C. METHODS: Liver cancer cell line Huh-7 were divided into 4 groups, control, amarogentin, amarogentin+H89 and amarogentin+H7 group (n=8). The cells were treated with amarogentin (30 mmolL-1) for 6 h besides control group. The amarogentin+H89 and amarogentin+H7 group cells were treated with corresponding compounds at the last 3 h (H89 at 10 mmolL-1 and H7 10 mmolL-1). The apoptotic proteins and MEK/ERK signaling pathway related proteins were detected by Western blotting. The Caspase 3 and Caspase 9 were also be assayed by immune-cytochemistry. At the meaning time, the apoptosis state was assayed by DAPI. RESULTS: The results showed that the Bax, Caspase 3 and Caspase 9 were increased (P>0.05) while the Bcl2 were decreased (P>0.05) expressed greatly after the medication of amarogentin when compared with control. At the same time, the expression of Ras, Rsf, MEK and ERK1/2 were increased (P>0.05) greatly after the medication of amarogentin when compared with control. Those abnormalities were normalized greatly by the medication of H89 (P>0.05) but not H7(P>0.05). CONCLUSION: Amarogentin could promote the apoptosis of liver cancer cell line Huh-7 which is mediated by PKA.
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Objective: To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol. Methods: SW620 cell lines were divided into 5 groups, namely, control group, PD98059 group, low-dose resveratrol group, mid-dose resveratrol group and high-dose resveratrol group. The inhibition rate of cell proliferation was detected by MTT method. The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by real-time PCR and Western blotting. Results: Compared with control group, the proliferation of cells treated with resveratrol was significantly inhibited. In the case of apoptotic molecules, the expression of Bax, Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dose-dependent manner. In the case of molecules in MEK/ERK signaling pathway, the expression of Ras, Raf, MEK and ERK1/2 was decreased significantly in resveratrol groups with a dose-dependent manner. Conclusions: PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.
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AIM:To investigate the myocardial protective effect of endometrial stem cell ( EnSC)-derived cyto-kine cocktail ( EdCC) on myocardial ischemic reperfusion injury and the MEK-ERK signaling pathway.METHODS: A mouse model of myocardial ischemic reperfusion injury was established.Infarct area, cell apoptosis, and expression of cleaved caspase-3 and phosphorylatied ERK1/2 were determined by TTC/Evans blue staining, TUNEL assay and Western blot, respectively.RESULTS:The mesenchymal characteristics were observed in the EnSCs with expressing CD90 and in absence of CD34 and CD45.EdCC contained (6 811 ±312) ng/g epidermal growth factor (EGF) protein.The phospho-rylation of ERK1/2 markedly increased after injection of EdCC, but was abolished by MEK1 inhibitor PD98059 ( 5 mg/kg) .EdCC decreased the infarct area and apoptotic cell number in the border zone and inhibited caspase-3 activation. However, the effects were abolished by MEK1 specific inhibitor PD98059.EGF did not decrease the infarct area, but the EGF receptor antagonist AG-1487 (6 mg/kg) partly abolished the myocardial protective effect of EdCC.CONCLUSION:EdCC protects the myocardium from ischemic reperfusion injury via activating MEK1-ERK signaling pathway, indicating an essential role in the transmission of stem cell therapy from the cell transplantation to cytokine based strategy.
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OBJECTIVE:To study the effects and mechanism of propranolol on the myocardial abnormal electrophysiology sta-tion in diabetic model rats. METHODS:SD rats were randomly divided into normal control(normal saline)group,diabetic(nor-mal saline)group,PD98059(ERK inhibitor,10 mg/kg)group and propranolol low-dose,medium-dose and high-dose(1,20,50 mg/kg)groups,with 8 rats in each group. Except for normal control group,rats were given alloxan(20 mg/kg)intravenously via tail vein to induce diabetic model. They were given relevant medicine intragastrically,once a day,for consecutive 42 days. The car-diac index,electrocardiogram and action potential durations (APD) of rats were analyzed;the expression of TNF-α,IL-2,IL-6 and IL-10 protein in serum were detected,and the expression of Ras,Raf,ERK kinase(MEK)and ERK1/2 in myocardial tissue were detected. RESULTS:Compared with normal control group,cardiac index increased in diabetes group;heart rate decreased;QT interval and APD were prolonged;the relative expression of TNF-α,IL-2,IL-6,IL-10,Ras,Raf,MEK and ERK1/2 protein increased (P<0.01). Compared with diabetes group,cardiac index decreased in propranolol medium-dose and high-dose groups and PD98059 group,heart rate increased,QT interval and APD were shortened;the relative expression of TNF-α,IL-2,IL-6, IL-10,Ras,Raf,MEK and ERK1/2 protein decreased(P<0.05 or P<0.01). CONCLUSIONS:Propranolol can improve myocar-dial abnormal electrophysiology station of diabetic model rats by down-regulating inflammatory reactions in serum and inhibiting the activation of MEK/ERK signaling pathway.
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OBJECTIVE@#To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol.@*METHODS@#SW620 cell lines were divided into 5 groups, namely, control group, PD98059 group, low-dose resveratrol group, mid-dose resveratrol group and high-dose resveratrol group. The inhibition rate of cell proliferation was detected by MTT method. The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by real-time PCR and Western blotting.@*RESULTS@#Compared with control group, the proliferation of cells treated with resveratrol was significantly inhibited. In the case of apoptotic molecules, the expression of Bax, Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dose-dependent manner. In the case of molecules in MEK/ERK signaling pathway, the expression of Ras, Raf, MEK and ERK1/2 was decreased significantly in resveratrol groups with a dose-dependent manner.@*CONCLUSIONS@#PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.
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Objective To investigate the mechanism of matrine in inhibition of proliferation the proliferation of human chronic myeloid leukemia (CML) K562 cells via MEK-ERK signaling pathway. Methods Western blot was used to detect the expression of MEK1, ERK1/2, Shc and SHP2 (the signal effect molecules of MEK-ERK pathway) in K562 cells. The transcription and translation of bcr-abl and target protein (bcl-xL, Cyclin D1, c-myc and p27) were detected by RT-PCR and Western blot. Results Matrine was able to significantly inhibit the phosphorylation of MEK1, ERK1/2, Shc and SHP2 in K562 cells and suppress the protein and mRNA expression of bcr-abl. Moreover, the expressions of bcl-xL, Cyclin D1 and c-myc were down-regulated significantly, while the expression level of p27 (a negative regulator of cell cycle progression) was increased markedly after matrine treatment. Conclusions Suppression of the growth of human CML K562 cells is related to the inhibition of bcr-abl-mediated MEK-ERK pathway activity. The down-regulation of phosphorylated proteins or protein kinases activity in signaling pathways might be an important molecular mechanism in control the activity of MEK-ERK pathway.
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Objective To explore the effect of inhibitor of MEK/ERK pathway on the behaviors of autistic rats .Methods Autistic rats were made by intraperitoneal injection of sodium valproate (VPA) after pregnancy for 12 .5 days .After VPA injec-tion ,pregnant rats were treated with U0126 via oral at 400 μg/kg dose per day until weaning .Young rats were divided to 4 groups :control group ,VPA group ,U0126 group ,VPA combined U0126 group .The social interaction and behaviors of young rats were e-valuated at 35 days after bornning .The levels of MEK and phosphorylated ERK in brain tissues were investigated by Western blot . Results The autistic rat mode was prepared successfully .Compared with control rats ,the rats treated with VPA showed low the social interaction ,long moving time in central area and reducing standing times .Treatment with U0126 alone didn′t change the so-cial interaction and behaviors of young rats ,but VPA combined U0126 group could improve VPA-induced autistic-like behaviors . Western blot results show that compared with the control group ,the rats treated with VPA could enhance the prefrontal cortex of rats ,the hippocampus and cerebellum in the organization of MEK and ERK phosphorylation level ;while VPA combined U0126 group could inhibit the brain tissue of MEK and ERK phosphorylation level .Conclusion U0126 can improve the model rats of au-tism disorders behavior ,the mechanism may be related to the inhibition of MEK/ERK signaling pathway in the brain .
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AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway , thus enhancing drug resistance of K 562/A02 human leukemia multidrug resistant cell line.METHODS:siRNA targeting GCS was transfected into K562/A02 cells.Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting .After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting , respectively.The viability of the cells was evaluated by CCK-8 assay.RESULTS:The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K 562/A02 cells by GCS siRNA transfection compared with negative control group .Inactivation of MEK/ERK signaling due to U0126 treatment de-creased Bcl-2 mRNA and protein levels in a concentration-dependent manner , and sensitized K562/A02 cells to adriamy-cin.CONCLUSION:GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway , thus regulating multidrug resistance of human leukemia K 562/A02 cells.