MiRNA-145-5p inhibits gastric cancer progression via the serpin family E member 1-extracellular signal-regulated kinase-1/2 axis

BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.

Role of extracellular signal-regulated kinase 1/2 in cigarette smoke-induced mucus hypersecretion in a rat model

Background Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 （ERK 1/2） in airway mucus hypersecretion induced by cigarette smoke in rats. Methods A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks. Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 （p-ERK 1/2） were detected by RT-PCR, immunohistochemistry and Western blotting. Results Cigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke （P 〈0.05）. Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein （P 〈0.05）. Conclusions Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1 /2 pathway and cyclin D1 expression

Aim： To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods： The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase （ERK）1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor （GR） antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results： Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion： Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

BACKGROUND： The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE： To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN： Randomized controlled study. SETTING： Department of Anatomy, Sun Yat-Sen University. MATERIALS： A total of 60 healthy adult male Wistar rats weighing （250±10） g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS： The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups： normal group （n = 6）, sham operation group （n = 18）, model group （n = 18）, and electroacupuncture group （n = 18）. Middle cerebral artery occlusion （MCAO） was performed in the model group and electroacupuncture group. Zea Longa＇s grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between l and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery （CCA） was isolated, and the external carotid artery （ECA） was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture （People＇s Publishing House; 1997; First Edition）. The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves （sparse waves were 30 Hz, dense waves were 100 Hz）, with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES： Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS： All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group （P 〉 0.05）. However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment （P 〈 0.05）. CONCLUSION： Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinasesl/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim:To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2(ERK1/ 2),c-Jun N-terminal kinases(JNK)and p38 mitogen-activated protein kinases(MAPK)in response to heat stress in the cryptorchid testis,and to investigate a possible relation to Sertoli cell dedifferentiation.Methods:Immunohis- tochemistry and western blot were used to examine the expression and activation of ERK1/2,p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism.Results:The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis.Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK.Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis.Changes in the spatiotemporal expression of cytokeratin 18(CK18),a marker of immature or undifferentiated Sertoli cells,were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Conclusion:The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Sphingosine-1-Phosphate Protects Against the Development of Cardiac Remodeling via Sphingosine Kinase 2 and the S1PR2/ERK Pathway

Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac protection.Our previous work found that sphingosine-1-phosphate(S1P)could ameliorate cardiac hypertrophy.In this study,we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.Methods:Eight-week-old male C57BL/6 mice were randomly divided into a sham,transverse aortic constriction(TAC)or a TAC+S1P treatment group.Results:We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease inα-smooth muscle actin(α-SMA)and collagen type I(COL I)expression compared with the TAC group.We also found that one of the key S1P enzymes,sphingosine kinase 2(SphK2),which was mainly distributed in cytoblasts,was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro.In addition,our in vitro results showed that S1P treatment activated extracellular regulated protein kinases(ERK)phosphorylation mainly through the S1P receptor 2(S1PR2)and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.Conclusion:These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart.This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.

IL-1β通过激活ERK1/2信号通路抑制人脐带间充质干细胞CD200表达抑制巨噬细胞M2极化

目的探究白细胞介素1β(IL-1β)调控人脐带间充质干细胞CD200表达及其对巨噬细胞极化的影响及作用机制。方法无血清培养基分离培养获得人脐带间充质干细胞(hUC-MSC),形态学观察及流式细胞术检测CD73、CD90、CD105、CD14、CD34、CD45、人类白细胞抗原DR(HLA-DR)的表达,确定间充质干细胞属性;20 ng/mL IL-1β处理hUC-MSC 24 h,流式细胞术检测CD200阳性细胞率,实时定量PCR和Western blot法检测CD200 mRNA和蛋白表达水平;佛波酯(PMA)诱导THP-1巨噬细胞活化,并与IL-1β处理感染CD200过表达慢病毒的hUC-MSC共培养,流式细胞术检测CD11c和CD206阳性细胞比例;IL-1β联合细胞外信号调节激酶1/2(ERK1/2)特异性抑制剂PD98059处理hUC-MSC,Western blot法检测细胞丝裂原激活蛋白激酶(MAPK)信号分子与CD200的表达。结果IL-1β显著下调hUC-MSC CD200蛋白表达与CD200阳性细胞率;过表达CD200显著上调hUC-MSC CD200表达,且CD200过表达hUC-MSC提高巨噬细胞CD206阳性细胞比率;IL-1β激活hUC-MSC的ERK1/2信号通路,PD98059上调IL-1β处理后hUC-MSC中CD200的蛋白表达。结论IL-1β通过激活ERK1/2信号通路抑制CD200的表达,进而抑制hUC-MSC对巨噬细胞向M2型极化的促进作用。

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

SOX7靶向ERK1/2/PD-L1通路抑制结直肠癌血管生成

目的探讨性别决定区Y框蛋白7(SOX7)对结直肠癌血管生成的影响及潜在作用机制。方法应用免疫荧光检测结直肠癌患者组织样本中SOX7表达水平,之后通过裸鼠、转染SOX7 mimic的人结直肠癌细胞系SW480细胞和人脐静脉内皮细胞(HUVEC)共培养进一步研究。用Western-blot验证SOX7与ERK1/2/PD-L1对结直肠癌细胞的相关蛋白表达的影响。用CCK8检测SOX7与ERK1/2/PD-L1对HUVEC增殖的影响。通过体外内皮细胞成管实验测定SOX7与ERK1/2/PD-L1对肿瘤血管生成的影响。结果SOX7在人结直肠癌组织中表达被抑制(P<0.01),同时SOX7的过表达抑制了小鼠体内肿瘤生长(P<0.01)。SW480细胞中SOX7的过表达抑制了ERK1/2、c-Jun的表达,并在ERK1/2的激动剂Senkyunolide I的作用下上调了SW480细胞的ERK1/2、c-Jun蛋白表达(P<0.01),逆转了SOX7对SW480细胞中ERK1/2、c-Jun蛋白表达的影响(P<0.01)。HUVEC中SOX7抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,Senkyunolide I上调了HUVEC的PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,并逆转了SOX7对HUVEC中上述相关蛋白表达的影响(P<0.01)。PD-1/PD-L1 Inhibitor 3抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,SOX7过表达在PD-1/PD-L1 Inhibitor 3的影响下并没有表现出抑制作用。CCK8实验结果显示SOX7过表达显著抑制了HUVEC的增殖能力,Senkyunolide I作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显上升,PD-1/PD-L1 Inhibitor 3作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显下降,以上均有明显统计学差异(P<0.01)。成管实验结果显示SOX7过表达抑制了HUVEC的血管生成,Senkyunolide I强烈加速了血管生成,而PD-1/PD-L1 Inhibitor 3血管生成则被显著抑制,以上均有明显统计学差异(P<0.01)。结论SOX7通过ERK1/2/PD-L1通路抑制结直肠肿瘤的增殖和血管生成,SOX7可能是晚期CRC患者临床治疗中潜在的抗血管生成靶点。

Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF-induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes

Objective Intravenous administration of basic fibroblast growth factor （bFGF） is effective to reduce the volume of cerebral infract due to ischemia. This study was designed to investigate the molecular mechanism, especially the signal transduction pathways, involved in this protective role of bFGF. Methods Anoxia-reoxygenation treated atrocytes were used to study the role of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase （MAPK/ERK kinase, MEK）-ERK signaling pathway after exogenous bFGF administration by Western blot. Electrophoretic mobile shift assay was used to detect the binding activity of early growth response factor-1 （Egr-1）, an important transcription factor for endogenous bFGF. Results bFGF could protect some signal transduction proteins from the oxygen-derived free radicals induced degradation. ERK1/2 was activated and involved in Egr-1 binding activity enhancement induced by exogenous bFGF. Conclusion MEK-ERK MAPK cascade may be an important signal transduction pathway contributed to bFGF induced enhancement of Egr-1 binding activity in anoxia-reoxygenation injured astrocytes.

补骨脂酚通过抑制ERK1/2磷酸化并上调ABCA1表达减少巨噬细胞源性泡沫细胞形成

[目的]探讨补骨脂酚(BAK)对巨噬细胞源性泡沫细胞脂质蓄积的影响及机制。[方法]MTT筛选BAK对泡沫细胞药物毒性浓度;油红O染色、NBD胆固醇、Dil-ox-LDL检测泡沫细胞内脂质蓄积情况;使用RT-qPCR和Western blot检测mRNA和蛋白表达。[结果]BAK可以促进胆固醇流出并减少泡沫细胞内脂质蓄积。BAK可以上调三磷酸腺苷结合盒转运体A1(ABCA1)的mRNA和蛋白表达水平,同时可下调细胞外信号调节激酶1/2(ERK1/2)磷酸化水平。使用ERK1/2激动剂Ro 67-7476处理发现,与BAK处理组相比,加入Ro 67-7476处理后ABCA1蛋白表达下降。[结论]BAK通过抑制ERK1/2的磷酸化,上调ABCA1的表达并促进胆固醇的流出,减少泡沫细胞中的脂质蓄积,从而抑制泡沫细胞的形成。

Cross-Talk between Extracellular S1P/S1P2 and P42/44 MAPK in bcr/abl Positive Chronic Myeloid Leukemia Cells

Objective： BCR/ABL oncoprotein-expression is associated with uncontrolled cell growth. Sphingosine kinase 1 （SPK1） regulates the production of sphingosine 1-phosphate （S1P）, a key lipid signal molecular in cell proliferation and survival. The objective of this study was to elucidate the roles of S1P and its receptors in bcr/abl positive chronic myeloid leukemia （CML） cells. Methods： The expressions of SIP receptors： S1P1, S1P2 and S1P3 in CML cells were detected by RT-PCR. SPK1 expression, activity and extracellular S1P were determined in ECV304 and HL-60 cells which were transfected with bcr/abl gene. To elucidate the relationship between the BCR/ABL, ERK/MAPK （extracellular signal-regulated kinase/mitogen-activated protein kinase）, SPK/S 1P and S 1P/S 1 P2 signal pathways, bcr/abl positive CML cell line K562 was treated with STI571, PD98059, N,N-dimethyl sphingosine （DMS） and JTE-013. Results： Retrovirus-mediated overexpression of bcr/abl gene in ECV304 and HL-60 cells resulted in upregulation of the expression, activity of SPK1 and increase of the secretion of SIP, whereas treatment of STI571 and PD98059 decreased the BCR/ABL-induced S1P secretion. Treatment of DMS reduced S1P secretion and P42/44MAPK phosphorylation. S1P2-selective antagonist JTE-013 could also decrease P42/44MAPK phosphorylation. Conclusion： These results suggest that BCR/ABL up-regulates extracellular sphingosine 1-phosphate through sphingosine kinase 1 and there is cross-talk between SPK1/S1P/S1P2 and P42/44MAPK in bcr/abl positive CML cells.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

Glucocorticoid modulation of extracellular signal-regulated protein kinase 1/2 and p38 in human ovarian cancer HO-8910 cells

Objective To investigate the signaling pathway through testing the effects of dexamethasone (Dex) on the activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 kinase (p38) in HO-8910 cells.Methods Activation of the ERK1/2 and p38 was detected by Western blotting using the antibodies against the total ERK1/2 and p38 mitogen-activated protein kinases (MAPKs) protein and the phosphorylated forms of them. Results Dex could suppress the activation of ERK1/2, while enhance the activation of p38 rapidly and strongly in a dose- and time- dependent manner. Neither effect could be blocked by RU486, the antagonist of glucocorticoid receptor (GR).Conclusion Dex has rapid effects on the activation of ERK1/2 and p38, and these effects are not mediated by GR.

子宫内膜癌中ERK1/2信号转导通路与雌、孕激素受体的相关性

目的检测丝裂原活化蛋白激酶(MAPK)信号通路中细胞外信号调控激酶1/2(ERK1/2)在子宫内膜癌中的表达,探讨其与雌激素受体(ER)和孕激素受体(PR)表达间的相关性。方法用免疫组化方法(SP法)检测30例子宫内膜癌石蜡标本中ERK1/2以及ER和PR的表达;同法检测20例正常子宫内膜、13例增生过长子宫内膜石蜡标本中ERK1/2的表达;对标本的染色情况做半定量分析。结果ERK1/2在正常子宫内膜、增生过长子宫内膜和子宫内膜癌中的表达水平比较,差异无统计学意义(P>0.05)。磷酸化的ERK1/2(p-ERK1/2)在ER阳性与ER阴性子宫内膜癌中的高表达率比较(76.5%vs30.8%),差异有统计学意义(P<0.05);且p-ERK1/2的高表达率与ER的表达水平呈正相关(r=0.457,P<0.05);p-ERK1/2的高表达率与PR的表达水平无相关性(P>0.05)。结论ERK1/2信号转导通路与子宫内膜恶变过程无相关性,其活化与ER表达水平呈正相关。ER可能通过ERK1/2信号通路发挥其在子宫内膜癌中的调控作用。

人参皂苷Rg1对局灶性脑缺血再灌注损伤大鼠海马p-ERK1/2与p-JNK表达的影响

目的探讨人参皂苷Rg1抗脑缺血再灌注(ischemia reperfusion,I/R)损伤大鼠海马神经元凋亡的可能机制。方法成年健康雌性SD大鼠120只随机分为脑缺血再灌注模型组(模型组)、人参皂苷Rg1低(10mg/kg)、中(20mg/kg)、高剂量(40mg/kg)组及假手术组,每组18只。各组均腹腔注射给药,假手术组及模型组腹腔注射等量生理盐水,每天1次,连续7天,末次给药后30min,大鼠右侧大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)2h再灌注24h制备I/R模型。以LongaEZ法评定神经功能,尼氏染色、TUNEL染色观察海马锥体神经细胞的损伤情况,并计算神经细胞凋亡率。采用Westernblot法检测细胞外信号调节蛋白激酶1/2(extracellular signal-regulated kinase1/2,ERK1/2)及磷酸化细胞外信号调节蛋白激酶1/2(phosphorylated extracellular signal-regulated kinase1/2,p-ERK1/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinases,JNK)及磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinases,p-JNK)表达。结果与假手术组比较,模型组神经功能评分、细胞凋亡率、p-JNK、p-ERK1/2蛋白表达升高(P<0.05,P<0.01),锥体细胞存活数减少(P<0.01);与模型组比较,人参皂苷Rg1各剂量组神经功能评分、细胞凋亡率降低(P<0.05,P<0.01),人参皂苷Rg1中、高剂量组大鼠锥体细胞存活数增加,海马CA1区p-JNK蛋白表达降低,p-ERK1/2表达升高(P<0.05,P<0.01)。假手术组海马CA1区有3-4层锥体细胞,排列整齐、紧密,高倍镜下细胞核大而圆,有1～2个核仁。脑组织缺血损伤后,海马区神经细胞受损严重,CA1区失去正常结构,细胞排列散乱,细胞数量减少。部分神经元皱缩,核固缩、深染,呈三角形、长条形、梭形或不规则形,核染色聚集,核仁不清晰。与人参皂苷Rg1低剂量组比较,人参皂苷Rg1中、高剂量组神经功能评分、细胞凋亡率及p-JNK蛋白表达降低(P<0.05,P<0.01),锥体细胞存活数增加,p-ERK1/2表达升高(P<0.05,P<0.01)。人参皂苷Rg1中、高剂量能够改善缺血神经细胞形态,减少神经细胞的丢失,其中,高剂量组作用强于低剂量组。JNK蛋白条带分为两个亚带,JNK1是分子量为46kD的蛋白,JNK2分子量为54kD。ERK蛋白条带也分为两个亚带,ERK1是分子量为44kD的蛋白,ERK2分子量为42kD的蛋白。结论人参皂苷Rg1对I/R大鼠的保护作用与抑制海马神经元凋亡,调节p-JNK及p-ERK1/2表达水平有关。

ERK信号转导通路在CXCL12促进子宫内膜癌细胞增殖和侵袭中的作用

目的:探讨趋化因子CXCL12及其受体CXCR4(CXCL12/CXCR4)生物学轴通过细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)信号转导通路发挥促子宫内膜癌细胞增殖和侵袭的作用。方法:应用外源性CXCL12处理子宫内膜癌Ishikawa细胞株,通过Western blotting检测不同时间位点ERK1/2的磷酸化水平和Survivin蛋白的表达;通过ELISA检测细胞培养上清液中MMP-2的分泌水平。同时分析AMD3100和PD98059对细胞ERK1/2磷酸化水平、Survivin蛋白水平和MMP-2分泌水平的影响。结果:外源性CXCL12刺激后,可迅速上调ERK1/2的磷酸化水平(t=0.887,P<0.01),促进Survivin蛋白和MMP-2蛋白的表达(t=0.861,P<0.01;t=0.297,P<0.01),且三者均呈时间依赖性。PD98059和AMD3100均能明显抑制外源性CXCL12诱导后ERK1/2的磷酸化水平,而且在两者共同作用下,能完全抑制ERK1/2的磷酸化水平,阻断ERK通路的激活,下调Survivin蛋白和MMP-2蛋白的表达。结论:CXCL12/CXCR4生物学轴通过激活ERK通路上调Survivin蛋白和MMP-2蛋白表达,从而引发Ishikawa细胞一系列增殖和侵袭的生物学效应。

针康法对脑缺血大鼠神经功能和细胞外信号调节激酶1/2信号通路的影响

目的探讨针康法对脑缺血大鼠神经功能预后和细胞外信号调节激酶1/2(ERK1/2)信号通路的影响。方法 90只雄性Sprague-Dawley大鼠随机分入假手术组(n=18)、模型组(n=18)、针刺组(n=18)、康复组(n=18)及针康组(n=18),再分为术后3 d、7d、14 d三个亚组(n=6)。线栓法制备永久性局灶性脑缺血模型。假手术组和模型组不进行治疗,针刺组采用头穴丛刺针法治疗,康复组给予电动跑台训练,针康组采用针康法治疗。各时间点,采用改良神经损害严重程度评分进行评定,Western blotting法检测缺血半暗区皮层ERK1/2、p-ERK1/2蛋白表达。结果术后各时间点,与模型组比较,各治疗组神经缺损评分降低(P<0.05);术后7 d、14 d,与针刺组、康复组比较,针康组评分降低(P<0.05)。术后各时间点,与模型组比较,各治疗组p-ERK1/2蛋白表达增加(P<0.05),(p-ERK1/2)/(ERK1/2)增加(P<0.05);与针刺组、康复组比较,针康组p-ERK1/2蛋白表达升高(P<0.05),(p-ERK1/2)/(ERK1/2)增加(P<0.05)。结论针康法治疗可进一步改善脑缺血大鼠神经行为,可能与ERK1/2信号通路激活有关。