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.

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。

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.

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.

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.

microRNA125a-3p对滋养层细胞功能的调控作用及机制

目的观察microRNA125a-3p(miR-125a-3p)在滋养层细胞中的表达,探讨其对滋养层细胞增殖、侵袭和凋亡的调控及机制。方法荧光实时定量PCR检测人滋养层细胞系HTR-8/SVneo、绒癌细胞系JAR和JEG-3中miR-125a-3p的表达情况。以HTR-8/SVneo和JEG-3细胞为实验对象,分为3组:空白对照组(CK组),未做任何处理;阴性对照组(NC组),转染NC-inhibitor;实验组(inhibitor组),转染miR-125a-3p inhibitor。以Transwell、流式细胞仪、CCK8法分别检测细胞的侵袭、凋亡及增殖能力。Western blot检测Fyn蛋白表达情况及ERK1/2、STAT3磷酸化水平。荧光实时定量PCR检测Fyn mRNA水平,免疫共沉淀法检测Fyn活性水平。结果miR-125a-3p mRNA表达水平在HTR-8/SVneo、JAR和JEG-3细胞中依次降低,两两比较均有统计学差异(P<0.01)。抑制HTR-8/SVneo和JEG-3中miR-125a-3p后,细胞的凋亡水平明显降低,侵袭和增殖能力均明显升高(P<0.05);Fyn mRNA和蛋白的表达及活性水平均明显升高(P<0.05);ERK1/2及STAT3的磷酸化水平均不同程度增加(P<0.05)。结论本研究首次在滋养层细胞中检测到miR-125a-3p的表达。miR-125a-3p通过作用于Fyn和ERK1/2-STAT3信号通路可抑制滋养层细胞的增殖、侵袭,促进其凋亡。

MicroRNA-133b调节FGFR1-ERK1/2-SOX2信号通路对裸鼠肺癌NCI-H1975细胞移植瘤生长的影响

目的探讨microRNA-133b(miR-133b)对裸鼠肺癌NCI-H1975细胞移植瘤生长的抑制作用以及对成纤维细胞生长因子受体1-细胞外信号调节激酶1/2-性别决定区Y-box蛋白2信号通路(FGFR1-ERK1/2-SOX2)的影响。方法q RT-PCR检测人肺成纤维细胞、肺癌细胞株miR-133b表达。miR-133b过表达NCIH1975细胞。将NCI-H1975细胞分为对照组、mimic NC组、miR-133b mimic组、miR-133b mimic+pcDNA3.1组、miR-133b mimic+pcDNA3.1 FGFR1组。CCK-8法检测NCI-H1975细胞增殖抑制率,Transwell实验观察NCI-H1975细胞侵袭、迁移情况。复制裸鼠移植瘤模型并分组,将裸鼠分为对照组、mimic NC组、miR-133b mimic组、miR-133b mimic+AZD4547组,观察各组裸鼠肿瘤体积与重量,HE染色观察各组裸鼠肿瘤组织变化,TUNEL检测肿瘤组织细胞凋亡情况,免疫组织化学法观察裸鼠肿瘤组织Ki-67、Cyclin D1、VEGF-A的表达,Western blotting检测各组肿瘤组织FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量。结果与人肺成纤维细胞HLF-α比较,肺癌细胞株NCI-H1975、A427、NGE-1、A549中miR-133b mRNA相对表达量降低(P<0.05),其中以NCI-H1975细胞中miR-133b mRNA相对表达量最低。miR-133b mimic组miR-133b mRNA相对表达量较对照组和mimic NC组升高(P<0.05)。miR-133b可通过负调控FGFR1抑制肺癌NCIH1975细胞增殖和迁移。miR-133b mimic组移植瘤重量较对照组降低、体积缩小,miR-133b mimic+AZD4547组移植瘤重量较miR-133b mimic组降低、体积缩小(P<0.05)。miR-133b mimic组空泡样变性程度较对照组、mimic NC组减轻(P<0.05),miR-133b mimic+AZD4547组空泡样变性程度较miR-133b mimic组减轻(P<0.05)。miR-133b mimic组肿瘤组织细胞凋亡率较对照组升高(P<0.05),miR-133b mimic+AZD4547组肿瘤组织细胞凋亡率较miR-133b mimic组升高(P<0.05)。miR-133b mimic组VEGF-A、Cyclin D、Ki-67阳性细胞比例较对照组降低(P<0.05),miR-133b mimic+AZD4547组VEGF-A、Cyclin D、Ki-67阳性细胞比例较miR-133b mimic组降低(P<0.05)。miR-133b mimic组FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量较对照组降低(P<0.05),miR-133b mimic+AZD4547组FGFR1、p-ERK1/2/ERK1/2、SOX2蛋白相对表达量较miR-133b mimic组降低(P<0.05)。结论miR-133b过表达可能通过抑制FGFR1-ERK1/2-SOX2轴,抑制裸鼠肺癌NCI-H1975细胞移植瘤生长。

miR-17-5p对缺血性脑卒中大鼠的神经保护作用及其对细胞外调节蛋白激酶1/2信号通路的影响

目的:观察miR-17-5p对缺血性脑卒中大鼠的神经保护作用及其对胞外调节蛋白激酶1/2(ERK1/2)信号通路的影响。方法:将大鼠随机数字法分为模型组、miR-17-5p mimics组、miR-17-5p inhibitor组。建模后,模型组经尾静脉注射10 mL/kg生理盐水,miR-17-5p mimics组、miR-17-5p inhibitor组分别经尾静脉注射7μL miR-17-5p mimics、miR-17-5p inhibitor与RNAi脂质体转染试剂的混合溶液。各组大鼠苏醒后的24 h进行神经功能评分,测定脑梗死体积、大鼠脑组织含水量,检测缺血侧大脑皮质细胞凋亡率,caspase-3、Bax、Bcl-2和ERK1/2蛋白表达。结果:miR-17-5p mimics组的神经功能障碍评分、脑梗死体积、脑组织含水量、缺血侧大脑皮质细胞凋亡率明显低于模型组,miR-17-5p inhibitor组的神经功能障碍评分、脑梗死体积、脑组织含水量、缺血侧大脑皮质细胞凋亡率显著高于其他2组;miR-17-5p mimics组大鼠缺血脑组织中caspase-3、Bax蛋白表达明显低于模型组,Bcl-2、p-ERK1/2蛋白表达高于模型组;miR-17-5p inhibitor组大鼠缺血脑组织中caspase-3、Bax、p-ERK1/2蛋白表达显著高于其他2组,Bcl-2蛋白表达低于其他2组。结论:miR-17-5p对缺血性脑卒中大鼠的脑损伤具有一定的保护作用,miR-17-5p可抑制ERK1/2信号通路激活。

ERK1/2通过调控NADPH氧化酶和线粒体分裂在结肠炎中的作用

目的研究细胞外信号调节激酶1和2(ERK1/2)通过调控NADPH氧化酶(Nox)和线粒体分裂在结肠炎中的作用。方法3%葡聚糖硫酸钠(DSS)诱导小鼠急性结肠炎。将30只C57BL/6J小鼠用随机数表法分为6组:Control组、3%DSS组、1%二甲亚砜(DMSO)组、ERK1/2抑制剂(PD98059)组、3%DSS+1%DMSO组、3%DSS+PD98059组,每组5只。评估Control组和3%DSS组小鼠体重变化、结肠长度改变、疾病活动指数和结肠组织病理学改变,检测小鼠结肠黏膜ERK1/2、磷酸化(p)-ERK1/2、Nox1和Nox2表达水平。1%DMSO组、3%DSS+1%DMSO组给予腹腔注射1%DMSO;PD98059组、3%DSS+PD98059组小鼠给予腹腔注射PD98059。评估4组小鼠结肠组织病理学改变,检测Nox1、Nox2、动力相关蛋白1(DRP1)、p-DRP1-S616和p-DRP1-S637等线粒体分裂相关蛋白表达水平的改变。透射电镜观察Control组和3%DSS组小鼠结肠上皮细胞线粒体分裂情况。免疫荧光双染分析2组小鼠结肠黏膜中Nox2与线粒体外膜转位酶TOM复合体(TOMM20)共定位情况。分析2组小鼠结肠黏膜DRP1与Nox2 mRNA相对表达量的相关性。结果与Control组相比,3%DSS组小鼠体重下降、结肠长度缩短、疾病活动指数增加和结肠组织病理学评分升高,结肠黏膜p-ERK1/2、Nox1和Nox2表达增加(P均<0.05)。结肠炎小鼠结肠上皮细胞中的线粒体分裂增加,结肠黏膜的DRP1和Nox2共定位增加,两者mRNA相对表达呈正相关(r=0.678,P<0.05)。ERK1/2抑制剂PD98059改善结肠炎小鼠结肠组织病理学变化,并且下调结肠黏膜Nox1、Nox2、DRP1、p-DRP1-S616的表达。结论抑制ERK1/2可能通过减轻Nox表达和线粒体分裂,改善结肠炎。

从脑肠轴探讨黄芪建中汤对胃溃疡大鼠肝细胞生长因子及ERK1/2和TFF3蛋白表达的影响

目的观察黄芪建中汤对脾胃虚寒型胃溃疡大鼠胃组织中肝细胞生长因子(HGF)及下丘脑和海马区中磷酸化细胞外信号调节激酶1/2(p-ERK1/2)及肠三叶因子3(TFF3)蛋白表达的影响,探究黄芪建中汤治疗脾胃虚寒型胃溃疡的作用及可能机制。方法用随机数字表法将60只大鼠分为正常组、模型组、奥美拉唑组和黄芪建中汤组,每组15只。正常组隔日蒸馏水灌胃,每日不限饮食;其余组大鼠先以小承气汤结合饥饱失常法复制脾胃虚寒证模型,实验第11天采用冰醋酸法建立胃溃疡模型。之后模型组继续隔日上午给予小承气汤并当日禁食,次日恢复饮食,共持续20 d;奥美拉唑组和黄芪建中汤组大鼠除同模型组的每日处理外,每日下午分别给予4.2 mg/(kg·d)奥美拉唑和6.8 g/(kg·d)黄芪建中汤灌胃;正常组隔日上午及每日下午给予蒸馏水灌胃。实验结束后摘取各组大鼠胃,记录溃疡大小并计算胃溃疡指数,HE染色观察胃组织病理形态,免疫组化染色检测胃组织中HGF及下丘脑和海马区中p-ERK1/2、TFF3蛋白阳性表达情况。结果正常组大鼠胃黏膜正常,未见溃疡点及糜烂斑等;模型组大鼠胃黏膜皱襞存在中断,有点状溃疡、糜烂及大量炎性细胞浸润;黄芪建中汤组与奥美拉唑组胃黏膜损伤较模型组轻,有少量炎性细胞浸润,未见明显溃疡。奥美拉唑组和黄芪建中汤组大鼠的胃溃疡指数均明显低于模型组(P均<0.05),胃组织中HGF及下丘脑和海马区中p-ERK1/2、TFF3蛋白表达平均光密度均明显高于模型组(P均<0.05)。结论黄芪建中汤能促进脾胃虚寒型胃溃疡大鼠溃疡愈合,上调HGF、ERK1/2及TFF3的表达可能是其基于脑肠轴治疗脾胃虚寒型胃溃疡的作用机制之一。

细胞外信号调节激酶1/2信号通路调控细胞侵袭性的研究进展

细胞外信号调节激酶(ERK)1/2是一种蛋白丝氨酸/苏氨酸激酶,参与Ras-Raf有丝分裂原激活蛋白激酶-ERK的信号转导级联,通过影响基因的转录和表达,参与细胞的生长、增殖甚至侵袭作用。肺癌、肝癌、卵巢癌、宫颈癌、子宫内膜异位症及子痫前期等多种疾病的发生,以及其疾病的转移和病情的进展,均与ERK1/2信号通路调控细胞侵袭性密切相关。因此通过探索ERK1/2信号通路侵袭性在相关疾病发病过程中可能发挥的重要作用,从而寻找更加有效的治疗方案。本文根据近些年国内外的最新研究,介绍ERK1/2信号通路侵袭性这一特性在各个疾病中所发挥的相关调控作用,以期为相关疾病的临床治疗研究提供新启示。

2型糖尿病合并结直肠癌患者癌组织中Ras ERK1/2蛋白表达及意义

目的:研究目的通过检测2型糖尿病合并结直肠癌中Ras-ERK1/2信号表达水平,分析它们与淋巴结转移的关系及其与生存状况的相关性。方法:选择石家庄市人民医院2019年3月到2021年1月期间行结肠癌手术治疗患者59例,按是否同时伴发糖尿病,分为糖尿病组(n=29)和非糖尿病组(n=30)。通过免疫组织化学方法检测两组患者手术切除的癌组织中Ras、ERK1/2蛋白的表达水平。运用Log-rank法分析不同表达情况下淋巴结转移的差异。所有患者随访36个月,统计并比较两组间患者的生存情况,采用spearman相关性分析法分析Ras、ERK1/2表达与生存期相关性。结果:Ras、ERK1/2在结直肠癌组和结直肠癌合并2型糖尿病中均有表达,且主要集中在癌细胞浆中;与非糖尿病组相比,结直肠癌合并2型糖尿病组中Ras、ERK1/2阳性表达率显著升高(P<0.05);结直肠癌合并2型糖尿病组患者淋巴结转移率较单纯结直肠癌组明显升高(P<0.05);与Ras、ERK1/2阴性表达组相比,Ras、ERK1/2阳性表达组患者的淋巴结转移率明显升高(P<0.05)。经spearman相关性分析结果显示,结直肠癌患者Ras、ERK1/2蛋白表达与中位生存时间呈负相关(P<0.05)。结论:Ras-ERK1/2蛋白阳性表达与淋巴结转移正相关,与患者生存期呈负相关,可将其作为早期诊断结直肠癌、判断预后的重要靶点。

PMS2通过ERK/ERCC1通路对结肠癌SW480细胞生物学行为的影响

目的:探讨减数分裂后分离蛋白2(PMS2)表达对结肠癌SW480细胞生物学行为的影响,阐明PMS2与切除修复交叉互补组1(ERCC1)和细胞外调节蛋白激酶(ERK)信号转导通路的关系。方法:将PMS2 siRNA质粒和PMS2过表达质粒分别转染入结肠癌SW480细胞(分别为PMS2敲减组和PMS2过表达组),同时设PMS2敲减对照组(siRNA-NC组)和PMS2过表达对照组(PMS2 control组)。采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中PMS2 mRNA表达水平,Western blotting法检测各组细胞中PMS2蛋白表达水平,CCK-8法检测各组细胞增殖活性,细胞划痕实验检测各组细胞迁移率,Transwell小室实验检测各组细胞中侵袭细胞数,流式细胞术检测顺铂作用后各组细胞凋亡率。通过String数据库,对PMS2、ERCC1和ERK上下游蛋白的关系进行生物信息学分析。SW480细胞分别采用3条siRNA进行PMS2和ERCC1敲减,采用RT-qPCR法验证PMS2与ERCC1的相互作用,采用Western blotting法检测各组细胞中PMS2、细胞外调节蛋白激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)蛋白表达水平。结果:RT-qPCR法和Western blotting法检测,PMS2基因敲减和过表达细胞模型构建成功。与siRNA-NC组比较,PMS2敲减组细胞增殖活性和细胞迁移率明显升高(P<0.05或P<0.01),侵袭细胞数明显增加(P<0.01),顺铂作用后细胞凋亡率明显降低(P<0.01);与PMS2 control组比较,PMS2过表达组细胞增殖活性和细胞迁移率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),顺铂作用后细胞凋亡率明显升高(P<0.01)。蛋白-蛋白互作(PPI)富集P值为2.09e-07,包含ERCC1和ERK1/2等相互作用节点数共有13个,提示PMS2、ERCC1和ERK1/2之间可能存在调控作用。与siRNA-NC组比较,各PMS2敲减组细胞中ERCC1 mRNA表达水平明显降低(P<0.05或P<0.01);与siERCC1-NC组比较,各ERCC1敲减组细胞中PMS2 mRNA表达水平差异无统计学意义(P>0.05)。与siRNA-NC组比较,PMS2敲减组细胞中PMS2、ERK1/2和p-ERK1/2蛋白表达水平均明显降低(P<0.05或P<0.01);与PMS2 control组比较,PMS2过表达组细胞中PMS2、ERK1/2和p-ERK1/2蛋白表达水平均明显升高(P<0.01)。结论:PMS2表达可影响结肠癌SW480细胞增殖、迁移、侵袭和抗凋亡能力。PMS2与ERCC1存在互相作用关系,并可通过调节ERCC1参与ERK信号转导通路。

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.

CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响

目的探索CX3CR1对创伤性骨髓炎大鼠骨骼肌微纤维、ERK/MAPK信号通路及炎症反应的影响。方法选取30只SPF级SD雄性大鼠,依据随机数字表法分为健康组、模型组、CX3CR1抑制组,每组10只。除健康组外,其余各组均建立创伤性骨髓炎模型。其中健康组、模型组大鼠均每日常规腹腔注射生理盐水,CX3CR1干预组向残腔内注射CX3CR1中和抗体进行处理。采用ELISA法检测血清中IL-6、IL-10、IL-1β、TGF-β水平,应用改良X线Norden评分检测骨骼肌微纤维,HE染色观察病理变化,免疫印迹及PCR检测股骨组织中细胞外信号调节蛋白激酶(Extracellular regulated protein kinase,ERK1/2)、丝裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)蛋白及mRNA表达。结果与健康组比较,模型组TGF-β、IL-1β、IL-10、IL-6等炎症因子含量均升高(P<0.05);与模型组比较,CX3CR1抑制组炎症因子含量降低(P<0.05)。与健康组比较,模型组随时间推移X线Norden评分升高(P<0.05);与模型组比较,CX3CR1抑制组X线Norden评分降低(P<0.05)。HE染色显示,健康组骨质完好;模型组可见大量炎性细胞浸润、灶性脓肿及坏死灶;CX3CR1抑制组大鼠的骨质明显改善,炎症反应降低。与健康组比较,模型组ERK1/2、MAPK蛋白及mRNA表达升高(P<0.05);与模型组比较,CX3CR1抑制组ERK1/2、MAPK蛋白及mRNA表达降低(P<0.05)。结论抑制CX3CR1可改善创伤性骨髓炎大鼠的疾病反应,可能与降低炎症反应、ERK/MAPK信号通路以及改善骨骼肌微纤维相关。

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.