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.

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.

Regulating effect of glycyrrhetinic acid on bronchial asthma smooth muscle proliferation and apoptosis as well as inflammatory factor expression through ERK1/2 signaling pathway

Objective: To study the influence of glycyrrhetinic acid(GA) on bronchial asthma(BA)smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism.Methods: Male SD guinea pigs were selected and made into asthma models, bronchial asthma smooth muscle cells were cultured and divided into BA group, GA group and GA + LM group that were treated with serum-free RPMI1640 culture medium, serumfree RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid, serum-free RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid and 100 ng/mL LM22B-10 respectively; normal guinea pigs were collected and bronchial smooth muscle cells were cultured as control group. The cell proliferation activity as well as the expression of proliferation and apoptosis genes, inflammatory factors and p-ERK1/2 was determined.Results: Proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6,YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in BA group were significantly higher than those of control group while m RNA expression levels of Bax,caspase-9 as well as caspase-3 were significantly lower than that of control group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in GA group were significantly lower than those of BA group(P < 0.05) while the m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly higher than those of BA group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40 of airway smooth muscle cell in GA + LM group were significantly higher than those of GA group(P < 0.05) while m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower that of GA group(P < 0.05).Conclusion: GA can inhibit the proliferation of bronchial smooth muscle cells and reduce the expression of inflammatory factors by inhibiting the phosphorylation of ERK1/2.

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.

Human ciliary muscle cell responses to kinins:Activation of ERK1/2 and pro-matrix metalloproteinases secretion

AIM To study activation of extracellular signal-regulated kinase-1/2(ERK1/2) and pro-matrix metalloproteinases(pro-MMPs) secretion from isolated primary human ciliary muscle(h-CM) cells in response to bradykinin(BK) and other agonists. METHODS Serum-starved h-CM cells were challenged with vehicle, BK agonists or antagonists. Cell lysates were evaluated for phosphorylated ERK1/2 using homogeneous timeresolved fluorescence technology based on a sandwich immunoassay. Rabbit polyclonal anti-pro-MMP antibodies were used to measure pro-MMPs using immunoblot analysis.RESULTS A 10 min incubation time using 5 × 104 h-CM cells/well was optimum condition for studying stimulation of ERK1/2 phosphorylation. BK(100 nmol/L) caused a 1.86 ± 0.26 fold(n = 3) increase in ERK1/2 phosphorylation above baseline. BK analogs, Met-Lys-BK and RMP-7(100 nmol/L), also stimulated ERK1/2 phosphorylation by 1.57 ± 0.04 and 1.55 ± 0.09 fold, respectively. However, DesArg9-Bradykinin, a B1 receptor-selective agonist(0.1-1 μmol/L), was essentially inactive. HOE-140 or WIN-64338(B2-antagonists) appreciably blocked phosphorylation of ERK1/2 induced by various BK agonists. Pre-treatmentof cells with a prostaglandin(PG) synthase inhibitor(bromfenac; 1 μmol/L) failed to alter kinin-induced ERK1/2 activation. BK and a non-peptide BK agonist(FR-190997)(10 nmol/L-1 μmol/L) also enhanced pro-MMPs secretion(pro-MMP-1 > pro-MMP-3 > pro-MMP-2; 1.45-1.75-fold over baseline) from h-CM cells. CONCLUSION These collective data suggest that B2 kinin receptors initiate signaling in h-CM cells by a relatively rapid mechanism(within minutes) involving ERK1/2 activation which in turn regulates MMPs production(within hours). The latter process does not involve PGs.

Emodin regulating excision repair cross-complementation group 1 through fibroblast growth factor receptor 2 signaling

AIM: To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. METHODS: After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cells, the inhibition rate (IR), 50% inhibitory concentration (IC 50 ) and reversal index (IC 50 in experimental group/IC 50 in control group) were calculated. For HepG2, HepG2/OXA, HepG2/OXA/T, each cell line was divided into a control group, OXA group, OXA + fibroblast growth factor 7 (FGF7) group and OXA + emodin group, and the final concentrations of FGF7, emodin and OXA in each group were 5 ng/mL, 10 μg/mL and 10 μmol/L, respectively. Single-cell gel electrophoresis was conducted to detect DNA damage, and the fibroblast growth factor receptor 2 (FGFR2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and excision repair cross-complementing gene 1 (ERCC1) protein expression levels in each group were examined by Western blotting. RESULTS: Compared with the IC50 of 120.78 μmol/L in HepG2/OXA cells, the IC 50 decreased to 39.65 μmol/L after treatment with 10 μmol/L emodin; thus, the reversal index was 3.05. Compared with the control group, the tail length and Olive tail length in the OXA group, OXA + FGF7 group and OXA + emodin group were significantly increased, and the differences were statistically significant (P < 0.01). The tail length and Olive tail length were lower in the OXA + FGF7 group than in the OXA group, and this difference was also statistically significant. Compared with the OXA + FGF7 group, the tail extent, the Olive tail moment and the percentage of tail DNA were significantly increased in the OXA + emodin group, and these differences were statistically significant (P < 0.01). In comparison with its parental cell line HepG2, the HepG2/OXA cells demonstrated significantly increased FGFR2, p-ERK1/2 and ERCC1 expression levels, whereas the expression of all three molecules was significantly inhibited in HepG2/ OXA/T cells, in which FGFR2 was silenced by FGFR2 shRNA. In the examined HepG2 cells, the FGFR2, p-ERK1/2 and ERCC1 expression levels demonstrated increasing trends in the OXA group and OXA + FGF7 group. Compared with the OXA group and OXA + FGF7 group, the FGFR2, p-ERK1/2, and ERCC1 expression levels were significantly lower in the OXA + emodin group, and these differences were statistically significant. In the HepG2/OXA/T cell line that was transfected with FGFR2 shRNA, the FGFR2, p-ERK1/2 and ERCC1 expression levels were significantly inhibited, but there were no significant differences in these expression levels among the OXA, OXA + FGF7 and OXA + emodin groups. CONCLUSION: Emodin markedly reversed OXA resistance by enhancing OXA DNA damage in HepG2/OXA cells, and the molecular mechanism was related to the inhibitory effect on ERCC1 expression being mediated by the FGFR2/ERK1/2 signaling pathway.

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.

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.

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.

Cytokine receptor-like factor 1(CRLF1)promotes cardiac fibrosis via ERK1/2 signaling pathway

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease.Anti-fibrosis treatment is a significant therapy for heart disease,but there is still no thorough understanding of fibrotic mechanisms.This study was carried out to ascertain the functions of cytokine receptor-like factor 1(CRLF1)in cardiac fibrosis and clarify its regulatory mechanisms.We found that CRLF1 was expressed predominantly in cardiac fibroblasts.Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction,but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-β1(TGF-β1).Gain-and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts(NMCFs)with or without TGF-β1 stimulation.CRLF1 overexpression increased cell viability,collagen production,cell proliferation capacity,and myofibroblast transformation of NMCFs with or without TGF-β1 stimulation,while silencing of CRLF1 had the opposite effects.An inhibitor of the extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathway and different inhibitors of TGF-β1 signaling cascades,comprising mothers against decapentaplegic homolog(SMAD)-dependent and SMAD-independent pathways,were applied to investigate the mechanisms involved.CRLF1 exerted its functions by activating the ERK1/2 signaling pathway.Furthermore,the SMAD-dependent pathway,not the SMAD-independent pathway,was responsible for CRLF1 up-regulation in NMCFs treated with TGF-β1.In summary,activation of the TGF-β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression.CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway.CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

参苓白术散通过ERK/p38 MAPK信号通路干预溃疡性结肠炎大鼠结肠组织AQP3、AQP4的表达

目的探讨细胞外信号调节激酶/p38丝裂原活化蛋白激酶(ERK/p38MAPK)信号通路在参苓白术散调控脾虚湿困型溃疡性结肠炎大鼠结肠组织水通道蛋白(aquaporin,AQP)3、AQP4表达中的作用。方法将60只Wistar大鼠按照随机数字表均分为正常组、模型组(氯化钠注射液)、参苓白术散组、U0126+参苓白术散组、SB203580+参苓白术散组。除正常组外,脾虚湿困型溃疡性结肠炎大鼠采用2,4,6-三硝基苯磺酸/乙醇灌肠,结合环境与饮食干预复制。14 d后采用蛋白质印迹法检测各组大鼠结肠组织AQP3、AQP4蛋白的表达,实时荧光定量PCR法检测其mRNA表达情况。结果模型组大鼠AQP3、AQP4蛋白表达及其mRNA的表达水平明显低于正常组(P<0.05),参苓白术散组大鼠结肠组织AQP3、AQP4蛋白及mRNA表达较模型组明显升高,组间比较有显著性差异(P<0.05),SB203580+参苓白术散组及U0126+参苓白术散组大鼠结肠组织AQP3、AQP4蛋白及mRNA表达较模型组有较小幅度升高,与正常组和参苓白术散组相比均有显著性差异(P<0.05)。结论参苓白术散可显著改善大鼠结肠组织AQP3、AQP4蛋白及mRNA表达,ERK/p38 MAPK信号通路参与了参苓白术散对脾虚湿困型溃疡性结肠炎大鼠结肠组织AQP3、AQP4表达的调节作用。

碘缺乏和甲状腺功能减退对大鼠仔鼠海马ERK1/2表达的影响

目的观察碘缺乏和甲状腺功能减退对大鼠仔鼠海马细胞外信号调节蛋白激酶1及2(ERK1/2)表达的影响。方法健康2月龄孕Wistar大鼠28只,按体重随机分成对照组、甲状腺功能减退组[按饮水中含丙基硫尿嘧啶(PTU)剂量分为5mg/L组和15mg/L组]和碘缺乏组,每组7只。分别于出生后第7、14、21、28和42天每组随机取5只仔鼠,灌流固定大脑,用组织病理切片和免疫组化染色观察分析海马的ERK1/2表达。结果在出生后14、21、28和42天时,海马CA1和CA3区的ERK1/2表达在PTU5mg/L组、PTU15mg/L组和碘缺乏组显著低于对照组(P<0.05)。DG区的ERK1/2表达与对照组相比差异无显著性。出生后7天时,各组间ERK1/2表达差异无显著性。结论碘缺乏和甲状腺功能减退可降低海马CA1和CA3区的ERK1/2表达。

Salvianolate Reduces Murine Myocardial Ischemia and Reperfusion Injury via ERK1/2 Signaling Pathways in vivo

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

MAPK/ERK通路相关蛋白在肺癌组织中的表达及其与肺叶切除术患者预后关系

目的MAPK/ERK通路相关蛋白在肺癌组织中的表达及其在肺叶切除术肺癌患者预后评估中的作用鲜有报道。文章旨在分析MAPK/ERK通路相关蛋白细胞外信息调节蛋白激酶1/2(ERK1/2)蛋白及其磷酸化(p-ERK1/2)蛋白在肺癌组织中的表达情况,并探讨其与肺叶切除术患者临床预后的关系。方法回顾性收集2014年1月至2016年10月于武汉市第三医院胸外科行全胸腔镜肺叶切除术治疗的62例肺癌患者的肺癌组织标本及同一患者距癌变组织>5 cm的癌旁组织标本各62份。采用免疫组化法检测患者肺癌组织及癌旁组织中ERK1/2、p-ERK1/2表达阳性率,采用χ^(2)检验分析肺癌组织中ERK1/2、p-ERK1/2与患者临床病理特征的关系。随访至2021年10月,共57例患者获得随访。采用Kaplan-Meier生存分析法分析ERK1/2、p-ERK1/2表达与患者预后的关系,并拟合Cox模型评价不同指标与患者预后的关系。结果ERK1/2、p-ERK1/2在肺癌组织中主要定位于肿瘤细胞的细胞膜和细胞浆中,阳性肿瘤细胞弥漫分布;ERK1/2在癌旁正常组织中表达较少,p-ERK1/2在癌旁正常组织中呈阴性表达。肺癌组织中ERK1/2、p-ERK1/2蛋白表达阳性率均高于癌旁组织(P<0.05)。ERK1/2在不同分化程度、淋巴结有无转移患者中表达阳性率差异有统计学意义(P<0.05);p-ERK1/2在不同分化程度、临床病理分期、淋巴结有无转移患者中表达阳性率差异有统计学意义(P<0.05)。Kaplan-Meier生存曲线及log-rank分析显示,ERK1/2、p-ERK1/2表达阳性患者与其表达阴性患者术后累积生存率比较,差异有统计学意义(P=0.021、0.018)。多因素Cox比例风险模型显示,肿瘤分化程度低[HR:1.887(1.149~2.684)]、淋巴结转移[HR:2.348(1.109~3.527)]、p-ERK1/2表达阳性[HR:3.258(1.236~5.148)]是影响肺癌患者预后的风险因素(P<0.05)。结论MAPK/ERK通路相关蛋白ERK1/2、p-ERK1/2在肺癌组织中呈现高表达,其中p-ERK1/2表达与肺癌患者预后关系密切,可作为评判肺癌患者预后的重要指标。

The Role of SDF-1/CXCR4 Axis in Ovarian Cancer Metastasis

This study was aimed to explore the role of stromal-derived factor 1 （SDF-1）/CXC chemokine receptor 4 （CXCR4） axis in mediating the metastasis of ovarian cancer cells through activation of extracellular signal-regulated kinase-1/2 （ERK-1/2） signaling pathway. A highly metastatic ovarian cancer cell line, SKOV3, was used in the study. Intracellular calcium mobilization was detected by using laser scanning confocal fluorescence microscopy. Western blotting was used to detect the phosphorylation of ERK1/2 in SDF-1α-treated SKOV3 cells. Adhesion capability and matrix metalloproteinase （MMP） activity of ovarian cancer cells after exposure to SDF-1 a were measured by adhesion assay and gelatin zymography. The results showed that SDF-1α induced rapid intracellular calcium mobilization in SKOV3 cells, as well as the phosphorylation of ERK-1/2. The adhesion of ovarian cancer cells to fibronectin and collagen Ⅳ was increased after SDF-1α treatment. An inhibitor of ERK-1/2 signaling, PD98059, could antagonize such effects of SDF-1α. SDF-1α could also increase the secretion of active MMP-2 and MMP-9. It was concluded that the SDF-1/CXCR4 axis played a critical role in the metastasis of human ovarian cancer by increasing the adhesion capability of cancer cells and the activity of MMP-2 and MMP-9 via ERK1/2 signaling pathway.

AcSDKP经由PDGF介导的细胞外信号调节激酶1/2的调节通路在大鼠矽肺纤维化形成中的作用

探讨抗纤维化短肽N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)经由血小板源性生长因子(PDGF)介导的细胞外信号调节激酶1/2(ERK1/2)的调节通路在矽肺纤维化形成中的作用。采用一次性支气管内灌注二氧化硅(SiO2)粉尘制作矽肺大鼠模型,将其分为对照组(4周组和8周组)、矽肺模型组(4周和8周组)、AcSDKP治疗组(抗纤维化治疗组和预防治疗组)。采用贴块法进行肺成纤维细胞的原代和传代培养。HE染色观察肺组织形态学变化,Western blot法检测肺组织、肺成纤维组织Ⅰ型、Ⅲ型胶原蛋白和ERK1/2及磷酸化-ERK1/2蛋白的表达以及肺组织PDGF及其受体蛋白的表达。与对照比较,矽肺大鼠肺内PDGF及其受体蛋白、Ⅰ型和Ⅲ型胶原蛋白以及磷酸化-ERK1/2蛋白表达均增强,而AcSDKP治疗组则见上述蛋白表达减弱;在培养的肺成纤维细胞,PDGF能够刺激其Ⅰ型、Ⅲ型胶原蛋白表达增加,同时上调磷酸化-ERK1/2蛋白的表达,而AcSDKP则显示出与PD98059(细胞外信号调节激酶通路特异性抑制剂)相同的作用,即能够抑制PDGF刺激成纤维细胞Ⅰ型、Ⅲ型胶原蛋白表达和上调磷酸化-ERK1/2蛋白表达。提示AcSDKP可能通过阻断PDGF介导的ERK1/2信号转导通路抑制矽肺纤维化的形成与发展。

碘缺乏和甲状腺功能减退对大鼠仔鼠小脑细胞外信号调节激酶1／2蛋白表达的影响

目的观察碘缺乏和甲状腺功能减退（甲减）对大鼠仔鼠小脑细胞外信号调节激酶1／2（extracellular signal—regulate kinase，ERK1／2）蛋白表达的影响。方法健康Wistar大鼠28只，雌性，60日龄。将大鼠按体质量随机分为对照组、碘缺乏病组、甲减组，甲减组根据饮水中含丙基硫尿嘧啶（PrrU）剂量分为5mg／L组和15mg／L组。每组各7只。母鼠妊娠后，分别选用低碘饲料及PrrU饮水诱导建立低碘及甲减大鼠动物模型。仔鼠出生7、14、21、28、42d时分别处死，测定仔鼠小脑质量；取小脑组织进行镀银染色和免疫组织化学链霉菌抗生物素蛋白-过氧化物酶（S—P）染色，光镜下观察小脑形态学变化，图像分析仪下观察ERK1／2蛋白表达。结果仔鼠出生14、21、28、42d时，小脑质量各组间比较差异有统计学意义（F=6．325、8．870、16．191、21．574，P均〈0．05）；碘缺乏病组仔鼠小脑质量[（0．0945±0．0233）、（0．1347±0．0046）、（0．1542±0．0094）、（0．1949±0．0048）g]明显低于对照组[（0．1856±0．0123）、（0．2049±0．0098）、（0．2268±0．0065）、（0．2606±0．0086）g，P均〈0．05]和甲减组[5mg／L组：（0．1741±0．0172）、（0．1927±0．0103）、（0．2181±0．0064）、（0．2583±0．0054）g，P均〈0．05；15mg／L组：（0．1604±0．0083）、（0．1682±0．0103）、（0．1996±0．0073）、（0．2579±0．0067）g，P均〈0．05]。出生7d时，对照组、甲减组仔鼠小脑皮质具有典型的层状结构，各层之间界限清晰，但碘缺乏病组各层之间界限模糊；出生21d时，与对照组比较，15mg／L组和碘缺乏病组仔鼠小脑外颗粒细胞消失延迟，仍有2～3层外颗粒细胞；出生28、42d时。甲减组和碘缺乏病组仔鼠小脑分子层厚度低于对照组。小脑组织ERK1／2平均积分光密度，在出生7d时，组间比较差异无统计学意义（F=1．102，P〉0．05）；在出生14、21、28、42d时，各组间比较差异有统计学意义（F=16．131、13．543、26．953、41．583，P均〈0．01）；碘缺乏病组（7．3245±0．5070）、（8．3606±1．0683）、（9．1217±1．0402）、（12．1587±0．7581）和甲减组[5mg／L组：（11．4307±15200）、（14．919±0．8497）、（16．0082±1．1130）、（15．7721±0．8293）；15mg／L组：（7．8538±0．9775）、（11．2461±0．8138）、（12．7800±1．3783）、（13．0871±1．1450）]明显低于对照组[（16．2831±0．5143）、（20．2653±0．9551）、（22．7485±1．0267）、（22．1725±0．9939），P均〈0．01]。结论碘缺乏和甲减可使仔鼠小脑产生明显的形态学改变，降低大鼠仔鼠小脑组织ERK1／2蛋白表达，影响神经系统的发育。

Poly-L-ornithine blocks the inhibitory effects of fibronectin on oligodendrocyte differentiation and promotes myelin repair

The extracellular matrix surrounding oligodendrocytes plays an important role during myelination and remyelination in the brain.In many cases,the microenvironment surrounding demyelination lesions contains inhibitory molecules,which lead to repair failure.Accordingly,blocking the activity of these inhibitory factors in the extracellular matrix should lead to more successful remyelination.In the central nervous system,oligodendrocytes form the myelin sheath.We performed primary cell culture and found that a natural increase in fibronectin promoted the proliferation of oligodendrocyte progenitors during the initial stage of remyelination while inhibiting oligodendrocyte differentiation.Poly-L-ornithine blocked these inhibitory effects without compromising fibronectin’s pro-proliferation function.Experiments showed that poly-L-ornithine activated the Erk1/2 signaling pathway that is necessary in the early stages of differentiation,as well as PI3K signaling pathways that are needed in the mid-late stages.When poly-L-ornithine was tested in a lysolecithin-induced animal model of focal demyelination,it enhanced myelin regeneration and promoted motor function recovery.These findings suggest that poly-L-ornithine has the potential to be a treatment option for clinical myelin sheath injury.