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.

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.

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.

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.

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。

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.

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.

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。

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。

17β-雌二醇对子宫内膜异位症患者在位子宫内膜间质细胞ERK1/2信号转导通路活化的影响

目的研究17β-雌二醇（17β-E2）对子宫内膜异位症（内异症）患者在位子宫内膜间质细胞胞外信号调节激酶1/2（ERK1/2）信号转导通路活化的影响,探讨ERK1/2信号转导通路在介导雌激素促进内异症发生发展中的作用。方法体外分离培养内异症患者在位子宫内膜间质细胞。用1×10^-6mol/L 17β-E2处理子宫内膜间质细胞不同时间（0~120 min）,然后用MTT法和免疫印迹法（Western blot）检测子宫内膜间质细胞的活性和ERK1/2的活化情况,检测出子宫内膜间质细胞活性最强的时间点。用Western blot和MTT法分别检测不同浓度ERK1/2抑制剂PD98059对1×10^-6mol/L 17β-E2作用20 min后子宫内膜间质细胞活性及ERK1/2活性的变化。结果 1×10^-6mol/L的17β-E2作用20 min时,子宫内膜间质细胞的活性最强,ERK1/2活化在17-βE2作用10 min后出现,在20 min时达高峰;随着PD98059作用浓度的增加,ERK1/2活化水平逐渐下降,浓度为50μmol/L时已被完全阻断;随着PD98059作用浓度的增加,子宫内膜间质细胞活性逐渐下降,在50μmol/L和100μmol/L时,活性最低,但此时仍高于空白对照组。结论 17β-E2增强子宫内膜间质细胞的活性可能与17β-E2通过非转录机制,迅速激活子宫内膜间质细胞的ERK1/2信号转导通路有关。

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。

针刺对大鼠局部筋膜和脊髓细胞外信号调节激酶1/2和P38丝裂酶原活化蛋白激酶信号通路的影响

目的:观察针刺捻转拉伸大鼠皮下筋膜对局部筋膜和脊髓背角细胞外信号调节激酶1/2(ERK1/2)和丝裂原活化蛋白激酶P38(P38MAPK)信号通路的影响及筋膜结缔组织的形态学变化。方法:20只SD大鼠通过随机分组,每组5只,针刺后三里组和针刺非穴位组进行手针捻转,拉伸刺激组进行拉伸刺激,采用免疫组化技术观察筋膜和脊髓组织中细胞信号蛋白的变化;利用相差显微镜观察拉伸刺激组局部皮下筋膜形态学变化。结果:组织学改变:拉伸刺激组皮下筋膜的纤维以拉伸点为中心呈向心性分布,单位面积内细胞密度增大,细胞骨架和胞核重构成"扁梭形"。细胞信号蛋白变化:针刺组筋膜结缔组织ERK1/2和P38MAPK表达与对照组相比均有增加,但以非穴组增加显著;ERK1/2与P38MAPK在脊髓中的表达位置由胞质转向胞核,ERK1/2与空白对照组相比差异没有显著性意义;P38MAPK的表达有所增加。结论:针刺对局部浅筋膜的ERK1/2和P38有上调作用,但与脊髓中的信号蛋白增加幅度并不完全一致,提示筋膜结缔组织支架可能在微观的信号转导层面对局部细胞分化与增殖具有促进作用。

TROP2、p-ERK1/2和Cyclin D1在胆囊癌组织中的表达及临床意义

目的探讨胆囊癌组织中人滋养层细胞表面抗原(TROP)2、磷酸化细胞外信号调节激酶(p-ERK)1/2和细胞周期蛋白D1(Cyclin D1)的表达及其与临床病理参数之间的关系,并分析其与胆囊癌患者预后的关系。方法搜集第二军医大学附属长征医院2005年6月-2010年6月确诊并获取病理标本的胆囊癌患者88例,采用免疫组化法检测88例胆囊癌组织及15例癌旁组织中TROP2、p-ERK1/2和Cyclin D1蛋白的表达。计数资料采用χ~2检验,Spearman检验法分析TROP2、p-ERK1/2和Cyclin D1间相关性;单因素和多因素Cox回归分析胆囊癌患者预后的影响因素;Kaplan-Meier法绘制患者的生存曲线。结果 TROP2、p-ERK1/2和Cyclin D1蛋白在胆囊癌组织中的阳性表达率分别为74.30%、58.40%和55.30%,明显高于癌旁组织中的表达(5.42%、35.67%和39.87%)(P值均<0.05)。TROP2、p-ERK1/2和Cyclin D1蛋白的表达与胆囊结石、肿瘤直径、分化程度、血管神经侵犯、淋巴结转移、手术方式及TNM分期相关(χ~2=4.300~53.315,P值均<0.05)。TROP2与p-ERK1/2、Cyclin D1的表达呈正相关(rs值分别为0.402、0.742,P值均<0.001),且p-ERK1/2与Cyclin D1的表达也呈正相关(rs=0.242,P=0.023)。多因素生存分析提示,TROP2的阳性表达是患者3年生存率的独立危险因素(相对危险度=2.412,95%可信区间:1.186~5.126,P=0.010)。结论 TROP2的高表达可能是胆囊癌恶性进展的重要原因,高表达的TROP2可能介导p-ERK1/2和Cyclin D1的高表达,导致胆囊癌恶性进展。TROP2是判断胆囊癌患者预后的独立危险因素,有可能是临床干预的有效靶点。

黄连素抑制ERK1/2途径减轻大气细颗粒物对EA.hy926内皮细胞损伤的研究

目的:探讨大气细颗粒物(PM2.5)对EA.hy926型人脐静脉内皮细胞的损伤及黄连素的保护作用及其机制。方法:采集大气PM2.5并分别以0、20、200、400 mg/L染毒EA.hy926细胞24 h,MTT法检测细胞存活率、流式细胞术检测细胞凋亡、Western blot法检测p-ERK1/2、BAX、BCL-2蛋白表达,ELISA法测IL-6、TNF-α、MDA含量及SOD、LDH活性;分别加入黄连素(10、50、100μmol/L)和ERK1/2通路特异性阻滞剂PD98059 20μmol/L检测黄连素的干预作用。结果:与对照组比较,PM2.5染毒后呈剂量依赖性降低细胞存活率,并上调p-ERK1/2蛋白水平及BAX/BCL-2蛋白比率以促进细胞凋亡、诱导分泌IL-6、TNF-α及升高MDA含量、降低SOD活性、升高LDH活性(P<0.05);黄连素呈剂量依赖性升高PM2.5作用下细胞存活率、下调p-ERK1/2蛋白水平及BAX/BCL-2蛋白比率以抑制细胞凋亡、降低IL-6、TNF-α及MDA含量、升高SOD活性、降低LDH活性(P<0.05)。结论:黄连素能通过抑制ERK1/2通路,减轻PM2.5对EA.hy926细胞的损伤。

ERK1/2介导依达拉奉保护H9c2心肌细胞对抗异丙肾上腺素诱导的损伤作用

目的探讨胞外信号调节激酶1/2(ERK1/2)在依达拉奉(EDA)保护H9c2心肌细胞对抗异丙肾上腺素(ISO)损伤中的作用。方法用不同浓度的ISO处理H9c2心肌细胞,建立β1肾上腺素受体持续兴奋诱导心脏毒性的体外模型。EDA在ISO处理心肌细胞前1h加入培养基中作为预处理。PD-98059(ERK1/2抑制剂)在应用EDA前加入培养基中并作用1h,以抑制ERK1/2的磷酸化。CCK-8比色法检测细胞存活率;Western blot法检测总量及磷酸化ERK1/2蛋白的表达;罗丹明123(Rh123)染色荧光显微镜照相检测线粒体膜电位(MMP)。结果 80μmol/L ISO处理H9c2心肌细胞24h,可使磷酸化的ERK1/2及MMP的水平明显降低。EDA在10～40μmol/L浓度范围内预处理1h可以剂量依赖性地减弱80μmol/L ISO处理H9c2心肌细胞48h引起的毒性反应;40μmol/L EDA预处理1h可明显抑制ISO作用24h引起的ERK1/2磷酸化水平降低及MMP受损。ERK1/2抑制剂,PD-98059,可以取消EDA诱导的细胞保护作用,使细胞毒性及MMP受损加重。结论 EDA可保护H9c2心肌细胞对抗ISO诱导的损伤作用,其机制之一可能与拮抗ISO对ERK1/2的磷酸化抑制作用有关。

紫草素抑制ERK1/2-NF-κB通路对大鼠心房颤动的改善作用

目的探讨紫草素通过细胞外信号调节蛋白激酶1/2(ERK1/2)/核转录因子-κB(NF-κB)介导心肌细胞纤维化对大鼠心房颤动(简称房颤)的改善作用。方法30只SD大鼠随机分为正常对照组、模型组、紫草素组,每组10只。紫草素组大鼠以4 mg/kg紫草素灌胃给药,正常对照组和模型组大鼠等量生理盐水灌胃,1次/d,连续7 d;除正常对照组外,其余组大鼠按照1 ml/kg的剂量尾静脉注射乙酰胆碱-氯化钙(ACh-CaCl2)混合液1次/d、连续7 d建立房颤模型,正常对照组大鼠尾静脉注射等量生理盐水,7 d后取材。心电图检测各组大鼠房颤诱发时间和持续时间;超声心动图检测各组大鼠心功能;HE染色观察各组大鼠心肌组织病理学改变;Masson染色检测各组大鼠心肌组织胶原面积百分比;Western blotting检测各组大鼠胶原蛋白Ⅰ(ColⅠ)、ERK1/2、p-ERK1/2、NF-κB p65和p-NF-κB p65蛋白表达情况。结果正常对照组大鼠心肌细胞排列致密、整齐;模型组大鼠心肌细胞排列紊乱、肥大、间隙增宽;紫草素组大鼠心肌细胞形态和结构较模型组有所改善。与正常对照组比较,模型组及紫草素组大鼠左心室射血分数(LVEF)、左心室缩短分数(LVFS)降低,左心室收缩末期内径(LVESD)和左心室舒张末期内径(LVEED)增高,胶原面积百分比增加,ColⅠ、p-ERK1/2和p-NF-κB p65蛋白表达升高(P<0.05);与模型组比较,紫草素组大鼠房颤诱发时间延长、持续时间缩短,LVEF、LVFS升高,LVESD、LVEED缩短,胶原面积百分比减小,ColⅠ、p-ERK1/2和p-NF-κB p65蛋白表达明显降低(P<0.05)。结论紫草素可能通过抑制ERK1/2-NF-κB信号通路而减轻心肌纤维化,抑制房颤的发生,并改善心功能。

TGF-β_1和ERK_1/ERK_2的相互作用及对人胰腺癌细胞生长的影响

目的:研究转化生长因子β1(TGF β1)和细胞外信号调节激酶K1/细胞外信号调节激酶K2 (ERK1/ERK2)对胰腺癌生长的影响及其相互关系,并探讨它们对胰腺癌作用的可能机制。方法:应用免疫组织化学技术检测临床胰腺癌患者病理组织中TGF β1和ERK1/ERK2 蛋白表达情况;MTT法观察不同剂量TGF β1对人胰腺癌Panc 1细胞生长的影响;Western印迹法观察不同剂量TGF β1对ERK1/ERK2 蛋白表达的影响。结果:胰腺癌患者组织TGF β1、ERK1 和ERK2 蛋白均呈显著性高表达,其阳性表达率分别为66.67%(30/45)、86.70%(39/45)和84.44%(38/45),而在对照组织的阳性表达率分别为30%(6/20)、30%(6/20)和20%(4/20),两者间存在显著差异(P<0.05);TGF β1抑制Panc 1细胞生长,随着剂量的增加和作用时间的延长,其抑制作用逐渐增强,到第4 天抑制作用达高峰,随后抑制作用不同程度的减弱,细胞生长速度加快;外源TGF β1不能改变ERK1/ERK2 蛋白的高表达状态。结论:胰腺癌患者病理组织强表达TGF β1 和ERK1/ERK2 蛋白;外源TGF β1在一定阶段抑制胰腺癌细胞系生长,但不改变ERK1/ERK2 的表达;胰腺癌细胞逃避TGF β1 抑制作用的机制之一可能是高水平ERK1/ERK2 的表达。