MEK/ERK signaling pathway in apoptosis of SW620 cell line and inhibition effect of resveratrol

Objective:To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol.Methods:SW620 cell lines were divided into 5 groups,namely,control group.PD98059 group,low-dose resveratrol group,mid-dose resveratrol group and high-dose resveratrol group.The inhibition rate of cell proliferation was detected by MTT method.The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by realtime PCR and Western blotting.Results:Compared with control group,the proliferation of cells treated with resveratrol was significantly inhibited.In the case of apoptotic molecules,the expression of Bax,Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dosedependent manner.In the case of molecules in MEK/ERK signaling pathway,the expression of Ras,Raf,MEK and ERKl/2 was decreased significantly in resveratrol groups with a dose-dependent manner.Conclusions:PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.

Longan Aril Reverses H2O2 Cytotoxicity in PC12 Cells via RAS/MEK/ERK Signaling Pathway

[Objectives]To explore the neuroprotective effects and mechanism of Longan Aril(LA)effective parts on PC12 cells injured by H2O2.[Methods]The neuroprotective effects of LA were evaluated by the cell viability,SOD and MDA content,apoptosis assay and relative protein expression of Aβand p-Tau.The neuroprotective mechanism of LA was studied by using metabolomics and network pharmacology,and the expressions of RAS/MEK/ERK signaling pathway-related proteins were detected by western blotting.[Results]LA could improve the cell survival rate and SOD content,and reduce apoptosis and expression of Aβand p-tau.Inhibition of RAS/MEK/ERK signaling pathway is a possible mechanism of LA neuroprotective effects.[Conclusions]LA has a neuroprotective effects in vitro and be likely to inhibit the process of AD by inhibition of RAS/MEK/ERK signalling pathway.

Effect of Guizhi Fuling Pill combined with GnRH analog on cell proliferation and invasion as well as MEK/ERK pathway in endometriosis lesions

Objective: To study the effect of Guizhi Fuling Pill combined with gonadotropin-releasing hormone analog (GnRH-a) on cell proliferation and invasion as well as MEK/ERK pathway in endometriosis lesions. Methods: Patients who were diagnosed with endometriosis in Bazhong Hospital of Traditional Chinese Medicine between November 2014 and March 2017 were selected as the research subjects and randomly divided into two groups, observation group received preoperative Guizhi Fuling Pill combined with GnRH analog therapy, and control group received preoperative GnRH analog monotherapy. After surgical resection, the endometriosis lesion was collected to determine the mRNA expression of proliferation and invasion-related genes as well as the protein expression of MEK/ERK pathway molecules. Results: Id-1, Sema3A, c-IAP1, OPN and uPA mRNA expression as well as p-MEK, p-EKR1/2, caspase-3 and MMP2 protein expression in endometriosis lesion of observation group were significantly lower than those of control group while Bak, Smac, PAI-1, TIMP1 and TIMP2 mRNA expression as well as caspase-3 protein expression were significantly higher than those of control group. Conclusion: Guizhi Fuling Pill combined with GnRH analog can inhibit the cell proliferation and invasion as well as the MEK/ERK pathway activation in endometriosis lesions.

三萜皂苷Saxifragifolin D抑制人肝癌耐药细胞HepG2/ADM生长并诱导细胞凋亡作用研究

目的研究Saxifragifolin D(SD)对人肝癌耐药细胞HepG2/ADM的生长抑制及诱导凋亡作用。方法采用MTT法观察SD对HepG2/ADM细胞的增殖抑制作用,应用流式细胞仪分析SD对细胞周期的影响,AnnexinⅤ-FITC/PI双染检测凋亡细胞比率,JC-1染色观察SD对细胞内线粒体膜电位的影响,Western blot检测凋亡相关蛋白caspase-9,caspase-3和PARP的激活及c-Raf,MEK和ERK蛋白的表达和磷酸化水平。结果 SD可以明显抑制人肝癌耐药细胞HepG2/ADM的增殖。细胞周期检测发现SD诱导细胞产生亚二倍体凋亡峰,同时细胞凋亡率也由对照组的5.3%增加到34.8%和47.8%。线粒体膜电位检测结果显示SD导致细胞内线粒体膜电位的明显降低。Western blot检测结果表明caspase-9,caspase-3被激活,PARP被剪切活化,cytochrome C由线粒体释放至胞质,c-Raf、MEK和ERK蛋白的磷酸化水平降低。结论 SD可以抑制人肝癌耐药细胞HepG2/ADM增殖并诱导其凋亡,作用机制可能与线粒体功能障碍及抑制c-Raf/MEK/ERK通路的活化有关。

Apoptosis in glioma-bearing rats after neural stem cell transplantation

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of gJioblastoma treatment by neural stem ceiJ trans- plantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in sus- pension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly in-creased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.

参芪扶正注射液通过下调MMP-2表达抑制口腔鳞状细胞癌转移

【目的】探讨参芪扶正注射液对口腔鳞状细胞癌转移的抑制作用及机制。【方法】①体外实验:首先测定参芪扶正注射液对SCC-25细胞无毒性的浓度范围。其次观察参芪扶正注射液对SCC-25细胞迁移、侵袭,基质金属蛋白酶2(MMP2)/β-actin、p-c-Raf/c-Raf、磷酸化丝裂原活化蛋白激酶激酶(p-MEK)/丝裂原活化蛋白激酶激酶(MEK)、磷酸化细胞外调节蛋白激酶(p-ERK)/细胞外调节蛋白激酶(ERK)蛋白相对表达量的影响。最后应用LipofectamineTM 2000试剂将pc-MMP2质粒转染至SCC-25细胞后,观察参芪扶正注射液对MMP2高表达SCC-25细胞迁移、侵袭,MMP2/β-actin、p-c-Raf/c-Raf、p-MEK/MEK、p-ERK/ERK蛋白相对表达量的影响。②体内实验:于裸鼠右后肢腹侧皮下注射SCC-25细胞复制口腔鳞状细胞癌裸鼠移植瘤模型。测量移植瘤体积和质量。观察参芪扶正注射液对MMP2高表达移植瘤上皮钙黏素(E-cadherin)阳性细胞的比率,MMP2/β-actin、p-c-Raf/c-Raf、p-MEK/MEK、p-ERK/ERK蛋白相对表达量的影响。【结果】参芪扶正注射液在0~40μg·mL-1范围内,对SCC-25细胞无毒性。参芪扶正注射液可降低SCC-25细胞和MMP2高表达的SCC-25细胞迁移率和侵袭细胞数目,下调细胞MMP2/β-actin、p-c-Raf/c-Raf、p-MEK/MEK、p-ERK/ERK蛋白相对表达量。参芪扶正注射液可降低裸鼠移植瘤的质量和体积,升高MMP2高表达SCC-25细胞移植瘤中E-cadherin的阳性细胞比率,下调MMP2/β-actin、p-c-Raf/c-Raf、p-MEK/MEK、p-ERK/ERK蛋白相对表达量。【结论】参芪扶正注射液可抑制口腔鳞状细胞癌转移,其机制可能与下调MMP2活性进而抑制c-Raf/MEK/ERK通路激活有关。

Apolipoprotein A1 suppresses the hypoxia-induced angiogenesis of human retinal endothelial cells by targeting PlGF

AIM:To investigate the anti-angiogenic effect of apolipoprotein A1(apoA1)on primary human retinal vascular endothelial cells(HRECs)and explore the possible mechanism.METHODS:The primary HRECs were transfected with apoA1-GFP recombinant lentiviral and were compared with cells undergoing transfection with empty lentiviral vectors.Hypoxia chambers were used to simulate the anoxic environment of cells under pathological condition.The concentrations of secreted vascular endothelial growth factor(VEGF)and placental growth factor(PlGF)were measured by enzyme-linked immunosorbent assay(ELISA).Cell migration ability was detected by wound healing assay.The sprouting of HRECs was determined by tube formation assay.The protein levels of extracellular signal regulated kinase 1/2(ERK1/2)and phosphor ylated ERK1/2(p-ERK1/2)were measured by Western blot.RESULTS:Overexpressed apoA1 in hypoxia-induced HRECs significantly suppressed PlGF(0.67±0.10 folds,P=0.007).Overexpressed apoA1 also attenuated hypoxiainduced cell migration(0.32±0.11 folds,P<0.0001),tube formation(0.66±0.01 folds,P<0.0001)and the phosphorylation levels of ERK(0.6±0.11 folds,P=0.025).Pretreatment of mitogen-activated protein kinase kinase(MEK)inhibitor(U0126)further reduced the PlGF and angiogenesis in hypoxia-induced HRECs.CONCLUSION:ApoA 1 inhibits the angiogenesis at least in part by inactivating ERK1/2 in hypoxia-induced HRECs.Moreover,apoA1 suppresses the PlGF expression,which selectively associated with pathological angiogenesis.

Enhancement of effect of risperidone on cognitive function of schizophrenic mice by fluoxetine

Objective To explore the molecular mechanism of enhancing the effect of risperidone on the cognitive function of schizophrenic mice by fluoxetine. Methods Schizophrenic mice were injected with M K-801 and drugs( fluoxetine and / or risperidone) were adm inistrated orally 30 min before the injection of M K-801.Mice of the control group adm inistrated the same dose of norm al saline. The cognitive function, distance of activity, and stereotyped behavior of mice were observed. The expressions of key param eters of MEK / ERK pathway in hippocam pus were detected by Western blotting or real-time qPCR. The participation of MEK / ERK pathway in isolated hippocam palneuron injuries induced by M K-801 mediation was also analyzed. Results The mice of M K-801 group showed prolonged activity distance, increased stereotyped behavior scores, and cognitive im pairm ent. Expressions of pMEK/MEK, pERK/ERK1 /2, MMP2 /9, and TIMP1 /2 of the MK-801 group increased. The aforem entioned proteins of drug adm inistration groups decreased dram atically. For schizophrenic m ice, fluoxetine enhanced effects of risperidone in norm alizing abnorm al behaviors, affecting cognitive function, and unusual protein expressions( P < 0. 05). Results of in vitro experim ents were sim ilar to those of in vivo experim ents. Conclusion Fluoxetine enhances the anti-schizophrenic effect of risperidone by norm alizing the activated MEK / ERK signaling pathway.

PLX8394,a RAF inhibitor,inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling

Enterovirus 71(EV71)poses a serious threat to human health,with scattered outbreaks worldwide.There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection.Therefore,it is urgent and of significance to develop anti-EV71 drugs.Here,we found that PLX8394,a RAF inhibitor,possesses high antiviral activity against EV71 in vitro,being superior to the traditional clinical drug ribavirin.Moreover,PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses.Notably,in a suckling mouse model,PLX8394 provided a 70%protection rate for EV71-infected mice,reduced the viral load in liver and heart tissues,and relieved the inflammatory response.A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway.Thus,PLX8394 lays a foundation for the development of new drugs against EV71.

Enhancing osteogenic bioactivities of coaxial electrospinning nanoscaffolds through incorporating iron oxide nanoparticles and icaritin for bone regeneration

Bone tissue engineering provides a promising strategy for the treatment of bone defects.Nonetheless,the clinical utilization of biomaterial-based scaffolds is constrained by their inadequate mechanical strength and absence of osteo-inductive properties.Here,we proposed to endow nano-scaffold(NS)constructed by coaxial electrospinning technique with enhanced osteogenic bioactivities and mechanical properties by incorporating biocompatible magnetic iron oxide nanoparticles(IONPs)and icaritin(ICA).Four types of nano-scaffolds(NS,ICA@NS,NS-IONPs and ICA@NS-IONPs)were prepared.The incorporation of ICA and IONPs minimally impact their surface morphological and chemical properties.IONPs enhanced the mechanical properties of NS scaffolds,including hardness,tensile strength,and elastic modulus.In vitro assessments demonstrated that ICA@NS-IONPs exhibited enhanced osteogenic bioactivities towards mouse calvarial pre-osteoblast cell line MC3T3-E1 as evidenced by detecting the alkaline phosphatase(ALP)activity level,expressions of osteogenesis-related genes and proteins as well as mineralized nodule formation.Mechanistic investigations revealed that MEK/ERK(MAP kinase-ERK kinase(MEK)/extracellularsignal-regulated kinase(ERK))signaling pathway could offer a plausible explanation for the osteogenic differentiation of MC3T3-E1 cells induced by ICA@NS-IONPs.Furthermore,the implantation of nano-scaffolds in rat skull defects exhibited a substantial improvement in in vivo bone regeneration.Therefore,IONPs and ICA incorporated coaxial electrospinning nano-scaffolds present a novel strategy for the optimization of scaffolds for bone tissue engineering.

异鼠李素与索拉非尼联合对肾癌的抑制作用及作用机制

本研究主要探讨黄芪成分异鼠李素与索拉非尼联合使用后对肾癌的生长抑制作用及其作用机制。MTT法检测异鼠李素及与索拉非尼联合后的抗肿瘤活性;采用小鼠Renca移植瘤模型观察其对肾癌生长的抑制作用。Western blot初步探讨异鼠李素及与索拉非尼联合后抗肾癌作用机制。淋巴细胞增殖实验检测体内异鼠李素及与索拉非尼联合后对小鼠脾淋巴细胞增殖影响。结果显示,异鼠李素对不同肾癌细胞增殖有较弱的抑制作用,其作用于A498细胞、786-O细胞和Renca细胞120 h后的半数抑制浓度(IC50)分别是31.7、28.8和106.0μmol·L^-1,异鼠李素联合索拉非尼可增加索拉非尼对Renca细胞的生长抑制作用,索拉非尼的IC50由26.1μmol·L^-1降至12.0μmol·L^-1;单独使用50.0 mg·kg^-1异鼠李素能较弱地抑制小鼠Renca肾癌的生长,抑制率仅为26.9%,而此剂量的异鼠李素和20.0 mg·kg-1索拉非尼联合使用时抑制率可达60.7%,提示异鼠李素可增加索拉非尼的抗肾癌活性。Western blot结果表明,异鼠李素可通过抑制c-Raf/MEK/ERK、AKT/mTOR等信号通路发挥抑制肾癌生长的作用。与对照组相比,异鼠李素与索拉非尼体内联合后能促进未活化的脾淋巴细胞增殖及刀豆蛋白A和脂多糖刺激后的淋巴细胞增殖。综上所述,异鼠李素与索拉非尼联合可增强索拉非尼的抗肾癌活性,其作用机制可能与抑制c-Raf/MEK/ERK和AKT/mTOR信号通路有关。动物实验中对动物的处理均遵循中国医学科学院药物研究所动物实验中心标准操作规程。