mTOR和ERK/MAPK信号通路调控自噬在孤独症发病中的作用

孤独症是一种以重复刻板样行为和社交缺陷为主要特征的神经发育障碍性疾病,发病率高的特点使其逐渐成为研究的热点。中国与西方国家孤独症的发病率相似,约为1%,位于儿童精神疾病的前列^([1])。目前认为孤独症由环境和遗传因素共同决定,病因复杂,具体机制尚不明确。随着研究的深入,自噬在孤独症发病机制中的作用受到广泛关注。

Silvestrol alleviates glioblastoma progression through ERK pathway modulation and MANBA and NRG-1 expression

Background:Glioblastoma,a notably malignant tumor within the central nervous system,is distinguished by its aggressive behavior.Silvestrol,a robust inhibitor of the RNA helicase eukaryotic initiation factor 4A(eIF4A),has shown significant potential as an anticancer compound.Yet,the impact of silvestrol on glioblastoma,especially its molecular mechanisms,has not been fully elucidated.Methods:This investigation employed a variety of in vitro assays,such as cell counting kit-8(CCK-8),clonogenic,5-ethynyl-2′-deoxyuridine(EDU),wound healing,and flow cytometry,to evaluate cell cycle progression,apoptosis,cell viability,and migration.Western blot analysis was also performed to study the apoptosis and extracellular regulated kinase(ERK)pathways.After the ERK pathway was inhibited,differentially expressed genes(DEGs)in U87 cells were identified,followed by an analysis of target genes using the gene expression profiling interactive analysis(GEPIA)database.Results:Silvestrol significantly suppressed the proliferation,migration,and colony formation of glioma cells.It caused cell cycle arrest and enhanced apoptosis in these cells.Additionally,silvestrol stimulated the ERK pathway,with these effects being reversible by an ERK phosphorylation inhibitor.Transcriptome combined with GEPIA,GSCA,UALCAN,TIMER database screened 4 potential drug targets of silvestrol:chromosome 1 open reading frame 226(C1ORF226),mannosidase beta A(MANBA),IQ motif and Sec7 domain 2(IQSEC2),neuregulin 1(NRG-1).Among them,C1ORF226 was lower risk gene while MANBA,IQSEC2,and NRG-1 were high-risk genes.Furthermore,silvestrol notably reduced MANBA mRNA levels,which could be reversed by inhibiting ERK phosphorylation.Furthermore,silvestrol markedly decreased NRG-1 protein levels,with an additional reduction observed when the ERK pathway was blocked.Conclusion:Silvestrol’s anti-glioma effects are primarily due to the suppression of MANBA expression via the ERK pathway and possibly by hindering the translation of NRG-1 protein,thus reducing its expression.The downregulation of MANBA and NRG-1 proteins may be crucial in hindering glioma development and progression.These results highlight the intricate relationship between the ERK pathway and gene expression regulation in silvestrol’s therapeutic effectiveness against glioma.

Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

Alleviatory effect of isoquercetin on benign prostatic hyperplasia via IGF-1/PI3K/Akt/mTOR pathway

We evaluated the effect of isoquercetin(quercetin-O-3-glucoside-quercetin,IQ)as a functional component of Abeliophyllum disistichum Nakai ethanol extract(ADLE)on prostate cell proliferation and apoptosis and its effects on the IGF-1/PI3K/Akt/mTOR pathway in benign prostatic hyperplasia(BPH).Metabolites in ADLE were analyzed using UHPLC-qTOF-MS and HPLC.IQ was orally administered(1 or 10 mg/kg)to a testosterone propionate-induced BPH rat model,and its effects on the prostate weight were evaluated.The effect of IQ on androgen receptor(AR)signaling was analyzed in LNCaP cells.Whether IGF-1 and IQ affect the IGF-1/PI3K/Akt/mTOR pathway in BPH-1 cells was also examined.The metabolites in ADLE were identified and quantified,which confirmed that ADLE contained abundant IQ(20.88 mg/g).IQ significantly reduced the prostate size in a concentration-dependent manner in a BPH rat model,and significantly decreased the expression of AR signaling factors in the rat prostate tissue and LNCaP cells in a concentration-dependent manner.IQ also inhibited the PI3K/AKT/mTOR pathway activated by IGF-1 treatment in BPH-1 cells.In BPH-1 cells,IQ led to G0/G1 arrest and suppressed the expression of proliferation factors while inducing apoptosis.Thus,IQ shows potential for use as a pharmaceutical and nutraceutical for BPH.

香芹酚调节ERK/mTOR途径抑制宫颈癌细胞增殖及诱导细胞凋亡的作用研究

目的:探讨香芹酚可否通过介导ERK/mTOR途径抑制宫颈癌细胞增殖并诱导其凋亡。方法:宫颈癌HeLa和caski细胞分为空白对照组及香芹酚低(50μmol/L)、中(150μmol/L)、高(450μmol/L)浓度组,培养24 h, CCK-8法检测细胞增殖抑制率;克隆形成实验检测细胞的集落形成能力;AnnexinV-FITC/PI双染法检测细胞凋亡率;Hoechst 33258荧光染色检测细胞凋亡形态;Western Blot法检测细胞中p-ERK1/2/ERK1/2、p-mTOR/mTOR和LC3Ⅰ/LC3Ⅱ水平及cleaved Caspase-3、Bcl-2、Bax、Beclin-1、P62蛋白表达。结果:与空白对照组比较,香芹酚各浓度组HeLa和caski细胞增殖抑制率、凋亡率及cleaved Caspase-3、Bax、Beclin-1蛋白表达显著升高,细胞集落形成数显著减少,Bcl-2、P62蛋白表达及p-ERK1/2/ERK1/2、p-mTOR/mTOR、LC3Ⅰ/LC3Ⅱ水平显著降低(P<0.05)。结论:香芹酚可抑制宫颈癌细胞增殖并诱导凋亡,其作用机制可能与抑制ERK/mTOR途径进而促进癌细胞自噬发生有关。

基于ERK/mTOR信号通路探讨六味地黄丸对氧化应激状态下成骨细胞自噬的影响

目的从ERK/mTOR信号通路观察六味地黄丸对氧化应激状态下MC3T3-E1细胞自噬的影响,探讨其治疗绝经后骨质疏松症的作用机制。方法用过氧化氢(H 2O 2)模拟氧化应激状态,六味地黄丸(LWDH)含药血清干预MC3T3-E1细胞,将细胞分为Model组(H 2O 2干预)、Blank组(空白血清)、LWDH组(LWDH含药血清)、Rap组(mTOR通路抑制剂)、U0126组(ERK通路抑制剂)、Rap+LWDH组(mTOR通路抑制剂、LWDH含药血清)、U0126+LWDH组(ERK通路抑制剂、LWDH含药血清);以不做干预的Control组作为对照。通过细胞活性氧(reactive oxygen species,ROS)检测细胞ROS水平;检测自噬蛋白LC3B及ERK/mTOR信号通路相关蛋白mTOR、p-mTOR、ERK1/2和p-ERK1/2的表达。结果ROS结果显示,与Model组相比,LWDH组、Rap组、U0126等各组细胞的ROS水平皆降低(P<0.05)。Western blot结果显示,与Model组相比,LWDH组、Rap组、LWDH+Rap组蛋白LC3Ⅱ/Ⅰ增高(P<0.05);与Model组相比,LWDH组、Rap组与Rap+LWDH组的p-mTOR蛋白表达下调(P<0.05),LWDH组、U0126组的p-ERK1/2蛋白表达下调(P<0.05)。结论六味地黄丸治疗绝经后骨质疏松症的机制可能与抑制ERK/mTOR信号通路诱导氧化应激状态下成骨细胞自噬相关。

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral

Background:Oral squamous cell carcinoma(OSCC)is a common malignant tumor.Recently,Laminin Gamma 2(LAMC2)has been shown to be abnormally expressed in OSCC;however,how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored.This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC.Methods:To explore the mechanism by which LAMC2 is highly expressed in OSCC,we used small interfering RNA(siRNA)to knock down LAMC2 to further observe the changes in the signaling pathway.Furthermore,we used cell proliferation assays,Transwell invasion assays,and wound-healing assays to observe the changes in OSCC proliferation,invasion,and metastasis.RFP-LC3 was used to detect the level of autophagy intensity.A cell line-derived xenograft(CDX)model was used to detect the effect of LAMC2 on tumor growth in vivo.Results:This study found that the level of autophagy was correlated with the biological behavior of OSCC.The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation,invasion,and metastasis via inhibiting the PI3K/AKT/mTOR pathway.Moreover,autophagy has a dual effect on OSCC,and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis,invasion,and proliferation via the PI3K/AKT/mTOR pathway.Conclusions:LAMC2 interacts with autophagy to regulate OSCC metastasis,invasion,and proliferation via the PI3K/AKT/mTOR pathway.LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration,invasion,and proliferation.

How is the AKT/mTOR pathway involved in cell migration and invasion?

As a pathway that plays a role in nutrient absorption,anabolic response,cell growth and survival,the important role of AKT/mTOR in tumorigenesis has also come to light.For cancer patients,most deaths are caused by the growth of metastatic tumors outside the primary focus.Therefore,migration and invasion in the late stage of tumor progression are the main unresolved issues in the study of tumor pathogenesis,and AKT/mTOR has been found to participate in the migration and invasion of cancer cells,which means that the study of this pathway may contribute to a solution for the problem.Because of its extensive and complex functions in the organism,this pathway can be regulated by a variety of different signals in the body,and then realize its function through different downstream signal molecules.This article reviews the proteins that can indirectly affect this pathway by regulating the common upstream signaling molecules of this pathway,and the proteins that can directly affect the level of phosphorylation of AKT/mTOR in cancer cells.We also review the proteins that can co-regulate this pathway and its downstream pathways.Through this study,we hope to gain a deeper understanding of the regulatory mechanism of the AKT/mTOR pathway in cancer cells,in hopes of finding effective and harmless cancer treatment targets in the future.

Echinoside A from Pearsonothuria graeffei Exert the Cytotoxicity to MDA-MB-231 Cells via Mitochondrial Membrane and Modulation of PI3K/Akt/mTOR Pathway

A kind of triterpene glycosides echinoside A(EA)was extracted from sea cucumber Pearsonothuria graeffei,and its yield was about 0.78%.The purity of EA was 99.0%,and its molecular weight was 1206 Da.EA was a linear tetrasaccharide attached to a pentacyclic triterpene aglycon.It inhibited the growth of MDA-MB-231 cells in vitro.The antitumor effect was related to elevate ROS level,decrease mitochondrial membrane potential,enhance caspase-3 expression,induce cells apoptosis and arrest cell cycle at G2/M phase.EA also dose-dependently suppressed the expressions of phophorylation proteins p-PI3K,p-Akt,and p-mTOR as analyzed by western blotting.These results suggested that EA caused MDA-MB-231 cells apoptosis via intrinsic mitochondrial and PI3K/Akt/mTOR pathway.EA can be a potential anti-breast cancer agent to enhance the clinical efficacy.

Acupuncture at Back-Shu point improves insomnia by reducing inflammation and inhibiting the ERK/NF-κB signaling pathway

BACKGROUND Insomnia is a disease where individuals cannot maintain a steady and stable sleep state or fail to fall asleep.Western medicine mainly uses sedatives and hypnotic drugs to treat insomnia,and long-term use is prone to drug resistance and other adverse reactions.Acupuncture has a good curative effect and unique advantages in the treatment of insomnia.AIM To explore the molecular mechanism of acupuncture at Back-Shu point for the treatment of insomnia.METHODS We first prepared a rat model of insomnia,and then carried out acupuncture for 7 consecutive days.After treatment,the sleep time and general behavior of the rats were determined.The Morris water maze test was used to assess the learning ability and spatial memory ability of the rats.The expression levels of inflammatory cytokines in serum and the hippocampus were detected by ELISA.qRTPCR was used to detect the mRNA expression changes in the ERK/NF-κB signaling pathway.Western blot and immunohistochemistry were carried out to evaluate the protein expression levels of RAF-1,MEK-2,ERK1/2 and NF-κB.RESULTS Acupuncture can prolong sleep duration,and improve mental state,activity,diet volume,learning ability and spatial memory.In addition,acupuncture increased the release of 1L-1β,1L-6 and TNF-αin serum and the hippocampus and inhibited the mRNA and protein expression of the ERK/NF-κB signaling pathway.CONCLUSION These findings suggest that acupuncture at Back-Shu point can inhibit the ERK/NF-κB signaling pathway and treat insomnia by increasing the release of inflammatory cytokines in the hippocampus.

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway

The current study aimed to assess the effect of timosaponin AⅢ(T-AⅢ)on drug-metabolizing enzymes during anticancer therapy.The in vivo experiments were conducted on nude and ICR mice.Following a 24-day administration of T-AⅢ,the nude mice exhibited an induction of CYP2B10,MDR1,and CYP3A11 expression in the liver tissues.In the ICR mice,the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration.The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6,MDR1,and CYP3A4,along with constitutive androstane receptor(CAR)activation.Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression.Furthermore,other CAR target genes also showed a significant increase in the expression.The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice.Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation,with this effect being partially reversed by the ERK activator t-BHQ.Inhibition of the ERK1/2 signaling pathway was also observed in vivo.Additionally,T-AⅢ inhibited the phosphorylation of EGFR at Tyr1173 and Tyr845,and suppressed EGF-induced phosphorylation of EGFR,ERK,and CAR.In the nude mice,T-AⅢ also inhibited EGFR phosphorylation.These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

mTOR抑制剂联合熊去氧胆酸对肝癌大鼠抑制、免疫逃逸及Ras/ERK信号通路的影响

目的探究mTOR抑制剂联合熊去氧胆酸(UDCA)对肝癌大鼠抑制、免疫逃逸及Ras/ERK信号通路的影响。方法SPF级SD雄性大鼠60只,10只作为正常(NO)组,50只腹腔注射二乙基亚硝铵进行肝癌建模。建模成功后,取40只大鼠随机分为模型(MO)组,mTOR抑制剂(MI)组,UDCA(UD)组,mTOR抑制剂联合UDCA(UN)组,每组10只。MI组灌胃mTOR抑制剂西罗莫司2 mg/kg,UD组灌胃UDCA 30 mg/kg,UN组灌胃mTOR抑制剂西罗莫司2 mg/kg及30 mg/kg UDCA,NO组、MO组灌胃同体积0.9%NaCl溶液。观察大鼠肿瘤生长情况、肝组织病理形态,ELISA法检测免疫逃逸相关因子,免疫印迹法检测Ras/ERK信号通路相关蛋白表达。结果与NO组相比,MO组体重显著降低(P<0.05),肝重、肝脏系数显著升高(P<0.05);与MO组相比,MI组体重明显升高(P<0.05),肝重、肝脏系数显著降低(P<0.05);与MI组相比,UD组体重、肝重、肝脏系数无明显差异(P>0.05);与UD组相比,UN组体重明显升高(P<0.05),肝重、肝脏系数显著降低(P<0.05)。与MO组相比,MI组抑瘤曲线明显升高(P<0.05);与MI组相比,UD组抑瘤曲线无明显差异(P>0.05);与UD组相比,UN组抑瘤曲线明显升高(P<0.05)。NO组肝组织及肝小叶结构清晰完整,肝细胞分界清晰且呈放射状排列,未见增生坏死细胞及炎性细胞,MO组肝组织肝索结构不清,肝细胞排列紊乱且细胞肿胀,可见癌巢及大量炎性细胞浸润。与MO组比较,MI组、UD组、UN组病理结构明显改善,炎性细胞及癌细胞明减少。与NO组比较,MO组大鼠血清IL-4、IL-10、TGF-β1含量显著升高(P<0.05);与MO组比较,MI组、UD组、UN组大鼠血清IL-4、IL-10、TGF-β1含量显著降低(P<0.05),且UD组与MI组相比基本无差异(P>0.05),UN组比UD组降低显著(P<0.05)。NO组、MO组、MI组、UD组、UN组Ras蛋白表达分别为1.16±0.11、2.26±0.29、1.51±0.17、1.55±0.18、1.19±0.14,p-ERK蛋白表达分别为1.05±0.10、2.94±0.28、1.66±0.14、1.68±0.16、1.14±0.11;与NO组比较,MO组大鼠肝组织Ras、p-ERK蛋白表达显著升高(P<0.05);与MO组比较,MI组、UD组、UN组大鼠肝组织Ras、p-ERK蛋白表达显著降低(P<0.05),且UD组与MI组相比基本无差异(P>0.05),UN组比UD组降低显著(P<0.05)。结论mTOR抑制剂联合UDCA可显著抑制肝癌大鼠肿瘤生长,抑制免疫逃逸及Ras/ERK信号通路激活。

Research progress of TCM regulating mTOR signaling pathway in the treatment of Alzheimer's disease

Alzheimer's disease(AD)is a degenerative disease of the central nervous system.The pathogenesis of AD is complex and diverse,and its occurrence and development is the result of the interaction of multiple factors.A number of studies have shown that mTOR signaling pathway is closely related to AD.In recent years,people in exploring relevant methods for the treatment of AD and the process of drugs,more and more studies have found that traditional Chinese medicine compound traditional Chinese medicine monomer,and can be applied to mTOR signaling pathway to improve symptoms in patients with AD.This paper will review the mechanism of action and treatment of TCM in Alzheimer's disease based on mTOR signaling pathway in recent years,so as to provide reference and expand thinking for the prevention and treatment of AD.

青藤碱通过MAPK/ERK/mTOR通路诱导自噬抑制人食管癌TE-1细胞增殖

目的:研究青藤碱对人食管癌TE-1细胞增殖、自噬的作用和机制。方法:将TE-1细胞分成对照组、青藤碱200 mg/L组、青藤碱400 mg/L组、青藤碱800 mg/L组。对照组不做处理,青藤碱200 mg/L组、青藤碱400 mg/L组、青藤碱800 mg/L组分别予以相应浓度的青藤碱干预。光学显微镜观察细胞形态改变,Hochest染色检测活细胞数,CCK8法检测细胞抑制率,流式细胞术检测细胞凋亡情况,透射电镜观察自噬小体情况,免疫荧光检测LC3表达情况,Western blotting检测LC3-Ⅰ、LC3-Ⅱ、Beclin-1、ERK1/2、p-ERK1/2、mTOR、p-mTOR蛋白相对表达量。结果:在200、400、800 mg/L青藤碱的作用下,TE-1细胞皱缩、变圆,漂浮在培养液中,活细胞数不断减少。青藤碱200 mg/L组、青藤碱400 mg/L组、青藤碱800 mg/L组活细胞数比例显著低于对照组(P<0.01),细胞抑制率、细胞凋亡率显著高于对照组(P<0.01);青藤碱200 mg/L组、青藤碱400 mg/L组、青藤碱800 mg/L组TE-1细胞内自噬小体多于对照组;青藤碱200 mg/L组、青藤碱400 mg/L组、青藤碱800 mg/L组TE-1细胞LC3表达、LC3-Ⅱ/LC3-Ⅰ比值、Beclin-1蛋白相对表达量均显著高于对照组(P<0.01),p-ERK1/2/ERK1/2及p-mTOR/mTOR比值均显著低于对照组(P<0.01)。结论:青藤碱能抑制人食管癌TE-1细胞增殖,其机制与抑制MAPK/ERK/mTOR通路和诱导自噬有关。

观察ERK/mTOR通路在六味地黄丸调控自噬骨细胞应激反应的影响

目的:观察以ERK/mTOR通路的角度分析六味地黄丸含药血清的调控自噬,对改善骨细胞应激反应的影响。方法:将60只健康SPF级SD的大鼠作为研究对象,研究人员于模型组大鼠体外培养MC3T3-E1细胞,并进行24h的0.04~0.79mmol/LH 2O细胞干预随机分为3组:六味地黄丸组(给予六味地黄丸治疗)、紫檀芪组(给予紫檀芪治疗)及对照组(无任何治疗方案),各20只,治疗14周。给药结束后检测氧化应激指标、各组自噬相关LC3B mRNA含量、ERK1/2及p-ERK1/2蛋白表达水平。结果:研究结果显示,伴随时间延长,与对照组相比,六味地黄丸组及紫檀芪组的SOD水平数值均得到显著提升,MDA水平数值明显下降(P<0.05)。且六味地黄丸组SOD水平数值高于紫檀芪组,MDA水平数值低于紫檀芪组(P<0.05);与对照组和紫檀芪组相比,六味地黄丸组的自噬相关LC3B mRNA含量明显提升,且紫檀芪组高于对照组,差异存在统计学意义(P<0.05)。说明六味地黄丸可改善应激状态下的成骨细胞自噬水平的提升;与对照组相比,六味地黄丸组和紫檀芪组的ERK1/2及p-ERK1/2蛋白表达水平下降,差异存在统计学意义(P<0.05)。但六味地黄丸组和紫檀芪组的ERK1/2及p-ERK1/2蛋白表达水平对比无统计学意义(P>0.05)。说明六味地黄丸可利用压制ERK/mTOR通路以诱导自噬细胞降低成骨细胞的损伤。结论:六味地黄丸可改善机体应激指标,提升MC3T3-E1细胞的细胞活性,提升LC3B mRNA含量,降低的ERK1/2及p-ERK1/2蛋白表达水平。

Novel insights into mTOR signalling pathways: A paradigm for targeted tumor therapy

As a crucial protein kinase,the mammalian target of rapamycin(mTOR)intimately controls essential cellular processes like cell development,proliferation,metabolism,and other crucial activities.Different cancers and disorders have been linked to imbalances in mTOR's regulatory systems.Multiple mTOR inhibitor therapy has recently acquired popularity as a method of treating cancers brought on by abnormal signal transduction pathways.We also explore potential processes behind tumor cell resistance to mTOR inhibitors and suggest workarounds to overcome this challenge.We hold the potential to pioneer cutting-edge methods for tumor therapy by methodically examining the complex mTOR signaling system and its regulatory complexity.Increasing our knowledge of mTOR-related mechanisms not only creates opportunities for cutting-edge methods to target and treat cancers but also has the potential to improve patient outcomes and general quality of life significantly.This review paper explores the most recent developments in understanding mTOR signaling pathways and the use of mTOR inhibitors in treating tumors.

运动骨骼肌ERK1/2与mTOR通路关系的研究

目的:通过阻断ERK1/2和m TOR通路,探讨运动中ERK1/2通路和m TOR通路在促进骨骼肌蛋白质合成中的关系。方法:7w龄SD雄性大鼠随机分为9组:安静对照组(C)、m TOR阻断组(R)、ERK1/2阻断组(P)、m TOR+ERK1/2阻断组(RP)、运动组(E)、运动m TOR阻断组(ER)、运动ERK1/2阻断组(EP)、运动+m TOR+ERK1/2阻断组(ERP)、DMSO组(D)。m TOR阻断干预组于运动前2h以1.5mg/kg剂量腹腔注射雷帕霉素,ERK1/2阻断干预组于运动前30min以5mg/kg剂量腹腔注射PD98059,连续注射10d。于末次运动后6h取趾长伸肌,测定其湿重以及ERK1/2通路和m TOR通路信号分子的磷酸化表达。结果:与C组相比,R组趾长伸肌湿重减少了15.9%(P<0.01),P组未有显著变化,RP组减少了20.3%(P<0.01),E组增加了5.3%,但无显著性差异;与E组相比,ER组减少了16.1%(P<0.01),ERP组减少了19.8%(P<0.01),而EP组未有显著变化。与C组相比,P组m TOR通路的蛋白磷酸化表达均显著下降(P<0.01-0.05),R组MEK1/2的磷酸化表达增强(P<0.01),RP组ERK1/2通路和m TOR通路的磷酸化表达均显著下降(P<0.01-0.05),E组m TOR通路和ERK1/2通路的磷酸化表达显著升高(P<0.01-0.05)。与E组相比,EP组m TOR通路的蛋白磷酸化表达显著下降(P<0.01-0.05),而ER组ERK1/2通路的磷酸化表达未有显著变化,ERP组ERK1/2通路和m TOR通路的磷酸化表达均显著下降(P<0.01)。结论:在运动中m TOR通路和ERK1/2通路可协同促进骨骼肌蛋白质合成;并且ERK1/2通路位于m TOR通路的上游,可正向调节m TOR通路的活性。

Discovering differential protein expression caused by CagA-induced ERK pathway activation in AGS cells using the SELDI-ProteinChip platform

AIM： To identify the protein expression differences related to the CagA-induced ERK pathway activation in AGS cells. METHODS： Human AGS cells transfected with cagA and blank vector were treated with specific mitogenactivated protein kinase kinase （MEK） inhibitor. Total cell proteins were combined by strong anion exchange （SAX2） and weak cation exchange （CM10） ProteinChip arrays and analyzed using surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry （SELDI-TOF- MS） proteomics technology. Protein expression profiles were compared with those of inhibitor-untreated cagA transfectants. SwissProt/TrEMBL database searching for differentially expressed proteins was carried out using the TagIdent tool with the pI and mass information. RESULTS： When a total of 16 proteins that showed expression differences in inhibitor-untreated cagA transfectants were compared with vector transfectants, three proteins with m/z 4229, 8162 and 9084 were found to have no expression differences after treatment with MEK inhibitor, while the other 13 maintained the same expression differences after inhibitor treatment. Seven pieces of meaningful matching information for the three proteins were obtained from database searching. CONCLUSION： Biomarkers with m/z 4229, 8162 and 9084 are ERKI/2 phosphorylation dependent, andtherefore are the downstream molecules of ERK1/2 in the ERK/MAPK signaling pathway. The three biomarkers may be important cancer-associated proteins according to SwissProt/TrEMBL database information.

Estrogen up-regulates MMP2/9 expression in endometrial epithelial cell via VEGF-ERK1/2 pathway

Objective:To study the effect of estrogen on anovulatory dysfunctional uterine bleeding(ADUB).Methods:Primary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolylic activity of gelalinase was determined by gelatin zymography analysis.Cellular mRNA and protein synthesis was blocked respectively to determine whether the increased expression of MMP-2/9 was induced by estrogen.The expression of VEGF was blocked by siRNA.After treatment with various factors.MMP-9,VEGF,total Erk and phosphorylated Erk expression in primary uterine epithelial cells was detected by Western blotting analysis.Cell MMP-2/9mRNA levels was measured by real-time RT-PCR.Results:The activity and expression of MMP2/9 was inereased in the endometrium of patients with ADUB.Estrogen could up-regulate the expression of VEGF and activate Erk 1/2-Elk1 signal path.After interference by siRNA,ERK1/2 pathway was blocked in cells,and the expression of MMP-2/9 was down-regulated.ERK1/2 specific blocker U0126 blocked ERK phosphorylation,and it could down-regulate the expression of MMP-2/9.Conclusions:The results showed that the estrogen can increase the expression of VEGF,and thus activate ERK1/2 pathway to induce MMP-2/9 expression.

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.