葫芦素B通过AKT/mTOR和MAPK信号通路诱导非小细胞肺癌细胞自噬

目的:观察葫芦素B(Cucurbitacin B,CuB)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞自噬的影响,探讨其可能的机制。方法:应用CCK-8法测定不同浓度CuB对A549细胞及H1299细胞增殖能力的影响。应用丹酰戊二胺(monodansylcadaverine,MDC)染色,荧光显微镜下观察细胞自噬,应用透射电镜观察自噬小体和自噬溶酶体。另外,应用自噬双标腺病毒(mRFP-GFP-LC3)进行转染,使用共聚焦显微镜观察药物处理后的自噬流变化。Western blot方法检测自噬相关标志物LC3II/I、p62及Beclin-1的表达水平变化。应用Western blot考察AKT、mTOR、ERK、p38、JNK蛋白及其磷酸化蛋白表达水平变化。结果:CuB呈剂量依赖抑制A549及H1299细胞增殖。MDC染色荧光显微镜下观察,药物处理组可见明显绿色致密斑点;药物处理48 h后透射电镜观察可见自噬溶酶体;mRFP-GFP-LC3转染A549及H1299细胞后显示CuB组红色荧光增加,提示自噬流增加;0.04μmol/L的CuB处理A549及1299细胞48 h后,LC3II/I及Beclin-1的蛋白表达增加,p62表达水平降低,AKT及mTOR蛋白磷酸化水平表达降低,ERK、JNK、p38 MAPK磷酸化蛋白水平升高。结论:本研究首次阐明了CuB在非小细胞肺癌中具有诱导自噬的能力,这种作用可能与抑制AKT/mTOR、激活MAPK信号通路有关。

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。

Cyanidin-3-glucoside protects the photooxidative damage of retinal pigment epithelium cells by regulating sphingolipid signaling and inhibiting MAPK pathway

Cyanidin-3-glucoside(C3G)is the most common anthocyanin in dark grains and berries and is a food functional factor to improve visual health.However,the mechanisms of C3G on blue light-induced retinal pigment epithelial(RPE)cell photooxidative damage needs further exploration.We investigated the effects of C3G on blue light-irradiated A2E-containing RPE cells and explored whether sphingolipid,mitogen-activated protein kinase(MAPK),and mitochondria-mediated pathways are involved in this mechanism.Blue light irradiation led to mitochondria and lysosome damage in RPE cells,whereas C3G preserved mitochondrial morphology and function and maintained the lysosomal integrity.C3G suppressed the phosphorylation of JNK and p38 MAPK and mitochondria-mediated pathways to inhibit RPE cell apoptosis.Lipidomics data showed that C3G protected RPE cells against blue light-induced lipid peroxidation and apoptosis by maintaining sphingolipids balance.C3G significantly inhibited ceramide(Cer d18:0/15:0,Cer d18:0/16:0 and Cer d18:0/18:0)accumulation and elevated galactosylceramide(GalCer d18:1/15:0 and GalCer d18:1/16:0)levels in the irradiated A2E-containing RPE cells.Furthermore,C3G attenuated cell membrane damage by increasing phosphatidylcholine and phosphatidylserine levels.C3G inhibited apoptosis and preserved the structure of mitochondria and lysosome by regulating sphingolipid signaling and suppression of MAPK activation in RPE cells.Thus,dietary supplementation of C3G prevents retinal photooxidative damage.

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.

Effect of ginsenoside Rg1 on hematopoietic stem cells in treating aplastic anemia in mice via MAPK pathway

BACKGROUND Aplastic anemia(AA)presents a significant clinical challenge as a life-threatening condition due to failure to produce essential blood cells,with the current the-rapeutic options being notably limited.AIM To assess the therapeutic potential of ginsenoside Rg1 on AA,specifically its protective effects,while elucidating the mechanism at play.METHODS We employed a model of myelosuppression induced by cyclophosphamide(CTX)in C57 mice,followed by administration of ginsenoside Rg1 over 13 d.The invest-igation included examining the bone marrow,thymus and spleen for pathological changes via hematoxylin-eosin staining.Moreover,orbital blood of mice was collected for blood routine examinations.Flow cytometry was employed to identify the impact of ginsenoside Rg1 on cell apoptosis and cycle in the bone marrow of AA mice.Additionally,the study further evaluated cytokine levels with enzyme-linked immunosorbent assay and analyzed the expression of key proteins in the MAPK signaling pathway via western blot.RESULTS Administration of CTX led to significant damage to the bone marrow’s structural integrity and a reduction in hematopoietic cells,establishing a model of AA.Ginsenoside Rg1 successfully reversed hematopoietic dysfunction in AA mice.In comparison to the AA group,ginsenoside Rg1 provided relief by reducing the induction of cell apoptosis and inflammation factors caused by CTX.Furthermore,it helped alleviate the blockade in the cell cycle.Treatment with ginsenoside Rg1 significantly alleviated myelosuppression in mice by inhibiting the MAPK signaling pathway.CONCLUSION This study suggested that ginsenoside Rg1 addresses AA by alleviating myelosuppression,primarily through modulating the MAPK signaling pathway,which paves the way for a novel therapeutic strategy in treating AA,highlighting the potential of ginsenoside Rg1 as a beneficial intervention.

Investigation on the mechanism of Qiangxinhuoli prescription in the treatment of chronic heart failure based on p38-MAPK signaling pathway

Background:The aim of this study is to investigate the mechanism of action underlying the therapeutic effects of the national patent Chinese medicine compound“Qiangxinhuoli prescription(QXHLF)”on chronic heart failure(CHF).Methods:In vitro,the H_(9)C_(2) cell model was induced by ANGII,and cell proliferation and related protein expression were detected by Cell Counting Kit-8 and Western blot.In vivo,A rat model of CHF was prepared by ligation of the left anterior descending coronary artery.The effects of QXHLF on cardiac function in CHF rats were evaluated by cardiac index,hemodynamic changes,enzyme-linked immunosorbent assay,hematoxylin-eosin staining,immunohistochemistry,Western blot and RT-PCR.The expression of pro-apoptotic factors and anti-apoptotic factors,as well as TGFβ1,p-p38,TAK 1 mRNA,and protein,were detected.Results:In vitro,QXHLF has a significant inhibitory effect on the proliferation of H_(9)C_(2) cells.QXHLF can reduce the expression levels of TAK 1,TGFβ1,p-p38,Caspase3 and BAX proteins in H_(9)C_(2) cells,and increase the expression level of BCL_(2) protein.In vivo,QXHLF has the potential to increase left ventricular systolic pressure,m aximum rate of change in left ventricular pressure while decreasing left ventricular end diastolic pressure,and inhibiting the serum levels of brain natriuretic peptide.Moreover,QXHLF exhibits significant improvements in the pathological alterations of myocardial cells and fibers in CHF rats,leading to enhanced myocardial tissue morphology and notable advantages in combating myocardial fibrosis.QXHLF can reduce the levels of BAX and Caspase3 and up-regulate the expression of BCL_(2),thereby inhibiting cardiomyocyte apoptosis.Furthermore,QXHLF demonstrates inhibitory effects on the mRNA and protein expression levels of TGFβ_(1),TAK_(1),and p-p38 in the heart tissue of the CHF rat model.Conclusion:These findings indicate that QXHLF has a therapeutic effect on CHF by inhibiting the p38-MAPK signaling pathway,reducing myocardial fibrosis,preventing apoptosis,inhibiting cell proliferation,and restoring myocardial injury.

阿霉素通过调节MAPK/mTOR通路抑制弥漫大B细胞淋巴瘤的发生

目的探讨阿霉素(Adriamycin,ADM)抑制弥漫大B细胞淋巴瘤(DLBCL)的发生过程,及MAPK/m TOR通路在此过程中的作用。方法 DLBCL细胞给予0、5和10μmol/L ADM培养12 h、24 h和48 h后,细胞计数CCK-8法检测DLBCL细胞的存活率;TUNEL法检测检测DLBCL细胞凋亡发生;Caspase 3活性检测试剂盒检测细胞凋亡蛋白Caspase 3活性;Western blot法检测DLBCL中磷酸化蛋白p38、JNK、ERK和m TOR蛋白表达水平。结果与对照组相比,阿霉素治疗组中DLBCL细胞存活率显著降低,而Caspase 3活性显著增加,差异具有统计学意义(P<0.05);与对照组相比,阿霉素治疗组中p38和JNK mRNA水平和磷酸化蛋白表达含量均明显增加,ERK mRNA水平和磷酸化蛋白表达无明显变化,而AKT mRNA水平和磷酸化蛋白的表达含量均明显下降,差异具有统计学意义(P<0.05)。结论 MAPK/m TOR信号通路在阿霉素抑制DLBCL细胞生长,并诱导细胞凋亡发生的过程中起到重要作用。

紫杉醇联合卡培他滨调控MAPK/mTOR信号抑制食管癌的机制研究

目的探讨紫杉醇联合卡培他滨调控MAPK/mTOR信号抑制食管癌的机制研究。方法选取2017年1月—2018年1月本院收集的80例晚期食管癌患者给予紫杉醇联合卡培他滨治疗2疗程,评价化疗临床疗效。免疫组织化学法检测化疗前后患者食管癌组织内MAPK和mTOR表达。选取食管癌Ec-9706细胞给予紫杉醇联合卡培他滨培养24 h、48 h和72 h,细胞计数CCK-8法检测食管癌Ec-9706细胞的存活率;Capase-3活性试剂盒检测Capase-3活性;Western blot法检测患者食管癌组织内和食管癌Ec-9706细胞中磷酸化蛋白p-MAPK、p-AKT、mTOR、NF-kB、BCL-2和cleaved-Capase-3蛋白表达水平。结果食管癌患者应用紫杉醇联合卡培他滨给药化疗后疗效为65.00%(52/80)。免疫组化法结果显示食管癌患者化疗后MAPK含量显著上升,mTOR含量显著下降,差异有统计学意义(P<0.05)。Western blot结果显示,食管癌患者应用紫杉醇联合卡培他滨给药化疗后,患者组织内p-MAPK、NF-kB和cleaved-Capase-3蛋白含量显著增加,p-AKT、mTOR和BCL-2蛋白含量显著降低,差异有统计学意义(P<0.05)。给予紫杉醇联合卡培他滨治疗后,食管癌细胞株的细胞存活率随时间的延长明显降低,Caspase-3活性随时间的延长显著升高,食管癌EC9706细胞p-MAPK、NF-kB和cleaved-Capase-3蛋白含量随时间的延长呈显著增加,p-AKT、mTOR和BCL-2蛋白含量随时间的延长呈显著降低,差异有统计学意义(P<0.05)。结论紫杉醇联合卡培他滨可能通过上调MAPK/mTOR信号抑制食管癌的发生与发展。

IGF-Ⅰ对大鼠骨骼肌AR、mTOR和MAPK信号通路的影响

目的研究通过注射外源性IGF-Ⅰ,研究其对骨骼肌雄激素受体(AR)、mTOR通路和MAPK通路的影响,探讨IGF-Ⅰ促进骨骼肌肥大的信号转导机制。方法 24只雄性成熟SD大鼠在适应性训练后,随机分为安静对照组(S组)、20 min组、60 min组和120 min组。腓肠肌内注射IGF-Ⅰ,分别于注射后20 min、60 min和120 min取白腓肠肌,并检测IGF-Ⅰ和AR基因表达,MHC含量及AR、MEK、ERK、p90RSK、mTOR、p70S6K和4EBP1磷酸化。结果(1)在注射IGF-Ⅰ 20 min后,AR基因和IGF-Ⅰ基因分别增至3.36倍和5.07倍,随后均逐渐回落;(2)肌肉注射IGF-Ⅰ未使MHC出现明显变化,注射60 min后,AR磷酸化增至1.43倍,然后回落;(3)在注射IGF-Ⅰ 20 min后,MEK、ERK和p90RSK磷酸化分别增至1.5倍、1.57倍和1.63倍,然后均逐渐回落;(4)注射IGF-Ⅰ20 min后,mTOR、p70S6K和4EBP1磷酸化分别增至1.53倍、1.45倍和1.42倍,而后逐渐回落。结论 (1)IGF-Ⅰ可使骨骼肌AR合成增强,且AR活性于注射后60 min达到峰值;(2)IGF-Ⅰ可使骨骼肌MAPK通路和mTOR通路活性迅速增强,且均于注射后20 min达到峰值。

MAPK与PI3K-AKT-mTOR通路双重抑制对去势抵抗性前列腺癌的作用机制研究

目的探讨丝裂原活化蛋白激酶(MAPK)与磷脂酰肌醇3-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-m TOR)通路双重抑制对去势抵抗性前列腺癌(CRPC)的作用机制。方法采用Western blot检测细胞中前列腺癌细胞(DU145和LNCa P)的细胞外信号调节激酶5(ERK5)、m TOR含量。应用RNA干扰(RNAi)技术建立基因降表达细胞模型,分别建立ERK5降表达、m TOR降表达、ERK5和m TOR同时降表达细胞模型。MTT法检测不同基因降表达对前列腺癌细胞的增殖的影响。比较CYP17A1在m TOR降表达DU145细胞和正常细胞中的表达。MTT法检测分别加入m TOR抑制剂(替西罗莫司)和CYP17A1抑制剂(阿比特龙)的DU145细胞的抑制率。采用半定量逆转录聚合酶链式反应(RT-PCR)测定ERK5和m TOR在前列腺癌、前列腺增生和CRPC组织中的表达。结果m TOR蛋白在DU145细胞中的表达显著高于LNCa P细胞(P<0.05)。ERK5降表达组、m TOR降表达组、ERK5+m TOR降表达组的LNCa P细胞的增殖未受显著抑制(P>0.05)。m TOR降表达组、ERK5+m TOR降表达组的DU145细胞抑制率显著高于ERK5降表达组(P<0.05)。CYP17A1在m TOR降表达DU145细胞中的表达显著低于正常DU145细胞,且呈剂量依赖(P<0.05)。替西罗莫司1μmol/L+阿比特龙0.5μmol/L组的DU145细胞抑制率显著高于单用替西罗莫司组和单用阿比特龙组(P<0.05)。ERK5和m TOR在前列腺癌和CRPC组织中均高表达,m TOR在CRPC组中的表达水平显著高于前列腺增生组和前列腺癌组(P<0.05)。结论m TOR蛋白在DU145细胞中、CRPC组织中表达显著高于ERK5,m TOR降表达能显著抑制DU145细胞增殖。提示CRPC以PI3K-Akt-m TOR通路高表达为主,m TOR对DU145细胞的抑制作用与其对CYP17A1的抑制作用有关,m TOR联合CYP17A1的抑制作用优于m TOR或CYP17A1的单独抑制。

硒对金黄色葡萄球菌感染的奶牛乳腺上皮细胞Nod2/MAPK/mTORs信号通路中关键因子mRNA转录水平的影响

为探究硒(Se)对金黄色葡萄球菌(S.aureus)感染的奶牛乳腺上皮细胞(bMECs) Nod2/MAPK/mTORs信号通路的调控机制,本研究首先用不同浓度硒(2μmol/L、4μmol/L和8μmol/L)对bMECs进行预孵育,12 h后再经S.aureus感染处理。分别于感染后6 h、8 h和10 h收集bMECs提取其RNA,应用q PCR方法检测bMECs中Nod2、RIP2、JNK、AKT、mTOR、IL-8和IL-10 mRNA的转录水平。结果显示,S.aureus能显著提高bMECs中Nod2、RIP2、JNK、AKT和mTOR mRNA的转录水平(p<0.01),而硒能不同程度的抑制这些因子mRNA的转录水平(p<0.05或p<0.01)。此外,S.aureus能显著或极显著提高bMECs中IL-8和IL-10 mRNA的转录水平(p<0.05或p<0.01),而硒对S.aureus感染的bMECs中IL-8和IL-10 mRNA的转录水平有明显抑制作用(p<0.05或p<0.01)。上述结果表明,硒可通过抑制Nod2/MAPK/mTORs信号通路的转导而减轻S.aureus诱导的bMECs的炎症反应。本研究为阐明硒能减轻S.aureus诱导的bMECs炎症反应的机制提供试验依据。

硒对S.aureus诱导的奶牛乳腺上皮细胞Nod2/MAPK/mTORs信号通路关键蛋白表达的影响

【目的】硒(Se)能否通过Nod2/MAPK/mTOR途径调控金黄色葡萄球菌诱导的奶牛乳腺上皮细胞炎性损伤,有待于进一步研究。因此本研究将探究硒对金黄色葡萄球菌(S. aureus)感染的奶牛乳腺上皮细胞(bMECs)Nod2/MAPK/mTORs信号通路中关键蛋白表达的影响,从而为阐明硒的免疫调控机制提供理论依据。【方法】首先将bMECs以10^6细胞/孔接种于6孔板中,当细胞超过80%的汇合度时,用含2、4和8μmol·L^-1浓度硒的培养基替换原来的培养基,继续孵育12 h,然后用PBS洗涤每孔3次,将S. aureus按MOI=1:1的比例加入6孔板中,继续培养0.5 h,然后收集bMECs细胞进行相关蛋白的检测。本试验共分3大组,即对照(Con)组(bMECs)、模型(Mod)组(bMECs+S. aureus)和试验组。其中试验组又分3个亚剂量组,即Low组(bMECs+2μmol·L^-1 Se+S. aureus)、Mid组(bMECs+4μmol·L^-1 Se+S. aureus)和Hig组(bMECs+8μmol·L^-1 Se+S. aureus),每组设3个重复。利用BCA蛋白测定试剂盒对收集的bMECs细胞进行总蛋白提取。应用Western blotting技术检测bMECs中Nod2和RIP2蛋白表达水平及JNK,AKT和mTOR蛋白磷酸化水平。将蛋白样品加到10%的SDS聚丙烯酰胺凝胶电泳中,上样量为20μg/孔,之后将蛋白转移到聚偏氟乙烯(PVDF)膜上。将PVDF膜用5 mL 5%脱脂乳阻断2 h,脱脂乳脱脂后用TBST清洗后,分别用5 mL的Nod2、RIP2、JNK、AKT、mTOR和β-actin的一抗孵育过夜,回收一抗。之后在PVDF膜中分别加入5 mL上述蛋白的二抗,室温孵育2 h,回收二抗。PVDF用TBST洗涤5次,最后在暗室条件下进行化学显影。【结果】S. aureus能显著提高bMECs中Nod2和RIP2蛋白表达水平及JNK,AKT和mTOR蛋白磷酸化水平(P<0.01)。S. aureus感染0.5 h后,Nod2蛋白水平显著升高(P<0.01)。在培养基里添加2μmol·L^-1的硒可极显著抑制Nod2蛋白的表达(P<0.01),在培养基里添加8μmol·L^-1的硒可显著抑制Nod2的表达(P<0.05);S. aureus感染0.5 h后,RIP2蛋白水平显著升高(P<0.05),而在培养基里添加8μmol·L^-1硒可显著抑制RIP2蛋白的表达(P<0.05);S. aureus感染0.5 h后,与对照组相比,模型组JNK蛋白磷酸化水平显著升高(P<0.01)。在培养基里添加4μmol·L^-1的硒能显著抑制JNK蛋白的磷酸化水平(P<0.05),在培养基里添加8μmol·L^-1的硒能显著抑制JNK蛋白的磷酸化水平(P<0.01);S. aureus感染0.5 h后,与对照组相比,模型组AKT蛋白磷酸化水平显著升高(P<0.01)。在培养基里添加4μmol·L^-1硒可极显著抑制JNK蛋白的磷酸化水平(P<0.01),在培养基里添加8μmol·L^-1硒可显著抑制AKT蛋白的磷酸化水平(P<0.05);S. aureus感染0.5 h后,模型组mTOR蛋白磷酸化水平显著升高(P<0.01)。在培养基里分别添加4μmol·L^-1和8μmol·L^-1硒均能显著抑制mTOR蛋白磷酸化水平(P<0.05)。【结论】硒可通过抑制bMECs Nod2/MAPK/mTORs信号通路中关键因子蛋白的表达而减轻S. aureus诱导的bMECs炎症反应。

MIF May Participate in Pathogenesis of Polycystic Ovary Syndrome in Rats through MAPK Signalling pathway

The polycystic ovary syndrome (PCOS) model was established in fats and correlation between the expression of macrophage migration inhibitory factor (MIF) and cytokinesis with the MAPK signalling pathway in the rat ovary was measured. The PCOS model in rats was established by dehydroepiandrosterone (DHEA).Thirty sexually immature female Sprague-Dawley rats were randomly and equally assigned to three groups:control group,PCOS group,and PCOS with high-fat diet (HFD) group.Serum hormones were assayed by radioimmunoassay (RIA).The ovaries'were immunohistochemically stained with MIF,and the expression of MIF,p-JNK and p-p38 was detected by Western blotting in ovaries.The serum testosterone level,LH concentration,LH/FSH ratio,fasting insulin level and HOMA IR index in the PCOS group (6.077±0.478,13.809±1.701,1.820±0.404,10.83±1.123 and 1.8692±0.1096)and PCOS with HFD group (6.075±0.439,14.075±1.927,1.779±0.277,10.20±1.377 and 1.7736±0.6851)were significantly higher than those in the control group (4.949±0.337, 2.458±0.509,1.239±0.038,9.53±0.548 and 1.5329±0.7363),but there was no significant difference between the PCOS group and PCOS with HFD group.The expression levels of MIF,p-JNK,and p-p38 in the PCOS group (0.4048±0.013,0.6233±0.093 and 0.7987±0.061)and PCOS withHFD group (0.1929±0.012,0.3346±0.103 and 0.3468±0.031)were obviously higher than those in control group (0.2492±0.013, 0.3271±0.093 and 0.3393±0.061),but no Significant difference was observed between PCOS group and PCOS with HFD group.It was suggested that MIF may participate in the pathogenesis of PCOS through the MAPK signalling pathway in PCOS rats induced by DHEA.

The mechanisms of melanogenesis inhibition by glabridin:molecular docking,PKA/MITF and MAPK/MITF pathways

Glabridin is the main ingredient of hydrophobic fraction in licorice extract and has been shown to have anti-melanogenesis activity in skins.However,the underlying mechanism(s)remain not completely understood.The aim of this study is thus to elucidate the possible mechanisms related to the melanogenesis suppression by glabridin in cultured B16 murine melanoma cells and in UVA radiation induced hyperpigmentation model of BALB/c mice as well.Molecular docking simulations revealed that between catalytic core residues and the compound.The treatment by glabridin significantly downregulated both transcriptional and/or protein expression of melanogenesis-related factors including melanocyte stimulating hormone receptor(MC1R),microphthalmia-associated transcription factor(MITF),tyrosinase(TYR),TYR-related protein-1(TRP-1)and TRP-2 in B16 cells.Both PKA/MITF and MAPK/MITF signaling pathways were found to be involved in the suppression of melanogenesis by glabridin in B16 cells.Also in vivo glabridin therapy significantly reduced hyperpigmentation,epidermal thickening,roughness and inflammation induced by frequent UVA exposure in mice skins,thus beneficial for skin healthcare.These data further look insights into the molecular mechanisms of melanogenesis suppression by glabridin,rationalizing the application of the natural compound for skin healthcare.

Resveratrol Induces Apoptosis and Autophagy in T-cell Acute Lymphoblastic Leukemia Cells by Inhibiting Akt/mTOR and Activating p38-MAPK

Objective To explore the effects of resveratrol-induced apoptosis and autophagy in T-cell acute lymphoblastic leukemia （T-ALL） cells and potential molecular mechanisms. Methods The anti-proliferation effect of resveratrol-induced, apoptosis and autophagy on T-ALL cells were detected by using MTI- test, immunofluorescence, electronic microscope, and flow cytometry, respectively. Western blotting was performed for detecting changes of apoptosis-associated proteins, cell cycle regulatory proteins and state of activation of Akt, mTOR, p70S6K, 4E-BP1, and p38-MAPK. Results Resveratrol inhibited the proliferation and dose and time-dependent manner. It also induced cyclin-dependent kinase （CDK） inhibitors p21 and induced apoptosis and autophagy in T-ALL cells in a cell cycle arrest at G0/G1 phase via up regulating p27 and down regulating cyclin A and cyclin D1. Western blotting revealed that resveratrol significantly decreased the expression of antiapoptotic proteins （Mcl-1 and Bcl-2） and increased the expression of proapoptotic proteins （Bax, Bim, and Bad）, and induced cleaved-caspase-3 in a time-dependent manner. Significant increase in ratio of LC3-11/LC3-1 and Beclin 1 was also detected. Furthermore, resveratrol induced significant dephosphorylation of Akt, mTOR, p70S6K, and 4E-BP1, but enhanced specific phosphorylation of p38-MAPK which could be blocked by SB203580. When autophagy was suppressed by 3-MA, apoptosis in T-ALL cells induced by resveratrol was enhanced. Conclusion Our findings have suggested that resveratrol induces cell cycle arrest, apoptosis, and autophagy in T-ALL cells through inhibiting Akt/mTOR/p7OS6K/4E-BP1 and activating p38-MAPK signaling pathways. Autophagy might play a role as a self-defense mechanism in T-ALL cells treated by resveratrol. Therefore, the reasonable inhibition of autophagy in T-ALL cells may serve as a promising strategy for resveratrol induced apoptosis and can be used as adjuvant chemotherapy for T-ALL.

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.

欧前胡素调节MAPK/mTOR/p70S6K信号通路对急性髓系白血病细胞恶性生物学行为的影响

目的探讨欧前胡素(IMP)对急性髓系白血病(AML)细胞恶性生物学行为的影响以及对MAPK/mTOR/p70S6K信号通路的调节机制。方法将人AML细胞HL-60进行培养传代,并分为对照组(未处理组),ERK抑制剂组(PD98059组),IMP低、中、高剂量组,IMP高剂量+ERK激活剂组(IMP高+Cearoin组),每组均设置6个重复。MTT法检测IMP对HL-60细胞的毒性;MTT法、软琼脂克隆形成实验检测HL-60细胞的增殖活性;流式细胞仪术检测HL-60细胞凋亡;Transwell小室检测HL-60细胞迁移和侵袭能力;ELISA检测HL-60细胞培养液中TNF-α、IL-1β、IL-6的水平;Western blot检测通路相关蛋白及Bcl-2、Beclin-1、LC3Ⅱ/Ⅰ蛋白的表达。结果与未处理组比较,PD98059组和IMP低、中、高剂量组HL-60细胞的存活率、克隆形成数量、迁移和侵袭数量、促炎因子TNF-α、IL-1β、IL-6水平以及ERK1/2、mTOR、p70S6K磷酸化水平、Bcl-2表达显著降低(P<0.05),细胞凋亡率、自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ显著升高(P<0.05);与IMP高剂量组比较,IMP高+Cearoin组中ERK激活剂明显消除了IMP对上述指标的影响(P<0.05)。结论IMP可能通过抑制MAPK/mTOR/p70S6K信号通路,抑制AML细胞增殖、迁移和侵袭,促进细胞自噬和凋亡。

Immunoregulatory polysaccharides from Apocynum venetum L.flowers stimulate phagocytosis and cytokine expression via activating the NF-κB/MAPK signaling pathways in RAW264.7 cells

Two immunomodulatory polysaccharides(Vp2a-Ⅱ and Vp3) were isolated and identified from Apocynum venetum L. flowers, and their innate immune-stimulating functions and working mechanisms were evaluated in RAW264.7 cells. Both the level of released nitric oxide(NO) and expression of inducible nitric oxide synthase(iNOS) m RNA were significantly enhanced in the RAW264.7 macrophages cells treated by Vp2a-Ⅱ and Vp3. Vp2a-Ⅱ(100–800 μg/m L) and Vp3(400 μg/mL) could significantly increase the phagocytic activity of RAW264.7 cells and the secretion and m RNA expression of TNF-α and IL-6 in a concentrationdependent manner through affecting mitogen-activated protein kinase(MAPK) activity and nuclear factor κB(NF-κB) nuclear translocation. Vp2a-Ⅱ might activate the MAPK signaling pathways and induce the nuclear translocation of NF-κB p65, whilst Vp3 likely activated the NF-κB and MAPK signaling pathways without influencing the p38 MAPK route.

Selenium-enriched oolong tea(Camellia sinensis)extract exerts anti-inflammatory potential via targeting NF-κB and MAPK pathways in macrophages

Both tea polyphenols and selenium(Se)have been suggested to exert the health benefits via the regulatory capacities of chronic inflammation,which make Se-enriched oolong tea a promising beverage as an anti-inflammatory diet.The aim of this study is to investigate the anti-inflammatory effects of Se-enriched oolong tea extract(Se-TE)and underlying mechanism in lipopolysaccharide(LPS)-induced RAW264.7 cells.Se-TE treatments(50 and 150μg/m L)significantly suppressed the over-production of nitric oxide(NO)and prostaglandin E2(PGE2)in LPS-stimulated macrophages via downregulating the expression of nitric oxide synthase(i NOS)and cyclooxygenase-2(COX-2).Moreover,Se-TEs also effectively inhibited the productions of inflammatory cytokines,such as tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β).Furthermore,Se-TE could block mitogen-activated protein kinase(MAPK)and nuclear factor-kappa B(NF-κB)signaling pathways through the inhibition of the phosphorylation of key proteins(IκB-α,p65,p38,ERK,and JNK)and the translocation of the p65 subunit into the nucleus.Collectively,our results indicated that Se-TE may have the potential to be used as a novel food ingredient for the development of various anti-inflammatory foods and the treatment and prevention of chronic inflammation-related diseases.