Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Human neural stem cell-derived extracellular vesicles protect against ischemic stroke by activating the PI3K/AKT/mTOR pathway

Human neural stem cell-derived extracellular vesicles exhibit analogous functions to their parental cells,and can thus be used as substitutes for stem cells in stem cell therapy,thereby mitigating the risks of stem cell therapy and advancing the frontiers of stem cell-derived treatments.This lays a foundation for the development of potentially potent new treatment modalities for ischemic stroke.However,the precise mechanisms underlying the efficacy and safety of human neural stem cell-derived extracellular vesicles remain unclear,presenting challenges for clinical translation.To promote the translation of therapy based on human neural stem cell-derived extracellular vesicles from the bench to the bedside,we conducted a comprehensive preclinical study to evaluate the efficacy and safety of human neural stem cell-derived extracellular vesicles in the treatment of ischemic stroke.We found that administration of human neural stem cell-derived extracellular vesicles to an ischemic stroke rat model reduced the volume of cerebral infarction and promoted functional recovery by alleviating neuronal apoptosis.The human neural stem cell-derived extracellular vesicles reduced neuronal apoptosis by enhancing phosphorylation of phosphoinositide 3-kinase,mammalian target of rapamycin,and protein kinase B,and these effects were reversed by treatment with a phosphoinositide 3-kinase inhibitor.These findings suggest that human neural stem cell-derived extracellular vesicles play a neuroprotective role in ischemic stroke through activation of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.Finally,we showed that human neural stem cell-derived extracellular vesicles have a good in vivo safety profile.Therefore,human neural stem cell-derived extracellular vesicles are a promising potential agent for the treatment of ischemic stroke.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection

Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

基于PI3K/AKT/mTOR信号通路探讨化瘀通络灸促血管性痴呆大鼠髓鞘再生的作用机制

目的观察化瘀通络灸对血管性痴呆(vascular dementia,VD)大鼠胼胝体磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的影响,探讨化瘀通络灸促VD大鼠髓鞘再生的作用机制。方法经Morris水迷宫筛选后,随机选取12只大鼠纳入假手术组,剩余大鼠复制VD模型成功后,随机分为模型组、艾灸组、艾灸+LY294002组,每组12只。艾灸组予以化瘀通络灸干预,艾灸+LY294002组在化瘀通络灸干预的基础上予以PI3K抑制剂LY294002腹腔注射,采用Longa评分法评价各组大鼠神经功能损伤程度,Morris水迷宫实验检测各组大鼠学习记忆能力,Western blot法检测各组大鼠PI3K/AKT/mTOR信号通路相关蛋白的表达水平,神经髓鞘固蓝染色法观察各组大鼠胼胝体髓鞘的形态,透射电子显微镜观察各组大鼠髓鞘超微结构。结果与假手术组比较,模型组和艾灸+LY294002组大鼠的Longa评分显著升高(P<0.05),逃避潜伏期显著延长(P<0.05),PI3K/AKT/mTOR通路相关蛋白表达水平显著降低(P<0.05),胼胝体内髓鞘纹理不清,排列混乱,边缘呈空泡或空网状改变,髓鞘线圈样结构离散,部分膨出和崩解,有髓神经轴突数量显著减少(P<0.05);与模型组和艾灸+LY294002组比较,艾灸组大鼠Longa评分显著下降(P<0.05),逃避潜伏期显著缩短(P<0.05),PI3K/AKT/mTOR通路相关蛋白表达水平显著提高(P<0.05),胼胝体内髓鞘结构有所恢复,排列整齐,边缘结构较为致密,有髓神经轴突数量显著增加(P<0.05)。结论化瘀通络灸可能通过激活PI3K/AKT/mTOR通路,修复VD大鼠损伤髓鞘并促进其重塑,恢复脑白质功能。

PI3K/Akt信号通路调控急性髓系白血病机制及中医药治疗研究进展

急性髓系白血病(AML)是一种极具侵袭力的血液恶性肿瘤,其特征表现为未成熟的髓系白血病细胞快速增殖。随着基因测序、蛋白组学等现代科学技术的快速发展,AML的发病及预后分子机制的研究逐渐深入,但仍未打破目前AML治疗复发率高、免疫逃逸、微小残留、放化疗副反应大、患者家庭经济及心理负担重的局面。磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)/哺乳动物雷帕霉素靶点是致癌通路中最为经典的一条,不少研究通过该条通路研发出药物以应对AML。近些年,中医药因其具有多层次、多靶点、低不良反应等优势,在肿瘤治疗领域大展身手,发挥重要作用,受到医学界广泛关注与认可。因此,该综述概述了PI3K/Akt信号通路与AML的关系,归纳并发现中药单体和中药复方能介导PI3K/Akt信号通路,抑制肿瘤细胞增殖、迁移、侵袭及血管新生,进而影响AML的病理发展。

大黄素干预PI3K/Akt/mTOR信号通路影响肝癌前病变大鼠铁死亡的作用机制

目的 探究大黄素对二乙基亚硝胺(diethylnitrosamine, DEN)诱导的肝癌前病变大鼠磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)/蛋白激酶B(protein kinase B, Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号转导通路的调控作用及对铁死亡的影响。方法 采用DEN诱发大鼠肝癌前病变进行肝癌前病变造模。随机将50只雄性SD大鼠分为对照组[未造模+10ml/(kg·d)生理盐水灌胃)]、DEN模型组[肝癌前病变造模+10 ml/(kg·d)生理盐水灌胃]、DEN+大黄素组[肝癌前病变造模+80 mg/(kg·d)大黄素浓缩液灌胃]、DEN+护肝片组[肝癌前病变造模+900 mg/kg护肝片灌服]和DEN+大黄素+护肝片组[肝癌前病变造模+80 mg/(kg·d)大黄素浓缩液灌胃+900 mg/kg护肝片灌服],每组各10只大鼠。各组大鼠连续干预治疗12周。生化分析法检测各组大鼠血清丙氨酸氨基转移酶(alanine aminotransferase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)、白蛋白(albumin, ALB)水平;比较各组大鼠的肝脏指数。酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)法检测各组大鼠血清白细胞介素1β(interleukin 1 beta, IL-1β)、肿瘤坏死因子α(tumor necrosis factor alpha, TNF-α)、IL-10含量。苏木精-伊红(hematoxylin-eosin, HE)染色法观察各组大鼠肝组织病理形态学改变;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling, TUNEL)分析各组大鼠肝组织细胞凋亡情况。免疫组织化学法检测各组大鼠肝组织中铁死亡中心调节因子谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白表达。马松三色染色(Masson′s trichrome staining, MASSON)法分析各组大鼠肝组织病理改变。逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)法检测各组大鼠肝组织中PIK3、Akt、mTOR的mRNA水平。结果 与对照组比较,DEN模型组大鼠ALT水平、AST水平、肝脏指数显著升高,ALB水平显著降低(P均<0.05),血清IL-1β、TNF-α、IL-10含量明显升高(P均<0.05);肝细胞明显炎性浸润,细胞免疫应答增加,肝细胞形态畸变并出现变性死亡;肝组织中GPX4蛋白表达量明显下降(P<0.05),PI3K、Akt、mTOR的mRNA表达量明显上升(P均<0.05)。与DEN模型组比较,DEN+大黄素组、DEN+护肝片组、DEN+大黄素+护肝片组大鼠ALT水平、AST水平、肝脏指数显著降低,ALB显著升高(P均<0.05),血清IL-1β、TNF-α、IL-10含量明显降低(P均<0.05);肝细胞排列趋向正常,炎性浸润减轻,肝细胞弥漫性纤维化病变减弱,血管及胆管周围肝细胞形态逐渐好转,蓝色胶原纤维组织减少;肝组织中GPX4蛋白表达量明显上升(P均<0.05),PI3K、Akt、mTOR的mRNA表达量明显下降(P均<0.05),且上述变化DEN+大黄素+护肝片组改善效果明显优于DEN+大黄素组和DEN+护肝片组(P均<0.05)。结论 大黄素可通过促进组织中铁死亡相关蛋白GPX4表达来改善大鼠肝癌前病变,其作用机制可能与大黄素调控PI3K/Akt/mTOR信号通路有关。

黄角颗粒通过刺激PI3K/AKT/mTOR信号通路减轻脑缺血再灌注大鼠的脑损伤

目的探讨黄角颗粒对脑缺血再灌注损伤大鼠的作用及对磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/AKT/mTOR)信号通路的影响。方法采用线栓法构建大鼠脑缺血再灌注损伤模型,采用黄角颗粒进行处理。Zea Longa评分法进行大鼠神经功能评分、氯化三苯四唑(TTC)染色检测脑梗死百分比、HE染色观察脑组织病变情况、ELISA检测脑组织中白细胞介素10(IL-10)、IL-1β、肿瘤坏死因子α(TNF-α)含量,Western blot法检测PI3K、AKT、磷酸化的AKT(p-AKT)、mTOR和磷酸化的mTOR(p-mTOR)蛋白水平。结果黄角颗粒可显著减轻模型大鼠的神经功能缺损程度,减少脑梗死百分比,减轻脑组织病理损伤,显著上调脑组织中IL-10、PI3K、p-AKT和p-mTOR的水平,显著下调IL-1β和TNF-α的水平。结论黄角颗粒对脑缺血再灌注损伤的保护作用可能与促进PI3K/AKT/mTOR信号通路的激活相关。

阿托伐他汀通过激活PI3K/Akt/mTOR信号转导而促进神经元突起生长

目的探讨阿托伐他汀(Ato)对体外培养大鼠皮质神经元突起生长促进作用的信号转导机制。方法 取培养7 d大脑皮质神经元,分为Ato 10μmo.lL-1作用48 h组和阻断剂+Ato组,先分别加入阻断剂PD98059 50μmo.l L-1、LY294002 30μmol.L-1、曲西立滨(TCBN)2.5μmol.L-1和西罗莫司(雷帕霉素,Rapa)100 nmo.l L-1作用1 h,再加入Ato共同作用48 h。应用倒置相差显微镜观察神经元突起生长状况;Western印迹法检测磷酸化的磷酸肌醇依赖激酶1(PDK1)、磷酸化蛋白激酶B(Akt)、磷酸化西罗莫司靶蛋白(mTOR)、磷酸化的核糖体S6激酶(p70S6K)和磷酸化的真核翻译起始因子4E结合蛋白1(p-4E-BP1)的表达。结果 形态学观察结果显示,Ato 10μmo.lL-1组可明显促进突起生长,表现为突起总长度增加、一级突起数目增多、末端分支数增多及胞体面积增大。PD98059,LY294002,TCBN和Rapa均可阻断Ato对神经元突起生长的促进作用。Western印迹结果显示,Ato 10μmo.lL-1可显著上调p-PDK1,p-Akt(Ser473),p-mTOR,p-p70S6K和p-4E-BP1蛋白表达水平(P<0.01)。LY294002可显著阻断Ato引起的p-PDK1,p-Akt(Ser473)蛋白表达水平增加(P<0.01)。TCBN可显著阻断Ato引起的p-mTOR蛋白表达水平增加(P<0.01)。Rapa可明显阻断Ato引起的p-p70S6K和p-4E-BP1蛋白表达水平增加(P<0.01)。结论 Ato对体外培养皮质神经元突起发育的促进作用可能与激动MEK/ERK信号转导通路有一定的关系,主要可能与通过激活PI3K/Akt/mTOR信号转导通路有关。

Scorpiones,Scolopendra and Gekko Inhibit Lung Cancer Growth and Metastasis by Ameliorating Hypoxic Tumor Microenvironment via PI3K/AKT/mTOR/HIF-1αSignaling Pathway

Objective:To investigate whether Buthus martensii karsch(Scorpiones),Scolopendra subspinipes mutilans L.Koch(Scolopendra)and Gekko gecko Linnaeus(Gekko)could ameliorate the hypoxic tumor microenvironment and inhibit lung cancer growth and metastasis by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin/hypoxia-inducible factor-1α(PI3K/AKT/mTOR/HIF-1α)signaling pathway.Methods:Male C57BL/6J mice were inoculated with luciferase labeled LL/2-luc-M38 cell suspension to develop lung cancer models,with rapamycin and cyclophosphamide as positive controls.Carboxy methyl cellulose solutions of Scorpiones,Scolopendra and Gekko were administered intragastrically as 0.33,0.33,and 0.83 g/kg,respectively once daily for 21 days.Fluorescent expression were detected every 7 days after inoculation,and tumor growth curves were plotted.Immunohistochemistry was performed to determine CD31 and HIF-1αexpressions in tumor tissue and microvessel density(MVD)was analyzed.Western blot was performed to detect the expression of PI3K/AKT/mTOR/HIF-1αsignaling pathway-related proteins.Enzyme-linked immunosorbent assay was performed to detect serum basic fibroblast growth factor(bFGF),transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)in mice.Results:Scorpiones,Scolopendra and Gekko prolonged the survival time and inhibited lung cancer metastasis and expression of HIF-1α(all P<0.01).Moreover,Scorpiones,Scolopendra and Gekko inhibited the phosphorylation of AKT and ribosomal protein S6 kinase(p70S6K)(P<0.05 or P<0.01).In addition,they also decreased the expression of CD31,MVD,bFGF,TGF-β1 and VEGF compared with the model group(P<0.05 or P<0.01).Conclusion:Scorpiones,Scolopendra and Gekko all showed beneficial effects on lung cancer by ameliorating the hypoxic tumor microenvironment via PI3K/AKT/mTOR/HIF-1αsignaling pathway.

PI3K/AKT/mTOR信号通路在卵巢癌中的研究进展

卵巢癌的发生过程中存在磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/AKT/m TOR)信号通路的激活,该通路的异常激活可促进细胞增殖、抑制细胞凋亡、促进肿瘤侵袭和转移。PI3K/AKT/m TOR通路靶向抑制剂对卵巢癌显示出一定的疗效,目前该靶向药物的临床试验已开展,并显示出良好的安全性和有效性。针对PI3K/AKT/m TOR的靶向药物将为卵巢癌的治疗指明新方向。

养心定悸胶囊治疗帕金森病合并抑郁患者的临床疗效及对PI3K、AKT、mTOR蛋白水平的影响

目的基于磷脂酰肌醇-3-激酶/蛋白激酶B/雷帕霉素靶体蛋白(PI3K/AKT/mTOR)信号通路探讨养心定悸胶囊对帕金森病(Parkinson′s disease,PD)合并抑郁患者的疗效。方法选取2018年1月—2021年1月期间河北中医学院第二附属医院收治的PD合并抑郁患者102例,随机分为对照组和研究组,每组各51例。对照组接受常规西药治疗,研究组在对照组基础上加用养心定悸胶囊。治疗12周后,观察比较两组患者临床疗效,治疗前后统一帕金森病评分量表(Unified Parkinson′s Disease Rating Scale,UPDRS)评分、帕金森病自主神经症状量表(SPOCA-AUT)评分、汉密尔顿抑郁量表(Hamilton depression scale,HAMD)评分及血清PI3K、AKT、mTOR蛋白水平情况。结果治疗后研究组总有效率92.16%(47/51)较对照组76.47%(39/51)明显升高,差异有统计学意义(P<0.05)。治疗后两组患者各项UPDRS评分及总分均低于治疗前,差异有统计学意义(P<0.05);且研究组各项UPDRS评分及总分低于对照组,差异有统计学意义(P<0.05)。治疗后研究组消化、心血管系统、体温调节、瞳孔调节评分及SCOPA-AUT总分均低于治疗前,差异有统计学意义(P<0.05);且研究组消化、心血管系统、体温调节、瞳孔调节评分及SCOPA-AUT总分均低于对照组,差异有统计学意义(P<0.05)。治疗后研究组HAMD评分低于治疗前,差异有统计学意义(P<0.05);且研究组HAMD评分低于对照组,差异有统计学意义(P<0.05)。治疗后研究组PI3K、AKT、mTOR蛋白表达水平均低于对照组,差异均有统计学意义(P<0.05)。结论养心定悸胶囊对PD合并抑郁患者具有较好的疗效,可缓解病情发展,改善非运动症状,并能减轻抑郁情绪,可能是通过影响PI3K/AKT/mTOR信号通路而发挥作用。

miR-383-3p靶向PTEN/PI3K/Akt/mTOR信号通路对冠心病大鼠内皮细胞凋亡的影响

目的:探究微小RNA-383-3p(miR-383-3p)靶向调节人第10号染色体缺失的磷酸酶/磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白(PTEN/PI3K/AKT/mTOR)信号通路对冠心病(CHD)大鼠内皮细胞凋亡的影响。方法:将SD大鼠分为control组、CHD组、mimic NC组、miR-383-3p mimic组、miR-383-3p mimic+pcDNA3.1组、miR-383-3p mimic+pcDNA3.1-PTEN组,每组15只;除control组外其余组大鼠通过高脂饲料喂养及垂体后叶素注射构建CHD模型,造模前24h,将慢病毒液或质粒载体注射到相应组大鼠中;实时荧光定量PCR(qRT-PCR)检测冠状动脉miR-383-3p表达;全自动生化分析仪测定大鼠血脂水平;酶联免疫吸附(ELISA)法检测血清内皮素-1(ET-1)、血管紧张素Ⅱ(AngⅡ)、血管内皮生长因子(VEGF)、一氧化氮(NO)水平;HE染色及TUNEL法观察冠状动脉组织病理变化及内皮细胞凋亡情况;双荧光素酶报告基因实验验证miR-383-3p与PTEN靶向关系;Western blot检测冠状动脉组织Bcl-2、Bax及PTEN/PI3K/Akt/mTOR通路蛋白表达。结果:与control组相比,CHD组大鼠miR-383-3p、高密度脂蛋白胆固醇(HDL-C)、VEGF、NO、Bcl-2、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR水平显著减少,三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、总胆固醇(TC)、ET-1、AngⅡ水平、冠状动脉组织病理损伤程度、内皮细胞凋亡指数(AI)、Bax、PTEN表达显著升高(P<0.05);与CHD组相比,miR-383-3p mimic组miR-383-3p、HDL-C、VEGF、NO、Bcl-2、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR水平显著增加,TG、LDL-C、TC、ET-1、AngⅡ水平、冠状动脉组织病理损伤程度、内皮细胞AI、Bax、PTEN表达显著减少(P<0.05);过表达PTEN可逆转miR-383-3p高表达对CHD大鼠内皮细胞凋亡及功能损伤的改善作用;PTEN是miR-383-3p的靶向基因。结论:miR-383-3p可通过靶向抑制PTEN表达来激活PI3K/Akt/mTOR信号通路,与抑制CHD大鼠内皮细胞凋亡密切相关。

细胞自噬与PI3K/Akt/mTOR信号通路在黑色素瘤耐药性中的研究进展

黑色素瘤是一种恶性皮肤肿瘤,对手术、放疗、化疗等治疗的反应差,虽然基因靶向药物对黑色素瘤有明显的治疗效果,但多数患者很快产生耐药,限制了靶向药物在黑色素瘤临床治疗中的应用。因此,深入研究黑色素瘤细胞产生耐药的分子机制非常必要。细胞自噬的异常是肿瘤发生耐药的一种重要机制,磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路是细胞自噬的主要调控通路,黑色素瘤细胞中存在细胞自噬现象,其耐药性的产生可能与细胞自噬及PI3K/Akt/mTOR信号通路密切相关。

野生型肺炎链球菌溶血素通过PI3K-I/Akt/mTOR途径诱导A549细胞自噬

目的:研究肺炎链球菌(Streptococcus pneumoniae,Sp)诱导人肺上皮细胞自噬发生的现象及其机制。方法:常规培养人肺上皮A549细胞与野生型Sp;将GFP-LC3真核载体转染A549细胞,分为实验组(Sp,MOI=30∶1)、阴性对照组[渥曼青霉素(wortmannin,WT),2μmol/L 2 h,自噬抑制剂]、阳性对照组[雷帕霉素(rapamycin,Rapa),1 mmol/L 10 h,mTOR抑制剂]和参照组(WT和Sp共同处理),各组处理时间均为1、2、3和4 h;荧光显微镜观察各组自噬点形成,透射电镜观察各组细胞自噬体结构,Western blot检测各组微管相关蛋白轻链-Ⅰ/Ⅱ(microtuble-associated protein light chain 3-Ⅰ/Ⅱ,LC3-Ⅰ/Ⅱ)、磷酸化UNC-51-K激酶1(phosphorylated UNC-51-like kinase1,P-ULK1)和螯体1(sequestosome 1,P62)的表达;将溶血素表达载体RFP-PLY(red fluorescent protein-pneumolysin)、GFP-LC3或(和)RFP-PLY转染/共转染A549细胞,Western blot检测各组磷酸肌醇3-激酶-Ⅰ/Ⅲ(phosphoinositide 3-kinase-Ⅰ/Ⅲ,PI3K-Ⅰ/Ⅲ)、蛋白激酶B(protein kinase B,Akt)、Beclin-1蛋白(Beclin 1 protein,Beclin-1)和哺乳动物雷帕霉素靶(mammalian target of rapamycin,mTOR)的表达。结果:处理A549细胞3 h后,与阴性对照组(WT)相比,Sp感染后,自噬点明显增多(t=41.313,P=0.001),电镜观察到典型的自噬体结构,LC3-Ⅱ蛋白表达明显上调(t=121.592,P=0.000);A549细胞转染RFP-PLY处理3 h后,与阴性对照组(WT)相比,Sp感染同样观察到明显的自噬现象,PI3K-I、Akt蛋白和mTOR表达下调(tPI3K-I=16.544,PPI3K-I=0.004;tAkt=22.679,PAkt=0.002;tmTOR=19.503,PmTOR=0.003),而PI3K-Ⅲ和Beclin-1水平无明显差异(tPI3K-Ⅲ=3.572,PPI3K-Ⅲ=0.070;tBeclin-1=0.799,PBeclin-1=0.508)。结论:野生型Sp诱导A549细胞自噬可能通过PI3K-I/Akt/mTOR信号途径。

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

Treating non-small cell lung cancer by targeting the PI3K signaling pathway

The phosphosphatidylinositol-3-kinase(PI3K)signaling pathway is one of the most important intracellular signal transduction pathways affecting cell functions,such as apoptosis,translation,metabolism,and angiogenesis.Lung cancer is a malignant tumor with the highest morbidity and mortality rates in the world.It can be divided into two groups,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for>85%of all lung cancers.There are currently many clinical treatment options for NSCLC;however,traditional methods such as surgery,chemotherapy,and radiotherapy have not been able to provide patients with good survival benefits.The emergence of molecular target therapy has improved the survival and prognosis of patients with NSCLC.In recent years,there have been an increasing number of studies on NSCLC and PI3K signaling pathways.Inhibitors of various parts of the PI3K pathway have appeared in various phases of clinical trials with NSCLC as an indication.This article focuses on the role of the PI3K signaling pathway in the occurrence and development of NSCLC and summarizes the current clinical research progress and possible development strategies.

白藜芦醇对颞下颌关节骨关节炎的治疗作用及其机制

目的:探讨白藜芦醇对颞下颌关节骨关节炎(TMJOA)的治疗作用,并阐明相关作用机制。方法:45只SD大鼠随机分为对照组、模型组和白藜芦醇组,每组15只。模型组和白藜芦醇组大鼠关节腔内注射20 g·L^(-1)碘乙酸钠(MIA)50μL,构建TMJOA大鼠模型;对照组大鼠注射等量生理盐水。造模3周后,白藜芦醇组大鼠注射80μL白藜芦醇溶液,每周1次,连续3周,对照组和模型组大鼠注射等量生理盐水。微型计算机断层扫描技术(Micro-CT)系统检测各组大鼠髁突结构,计算感兴趣区的骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁间距(Tb.p)和骨小梁数(Tb.N)。HE染色和甲苯胺蓝染色观察各组大鼠颞下颌关节(TMJ)组织病理形态表现,免疫组织化学法检测各组大鼠TMJ组织中性别决定区Y框蛋白(SOX)-9、基质金属蛋白酶(MMP)-13、沉默信息调节因子(Sirt)1、磷脂酰肌醇3激酶(PI3K)、磷酸化蛋白激酶B(p-Akt)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)蛋白表达水平,实时荧光定量PCR(RT-qPCR)法检测各组大鼠TMJ组织中SOX-9、MMP-13、Sirt1、PI3K、哺乳动物雷帕霉素靶蛋白(mTOR)和蛋白激酶B(Akt)mRNA表达水平。结果:造模3周后,大鼠髁突骨质破坏明显,表面粗糙不平,延续性中断,表明TMJOA大鼠模型造模成功。Micro-CT系统检测,对照组大鼠髁突表面光滑,形态规则,骨质连续完整;模型组大鼠髁突破坏明显,骨质连续性受到不同程度破坏,表面粗糙可伴有不同程度骨质缺损;白藜芦醇组大鼠髁突病变减轻,髁突形态外观得到一定的改善。与对照组比较,模型组大鼠BV/TV和Tb.Th均明显降低(P<0.05),Tb.Sp明显增加(P<0.05);与模型组比较,白藜芦醇组大鼠BV/TV和Tb.Th均明显升高(P<0.05),Tb.Sp明显减少(P<0.05)。HE染色观察,对照组大鼠髁突层次清晰,软骨细胞排列规则,整齐有序;模型组大鼠髁突表面粗糙不平,缺损明显,呈现典型的TMJOA表现;白藜芦醇组大鼠髁突表面略微粗糙,整体分层大致清晰,细胞排列较为有序。甲苯胺蓝染色观察,对照组大鼠髁突肥大层软骨细胞呈蓝紫色,染色明显且均匀;模型组大鼠髁突肥大层软骨细胞淡染,部分区域甚至失染;白藜芦醇组大鼠髁突肥大细胞层染色基本明显且较为均匀。免疫组织化学法检测,与对照组比较,模型组大鼠TMJ组织中MMP-13、PI3K、p-Akt和p-mTOR蛋白表达水平均明显升高(P<0.05),SOX-9和Sirt1蛋白表达水平均明显降低(P<0.05);与模型组比较,白藜芦醇组大鼠TMJ组织中SOX-9和Sirt1蛋白表达水平均明显升高(P<0.05),MMP-13、PI3K、p-Akt和p-mTOR蛋白表达水平均明显降低(P<0.05)。RT-qPCR法检测,与对照组比较,模型组大鼠TMJ组织中MMP-13、PI3K、Akt和mTOR mRNA表达水平均明显升高(P<0.05),SOX-9和Sirt1 mRNA表达水平均明显降低(P<0.05);与模型组比较,白藜芦醇组大鼠TMJ组织中SOX-9和Sirt1 mRNA表达水平均明显升高(P<0.05),MMP-13、PI3K、Akt和mTOR mRNA表达水平均明显降低(P<0.05)。结论:白藜芦醇对TMJOA有治疗作用,其作用机制可能是通过激活Sirt1、抑制PI3K-Akt-mTOR信号通路实现的。