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.

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.

Adenosine triphosphate promotes locomotor recovery after spinal cord injury by activating mammalian target of rapamycin pathway in rats

The mammalian target of rapamycin （mTOR） pathway plays an important role in neuronal growth, proliferation and differentiation. To better understand the role of mTOR pathway involved in the induction of spinal cord injury, rat models of spinal cord injury were established by modified Allen＇s stall method and interfered for 7 days by intraperitoneal administration of mTOR activator adenosine triphosphate and mTOR kinase inhibitor rapamycin. At 1-4 weeks after spinal cord injury induction, the Basso, Beattie and Bresnahan locomotor rating scale was used to evaluate rat locomotor function, and immunohistochemical staining and western blot analysis were used to detect the expression of nestin （neural stem cell marker）, neuronal nuclei （neuronal marker）, neuron specific enolase, neurofilament protein 200 （axonal marker）, glial fibrillary acidic protein （astrocyte marker）, Akt, mTOR and signal transduction and activator of transcription 3 （STAT3）. Results showed that adenosine triphosphate-mediated Akt/mTOR/STAT3 pathway increased endogenous neural stem cells, induced neurogenesis and axonal growth, inhibited excessive astrogliosis and improved the locomotor function of rats with spinal cord injury.

淫羊藿苷调控mTOR/Akt/CREB通路对高糖诱导的足细胞自噬及凋亡的影响

目的 探讨淫羊藿苷对高糖诱导的足细胞自噬、凋亡及哺乳动物雷帕霉素靶蛋白(mTOR)/丝氨酸苏氨酸蛋白激酶(Akt)/环磷酸腺苷反应元件结合蛋白(CREB)通路的影响。方法 将小鼠足细胞MPC5分为5组:正常对照组(5.5 mmol·L^(-1)葡萄糖)、高糖组(30 mmol·L^(-1)葡萄糖)、淫羊藿苷组(30 mmol·L^(-1)葡萄糖+5μmol·L^(-1)淫羊藿苷)、GDC-0349组(30 mmol·L^(-1)葡萄糖+50μmol·L^(-1)GDC-0349)、淫羊藿苷+GDC-0349组(30 mmol·L^(-1)葡萄糖+5μmol·L^(-1)淫羊藿苷+50μmol·L^(-1)GDC-0349)。培养48 h后,噻唑蓝法检测MPC5细胞活力;吖啶橙染色观察MPC5细胞自噬情况;流式细胞术检测MPC5细胞凋亡;蛋白印迹法检测MPC5细胞自噬[微管相关蛋白1轻链3(LC3)Ⅱ、LC3Ⅰ、自噬相关蛋白(Beclin-1)]、凋亡[Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)]和mTOR/Akt/CREB通路相关蛋白的表达。结果 与正常对照组比较,高糖组MPC5细胞活力、Bcl-2、磷酸化mTOR(p-mTOR)/mTOR、磷酸化Akt(p-Akt)/Akt、磷酸化CREB(p-CREB)/CREB蛋白表达水平显著降低(P<0.05),自噬能力增强,自噬体表现出橙色荧光,细胞凋亡率、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bax蛋白表达水平显著升高(P<0.05)。与高糖组比较,淫羊藿苷组MPC5细胞活力、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bcl-2、p-mTOR/mTOR、p-Akt/Akt、p-CREB/CREB蛋白表达水平显著升高,自噬能力进一步增强,自噬体数量增多,自噬体呈现出砖红色荧光(P<0.05),细胞凋亡率、Bax蛋白表达水平显著降低(P<0.05);GDC-0349组MPC5细胞活力、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bcl-2、p-mTOR/mTOR、p-Akt/Akt、p-CREB/CREB蛋白表达水平显著降低,自噬能力减弱,自噬体数量减少,自噬体表现出橙色荧光(P<0.05),细胞凋亡率、Bax蛋白表达水平显著升高(P<0.05);淫羊藿苷+GDC-0349可逆转淫羊藿苷对高糖诱导MPC5细胞的作用效果(P<0.05)。结论 淫羊藿苷通过激活mTOR/Akt/CREB通路促进高糖诱导的足细胞自噬抑制细胞凋亡。

下调HMGB2表达对肝癌LM3细胞上皮-间质转化的抑制作用及其AKT/mTOR信号通路机制

目的:探讨下调肝癌细胞中高迁移率族框蛋白2 (HMGB2)表达对肝癌细胞生物学行为及上皮-间质转化(EMT)进程的影响,并阐明其作用机制。方法:对数生长期的人肝癌LM3细胞分为阴性对照组和HMGB2 RNA干扰组(HMGB2 siRNA组),分别以Lipofectamin 2000为载体转染无关序列的RNA寡核苷酸(RNA oligo)和敲除HMGB2序列的RNA oligo。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测2组细胞中HMGB2 mRNA和蛋白表达水平,分别采用细胞划痕实验和Transwell小室实验检测2组细胞的迁移和侵袭能力,采用Western blotting法检测2组细胞中E-钙黏蛋白(E-cadherin)、 N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达水平。结果:与阴性对照组比较,HMGB2 siRNA组细胞中HMGB2 mRNA和蛋白表达水平均明显降低(P<0.05),HMGB2 siRNA组细胞划痕愈合率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),细胞中E-cadherin蛋白表达水平明显升高(P<0.01),N-cadherin、Vimentin、mTOR、AKT和磷酸化AKT (p-AKT)蛋白表达水平明显降低(P<0.05或P<0.01)。结论:下调HMGB2的表达可降低肝癌LM3细胞迁移和侵袭能力并抑制EMT,其作用机制可能与参与调节AKT/mTOR通路相关蛋白表达有关。

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.

LRP6 Bidirectionally Regulates Insulin Sensitivity through Insulin Receptor and S6K Signaling in Rats with CG-IUGR

Objective Intrauterine growth restriction followed by postnatal catch-up growth(CG-IUGR)increases the risk of insulin resistance-related diseases.Low-density lipoprotein receptor-related protein 6(LRP6)plays a substantial role in glucose metabolism.However,whether LRP6 is involved in the insulin resistance of CG-IUGR is unclear.This study aimed to explore the role of LRP6 in insulin signaling in response to CG-IUGR.Methods The CG-IUGR rat model was established via a maternal gestational nutritional restriction followed by postnatal litter size reduction.The mRNA and protein expression of the components in the insulin pathway,LRP6/β-catenin and mammalian target of rapamycin(mTOR)/S6 kinase(S6K)signaling,was determined.Liver tissues were immunostained for the expression of LRP6 andβ-catenin.LRP6 was overexpressed or silenced in primary hepatocytes to explore its role in insulin signaling.Results Compared with the control rats,CG-IUGR rats showed higher homeostasis model assessment for insulin resistance(HOMA-IR)index and fasting insulin level,decreased insulin signaling,reduced mTOR/S6K/insulin receptor substrate-1(IRS-1)serine307 activity,and decreased LRP6/β-catenin in the liver tissue.The knockdown of LRP6 in hepatocytes from appropriate-for-gestational-age(AGA)rats led to reductions in insulin receptor(IR)signaling and mTOR/S6K/IRS-1 serine307 activity.In contrast,LRP6 overexpression in hepatocytes of CG-IUGR rats resulted in elevated IR signaling and mTOR/S6K/IRS-1 serine307 activity.Conclusion LRP6 regulated the insulin signaling in the CG-IUGR rats via two distinct pathways,IR and mTOR-S6K signaling.LRP6 may be a potential therapeutic target for insulin resistance in CG-IUGR individuals.

弥漫大B细胞淋巴瘤中铁死亡相关基因的表达及其与免疫细胞和信号通路的关系

目的通过癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)中铁死亡相关基因的表达及其与程序性死亡受体配体-1(programmed death ligand-1,PD-L1)和免疫细胞的关系,为DLBCL的治疗提供新的靶标。方法通过TCGA数据库查找获得22个铁死亡相关基因。从TCGA数据库获取48例DLBCL(DLBCL组)及54例反应性淋巴结增生患者(对照组)淋巴结标本的铁死亡相关基因以及PD-L1的表达数据。使用Wilcoxon秩和检验进行组间差异性表达分析。基因表达相关性分析采用Spearman相关性分析。采用R软件包pheatmap分析DLBCL中铁死亡相关基因表达与免疫细胞的相关性。采用R软件GSVA包分析铁死亡相关基因表达与磷脂酰肌醇-3-激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(phosphatidylinositol 3 kinase-protein kinase B-mammalian target of rapamycin,PI3K-Akt-mTOR)信号通路的相关性。结果DLBCL中周期素依赖性激酶抑制因子1A(cyclin dependent kinase inhibitor 1A,CDKN1A)、70 kDa热休克蛋白5(heat shock 70 kDa protein 5,HSPA5)、内质膜蛋白复合体亚基2(endoplasmic membrane protein complex subunit 2,EMC2)、溶质载体家族7成员11(solute carrier family 7,member 11,SLC7A11)、金属硫蛋白1G(metallothionein 1G,MT1G)、热休克蛋白B1(heat shock protein B1,HSPB1)、谷胱甘肽过氧化酶4(glutathione peroxidase4,GPX4)、范可尼贫血互补群D2(Fanconi anemia complementary group D2,FANCD2)、柠檬酸合成酶(citrate synthase,CS)、CDGSH铁硫结构域1(CDGSH iron sulfur domain 1,CISD1)、法尼基二磷酸法尼基转移酶1(farnesyl diphosphate farnesyltransferase 1,FDFT1)、SLC1A5、转铁蛋白受体(transferrin receptor,TFRC)、核糖体蛋白L8(ribosomal protein L8,RPL8)、核受体共激活因子4(nuclear receptor coativator 4,NCOA4)、二肽基肽酶Ⅳ(dipeptidyl peptidaseⅣ,DPP4)和花生四烯酸15脂氧合酶(arachidonate-15-lipoxygenase,ALOX15)基因表达均上调(均P<0.05)。免疫细胞相关分析显示,铁死亡相关基因可激活体内巨噬细胞M1(P<0.05)。DLBCL中长链脂酰辅酶A合成酶4(acyl-CoA synthetase long chain family member 4,ACSL4)、CDKN1A、DPP4、EMC2、谷氨酰胺酶2(glutaminase 2,GLS2)、HSPA5、溶血卵磷脂酰基转移酶3(lysophosphatidylcholine acyltransferase 3,LPCAT3)、MT1G、NCOA4、红细胞衍生核因子2样蛋白2(nuclear factor erythroid 2-like-2,NFE2L2)、精脒/精胺N1-乙酰基转移酶1(spermidine/spermine N1-acetyltransferase 1,SAT1)、SLC7A11和TFRC这些铁死亡相关基因的表达均与PD-L1表达呈正相关(均r>0.4,均P<0.05)。铁死亡相关基因LPCAT3、NCOA4和TFRC的表达均与PI3K-AktmTOR通路呈正相关(均r>0.4,均P<0.05)。结论多数铁死亡相关基因在DLBCL组织中高表达,且与PD-L1、免疫浸润及PI3K-Akt-mTOR通路有关。

替米沙坦通过磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路抑制硬化性胃癌细胞增殖

目的研究替米沙坦对硬化性胃癌(SGC)细胞增殖的影响,并探讨其作用机制。方法常规培养胃癌细胞MKN1和SGC细胞HSC45。采用细胞计数盒8实验检测替米沙坦对MKN1和HSC45增殖能力的影响;流式细胞仪检测替米沙坦对HSC45凋亡和细胞周期的影响;蛋白质印迹法检测激活替米沙坦对HSC45自噬和磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白表达的影响;采用PI3K/AKT/mTOR信号通路激活剂SC79和替米沙坦共同处理HSC45,分别检测激活PI3K/AKT/mTOR信号通路后,替米沙坦对HSC45增殖、凋亡、自噬和细胞周期的影响。结果替米沙坦呈浓度和时间依赖性抑制MKN1和HSC45细胞增殖(均P<0.001),且对HSC45细胞增殖抑制效果更为显著(P<0.05)。替米沙坦组HSC45早期凋亡率、LC3Ⅱ/Ⅰ蛋白表达量、G_(0)/G_(1)期细胞周期比例均高于对照组[(26.2±2.6)%比(1.3±0.4)%、(1.02±0.09)比(0.29±0.04)、(53.4±3.4)%比(38.1±2.9)%],磷酸化PI3K、磷酸化AKT和磷酸化mTOR蛋白表达均低于对照组(均P<0.05)。替米沙坦组和替米沙坦+SC79组HSC45细胞增殖抑制率、细胞凋亡率、LC3Ⅱ/Ⅰ蛋白表达及G_(0)/G_(1)期细胞比例均高于对照组,但替米沙坦+SC79组均低于替米沙坦组(均P<0.05)。结论替米沙坦下调PI3K/AKT/mTOR信号通路,促进SGC细胞凋亡、自噬和周期阻滞,进而抑制SGC细胞增殖能力。

基于磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白通路探讨大株红景天注射液对缺氧H9C2心肌细胞损伤的影响

目的探讨大株红景天注射液对缺氧H9C2心肌细胞的影响及保护机制。方法取H9C2心肌细胞缺氧造模,实验分为正常组、模型组、大株红景天注射液组(简称:SI组)、尼可地尔组。采用RT-qPCR检测磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)、蛋白激酶B(protein kinase B,AKT)、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)的mRNA;免疫蛋白印记法检测PI3K、AKT、mTOR、p-PI3K、p-AKT、p-mTOR的蛋白表达;流式细胞仪检测细胞凋亡水平。结果与模型组比较,SI组及尼可地尔组PI3K、AKT、mTOR的mRNA表达均显著降低(P<0.05)。与模型组比较,SI组和尼可地尔组PI3K、AKT、mTOR蛋白表达无明显差异(P>0.05)。经磷酸化处理后,与模型组比较,SI组和尼可地尔组p-PI3K、p-AKT、p-mTOR的蛋白表达显著下降(P<0.05);与尼可地尔组相比,SI组p-PI3K、p-AKT的蛋白表达下降(P<0.05),p-mTOR的蛋白含量无明显差异(P>0.05)。结论大株红景天注射液通过下调PI3K/AKT/mTOR通路,起到了抑制缺氧心肌细胞过度自噬,改善心肌细胞损伤的作用。

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.

SREBP2及PI3K/Akt/mTORC2在喉癌中的表达及相关性研究

目的探究磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白C2(mTORC2)信号通路分子及人胆固醇调节元件结合蛋白2(SREBP2)分子在喉癌组织中的表达情况。方法选取本院2019年1月至2022年6月收治的原发性喉癌患者60例,取其肿瘤组织及癌旁组织样本,以免疫组织化学法测定PI3K、AKT、mTORC2及SREBP2表达情况并进行比较。结果肿瘤组织PI3K(88.3%vs.40.0%)、AKT(76.7%vs.31.7%)、mTORC2(78.3%vs.21.7%)、SERBP2(65.0%vs.8.3%)阳性表达率高于癌旁组织,差异均有统计学意义(P<0.05)。肿瘤直径大于或等于3 cm患者AKT、mTORC2、SERBP2阳性表达率高于肿瘤直径小于3 cm患者,差异均有统计学意义(P<0.05);临床分期Ⅲ~Ⅳ期患者AKT、SERBP2阳性表达率高于Ⅰ~Ⅱ期患者,差异均有统计学意义(P<0.05);不同分化程度患者mTORC2、SERBP2阳性表达率比较,差异均有统计学意义(P<0.05)。PI3K、AKT、mTORC2、SERBP2阳性表达患者2年生存率低于阴性表达患者,但差异无统计学意义(P>0.05)。结论喉癌患者肿瘤组织中PI3K/AKT/mTORC2信号通路分子及SREBP2均呈高表达,与肿瘤直径、临床分期及分化程度有相关性,是潜在的喉癌靶向代谢重编程治疗的靶点。

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.

磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白自噬通路在支气管肺发育不良中的作用

目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)自噬通路在支气管肺发育不良中的作用。方法将A549细胞(腺癌人类肺泡基底上皮细胞)一定条件培养后分成对照组、支气管肺发育不良组、雷帕霉素干预组。支气管肺发育不良组、雷帕霉素干预组高氧环境培养,置于37℃、85%氧气孵化箱中培养48 h,前者不加药剂、后者加10μmol/L的雷帕霉素干预;对照组常规环境培养,置于37℃、5%二氧化碳孵化箱中培养48 h,不加药剂。利用高氧构建支气管肺发育不良的细胞模型,利用蛋白质印迹实验和实时荧光定量聚合酶链反应(RT-qPCR)实验验证自噬对高氧处理后A549细胞肺泡表面标志物合成蛋白复合物(SPC)、水通道蛋白5(AQP5)的影响,利用5-乙炔基-2′-脱氧尿苷(EdU)实验检测自噬对高氧处理后A549细胞增殖的影响,利用蛋白质印迹实验验证PI3K/Akt/mTOR自噬通路在支气管肺发育不良中的作用。结果蛋白质印迹实验和RT-qPCR实验表明自噬激活剂雷帕霉素挽救了高氧处理后A549细胞SPC、AQP5表达的下调。EdU实验表明雷帕霉素显著促进了高氧处理后A549细胞的增殖,EdU阳性细胞百分比高于支气管肺发育不良组[(0.48±0.06)比(0.36±0.02)](P<0.05)。蛋白质印迹实验显示雷帕霉素干预后的A549细胞中的磷酸化-PI3K、磷酸化-Akt、磷酸化-mTOR、自噬选择性底物P62的表达水平显著降低,微管相关蛋白1轻链3的表达水平显著上升(均P<0.05)。结论PI3K/Akt/mTOR自噬通路在支气管肺发育不良中发挥着重要作用。

栀子苷调节PI3K/AKT/mTOR信号通路在动脉粥样硬化形成过程中对Th17/Treg功能的影响

目的:观察栀子苷对载脂蛋白E缺乏(ApoE^(-/-))小鼠Th17/调节性T(Treg)细胞失衡的影响及其作用机制。方法:将50只纯合子ApoE^(-/-)雌性小鼠随机分为对照组、模型组和栀子苷低剂量组、栀子苷中剂量组、栀子苷高剂量组。对照组小鼠喂养普通饲料,模型组和栀子苷组小鼠喂养高脂饲料。从第8周开始,栀子苷各剂量组每日灌胃栀子苷(25、50、100 mg/kg),连续8周。试验结束时,采用油红O染色评估主动脉及其根部动脉粥样硬化(AS)病变面积比。采用定量逆转录聚合酶链式反应(RT-PCR)分析主动脉组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-17A和IL-10 mRNA表达;采用流式细胞仪分析脾脏中Th17和Treg细胞百分比;蛋白免疫印迹法(Western Blot)检测主动脉组织磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白表达。结果:油红O染色病变显示,栀子苷中剂量组、栀子苷高剂量组病变百分比低于模型组(P<0.05)。与对照组比较,模型组主动脉TNF-α、IL-6和IL-17A mRNA表达水平升高(P<0.05);栀子苷各剂量组主动脉TNF-α、IL-6和IL-17A mRNA表达水平降低(P<0.05)。与对照组比较,模型组主动脉抗炎细胞因子IL-10 mRNA表达水平降低(P<0.05);栀子苷各剂量组主动脉抗炎细胞因子IL-10 mRNA表达水平升高(P<0.05)。与对照组比较,模型组小鼠脾脏中Th17细胞百分比升高,Treg细胞百分比降低(P<0.05)。栀子苷处理恢复了AS小鼠Th17和Treg细胞的平衡。栀子苷抑制PI3K的表达及AKT和mTOR的磷酸化,MHY1485(mTOR活化剂)减弱了栀子苷对T细胞分化的影响。结论:栀子苷抗AS作用机制可能与抑制PI3K/AKT/mTOR信号引起的Treg细胞增多和Th17细胞减少有关。

LncRNA HOXB-AS1对乳腺癌MCF-7细胞增殖、迁移、侵袭和凋亡的影响机制研究

目的 探讨长链非编码RNA(LncRNA)同源异型盒基因B-AS1(HOXB-AS1)通过Akt-mTOR自噬通路对乳腺癌MCF-7细胞增殖、迁移、侵袭和诱导凋亡的影响及机制。方法 人乳腺癌MCF-7细胞于2020年6月20购自中国科学院分子细胞科学卓越创新中心。将乳腺癌MCF-7细胞分为对照组(Con组,n=3)、阴性对照组(NC组,n=3)和HOXB-AS1过表达组(HOXB-AS1组,n=3),通过实时定量聚合酶链反应(qRT-PCR)法检测HOXB-AS1的表达;用CCK-8法检测MCF-7细胞的增殖;用细胞迁移实验、细胞Transwell实验检测MCF-7细胞的迁移及侵袭;用流式细胞术检测细胞的凋亡;用Western blot法检测基质金属蛋白酶(MMP)-2、MMP-9、Beclin 1、LC3B、p62、p-Akt、p-mTOR,并对检测结果进行统计分析。结果 乳腺癌MCF-7细胞转染后,Con组的LncRNA HOXB-AS1相对表达量为0.15±0.05、NC组为0.13±0.03、HOXB-AS1组为0.75±0.04,HOXB-AS1组的LncRNA HOXB-AS1相对表达量显著高于Con组和NC组(t=16.030,P=0.000)。MCF-7细胞经HOXB-AS1处理24h后,Con组的MCF-7细胞活力为(100.00±9.13)%、NC组为(100.00±2.94)%、HOXB-AS1组为(60.50±5.72)%,HOXB-AS1组的MCF-7细胞活力显著低于Con组和NC组(t=7.428,P=0.000)。转染HOXB-AS1后,Con组的划痕愈合速度为(61.00±3.61)%、NC组为(59.83±2.26)%、HOXB-AS1组为(39.03±3.61)%,HOXB-AS1组的划痕愈合速度显著低于Con组和NC组(t=16.090,P=0.002)。转染HOXB-AS1后,Con组的MCF-7细胞侵袭数为(343.36±20.82)个、NC组为(316.00±29.46)个、HOXB-AS1组为(105.02±15.00)个,HOXB-AS1组的MCF-7细胞侵袭数显著低于Con组和NC组(t=16.093,P=0.000)。转染HOXB-AS1后,Con组的MCF-7细胞凋亡率为(93.47±4.72)%、NC组为(90.67±7.37)%、HOXB-AS1组为(31.33±2.59)%,HOXB-AS1组的MCF-7细胞凋亡率显著低于Con组和NC组(t=19.992,P=0.000)。HOXB-AS1降低了p-Akt、p-mTOR蛋白的表达,上调了自噬相关蛋白Beclin 1、LC3B的表达,下调了自噬相关蛋白p62的表达,增强了MCF-7细胞的自噬活性。结论 HOXB-AS1抑制了乳腺癌MCF-7细胞的增殖、迁移、侵袭和凋亡,其作用可能与抑制Akt/mTOR通路介导的自噬有关。

针刺联合督灸治疗强直性脊柱炎的疗效观察

目的 观察针刺联合督灸治疗强直性脊柱炎的临床疗效及对AKT/PI3K/mTOR信号通络的影响。方法将96例强直性脊柱炎患者随机分为两组(观察组和对照组),每组48例。对照组口服美洛昔康治疗,观察组予针刺联合督灸治疗。观察两组治疗前后巴氏强直性脊柱炎病情评测指数(Bath ankylosing spondylitis disease activity index,BASDAI)和巴氏强直性脊柱炎活动能力量表(Bath ankylosing spondylitis functional index,BASFI)评分、血清炎性因子[核因子-κB(nuclear factor-κB,NK-κB)、巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)、CC趋化因子受体6(CC chemokine receptor 6,CCR6)和单核细胞趋化蛋白-4(monocyte chemokine protein-4,MCP-4)]水平、骨化相关蛋白[基质金属蛋白酶组织抑制剂-2(matrix metalloproteinase tissue inhibitor-2,TIMP-2)、高迁移率族蛋白B1(high mobility group protein box 1,HMGB1)、Dickkopf相关蛋白-1(dickkopf-related protein-1,DKK-1)和骨硬化蛋白(sclerostin,SOST)]水平以及AKT/PI3K/mTOR信号通路[蛋白激酶B(protein kinase B,AKT)、磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)和雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)]水平的变化。比较两组临床疗效及不良反应发生情况。结果 观察组总有效率为97.9%,高于对照组的81.8%(P<0.05)。观察组治疗后、随访3个月及6个月BASDAI和BASFI评分均低于对照组(P<0.05)。观察组治疗后血清NK-κB、MIF、CCR6、MCP-4、TIMP-2、HMGB1、AKT、PI3K和mTOR水平均低于对照组(P<0.05),DKK-1和SOST水平高于对照组(P<0.05)。结论 针刺联合督灸可明显缓解强直性脊柱炎患者的临床症状,疗效优于口服美洛昔康治疗,并可调节AKT/PI3K/mTOR信号通络的表达。

芍药苷通过调控PI3K/AKT/mTOR信号通路对盐敏感性高血压大鼠血压和血管内皮功能的影响

目的:探讨芍药苷对盐敏感性高血压(SSH)大鼠血压和血管内皮功能的影响及其相关作用机制。方法:将50只Dahl盐敏感大鼠随机分为正常对照组(Control组)、高盐组(SSH组)、芍药苷组(PF组)、磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路激活剂组(740Y-P组)、芍药苷+740Y-P组(PF+740Y-P组),每组10只。各组大鼠进行4周给药干预。采用动物无创血压仪测量大鼠尾动脉收缩压、舒张压;酶联免疫吸附法(ELISA)测定大鼠血清内皮素-1(ET-1)、一氧化氮(NO)、血栓素B2(TXB2)水平;苏木精-伊红(HE)染色观察大鼠主动脉病理变化;免疫组织化学染色检测大鼠主动脉组织中内皮型一氧化氮合酶(eNOS)表达;蛋白质免疫印迹法(Western Blot)检测大鼠主动脉组织中PI3K/AKT/mTOR信号通路蛋白表达。结果:与Control组比较,SSH组和740Y-P组大鼠主动脉血管内皮不完整,部分血管内皮脱落,且内膜明显增厚、外膜有大量沉积物;PF组大鼠主动脉血管病理损伤较SSH组明显减轻;PF+740Y-P组大鼠主动脉血管病理损伤较740Y-P组明显减轻,但较PF组明显加重。与Control组比较,SSH组大鼠收缩压、舒张压、血清ET-1、TXB2水平均升高,血清NO水平降低(P<0.05);主动脉组织中eNOS表达水平降低,磷酸化(p)-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值均升高(P<0.05)。与SSH组比较,PF组大鼠收缩压、舒张压、血清ET-1、TXB2水平均降低,血清NO水平升高(P<0.05);主动脉组织中eNOS表达水平升高,p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值均降低(P<0.05)。与PF组比较,PF+740Y-P组大鼠收缩压、舒张压、血清ET-1、TXB2水平均升高,血清NO水平降低(P<0.05);主动脉组织中eNOS表达水平降低,p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值均升高(P<0.05)。与740Y-P组比较,PF+740Y-P组大鼠收缩压、舒张压、血清ET-1、TXB2水平均降低,血清NO水平升高(P<0.05);主动脉组织中eNOS表达水平升高,p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值均降低(P<0.05)。结论:芍药苷可以有效降低SSH大鼠血压,并改善大鼠血管内皮功能,其作用机制可能与抑制PI3K/AKT/mTOR信号通路激活有关。

紫草素对人早幼粒细胞白血病细胞自噬和凋亡的影响

目的:探讨紫草素对人早幼粒细胞白血病细胞自噬和凋亡的影响及可能的机制。方法:取对数生长期的人早幼粒细胞白血病细胞NB4,将其分为对照组(未处理的NB4细胞)、紫草素组(0.3μmol/L紫草素处理)、740Y-P组(15μmol/L PI3K/Akt/m TOR通路激活剂740Y-P处理)、紫草素+740Y-P组(0.3μmol/L紫草素与15μmol/L 740Y-P共同处理),处理24 h后,用于后续实验,CCK-8法检测各组细胞活力,单丹磺酰戊二酸染色检测细胞自噬囊泡的聚集情况,流式细胞术检测细胞凋亡,Western blot检测各组细胞中Beclin1、LC3、p62、Bax、cleaved caspase-3、Bcl-2及PI3K/Akt/m TOR通路相关蛋白的表达。结果:与对照组比较,紫草素组NB4细胞内紫色点状荧光强度、细胞凋亡率及Beclin1、LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3、Bax蛋白相对表达量升高,OD_(450)值(24、48 h)及Bcl-2、p62蛋白相对表达量降低(均P<0.05);与对照组比较,740Y-P组NB4细胞内紫色点状荧光强度、细胞凋亡率及Beclin1、LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3、Bax蛋白相对表达量降低,OD_(450)值(24、48 h)及Bcl-2、p62蛋白相对表达量升高(均P<0.05);与紫草素组比较,紫草素+740Y-P组NB4细胞内紫色点状荧光强度、细胞凋亡率及Beclin1、LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3、Bax蛋白相对表达量降低,OD_(450)值(24、48 h)及Bcl-2、p62蛋白相对表达量升高(均P<0.05)。与对照组比较,紫草素组NB4细胞中PI3K/Akt/m TOR通路相关蛋白p-PI3K、p-Akt、p-m TOR蛋白表达显著降低,而740Y-P组显著升高(均P<0.05);与紫草素组比较,紫草素+740Y-P组NB4细胞中p-PI3K、p-Akt、p-m TOR蛋白表达显著升高(均P<0.05)。结论:紫草素可能通过抑制PI3K/Akt/mTOR通路促进NB4细胞自噬与凋亡。