PC-1解除S6K对AKT信号通路的负反馈抑制

目的:通过建立过表达PC-1的前列腺癌LNCaP细胞系及敲低PC-1表达的C4-2细胞系,探究PC-1激活AKT信号通路的分子机制。方法:将PC-1基因及针对PC-1的siRNA序列,分别克隆至慢病毒表达载体pCDH-EF1-Myc-MCS-T2A-Puro及干扰载体pSIH1-H1-Puro,包装成慢病毒后分别感染前列腺癌LNCaP及C4-2细胞,通过Western印迹鉴定PC-1过表达及敲低效果,并检测PI3K/AKT/mTOR信号通路相关蛋白S6K、AKT的磷酸化水平。结果:PC-1过表达时,S6K磷酸化水平下降,而AKT的磷酸化水平上升。结论:PC-1可以通过抑制S6K激酶活性,解除其对AKT的负反馈抑制作用,从而激活AKT激酶的活性。     

Prosaposin对细胞增殖和凋亡的调控及其分子机制

为研究鞘脂激活蛋白原(Prosaposin)对细胞增殖、细胞凋亡的调控及其可能的分子机制, 以pcDNA3.1 in NIH3T3阴性对照细胞株和过表达prosaposin的Psap-Myc in NIH3T3细胞株为模型, 噻唑蓝(MTT)比色法检测prosaposin对细胞增殖的影响; Annexin V联合碘化丙啶(Propidium iodide, PI)法检测血清饥饿状态下prosaposin对细胞凋亡的影响; Western blotting检测PI3K/Akt信号通路中蛋白磷酸化水平的变化; Real-time PCR检测PI3K/Akt信号通路下游靶分子表达水平的改变。结果表明prosaposin可活化PI3K/Akt信号通路, 提高Akt^Ser473的磷酸化水平, 抑制细胞周期抑制基因P27^KIP1的表达, 上调细胞周期蛋白Cyclin D1的表达, 促进细胞周期从G1→S期进展; 诱导survival基因cIAP1、cIAP2的表达, 促进细胞存活。这些结果提示, prosaposin对细胞增殖和凋亡的调控可能是通过PI3K/Akt信号通路及其下游靶分子进行的。     

AD认知功能障碍的代谢紊乱机制:脑胰岛素抵抗及其介导的PI3K/Akt信号通路受损

阿尔茨海默病(Alzheimer’s disease, AD)是一种以进行性痴呆为主要特征的中枢神经系统退行性疾病,其认知功能障碍可能与Ⅱ型糖尿病(type 2 diabetes, T2DM)诱发的胰岛素抵抗所损伤的PI3K/Akt胰岛素信号级联通路相关。胰岛素是调节机体新陈代谢的重要激素,通过与神经细胞表面的胰岛素受体结合激活PI3K/Akt信号通路,以调控葡萄糖、脂质的代谢。任何中间媒介功能紊乱所导致的脑胰岛素水平和胰岛素敏感性的降低都会损坏PI3K/Akt信号通路,诱发脑能量代谢障碍、Aβ沉积、Tau蛋白过度磷酸化,引起并加重AD认知功能障碍。因此,本文以PI3K/Akt胰岛素信号通路为主线,揭示了T2DM中脑胰岛素抵抗(insulin resistance, IR)与AD之间的复杂机制,旨在加深对脑IR介导的AD病理过程的系统性理解,借此为延缓或治疗AD的认知功能障碍提供理论基础。     

葡萄籽原花青素通过PI3K/Akt信号通路抑制氧化应激诱导的H9c2心肌细胞凋亡

为了探讨葡萄籽原花青素(grape seed proanthocyanidin, GSP)对心肌细胞的保护作用及机制,通过CCK-8法评估细胞活力,采用Western-blot分析评估GSP对凋亡相关蛋白质(cleaved caspase-3、Bax和Bcl-2)和PI3K/Akt通路相关蛋白质(p-PI3K、PI3K、p-Akt和Akt)表达水平的影响,并使用TUNEL染色和Hoechst 33258染色评估H9c2心肌细胞凋亡情况。结果显示, GSP可以抑制H₂O₂诱导的H9c2心肌细胞的细胞毒性和凋亡,使促凋亡蛋白cleaved caspase-3和Bax表达下降,并使抗凋亡蛋白Bcl-2表达水平升高; GSP作用于H9c2细胞后, PI3K和Akt的磷酸化水平增加,使PI3K/Akt信号通路激活。实验结果初步表明, GSP可抑制氧化应激诱导的H9c2心肌细胞凋亡,其作用机制可能与激活PI3K/Akt信号通路有关。     

木兰醇通过激活AKT信号通路改善心肌细胞胰岛素抵抗

目的:探究木兰醇对胰岛素抵抗的心肌细胞糖代谢的影响。方法:通过MTT法和LDH检测试剂盒检测木兰醇对心肌细胞的细胞毒性;100 n M的胰岛素刺激SD大鼠乳鼠心肌细胞24 h构建心肌胰岛素抵抗模型;葡萄糖检测试剂盒、糖摄取检测试剂盒检测心肌细胞的糖代谢情况;通过Western blot检测糖代谢相关信号通路蛋白磷酸化AKT的表达情况。结果:木兰醇对心肌细胞无明显毒性,且剂量依赖性的增加胰岛素敏感和非敏感型心肌细胞糖代谢。30μM的木兰醇孵育心肌细胞1 h,显著激活细胞磷酸化AKT信号通路。100 n M的胰岛素刺激心肌细胞24 h后,再次给予100 n M的胰岛素刺激后,心肌细胞的糖代谢水平无明显变化。30μM的木兰醇孵育心肌细胞24 h显著增加胰岛素抵抗心肌细胞的糖代谢水平。PI3K抑制剂Wortmanmin完全抑制木兰醇的上述作用。结论:木兰醇可通过激活AKT信号通路改善心肌细胞胰岛素抵抗。     

FoxO1对下丘脑摄食中枢的影响研究进展

下丘脑是人体的摄食中枢,它通过抑制食欲的阿黑皮素原(POMC)神经元和促进食欲的神经肽相关蛋白(AgRP)神经元调节摄食及能量代谢。叉头转录因子O亚族1(FoxO1)是胰岛素信号通路和瘦素信号通路中重要的调节蛋白,FoxO1的生理作用是促进下丘脑Agrp基因表达、抑制Pomc基因表达,抑制瘦素信号通路的转录激活因子3(STAT3)蛋白对Pomc基因转录的促进作用,从而促进食欲。瘦素和胰岛素均可激活经典的IRS/PI(3)K/Akt信号通路,使FoxO1磷酸化失去活性,抑制食欲。此外,沉默信息调节因子Sirt1也可以通过去乙酰化,影响FoxO1的转录活性。本文综述了胰岛素、瘦素、Sirt1通过FoxO1调节下丘脑摄食中枢的作用机制。     

MicroRNA-185改善非酒精性脂肪肝小鼠胰岛素敏感性并调节脂代谢基因的表达

目的:探讨micro RNA-185(miR-185)对高脂饮食的小鼠模型的HepG2肝细胞脂质代谢和胰岛素信号通路的调节作用。方法:应用定量反转录聚合酶链反应评估过表达或抑制miR-185表达脂质合成相关基因的mRNA水平。此外,应用Western Blot方法测定转染HepG2细胞pre-mir-185后的关键信号通路组分(IRS-1,IRS-2,PI3K、AKT2)和磷酸化PI3K和AKT2的表达情况。结果:诱导的人类HepG2细胞的软脂酸对mir-185水平的下降具有时间和剂量依赖性。经过mir-185转染的HepG2细胞显著降低脂肪酸合成酶,3-hydroxy-3-methylglutaryl-coa还原酶,固醇调节元件结合蛋白和固醇调节元件结合蛋白-1c的mRNA水平,而使用anti-mir-185寡核苷酸抑制mir-185在HepG2细胞中产生相反的作用。在高脂饮食的小鼠模型,与对照组动物相比,mir-185处理后脂质积累明显改善。mir-185诱导后通过上调胰岛素受体底物2增强胰岛素信号通路。结论:miR-185在体内和体外调节肝细胞脂肪酸代谢和胆固醇平衡,以及在改善胰岛素敏感性中起重要作用,miR-185可能成为非酒精性脂肪肝和胰岛素抵抗的新靶点和治疗非酒精性脂肪肝药物作用新靶标。     

EGCG通过PI3-K/Akt信号通路诱导人甲状腺乳头状癌细胞K1增殖和凋亡的影响

研究表没食子儿茶素没食子酸酯(EGCG)通过PI3-K/Akt信号通路对人甲状腺乳头状癌细胞K1增殖和凋亡的影响。利用MTT法研究不同剂量EGCG对K1细胞的增殖作用;采用流式细胞术分析EGCG对K1细胞周期和凋亡影响;Westernblot方法检测分析EGCG对人甲状腺乳头状癌K1细胞PI3-K、Akt/p-Akt、mTOR、cyclin D1、CDK4、Bcl-2/Bad、cleaved-caspase-3蛋白表达影响。MTT结果显示EGCG作用后K1细胞增殖显著受到抑制,且表现出明显的剂量依赖性和时间依赖性(P0.001);流式细胞术结果显示EGCG能够将K1细胞阻滞于G1期,并且产生剂量依赖性诱导K1细胞凋亡(P0.001);Westernblot结果显示EGCG能够上调K1细胞促凋亡蛋白Bad及cleaved-caspase-3的表达,下调PI3-K、mTOR、cyclin D1、CDK4蛋白表达,降低AKT蛋白磷酸化及抑凋亡蛋白Bcl-2表达(P0.05)。结果证明,EGCG能够通过PI3-K/Akt信号通路,诱导G1阻滞及细胞凋亡,抑制人甲状腺乳头状癌细胞K1增殖。     

PI3K/AKT信号通路调控Myogenin和MCK基因的表达

骨骼肌分化过程受多个信号通路调控, PI3K/AKT信号通路是其中最重要的信号转导通路之一。PI3K/AKT信号通路可以调控骨骼肌分化, 但在染色质水平上的调控机制还不是很清楚。文章以小鼠成肌细胞(C2C12)为研究材料, 采用免疫印迹、染色质免疫共沉淀(Chromatin immunoprecipitation, ChIP)、定量PCR (Q-PCR)的方法研究PI3K/AKT信号通路调控Myogenin和MCK基因的表达。研究发现, C2C12细胞分化过程中添加PI3K/AKT信号通路激活剂处理24 h, Myogenin和MCK蛋白表达水平显著升高, 组蛋白H3K27me3去甲基化酶UTX的表达也升高, H3K27me3在Myogenin基因启动子区和MCK基因启动子及增强子区的富集与对照组相比显著降低。用PI3K/AKT信号通路抑制剂处理, 结果相反。因此, PI3K/AKT信号通路可能通过调控组蛋白去甲基化酶UTX的表达活性改变靶基因的H3K27me3的富集进而调控骨骼肌分化。     

急、慢性运动对2型糖尿病大鼠脂肪PI3K/AKT/GLUT4通路的影响

目的:探讨急性和慢性运动对2型糖尿病(T2DM)大鼠脂肪组织明磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/葡萄糖运载体4(GLUT4)信号通路的影响。方法:15月龄SD雄性大鼠52只随机分为正常对照组(n=13)和高脂组(n=39),分别喂养普通和高脂饲料。8周后,高脂组体重＞正常对照组20％,注射小剂量STZ后,血糖＞16.7 mmol/l,造模成功。将糖尿病模型组随机分为糖尿病对照组(DC,n=13),糖尿病慢性运动组(DCE,n=13),糖尿病急性运动组(DAE,n=13)。DCE组进行8周的游泳运动,DAE组进行一次性游泳运动。测定血脂,血糖和血清胰岛素,Western blot法测定脂肪PI3K、AKT和GLUT4蛋白含量。结果:糖尿病组体重、血脂、血糖、胰岛素显著高于正常对照组(P均＜0.01);高密度脂蛋白胆固醇(HDL-C)水平降低(P＜0.05),脂肪组织中PI3K、AKT和GLUT4蛋白表达下降(P均＜0.01)。糖尿病慢性运动组体重、血脂、血糖、胰岛素均出现显著性下降(P均＜0.01);HDL-C升高(P＜0.05),脂肪PI3K、AKT和GLUT4蛋白表达上升(P＜0.01)。糖尿病急性运动组血脂、血糖、胰岛素下降(P均＜0.05);HDL-C升高(P＜0.05),脂肪PI3K、AKT和GLUT4含量显著上升(P均＜0.05)。结论:①高脂饮食结合小剂量STZ诱导的T2DM大鼠脂肪组织PI3K/AKT通路受损,降低了胰岛素的敏感性。②急性、慢性有氧运动,均可以通过PI3K/AKT通路,改善糖脂代谢紊乱,慢性运动略优于急性运动。     

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.     

The effect of 4-isothiocyanatobenzoic acid on the oxygen-linked chloride binding sites in human hemoglobin: Influence on the alkaline Bohr effect

Human oxyhemoglobin reacted with 4-isothiocyanatobenzoic acid shows a decreased oxygen affinity that does not change with increasing chloride concentration indicating that all of the oxygen-linked chloride binding sites are blocked in the modified protein. By contrast, reaction of oxyhemoglobin with 4-isothiocyanatobenzenesulfonamide produces a modified protein with increased oxygen affinity below pH 7.3 that shows the expected decrease in oxygen affinity with increasing chloride concentration. The latter result demonstrates the importance of the negatively charged moiety in producing both the decrease in oxygen affinity and the effect on the oxygen-linked chloride binding sites produced by 4-isothiocyanatobenzoic acid. Reduction in the alkaline Bohr effect by 50% in the protein modified by 4-isothiocyanatobenzoic acid indicates that contribution to the alkaline Bohr effect is evenly divided between chloride dependent and chloride independent groups.     

Acquirement of fetal properties in hepatoma on glutathione metabolism

Glutathione peroxidase/glutathionè reductase activity ratio was determined in the high-speed supernatant fraction of the rat livers. The ratio was dependent on age and the ratio increase gradually with the increase in age. The fetal liver showed a ratio of 1.5–2.0, which was almost the same value to those of the 4-dimethylaminoazobenzene-induced primary hepatoma and some transplantable hepatomas originating from the azodye-induced hepatoma. Four cell lines of transplantable ascites hepatoma examined in this study showed the value of 1.2–1.8 for the activity ratio, however, the values of two strains were found to be 2.8–3.0, even though these cell lines were also originated from the azodye-induced hepatoma.Glutathione contents of azodye-induced hepatoma and ascites hepatomas were also similar with those of fetal rat livers.The acquirement of the fetal properties in hepatoma was discussed in relation to glutathione metabolism.     

Identification of 4-acetoxyaminoquinoline from the hydrolysis of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline,in vitro and in vivo properties

4-Acetoxyaminoquinoline (Ac-4-HAQ) (1) was identified as a hydrolysis product of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline (diAc-4-HAQO). The reaction allowing the obtention of (1) obeys to a reduction mechanism implying the N₁-O cleavage. The carcinogenic properties of (1) observed by Sato et al. (Japan J. Exp. Med., 40 (1970) 475) in mice were studied in rats with the in vivo system we used previously with 4-nitroquinoline-1-oxide (4-NQO) and 4-hydroxyaminoquinoline-1-oxide (4-HAQO). In rats (1) does not covalently bind DNA. It was, therefore, possible to propose an interpretation of the results obtained by Enomoto et al. (Proc. Soc. Exp. Biol. Med., 136 (1971) 1206) who injected diAc-4-HAQO s.c. to mice and rats. Compound 1 could be responsible for the carcinogenic effects observed through the following pathway: (1) should be formed by hydrolysis of diAc-4-HAQO and reactivated by an enzymatic system to N-oxide derivative, the 4-acetoxyaminoquinoline-1-oxide (Ac-4-HAQO), which constitutes an ultimate carcinogen model of 4-NQO.     

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, β-sitosterol, Δ5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1515 mg/100 g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100 g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100 g oil) was detected in H52 at 14 DAF. During linseed development, β sitosterol (830 mg/100 g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100 g oil) and stigmasterol (265 mg/100 g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Synthesis of metabolism-resistant substrates for the transport system for cationic amino acids; their stimulation of the release of insulin and glucagon, and of the urinary loss of amino acids related to cystinuria

Several amino acids have been synthesized as model transport substrates building on the piperidine and cyclohexane rings. Only when the distal N atom is part of an unambiguously cationic structure are these compounds transported predominantly by the cationic amino acid system. These amino acids in labeled form are excreted rather slowly in unmodified state, very little ¹⁴CO₂ being released. Those which are unambiguously cationic (including also homoarginine) led to a greatly increased excretion of arginine, lysine, ornithine and citrulline. Those which might be expected to act as lysine analogs had little effect on the excretion of the basic amino acids, although the excretion of citrulline and the sum of glutamine plus asparagine was accelerated. Certain of the analogs intensified the excretion of citrulline in dissociation from effects on resorption of the basic amino acids, also in dissociation from effects on cystine resorption. These results indicate citrulline resorption does not occur principally by the same agency serving for the basic amino acids, nor by the agency serving for cystine, despite the observed interactions for resorption. The injection of either of three transport analogs for arginine into the rat leads to early increases in the circulating levels of immunologically reactive insulin and glucagon.     

Ontogenic expression pattern and genetic polymorphisms of the retinol-binding protein 4 (RBP4) gene in Erlang mountainous chickens

Retinol-binding protein 4 (RBP4) is the only circulatory transport protein for vitamin A. Based on the essential role of vitamin A in chicken reproduction, we measured RBP4 mRNA abundance in Erlang mountainous chickens. We also identified and analyzed the gene polymorphism and its effect on reproduction traits among 349 chickens. The expression of RBP4 mRNA showed specific developmental changes and striking differences among tissues. The mRNA abundance was greatest (P < 0.05) in the liver, intermediate in the ovary, kidney, small intestine, oviduct and heart, and lowest in the hypothalamus and pituitary, as compared to all other tissues (P < 0.05). We detected one single nucleotide polymorphism (g.19942455C>G) in intron 2 of the RBP4 gene. Three genotypes (CC, CG and GG) were identified, with a significant effect of genotype on the age at first egg (AFE), first egg weight (FEW), total eggs at 300 days (TE300), highest continuous laying days (HCLD) and average laying interval (ALI). The GG genotype, where chickens display earlier AFE, more TE300, longer HCLD and shorter ALI, would be genetically advantageous and its selection may improve reproduction traits. These results suggested that the RBP4 gene might play an important role in reproduction traits in chickens.     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。