Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury

The signaling mechanisms underlying ischemia-induced nerve cell apoptosis are poorly understood. We investigated the effects of apoptosis-related signal transduction pathways following ischemic spinal cord injury, including extracellular signal-regulated kinase（ERK）, serine-threonine protein kinase（Akt） and c-Jun N-terminal kinase（JNK） signaling pathways. We established a rat model of acute spinal cord injury by inserting a catheter balloon in the left subclavian artery for 25 minutes. Rat models exhibited notable hindlimb dysfunction. Apoptotic cells were abundant in the anterior horn and central canal of the spinal cord. The number of apoptotic neurons was highest 48 hours post injury. The expression of phosphorylated Akt（pAkt） and phosphorylated ERK（p-ERK） increased immediately after reperfusion, peaked at 4 hours（p-Akt） or 2 hours（p-ERK）, decreased at 12 hours, and then increased at 24 hours. Phosphorylated JNK expression reduced after reperfusion, increased at 12 hours to near normal levels, and then showed a downward trend at 24 hours. Pearson linear correlation analysis also demonstrated that the number of apoptotic cells negatively correlated with p-Akt expression. These findings suggest that activation of Akt may be a key contributing factor in the delay of neuronal apoptosis after spinal cord ischemia, particularly at the stage of reperfusion, and thus may be a target for neuronal protection and reduction of neuronal apoptosis after spinal cord injury.

Metformin promotes anti-tumor immunity in STK11 mutant NSCLC through AXIN1-dependent upregulation of multiple nucleotide metabolites

Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/threonine kinase 11(STK11)mutant non-small cell lung cancer(NSCLC)through an axis inhibition protein 1(AXIN1)-dependent manner.However,the alterations of tumor metabolism and metabolites upon metformin administration remain unclear.Methods:We performed untargeted metabolomics using liquid chromatography(LC)-mass spectrometry(MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis.Results:According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database,most metabolites were annotated into metabolism,including nucleotide metabolism.Next,the differentially expressed metabolites in H460(refers to H460 cells),H460_met(refers to metformin-treated H460 cells),and H460_KO_met(refers to metformin-treated Axin1-/-H460 cells)were distributed into six clusters based on expression patterns.The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis.We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated.Furthermore,these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes(STING)pathway independently of AXIN1.Conclusion:Relying on AXIN1,metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC,indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.

小麦STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类催化结构域Ⅰ和Ⅷ氨基酸保守序列设计简并引物,以TcLr19、TcLr35和感病对照Thatcher的cDNA为模板进行抗病基因同源序列的PCR扩增,得到了6条通读的抗病基因同源序列(RGAs)Lr19-RGA1、Lr19-RGA2、Lr19-RGA3、Lr35-RGA1、Lr35-RGA2和TC-RGA。在NCBI中用BLASTp比对发现,Lr19-RGA1、Lr19-RGA2、Lr19-RGA3、Lr35-RGA1和Lr35-RGA2编码的氨基酸序列具有丝氨酸/苏氨酸蛋白激酶(Serine-thre-onine kinase,STK)的催化结构域Ⅱ-Ⅷ。对序列分析还发现,它们与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小麦抗叶锈病相关基因提供了依据。

甜瓜STK类R基因同源序列的克隆与分析

以植物丝氨酸/苏氨酸蛋白激酶类(serine-threonine kinase,STK)抗病基因产物催化结构域Ⅰ和Ⅸ的保守氨基酸序列(FGK/V/L/SVYK/RG,DY/IYSF/YGV/I/M)设计简并引物,对甜瓜(Cucumis melo L.)基因组DNA进行PCR扩增,得到大约500 bp的目的条带,通过重组质粒克隆并经PCR检测后得到12条不同的DNA序列,命名为tg1～tg12,其中tg2、tg5、tg9和tg12(Genbank登录号为JN646853～JN646856)可以编码完整的氨基酸序列。Blast分析结果显示:4条序列均具有ATP结合部位、底物结合部位和激酶结构域的活化环(A-loop)等,属于典型的蛋白激酶基因家族,可能是STK类R基因的同源序列片段;4条序列与蓖麻(Ricinus communis L.)的STK同源性均较高。氨基酸序列比对结果显示tg2、tg5、tg9和tg12均具有R基因的9个保守结构域,为STK类候选抗病基因类序列。分子系统树显示tg2、tg5、tg9和tg12与已知的R基因(Pto、Lr10和Lectin)在氨基酸水平上的相似性仅为33.5%～53.4%,且4个甜瓜同源序列的氨基酸相似性也较低,表明甜瓜RGAs标记可能具有较高的特异性。

Active compounds of Caodoukou(Semen Alpinia Katsumadai)inhibit the migration,invasion and metastasis of human pancreatic cancer cells by targeting phosphoinosmde-3-kinase/protein kinase B/mammalian target of rapamycin pathway

OBJECTIVE:To detect the effects of active compounds of Caodoukou(Semen Alpinia Katsumadai)(ACAK)on the proliferation,migration and invasion of pancreatic cancer,and explain the possible molecular mechanism of ACAK interacting with these processes.Methods:Cell counting kit-8 method,cell scratch repair experiment,Transwell migration and invasion experiment,immunohistochemistry,western blot assay and real-time polymerase chain reaction experiment were used to evaluate the effect of ACAK on the proliferation,migration and invasion of pancreatic cancer cells.The levels of active molecules involved in the phosphoinosmde-3-kinase(PI3K)/Akt/the mammalian target of rapamycin(m TOR)signal transduction were detected by Western blot assay.In addition,the function of ACAK in vivo was evaluated by xenotransplantation tumor model in nude mice.Results:The inhibitory effect of ACAK on the proliferation of pancreatic cancer cells showed certain time-dose dependence.The results of scratch repair test,Transwell test,Western blotting and real time polymerase chain reaction assay showed that ACAK could inhibit the migration and invasion of pancreatic cancer cells in vitro.In addition,the regulatory effect of ACAK on epithelialmesenchymal transition(EMT)is partly attributed to PI3K/Akt/mT OR signaling pathway.The experimental results in vivo showed that ACAK regulated the development of pancreatic cancer.Conclusions:ACAK can partly inhibit the activity of EMT and matrix metallopeptidases by down-regulating the downstream proteins of PI3K/Akt/mTOR signal pathway,thus inhibiting the ability of migration and invasion of pancreatic cancer.

云南3种野生稻中抗病基因同源序列的克隆及序列分析

根据已报道的NBS LRR类和STK类抗病基因结构中的氨基酸保守区域 ,设计简并引物 ,通过PCR扩增及克隆 ,从普通野生稻 (OryzarufipogonGriff.)、药用野生稻 (OryzaofficinalisWall.)、疣粒野生稻 (Oryzameye rianaBaill.)中共获得 14类NBS LRR类抗病基因同源序列 ,其中普通野生稻中的有 7类 ,药用野生稻中的有 2类 ,疣粒野生稻中的有 6类。药用野生稻中TO12代表序列与普通野生稻中TR19代表序列 ,同属一类 ,且具有 10 0 %的同源性 ,说明不同种野生稻中的同一类 (聚类 )抗病基因同源序列是完全一致的。同时 ,还获得 5类STK类抗病基因同源序列 ,其中普通野生稻中的有 4类 ;药用野生稻中的有 1类。通过氨基酸同源性比较分析 ,发现笔者克隆到的抗病基因同源序列与已克隆的抗病基因L6、N、Bs2、Prf、Pto、Lr10和Xa2 1等的氨基酸同源性都相当低 (均低于 2 5 % ) ,暗示了这些抗病基因同源序列可能是目前尚未报道的抗病基因的同源序列。

电项针对全脑缺血VD模型大鼠PI3K/AKT/GSK-3β信号通路的影响

目的研究电项针对全脑缺血血管性痴呆(vascular dementia,VD)模型大鼠磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸蛋白激酶/糖原合成酶激酶-3β(Phosphatidylinositol-3 kinase/serine-threonine kinase/glycogen synthase kinase-3β,P13K/AKT/GSK-3β)信号通路的影响。方法采用四血管阻断方法制备VD模型大鼠,电项针组取双侧风池穴、供血穴,电针30 min/次,1次/d,治疗14d。采用Y迷宫评价大鼠学习记忆能力;荧光定量PCR(RT-PCR)、Western blot法检测大鼠海马组织中磷酸化蛋白激酶B(phosphorylatedproteinkinaseB,p-AKT)、磷酸化糖原合成酶激酶-3β(Phosphorylated GSK-3β,P-GSK-3β)mRNA和p-AKT、p-GSK-3β蛋白的表达。结果与模型组比较,电项针组可显著提高VD大鼠Y迷宫学习与记忆正确次数(P<0.01)。与模型组比较,电项针组大鼠海马组织中p-AKT、p-GSK-3βmRNA和p-AKT、p-GSK-3β蛋白表达均有不同程度的升高(P<0.01)。结论电项针能够改善VD模型大鼠学习记忆能力,具体机制可能是激活PI3K/AKT/GSK-3β信号通路,发挥抗凋亡作用,起到对缺血海马神经元的保护作用。

甘蔗不同组织丝氨酸/苏氨酸蛋白激酶基因家族的表达分析

本研究通过光合参数和荧光定量PCR的测定来分析甘蔗不同组织丝氨酸/苏氨酸蛋白激酶基因家族的表达。研究结果表明,乙烯利对九月龄甘蔗光合作用影响不大,对光合产物的积累和转化,糖代谢的信号调控的影响微弱;通过分析STK基因家族在甘蔗各组织中的组织表达差异时发现,多数基因是在鞘和嫩叶、嫩茎中表达量较高,且STK2、STK7在茎中表达量高,STK3在老叶中表达量高,STK5的嫩叶表达量高。

Electroacupuncture Pretreatment at Neiguan (PC 6) attenuates autophagy in rats with myocardial ischemia reperfusion through the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway

OBJECTIVE: To investigate the protective efficacy of electroacupuncture(EA) pretreatment at Neiguan(PC6) on myocardial ischemia-reperfusion(I/R) in rats.METHODS: Fifty rats were randomly divided into five groups(n = 10): sham operation group, model group(underwent in vivo myocardial I/R), EA pretreatment group [EA at Neiguan(PC 6) 1 week before I/R], wortmannin group(1 week before I/R, the PI3K inhibitor, wortmannin, was injected), EA pretreatment + wortmannin group(both pretreatments were performed simultaneously). After establishing the I/R model, 2,3,5-triphenyltetrazolium chloride(TTC) staining was used to analyze the weight and area of the myocardial infarction tissue.The biosignal and pressure test system was used to determine the left ventricular systolic mean pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), fractional shortening(FS), and ejection fraction(EF). Ultraviolet spectrophotometry was used to determine the expression of creatine kinase(CK)-MB, inducible nitric oxide synthase(i NOS), and total antioxidant capacity(T-AOC) in the serum. The expression of autophagy-related protein 13(ATG13), mammalian target of rapamycin(m TOR), and phosphatidylinositol 3-kinase(PI3K) in cardiac muscle cells was determined by immunofluorescence. Hematoxylin and eosin staining was used to observe autophagy-related pathological changes in rat cardiomyocytes, and ultrastructural changes of cardiomyocytes were examined by transmission electron microscopy.RESULTS: In this study, the infarction size and tissue weight of the EA pretreatment group were decreased compared with the model group(P <0.0001). Furthermore, compared with the model group, the LVEDP values of the EA pretreatment group were significantly reduced(P = 0.0091), and the LVSP, FS, and EF values were slightly increased (P = 0.0007, 0.0020, 0.0031). EA pretreatment also significantly decreased the expression of CK-MB and i NOS, while it increased the expression of T-AOC in the serum of rats with I/R injury(P <0.0001). Furthermore, EA pretreatment slightly widened the myocardial fiber space, reduced necrosis and myocardial cell swelling and maintained the nucleus and mitochondria structure intact.CONCLUSION: EA pretreatment promoted autophagy flux and alleviated myocardial I/R injury through the PI3K-Akt-m TOR pathway.

Efficacy of self-made Gengnian decoction on phosphatidylinositol 3-kinases/protein kinase B/mammalian target of rapamycin signaling pathway in perimenopausal rats

OBJECTIVE:To investigate the efficacy of self-made Gengnian decoction on expressions of phosphatidylinositol 3-kinase(PI3 K),protein kinase B(Akt)and mammalian target of rapamycin(m TOR)in ovarian tissues of perimenopausal rats.They were identified with symptom pattern of kidney-Yang deficiency in terms of Traditional Chinese Medicine.METHODS:Female Sprague-Dawley rats aged10-12 months were selected.Estrous cycle was observed by vaginal smears of keratinocytes to screen the perimenopausal model rats.The chosen rats were randomly divided into five groups,including perimenopausal model of kidney-Yang deficiency group(24 rats),self-made Gengnian decoction of high-dose group(24 rats),self-made Gengnian decoction of middle-dose group(24 rats),self-made Gengnian decoction of low dose group(24 rats)and tibolone control group(24 rats).In addition,rats aged 4-6 months were selected as young control group.The perimenopausal model rats of kidney-Yang deficiency were prepared by alternative intramuscular injection of hydrocortisone 5 mg·kg^-1·d^-1The successfully prepared models in self-made Gengnian decoction of high-dose,middle-dose and low-dose groups and tibolone control group were given self-made Gengnian decoction 26.4,13.2 and 6.6 mg·kg^-1·d^-1,and tibolone tablets solvent 0.22 mg·kg^-1·d^-1,respectively,through intragastric administration.Models group and young control group were given the same dose of normal saline,1 time a day for 15 consecutive days.24 h after the last administration,blood and ovarian tissues were collected after anesthesia with 20%ethyl carbamate.The follicles of different levels in ovarian tissue were observed and counted by histopathological hematoxylin-eosin staining.Enzyme linked immunosorbent assay was applied to test insulin-like growth factor-1(IGF-1)level in the serum of experimental rats.The expression levels of PI3 K,phosphorylated-Akt(p-Akt)and phosphorylated-m TOR(p-m TOR)m RNA in ovarian tissue were detected by quantitative real-time polymerase chain reaction.RESULTS:The total follicle counts of perimenopausal model rats with kidney-Yang deficiency were significantly reduced,and the number of follicles(mainly increased in preantral follicles and antral follicles)in perimenopausal model rats with kidney-Yang deficiency was significantly increased after intervention of high and middle doses of Gengnian decoction and tibolone(P<0.05).Compared with normal rats in young control group,the levels of IGF-1 in serum of perimenopausal rats with kidney-Yang deficiency were significantly decreased(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated.The relative expression levels of PI3 K,p-Akt,p-m TOR m RNA in ovarian tissues of perimenopausal rats with kidney-Yang deficiency were significantly lower than those of young rats(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated(P<0.05).CONCLUSION:Self-made Gengnian decoction can increase the levels of IGF-1,PI3 K,Akt and m TOR m RNA expression in serum.

Effect of Shenzhu Tiaopi granule(参术调脾颗粒) on hepatic insulin resistance in diabetic Goto-Kakizakirats via liver kinase B1/adenosine 5’-monophosphate/mammalian target of rapamycin signaling pathway

OBJECTIVE: To observe the therapeutic effect of Shenzhu Tiaopi granule(参术调脾颗粒, STG) on insulin resistance(IR) in the liver of diabetic Goto-Kakizaki(GK) rat and investigate underlying mechanisms.METHODS: Ten 12-week-old male Wistar rats were assigned as normal control(NC) group, while 4012-week-old male specific-pathogen-free GK rats were randomly divided into four experimental groups, 10 diabetic rats each. Animals were fed with a normal diet. Fasting blood glucose(FBG), water intake, and body weight were recorded during6 weeks of daily single-dose treatment: STG low-dose group, 4.5 g/kg(STG-L);STG high-dose group,9 g/kg(STG-H);metformin group, 0.1 g/kg(MET);model control(MC) and NC groups, equal volume of 0.9% Na Cl solution. The serum fasting insulin(FINS), C-Peptide and IR index(HOMA-IR) were detected every 2 weeks during treatment and glucose tolerance was measured in the 3 rd day before the material was taken. After the 6-week STG treatment, Liver tissues were processed for hematoxylin-eosin staining to perform light microscopy analysis and for assessing expression and distribution of insulin receptor substrates(IRS-1) and glucose transporter(GLUT-4) by immunohistochemistry analysis. Expression levels of liver kinase B1(LKB1)/adenosine 5’-monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway proteins, including LKB1, phospho-AMPK(p-AMPK)/AMPK, phospho-mTOR(p-mTOR)/mTOR, and ribosomal protein S6 kinase polypeptide 1(S6 K1),were detected by Western blotting.RESULTS: STG significantly reduced the FBG level and liver fat deposition in diabetic GK rats. After STG treatment completion, FINS, HOMA-IR, C-Peptide and area under blood glucose curve(AUC)were lower in STG groups than in the MC group, indicating that IR was reduced and liver fat lesions were resolved. In liver tissues, STG groups displayed significantly higher IRS-1 and GLUT-4 expression than the MC group, along with increased LKB1 and p-AMPK/AMPK expression and decreased p-mTOR/mTOR and phospho-S6 K1 expression, suggesting that STG stimulated LKB1 activation of AMPK and suppressed themTOR/S6 K1 downstream pathway.CONCLUSION: Growing GK rats developed hepatic IR, but STG treatment significantly improved hyperglycemia and IR and resolved hepatic fatty lesions.Interestingly, STG treatment stimulated the expression of IRS-1 and GLUT-4 in the liver of diabetic GK rats, indicating a potential involvement in the regulation of the LKB1/AMPK/mTOR signaling pathway.

Cloning and Sequence Analysis of Disease Resistance Gene Analogues from Three Wild Rice Species in Yunnan

Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine protein kinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified from three wild rice species in Yunnan Province, China. The DIN A fragments from amplification have been cloned into the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. , and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Oryza officinalis Wall, and TR19 from Oryza rufipogon Griff, belong to the same class and homology of their sequences are 100%. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. 5 classes STK disease resistance gene analogues were also obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. By comparison analysis of amino acid sequences. we found that the obtained disease resistance gene analogues have very low identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative disease resistance genes that have not been isolated so far.

Growth Hormone Prevents the Memory Deficit Caused by Oxidative Stress in Early Neurodegenerative Stage in Rats

Oxidative stress has been involved in neurodegenerative diseases. The growth hormone (GH) counteracts the levels of reactive oxygen species. Previously, we showed that the prolonged exposure to ozone causes oxidative stress in the hippocampus and memory deficits. In this work, we analyzed the effects of the growth hormone on the memory deficit generated by ozone exposure, growth hormone effects on the Insulin-like growth factor I (IGF-I), and the serinethreonine protein kinase (Akt) activation in the dentate gyrus. Our results show that GH prevents memory deficits in early stages of the neurodegenerative process.

黑子南瓜中STK类抗病基因同源序列的克隆及序列分析

黑子南瓜是一种具有较强抗病性的葫芦科植物,依据所克隆的植物丝氨酸/苏氨酸蛋白激酶类抗病基因中的保守氨基酸序列合成相应的简并引物,从黑子南瓜基因组DNA中通过PCR克隆到了一些STK类的R-片段,经相关软件分析发现其中之一可以编码完整的氨基酸序列,可能来源于黑子南瓜中某个抗病或抗病相关基因.该基因片段与来源于其他已知基因的相应序列比较发现同源性都比较低,属于一个新的STK类R-片段家族成员.从黑子南瓜中克隆STK类片段,将为进一步从该植物材料中获得STK类抗病基因提供新线索.

RNA干扰LKB1基因对前列腺癌细胞转化生长因子-β1表达的影响

目的探讨沉默肝激酶B1/丝氨酸-苏氨酸激酶11(liver kinase B1/serine-threonine kinase 11,LKB1/STK11),基因对前列腺癌细胞株RWPE-1中转化生长因子-β1(transforming growth factor-β1,TGF-β1)的调控作用。方法构建3个sh RNA LKB1重组载体,转染至高表达LKB1的前列腺癌细胞株RWPE-1中,RT-PCR、Western blot法分别检测LKB1及TGF-β1 m RNA及蛋白表达水平;ELISA法检测细胞培养液上清中TGF-β1的分泌水平。结果构建获得的3种sh RNA LKB1表达载体,均能抑制LKB1基因的表达,其中以LKB1-sh RNA1沉默效果最强,其对LKB1 m RNA水平的抑制率为74.20%,对蛋白水平的抑制率为71.66%;转染重组质粒LKB1-sh RNA1后,TGF-β1的m RNA、蛋白表达及分泌水平均明显增加(P﹤0.05)。结论 LKB1基因沉默后,TGF-β1的表达明显增高,LKB1参与调节前列腺癌细胞中TGF-β1的表达,提示LKB1基因可作为研究前列腺癌发生发展的分子机制的新靶点。

乙酰胆碱受体对胞壁酰二肽激活小鼠巨噬细胞NLR2/RIP2通路的调控作用

目的 评价乙酰胆碱受体对胞壁酰二肽（MDP）激活小鼠巨噬细胞Nod样受体2/受体相互作用蛋白2（NLR2/RIP2）通路的调控作用.方法 RAW264.7细胞长至对数生长期时,接种于12孔培养板（细胞密度1＆#215;106个/ml,2 ml/孔）,108个培养孔.采用随机数字表法,将其分为3组（n=36）,正常对照组（C组）常规培养;M组加入MDP,终浓度为10 μg/ml;G组同时加入MDP和α7烟碱型乙酰胆碱受体特异性激动剂GTS-21,终浓度分别为10、50 μg/ml.分别于MDP孵育1、6、24h时取12个培养孔,取细胞悬液,采用实时荧光定量PCR法检测NLR2 mRNA表达,采用Western blot印迹法检测RIP2表达,采用ELISA法检测培养液TNF-α和高迁移率族蛋白-1（HMGB1）的浓度.结果 与C组比较,M组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平升高（P＜0.05）;与M组比较,G组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平降低（P＜0.05）.结论 乙酰胆碱受体可抑制MDP激活小鼠巨噬细胞NLR2/RIP2通路转导.

Bushenhuoxue improves cognitive function and activates brain-derived neurotrophic factor-mediated signaling in a rat model of vascular dementia

OBJECTIVE:To explore the protective mechanisms of the Traditional Chinese Medicine Bushenhuoxue(BSHX)in a rat model of vascular dementia(VD).METHODS:A rat model of VD was developed using bilateral common carotid artery occlusion(BCCAO).Rats were administered BSHX(10.14 or 5.07 g/kg),nimodipine(11.06 mg/kg;positive control),or saline(control)by gavage daily for 30 d post-surgery.Learning and memory abilities were assessed using the Morris water maze.Morphological changes in the hippocampus were observed using light microscopy(hematoxylin and eosin staining)and transmission electron microscopy(TEM).The m RNA and protein expression levels of brain-derived neurotrophic factor(BDNF),tyrosine receptor kinase B(Trk B),phosphatidyl inositol 3-kinase(PI3 K),serine/threonine kinase(AKT),and c AMP response element binding protein(CREB)were measured by real-time polymerase chain reaction(RT-PCR)and Western blot,respectively.RESULTS:Compared with the sham group,rats with BCCAO exhibited impaired learning and memory abilities(Morris water maze)and showed abnormalities in neuronal morphology(light microscopy)and ultrastructure(TEM)in the hippocampus.They also had decreased m RNA and protein expressions of BDNF,Trk B,PI3 K,AKT,and CREB in hippocampal tissue(all P<0.05).In rats with BCCAO,administration of BSHX attenuated deficits in learning and memory,improved the morphology and ultrastructure of hippocampal neurons,and enhanced m RNA and protein expression levels of BDNF,Trk B,PI3 K,AKT,and CREB(all P<0.05).CONCLUSION:BSHX may protect hippocampal neurons and improve learning and memory abilities,at least in part via the activation of BDNF/Trk B/PI3 K/AKT/CREB signaling.

Effect of aqueous extract of Astragalus membranaceus on behavioral cognition of rats living at high altitude

OBJECTIVE:To investigate the effect of aqueous extract of Astragalus membranaceus on cognitive ability of rats living at high altitude.METHODS:Rats were exposed to a simulated highaltitude hypobaric hypoxia chamber.The behavior of rats was tested by eight-arm maze.The contents of malondialdehyde(MDA),glutathione(GSH),reactive oxygen species(ROS)and activity of total superoxide dismutase(T-SOD)in hippocampus were measured.The expressions of mammalian target of rapamycin(mTOR)and cleaved capase-3 in hippocampus were determined by reverse transcription-polymerase chain reaction and Western blot.RESULTS:The behavioral cognitive ability of the hypoxic control group was significantly lower than that of the normoxic control group.Under hypoxic environment,after the administration of aqueous extract of Astragalus membranaceus,the behavioral cognitive ability of rats was significantly improved.In hippocampal tissue,the content of MDA and ROS were significantly decreased,while the content of GSH and activity of T-SOD in hippocampus were significantly increased.The m RNA expression of mTOR and P70S6K and the protein expression of p-mTOR were significantly increased;the m RNA expression of 4E-binding protein 1(4E-BP1)and the protein expression of phosphorylated-4E-BP1(p-4EBP1)and cleaved capase-3 were significantly decreased.CONCLUSION:When the rats are exposed to high altitude hypoxia,the behavioral cognitive ability could be significantly reduced.Aqueous extract of Astragalus membranaceus can significantly improve cognitive function in rats under hypoxia.The potential mechanism is related to improving oxidative stress,reducing the accumulation of free radicals and metabolites,and activating mTOR signaling pathway.

Sanguinarine suppresses cell proliferation, migration and invasion in nasopharyngeal carcinoma via inhibiting mTOR signaling

OBJECTIVE: To confirm the anti-NPC effect of sanguinarine(SA) through a series of wet experiments.METHODS: NPC cell viability was determined by proliferation experiment. Cell clone formation experiment, cell scratch test, transwell migration and invasion experiment and flow cytometry-based cell apoptosis assay were further performed. In addition, Western blotting was performed to investigate the cell signaling pathway. All the relevant experimental data were statistically processed using SPSS 16.0 software.RESULTS: The results showed that sanguinarine represented a time and dose dependent inhibition effects on NPC cell proliferation including the low differentiated CNE2 cells and high metastatic 5-8F cells, along with the cell cloning ability reduction. In addition, sanguinarine has a certain inhibitory effect on the invasion and migration of NPC cells. Mechanistically, sanguinarine displayed the anti-NPC effects mainly involved into the suppression of m TOR signaling and cell apoptosis, which is closely associated with the tumor growth and metastatic malignancy. CONCLUSIONS: Collectively, we discover that sanguinarine is a new high-efficiency anti-NPC monomer of Chinese medicine, with a value for the follow-up preclinical research.

Effects of Gas1 on gliomas:a review on current preclinical studies

Glioblastoma multiforme(GBM)is the most common and lethal brain tumor.Its prognosis remains very poor,despite the use of combined treatments such as surgical resection,radiation and chemotherapy.The major limitations for the treatment of GBM are its high invasiveness,tumor recurrence and resistance to treatments.Therefore,gene therapy appears as a relevant strategy for its treatment.Thus,we have investigated the use of growth-arrest-specific 1(Gas1)for the treatment of GBM.Gas1 is a tumor suppressor protein that inhibits glioma growth by inducing arrest and apoptosis of tumor cells.Moreover,we have shown that a soluble form of Gas1 acting in both autocrine and paracrine manners is also effective inhibiting tumor growth in animal models,indicating its potential as an adjuvant for the treatment of GBM.