基于PI3K/AKT/GSK-3β信号通路探讨EA改善APP/PS1双转基因小鼠认知功能障碍的内在机制

目的探讨鞣花酸(ellagicacid,EA)对APP/PS1双转基因小鼠认知功能的影响,并基于磷脂酰肌醇3-激酶/蛋白激酶B/糖原合成酶激酶-3(PI3K/AKT/GSK-3β)信号通路探讨鞣花酸对双转基因小鼠海马氧化应激水平的调节机制。方法将32只SPF级6月龄APP/PS1双转基因小鼠随机分为4组,即APP/PS1组、APP/PS1+EA组、APP/PS1+LY294002组、APP/PS1+EA+LY294002组,每组8只,另外选取8只SPF级C57BL/6J野生型小鼠(Wildtype)作为空白对照组,即WT组。APP/PS1+EA组给予50mg·kg^(-1)·d^(-1)灌胃EA;APP/PS1+LY294002组予以1.5mg·kg^(-1)·d^(-1)腹腔注射PI3K抑制剂LY294002;APP/PS1+EA+LY294002组予以50mg·kg^(-1)·d^(-1)灌胃EA,同时按1.5mg·kg^(-1)·d^(-1)腹腔注射LY294002;WT组和APP/PS1组于相同时间点灌胃等体积10%二甲基亚砜(DMSO)。每日给药1次,连续给药60天。Morris水迷宫检测小鼠学习和记忆能力,免疫组化、蛋白免疫印迹法检测PI3K、AKT、GSK-3β相关蛋白的表达,透射电镜观察小鼠海马组织超微结构变化。结果与WT组相比,其他四组的逃避潜伏期均增长(P<0.05),穿越平台次数明显减少(P<0.01);APP/PS1组、APP/PS1+LY294002组和APP/PS1+EA+LY294002组中的PI3K、AKT蛋白表达量显著降低(P<0.01),GSK-3β表达量显著升高(P<0.01);APP/PS1+EA组的PI3K表达量降低(P<0.05),AKT表达量显著降低(P<0.01),GSK-3β表达量升高(P<0.05);与WT组相比,APP/PS1组海马神经元细胞数目较少,线粒体结构破坏,大部分线粒体出现肿胀,线粒体的内膜和外模不完整,部分线粒体嵴消失,微管、微丝缠结,排列紊乱,而APP/PS1+EA组神经元细胞数较APP/PS1组增多,线粒体结构较清晰,可见清楚的线粒体嵴,线粒体轻度水肿。微管、微丝排列较整齐有序。结论鞣花酸改善AD模型小鼠的学习和记忆能力、减少海马神经元细胞损伤和凋亡,其作用机制可能是通过调节PI3K、AKT、GSK-3β等相关蛋白降低AD模型小鼠海马氧化应激水平。

黄芪阳和汤调控PI3K/AKT/NF-κB信号通路促进糖尿病足溃疡大鼠创面愈合

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子-κB(NF-κB)信号通路探究黄芪阳和汤对糖尿病足溃疡(DFU)大鼠创面愈合的影响。方法构建DFU大鼠模型,将建模成功的48只大鼠随机分为模型组,黄芪阳和汤低(8.5 g/kg)、高(17 g/kg)剂量组,黄芪阳和汤高剂量(17 g/kg)+LY294002(PI3K/AKT通路抑制剂,0.3 mg/kg)组;每组12只;另取12只大鼠为对照组。各组大鼠给予对应药物干预,连续4周。第14、28天给药后,观察大鼠一般状态及创面变化,计算创面愈合率,检测大鼠空腹血糖(FBG)水平和大鼠创面周围组织经皮氧分压(TcpO2);酶联免疫吸附试验检测大鼠血清血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)、C反应蛋白(CRP)、白细胞介素(IL)-6水平;苏木素-伊红染色观察大鼠创面组织病理学变化;免疫组织化学染色测定大鼠创面组织微血管密度;蛋白免疫印迹法检测大鼠创面组织中PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB抑制蛋白α(IκB-α)蛋白表达。结果对照组大鼠毛色光滑,饮食、饮水、排泄均正常,较活跃,创面愈合快,创面组织炎症反应较轻,新生血管较多,肉芽组织中成纤维细胞及胶原基质丰富;模型组大鼠毛色暗淡无光泽,活动减少,且出现多饮、多食、多尿症状,创面颜色较深,且周围组织出现水肿、溃疡,创面组织可见大量炎性细胞浸润,伴组织坏死、渗出,新生血管及成纤维细胞较少,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平降低,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达升高(P<0.05);与模型组相比,黄芪阳和汤低、高剂量组大鼠状态逐渐改善,创面组织病变程度依次减轻,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平依次升高,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达依次降低(P<0.05);LY294002能部分逆转高剂量黄芪阳和汤对DFU大鼠的治疗作用(P<0.05)。结论黄芪阳和汤能调控PI3K/AKT/NF-κB信号通路,抑制DFU大鼠炎症反应,促进血管新生,从而促进创面愈合。

葛根芩连汤通过IRS-1/PI3K/AKT通路对胃肠湿热型2型糖尿病大鼠糖脂代谢的影响

目的探究葛根芩连汤通过胰岛素受体底物-1(IRS-1)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路对胃肠湿热型2型糖尿病大鼠糖脂代谢的影响。方法将40只SD大鼠随机分为正常组(2 mL生理盐水灌胃)、造模组(2 mL生理盐水灌胃)、二甲双胍组(4.17 mg/100 g二甲双胍灌胃)和葛根芩连汤组(1 g/100 g葛根芩连汤灌胃),每组10只。采用高脂高糖饲料加腹腔注射链脲佐菌素(STZ)构建2型糖尿病大鼠模型,随后喂食油脂、42°白酒及蜂蜜水构建胃肠湿热型2型糖尿病大鼠模型。测量各组大鼠不同时间节点体质量,血糖仪测定空腹血糖(FBG);ELISA检测空腹胰岛素(FINS)、三酰甘油(TG)、总胆固醇(TC)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平变化、计算胰岛素抵抗指数(HOMA-IR);HE染色检测肝组织病理学变化;检测肝组织过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量变化。Western blot检测肝组织IRS-1、PI3K、p-PI3K、AKT及p-AKT蛋白变化。结果与正常组比较,造模组大鼠体质量、FBG、FINS及HOMA-IR、GSH-Px、CAT、SOD、IRS-1、p-PI3K/PI3K及p-AKT/AKT水平均明显下降(P<0.05)、TG、TC、IL-6、TNF-α及MDA含量均显著升高(P<0.05),可见局灶性肝实质损失。与造模组比较,二甲双胍组及葛根芩连汤组大鼠体质量、FBG、FINS及HOMA-IR、GSH-Px、CAT、SOD、IRS-1、p-PI3K/PI3K及p-AKT/AKT水平均明显升高(P<0.05)、TG、TC、IL-6、TNF-α及MDA含量均显著降低(P<0.05),显示正常的肝实质。结论葛根芩连汤可明显改善胃肠湿热型2型糖尿病糖脂紊乱,可能是通过IRS-1/PI3K/AKT通路发挥作用。

LncRNA MALAT1通过靶向miR-146a调节PI3K/Akt信号通路影响胃癌细胞的增殖、迁移和侵袭

目的 探究长链非编码RNA(long non-coding RNA,LncRNA)肺腺癌转移相关转录子1(metastasis-associated lung adenocarcinoma transcript 1,MALAT1)通过调节miR-146a对胃癌(gastric cancer, GC)细胞的增殖、迁移和侵袭的影响及其机制。方法 收集GC组织和配对正常胃上皮组织,将GC细胞MNK-45分为空白对照(blank control, BC)组(未转染)、MALAT1 siRNA-NC组(转染MALAT1 siRNA-NC)、MALAT1 siRNA组(转染MALAT1 siRNA)、miR-146a mimics-NC组(转染miR-146a mimics-NC)、miR-146a mimics组(转染miR-146a mimics)、MALAT1 siRNA+miR-146a inhibitor-NC组(共转染MALAT1 siRNA+miR-146a inhibitor-NC)、MALAT1 siRNA+miR-146a inhibitor组(共转染MALAT1 siRNA+miR-146a inhibitor)。定量荧光PCR(qRT-PCR)检测胃组织或细胞中MALAT1、miR-146a表达量;CCK-8法和克隆形成实验检测细胞增殖能力;Transwell法检测细胞侵袭和迁移能力;RNA pull down实验、双荧光素酶报告实验分析MALAT1和miR-146a的结合情况;Western blot检测磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase, PI3K)/蛋白激酶B(protein kinase B,Akt)通路蛋白及c-Myc、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)蛋白表达量。结果 转染MALAT1 siRNA可明显降低MNK-45细胞的MALAT1表达量,敲低MALAT1或过表达miR-146a可降低细胞活力、克隆能力、迁移和侵袭,增加miR-146a表达,降低PI3Kp85α、PI3Kp85β、c-Myc、MMP9蛋白表达量及p-Akt/Akt水平;MALAT1可结合并靶向下调miR-146a表达;低表达miR-146a可逆转敲低MALAT1对MNK-45细胞增殖、迁移和侵袭的抑制效应。结论 MALAT1可能作为ceRNA吸附并降解miR-146a,敲低MALAT1可上调miR-146a表达,并通过PI3K/Akt通路抑制GC细胞增殖、迁移和侵袭。

Micro RNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of micro RNA-21(mi R-21), phosphatase gene(p TEN), and its downstream proteins, protein kinase B(AKT) and phosphatidylinositol 3-kinase(p I3K), in colorectal cancer(CRC) cells. METHODS: Quantitative real-time p CR(q RT-p CR) and Western blot were used to detect the expression levels of mi R-21 and p TEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of p TEN m RNA and its downstream proteins AKT and p I3 K in HCT116 cells after downregulating mi R-21 were investigated. RESULTS: Comparing the mi R-21 expression in CRC cells, the expression levels of mi R-21 were highest in HCT116 cells, and the expression levels of mi R-21 were lowest in SW480 cells. In comparing mi R-21 and p TEN expression in CRC cells, we found that the protein expression levels of mi R-21 and p TEN were inversely correlated(p < 0.05); when mi R-21 expression was reduced, m RNA expression levels of p TEN did not significantly change(p > 0.05), but the expression levels of its protein significantly increased(p < 0.05). In comparing the levels of p TEN protein and downstream AKT and p I3 K in HCT116 cells after downregulation of mi R-21 expression, the levels of AKT and p I3 K protein expression significantly decreased(p < 0.05). CONCLUSION: p TEN is one of the direct target genesof mi R-21. Thus, phosphatase gene and its downstream AKT and p I3 K expression levels can be regulated by regulating the expression levels of mi R-21, which in turn regulates the development of CRC.

Phosphatidylinositol 3-kinase/Akt pathway regulates hepatic stellate cell apoptosis

AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC). METHODS: An activated HSC cell line was used in this study. LY 294002, the PI 3-K/Akt signal pathway block-er was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this path-way in HSC apoptosis. Immunocytochemistry, Western blot and reverse transcription polymerase chain reac-tion (RT-PCR) analysis were applied to detect the ex-pression of PI 3-K, and simultaneously phosphorylated-Akt (p-Akt) and total-Akt were determined by Western blot. The HSC apoptosis was examined by annexin-V/ propidium iodide double-labelled flow cytometry and transmission electron microscopy. RESULTS: The apoptosis rates in LY 294002 (30.82% ± 2.90%) and LY 294002 + PDGF-BB (28.16% ± 2.58%) groups were signif icantly increased compared with those of control (9.02% ± 1.81%) and PDGF-BB (4.35% ± 1.18%). PDGF-BB augmented PI 3-K and p-Akt expres-sion. LY 294002 signif icantly reduced the contents of PI 3-K and p-Akt. mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression. CONCLUSION: Inhibition of PI 3-K/Akt signaling path-way induces apoptosis in HSC.

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM: To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action. METHODS: The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential (△Ψm) were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases (PI3K). RESULTS: Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time-and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 μmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt. CONCLUSION: Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.

Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer

AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment.The patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical records.Expression of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM kit.Standard criteria for HER2 positivity(0,1+,2+,and 3+) were used.Tumors that scored 3+ were considered HER2-positive.Expression of phospho Akt(pAkt) was also analyzed by IHC.Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more.PI3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by pyrosequencing.Epstein-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA probe.Microsatellite instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.RESULTS:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,respectively.HER2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 cancers.Mutations in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,respectively.Two new types of PIK3CA mutation,R88Q and R108H,were found in exon1.All PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in exon20.Eighteen cancers(8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type(13/18 vs 93/198,P = 0.04).There were 7 cases of lymphoepithelioma-like carcinomas(LELC) and 6 of those cases were EBV-positive(percent/EBV:6/18,33%;percent/all LELC:6/7,86%).pAkt expression was positive in 119(53%) cases but showed no correlation with clinicopathological characteristics.pAkt expression was significantly correlated with HER2 overexpression(16/20 vs 103/211,P < 0.01) but not with PIK3CA mutations(12/20 vs 107/211,P = 0.37) or EBV infection(8/18 vs 103/211,P = 0.69).The frequency of pAkt expression was higher in cancers with exon20 mutations(100%) than in those with exon1(40%) or exon9(56%) mutations.One case showed both HER2 overexpression and EBV infection and 3 cases showed both PIK3CA mutations and EBV infection.However,no cases showed both PIK3CA mutations and HER2 overexpression.One EBVpositive cancer with PIK3CA mutation(H1047R) was MSI-positive.Three of these 4 cases were positive for pAkt expression.In survival analysis,pAkt expression significantly correlated with a poor prognosis(hazard ratio 1.75;95%CI:1.12-2.80,P = 0.02).CONCLUSION:HER2 expression,PIK3CA mutations and EBV infection in gastric cancer were characterized.pAkt expression significantly correlates with HER2 expression and with a poor prognosis.

Analysis of Phosphatidylinositol 3-kinase Activation in the Adipose Tissue of Gestational Diabetes Mellitus Patients and Insulin Resistance

The P85 regulatory subunit protein and gene expression and P110 catalylic subunit activity of phosphatidylinositol 3-kinase (PI-3K) were investigated in adipose tissue of patients with gestational diabetes mellitus (GDM) in order to explore the molecular mechanisms of insulin resistance (IR) of GDM. Samples from patients with GDM (n=50), and controls (n=50) were collected. Fasting insulin (FIN) was determined by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Western blot technique was used to detect the levels of PI-3K P85 subunit in adipose tissues of patients with GDM. The mRNA expression of PI-3K P85 subunit was detected by reverse transcription polymerase chain reaction (RT-PCR) method in the adipose tissue. PI-3K activity was examined by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed statistically. It was found that the levels of FPG, FIN and HOMA-IR in GDM group were significantly higher than those in control group (all P0.05). PI-3K activity was significantly decreased to 82.89% in GDM group as compared with control group (P<0.01) and negatively correlated with HOMA-IR (r=-0.75, P<0.01). It was concluded that PI-3K in GDM patients may be involved in the insulin signaling pathway, resulting in IR of GDM.

Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury

BACKGROUND: Acute lung injury(ALI) is a common and serious complication of severe acute pancreatitis(SAP). The study aimed to investigate the protective effect and mechanism of phosphatidylinositol-3 kinase(PI3K) inhibitor Wortmannin in SAP associated with ALI.METHODS: Ninety rats were randomly divided into three groups: sham operation(SO) group(n=30), SAP group(n=30), and SAP+Wortmannin(SAP+W) group(n=30). SAP model was induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct of rats. The rate of lung water content, myeloperoxidase(MPO), matrix metalloproteinase 9(MMP-9), protein kinase B(PKB), abdphosphorylation of protein kinase B(P-PKB) activity in the lung tissue were evaluated.RESULTS: In the SAP group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours(P<0.05); the rate of lung water content, MPO and TNF-α activity were also gradually increased, and the degree of lung lesion gradually increased(P<0.05). In the SAP+Wortmannin group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours; it was higher than that in the SO group(P<0.05), but significantly lower than that in the SAP group(P<0.05). The rest indicators in the SAP+Wortmannin group were also significantly decreased as compared with the SAP group(P<0.05).CONCLUSIONS: The expression of phosphatidylinositol-3 kinase/protein kinase B was elevated in severe pancreatitis rats with lung injury. This suggested that PI3 K signal transduction pathway is involved in the control and release of proinfl ammatory cytokines TNF-α, which may play an important role in the pathogenesis of severe acute pancreatitis associated with lung injury. This finding indicated that Wortmannin can block the PI3 K signal transduction pathway, and inhibit the release of infl ammatory factor TNF-α.

Neural stem cell transplantation in the hippocampus of rats with cerebral ischemia/reperfusion injury Activation of the phosphatidylinositol-3 kinase/Akt pathway and increased brain-derived neurotrophic factor expression

The phosphatidylinositol-3 kinase （PI3K）/Akt pathway and brain-derived neurotrophic factor （BDNF） are involved in neurological functional recovery following cerebral ischemia. Therefore, we hypothesized that mechanisms of neuroprotection by transplantation of neural stem cells （NSCs） on cerebral ischemia contributed to activation of the PI3K/Akt pathway and enhanced BDNF expression. In the present study, Wortmannin （a specific, covalent inhibitor of PI3K） was administered adjacent to ischemic hippocampus by stereotactic transplantation to further confirm the neuroprotective mechanisms of NSC transplantation following cerebral ischemia. Results showed that focal infarct volume was significantly smaller in the NSCs group, but the neurological behavior score in the NSC group was significantly greater than the middle cerebral artery occlusion model group, Wortmannin treatment group, and NSCs ＋ Wortmannin treatment group. Protein expression of BDNF was significantly greater in the NSC group compared with the Wortmannin treatment group and NSCs ＋ Wortmannin treatment group. These results suggest that the neuroprotective role of NSC transplantation in the cerebral ischemia activated the PI3K/Akt pathway and upregulated BDNF expression in lesioned brains.

Glycine-extended gastrin activates two independent tyrosine-kinases in upstream of p85/p110 phosphatidylinositol 3-kinase in human colonic tumour cells

瞄准：为了调查 Src， JAK2 和 phosphatidylinositol 3-kinase (PI3K ) 小径是否涉及人的结肠的瘤房间的增长，由扩大 glycine 的促胃液素导致了(G-gly ) ，先锋在 ated 促胃液素中并且到成熟阐明在响应这肽的这三家族 ases 之间的分子的相互作用。方法：用人的结肠的瘤，房间作为一个模型衬里 HCT116，我们首先测量了 PI3K， p60-Src 和 JAK2 的激活响应 G-gly 由在试管内激酶试金。然后，我们由 MTT 测试在 G-gly-induced 房间增长调查了这些家族 ases 的参与。结果：G-gly，刺激在 HCT116 导致了 p60-Src， JAK2 和 PI3K 激活。不同小径涉及房间导致了由的人的结肠癌的增长 G-gly。而且，我们发现 Src 和 JAK2 对由这肽的 PI3K 规定必要。然而，我们没发现在二酷氨酸家族 ases 之间的任何串音。结论：我们的结果建议 p60-Src/PI3K 和 JAK2/PI3K 小径独立地行动调停人的结肠的瘤房间上的 G-gly 增生的的效果。

Phosphatidylinositol 3-kinase CB association with preoperative radiotherapy response in rectal adenocarcinoma

AIM:To examine the correlation of phosphatidylinositol3-kinase(PIK3)CB expression with preoperative radiotherapy response in patients with stageⅡ/Ⅲrectal adenocarcinoma.METHODS:PIK3CB immunoexpression was retrospectively assessed in pretreatment biopsies from 208 patients with clinical stageⅡ/Ⅲrectal adenocarcinoma,who underwent radical surgery after 30-Gy/10-fractionpreoperative radiotherapy.The relation between PIK3CB expression and tumor regression grade,clinicopathological characteristics,and survival time was statistically analyzed.Western blotting and in vitro clonogenic formation assay were used to detect PIK3CB expression in four colorectal cancer cell lines(HCT116,HT29,Lo Vo,and LS174T)treated with 6-Gy ionizing radiation.Pharmacological assays were used to evaluate the therapeutic relevance of TGX-221(a PIK3CB-specific inhibitor)in the four colorectal cancer cell lines.RESULTS:Immunohistochemical staining indicated that PIK3CB was more abundant in rectal adenocarcinoma tissues with poor response to preoperative radiotherapy.High expression of PIK3CB was closely correlated with tumor height(P<0.05),yp T stage(P<0.05),and high-degree tumor regression grade(P<0.001).High expression of PIK3CB was a potential prognostic factor for local recurrence-free survival(P<0.05)and metastasis-free survival(P<0.05).High expression of PIK3CB was also associated with poor therapeutic response and adverse outcomes in rectal adenocarcinoma patients treated with 30-Gy/10-fraction preoperative radiotherapy.In vitro,PIK3CB expression was upregulated in all four colorectal cancer cell lines concurrently treated with 6-Gy ionizing radiation,and the PIK3CB-specific inhibitor TGX-221 effectively inhibited the clonogenic formation of these four colorectal cancer cell lines.CONCLUSION:PIK3CB is critically involved in response to preoperative radiotherapy and may serve as a novel target for therapeutic intervention.

黄芩素对人乳腺癌细胞系MDA-MB-231侵袭、迁移、上皮间充质转化的调控作用及其机制

目的观察黄岑素对人乳腺癌细胞系MDA-MB-231的侵袭、迁移及上皮间充质转化(EMT)的调控作用,探讨其可能作用机制。方法取对数生长期MDA-MB-231细胞分为一组、二组、三组及对照组,一组、二组、三组分别加入2.5、5、10μmol/L的黄岑素,对照组不做任何处理。培养48 h时采用划痕修复实验观察四组细胞迁移能力、采用Transwell侵袭实验观察四组细胞侵袭能力,采用Western Blotting法检测细胞EMT标志物波形蛋白(vimentin)及E-钙黏蛋白(E-cadherin)、整合素αv、β3、磷酸化黏着斑激酶(p-FAK)、磷酸化磷脂酰肌醇3激酶(整合素p-PI3K)。结果与对照组相比,黄岑素组细胞迁移率降低、侵袭细胞数少,细胞E-cadherin相对表达量高,vimentin、整合素αv、整合素β3、p-FAK、p-PI3K蛋白相对表达量低,且呈剂量依赖性(P均<0.05)。结论黄芩素抑制MDA-MB-231细胞的侵袭、迁移及EMT。黄岑素可能通过抑制整合素αv、整合素β3表达,进一步抑制p-FAK、p-PI3K蛋白表达,抑制MDA-MB-231的侵袭、迁移及EMT。

miR-375靶向PI3K/AKT通路在高糖诱导的人视网膜内皮细胞增殖和血管生成中的作用

目的探讨miR-375靶向磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路对高糖诱导的人视网膜内皮细胞(hRECs)增殖和血管生成的影响机制。方法体外培养hRECs,对hRECs进行转染及双荧光素酶检测。hRECs分为对照组、高糖组、高糖+miR-375组、高糖+miR-375+LM22B-10组。采用CCK-8实验检测细胞增殖能力,采用血管形成实验检测细胞血管形成能力,采用RT-qPCR检测hRECs内miR-375和PI3K mRNA的表达情况,采用Western blot检测hRECs内PI3K、p-AKT/AKT蛋白表达的情况。结果与对照组比较,高糖组、高糖+miR-375组、高糖+miR-375+LM22B-10组培养48 h和72 h时hRECs增殖活力、PI3K和p-AKT/AKT的蛋白表达水平、血管形成能力和PI3K mRNA表达均显著升高,而miR-375表达降低(均为P<0.05);与高糖组比较,高糖+miR-375组培养48 h和72 h时hRECs增殖活力、PI3K mRNA和蛋白以及p-AKT/AKT的蛋白表达水平、血管形成能力均降低,而miR-375表达升高(均为P<0.05);与高糖+miR-375组比较,高糖+miR-375+LM22B-10组培养48 h和72 h时hRECs增殖活力、血管形成能力和p-AKT/AKT蛋白表达水平均升高(均为P<0.05),而miR-375、PI3K mRNA差异均无统计学意义(均为P>0.05)。转染miR-375模拟物和PI3K野生型载体后,hRECs中相对荧光素酶活性分别较转染miR-375阴性对照、转染PI3K突变型载体后显著降低(均为P<0.05)。结论miR-375靶向抑制PI3K/AKT通路可抑制高糖诱导的hRECs增殖和血管生成,进而缓解DR。

血管生成素-1激活tyrosine kinase／PI3K增加血管内皮细胞[Mg^2＋]i

目的:探讨血管生成素-1(Ang-1)对人脐静脉内皮细胞(HUVECs)内游离镁离子浓度([Mg2+]i)的调节机制。方法:我们采用荧光指示剂mag-fura-2,运用PTi阳离子测定系统动态测HUVECs的[Mg2+]i。结果:Ang-1诱导的[Mg2+]i增加与细胞外Mg2+浓度无关。Ang-1诱导的[Mg2+]i增加与细胞内Ca2+浓度无关。经酪氨酸激酶阻断剂(tyrphostin A23和genistein),磷脂酰3激酶阻断剂(wortmannin和LY294002)预处理,均显著阻断Ang-1诱导的[Mg2+]i增加。但经活化丝裂原激活激酶阻断剂(SB202190和PD98059)预处理,不能阻断Ang-1诱导的[Mg2+]i增加。结论:Ang-1通过酪氨酸激酶/磷脂酰3激酶信号传递途径使细胞内的Mg2+库释放Mg2+,从而增加HUVECs的[Mg2+]i。

Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

薯蓣皂苷经PI3K/AKT通路对肝癌Bel-7402细胞增殖和凋亡的影响

目的分析薯蓣皂苷对肝癌Bel-7402细胞增殖和凋亡的影响并探讨其机制。方法肝癌Bel-7402细胞分为空白组和薯蓣皂苷低、中、高剂量组(给予1、2、8μmol/L的薯蓣皂苷)及薯蓣皂苷+抑制剂组(给予8μmol/L的薯蓣皂苷+10μmol/L的磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路抑制剂LY294002),于处理后12、24、36、48及72 h时采用四甲基偶氮唑盐(MTT)比色法测定细胞活力,于24 h时采用流式细胞术检测细胞凋亡情况、采用蛋白免疫印迹法(Western blot)检测p-PI3K和p-AKT的表达。结果与空白组相比较,薯蓣皂苷低、中、高剂量组细胞活力及p-PI3K、p-AKT表达均下降,凋亡率升高(P<0.05),且各剂量组间两两比较,上述指标水平差异均有统计学意义(P<0.05);与薯蓣皂苷高剂量组比较,薯蓣皂苷+抑制剂组细胞活力及p-PI3K、p-AKT表达下降,凋亡率升高(P<0.05)。结论薯蓣皂苷可能通过抑制PI3K/AKT通路抑制肝癌Bel-7402细胞增殖,诱导Bel-7402细胞凋亡。

眼镜蛇毒细胞毒素-1的分离纯化及其对HSC-LX2细胞PI3K/AKT 信号通路的影响

肝纤维化是多种刺激诱导的肝脏反复损伤的一种病理再生反应[1],其主要表征是肝脏中沉积着过量的细胞外基质(extracellular matrix,ECM)。ECM产生主要原因之一是由于肝星状细胞(hepatic stellate cell,HSC)被大量激活分化,磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)信号通路是一条重要的细胞内信号通路,能通过影响HSC的增殖和凋亡来调节肝纤维化[2-4]。

二苯乙烯苷调控PI3K/AKT信号通路干预SH-SY5Y细胞凋亡的机制研究

目的:探讨二苯乙烯苷(TSG)经PI3K/AKT通路对冈田酸诱导的人神经母细胞瘤细胞(SH-SY5Y)凋亡的影响及调控机制。方法:CCK-8法筛选OA最佳浓度,将SH-SY5Y细胞分为对照组、模型组、TSG组、LY294002组和LY294002+TSG组。通过CCK-8法和TUNEL法检测各组细胞增殖和凋亡情况;Western blotting法和qRT-PCR法检测PI3K、P-PI3K(Y607)、AKT、P-AKT(Ser473)、Bcl-2、Bax蛋白及mRNA表达情况,通路及凋亡蛋白相对表达量以P-PI3K(Y607)/PI3K、P-AKT(Ser473)/AKT、Bcl-2/Bax灰度比值表示。结果:OA最佳处理浓度为40 nmol/L。与对照组相比,模型组细胞活力降低,细胞凋亡率升高,通路及凋亡蛋白、PI3K、AKT、Bcl-2 mRNA表达水平降低,Bax mRNA表达水平升高(均P<0.05);与模型组相比,TSG组细胞活力升高,细胞凋亡率降低,通路及凋亡蛋白、PI3K、AKT、Bcl-2 mRNA相对表达水平升高,Bax mRNA表达水平降低(均P<0.05),LY294002组细胞活力降低,细胞凋亡率升高,P-PI3K(Y607)/PI3K蛋白表达水平显著降低(P<0.05)、P-AKT(Ser473)/AKT、Bcl-2/Bax蛋白表达水平较明显降低,但未有统计学意义,PI3K、AKT、Bcl-2 mRNA表达水平降低,Bax mRNA表达水平升高(均P<0.05);与LY294002组相比,LY294002+TSG组细胞活力升高,细胞凋亡率降低,通路及凋亡蛋白相对表达水平、PI3K、AKT、Bcl-2 mRNA升高,Bax mRNA表达水平降低(均P<0.05)。结论:二苯乙烯苷可能通过干预PI3K/AKT信号通路,进而调节Bcl-2、Bax等凋亡因子的表达,从而缓解冈田酸诱导的SH-SY5Y细胞凋亡。