Glucagon like peptide-1 mediated PI3K/Akt pathway antagonizes advanced oxidized protein products induced apoptosis of HTR-8/SVneo cells / 中国医师杂志

Objective:To explore the protective mechanism of phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway in glucagon like peptide-1 (GLP-1) antagonizing the apoptosis of gestational trophoblasts (HTR-8/SVneo) induced by advanced oxidized protein products (AOPP).Methods:Pregnant trophoblast HTR-8/SVneo were cultured in vitro. The cells were divided into control group, AOPP group, GLP-1 group, AOPP + GLP-1 group and AOPP + GLP-1 + LY294002 group. The control group was cultured in 1640 medium; AOPP group was stimulated with 200 μg/ml AOPP; GLP-1 group was stimulated with 50-100 nmol/L GLP-1 for 1 h; AOPP + GLP-1 group was stimulated with 200 μg/ml AOPP for 48 hours, and then GLP-1 (50-100 nmol/L) was added for 1 hour; In AOPP + GLP-1 + LY294002 group, PI3K inhibitor LY294002 was added on the basis of the intervention of AOPP + GLP-1 group. The expression of PI3K/Akt pathway related protein p-Akt was detected by Western blot. Cell viability was detected by cell counting kit (CCK-8). Enzyme linked immunosorbent assay (ELISA) was used to detect the contents of apoptosis promoter protease caspase-9 and caspase-3, and the contents of apoptosis related proteins Bcl-2, Bax and Cyto-c. Results:After AOPP stimulation, the expression of p-Akt in AOPP group was lower than that in control group ( P<0.05); After 50 and 100 nmol/L GLP-1 intervention, the expression of p-Akt in AOPP + GLP-1 group was significantly higher than that in AOPP group (all P<0.05). After 24 and 48 hours of 100 nmol/L GLP-1 intervention, the expression of p-Akt in AOPP + GLP-1 group was significantly higher than that in AOPP group (all P<0.05). After AOPP stimulation, the cell viability of AOPP group was lower than that of control group ( P<0.05); After GLP-1 intervention, the cell viability of AOPP + GLP-1 group was significantly higher than that of AOPP group ( P<0.05). After adding PI3K inhibitor LY294002, the cell viability of AOPP + GLP-1 + LY294002 group was significantly lower than that of AOPP + GLP-1 group ( P<0.05). The results of ELISA showed that the contents of apoptosis promoter protein caspase-3, caspase-9, apoptosis related protein Bax and Cyto-c in AOPP group were higher than those in control group (all P<0.05), and the content of anti-apoptosis protein Bcl-2 was lower than that in control group ( P<0.05); After GLP-1 intervention, the contents of caspase-3, caspase-9, Bax and Cyto-c in AOPP + GLP-1 group were significantly lower than those in AOPP group ( P<0.05), and the content of anti-apoptosis protein Bcl-2 was higher than that in AOPP group ( P<0.05). After treatment with PI3K inhibitor LY294002, the contents of Bcl-2 in AOPP + GLP-1 + LY294002 group were lower than those in AOPP + GLP-1 group, and the contents of Bax and Cyto-c were higher than those in AOPP + GLP-1 group (all P<0.05). Conclusions:GLP-1 may mediate PI3K / Akt pathway to antagonize the apoptosis of HTR-8/SVneo induced by AOPP.

Effect of astragaloside IV on PI3K/Akt signaling pathway in substantia nigra of mice with Parkinson′s disease / 中华麻醉学杂志

Objective:To evaluate the effect of astragaloside IV on phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in substantia nigra of mice with Parkinson′s disease.Methods:Forty-five SPF healthy male C57BL/6 mice, aged 8 weeks, weighing 19-25 g, were divided into 3 groups ( n=15 each) using a random number table method: control group (group C), Parkinson′s disease group (group PD) and astragaloside IV group (group A). The mouse model of Parkinson′s disease was developed by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) 30 mg/kg everyday for 7 consecutive days.Astragaloside 20 mg/kg was intraperitoneally injected everyday at 30 min before MPTP injection for 7 consecutive days before the model was prepared in group A, and the equal volume of normal saline was given instead in group C. Behavior was measured at 1 day interval after completion of administration.The mice were then sacrificed, and the substantia nigra of the brain tissue were obtained for determination of the expression of tyrosine hydroxylase(TH), phosphorylated PI3K(p-PI3K), phosphorylated Akt(p-Akt), glial fibrillary acidic protein (GFAP) and brain-derived neurotrophic factor (BDNF) (by Western blot). Results:Compared with C group, the total distance of movement and latency of falling were significantly shortened, the hanging score was decreased, the step width was increased, the expression of TH, p-PI3K, p-Akt and BDNF in substantia nigra was down-regulated, and the expression of GFAP was up-regulated in PD group and A group ( P<0.05). Compared with PD group, the total distance of movement and the latency to fall were significantly prolonged, the hanging score was increased, the step width was reduced, and the expression of TH, p-PI3K, p-Akt and BDNF in the substantia nigra was up-regulated, and the expression of GFAP was down-regulated in group A ( P<0.05). Conclusions:The mechanism by which astragaloside IV improves motor dysfunction is related to the activation of PI3K/Akt signaling pathway, up-regulation of BDNF expression and inhibition of astrocyte activation in mice with Parkinson′s disease.

La vía canónica PI3K/AKT/mTOR y sus alteraciones en cáncer / The PI3K/AKT/mTOR canonical pathway and its alterations in cancer

RESUMEN La vía PI3K/AKT/mTOR participa en múltiples procesos celulares fundamentales para la célula. Algunas mutaciones genéticas de los componentes de esta vía se han asociado a diversas enfermedades humanas: las más importantes son los carcinomas de mama, tiroides y endometrio, el glioblastoma multiforme, el cáncer de próstata y los linfomas. La vía canónica PI3K/AKT/mTOR se ha estudiado ampliamente en los últimos años. Sin embargo, el conocimiento de la complejidad de sus componentes principales y su interrelación con los elementos de otras vías va en aumento. Por ello, es importantes actualizar cada cierto tiempo la información disponible para la comprensión de este mecanismo. Así mismo, se están y se han desarrollado numerosos ensayos con medicinas selectivas en búsqueda de un tratamiento más inteligente para las enfermedades asociadas a alteraciones de esta vía. Por tanto, realizamos una revisión de esta vía de transducción con el objetivo de tener una visión cercana de su funcionamiento, sus alteraciones y enumerar algunas moléculas promisorias para ser utilizadas en futuros tratamientos.

ABSTRACT The PI3K/AKT/mTOR pathway is involved in multiple cellular processes which are essential for the cells. Some genetic mutations of the components of this pathway have been associated with various human diseases, the most important of which are breast, thyroid and endometrium carcinomas; glioblastoma multiforme; prostate cancer and lymphomas. The PI3K/AKT/mTOR canonical pathway has been extensively studied in recent years. However, as the complexity of its main components and their correlation with the components of other pathways are increasing, it is important to update from time to time the available information to understand this mechanism. Furthermore, many trials have been conducted with selective medicines aimed to look for a more intelligent treatment for diseases associated with alterations in this pathway. Therefore, we review this transduction pathway to take a close look at its functioning and alterations, and to list some promising molecules for future treatments.

Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates / Papel de las proteínas de choque térmico en la infección de la línea celular de adenocarcinoma humano MCF-7 por aislados de rotavirus adaptados a tumores

Abstract Background: Viruses are being used as alternative and complementary tools for treating cancers. Oncolytic viruses exhibit tumor tropism, ability to enhance anti-tumor immunity and ability to be used in combination with conventional chemotherapy and radiotherapy. We have recently selected some rotavirus isolates which are adapted to efficiently infect and kill tumor cell lines. Aim: We tested five tumor cell-adapted rotavirus isolates for their ability to infect the human adenocarcinoma cell line MCF-7. Methods: Cell surface membrane-associated proteins mediating virus particle attachment were characterized using ELISA, immunoprecipitation, FACS analysis, and antibody blocking. Results: It was found that heat shock proteins (HSPs) such as Hsp90, Hsp70, Hsp60, and Hsp40 are expressed on the cell surface forming complexes with protein disulfide isomerase (PDI), integrin β3, and heat shock cognate protein 70 (Hsc70) in lipid raft microdomains. Interaction of rotavirus isolates with these cellular proteins was further confirmed by a competition assay and an inhibition assay involving the HSPs tested. Conclusion: Our findings suggest that the tumor cell-adapted rotavirus isolates studied here offer a promising tool for killing tumor cells, thus encouraging further research into this topic, including animal models.

Resumen Antecedentes: Los virus se utilizan como herramientas alternativas y complementarias para el tratamiento del cáncer. Los virus oncolíticos exhiben tropismo por tumores, capacidad para intensificar la inmunidad antitumoral y la capacidad para utilizarse en combinación con quimioterapia y radioterapia convencionales. Recientemente, hemos seleccionado algunos aislamientos de rotavirus que están adaptados para infectar y eliminar de manera eficiente líneas de células tumorales. Objetivo: Se ensayaron cinco aislamientos de rotavirus adaptados a células tumorales para determinar su capacidad para infectar la línea celular de adenocarcinoma humano MCF-7. Métodos: Las proteínas asociadas a la membrana de la superficie celular que median la unión de partículas de virus se caracterizaron mediante ELISA, inmunoprecipitación, análisis FACS y bloqueo de anticuerpos. Resultados: Se encontró que las proteínas de choque térmico (HSPs) como Hsp90, Hsp70, Hsp60 y Hsp40 se expresan en la superficie celular formando complejos con la proteína disulfuro isomerasa (PDI), la integrina β3 y la proteína análoga de choque térmico 70 (Hsc70) en microdominios lipídicos (rafts). La interacción de los aislamientos de rotavirus con estas proteínas celulares se confirmó adicionalmente mediante un ensayo de competición y un ensayo de inhibición que incluía las HSP ensayadas. Conclusión: Nuestros hallazgos sugieren que los aislamientos de rotavirus adaptados a las células tumorales estudiados aquí ofrecen una herramienta prometedora para eliminar las células tumorales, lo que estimula más investigaciones sobre este tema, incluidos los modelos animales.

MiR-26a promotes angiogenesis in rats with cerebral ischemia through PI3K/Akt pathway / 国际脑血管病杂志

Objective:To investigate the effect of miR-26a mediated phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway on angiogenesis in rats with cerebral ischemia.Methods:A total of 100 male SD rats were divided into sham operation group, model group, miR-NC group, and miR-26a group according to the random number table method. The miR-NC group and the miR-26a group were injected with 5 μl miR-26a simulant negative control and miR-26a simulant into the lateral ventricle respectively. The sham operation group and the model group were injected with the same amount of normal saline respectively. The middle cerebral artery occlusion model was induced by the modified intraluminal suture method. In the sham operation group, the thread was only inserted without ligation. Five rats in each group were injected intraperitoneally with 5-bromodeoxyuridine (BrdU) daily for 7 days. Rat brain microvascular endothelial cells (BMECs) cultured and transfected in vitro were divided into control group, oxygen glucose deprivation (OGD) group, miR-NC group, and miR-26a group. The dual luciferase experiment verified the regulatory effect of miR-26a on the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Longa score was used to detecte the neurological damage of rats. The volume of cerebral infarction was measured by triphenyltetrazolium chloride (TTC) staining. The methyl thiazolyl tetrazolium (MTT) staining, annexin Ⅴ fluorescein isothiocyanate/propidium iodide double staining and tubule formation experiment were used to detect the proliferation, apoptosis and angiogenesis of BMECs, respectively. Real-time fluorescence quantitative reverse transcription polymerase chain reaction was used to detect the miR-26a expression of ischemic brain tissue and BMECs. Immunofluorescence double labeling method (BrdU/von Willebrand factor [vWF]) was used to detect the proliferation of rat vascular endothelial cells. Western blot analysis was used to detect the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-2 (Ang-2), PTEN, PI3K and Akt protein in ischemic brain tissue.Results:Bioinformatics and dual luciferase experiments verified the targeted regulation of PTEN by miR-26a. Compared with the sham operation group, the expression of miR-26a, VEGF, bFGF, Ang-2, PI3K, AKT and the number of BrdU + /VWF + cells in ischemic brain tissue in the model group and miR-NC group increased, while the expression of PTEN decreased (all P<0.05). Compared with the model group, the effect of various indexes in the miR-26a group was more significant (all P<0.05). Compared with the control group, the proliferation and angiogenesis of BMECs in the OGD group and the miR-NC group were significantly increased, and the apoptosis was significantly reduced (all P<0.05). Compared with the OGD group, the effect of various indexes in the miR-26a group was more significant (all P<0.05). Conclusion:miR-26a can mediate the targeted inhibition of PTEN expression, up-regulate angiogenesis related factors (VEGF, bFGF and Ang-2), and promote vascular endothelial cell proliferation and angiogenesis in rats with cerebral infarction by activating PI3K/Akt signaling pathway.

Role of SPP1 in endogenous protective mechanism underlying neuropathic pain in mice with spinal cord injury: relationship with VEGF-AKT signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of secreted phosphoprotein 1 (SPP1) in endogenous protective mechanism underlying neuropathic pain (NP) in mice with spinal cord injury and the relationship with the vascular endothelial growth factor (VEGF)/kinase B (Akt) signaling pathway.Methods:Seventy-two clean-grade healthy female Kunming mice, aged 7-8 weeks, weighing 30-35 g, were divided into 4 groups ( n=18 each) using a random number table method: sham group (Sham group), NP caused by spinal cord injury group (NP group), NP caused by spinal cord injury+ SPP1-siRNA group (NS-siRNA group), and NP caused by spinal cord injury+ adeno-associated virus vector group (NP-EV group). A model of NP was established by a semi-transecting of the spinal cord.AAV-SPP1-siRNA-GFP adenovirus and AAV-vector-GFP adenovirus 7 μl were intrathecally injected in NS-siRNA group and NP-EV group, respectively, and 5 days later the model was established.At 1, 2 and 3 weeks after operation, 6 mice in each group were randomly selected to measure paw withdrawal threshold to mechanical stimulation (PWMT) and tail flick latency (TFL) to thermal stimuli.And then the mice were sacrificed and the ipsilateral injured spinal cord tissues were taken for determination of the expression of SPP1 mRNA (by real-time polymerase chain reaction) and expression of SPP1, VEGF, Akt and phosphorylated Akt (p-Akt) (by Western blot). Results:Compared with group Sham, PWMT was significantly decreased, TFL was shortened, and the expression of SPP1 mRNA, SPP1, VEGF and p-Akt protein was up-regulated at 1, 2 and 3 weeks after operation in NP, NS-siRNA and NP-EV groups( P<0.05). Compared with group NP, PWMT was significantly decreased, TFL was shortened, and the expression of SPP1 mRNA, SPP1, VEGF and p-Akt protein was down-regulated at 1, 2 and 3 weeks after operation in group NS-siRNA( P<0.05). Compared with group NS-siRNA, PWMT was significantly increased, TFL was prolonged, and the expression of SPP1 mRNA, SPP1, VEGF and p-Akt protein was up-regulated at 1, 2 and 3 weeks after operation in group NS-siRNA( P<0.05). Conclusion:SPP1 is involved in the endogenous protective mechanism underlying NP in mice with spinal cord injury, which may be related to the activation of the VEGF/AKT signaling pathway.

Neutral lactate promotes proliferation, migration and invasion of hepatocellular carcinoma cells by activating pi3k/akt signaling pathway / 肿瘤

Objective: To investigate the effects of neutral lactate on the proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells, and to explore its possible molecular mechanism. Methods: The HCC SMMC-7721 and HepG2 cells were treated with 0 (as the control), 2.5, 5 and 10 mmol/L neutral lactate, respectively. Then the proliferation of HCC cells was detected by MTT assay. The migration and invasion abilities of HCC cells were detected by Transwell chamber method. The expression levels of phosphoinositide 3-kinase (PI3K), protein kinase B (PBK, Akt), phospho-Akt (Ser473) [p-Akt (S473)], matrix metallopeptidase-2 (MMP-2) and MMP-9 were detected by Western blotting. The HCC cells SMMC-7721 and HepG2 were divided into four groups, and treated with equal volume of medium (as the control), neutral lactate, perifosine (an inhibitor of PI3K/Akt pathway), and neutral lactate combined with perifosine, respectively. Then MTT assay and Transwell chamber assay were used to detect the proliferation, migration and invasion abilities of HCC cells in each group. The expression levels of PI3K, Akt, p-Akt (S473), MMP-2 and MMP-9 proteins in HCC cells were detected by Western blotting. Results: The proliferation, migration and invasion abilities of SMMC-7721 and HepG2 cells in 5 and 10 mmol/L neutral lactate treatment groups were significantly enhanced as compared with the control group (all P < 0.01). The expression levels of PI3K, p-Akt (S473), MMP-2 and MMP-9 proteins in HCC cells treated with neutral lactate were significantly increased as compared with the control group (all P < 0.01). Compared with the control group, the proliferation, migration and invasion abilites of HCC cells in the perifosine treatment group were significantly decreased (all P < 0.05), and the expression levels of PI3K, p-Akt (S473), MMP-2 and MMP-9 proteins were also significantly down-regulated (all P < 0.05). Compared with the neutral lactate treatment group, the proliferation, migration and invasion abilities of HCC cells in the neutral lactate combined with perifosine treatment group were significantly reduced (all P < 0.01), and the expression levels of PI3K, p-Akt (S473), MMP-2 and MMP-9 proteins were significantly decreased (all P < 0.01). Conclusion: Neutral lactate promotes the proliferation, migration and invasion of HCC cells by activating PI3K/Akt pathway, while the PI3K/ Akt inhibitor perifosine can significantly reduce the stimulative effects of neutral lactate on the proliferation, migration and invasion of HCC cells.

Expression and significance of phosphorylated protein kinase B and spleen tyrosine protein kinase in different TNM stages of gastric cancer patients / 中国医师杂志

Objective@#To investigate the expression of phosphorylated protein kinase B (PKB/AKT) and spleen tyrosine protein kinase (Syk) in different tumor node metastasis (TNM) stages of gastric cancer patients.@*Methods@#From January 2015 to April 2018, 82 patients with gastric cancer confirmed by gastroscopy and surgical pathology were enrolled in this study. All patients were selected for cancer tissue, and 30 patients were randomly selected from normal gastric mucosa at 5 cm adjacent to the tumor. Immunohistochemistry was used to detect the expression of PKB/AKT and Syk protein. To compare the expression of PKB/AKT and Syk in gastric cancer and adjacent normal tissues, and to analyze the relationship between PKB/AKT and Syk expression and clinicopathological features in gastric cancer tissues, and the correlation between PKB/AKT and Syk and TNM staging of gastric cancer patients.@*Results@#The positive expression rate of PKB/AKT in gastric cancer tissues was higher than that in adjacent tissues (P<0.05). The positive expression rate of Syk was lower than that in adjacent tissues (P<0.05). PKB/Akt and Syk gene expression in gastric cancer were related to histological grade, tumor infiltration, TNM staging, lymph node metastasis and distant metastasis (P<0.05), and the expression of PKB/AKT was significantly increased with the increase of TNM staging in gastric cancer patients, and the positive expression of Syk was significantly decreased (P<0.05).@*Conclusions@#PKB/AKT is positively correlated with TNM staging of gastric cancer patients. Syk is negatively correlated with TNM staging of gastric cancer patients. The clinical expression of PKB/AKT and Syk can be used to determine the TNM staging of gastric cancer, which provides a strong basis for tumor treatment. It is of great significance in treatment.

Expression and significance of phosphorylated protein kinase B and spleen tyrosine protein kinase in different TNM stages of gastric cancer patients / 中国医师杂志

Objective To investigate the expression of phosphorylated protein kinase B (PKB/AKT) and spleen tyrosine protein kinase (Syk) in different tumor node metastasis (TNM) stages of gastric cancer patients.Methods From January 2015 to April 2018,82 patients with gastric cancer confirmed by gastroscopy and surgical pathology were enrolled in this study.All patients were selected for cancer tissue,and 30 patients were randomly selected from normal gastric mucosa at 5 cm adjacent to the tumor.Immunohistochemistry was used to detect the expression of PKB/AKT and Syk protein.To compare the expression of PKB/AKT and Syk in gastric cancer and adjacent normal tissues,and to analyze the relationship between PKB/AKT and Syk expression and clinicopathological features in gastric cancer tissues,and the correlation between PKB/AKT and Syk and TNM staging of gastric cancer patients.Results The positive expression rate of PKB/AKT in gastric cancer tissues was higher than that in adjacent tissues (P ＜ 0.05).The positive expression rate of Syk was lower than that in adjacent tissues (P ＜ 0.05).PKB/Akt and Syk gene expression in gastric cancer were related to histological grade,tumor infiltration,TNM staging,lymph node metastasis and distant metastasis (P ＜ 0.05),and the expression of PKB/AKT was significantly increased with the increase of TNM staging in gastric cancer patients,and the positive expression of Syk was significantly decreased (P ＜ 0.05).Conclusions PKB/AKT is positively correlated with TNM staging of gastric cancer patients.Syk is negatively correlated with TNM staging of gastric cancer patients.The clinical expression of PKB/AKT and Syk can be used to determine the TNM staging of gastric cancer,which provides a strong basis for tumor treatment.It is of great significance in treatment.

Induction and molecular mechanism of placental trophoblast cell autophagy in preeclampsia / 中华围产医学杂志

Objective To investigate the induction and regulatory mechanism of placental trophoblast cell autophagy in women with preeclampsia (PE).Methods Twenty gravidas with severe PE who underwent cesarean section in the Department of Obstetrics and Gynecology of Changzhou Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University from August 2016 to November 2016 were enrolled in PE group.An equal number of normotensive gravidas without proteinuria who also underwent cesarean section during the same period were randomly selected as control group.Placental tissue samples were collected from all gravidas.Ultrastructure of placental trophoblast cells and changes in autophagosome formation were observed by transmission electron microscope.Expressions ofmicrotubule associated protein 1 light chain 3B (MAP1LC3B,or LC3B) and Beclin 1 in placental tissue samples were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot.Activities of protein kinase B (PKB,also known as Akt)/mammalian target of rapamycin (mTOR) pathway in placental tissue samples were detected by Western blot.Two independent samples t-test or Mann-Whitney U test was used for statistical analysis.Results Sparse and disordered villi and many typical autophagosomes were observed in placental trophoblast cells from patients with severe PE.Significantly enhanced expression of LC3B at mRNA and protein levels and increased ratio of LC3-Ⅱ/LC3-Ⅰ were observed in the PE group as compared with the control group [3.37 (2.37-6.11) vs 0.62 (0.25-4.15),1.40±0.17 vs 1.00±0.13,1.57±0.25 vs 1.00±0.31,Zor t=--4.440,3.274 and 3.113,all P＜0.05].No significant difference in the expression ofBeclin 1 at mRNA or protein level in placental tissues was found between the two groups (both P＞0.05).Furthermore,Akt and mTOR phosphorylation in the PE group was significantly suppressed as compared with that in the control group (1.00±0.29 vs 0.64±0.21,1.00±0.32 vs 0.60±0.22,t=--3.672 and-2.895,both P＜0.05).However,the two groups showed no significant difference in the expression of Akt or mTOR protein (both P＞0.05).Conclusions Suppressed activity of Akt/mTOR pathway and enhanced induction of trophoblast cell autophagy are detected in placental tissues of patients with severe PE,indicating that excessive trophoblast cell autophagy,induced by decreased activity of Akt/mTOR pathway,may be the pathogenesis for PE.

siRNA-Mediated Suppression of Synuclein gamma Inhibits MDA-MB-231 Cell Migration and Proliferation by Downregulating the Phosphorylation of AKT and ERK / 한국유방암학회지

PURPOSE: Synuclein-gamma (SNCG), which was initially identified as breast cancer specific gene 1, is highly expressed in advanced breast cancers, but not in normal or benign breast tissue. This study aimed to evaluate the effects of SNCG siRNA-treatment on breast cancer cells and elucidate the associated mechanisms. METHODS: Vectors containing SNCG and negative control (NC) siRNAs were transfected into MDA-MB-231 cells; mRNA levels were determined by real-time polymerase chain reaction. Cell proliferation was evaluated using the MTT assay, cell migration was assessed by the Transwell assay, apoptosis and cell cycle analyses were conducted with the flow cytometer, and Western blot analysis was performed to determine the relative levels of AKT, ERK, p-AKT, and p-ERK expression. RESULTS: SNCG mRNA levels were significantly reduced in MDA-MB-231 cells transfected with SNCG siRNA. Our results indicate that in SNCG siRNA-treated cells, cell migration and proliferation decreased significantly, apoptosis was induced, and the cell cycle was arrested. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA-treated groups, than in the control and NC groups. CONCLUSION: SNCG siRNA could decrease the migration and proliferation of breast cancer cells by downregulating the phosphorylation of AKT and ERK.

PI3K/Akt signaling pathway and neuroprotection / 国际脑血管病杂志

PI3K/Akt pathway is a biological signal transduction pathway activated by phosphatidylinositol 3-kinase (PI3K).It plays a key role in cell proliferation,cell-cycle regulation,apoptosis initiation,and angiogenesis.In addition,the PI3K/Akt pathway is closely associated with the protection mechanisms of central nervous system damage.In-depth study of PI3K/Akt,downstream molecules and their regulation mechanisms,can provide some new ideas and methods for the treatment of brain injury.

Involvement of PI3K/Akt pathway in apoptosis induced by semaphorin 3A in cultured rat cortical neurons / 国际脑血管病杂志

Objective To investigate the effect of exogenous semaphorin 3A (Sema3A) on apoptosis in primary cultured rat cortical neurons and the roles of phosphoinositide 3-kinase (PI3K)/serine-threonine kinase (Akt) pathway in apoptosis induced by Sema3A.Methods Newborn Sprague-Dawley rat cortical neurons were cultured in vitro and they were identified by microtubule associated protein-2 (MAP-2) staining The cultured cortical neurons were treated with various concentrations of Sema3A (0,500,1 000,and 2 000 μg/ml) for 48hours.Neuronal survival rate was detected with CCK8 assay.Neuronal apoptosis was detected with Hoechst33342 staining and TUNEL staining.The expressions of P-Akt,Akt and Bcl-2 in cortical neurons were determined with Western blotting.Results The purity of cortical neurons culture was more than 95％.CCK8 assay showed that the survival rates of cortical neurons in the groups of 500,1 000and 2 000 μg/ml Sema3A were 80.9％ ± 5.3％,67.5％ ± 3.9％ and 50.2％ ± 4.4％ of the control group,respectively (F =165.042,P =0.000).Hoechst33342 staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 22.4％ ± 1.2％,34.0％ ± 1.2％,39.3％ ± 1.4％ and 47.3％ ±2.3％,respectively (F =103.237,P =0.000).TUNEL staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 23.9％ ± 1.1％,31.9％ ± 1.0％,40.1％ ± 1.5％ and 51.4％ ± 3.4％,respectively (F =103.118,P =0.000).Western blotting showed that the expressions of P-Akt (F =15.959,P =0.001) and Bcl-2 (F=18.776,P =0.001) decreased gradually,while the expression of Akt had no significant changes (F =0.590,P =0.639).Conclusions Sema3A can decrease the survival rate of the cultured cortical neurons,mainly by inducing apoptosis,and the mechanism of which might be related to the down-regulation of expressions of P-Akt and Bcl-2.

Neuroprotective effect of inhibition of soluble epoxide hydrolase on focal cerebral ischemia/reperfusion in Rats / 国际脑血管病杂志

Objective To investigate the neuroprotective effect of soluble epoxide hydrolase (sEH) inhibitor 12-(3-adamantan-l-yl-ureido) dodecanoic acid (AUDA) on focal cerebral ischemia/reperfusion in rats and its mechanisms.Methods Sixty male Sprague-Dawley rats were randomly divided into sham operation and saline control groups,as well as low-dose (0.157 ml/kg),medium-dose (0.235 ml/kg) and high-dose (0.314 ml/kg) AUDA groups (n =12 in each group).Four rats in each group were selected for infarct volume,cell apoptosis and p-Akt immunohistochemistry detection.A model of middle cerebral artery ischemia/ reperfusion was induced by the suture method.The corresponding dose AUDA or equal volume of saline was injected intraperitoneally before reperfusion in each AUDA group and the saline control group.Neurological deficit scores were performed at 24 h of reperfusion.2,3,5 triphenyltetrazolium chloride (TTC) staining was used to detect infarct volume.TdT-mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells of brain tissue in the periinfarction area.Immunohistochemical method was used to detect p-Akt expression of brain tissue in the peri-infarction area.Results TTC staining showed no infarction was observed in the sham operation group.The infarction volumes in the saline control group as well as the low-dose,medlum-dose and high-dose AUDA groups were 254.146 ± 25.481,212.679 ± 7.514,150.188 ± 33.997,and 99.563 ± 3.415 mm3,respectively.There were significant differences (F =39.637,P =0.000).The each dose AUDA group was significant less than the control group (all P=0.000).The medium-dose AUDA group was significantly less than the low-dose AUDA group (P=0.002),and the high-dose AUDA group was also significantly less than the low-dose AUDA group (P =0.000) and medium-dose AUDA group (P =0.006).TUNEL staining showed that a small number of apoptotic cells (6.400 ± 1.477/high-power field) were observed in the sham operation group.The numbers of apoptotic cells in the saline control group as well as in the low-dose,medium-dose and high-dose AUDA groups were 57.550 ± 13.067,47.030 ± 8.423,34.530 ± 4.393 and 26.400 ± 2.683/high power field,respectively.Each dose AUDA group was significantly less than the saline control group (all P ＜0.01).The medium-dose and high-dose AUDA groups were significantly less than the low-dose AUDA group (P ＜ 0.01),and the high-dose AUDA group was also significantly less than the medium-dose AUDA group (P ＜0.01).Immunohistochemistry showed that only a few p-Akt-positive cells (3.325 ± 1.438/high power field) were observed in the sham operation group.The numbers of p-Akt-positive cells in the saline control group as well as the low-dose,medium-dose and high-dose AUDA groups were 9.450 ±2.531,16.400 ± 3.865,22.875 ± 7.974,and 29.300 ± 3.203/high-power field,respectively.Each dose AUDA group was significantly more than the saline control group (all P ＜0.01).The medium-dose and high-dose AUDA groups were significantly more than the low-dose AUDA group (all P ＜0.01).The high-dose AUDA group was also significantly more than the medium-dose AUDA group (P ＜ 0.01).Conclusions The inhibition of sEH may decrease neuronal apoptosis and reduce infarct volume in the peri-infarction area by upregulating the PI3K/Akt pathway.It has a neuroprotective effect for focal cerebral ischemia/reperfusion in rats.

Influence of pertussis toxin on GPER-mediated activation of phosphatidylinositol 3-kinase/protein kinase R signaling induced by 17β-estradiol in endometrial carcinoma cells / 中华妇产科杂志

Objective To investigate the influence of pertussis toxin(PTX)on G protein-coupled estrogen receptor(GPER)-mediated activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt)signaling activated by 17 β-estradiol(17β-E2)in endometrial carcinoma cells.Methods Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells.Changes of levels of GPER,ERα and ERβ protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations(0,0.1,0.5,1.0 μg/ml),and then co-stimulated with with 1 × 10-6 mol/L 17β-E2 respectively at different time (Ishikawa 30 minutes,HEC-1A 15 minutes).Results(1)Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell.(2)After co-treated with PTX at different concentrations(0,O.1,0.5,1.0 μg/ml)and 10-6 mol/L 17β-E2,in Ishikawa cell,the ratio of pAkt/Akt was 0.74 ±0.54,0.34 ±0.06,0.18 ±0.03,0.07 ±0.15,the gray values of GPER was 0.872 ± 0.490,0.395 ± 0.054,0.145 ± 0.014,0.034 ± 0.008,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which was most obviously when the concentration was 1.0 μg/ml(F =63.729,P =0.0001;F =160.284,P =0.0001);ERα and ERβ protein had no significant change among different groups(P ＞0.05).In HEC-1A cell,the ratio of pAkt/Akt was 0.73 ±0.09,0.26 ±0.14,0.11 ±0.03,0,the Gray values of GPER is 0.927 ±0.134,0.485 ± 0.022,0.194 ± 0.004,0,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which were also completely inhibited when the concentration was 1 μg/ml(F =1039.321,P =0.0001;F =109.646,P =0.0001),ERα protein had no significant differences(P ＞ 0.05)among different groups.ERβ was negatively expressed.Conclusion The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.

Expressions of p-Akt (Ser473) and p-Bad (Ser136) and apoptosis in peri-infarction tissue following permanent middle cerebral artery occlusion in rats / 国际脑血管病杂志

Objective To investigate the relationship between the expression changes of p-Akt (Ser473),p-Bad (Ser136) and the cell apoptosis in peri-infarction tissue of permanent middle cerebral artery occlusion (MCAO) in rats.Methods Sixty male Sprague-Dawley rats were randomly allocated into sham operation,MCAO 3 h,MCAO 12 h,LY294002 intervention MCAO 3 h,and LY294002 intervention MCAO 12 h groups (n =12 in each group).A permanent MCAO model was induced by the modified suture method.At 15 minutes before modeling,the rats in the LY294002 intervention MCAO groups were administered via lateral ventricle.The neurological function score was scored by using Zea Longa method.2,3,5-triphenyltetrazolium chloride (TTC) staining was used to detect infarct volume.Immunohistochemical staining was use to detect pAkt (Ser473) and p-Bad (Ser136) expressions in peri-infaretion tissue.TUNEL assay was used to detect apoptotic cells in peri-infarction tissue.Results Three hours after modeling all the experimental rats awoke from anesthesia.The neurological deficit score in the sham operation group was 0,and the scores of the MCAO 3 h,MCAO 12 h,LY294002 intervention MCAO 3 h and LY294002 intervention MCAO 12 h groups were 2.25 ± 0.45,2.92 ± 0.99,3.00 ± 0.95,and 3.02 ± 0.36,respectively.There were significant differences among all groups (F =26.520,P =0.000).The score of the LY294002 intervention MCAO 3 h group was significantly higher than that of the MCAO 3 h group (P =0.009).There was no significant difference between the LY294002 intervention MCAO 12 h group and the MCAO 12 h group (P =0.354).TTC staining showed that no infarct was observed in the sham operation group.The infarct volumes of the MCAO 3 h,MCAO 12 h,LY294002 intervention MCAO 3 h and LY294002 intervention MCAO 12 h groups were 23.4 ± 1.4,40.3 ± 1.1,31.9 ±6.0 and 44.4 ±3.8 mm3,respectively.There were significant differences among the groups (F =30.440,P =0.000).The score of the LY294002 intervention MCAO 3 h group was significantly greater than that of the MCAO 3 h group (P =0.002).There was no significant difference between the LY294002 intervention MCAO 12 h group and the MCAO 12 h group (P=0.113).Compared with the sham operation group,the p-Akt (Ser473) expression in peri-infarction tissue in the MCAO 3 h group was significantly high,and it was significantly decreased in the MCAO 12 h group; the expression level of p-Bad (Ser136) showed a progressive decline with the passage of MCAO time,at the same time,the number of apoptotic cells increased progressively.After the LY294002 intervention,the expression levels of p-Akt (Ser473) and p-Bad (Ser136) in peri-infarction tissue decreased significantly at 3 h after MCAO,and the number of apoptotic cells increased significantly (P ＜0.05),but there was no significant effect on each index at 12 h after MCAO.Conclusions The activation of the PI3K/Akt signal transduction pathway in early cerebral infarction and the stress elevation of the key protein p-Akt (Ser473) of this pathway have brain protection,while the failure of this pathway activity and the drastical decrease of its key protein in the late cerebral infarction are associated with brain injury.

Roles of protein kinase B and mitogen-activated protein kinase pathways in the protection by nicotinic acid against ultraviolet B-induced damage in keratinocytes / 中华皮肤科杂志

Objective To investigate the intracellular signal transduction pathways involved in the protective effect of nicotinic acid against ultraviolet B(UVB)-induced damage in human skin keratinocytes.Methods Cultured human keratinocyte HaCaT cells were divided into several groups to be treated with nicotinic acid,UVB irradiation,LY294002(an inhibitor of Akt),U0126(an inhibitor of extracellular signal-regulated kinase(ERK)1/2),SB203580(an inhibitor of P38)alone or in combination for different durations.Then,Western blot was performed to quantify the phosphorylation levels of the protein kinase B(Akt)/MAPK pathwayassociated proteins including Akt,P38,JNK and ERK1/2,MTT assay to evaluate the activity of HaCaT cells,enzyme-linked immunosorbent assay to determine the levels of endothelin-1(ET-1)and basic fibroblast growth factor(bFGF)in the culture supernatant of HaCaT cells,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)to evaluate the apoptosis in HaCaT cells.Results As Western blot showed,phosphorylated Akt,P38,JNK and ERK1/2 were markedly activated within 60 minutes after pretreatment with nicotinic acid and irradiation with UVB(all P ＜ 0.01),and the activation was more significant for phosphorylated Akt,P38,and ERK1/2 within 2 hours(all P ＜ 0.01).Nicotinic acid effectively suppressed the UVB-induced cell death and apoptosis in HaCaT cells.The levels of supernatant ET-1 and bFGF were significantly decreased in HaCaT cells treated with the above 3 inhibitors followed by UVB irradiation than in those treated with the inhibitors alone(all P ＜ 0.05),and nicotinic acid pretreatment only reversed the decrease in supernatant bFGF in HaCaT cells treated with SB203580 followed by UVB irradiation.Conclusion The Akt signaling pathway may play a regulatory role in the protection by nicotinic acid against UVB-induced damage in HaCaT cells.

Effects of PI3K/Akt/NF-κB signal pathway on FSH facilitation on cell proliferation and invasion by human epithelial ovarian cancer / 中华妇产科杂志

ObjectiveTo explore the effects of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor-kappa B (NF-κB) signal pathway on the process of follicle-stimulating hormone (FSH) facilitating cell proliferation and invasion in human epithelial ovarian cancer. Methods Ovarian cancer cell lines SKOV3 and 3AO were cultured to exponential phase,then assigned to control group,FSH group,LY294002 group and FSH + LY294002 group,respectively.Cells were treated with different concentration of FSH and LY294002,respectively.The effects of FSH on cell proliferation were observed by methylthiazolyl tetrazolium (MTT).Morphological changes were observed by phase contrast microscope.The ability of cell invasion was investigated by transwell invasion assay.The expression of FSH receptor (FSHR),Akt1/2,phosphorylated-Akt (p-Akt) and NF-κB p65 protein were detected by western blot.Results( 1 ) FSH could promote the proliferation of SKOV3 and 3AO cells.When the cells were treated with 40 U/L FSH for 48 hours (SKOV3) and 24 hours (3AO),compared with those in control groups,they reached the highest proliferation rate (P ＜ 0.05 ),respectively.(2) The morphology of SKOV3 and 3AO cells in four groups:in control group,SKOV3 cells were short spindle and 3AO cells were long spindle,the nuclei of them were both roundness or oval,the cytoplasm were bright.In FSH group,the cells changed to slightly longer or polygonal,they were full in shape,meanwhile,the cell intensity were higher than control group.In LY294002 group,some cells changed from spindle to round,and began to shrink.The cell intensity diminished.The morphology of FSH + LY294002 group was similar with control group,but the cell intensity was lower than that in FSH group.(3)The number of SKOV3 cell that passed through the membrane in control group,FSH group,LY294002 group and FSH + LY294002 group was (26 ± 6),( 118 ± 19),( 18 ± 5) and ( 38 ± 7 ),respectively.The number of 3AO cell was ( 19 ± 4 ),( 134 ± 20),(12 ±3) and (58 ± 11 ),respectively.The results showed that the number of cells in FSH group was significantly higher than that in control group ( P ＜ 0.05 ),while the number of cell in FSH + LY294002 group was significantly fewer than that in FSH group (P ＜ 0.05 ).(4) There was no significant difference in the expression of FSHR and Akt1/2 between FSH group and control group (P ＞ 0.05 ),but FSH increased the expression of p-Akt and the ratio of NF-κB p65 in the nucleus versus cytoplasm in SKOV3 and 3AO cells,there were significant differences compared with control group ( P ＜ 0.05 ).LY294002 reversed the effects of FSH on increasing the expression of p-Akt and the ratio of NF-κB p65 in the nucleus versus cytoplasm,there were significant differences among LY294002 group,FSH + LY294002 group and FSH group (P ＜ 0.05 ).ConclusionThe effects of FSH on proliferation and invasion of ovarian cancer cell lines SKOV3 and 3AO may be realized by regulating the activity of NF-κB in PI3K/Akt signal pathway.

Expressions of Akt and glycogen synthase kinase-3β in the hippocampus after repetitive bilateral common carotid artery occlusion in mice / 国际脑血管病杂志

Objective To investigate the expressions of protein kinase B (PKB/Akt) and glycogen synthase kinasc-3β in the hippocampus in mice with vascular dementia (VaD) induced by repetitive bilateral common carotid artery occlusion.Methods Forty-eight healthy adult male C57B1/6 mice were randomly allocated into 3 group:normal group,sham operation group,and model group (n =16 in each group).A mouse VaD model was induced by intermittent blocking the bilateral common carotid artery for 3 times in the model group.The sham group only separated the bilateral common carotid artery,but did not block it.The normal group did not receive any treatment.The behavioral changes of the mice were observed using the water maze and step-down tests at 4 weeks after procedure.HE staining was used to observe the histopathological changes of hippocampal tissue.The Western blotting was used to detect the expressions of Akt,p-Akt (Ser473),GSK3β and p-GSK3β (Ser9) proteins.Results In the water maze test,the time of swimming the entire distance was prolonged at the learning stage and memory stage (learning stage:F =19.389,P ＜0.05; memory stage:F =27.929,P ＜ 0.05),the number of errors increased (learning stage:F =7.228,P ＜ 0.05; memory stage:F =21.189,P＜0.05) in the model group.In the step-down test,the response time was prolonged (F=19.162,P ＜0.05) at learning stage and the number of errors increased (F =6.562,P ＜ 0.05),the latency time was shortened (F=10.634,P＜0.05) and the number of errors increased (F=12.890,P＜0.05) in the model group.At the same time,HE staining showed the reduction of neurons and the proliferation of glial cells in the hippocampal CA1 region in the model group; p-Akt (Ser473) (F=37.849,P＜0.05) and p-GSK3β (Ser9)(F =67.725,P ＜0.05) protein expressions were up-regulated significantly (F =37.849,P ＜0.05; F =67.725,P＜0.05) at 4 weeks after procedure compared to those in the sham operation group,while there were no significant differences in Akt (F =1.004,P ＞0.05) and GSK3β(F =0.329,P ＞0.05) total protein expressions among all groups.Conclusions The repetitive bilateral common carotid artery occlusion may result in learning and memory impairment and severe damage in the hippocampus in mice.The Akt and GSK3β expressions may be involved in the mechanism of VaD.

Effect of GPER on the activation of PI3K/Akt induced by 17β-estradiol in endometrial carcinoma cells / 中华妇产科杂志

ObjectiveTo investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17β-estradiol (17β-E2 )in endometrial carcinoma cells,Ishikawa and HEC-1A.Methods Expressions of GPER,Erα and Erβ protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method.Levels of GPER,Erα and Erβ were examined by western blot in Ishikawa and HEC-1A cells after treated with 1 ×10-6 mol/L 17β-E2 at different time (0,15,30,60,120 minutes).ResultsGPER was positive expressed in Ishikawa and HEC-1A cells.Erα and Erβ were both positive expressed in Ishikawa cells.While,Erα was weakly expressed and Erβ was almost negatively expressed in HEC-1A cells.Western blot analysis showed that 1× 10-6 mol/L 17β-E2 treatment,the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P ＜ 0.05 ),which Ishikawa 30 minutes,when cells reached the highest level (0.192 ± 0.004),HEC-1A cells for 15 minutes and reached the highest level (0.184 ±0.006) ; Ishikawa and HEC-1A cells,Akt,activation of 15 minutes from the treatment start was significantly increased (P ＜ 0.05 ),which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021 ),HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1 A cells,Erα and Erβ protein expression did not change significantly ( P ＞ 0.05 ).Conclusion GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells,Ishikawa and HEC-1A.