Magnolol and 5-fluorouracil synergy inhibition of metastasis of cervical cancer cells by targeting PI3K/AKT/mTOR and EMT pathways / 中草药·英文版

OBJECTIVE@#This study is designed to investigate the mode of action of the synergistic effect of 5-fluorouracil (5-FU) and magnolol against cervical cancer.@*METHODS@#Network pharmacological approach was applied to predict the molecular mechanism of 5-FU combined with magnolol against cervical cancer. CCK-8 assay, colony formation assay, immunofluorescence staining, adhesion assay, wound healing mobility assay, cell migration and invasion assay and Western blot analysis were conducted to validate the results of in silico study.@*RESULTS@#Phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was identified as the key pathway in silico study. The experimental results showed that 5-FU combined with magnolol strongly inhibited cervical cancer cell proliferation, induced the morphological change of HeLa cells by down-regulating the expression of α-actinin, tensin-2 and vinculin. Moreover, magnolol enhanced inhibitory effect of 5-FU on the cell adhesion, migration and invasion. The phosphorylation of AKT and PI3K and the expression of mTOR were strongly inhibited by the combination of 5-FU and magnolol. Moreover, the expression of E-cadherin and β-catenin was upregulated and the expression of Snail, Slug and vimentin was down-regulated by the 5-FU together with magnolol.@*CONCLUSION@#Taken together, this study suggests that 5-FU combined with magnolol exerts a synergistic anti-cervical cancer effect by regulating the PI3K/AKT/mTOR and epithelial-mesenchymal transition (EMT) signaling pathways.

Wenyang Jieyu Prescription Regulates Hippocampal Neuron Apoptosis and Improves Synaptic Plasticity in Depressed Mice via BDNF/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomly assigned into a control group （n=10） and a modeling group （n=50）. Maternal separation combined with restraint stress was adopted to establish the mouse model of depression， and the modeled mice were randomized into model， Wenyang prescription， Jieyu prescription， Wenyang Jieyu prescription， and fluoxetine groups （n=10） on the weaning day （PD21）. From PD21 to PD111， the mice were fed with the diets mixed with corresponding medicines. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then conducted to evaluate the depression， memory， and learning abilities of mice. Immunohistochemistry （IHC） was employed to measure the atomic absorbance （AA） of postsynaptic density protein 95 （PSD95） in the hippocampus. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of hippocampal neurons. Western blot was employed to determine the protein levels of brain-derived neurotrophic factor （BDNF）， phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B （p-TrkB/TrkB）， phosphorylated protein kinase B/protein kinase B （p-Akt/Akt）， phosphorylated mammalian target of rapamycin/mammalian target of rapamycin （p-mTOR/mTOR）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cysteinyl aspartate-specific proteinase-3 （Caspase-3）， synaptophysin （Syn）， and PSD95. ResultCompared with the control group， the modeling decreased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.01）. Furthermore， it decreased the expression of PSD95， increased the neuron apoptosis in the hippocampus （P<0.01）， down-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and up-regulated the protein levels of Bax and Caspase-3 （P<0.05） in the hippocampus. Compared with the model group， Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. Moreover， the drugs increased the expression of PSD95， reduced the neuron apoptosis （P<0.01）， up-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and down-regulated the protein levels of Bax and Caspase-3 （P<0.01）. ConclusionWenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice. It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.

Eleutheroside B induces apoptosis and autophagy of lung cancer cells by regulating PI3K/Akt/mTOR pathway / 中国中药杂志

This study investigated the effect of eleutheroside B on apoptosis and autophagy of lung cancer A549 and H460 cells and its molecular mechanism. MTT assay was used to detect the cytotoxicity of eleutheroside B at 5, 10, 15, 20, 25, 30, 35, 40, and 45 mmol·L~(-1) on lung cancer cells. Trypan blue exclusion assay was used to detect the effect of eleutheroside B on the survival rate of lung cancer A549 and H460 cells at different time. Colony formation assay was used to detect the effect of eleutheroside B on the proliferation of lung cancer A549 and H460 cells. AO/EB fluorescence double staining and Hoechst 33342 fluorescence staining were used to detect the effect of eleutheroside B on apoptosis of lung cancer A549 and H460 cells, and Western blot was used to detect apoptosis-related proteins to explore the apoptosis-related molecular mechanism. AO fluorescence staining and Western blot were used to detect the expression of autophagic vesicles and autophagy-related proteins P62 and LC3. The results showed that compared with the control group, eleutheroside B inhibited the growth of lung cancer A549 and H460 cells in a concentration-dependent manner. The optimal effect time of eleutheroside B on lung cancer A549 and H460 cells was 24 h, and the optimal concentrations were 28.64 and 22.16 mmol·L~(-1), respectively. Eleutheroside B could inhibit the colony formation of A549 and H460 cells. Compared with the control group, eleutheroside B could promote the formation of apoptotic bodies and induce cell apoptosis, as well as induce the expression of mitochondrial pathway-related proteins. Under the effect of eleutheroside B, the acidic autophagy vacuole in lung cancer cells increased, LC3Ⅱ expression increased, P62 protein expression decreased, and PI3K, p-Akt, and p-mTOR protein expression decreased in the PI3K/Akt/mTOR pathway. Studies have shown that eleutheroside B can inhibit the growth of lung cancer cells, reduce colony formation, induce apoptosis of lung cancer cells through mitochondrial pathway, and induce autophagy. The mechanism may be related to the PI3K/Akt/mTOR pathway.

Progress on the impact of pathogenic gene mutations in epilepsy on treatment choices / 国际儿科学杂志

In the past decade, significant progress has been made in the study of epilepsy-causing genetic mutations and the molecular mechanisms of epilepsy clinical manifestations.A growing number of studies have shown that the mechanism of action of pathogenic genes related to clinical symptoms shows significant correlation.In the selection of antiepileptic drugs for patients with different gene mutation, early identification of pathogenic genes has guiding significance for the selection of antiepileptic drugs.This review summairzes common epilepsy pathogenic genes, including ion channels genes, cellular metabolism related genes and cell signaling pathway related genes, and research progress on therapeutic targets corresponding to pathogenic genes in recent years.As research deepens, specific gene defects and their machanisms of action provide a basis for studying new treatment methods.

FERM domain-containing protein FRMD6 activates the mTOR signaling pathway and promotes lung cancer progression / 医学前沿

FRMD6, a member of the 4.1 ezrin-radixin-moesin domain-containing protein family, has been reported to inhibit tumor progression in multiple cancers. Here, we demonstrate the involvement of FRMD6 in lung cancer progression. We find that FRMD6 is overexpressed in lung cancer tissues relative to in normal lung tissues. In addition, the enhanced expression of FRMD6 is associated with poor outcomes in patients with lung squamous cell carcinoma (n = 75, P = 0.0054) and lung adenocarcinoma (n = 94, P = 0.0330). Cell migration and proliferation in vitro and tumor formation in vivo are promoted by FRMD6 but are suppressed by the depletion of FRMD6. Mechanistically, FRMD6 interacts and colocalizes with mTOR and S6K, which are the key molecules of the mTOR signaling pathway. FRMD6 markedly enhances the interaction between mTOR and S6K, subsequently increasing the levels of endogenous pS6K and downstream pS6 in lung cancer cells. Furthermore, knocking out FRMD6 inhibits the activation of the mTOR signaling pathway in Frmd6-/- gene KO MEFs and mice. Altogether, our results show that FRMD6 contributes to lung cancer progression by activating the mTOR signaling pathway.

Ephedra Herb extract ameliorates adriamycin-induced nephrotic syndrome in rats via the CAMKK2/AMPK/mTOR signaling pathway / 中国天然药物

This study aimed to investigate the effect and mechanisms of Ephedra Herb (EH) extract on adriamycin-induced nephrotic syndrome (NS), providing an experimental basis for the clinical treatment of NS. Hematoxylin and eosin staining, creatinine, urea nitrogen, and kidn injury molecule-1 were used to evaluate the activities of EH extract on renal function. The levels of inflammatory factors and oxidative stress were detected by kits. The levels of reactive oxygen species, immune cells, and apoptosis were measured by flow cytometry. A network pharmacological approach was used to predict the potential targets and mechanisms of EH extract in the treatment of NS. The protein levels of apoptosis-related proteins and CAMKK2, p-CAMKK2, AMPK, p-AMPK, mTOR and p-mTOR in the kidneys were detected by Western blot. The effective material basis of EH extract was screened by MTT assay. The AMPK pathway inhibitor (compound C, CC) was added to investigate the effect of the potent material basis on adriamycin-induced cell injury. EH extract significantly improved renal injury and relieve inflammation, oxidative stress, and apoptosis in rats. Network pharmacology and Western blot results showed that the effect of EH extract on NS may be associated with the CAMKK2/AMPK/mTOR signaling pathway. Moreover, methylephedrine significantly ameliorated adriamycin-induced NRK-52e cell injury. Methylephedrine also significantly improved the phosphorylation of AMPK and mTOR, which were blocked by CC. In sum, EH extract may ameliorate renal injury via the CAMKK2/AMPK/mTOR signaling pathway. Moreover, methylephedrine may be one of the material bases of EH extract.

开放科学(资源服务)标识码(OSID) Effect of Tanshinone ⅡA on abnormal integration of granule cells in the dentate gyrus of mice with chronic epilepsy / 西安交通大学学报(医学版)

【Objective】 To clarify the role and molecular mechanism of Tanshinone ⅡA (TanⅡA) in the pathological integration of granule cells in the dentate gyrus (DG) by using the mouse model of temporal lobe epilepsy (TLE). 【Methods】 Status epilepticus (SE) was induced in the mice with pilocarpine and treated with TanⅡA 5 mg/kg. After two months, Morris water maze was used to examine the spatial learning and memory ability and video surveillance was used to monitor spontaneous seizures. The DG was removed for staining of Timm, Prox-1, DCX and SynⅠ. PTEN, p-AKT, and p-S6 expressions were observed by Western blotting. 【Results】 TanⅡA decreased Timm score, SynⅠ, PSD-95 and pS6 levels, and increased the level of PTEN in the DG, and attenuated the formation of mossy fiber sproutings and basal dendrites of the granule cells. Video surveillance showed that TanⅡA reduced the frequency of Racine’ grade 5 seizures. 【Conclusion】 TanⅡA can effectively attenuate the abnormal integration of the granule cells in the DG by regulating PTEN/AKT/mTOR pathway and thus plays an anti-epileptic role.

Methyl rosmarinate induces cell apoptosis in human hepatocellular carcinoma cells via inhibiting the Akt/mTOR signaling pathways / 西安交通大学学报(医学版)

【Objective】 To investigate the cell death-inducing effect of methyl rosmarinate (MR) on human hepatoma Hep-3B and SK-Hep1 cells and their potential mechanisms. 【Methods】 The effects of MR on the viability of Hep-3B, SK-Hep1 and MIHA cells were determined by cell counting kit-8 (CCK-8) assay. The morphological changes of three kinds of cells treated with different concentrations of MR were observed by optical microscopy. EdU assay and flow cytometry were used to detect the proliferation and apoptosis of Hep-3B and SK-Hep1 cells. Transwell assay was used to study the effects of MR on the migration and invasion of Hep-3B and SK-Hep1 cells. Western blotting was used to evaluate the protein expression levels of apoptosis, EMT and Akt/mTOR signaling pathways. 【Results】 After treated with different concentrations of MR (0~200 μmol/L) for 48 h, Hep-3B and SK-Hep1 cells activities were significantly decreased in a concentration-dependent manner (P<0.01), while there was no significant effect on MIHA cell activity (P>0.05), and the IC50 of Hep-3B and SK-Hep1 cells were 102.5 and 99.3 μmol/L, respectively. MR treatment (0-150 μmol/L) significantly inhibited the proliferation of Hep-3B and SK-Hep1 cells (P<0.05), while cell detachment and shrinkage were observed by optical microscopy on the Hep-3B and SK-Hep1 cells, while the morphology of MIHA cells was not changed. Compared with the control group, MR (100, 150 μmol/L) induced apoptosis in Hep-3B and SK-Hep1 cells, the expression levels of the pro-apoptotic proteins Bax and cleaved PARP were significantly increased (P<0.05), while the expression level of the anti-apoptotic protein Bcl-2 was significantly decreased (P<0.05). MR (100, 150 μmol/L) also inhibited the migration and invasion of HCC cells, significantly increased the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin compared with the control group (P<0.05). Finally, Western blotting results showed that the expressions of p-Akt and p-mTOR in Hep-3B and SK-Hep1 treated by MR were significantly reduced in a dose-dependent manner, suggesting that MR may play an anti-cancer role by inhibiting Akt/mTOR signaling pathway. 【Conclusion】 MR can promote apoptosis in Hep-3B and SK-Hep1 cells, which may be closely related to the inhibition of Akt/mTOR signaling pathway.

Effect and Mechanism of Atorvastatin on Reversing Drug Resistance in Leukemia by Regulating Glycolysis through PTEN/mTOR Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM.@*METHODS@#HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected.@*RESULTS@#CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance.@*CONCLUSION@#Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.

Delicaflavone inhibits the invasion and migration of gefitinib-resistant lung cancer PC-9/GR cells by regulating epithelial-mesenchymal transition via PI3K/Akt/mTOR pathway / 中国临床药理学与治疗学

AIM: To study the effect and mechanism of Delicaflavone on migration and invasion of gefitinib-resistant lung cancer cell line PC-9/GR. METHODS: MTT assay was used to detect cell viability. Transwell and scratch assays were used to detect cell invasion and migration abilities. Western blotting was used to detect the expressions of MMP-9, MMP-2, E-cadherin, N-cadherin, Vimentin and PI3K/Akt/mTOR pathway-related proteins in PC-9/GR cells. RESULTS: Compared with control group, 20 mg/L Delicaflavone could significantly inhibit the viability of PC-9/GR cells for 24 h (P<0.05), while Delicaflavone below 10 mg/L had no significant effect on cell proliferation. The number of invasive cells and migrated cells were decreased significantly by Delicaflavone in a concentration-dependent way (P<0.05 and P<0.01). Delicaflavone could concentration-dependently reduce the expression of MMP-9, MMP-2, N-cadherin, vimentin (P<0.01), meanwhile up-regulate the expression of E-cadherin (P<0.01). In addition, Delicaflavone also decreased the expression of p-PI3K, p-Akt and p-mTOR in a concentration-dependent manner (P<0.01). CONCLUSION: Delicaflavone can inhibit the migration and invasion of PC-9/GR cells by regulating epithelial-mesenchymal transition via PI3K/Akt/mTOR pathway.

Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage / 中国结合医学杂志

OBJECTIVE@#To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons.@*METHODS@#The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway.@*RESULTS@#MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01).@*CONCLUSION@#TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.

P4HA2 promotes occurrence and progression of liver cancer by regulating the PI3K/Akt/mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of proline 4-hydroxylase Ⅱ (P4HA2) in the occurrence and progression of liver cancer.@*METHODS@#GEPIA and Human Protein Atlas database were used to predict the expression of P4HA2 in hepatocellular carcinoma (HCC), and K-M plotter online database was used to analyze the relationship between P4HA2 expression and the prognosis of HCC. We also examined the expressions of P4HA2 in HCC cells and normal hepatocytes using qRT-PCR and Western blotting. With lentivirus-mediated RNA interference, P4HA2 expression was knocked down in hepatoma SNU-449 and Hep-3B cells, and the changes in cell proliferation, migration and invasion were assessed using cell counting kit-8 (CCK-8) assay, colony formation test, scratch test and Transwell assay. The changes in the expressions of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR signal pathway-related proteins were detected using Western blotting.@*RESULTS@#Online database analysis showed that the expression of P4HA2 was significantly higher in HCC tissues than in normal liver tissues (P < 0.05). The expression levels of P4HA2 mRNA and protein were also significantly higher in HCC cell lines than in normal hepatocytes (P < 0.01). Lentivirus-mediated RNA interference of P4HA2 significantly lowered the expression levels of P4HA2 mRNA and protein in the hepatoma cells (P < 0.05) and caused obvious inhibition of cell proliferation, migration and invasion. P4HA2 knockdown significantly increased the expression of E-cadherin protein, lowered the expressions of N-cadherin and Snail, and obviously decreased the expressions of phosphorylated PI3K, AKT and mTOR (P < 0.05).@*CONCLUSION@#P4HA2 enhances the proliferation, migration, invasion, and EMT of hepatoma cells by activating the PI3K/Akt/mTOR signaling pathway to promote the occurrence and progression of liver cancer.

Mechanism of Cucurbitacin B in Regulating Glycolysis and Inhibiting Proliferation of HuCCT1 Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of cucurbitacin B （CuB） in inhibiting cell proliferation and glycolysis. MethodCell counting kit-8 （CCK-8） was applied to investigate the effect of different concentrations of CuB （0， 40， 80， 120， 160， 200， 400， and 800 nmol·L-1） on the proliferation of HuCCT1 cells. The effect of different concentrations of CuB （50， 100， and 200 nmol·L-1） on the colony formation ability of HuCCT1 cells was detected by plate cloning assay. The effect of different concentrations of CuB （50， 100， 200 nmol·L-1） on the HuCCT1 cell cycle was analyzed by flow cytometry. Visible spectrophotometry was employed to detect the activity of key glycolytic enzymes hexokinase （HK） and pyruvate kinase （PK）） and changes in glucose consumption， lactate production， and adenosine triphosphate （ATP） production in HuCCT1 cells after administration of different concentrations of CuB （50， 100， 200 nmol·L-1）. Western blotting was used to assay the effect of CuB on the expression of cell cycle-related proteins， proliferation-related proteins， key glycolytic proteins， and Akt/mammalian target of rapamycin （mTOR） pathway-related proteins. ResultAs compared with the blank group， CuB at dose of 160-800 nmol·L-1 after 24 h administration and CuB at dose of 80-800 nmol·L-1 after 48 h administration inhibited the proliferation of HuCCT1 cells in a time- and dose-dependent manner （P<0.05， P<0.01）， and the median inhibitory concentration was 200 nmol·L-1 48 h after administration. CuB can restrain the colony formation ability of HuCCT1 cells in a dose-dependent manner （P<0.01）， and block HuCCT1 cell cycle in G2 phase （P<0.05， P<0.01）. CuB （100 and 200 nmol·L-1） can suppress the activities of HK and PK and reduce cell glucose consumption and production of lactate and ATP （P<0.05， P<0.01）. Western blot results showed that CuB （100 and 200 nmol·L-1） can inhibit the protein levels of cycle-related protein Cyclin B1， proliferating cell nuclear antigen （PCNA）， HK1， HK2， PKM1， PKM2， phosphorylated Akt （p-Akt）， phosphorylated mTOR （p-mTOR）， and phosphorylated ribosomal protein S6 （p-RPS6） （P<0.05， P<0.01）. ConclusionCuB can inhibit aerobic glycolysis in HuCCT1 cells via the Akt/mTOR pathway， thereby affecting cell proliferation.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Inhibition effect of active fraction from clove on PI3K/Akt/mTOR signaling pathway to induce apoptosis of human colon cancer HCT116 cells / 中国中药杂志

To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 μg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P<0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/β-actin after treatment of AFC(100 μg·mL~(-1)) were significantly different from those in the control group(P<0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 μg·mL~(-1)) were significantly lower than those in the control group(P<0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P<0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P<0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.

Effect of Bushen Tongluo Prescription on Synaptic Plasticity of Hippocampal Neurons of Vascular Dementia Model Rats Based on PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

Objective:To explore the effect of Bushen Tongluo prescription （BSTLP） on the synaptic plasticity of hippocampal neurons in vascular dementia （VD） model rats and its mechanism. Method:SD male rats of SPF grade were selected. The rat model of VD was established by permanent bilateral ligation of the common carotid artery several times. The model rats were randomly divided into a model group， an insulin-like growth factor-1 （IGF-1， 20 μg·kg^-1） group， high-dose （3 g·kg^-1）， medium-dose （1.5 g·kg^-1）， and low-dose （0.75 g·kg^-1） BSTLP groups. A sham operation group was also set. Drugs were administered to rats by gavage once a day for four weeks. The model group and the sham operation group received the same volume of normal saline. After the last administration， all the rats were detected for spatial learning and memory by the Morris water maze. The apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase （TdT） dUTP nick-end labeling （TUNEL） assay. The changes in synaptic morphological structure and the number of dendritic spines in hippocampal neurons were detected by Golgi's method. The expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （Akt）， mammalian target of rapamycin （mTOR）， synaptophysin （SYP）， and amyloid precursor protein （APP） in hippocampal neurons were detected by Western blot. Result:Compared with the sham operation group， the model group showed prolonged escape latency， lengthened swimming distance， dwindled the number of times for the platform crossing after platform removal （<italic>P</italic><0.05）， increased apoptotic cells （<italic>P</italic><0.05）， declining synaptic dendritic spines （<italic>P</italic><0.05）， down-regulated expression levels of PI3K， Akt， mTOR， and SYP proteins， and up-regulated expression level of APP protein in hippocampal neurons （<italic>P</italic><0.05）. Compared with the model group， the BSTLP groups and the IGF-1 group showed shortened escape latency and swimming distance， increased number of times for the platform crossing after platform removal （<italic>P</italic><0.05），declining apoptotic cells （<italic>P</italic><0.05）， up-regulated expression levels of PI3K， Akt， mTOR， and SYP proteins， and down-regulated expression level of APP protein in hippocampal neurons （<italic>P</italic><0.05）. Compared with the IGF-1 group， the high-dose BSTLP group showed no significant difference in the escape latency， swimming distance， the number of times for the platform crossing after platform removal， apoptotic cells， synaptic dendritic spines， and expression levels of PI3K， Akt， mTOR， SYP， and APP proteins in hippocampal neurons. However， the differences were significant in the medium-dose and low-dose BSTLP groups （<italic>P</italic><0.05）. Conclusion:BSTLP can improve the learning and memory of rats with VD. The mechanism is presumedly related to the activation of thePI3K/Akt/mTOR pathway and improvement of synaptic plasticity of hippocampal neurons.

Nicotine Induces Apoptosis of BEAS-2B Cells by Inhibiting Autophagy via the PI3K / Akt / mTOR Pathway / 中国生物化学与分子生物学报

Cigarette smoking is a major risk factor for chronic respiratory inflammatory diseases. Nicotine is the most important ingredient in tobacco smoke. The incidence rate of chronic respiratory diseases associated with nicotine is increasing rapidly. Therefore, it is urgent to find potential targets for nicotine-related chronic respiratory inflammatory diseases. This article hereby aims to investigate the effect of nicotine on apoptosis of BEAS-2B cells and its potential mechanism. The expressions of apoptosis, autophagy and PI3K/ Akt/ mTOR pathway-related proteins were detected by Western blotting. Flow cytometry and CCK-8 were used to detect cell apoptosis rate and cell viability. The results showed that nicotine induced apoptosis of BEAS-2B cells at 1, 2 and 4 mmol/ L, and the cell viability decreased with the increase of concentration. Compared with the control group, the expression of autophagy-related protein LC3Ⅱ and P62 increased significantly after nicotine treatment (P0. 05). Compared with the nicotine group, rapamycin pretreatment significantly decreased cell apoptosis and increased cell activity (P<0. 05). Compared with the nicotine group, the expression of p-Akt and p-mTOR decreased significantly and apoptosis decreased significantly after LY294002 pretreatment (P<0. 05). Furthermore, nicotine induces apoptosis of BEAS-2B cells by inhibiting autophagy may be via PI3K/ Akt/ mTOR pathway, which may be a potential target for the prevention and treatment of chronic respiratory inflammatory diseases.

Averrhoa Carambola L. Roots DMDD alleviates myocardial injury in diabetes mellitus mice by regulating ROS-mediated autophagy pathway / 中国药理学通报

Aim To evaluate the protective effect of Averrhoa Carambola L. Roots DMDD alleviating myocardial injury in diabetes mellitus (DM) mice and its mechanism. Methods SD mice were given high-glucose-high-fat diet combined with streptozotocin to induce DM model, and were administered with DMDD. The fasting blood glucose (FBG) was recorded. The left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), maximum upstroke velocity of left ventricular pressure (+ dp/dt

SR1001 Inhibits Clostridium difficile Infection by Interfering Phosphorylation of PI3K/AKT/mTOR Pathway / 胃肠病学

Background: Clostridium difficile infection (CDI) is the most commonly seen opportunistic infection. Although antibiotic therapy is the first-line treatment, there are still some problems existed such as poor therapeutic effect, recurrence of infection and recrudescence. Therefore, it is necessary to develop new anti-infectious drugs. Aims: To explore the possible intervention effect of SR1001 on CDI and its potential mechanism, and to explore its potential intervention targets. Methods: Clostridium difficile (C. difficile) was cultured with HT-29 cells, and were divided into control group, CDI group (infected with C. difficile) and SR1001 treatment group (infected with C. difficile+SR1001 treatment). Morphological changes of HT-29 cells were observed. Cell proliferation was detected by CCK-8 assay, expression of PI3K/AKT/mTOR signaling pathway was detected by Western blotting, and TcdB content in the cell supernatant was detected by ELISA. Results: Compared with the control group, cells in CDI group became brighter and rounder, apoptosis was obvious, cell proliferation ability was significantly decreased (P<0.05), and inhibition of cell proliferation increased with the extension of time; the expressions of PI3K/AKT/mTOR pathway phosphorylated proteins and the content of TcdB in the supernatant were significantly increased (P<0.05). After SR1001 treatment, the cells tended to be in normal 'paving stone'-like arrangement, apoptosis was improved, cell proliferation ability was significantly increased than in CDI group, and expressions of PI3K/AKT/mTOR pathway phosphorylated proteins and TcdB content in the supernatant were significantly decreased. Conclusions: SR1001 can reverse the effect of C. difficile on growth of colon cancer cells by interfering the phosphorylation of PI3K/AKT/mTOR pathway.

Mulberry polysaccharides improves the chemotherapy of liver cancer ascites tumor-bearing mice by regulating the PI3K/AKT/mTOR pathway / 中国临床药理学与治疗学

AIM: To study the synergistic and attenuating effects of mulberry polysaccharides on the chemotherapy of liver cancer ascites tumor-bearing mice by regulating the PI3K/AKT/mTOR pathway. METHODS: Ninety SPF male Kunming mice were randomly divided into normal group, model group, cyclophosphamide group, mulberry polysaccharide group and mulberry polysaccharide + cyclophosphamide group. Liver cancer ascites tumor-bearing mice were prepared, and each administration group was given 30 mg/kg of cyclophosphamide or (and) 200 mg/kg of mulberry polysaccharide and administered orally. The normal group and the model group were administrated with 10 mL/kg saline. The tumor suppression rate, liver, spleen and other indexes, tumor tissue VEGF and inflammatory factor content, and PI3K/AKT/mTOR pathway-related protein expression were observed in mice.RESULTS:Cyclophosphamide group, mulberry polysaccharide group and mulberry polysaccharide + cyclophosphamide group mice body weight, tumor mass, tumor tissue VEGF, TNF-α, IL-6, PI3K, AKT and mTOR phosphorylation levels were lower than the model group, and the level of IL-1β was higher than the model group; mulberry polysaccharide + cyclophosphamide group mice were observed with body weight, tumor inhibition rate, tumor tissue VEGF, TNF-α, IL-6, PI3K, AKT and The mTOR phosphorylation level higher than the mulberry polysaccharide group and cyclophosphamide group, and the tumor mass and IL-1β level were lower than the mulberry polysaccharide group and cyclophosphamide group (P<0.05). CONCLUSION: Morus alba polysaccharide combined with cyclophosphamide can effectively inhibit tumor proliferation of liver cancer ascites tumor-bearing mice and exert attenuating effect. The mechanism may be related to the down-regulation of PI3K /AKT/mTOR pathway expression.