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.

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.

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.

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.

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.

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.

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.

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

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.

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.

Effect of simvastatin pretreatment on human metapneumovirus infection and its mechanism / 解放军医学杂志

Objective: To investigate the effect of simvastatin pretreatment on human metapneumovirus (hMPV) infection and the related mechanism. Methods: Human bronchial epithelial cells (16HBE) were used in vitro experiments and BALB/c mice were used in vivo experiments. They were randomly divided into control group, simvastatin group, hMPV group, hMPV+simvastatin group. After corresponding treatment, the fluorescence of hMPV was observed with microscopy; virus titer and the expressions of autophagy-related gene recombinant human autophagy-related protein 5 (ATG5), Beclin1, and microtubule-associated protein 1 light chain 3 (LC3) were detected by Fluorescence Quantitative Real-time PCR; Western blotting was performed to detect autophagy-related protein ATG5, Beclin1, LC3 and serine/threonine kinase/mammalian rapamycin target protein (AKT/mTOR) pathway proteins. Results: In vitro experiment, the direct and indirect fluorescence expressions of hMPV and the virus titer were lower in hMPV+simvastatin group (0.260 ± 0.018) than those in hMPV group (1.241 ± 0.030), while the mRNA expression levels of autophagy-related gene ATG5, Beclin1 and LC3 were higher in hMPV+simvastatin group [(7.31±0.15), (8.67±0.88) and (6.55±0.30), respectively] than those in control group [(1.00±0.06), (1.00±0.05) and (1.10±0.06), respectively]; the mRNA and protein levels of autophagy-genes were the highest in hMPV+simvastatin group and the differences were statistically significant (P<0.05). The AKT/mTOR pathway was inhibited in hMPV group and hMPV+simvastatin group. In vivo experiment, the virus titer of the lung tissue and of the lung tissue supernatant inoculated in Vero E6 were lower in hMPV+simvastatin group (0.75±0.26 and 0.42±0.17, respectively) than those in hMPV group (2.46±0.53 and 1.80±0.40, respectively). The mRNA and protein expression levels of autophagy-related genes ATG5, Beclin1 and LC3 were the highest in hMPV+simvastatin group (3.89±0.42, 3.13±0.26 and 3.56±0.22, respectively), higher than those in control group and hMPV group, the differences were statistically significant (P<0.05), and the AKT/mTOR pathway was inhibited in this group; HE staining of lung tissue showed an inflammatory response in the hMPV group, and pre-treatment with simvastatin could reduce the inflammation. Conclusion: Pretreatment with simvastatin may reduce human metapneumovirus infection, which is associated with autophagy and the 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.

Momordica protein MAP30 promotes apoptosis and autophagy via AKT/mTOR pathway in multiple myeloma cell / 中国肿瘤生物治疗杂志

@#Objective: To investigate the role of momordica protein MAP30 in multiple myeloma (MM) and the possible mechanism. Methods: Human myeloma RPMI-8226, NCI-H929 and U266 cells were treated with MAP30 at different concentration (1-10 μmol/L) and then the proliferation rates of cells were detected by CCK-8 assay.Annexin V/PI flow cytometry was used to evaluate the apoptosis rate of myeloma cells, and the expressions of apoptosis-related protein (PARP), autophagy-related proteins (LC3II, P62) andAkt/mTOR pathway-related proteins in multiple myeloma cells were also detected via Wb. The changes in cell proliferation, apoptosis and autophagy after the treatment of MAP30 combined with autophagy agonist rapamycin (Rap) or autophagy inhibitor bafilomycin (Baf) were observed by CCK-8, flow cytometry and Wb, respectively. Results: MAP30 (1-10 μmol/L) inhibited the proliferation of myeloma cells in a time- and dose-dependent manner (P<0.05 or P<0.01). With MAP30 acting on myeloma cells alone, the apoptosis and autophagy of MM cells, as well as the expression of PARP cleavage and LC3II increased while the expression of P62 decreased significantly (all P< 0.05 or P<0.01). After being treated with MAP30+Baf, compared with MAP30 treatment alone, the cell proliferation was remarkably enhanced while cell apoptosis and cell autophagy were suppressed, besides, the expression of PARP cleavage and LC3II were decreased and P62 level was augmented (all P<0.05 or P<0.01). Conversely, after being treated with MAP30+Baf, compared with MAP30 treatment or Baf treatment alone, cell proliferation and P62 level were reduced, while apoptosis and autophagy as well as the expressions of PARP cleavage and LC3II level were increased (all P<0.05 or P<0.01). The expressions of p-AKT and p-mTOR were significantly reduced with the effect of MAP30 on myeloma cells (all P<0.05). Conclusion: MAP30 can promote the apoptosis and autophagy of myeloma cells throughAKT/mTOR pathway, which may provide a new therapeutic strategy for treatment of multiple myeloma.

Inhibition of peiminine on invasion and migration of human lung cancer A549 cells by decreasing epithelial-mesenchymal transition process via PI3K/Akt/mTOR pathway / 中草药

Objective: To detect the influence of peiminine on the invasion and migration of human lung cancer A549 cells. Methods A549 cells were treated with peiminine at the final concentrations of 0, 50, 100, and 200 μmol/L, respectively. The influence of peiminine on the invasion and migration of A549 cells and its underlying mechanisms were investigated by cell invasion experiment, cell scratch experiment, real-time quantitative PCR, ELISA, and Western blotting. Results Compared with 0 μmol/L peiminine group, the cell transmembrane number and scratch wound healing rate and expressions of MMP-9 and MMP-2 in the group treated with different concentrations of peiminine significantly decreased (P < 0.01). Compared with 0 μmol/L peiminine group, the mRNA expression of E-cadherin increased significantly (P < 0.01), while the mRNA expressions of N-cadherin and vimentin decreased significantly (P < 0.01). Compared with 0 μmol/L peiminine group, FN protein expression was significantly decreased in all the groups with different concentrations of peiminine group except treatment with 50 μmol/L peiminine after 24 h (P < 0.05, 0.01). Compared with 0 μmol/L peiminine group, the ratio of p-PI3K/PI3K and p-mTOR/mTOR in all concentrations of peiminine groups and p-Akt/Akt in 100 and 200 peiminine groups were significantly decreased (P < 0.05, 0.01). Conclusion Peiminine can inhibit the invasion and migration of A549 cells, which may be related to the activation of PI3K/Akt/mTOR pathway and decreasing the epithelial- mesenchymal transition process.

Polydatin protects H9c2 cells from hypoxia-induced injury via up-regulating long non-coding RNA DGCR5

Polydatin (PD), a monocrystalline polyphenolic drug mainly found in the roots of Polygonum cuspidatum, has various pharmacological activities. Long non-coding RNAs (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) was found to participate in the suppression of multiple cancers. Here, we proposed to study the effect of PD on myocardial infarction (MI) by inducing DGCR5. CCK-8 assay was performed to detect the viability of H9c2 cells. Flow cytometry was utilized to test apoptosis of H9c2 cells. These results determined the optimal concentration and effect time of hypoxia as well as PD. Si-DGCR5 was transfected into cells and the expression level was determined by qRT-PCR. Western blot was utilized to evaluate the expression of apoptosis-related proteins, Bcl-2, Bax, and cleaved-caspase-3, as well as autophagy-associated proteins including Beclin-1, p62, and LC3-II/LC3-I. As a result, PD efficiently attenuated hypoxia-induced apoptosis and autophagy in H9c2 cells. The expression of DGCR5 was down-regulated by hypoxia and up-regulated by PD. Besides, knocking-down the expression of DGCR5 inhibited the protection of PD in H9c2 cells. In addition, PD up-regulated the accumulation of DGCR5, DGCR5 decreased the expression of Bcl-2 and p62, raised the expression of Bax and cleaved-caspase-3, and the proportion of LC3-II/LC3-I. PD stimulated the PI3K/AKT/mTOR and MEK/ERK signaling pathways via up-regulating the expression of DGCR5. Our data demonstrated that PD reduced cell apoptosis and autophagy induced by hypoxia in cardiomyocytes. Moreover, PD activated PI3K/AKT/mTOR and MEK/ERK signaling pathways by up-regulating the expression of DGCR5.

Inhibitory effects of petasin on human colon carcinoma cells mediated by inactivation of Akt/mTOR pathway / 中华医学杂志(英文版)

Background:@#Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored.@*Methods:@#Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests.@*Results:@#Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm3 at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues.@*Conclusions:@#Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.

Pharmacological analysis of curdione on hypertrophic scar fibroblast / 中草药

Objective To investigate the effects of curdione on the HSF proliferation, transformation, and collagen secretion. Methods After the human HSF was treated with curdione, the proliferation inhibition ratio was measured using MTT method. Meanwhile, the TIMP-1, MMP-1, COL-I, and COL-III were detected by ELISA method, the α-SMA was analyzed by IHC technology, and the PI3K/Akt/mTOR and TGF-β1/Smads related molecular were evaluated by Western blotting. Results Curdione could reduce the proliferation inhibition ratio. Compared with control group, the TIMP-1, COL-I, and COL-III secretion were inhibited by curdione significantly (P < 0.05, P < 0.01), while the MMP-1 levels was significantly increased by curdione (P < 0.05, P < 0.01). The results also indicated that the expression levels of p-PI3K, p-Akt, p-mTOR, p-Smad3, TGF-β1, and α-SMA were significantly suppressed by curdione with concentration dependence (P < 0.05, P < 0.01). Conclusion Curdione could effectively improve the hypertrophic scar by inhibiting the HSF proliferation, transformation, and collagen secretion, and accelerating the collagen enzymolysis via PI3K/Akt/mTOR and TGF-β1/Smads pathways.

Mechanism research of safflower polysaccharide inducing apoptosis of human breast cancer MDA-MB-435 cells by interrupting PI3K/Akt/mTOR Pathway / 中草药

Objective To investigate the effect of safflower polysaccharide on apoptosis of human breast cancer MDA-MB-435 cells by blocking PI3K/Akt/mTOR pathway and explore its mechanism. Methods MDA-MB-435 cells were divided into control group and safflower polysaccharide 0.5 and 1.0 mg/mL groups. MTT assay and flow cytometry were used to detect the effects of different concentrations of safflower polysaccharide on the growth inhibition and apoptosis of MDA-MB-435 cells. RT-PCR and Western blotting were used to detect the effect of different concentrations of safflower polysaccharides on the mRNA and protein expression of PI3K, Akt, and mTOR in MDA-MB-435 cells. Results Compared with the control group, the inhibition rate of MDA-MB-435 cells in safflower polysaccharide 1.0 mg/mL group was (27.73 ± 3.75)%, which was significantly higher than that [(21.52 ± 2.43)%] in safflower polysaccharide 0.5 mg/mL group (P < 0.05). Compared with the control group, safflower polysaccharide could significantly increase the apoptosis rate of MDA-MB-435 cells in a dose-dependent manner (P < 0.01). The results of RT-PCR and Western blotting showed that, compared with the control group, safflower polysaccharide significantly decreased the mRNA and protein expression of PI3K, Akt, and mTOR in MDA-MB-435 cells (P < 0.05, 0.01). Conclusion Safflower polysaccharide can effectively inhibit the growth of MDA-MB-435 cells and promote their apoptosis, which may be achieved by blocking the PI3K/Akt/mTOR pathway.

Expression of PSAT1 in pancreatic cancer tissues and the mechanism underlying PSAT1-mediated cell proliferation and invasion / 中国肿瘤临床

Objectives: To investigate the expression of phosphoserine aminotransferase 1 (PSAT1) in pancreatic cancer tissues, and its potential role in pancreatic cancer. Methods: The expression of PSAT1 in 98 human pancreatic cancer tissues, which were collected from the People＇s Hospital of Guizhou, between July 2013 to July 2017, and the corresponding adjacent normal tissues was analyzed by immunohistochemical staining. Additionally, the relationship between the expression of PSAT1 and the clinicopathological parame-ters, overall survival (OS), and disease-free survival (DFS) of patients with pancreatic cancer was evaluated. The human pancreatic can-cer cell lines, BxPC-3 and SW1990, were transfected with PSAT1-siRNA, to investigate the effect of PSAT1 knockdown on cell prolifera-tion, migration, and invasion. Additionally, we performed Western blot to assess the expression of PI3K/Akt/mTOR-related proteins in PSAT1-knockdown cells. Results: The percentages of PSAT1-positive cells in pancreatic cancer and adjacent non-tumor tissues were 69.4% (68/98) and 5.0% (5/98), respectively, indicating a significantly higher expression of PSAT1 in pancreatic cancer tissues com-pared to adjacent non-tumor tissues (P<0.05). The increased expression of PSAT1 in pancreatic cancer tissues correlated with lymph node metastasis and TNM stage. Kaplan-Meier analysis suggested that a high expression of PSAT1 correlated with a poor OS and DFS compared to a low expression of PSAT1 (P<0.05). Cox regression analysis showed that the expression of PSAT1 is an independent prog-nostic marker for OS and DFS in pancreatic cancer patients (P<0.05, all). Transient transfection of BxPC-3 and SW1990 cells with PSAT1-siRNA markedly reduced the cell proliferation, migration, and invasion abilities of these cells compared to transfection with NC-siRNA (P<0.05). Knockdown of PSAT1 in pancreatic cancer cells also inhibited the expression of p-Akt and p-mTOR (P<0.05). Conclusions: The expression of PSAT1 increases in human pancreatic cancer tissues and cell lines. Additionally, PSAT1 regulates cell proliferation and in-vasion through the PI3K/Akt/mTOR pathway.