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.

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.