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.

Anti-angiogenetic effect of arenobufagin in vitro and in vivo / 药学学报

This study is to investigate the anti-angiogenetic effect of arenobufagin in vitro and in vivo. The anti-proliferation effect of arenobufagin on CNE-2, Hep2, SH-SY5Y, LOVO, PC-3 and DU145 cells as well as human umbilical vein endothelial cells (HUVECs) was determined by MTT assay. Cell morphological changes of LOVO and HUVECs after arenobufagin treatment were observed by microscopy. Arenobufagin inhibited the proliferation of CNE-2, Hep2, SH-SY5Y, LOVO, PC-3, DU145 and HUVECs in a dose-dependent manner. Furthermore, it was obviously observed that the subcytotoxic concentration of arenobufagin in human carcinoma cells induced a marked decrease in the viability of HUVECs. Chick embryo chorioallantoic membrane (CAM) model was used to detect the anti-angiogenetic effect of arenobufagin in vivo. Arenobufagin significantly suppressed the angiogenesis of CAM. Cell cycle analysis demonstrated that G2/M phase was arrested and the sub-G1 peak appeared with the increase of arenobufagin concentration. PI/Annexin V double staining assay further demonstrated that arenobufagin could induce apoptosis in a dose- and time-dependent manner. Mitochondrial potential collapse detected by flow cytometric analysis was increased after arenobufagin treatment. It also observed that PARP was cleaved to p85 active form by Western blotting. Taken together, arenobufagin has significant anti-angiogenetic effect in vitro and in vivo, and the action mechanisms behind its anti-angiogenesis may be associated with cell cycle arrest and apoptosis of vein endothelial cells.