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.

Oleanolic Acid Inhibits Neuronal Pyroptosis in Ischaemic Stroke by Inhibiting miR-186-5p Expression

Ischaemic stroke is a common condition leading to human disability and death. Previous studies have shown that oleanolic acid (OA) ameliorates oxidative injury and cerebral ischaemic damage, and miR-186-5p is verified to be elevated in serum from ischaemic stroke patients. Herein, we investigated whether OA regulates miR-186-5p expression to control neuroglobin (Ngb) levels, thereby inhibiting neuronal pyroptosis in ischaemic stroke. Three concentrations of OA (0.5, 2, or 8 μM) were added to primary hippocampal neurons subjected to oxygen–glucose deprivation/ reperfusion (OGD/R), a cell model of ischaemic stroke. We found that OA treatment markedly inhibited pyroptosis. qRT–PCR and western blot revealed that OA suppressed the expression of pyroptosis-associated genes. Furthermore, OA inhibited LDH and proinflammatory cytokine release. In addition, miR-186-5p was downregulated while Ngb was upregulated in OA-treated OGD/R neurons. MiR-186-5p knockdown repressed OGD/R-induced pyroptosis and suppressed LDH and inflammatory cytokine release. In addition, a dual luciferase reporter assay confirmed that miR-186-5p directly targeted Ngb. OA reduced miR-186-5p to regulate Ngb levels, thereby inhibiting pyroptosis in both OGD/R-treated neurons and MCAO mice. In conclusion, OA alleviates pyroptosis in vivo and in vitro by downregulating miR-186-5p and upregulating Ngb expression, which provides a novel theoretical basis illustrating that OA can be considered a drug for ischaemic stroke.