Mu-Xiang-You-Fang protects PC12 cells against OGD/R-induced autophagy via the AMPK/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Mu-Xiang-You-Fang (MXYF) is a classic prescription of Hui medicine. It is composed of five herbs and has been used to treat ischemic stroke for many years. However, the potential pharmacological mechanisms of MXYF remain unclear. The present research is aimed to investigate the protective effect and possible mechanisms of MXYF treatment in an in vitro model of cerebral ischemia-reperfusion injury. MATERIALS AND METHODS: An oxygen-glucose deprivation and reperfusion (OGD/R) model of PC12 cells was established. The effect of MXYF on the cell viability after OGD/R injury was determined using a cell counting kit (CCK-8) assay. The colorimetric method was used to determine the lactate dehydrogenase (LDH) leakage rate. The calcium concentration was determined by the chemical fluorescence method, and mitochondrial membrane potential was determined using flow cytometry. Monodansylcadaverine (MDC) staining and electron microscopic analysis were then conducted to detect autophagy after oxygen-glucose deprivation and reperfusion in PC12 cells. Immunofluorescence and western blot analyses were used to detect the expression of proteins associated with autophagy. RESULTS: It was found that MXYF (1, 2, 4 µg/mL) could significantly increase cell viability and mitochondrial membrane potential and decrease the calcium concentration and LDH release rate in PC12 cells. After OGD/R injury in PC12 cells, the number of autophagosomes and autophagolysosome significantly increased. MXYF (4 µg/mL) inhibited the autophagy induced by OGD/R and inhibited the expression of LC3, beclin1, p-AMPK, and ULK1. In contrast, the expression of p-mTOR, p-p70s6k, and p62 was significantly enhanced. CONCLUSIONS: These findings suggest that MXYF inhibits autophagy after OGD/R-induced PC12 cell injury through the AMPK-mTOR pathway. Thus, MXYF might have therapeutic potential in treating ischemic stroke.

Neuroprotective Effects of Radix Scrophulariae on Cerebral Ischemia and Reperfusion Injury via MAPK Pathways.

Ischemic stroke is a clinically common cerebrovascular disease whose main risks include necrosis, apoptosis and cerebral infarction, all caused by cerebral ischemia and reperfusion (I/R). Ischemia and reperfusion-induced injury or apoptosis inhibition in human brain tissue may exert an irreplaceable protective effect on ischemic nerves. This process has particular significance for the treatment of stroke patients. However, the development of neuroprotective drugs remains challenging. Radix Scrophulariae, traditionally considered a valuable medicine, has been discovered to have neuroprotective effects. To explore the neuroprotective effects of an aqueous extract of Radix Scrophulariae (RSAE) on cerebral ischemia/reperfusion and their underlying mechanisms, oxygen-glucose deprivation and reperfusion (OGD/R)-induced PC12 cells were used, and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was established. In vitro results showed that 12.5 µg/mL RSAE markedly improved cell viability; inhibited LDH leakage; increased SOD, GSH-Px and CAT enzyme activity; stabilized the mitochondrial membrane potential; and reduced OGD-induced cell injury and apoptosis. Additionally, in vivo results preliminarily suggested that in MCAO/R model mice, RSAE treatments attenuated infarct volume; reduced brain water content and nitric oxide (NO) and malondialdehyde (MDA) concentrations; inhibited I/R-induced neurological deficits; reduced the levels of lactate dehydrogenase (LDH) leakage release; improved antioxidant capacity by upregulating SOD, GSH-Px and CAT enzyme activity; and reduced neuronal apoptosis, necrosis and loss of neurons. Moreover, it was found that RSAE upregulated the expression of Bcl-2 and downregulated the expression of Bax. In addition, the phosphorylation levels of MAPK signal pathways were elucidated via western blot analysis and immunohistochemical evaluation. In summary, this study investigated the neuroprotective effects and potential mechanisms of RSAE on focal cerebral I/R injury in mice. Radix Scrophulariae has been previously identified as a potential neuroprotective natural plant. Hence, our results may offer insight into discovering new active compounds or drugs for the treatment of ischemic stroke. Many new natural active chemicals in this extract may be discovered by chemical separation and identification and may provide new insights into therapeutic targets in stroke patients.

Biospecific isolation and characterization of angiogenesis-promoting ingredients in Buyang Huanwu decoction using affinity chromatography on rat brain microvascular endothelial cells combined with solid-phase extraction, and HPLC-MS/MS.

Buyang Huanwu decoction (BHD) was reported to exert angiogenesis-promoting effects, but its active ingredients remain unknown. In this study, we developed a method to screen potential angiogenesis-promoting compounds in BHD, which involved biospecific isolation using live rat brain microvascular endothelial cells (rBMECs) and characterization using solid-phase extraction (SPE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Six compounds showed binding affinity to rBMECs and were further identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-ß-D-glucoside, galloylpaeoniflorin, formononetin-7-O-ß-D-glucoside, and (3R)-7,2'-hydroxy-3',4'-dimethoxy-isoflavan. The results indicated that five of them except 6-hydroxykaempferol-di-O-glucoside showed a protective effect against oxygen glucose deprivation/reperfusion injury in rBMECs and upregulated the secretion of vascular endothelial growth factor and basic fibroblast growth factor, suggesting a mechanism underlying their angiogenic activity. Our findings suggest that biospecific live cell-based isolation combined with SPE and HPLC-MS/MS is an effective method for screening potential bioactive components in traditional Chinese medicines.