Hydroxysafflor Yellow A promotes angiogenesis of brain microvascular endothelial cells from ischemia/reperfusion injury via glycolysis pathway in vitro.

Angiogenesis of brain microvascular endothelial cells (BMECs) after cerebral ischemia was conducive to improving the blood supply of ischemia tissues, which was upregulated by glycolysis. Hydroxysafflor Yellow A (HSYA) mends damaged tissues through increasing angiogenesis. HSYA treated proliferation, migration and angiogenesis of BMECs in vitro in vitro during OGD/R. HSYA regulated the key enzymes of glycolysis, such as hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), glucose uptake and products (pyruvate, ATP and lactate) were detected by western blot and kits, respectively. Scratch wound assay, transwell, tube formation and spheroid sprouting were used to explore the pathway that HSYA recovered migration and angiogenesis of BMECs. We evaluated the potential target of HSYA promoting glycolysis via molecular docking, drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). HSYA promoted the proliferation, migration, tube formation and spheroid sprouting of BMECs during OGD/R, and stimulated the expression of tip phenotype marker protein (CD34), and the receptor (Notch-1) that regulated the differentiation of endothelial cells into tip/stalk phenotype. In glycolysis, PFKFB3 expression was upregulated by HSYA; HSYA also improved ATP and pyruvate levels, as well as lactate release after OGD/R. Finally, upregulating VEGFA and p-VEGFR2 of HSYA was weakened because of suppressing glycolysis; the HSYA's improvement of BMECs migration and angiogenesis was attenuated under the inhibition of glycolysis, which confirmed that HSYA were upregulating angiogenesis and expression of VEGFA/VEGFR2 by glycolysis pathway. The result about molecular docking, DARTS and CETSA suggested that PFKFB3 was the possible target of HSYA. HSYA promotes angiogenesis of BMECs in vitro through the glycolysis mediated VEGFA/VEGFR2 pathway, and PFKFB3 is the potential target of HSYA to heighten glycolysis.

Ruan J ，Wang L ，Wang N ，Huang P ，Chang D ，Zhou X ，Seto S ，Li D ，Hou J ... -  《-》

Hydroxysafflor Yellow a Promotes Angiogenesis in Rat Brain Microvascular Endothelial Cells Injured by Oxygen-glucose Deprivation/reoxygenation(OGD/R) through SIRT1-HIF-1α-VEGFA Signaling Pathway.

Angiogenesis led by brain microvascular endothelial cells (BMECs) contributes to the remission of brain injury after brain ischemia reperfusion. In this study, we investigated the effects of hydroxysafflor yellow A(HSYA) on angiogenesis of BMECs injured by OGD/R via SIRT1-HIF-1α-VEGFA signaling pathway. The OGD/R model of BMECs was established in vitro by OGD for 2h and reoxygenation for 24h. At first, the concentrations of vascular endothelial growth factor (VEGF), Angiopoietin (ang) and platelet-derived growth factor (PDGF) in supernatant were detected by ELISA, and the proteins expression of VEGFA, Ang-2 and PDGFB in BMECs were tested by western blot; the proliferation, adhesion, migration (scratch healing and transwell) and tube formation experiment of BMECs; the expression of CD31 and CD34 were tested by immunofluorescence staining. The levels of sirtuin1(SIRT1), hypoxia-inducible factor-1α (HIF-1α), VEGFA mRNA and protein were tested. HSYA up-regulated the levels of VEGF, Ang and PDGF in the supernatant of BMECs under OGD/R, and the protein expression of VEGFA, Ang-2 and PDGFB was increased; HSYA could significantly alleviate the decrease of cell proliferation, adhesion, migration and tube formation ability of BMECs during OGD/R; HSYA enhanced the fluorescence intensity of CD31 and CD34 of BMECs during OGD/R; HSYA remarkably up-regulated the expression of SIRT1, HIF-1α, VEGFA mRNA and protein after OGD/R, and these increase decreased after SIRT1 was inhibited. SIRT1-HIF-1α-VEGFA signaling pathway is involved in HSYA improves angiogenesis of BMECs injured by OGD/R.

Ruan J ，Wang L ，Dai J ，Li J ，Wang N ，Seto S ... -  《-》

Hydroxysafflor yellow a protects brain microvascular endothelial cells against oxygen glucose deprivation/reoxygenation injury: Involvement of inhibiting autophagy via class I PI3K/Akt/mTOR signaling pathway.

The present study aimed to test whether Hydroxysafflor yellow A (HSYA) protects the brain microvascular endothelial cells (BMECs) injury induced by oxygen glucose deprivation/reoxygenation (OGD/R) via the PI3K/Akt/mTOR autophagy signaling pathway. Primary rat BMECs were cultured and identified by the expression of factor VIII-related antigen before being exposed to OGD/R to imitate ischemia/reperfusion (I/R) damage in vitro. The protective effect of HSYA was evaluated by assessing (1) cellular morphologic and ultrastructural changes; (2) cell viability and cytotoxicity; (3) transendothelial electrical resistance (TEER) of monolayer BMECs; (4) cell apoptosis; (5) fluorescence intensity of LC3B; (6) LC3 mRNA expression; (7) protein expressions of LC3, Beclin-1, Zonula occludens-1 (ZO-1), phospho-Akt (p-Akt), Akt, phospho-mTOR (p-mTOR) and mTOR. It was found that HSYA (20, 40, and 80 μM) and 3-MA effectively reversed the cellular morphological and ultrastructural changes, increased cell survival, normalized the permeability of BMECs, and suppressed apoptosis induced by OGD/R (2 h OGD followed by 24 h reoxygenation). Concurrently, HSYA and 3-MA also inhibited OGD/R-induced autophagy evidenced by the decreased number of autophagosomes and down-regulated levels of LC3 and Beclin-1 proteins and mRNAs. HSYA (80 μM), in combination with 3-MA showed a synergistic effect. Mechanistic studies revealed that HSYA (80 μM) markedly increased the levels of p-Akt and p-mTOR proteins. Blockade of PI3K activity by ZSTK474 abolished its anti-autophagic and pro-survival effect and lowered both Akt and mTOR phosphorylation levels. Taken together, these results suggest that HSYA protects BMECs against OGD/R-induced injury by inhibiting autophagy via the Class I PI3K/Akt/mTOR signaling pathway.

Yang G ，Wang N ，Seto SW ，Chang D ，Liang H ... -  《-》

Gnetupendin A protects against ischemic stroke through activating the PI3K/AKT/mTOR-dependent autophagy pathway.

Autophagy has been recently emerged as a prominent factor in the pathogenesis of ischemic stroke (IS) and is increasingly being considered as a potential therapeutic target for IS. Gnetum parvifolium has been identified as a potential therapeutic agent for inflammatory diseases such as rheumatism and traumatic injuries. However, the pharmacological effects of Gnetupindin A (GA), a stilbene compound isolated from Gnetum parvifolium, have not been fully elucidated until now. Here we identified the therapeutic potential of GA for IS, deeply exploring the possible mechanisms related to its regulation of autophagy. The mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R) and the oxygen-glucose deprivation reperfusion (OGD/R)-exposed cells served as models to study the protection of GA against IS. The adeno-associated virus (AAV) encoding shAtg5, in conjunction with autophagy inhibitor 3-Methyladenine (3-MA) were utilized to explore the role of GA in regulating autophagy following IS. Molecular docking, CETSA, and DARTS were used to identify the specific therapeutic target of GA. PI3K inhibitor LY294002 was employed to test the participation of PI3K in GA-mediated autophagy and neuroprotective effects following IS. Our findings revealed that treatment with GA significantly alleviated the brain infract volume, edema, improved neurological deficits and attenuated apoptosis. Mechanistically, we found that GA promoted autophagic flow both in vivo and in vitro after IS. Notably, neural-targeted knockdown of Atg5 abolished the neuroprotective effects mediated by GA. Inhibition of autophagy using 3-MA blocked the attenuation on apoptosis induced by GA. Moreover, molecular docking, CETSA, and DARTS analysis demonstrated that GA specifically targeted PI3K and further inhibited the activation of PI3K/AKT/mTOR signaling pathway. LY294002, which inhibits PI3K, reversed GA-induced autophagy and neuroprotective effects on OGD/R-treated cells. We demonstrated, for the first time, that GA protects against IS through promoting the PI3K/AKT/mTOR-dependent autophagy pathway. Our findings provide a novel mechanistic insight into the anti-IS effect of GA in regulating autophagy.

Mu D ，Liu J ，Mi Y ，Wang D ，Xu L ，Yang Y ，Liu Y ，Liang D ，Hou Y ... -  《-》

F-box and WD repeat domain-containing 7 (FBXW7) mediates the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway to affect hypoxic-ischemic brain damage in neonatal rats.

Sun L 《-》