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 ... -  《-》

[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

Shu MQ ，Dai YY ，Song LJ ，Ma D ，Liu KX ，Miao ZY ，Wei RH ，Yin JZ ，Ma CG ，Huang JJ ... -  《-》

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 ... -  《-》

The activation of the HIF-1α-VEGFA-Notch1 signaling pathway by Hydroxysafflor yellow A promotes angiogenesis and reduces myocardial ischemia-reperfusion injury.

Hydroxyl Safflower Yellow A (HSYA) is the primary bioactive compound derived from Safflower, which has been scientifically proven to possess anti-inflammatory, anti-apoptotic, and ameliorative properties against mitochondrial damage during acute myocardial ischemia-reperfusion injury (MIRI); however, its effects during the recovery stage remain unknown. Angiogenesis plays a crucial role in the rehabilitation process. The objective of this study was to investigate the long-term angiogenic effect of HSYA and its contribution to recovery after myocardial ischemia, as well as explore its underlying mechanism using non-targeted metabolomics and network pharmacology. The MIRI model in rat was established by ligating the left anterior descending branch of the coronary artery. The effect of HSYA was assessed based on myocardial infarction volume and histopathology. Immunofluorescence staining was employed to evaluate angiogenesis, while ELISA was used to detect markers of myocardial injury. Additionally, a rat myocardial microvascular endothelial cell (CMECs) injury model was established using oxygen-glucose deprivation/reoxygenation (OGD/R), followed by scratch assays, migration assays, and tube formation experiments to assess angiogenesis. Western blot analysis was conducted to validate the underlying mechanism. Our findings provide compelling evidence for the therapeutic efficacy of HSYA in reducing myocardial infarction size, facilitating cardiac functional recovery, and reversing pathological alterations within the heart. Furthermore, we elucidate that HSYA exerts its effects on promoting migration and generation of myocardial microvascular endothelial cells through activation of the HIF-1α-VEGFA-Notch1 signaling pathway. These results underscore how HSYA enhances cardiac function via angiogenesis promotion and activation of the aforementioned signaling cascade.

Ge C ，Meng D ，Peng Y ，Huang P ，Wang N ，Zhou X ，Chang D ... -  《-》

Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.

A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.

Wang L ，Li J ，Wang Y ，Ge C ，Huang Q ，Li L ，Wang N ，Chen Y ，Zhou X ，Chang D ，Li D ，Hou J ... -  《-》