7-3--dihydroxy-4--methoxyisoflavone and Reperfusion-Injury

Calycosin is a natural product extracted from some plant families and exhibits various biological properties. But the effect of calycosin on cerebral ischemia-reperfusion injury has not been fully elucidated. In this study, the neuroprotective effect of calycosin treatment on the differentiated SH-SY5Y cells exposed to OGD was evaluated using MTT and flow cytometry. Rats that were pretreatment with calycosin were subjected to MCAO, neurological behavior scores and brain infarct volume were evaluated. The protein expression of pERK/ERK were assessed using Western blot. siRNA-pERK and U0126 were administered to investigate the impact of the ERK pathway on calycosin preconditioning. The results demonstrated the neuronal viability in the calycosin-treated SH-SY5Y cells increased significantly, and the rate of apoptosis decreased compared with the Oxygen-glucose deprivation only SH-SY5Y cells. Calycosin pretreatment reduced infarct volume and improved neurological outcome in rats subjected to MCAO. Administration of calycosin increased the ratio of pERK/ERK expression, which was down-regulated in ischemia-reperfusion group. Down-regulation of pERK/ERK significantly attenuated the neuroprotective effect induced by calycosin pretreatment in vitro and in vivo. We concluded calycosin treatment could induce a neuroprotective effect against ischemia, which was related to the regulation of the ERK1/2 pathway.

Topics: Animals; Apoptosis; Brain Ischemia; Humans; MAP Kinase Signaling System; Neuroblastoma; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Ischemic stroke is the second leading cause of death globally. Calycosin is a typical phytoestrogen that protects against cerebral ischemia/reperfusion (I/R) injury. However, the role of ferroptosis in this effect remains unknown. In the present study, we investigated the ferroptosis mechanism of calycosin against cerebral I/R injury using transient middle cerebral artery occlusion/reperfusion (tMCAO/R)-exposed rats and oxygen-glucose deprivation/reperfusion (OGD/R)-stimulated PC12 cells. We found that calycosin treatment significantly improved neurological deficits, brain edema, blood-brain barrier (BBB) breakdown, infarction volume, and neuronal injuries in rats that underwent tMCAO/R; similar to ferrostatin-1 (a ferroptosis inhibitor), calycosin prevented cell viability loss in PC12 cells exposed to OGD/R stimulation. In addition, calycosin intervention decreased ferroptosis, as assessed by iron accumulation, malondialdehyde (MDA), superoxide dismutase (SOD), ceramide, and reactive oxygen species (ROS) levels, as well as ferroptosis-related protein expression (ACSL4, TfR1, FTH1, and GPX4). Furthermore, overexpression of ACSL4 reversed calycosin-induced beneficial efficacy in OGD/R-stimulated PC12 cells. The molecular docking analysis demonstrated that calycosin binds to ACSL4 by forming stable hydrogen bonds at G465, K690, and D573. Collectively, these findings indicate that calycosin ameliorates cerebral I/R injury by depressing ACSL4-dependent ferroptosis.

Topics: Animals; Brain Ischemia; Ferroptosis; Infarction, Middle Cerebral Artery; Molecular Docking Simulation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

As a common cerebrovascular disease (CVD) of the elderly, ischemic stroke (IS) is characterized by high disability and mortality. Excessive autophagy induced by IS is implicated in neuronal death, therefore, the inhibition of immoderate autophagy is viewed as a potential therapeutic avenue to treat IS. Calysoin (CA) is a bioactive component of Radix Astragali, which has been widely used to treat CVDs. However, the mechanism of the treatment of IS by CA is still problematic.. Based on the result of network pharmacology, whether CA inhibited autophagy by regulating the STAT3/FOXO3a pathway to alleviate cerebral ischemia-reperfusion injury (CIRI) was investigated in vivo and in vitro for the first time.. Integrate computational prediction and experimental validation based on network pharmacology.. In current study, network pharmacology was applied to predict the mechanism of the treatment of IS by CA, and it was shown that CA alleviated CIRI by inhibiting autophagy via STAT3/FOXO3a signaling pathway. One hundred and twenty adult male specific pathogen-free Sprague-Dawley rats in vivo and PC12 cells in vitro were used to verify the above prediction results. The rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by suture method, and oxygen glucose deprivation/re-oxygenation (OGD/R) model was used to simulate cerebral ischemia in vivo. The content of MDA, TNF-α, ROS and TGF-β1 in rat serum were detected by ELISA kits. The mRNA and protein expressions in brain tissue were detected by RT-PCR and Western Blotting. The expressions of LC3 in brain were detected immunofluorescent staining.. The experimental results demonstrated that administration of CA dosage-dependently improved rat CIRI as evidenced by the reduction in the cerebral infarct volume, amelioration of the neurological deficits. HE staining and transmission electron microscopy results revealed that CA ameliorated cerebral histopathological damage, abnormal mitochondrial morphology, and damaged mitochondrial cristae structure in MCAO/R rats. CA treatment exerted protective effects in CIRI by inhibiting inflammation response, oxidative stress injury, and cell apoptosis in rat and PC12 cells. CA relieved excessive autophagy induced by MCAO/R or OGD/R through downregulating the LC3Ⅱ/LC3Ⅰ ratio and upregulating the SQSTM1 expression. CA treatment also decreased p-STAT3/STAT3 and p-FOXO3a/FOXO3a ratio in the cytoplasm and modulated the autophagy-related gene expression both in vivo and in vitro.. Treatment with CA attenuated CIRI by reducing excessive autophagy via STAT3/FOXO3a signal pathway in rat and PC12 cells.

Topics: Animals; Apoptosis; Autophagy; Brain Ischemia; Infarction, Middle Cerebral Artery; Ischemic Stroke; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

Topics: Animals; Glucose; HMGB1 Protein; Humans; Microglia; NF-kappa B; Oxygen; Rats; Reperfusion Injury; Signal Transduction; Stroke; Toll-Like Receptor 4

Paeoniflorin (PF) and calycosin-7-glucoside (CG,. To investigate the synergistic effects of PF + CG on ischaemia/reperfusion injury. Male Sprague-Dawley rats were subjected to the middle cerebral artery occlusion/reperfusion (MCAO/R). After MCAO/R for 24 h, rats were randomly subdivided into 5 groups: sham, model (MCAO/R), study treatment (PF + CG, 40 + 20 mg/kg), LY294002 (20 mg/kg), and study treatment + LY294002. Males were given via intragastric administration; the duration of the. PF + CG significantly reduced neurobehavioral outcomes (21%), cerebral infarct volume (44%), brain edoema (1.6%) compared with the MCAO/R group. Moreover, PF + CG increased p-PI3K/PI3K (4.69%, 7.4%), p-AKT/AKT (6.25%, 60.6%) and Bcl-2/BAX (33%, 49%) expression. PF + CG showed a synergistic protective effect against ischaemic brain injury, potentially being a future treatment for ischaemic stroke.

Topics: Animals; Brain Ischemia; Glucosides; Glycogen Synthase Kinase 3 beta; Infarction, Middle Cerebral Artery; Ischemic Stroke; Isoflavones; Male; Monoterpenes; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Stroke

This study was designed to understand the pivotal anti-cerebral ischemia/reperfusion injury (CIRI) targets and pathways of calycosin through network pharmacology and molecular docking analyses. In this study, bioinformatics tools were employed to characterize and identify the pharmacological functions and mechanisms of calycosin for CIRI management. The network pharmacology data identified potential, merged CIRI-associated targets of calycosin including tumor protein p53 (TP53), protein kinase B (AKT1), vascular endothelial growth factor A (VEGFA), interleukin 6, tumor necrosis factor (TNF), and mitogen-activated protein kinase 1 (MAPK1). Molecular docking analysis indicated the binding efficacy of calycosin with three of the targets, namely TP53, AKT1, and VEGFA. The biological processes of calycosin for the treatment of CIRI are mainly involved in the improvement of endothelial cell proliferation and growth, inflammatory development, and cellular metabolism. In addition, the anti-CIRI actions of calycosin were primarily through suppression of the toll-like receptor, PI3K-AKT, TNF, MAPK, and VEGF signaling pathways. Taken together, the current bioinformatic findings revealed pivotal targets, biological functions, and pharmacological mechanisms of calycosin for the treatment of CIRI. In conclusion, calycosin, a functional phytoestrogen, can be potentially used for the treatment of CIRI in future clinical trials.

Topics: Algorithms; Brain Ischemia; Cluster Analysis; Humans; Isoflavones; Molecular Docking Simulation; Molecular Targeted Therapy; Reperfusion Injury; Signal Transduction

Calycosin-7-O-β-D-glucoside (CG), a calycosin derivative compound derived from Astragali Radix, has protective effect against ischemia/reperfusion injury as well as bacterial endotoxin-induced vascular cell injury. In the present study, we ascertained whether CG could reduce myocardial injury in heatstroke rats.. Heat stroke was induced by exposing anaesthetized rats to heat stress (43 °C for 70 min). Rats were given an i.p. dose of CG (26.8 mg/ml/kg) or vehicle solution (ml/kg) 15 min before the start of heat stress and immediately after termination of heat stress. Left ventricular performance, myocardial injury markers in the blood, and myocardial damage scores were assessed in heat stroke rats treated with or without CG. Additionally, cardiac levels of oxidative stress and inflammatory status were estimated simultaneously.. At the time point of heat stroke onset, compared with normothermic controls, group rats with vehicle solution had significantly decreased survival rate, increased hyperthermia, decreased left ventricular stress markers, and increased cardiac damage scores. Compared with group rats with vehicle solution, group rats with CG had significantly improved survival rate, decreased hyperthermia, decreased cardiac ischemic, inflammatory, and oxidative damage.. We thus conclude that myocardial injury can be a pressing need for the design of diagnostic and therapeutic modalities for heat stroke. In particular, our data indicate that CG protects against heat stroke in rats by mitigating myocardial injury.

Topics: Animals; Astragalus propinquus; Drugs, Chinese Herbal; Fever; Heat Stroke; Isoflavones; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Emerging evidence suggests that autophagy plays important roles in the pathophysiological processes of cerebral ischemia and reperfusion injury. Calycosin, an isoflavone phytoestrogen, possesses neuroprotective effects in cerebral ischemia and reperfusion in rats. Here, we investigated the neuroprotective effects of calycosin against ischemia and reperfusion injury, as well as related probable mechanisms behind autophagy pathways.. A cerebral ischemic and reperfusion injury model was established by middle cerebral artery occlusion in male Sprague-Dawley rats. Neurological scores, infarct volumes, and brain water content were assessed after 24 h reperfusion following 2 h ischemia. Additionally, the expression of the autophagy-related protein p62 and NBR1 (neighbor of BRCA1 gene 1), as well as Bcl-2, and TNF-α in rat brain tissues was measured by RT-PCR, western blotting and immunohistochemical analyses.. The results showed that calycosin pretreatment for 14 days markedly decreased infarct volume and brain edema, and ameliorated neurological scores in rats with focal cerebral ischemia and reperfusion. It was observed that levels of p62, NBR1 and Bcl-2 were greatly decreased, and levels of TNF-α significantly increased after ischemia and reperfusion injury. However, calycosin administration dramatically upregulated the expression of p62, NBR1 and Bcl-2, and downregulated the level of TNF-α.. All data reveal that calycosin exerts a neuroprotective effect on cerebral ischemia and reperfusion injury, and the mechanisms maybe associated with its anti-autophagic, anti-apoptotic and anti-inflammatory action.

Topics: Animals; Brain; Brain Ischemia; Disease Models, Animal; Down-Regulation; Infarction, Middle Cerebral Artery; Isoflavones; Male; Neuroprotective Agents; Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sequestosome-1 Protein; Tumor Necrosis Factor-alpha; Up-Regulation

Isoflavone calycosin is a typical phytoestrogen extracted from Chinese medical herb Radix Astragali. It has been reported that estrogens could provide neuroprotective effects, and dietary intake of phytoestrogens could reduce stroke injury in cerebral ischemia/reperfusion (I/R) animal models. In the present study, we investigate the molecular mechanisms underlying the neuroprotective effects of calycosin on middle cerebral artery occlusion (MCAO) rats. Focal cerebral ischemia was induced in male rats by MCAO, neurological deficits and brain edema was evaluated after 24h of reperfusion. The results shown calycosin significantly reduced the infarcted volume and the brain water content, and improved the neurological deficit. To provide insight into the functions of estrogen receptor (ER)-mediated signaling pathway in neuroprotection by calycosin, the expression of miR-375, ER-α, RASD1 (Dexamethasone-induced Ras-related protein 1) and Bcl-2 was determined by RT-PCR or western blot assay. Calycosin exhibited a downregulation of RASD1, and an upregulation of ER-α, miR-375 and Bcl-2. Our finding illustrated that calycosin had been shown neuroprotective effects in cerebral ischemia/reperfusion rats, and the molecular mechanisms may correlate with the positive feedback between ER-α and miR-375, along with the regulation of downstream targets.

Topics: Animals; Brain Ischemia; Dose-Response Relationship, Drug; Down-Regulation; Drugs, Chinese Herbal; Estrogen Receptor alpha; Isoflavones; Male; MicroRNAs; Neuroprotective Agents; Random Allocation; ras Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Treatment Outcome; Up-Regulation

Radix Astragali has been commonly used as traditional herbal medicine in China for reinforcing vital energy, strengthening superficial resistance and promoting the discharge of pus and the growth of new tissue.. The present study was to investigate the neuroprotective effect of calycosin isolated from the roots of Radix Astragali on cerebral ischemic/reperfusion injury.. After 24h of reperfusion following ischemia for 2h induced by middle cerebral artery occlusion (MCAO), Sprague-Dawley rats were intragastrically administered different doses of calycosin (7.5, 15, 30 mg/kg, respectively). Neurological deficit, infarct volume, histopathology changes and some oxidative stress markers were evaluated after 24h of reperfusion.. Treatment with calycosin significantly ameliorated neurologic deficit and infarct volume after cerebral ischemia reperfusion. Calycosin also reduced the content of malondialdehyde (MDA), protein carbonyl and reactive oxygen species (ROS), and up-regulated the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) in a dose-dependent manner. Moreover, calycosin can also inhibit the expression of 4-Hydroxy-2-nonenal (4-HNE).. These results suggest that calycosin has a neuroprotective effect against cerebral ischemia/reperfusion injury. The mechanism might be attributed to its antioxidant effects.

Topics: Aldehydes; Animals; Behavior, Animal; Brain; Catalase; Diagnostic Techniques, Neurological; Disease Models, Animal; Glutathione Peroxidase; Infarction, Middle Cerebral Artery; Isoflavones; Male; Malondialdehyde; Mitochondria; Movement; Neuroprotective Agents; Phytotherapy; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase