Calycosin alleviates cerebral ischemia/reperfusion injury by repressing autophagy via STAT3/FOXO3a signaling pathway.

BACKGROUND: 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. PURPOSE: 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. STUDY DESIGN: Integrate computational prediction and experimental validation based on network pharmacology. METHODS: 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-ß1 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. RESULTS: 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. CONCLUSION: Treatment with CA attenuated CIRI by reducing excessive autophagy via STAT3/FOXO3a signal pathway in rat and PC12 cells.

Combination of Radix Astragali and Safflower Promotes Angiogenesis in Rats with Ischemic Stroke via Silencing PTGS2.

Promotion of angiogenesis and restoration of the blood flow in the ischemic penumbra is an effective treatment for patients with ischemic stroke (IS). Radix astragali-safflower (AS), a classic herbal pair for accelerating blood circulation and dispersing blood stasis, has been used for thousands of years to treat patients with IS in China. Even so, the mechanism of the treatment of IS by AS is still undecipherable. In the current study, network pharmacology was firstly employed to unveil the mechanism of AS in treating IS, which showed that AS might promote angiogenesis associated with PTGS2 silence. Middle cerebral artery occlusion/reperfusion (MCAO/R) model rats were then used as the experimental animals to verify the prediction result. The experimental results revealed that treatment with AS improved the cerebral infarct volume, neurological damage, and cerebral histopathological damage; inhibited cell apoptosis; increased the contents of PDGF-BB, EPO, and TGF-ß1; and reduced the levels of PF4, Ang-2, and TIMP-1 in serum. Immunohistochemical staining demonstrated that the expression of PTGS2 was dramatically increased in the hippocampus and cerebral cortex of rats with MCAO/R, and this trend was reversed by the treatment of AS. Immunofluorescent staining expressed that AS reversed the down-regulation of VEGF and further promoted the expression of CD31, which indicated that AS promoted angiogenesis in MCAO/R rats. The abnormal protein or mRNA expression of PTGS2, PGI2, bFGF, TSP-1, and VEGF in the penumbra were transposed by AS or Celecoxib (an inhibitor of PTGS2). In conclusion, the protective mechanism of AS for IS promoted angiogenesis and was involved with PTGS2 silence.

An integrated strategy to evaluate active substances of Astragali Radix-Carthami Flos combination on the treatment of cerebral ischemia reperfusion injury based on TQSM polypharmacokinetics and pharmacodynamics.

As a classic herb pair, Astragali Radix-Carthami Flos (AR-CF) has revealed good biological activity in the treatment of cerebral ischemia/reperfusion injury (CI/RI), which remained to be further clarified together with the underlying efficacy related compounds for material basis. In this study, the nine formulations were obtained by L9 (34) orthogonal array design of four active fractions (saponin and flavonoid extracted from AR, safflower yellow and safflower red extracted from CF). The concentrations of eleven components and the levels of four biochemical indicators in rat plasma were continuously detected after intragastric administration of nine formulations, respectively. The collected data were analyzed by sigmoid-Emax function to understand the polypharmacokinetics and pharmacodynamics (PK-PD) behaviors of multi-components. Using the total quantum statistic moment polypharmacokinetics and its similarity method, the importance of four active fractions from AR-CF in relieving CI/RI was discussed and the Q-markers were screened. The results represented that a reliable and robust liquid chromatography tandem mass spectrometry method been successfully established to simultaneously determine the concentrations of eleven components in rat plasma. The AUC and MRT values of components from flavonoid fraction had the greatest contribution to AUCT and MRTT values. The transitivity in vivo of calycosin-7-O-ß-Dglucoside (CG), astragaloside IV (AIV) and hydroxysafflor yellow A (HYA) was closer to polypharmacokinetics behavior. All formulations up/down-regulated the levels of GSH-Px and ATP/ET and LDH to varying degrees, among which formulation 7 had the best regulating effect. By drawing the time-concentration-effect curve, clockwise hysteresis loops were presented in the time-concentration-effect relationships between eleven components and LDH/ET, while the relationship between eleven components and ATP/GSH-Px expressed as anticlockwise hysteresis loops. In conclusion, the combination based on the combination principle of formulation 7 produced the best alleviation effect on CI/RI, and flavonoid fraction might played key role in this process. The CG, AIV and HYA were identified as Q-markers. This research offered a novel strategy for exploring the active substances, and provided further understanding regarding the development of drugs for the treatment of cerebral ischemia-reperfusion injury.

LC-MS/MS combined with blood-brain dual channel microdialysis for simultaneous determination of active components of astragali radix-safflower combination and neurotransmitters in rats with cerebral ischemia reperfusion injury: Application in pharmacokinetic and pharmacodynamic study.

BACKGROUND: Astragali Radix-Safflower combination (ARSC) is widely utilized in clinic to treat cerebral ischemia/reperfusion injury (CI/RI). Whereas, there is no in-depth research of the pharmacokinetics (PK) and pharmacodynamics (PD) analysis of ARSC after intragastric administration in rats with CI/RI. PURPOSE: The purpose of this research is to investigate the PK characteristics of eight active ingredients (astragaloside IV, calycosin, calycosin-7-O-ß-glucoside, formononetin, ononin, hydroxysafflor yellow A, syringin and vernine) of ARSC, and the regulation of neurotransmitters disorders, revealing the pharmacodynamic substance basis and the mechanism of ARSC in treating CI/RI from the molecular level. METHODS: We established a new method which based on blood-brain dual channel microdialysis (MD) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to continuously gather, and determine the components of ARSC and neurotransmitters related to CI/RI in vivo. The collected data were analyzed by sigmoid-Emax function. The neurotransmitters primarily regulated in CI/RI rat were discussed by principal component analysis and the compound most associated with total pharmacodynamics was chosen by partial least squares regression. RESULTS: The validated LC-MS/MS method had specificity and selectivity to simultaneously analyze the concentration of eight active components of ARSC extract and five neurotransmitters of CI/RI rats. The recovery rates of brain MD probe and blood MD probe were stable within six hours. The MD probes recovery rates decreased with the increase of flow rates, but the solution concentration had little effect on the probes recovery rates. It was feasible to correct the recovery rates of probes in vivo by using reverse dialysis method. All eight active ingredients of ARSC could pass across the blood brain barrier after CI/RI. ARSC regulated the release of glutamate (Glu), γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and aspartic acid (Asp). Notably, astragaloside IV and hydroxysafflor yellow A might have better regulatory effect on neurotransmitters in comparison with other six measured components of ARSC, and Glu was the neurotransmitter mainly regulated in CI/RI rats. CONCLUSION: The ARSC was able to treat CI/RI through ameliorating neurotransmitters disorders. There was a hysteresis between the peaked drug concentration and maximum therapeutic effect of ARSC. The drug effective concentrations range of ASIV, calycosin, calycosin-7-O-ß-glucoside, syringin and vernine in blood microdialysate and calycosin, syringin, vernine in brain microdialysate were narrow, which need be paid attention in clinical use.

Optimization of smashing tissue and ultrasonic extraction of tanshinones and their neuroprotective effect on cerebral ischemia/reperfusion injury by inhibiting parthanatos.

A green smashing tissue and ultrasonic (STU) extraction method, which combines smashing tissue and ultrasonic-assisted extraction, was developed for the first time. The extraction of tanshinones from Salvia miltiorrhiza Bunge (SM) was taken as an example to discuss the practicability of this method. Taking the total yield of eight tanshinones as an evaluation index, response surface methodology (RSM) and artificial neural network (ANN) models were used to optimize the extraction parameters, and these two models were also compared by investigating the extract yield of tanshinones and the antioxidant activity of the obtained SM extract. The optimal STU conditions by ANN were as follows: an ethanol concentration of 73%, a liquid/solid ratio of 30 mL g-1, a smashing tissue time of 97 s and an ultrasonic time of 40 min. Under these optimal conditions, the yield of the eight components was 0.30% ± 0.12, which was greater than 0.28% ± 0.03 optimized by RSM. The IC50 values of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) of the obtained extract were 55.25 ± 3.72 µg mL-1 and 67.33 ± 2.62 µg mL-1, respectively, which were better than those of 75.49 ± 4.33 µg mL-1 and 112.10 ± 5.98 µg mL-1, respectively, optimized by RSM. Furthermore, the SM extract was found to exert neuroprotective effects by inhibiting parthanatos in middle cerebral artery occlusion/reperfusion (MCAO/R)-induced rats. The results supported the use of the SM extract, which was obtained by STU, as a potential product in the cosmetics, medicine, and food industries.

Simultaneous optimization of the extraction process of Yangyin Yiqi Huoxue prescription with natural deep eutectic solvents for optimal extraction yield and antioxidant activity: A comparative study of two models.

INTRODUCTION: Natural deep eutectic solvents (NaDESs) are green and effective solvents that are used to extract 3 flavonoids from Yangyin Yiqi Huoxue prescription, a traditional Chinese prescription. METHODS: A total of 6 types of NaDESs were systematically screened and evaluated for the total extraction yield of puerarin, calycosin, and formononetin by high-performance liquid chromatography. Then, a 4-factor-three-level experimental scheme designed by the Box-Benhnken Design was applied on the basis of a single experiment to determine the extraction yield and the antioxidant property. Finally, the extraction process was optimized through response surface methodology (RSM) and the genetic neural network (GNN), respectively. RESULTS: The use of betaine-lactic acid as an extractant displayed significant advantages in the screening process. The optimum extraction parameters provided by GNN were as follows: water content 25% (v/v), liquid to material ratio 190 mg/ml, extraction time 37 min, and extraction temperature 63 °C. Under this condition, the average experimental comprehensive evaluation values of the extraction yield and antioxidant properties were 3.12 mg/g and 86.27%, and the relative deviations to the predicted values were 0.30% and 1.44%, respectively. In addition, the experimental results of GNN were better than those of RSM (p < 0.01). CONCLUSIONS: We found the application of GNN to be effective and credible for bi-objective optimization of extraction yields and antioxidant activity in this study. Moreover, our results provide a reference and a theoretical basis for experimental and future industrial extraction for multi-objective situations.

Protective effects of polysaccharides on cerebral ischemia: A mini-review of the mechanisms.

Cerebral ischemia, a common cerebrovascular disease, is one of the great threats to human health. Nowadays, many drugs used in the treatment of cerebral ischemia such as clot busting drugs, antiplatelet drugs, and neuroprotective drugs have limits. It is urgent finding new effective treatments for the patients. Researches have confirmed that many kinds of polysaccharides from natural resources possess therapeutic effects on cerebral ischemia, but are still lack of a comprehensively understanding. In this paper, based on the pathophysiology of cerebral ischemic injury, we summarize the latest discoveries and advancements of 29 kinds of polysaccharides, focusing on their ameliorating effects on cerebral ischemia and the underlying mechanisms. Several mechanisms are involved, mainly including antioxidant activities, anti-inflammatory activities, regulating neuron apoptosis, as well as resisting nitrosative stress injury. Besides, polysaccharides show protective effects through certain signaling pathways including PI3K/Akt, MAPK, and NF-κB, PARP-1/AIF, JNK3/c-Jun/Fas-L, and Nrf2/HO-1 signaling pathways. The main goal of this mini-review is to emphasize the important roles of polysaccharides in attenuating cerebral ischemic injury through the elucidation of mechanisms.

A Study on Acetylglutamine Pharmacokinetics in Rat Blood and Brain Based on Liquid Chromatography-Tandem Mass Spectrometry and Microdialysis Technique.

Acetylglutamine (NAG) is the derivative of glutamine, which is the richest free amino acid in the human body. In this work, a novel reliable method of the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and microdialysis (MD) technique for the evaluation of NAG and its metabolites γ-aminobutyric acid (GABA) and glutamic acid (Glu) in rat blood and brain was proposed. A Zorbax SB-C18 column (2.1 × 100 mm, 3.5 µM) was applied to separate the analytes. The mobile phase was acetonitrile-water (70:30, v/v) containing 5 mM ammonium acetate and the flow rate was 0.3 ml/min. Based on the multiple reaction monitoring (MRM) mode of positive ion, the precursors of product ions chosen for NAG, Glu, GABA, and N-carbamyl-L-glutamic (NCG, IS) were (m/z) 189.1→130.0, 148.0→84.1, 104→87.1, and 191.0→130.1, respectively. All the validation data, including precision, accuracy, inter-day repeatability, matrix effect, and stability, were within the acceptable ranges according to the reference of Bioanalytical Method Validation Guidance for Industry (2018). Rats with microdialysis probes inserted into jugular vein and hippocampus were administered the low (75 mg/kg, NAG-L), medium (150 mg/kg, NAG-M), and high (300 mg/kg, NAG-H) doses of NAG and 10 ml/kg Guhong injection (GHI) by tail vein, respectively. In the blood test, the Cmax values of NAG-L group were markedly lower (P < 0.01) than those of NAG-M, NAG-H, and GHI groups, respectively. No differences were observed between NAG-M and GHI groups, while the Cmax values in GHI group were significantly upgraded compared with NAG-H group. There were notable differences in the Cmax values of NAG in brain dialysate after administration of NAG and GHI. The drug distribution coefficients of NAG, Glu, GABA in brain and blood at low, medium, high doses of NAG and GHI groups were 13.99, 27.43, 34.81, 31.37; 11.04, 59.07, 21.69, 2.69%; 212.88, 234.92, 157.59, and 102.65%, respectively. Our investigation demonstrates that NAG and its related metabolites in rat blood and brain can be simultaneously measured according to the above proposed method. Meanwhile, NAG has easy and dose-dependently access to the blood-brain barrier and exhibits a medium retention time in rat.