Filling the Gap in Understanding the Mechanism of GABAAR and Propofol Using Computational Approaches.

γ-Aminobutyric acid type-A receptors (GABAARs) play a critical role in neural transmission by mediating the inhibitory neural firing and are the target of many psychiatric drugs. Among them, propofol is one of the most widely used and important general anesthetics in clinics. Recent advances in structural biology revealed the structure of a human GABAAR in both open and closed states. Yet, the detailed mechanism of the receptor and propofol remains to be fully understood. Therefore, in this study, based on the previous successes in structural biology, a variety of computational techniques were applied to fill the gap between previous experimental studies. This study investigated the ion-conducting mechanism of GABAAR, predicted the possible binding mechanism of propofol, and revealed a new motion mechanism of transmembrane domain (TMD) helices. We hope that this study may contribute to future studies on ion-channel receptors, general anesthetics, and drug development.

Synthesis, antiepileptic effects, and structure-activity relationships of α-asarone derivatives: In vitro and in vivo neuroprotective effect of selected derivatives.

In the present study, we compared the antiepileptic effects of α-asarone derivatives to explore their structure-activity relationships using the PTZ-induced seizure model. Our research revealed that electron-donating methoxy groups in the 3,4,5-position on phenyl ring increased antiepileptic potency but the placement of other groups at different positions decreased activity. Besides, in allyl moiety, the optimal activity was reached with either an allyl or a 1-butenyl group in conjugation with the benzene ring. The compounds 5 and 19 exerted better neuroprotective effects against epilepsy in vitro (cell) and in vivo (mouse) models. This study provides valuable data for further exploration and application of these compounds as potential anti-seizure medicines.

The application of the MM/GBSA method in the binding pose prediction of FGFR inhibitors.

The success of a structure-based drug is highly dependent on a known binding pose of the protein-ligand system. However, this is not always available. In this study, we set out to explore the applicability of the popular and easy-to-use MD-based MM/GBSA method to determine the binding poses of known FGFR inhibitors. It was found that MM/GBSA combined with 100 ns of MD simulation significantly improved the success rate of docking methods from 30-40% to 70%. This work demonstrates a way that the MM/GBSA method can be more accurate than it is in ligand ranking, filling a gap in structure-based drug discovery when the binding pose is unknown.

Enhancing cytotoxicity of daunorubicin on drug-resistant leukaemia cells with microparticle-mediated drug delivery system.

Multidrug resistance is considered as a major obstacle for effective tumour chemotherapy. With the ability to deliver drugs into tumour cells, microparticles may act as a drug delivery vehicle to overcome drug resistance. In the present study, we developed an approach employing daunorubicin-loaded microparticles to surmount the drug resistance in leukaemia. The microparticles, derived from the drug-sensitive cells K562 and the drug-resistant cells K562/ADR, composed of cellular material, can effectively package drugs using intracellular and extracellular drug-loading method, respectively. The results demonstrated that the microparticles significantly improved the drug anti-tumour effect, which was influenced by the preparation methods and the source of donor cells. We further confirmed that the uptake of microparticles is mediated by an energy-driven endocytic process and mainly associated with clathrin-independent endocytosis and macropinocytosis. These results indicated that the microparticle could serve as a promising drug vehicle for the treatment of drug-resistant leukaemia.

Total Synthesis of Tanshinone I.

A novel total synthesis of tanshinone I (1) via the intermediate 3-hydroxy-8-methyl-1,4-phenanthrenedione (8) is described. The low overall yields and the use of expensive reagents in the synthesis process were minimized by the use of the Diels-Alder reaction to directly construct the 1,4-phenanthrenedione scaffold, providing tanshinone I (1) in only three steps.

[Preparation and antitumor effects of tanshinone â ¡A loaded albumin nanoparticles].

In this study, the tanshinone ⅡA loaded albumin nanoparticles were prepared by high pressure homogenization method. The formulation was optimized by central composite design-response surface method (CCD-RSM), with the particle size, encapsulation efficiency, and drug loading as indexes to investigate their in vitro anti-tumor effect. The results showed that the prepared nanoparticles had uniformly spherical morphology and uniform particle size distribution. The average particle size, encapsulation efficiency and drug loading of nanoparticles were about (175.7± 3.07) nm, 90.8%±1.47% and 5.52%±0.09%, respectively. Tanshinone ⅡA loaded albumin nanoparticles showed a more powerful antitumor effect than free tanshinone ⅡA for human promyelocytic leukemia NB4 cells. The preparation method of the drug-loaded albumin nanoparticles was simple and easy, and can significantly improve the solubility of tanshinone ⅡA, so it was helpful to extend its application in therapies against hematological malignancies.

[Preparation of CD123 mono-antibody modified tanshinone â ¡A loaded immunoliposome and its in vitro evaluation].

In the study, we developed a novel formulation, CD123 mono-antibody (mAb) modified tanshinone ⅡA loaded immunoliposome (CD123-TanⅡA-ILP) to achieve the targeted drug delivery for leukemia cells. Orthogonal test was used to optimize liposome preparation, and the TanⅡA-loaded PEGylated liposomes (TanⅡA-LP) of S100PC-Chol-(mPEG2000-DSPE)-TanⅡA at 19∶5∶1∶1 molar ratio were prepared by the thin film hydration-probe ultrasonic method. A post-insertion method was applied to prepare CD123-TanⅡA-ILP via thiolated mAb conjugated to the terminal of maleimide-PEG2000-DSPE. The cellular uptake assay was measured by flow cytometry, and the inhibitory effect of CD123-TanⅡA-ILP on NB4 cells proliferation was tested by using MTT assay. The results of cellular uptake assay showed that CD123-ILP could significantly increase the drug uptake of NB4 cells as compared with free drugs and LP. The IC50 values at 48 h incubation were 20.87, 11.71, 7.17 µmol•L⁻¹ respectively for TanⅡA，TanⅡA-LP and CD123-TanⅡA-ILP. CD123-ILP demonstrated a potential and promising targeted drug delivery strategy for acute myelogenous leukemia (AML) treatment.

Development of synthetic peptide-modified liposomes with LDL receptor targeting capacity and improved anticancer activity.

In this study, we report an active targeting liposomal formulation directed by a novel peptide (AA13) that specifically binds to the low density lipoprotein receptor (LDLR) overexpressed on acute myeloid leukemia (AML) cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of AA13-anchored liposomes on AML cells. AA13 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a postinsertion method. To study the effect of the peptide decoration and density on tumor cell targeting and internalization by AML cells (THP-1 and NB4), stealth liposomes bearing 3% (peptide/S100PC, molar ratio, LL) and 7% (peptide/S100PC, molar ratio, HL) AA13 were prepared, respectively. Higher uptake of LL (1.9- and 2.6-fold) and HL (2.3- and 3.6-fold) targeted liposomes occurred in THP-1 and NB4 cells, respectively, compared to untargeted liposomes. An LDLR inhibitor was used to confirm inhibition of the receptor-mediated cellular association of AA13 modified liposome in both cells. Daunorubicin (DNR) demonstrated a 2.2- and 3.5-fold higher cytotoxicity with the HL formulation and a 1.2- and 2.0-fold higher cytotoxicity with the LL formulation compared to the unmodified liposomal formulation in THP-1 and NB4 cells, respectively. Tumor drug accumulation of DNR-loaded HL was greater than that of the untargeted liposome in the biodistribution assay. The in vivo efficacy study in BALB/c nude mice bearing NB4 xenografts treated with DNR loaded HL also showed more tumor volume inhibition and a longer survival time compared to the untargeted formulation. In conclusion, the AA13-anchored liposomes demonstrated desirable potential as a promising vector for enhanced AML tumor drug targeting.

[Quality evaluation and stability investigation of asarone submicro emulsion injection].

The content of the asarone submicro emulsion injection was determind by HPLC method, and thereby a quality evaluation method was established based on indexes of pH value, particle size, peroxide value, methoxy aniline values, free fatty acid, lysophosphatidylcholine, visible foreign substances, insoluble particle, sterility, bacterial endotoxin and impurities, etc. The results showed that the injection exhibited uniform physical appearance and all the products were in milkwhite liquid. The content of the three batches products were respectively 102.9%, 100.8%, 97.70% of the labeled amount, with mean particle size of 210-250 nm, and other indexes all met with the standards. The reserved samples showed no obvious change in terms of detection indexes and indicated good stability after the accelerated stability test and long-term stability for 12 months. The quality evaluation method established in this study could be applied to quality control and stability investigation of asarone submicron emulsion injection, which laid a basis for further clinical research and application.

[Development of biphasic drug-loading lipid emulsion of Salvia miltiorrhiza and its quality evaluation].

The feasibility of simultaneously loading both liposoluble and water-soluble components of Salvia miltiorrhiza in emulsion was discussed, in order to provide new ideas in comprehensive application of effective components in S. miltiorrhiza in terms of technology of pharmaceutics. With tanshinone II (A) and salvianolic acid B as raw materials, soybean phospholipid and poloxamer 188 as emulsifiers, and glycerin as isoosmotic regulator, the central composite design-response surface method was employed to optimize the prescription. The coarse emulsion was prepared with the high-speed shearing method and then homogenized in the high pressure homogenizer. The biphasic drug-loading intravenous emulsion was prepared to investigate its pharmaceutical properties and stability. The prepared emulsion is orange-yellow, with the average diameter of 241 nm and Zeta potential of -35.3 mV. Specifically, the drug loading capacity of tanshinone II (A) and salvianolic acid B were 0.5 g x L(-1) and 1 g x L(-1), respectively, with a good stability among long-term retention samples. According to the results, the prepared emulsion could load liposoluble tanshinone II (A) and water-soluble salvianolic acid B simultaneously, which lays a pharmaceutical foundation for giving full play to the efficacy of S. miltiorrhiza.

Alpha-Asarone Ameliorates Neurological Dysfunction of Subarachnoid Hemorrhagic Rats in Both Acute and Recovery Phases via Regulating the CaMKII-Dependent Pathways.

Early brain injury (EBI) is the leading cause of poor prognosis for patients suffering from subarachnoid hemorrhage (SAH), particularly learning and memory deficits in the repair phase. A recent report has involved calcium/calmodulin-dependent protein kinase II (CaMKII) in the pathophysiological process underlying SAH-induced EBI. Alpha-asarone (ASA), a major compound isolated from the Chinese medicinal herb Acorus tatarinowii Schott, was proven to reduce secondary brain injury by decreasing CaMKII over-phosphorylation in rats' model of intracerebral hemorrhage in our previous report. However, the effect of ASA on SAH remains unclear, and the role of CaMKII in both acute and recovery stages of SAH needs further investigation. In this work, we first established a classic SAH rat model by endovascular perforation and intraperitoneally administrated different ASA doses (10, 20, and 40 mg/kg) 2 h after successful modeling. Then, the short- and long-term neurobehavioral performances were blindly evaluated to confirm ASA's efficacy against SAH. Subsequently, we explored ASA's therapeutic mechanism in both acute and recovery stages using histopathological examination, TUNEL staining, flow cytometry, Western-blot, double-immunofluorescence staining, and transmission electron microscopy (TEM) observation. Finally, KN93, a selective CaMKII inhibitor, was applied in oxyhemoglobin-damaged HT22 cells to explore the role of CaMKII in ASA's neuroprotective effect. The results demonstrated that ASA alleviated short- and long-term neurological dysfunction, reduced mortality and seizure rate within 24 h, and prolonged 14-day survival in SAH rats. Histopathological examination showed a reduction of neuronal damage and a restoration of the hippocampal structure after ASA treatment in both acute and recovery phases of SAH. In the acute stage, the Western-blot and flow cytometer analyses showed that ASA restored E/I balance, reduced calcium overload and CaMKII phosphorylation, and inhibited mitochondrion-involved apoptosis, thus preventing neuronal damage and apoptosis underlying EBI post-SAH. In the recovery stage, the TEM observation, double-immunofluorescence staining, and Western-blot analyses indicated that ASA increased the numbers of synapses and enhanced synaptic plasticity in the ipsilateral hippocampi, probably by promoting NR2B/CaMKII interaction and activating subsequent CREB/BDNF/TrkB signaling pathways. Furthermore, KN93 notably reversed ASA's neuroprotective effect on oxyhemoglobin-damaged HT22 cells, confirming CaMKII a potential target for ASA's efficacy against SAH. Our study confirmed for the first time that ASA ameliorated the SAH rats' neurobehavioral deterioration, possibly via modulating CaMKII-involved pathways. These findings provided a promising candidate for the clinical treatment of SAH and shed light on future drug discovery against SAH.

Formulation optimization of prostaglandin E1-loaded lipid emulsion: enhanced stability and reduced biodegradation.

The purpose of this study is to develop a new formulation for prostaglandin E1 (PGE1)-loaded lipid emulsion (Lipo-PGE1) with improved stability and reduced biodegradation. High-pressure homogenization was used to prepare the Lipo-PGE1, and high-performance liquid chromatography and accelerated test were used to evaluate its physicochemical stability. A tissue homogenate incubation test was firstly established and validated to assess its biodegradation. The factors influencing the stability of Lipo-PGE1, including oil phase, emulsifier, pH value, and drug concentration were systematically investigated. The optimized formulation consisting of poloxamer188 1.5% (w/v), egg lecithin 0.5% (w/v), soybean oil 10.0% (w/v), oleic acid 0.24% (w/v), and glycerol 2.2% (w/v), with the pH value at 4.0, was defined and characterized. When compared with the currently available commercial product of Lipo-PGE1, the degradation percentage of this optimized Lipo-PGE1 reduced by 47.1% after sterilization, the drug remaining percentage increased by 13.9% after storage at 4°C over 6 months. Also, a significant reduction in biodegradation of the optimized Lipo-PGE1 in comparison with the commercial Lipo-PGE1 was observed by a tissue homogenate incubation test. Overall, we provided a novel formulation for Lipo-PGE1 with a better physicochemical stability and a less biodegradation than the currently available commercial product of Lipo-PGE1, indicating its potential superiority in clinical application.

Neuroprotection of boropinol-B in cerebral ischemia-reperfusion injury by inhibiting inflammation and apoptosis.

Ischemic stroke is the leading cause of death and disability worldwide. The activation of gamma-aminobutyric acid A (GABAA) receptors can attenuate cerebral ischemia-reperfusion injury (CI/RI). Boropinol-B, originally isolated from Boronia pinnata Sm. (Rutaceae), has been proved the ability to activate GABAA receptors synergistically. However, whether boropinol-B has neuroprotection in CI/RI remains unknown. Here we reported the neuroprotective effect of boropinol-B on CI/RI and its underlying mechanism, focusing on inhibiting inflammation and apoptosis. The oxygen and glucose deprivation and reperfusion (OGD/R) cell model showed that boropinol-B could improve cell viability, mitigate cell injury, and inhibit apoptosis. In rats, the transient ischemic model was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. Our results indicated that boropinol-B improved neurological scores, reduced cerebral infarction and neuronal necrosis of rats 24 h after ischemia, and prolonged median survival time after continuous administration for 14 days. Furthermore, we found that boropinol-B inhibited the over-activation of microglia and astrocytes, reduced the release of pro-inflammatory factors, and down-regulated the expression of matrix metalloproteinases-3/9, thus alleviating cerebral edema and blood-brain barrier dysfunction. Also, it suppressed apoptosis by increasing Bcl-2 expression and decreasing the expression of Bax, Active Caspase-3, and Cytochrome C. In conclusion, boropinol-B demonstrated anti-inflammatory and anti-apoptotic properties that contributed to the neuroprotective effect against CI/RI, suggesting that it may be an up-and-coming drug candidate to treat ischemic stroke.

Co-Delivery of Daunorubicin and Homoharringtonine in Folic Acid Modified-Liposomes for Enhancing Therapeutic Effect on Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) remains a threatening disease due to severe complications, drug resistance, and high recurrence rates. Many drug combinations have demonstrated enhanced therapeutic effects in clinical practice. However, it requires complicated dosing regimens and is accompanied by increased toxicity. This study explored the combined effect of two therapeutic agents, daunorubicin (DNR) and homoharringtonine (HHT) in cell viability, apoptosis, and cell cycle in vitro and verified their synergistic effect. We encapsulated the two drugs into liposomes to construct a folic acid-modified co-delivery system (FA-DH-LP) to achieve an effective and safe therapeutic strategy. The FA-DH-LP was prepared by film hydration method. The resultant FA-DH-LP was homogeneously spherical and showed good blood compatibility with high encapsulation efficiency for DNR and HHT. The FA-DH-LP exhibited higher cellular uptake in HL60 and K562 cells and enhanced cytotoxicity than DNR/HHT co-delivery liposomes without folic acid modification (DH-LP) in vitro. In the HL60 subcutaneous xenotransplantation model, FA-DH-LP showed improved tumor targeting ability, anti-leukemia activity and safety profile superior to free combinational drugs and DH-LP after 18-day treatment. The results demonstrated that FA-DH-LP might present a promising delivery strategy to improve the efficacy of the two combinational chemotherapeutics while reducing toxicity.

Benzene, 1,2,4-Trimethoxy-5-(2-Methyl-1-Propen-1-yl) Attenuates D-galactose/AlCl3-induced Cognitive Impairment by Inhibiting Inflammation, Apoptosis, and improving Expression of Memory-Related Proteins.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by decreased learning ability and memory deficits. Our previous findings suggested that benzene, 1,2,4-trimethoxy-5-(2-methyl-1-propen-1-yl) (BTY) can ameliorate the dysfunction of GABAergic inhibitory neurons associated with neurological diseases. On this basis, we investigated the neuroprotective effect of BTY on AD and explored the underlying mechanism. This study included in vitro and in vivo experiments. BTY could maintain cell morphology, improve cell survival rate, reduce cell damage, and inhibit cell apoptosis in vitro experiments. Further, BTY has good pharmacological activity in vivo experiments, of which behavioral experiments showed that BTY could improve AD-like mice's learning and memory abilities. Besides, histopathological experiments indicated that BTY could maintain the morphology and function of neurons, reduce amyloid ß-protein 42 (Aß42) and phosphorylated tau (p-tau) accumulation, and decrease the levels of inflammatory cytokines. Finally, western blot experiments showed that BTY could inhibit the expression of apoptosis-related proteins and promote the expression of memory-related proteins. In conclusion, this study indicated that BTY may be a promising drug candidate for AD.

Sedative-hypnotic effects of Boropinol-B on mice via activation of GABAA receptors.

OBJECTIVES: Boropinol-B is a phenylpropanoid compound originally isolated from Boronia pinnata Sm. (Rutaceae). This study aimed to evaluate the sedative-hypnotic effects of Boropinol-B and explore the underlying mechanisms. METHODS: Pentobarbital sodium-induced sleep mouse model and caffeine-induced insomnia mouse model were used to investigate the sedative effects of Boropinol-B. Pharmacokinetics profiles of Boropinol-B in rats were evaluated by high-performance liquid chromatography. The effects of Boropinol-B on the γ-aminobutyric acid (GABA)ergic system were investigated using ELISA assay and patch-clamp technique. Immunohistochemistry and immunofluorescence were carried out to assess the effects of Boropinol-B on sleep-related brain nucleus. KEY FINDINGS: Boropinol-B showed significant sedative effects, including reduced sleep latency, increased sleep duration in pentobarbital sodium-treated mice and decreased locomotor activity in insomnia mice. Pharmacokinetics studies demonstrated that Boropinol-B had a rapid onset of action, a short half-life and no accumulation. It increased the GABA level in mice's brain, and promoted chloride ions influx mediated by the γ-aminobutyric acid type A (GABAA) receptors in neurons. Also, it increased the c-Fos positive ratio of GABAergic neurons in ventrolateral preoptic nucleus and decreased c-Fos expression in tuberomammillary nucleus. CONCLUSION: Boropinol-B showed significant sedative-hypnotic effects in mice by activating the GABAA receptors and stimulating the sleep-related brain nucleus.

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship.

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 µmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 µmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 µmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 µmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 µmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 µmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.

Benzene, 1,2,4-Trimethoxy-5-(2-Methyl-1-Propen-1-yl), a New Neuroprotective Agent, Treats Intracerebral Hemorrhage by Inhibiting Apoptosis, Inflammation, and Oxidative Stress.

The highest disability rates and mortality among neurodegenerative diseases were caused by intracerebral hemorrhage (ICH). We previously proved that Benzene, 1,2,4-trimethoxy-5-(2-methyl-1-propen-1-yl) (BTY) has an inhibitory effect on sodium ion channel and an activation effect on GABAA receptor, which were related to the brain injury. Based on this, we aimed to investigate BTY's neuroprotection on intracerebral hemorrhage and its underlying mechanism. In the in vivo study, a stereotactic injection of collagenase VII in Sprague Dawley rats (0.5 U) induced ICH and the BTY was intraperitoneally injected at 2 h after ICH. The neurological deficit scores, blood-brain barrier (BBB) permeability, and other indicators were assessed 24 h after ICH. The results showed that the BTY reduced brain edema and hematoma volume, improved neurological function and BBB permeability, and inhibited inflammatory factors and neuron apoptosis. The cell experiments proved that the BTY suppressed oxidative stress, cell apoptosis, intracellular calcium influx, and stabilized mitochondrial membrane potential by reducing glutamate's excitotoxicity. This study for the first time exhibited desirable neuroprotection of BTY, indicating it may be a promising neuroprotective agent for ICH therapy.

Alpha-asarone ameliorates neurological deterioration of intracerebral hemorrhagic rats by alleviating secondary brain injury via anti-excitotoxicity pathways.

BACKGROUND: Secondary brain injury (SBI) has been confirmed as a leading cause for the poor prognosis of patients suffering from intracerebral hemorrhage (ICH). SBI co-exists in ischemia and hemorrhagic stroke. Neuro-excitotoxicity is considered the initiating factor of ICH-induced SBI. Our previous research has revealed alpha-asarone (ASA)'s efficacy against cerebral ischemia-reperfusion stroke by mitigating neuro-excitotoxicity. It is not yet known if ASA exhibit neuroprotection against ICH. PURPOSE: This work aimed to investigate ASA's therapeutic effects and potential mechanisms of action against ICH in a classic rat model induced by collagenase Ⅶ injection. METHODS: An in vivo ICH model of Sprague-Dawley rats was established by collagenase Ⅶ injection. We administrated different ASA doses (10, 20, or 40 mg/kg, i.p.) at 2 h post-ICH. Then, rats' short- and long-term neurobehavioral function, bodyweight change, and learning and memory ability were blindly evaluated. Histological, Nissl, and flow cytometry were applied to assess the neuronal damage post-ICH. The wet/dry method and Evans blue extravasation estimated brain edema and blood-brain barrier function. Pathway-related proteins were investigated by immunofluorescence staining, enzyme-linked immunosorbent assay, and Western-blot analysis. RESULTS: The results demonstrated that ASA ameliorated neurological deterioration, bodyweight loss, and learning and memory ability of ICH rats. Histological, Nissl, and flow cytometry analyses showed that ASA reduced neuronal damage and apoptosis post-ICH. Besides, ASA probably mitigated brain edema and blood-brain barrier dysfunction via inhibiting astrocyte activation and consequent pro-inflammatory response. The mechanism investigation attributed ASA's efficacy to the following aspects: 1) promoting sodium ion excretion, thus blocking excitatory signal transduction along the axon; 2) preventing glutamate-involved pathways, i.e., decrease of N-methyl-d-aspartic acid receptor subunit 2B, increase of glutamate transporter-1, and alleviation of calcium-related cascades, mitochondrion-associated apoptosis, and neuronal autophagy; 3) enhancing the expression of GABAARs, thus abating neuronal excitotoxicity. CONCLUSION: Our study first confirmed the effect of ASA on ameliorating the neurobehavioral deterioration of ICH rats, possibly via alleviation of glutamate-involved neuro-excitotoxicity, i.e., calcium cascades, mitochondrion-involved apoptosis, neuronal autophagy, and astrocyte-related inflammation. These findings not only provided a promising drug candidate for clinical treatment of ICH but also shed light on the future drug discovery against ICH.

Neuroprotective Effect of Alpha-asarone on the Rats Model of Cerebral Ischemia-Reperfusion Stroke via Ameliorating Glial Activation and Autophagy.

Alpha-asarone, a major active component isolated from Acorus gramineus, can affect brain functions and behaviors by multiple mechanisms. However, the effect of alpha-asarone on cerebral ischemia-reperfusion (CIR) stroke has not been reported. The present study aimed to investigate the neuroprotective effect of alpha-asarone and the involved mechanisms against CIR stroke. Rats were subjected to middle cerebral occlusion (MCAO) for 2 h. Then the drug or drug-free vehicle was intravenously injected to corresponding groups. After reperfusion for 24 h, the infarct volume was evaluated by Triphenyl Tetrazolium Chloride (TTC) staining. The neurofunctional recovery and post-stroke epilepsy were evaluated. Nissl and Hematoxylin-Eosin (H&E) staining were used for histological observation. We investigated the protective mechanism of alpha-asarone against the stroke. The results showed that alpha-asarone exhibited a desirable neuroprotective effect, manifested as reducing infarct volume and post-stroke epilepsy and improving neurological function. Histological and flow cytometry analysis revealed that alpha-asarone treatment alleviated cell injury and apoptosis in vivo and in vitro. Furthermore, alpha-asarone decreased GFAP, Iba-1, and LC3II/LC3I expression and increased the expression of p62. These results suggested that alpha-asarone attenuated the CIR stroke injury via ameliorating glial activation and autophagy.