Preparation, characterization, and liver targeting evaluation of a novel sustained-release brucine self-assembled micelle mediated by glycyrrhetinic acid.

Background: Cancer is a serious threat to human life, health and social development. In recent years, nanomicelles, as an emerging drug carrier material, have gradually entered people's field of vision because of their advantages of improving bioavailability, maintaining drug levels, reducing systemic side effects and increasing drug accumulation at target sites. Methods: In this study, B-GPSG nano-micelles were prepared by film dispersion hydration method using brucine as model drug and glycyrrhetinic acid-polyethylene glycol-3-methylene glycol-dithiodipropionic acid-glycerol monostearate polymer as nano-carrier. The preparation process, characterization, drug release in vitro, pharmacokinetics and liver targeting were investigated. Results: The results showed that the range of particle size, polydispersion index and Zeta potential were 102.7 ± 1.09 nm, 0.201 ± 0.02 and -24.5 ± 0.19 mV respectively. The entrapment efficiency and drug loading were 83.79 ± 2.13% and 12.56 ± 0.09%, respectively. The drug release experiments in vitro and pharmacokinetic experiments showed that it had obvious sustained release effect. For pharmacokinetics study, it shows that both the B-GPSG solution group and the B-PSG solution group changed the metabolic kinetic parameters of brucine, but the B-GPSG solution group had a better effect. Compared with the B-PSG solution group, the drug was more prolonged in rats. The half-life in the body and the retention time in the body of B-GPSG are more helpful to improve the bioavailability of the drug and play a long-term effect. The tail vein injection results of mice indicate that B-GPSG can target and accumulate brucine in the liver without affecting other key organs. Cell uptake experiments and tissue distribution experiments in vivo show that glycyrrhetinic acid modified nano-micelles can increase the accumulation of brucine in hepatocytes, has a good liver targeting effect, and can be used as a new preparation for the treatment of liver cancer. Conclusion: The B-SPSG prepared in this experiment can provide a new treatment method and research idea for the treatment of liver cancer.

A novel nano-drug delivery system of glycyrrhetinic acid-mediated intracellular breakable brucine for enhanced anti-hepatitis B efficacy.

Background: Glycyrrhetinic acid-mediated brucine self-assembled nanomicelles enhance the anti-hepatitis B properties of brucine by improving its water solubility, short half-life, toxicity, and side effects. Brucine (B) is an indole alkaloid extracted from the seeds of Strychnos nux-vomica (Loganiaceae). Purpose: To assess the efficacy of the Brucine-Glycyrrhetnic acid-Polyethylene glycol-3,3'-dithiodipropionic acid-Glycerin monostearate (B-GPSG) in treating hepatitis B, its potential to protect against acute liver injury caused by d-galactosamine and its anti-hepatoma activities were studied. Research Design: The concentration of B-GPSG used in the in vivo and in vitro experiments was 0.63 mg/mL. The rats injected with d-GalN (450 mg/kg) were used as liver injury models. The rats were separated into normal, model, positive, positive control, B-PSG and B-GPSG groups. Hepatoma cells expressing HBV HepG2.2.15 were used for in vitro experiments. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, plate cloning, Hoechst staining and flow cytometry were conducted to explore the mechanism of B-GPSG against hepatitis B. Results: Compared with the model group, the liver coefficient of B-GPSG group decreased (4.59 ± 0.17 vs 5.88 ± 0.42), the content of MDA in rat liver homogenate decreased (12.54 ± 1.81 vs 23.05 ± 2.98), the activity of SOD increased, the activity of ALT and AST in rat serum decreased. In vitro, the IC50 values of B-GPSG group decreased. B-GPSG group effectively inhibited the proliferation and migration of HepG2.2.15 cells. Conclusions: The hepatoprotective effects of B-GPSG nanomicelles, which are attributed to their GA-mediated liver targeting and synergistic actions with brucine, suggest their therapeutic potential against hepatitis B. This development opens up new possibilities for the application of traditional Chinese medicine and nanomedicine in anti-hepatitis B.

Synthesis and Characterization of Graphene Oxide/Polyethylene Glycol/Folic Acid/Brucine Nanocomposites and Their Anticancer Activity on HepG2 Cells.

Background: Liver cancer is the sixth most prevalent form of cancer and the second major cause of cancer-associated mortalities worldwide. Cancer nanotechnology has the ability to fundamentally alter cancer treatment, diagnosis, and detection. Objective: In this study, we explained the development of graphene oxide/polyethylene glycol/folic acid/brucine nanocomposites (GO/PEG/Bru-FA NCs) and evaluated their antimicrobial and anticancer effect on the liver cancer HepG2 cells. Methodology: The GO/PEG/Bru-FA NCs were prepared using the co-precipitation technique and characterized using various techniques. The cytotoxicity of the GO/PEG/Bru-FA NCs was tested against both liver cancer HepG2 and non-malignant Vero cells using an MTT assay. The antimicrobial activity of the GO/PEG/Bru-FA NCs was tested against several pathogens using the well diffusion technique. The effects of GO/PEG/Bru-FA NCs on endogenous ROS accumulation, apoptosis, and MMP levels were examined using corresponding fluorescent staining assays, respectively. The apoptotic protein expressions, such as Bax, Bcl-2, and caspases, were studied using the corresponding kits. Results: The findings of various characterization assays revealed the development of GO/PEG/Bru-FA NCs with face-centered spherical morphology and an agglomerated appearance with an average size of 197.40 nm. The GO/PEG/Bru-FA NCs treatment remarkably inhibited the growth of the tested pathogens. The findings of the MTT assay evidenced that the GO/PEG/Bru-FA NCs effectively reduced the HepG2 cell growth while not showing toxicity to the Vero cells. The findings of the fluorescent assay proved that the GO/PEG/Bru-FA NCs increased ROS generation, reduced MMP levels, and promoted apoptosis in the HepG2 cells. The levels of Bax, caspase-9, and -3 were increased, and Bcl-2 was reduced in the GO/PEG/Bru-FA NCs-treated HepG2 cells. Conclusion: The results of this work demonstrate that GO/PEG/Bru-FA NCs suppress viability and induce apoptosis in HepG2 cells, indicating their potential as an anticancer candidate.

Brucine Inhibits Proliferation of Pancreatic Ductal Adenocarcinoma through PI3K/AKT Pathway-induced Mitochondrial Apoptosis.

INTRODUCTION: The purpose of this research was to settle the role of brucine in pancreatic ductal adenocarcinoma (PDAC) and the mechanisms involved. METHODS: The findings of this study suggest that brucine exerts inhibitory effects on cell growth, clonogenicity, and invasive potential of Panc02 and Mia Paca-2 cells. These effects may be linked to an increase in apoptotic-prone cell population. RESULTS: Gene sequencing data suggests that these effects are mediated through the induction of apoptosis. Experimental evidence further supports the notion that brucine reduces mitochondrial membrane potential and upregulates Bax expression while downregulating Bcl-2 expression. These effects are believed to be a result of brucine-mediated suppression of PI3K/Akt activity, which serves as a regulatory factor of mTOR, Bax, and Bcl-2. Suppression of PI3K activity enhances the tumor-suppressing effects of brucine. CONCLUSION: Overall, these findings suggest that brucine has therapeutic potential as a remedy option for PDAC.

Brucine D restrains colorectal cancer tumorigenesis and autophagy by downregulating circ_0068464.

Bruceine D (BD) from Brucea javanica (L) exerts an antitumor effect in several human cancers. At present, it has not been reported whether BD inhibits the malignancy of colorectal cancer (CRC) cells. Therefore, investigating the role and regulatory mechanisms of BD in CRC is the main thrust of this study. Effect of BD on CRC cell viability, proliferation, apoptosis, invasion, and autophagy was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell invasion, and western blotting assays. Expression changes of has_circ_0068464 (circ_0068464) were detected using real time quantitative polymerase chain reaction. The molecular mechanisms related to circ_0068464 were predicted through online prediction websites Starbase 2.0, circinteractome, and CircBank and validated using dual-luciferase reporter and RNA pull-down assays. The tumorigenic ability of BD and circ_0068464 on CRC was confirmed by xenograft experiments. The results showed that BD lessened CRC cell proliferation, invasion, autophagy, and prompted cell apoptosis. Circ_0068464 was overexpressed in CRC samples and cells. BD led to a significant reduction in circ_0068464 levels in cells of this carcinoma, but circ_0068464 overexpression partially rescued these effects urged by BD. Also, the combination of BD and circ_0068464 silencing decreased xenograft tumor growth compared to BD alone. Importantly, circ_0068464 could regulate ATG5 expression by functioning as a miR-520h molecular sponge. In conclusion, BD might suppress CRC growth by inhibiting the circ_0068464/miR-520h/ATG5 axis, providing a new perspective for the molecular pathogenesis of CRC and preliminarily indicating that BD may be a promising drug for CRC treatment.

Regulation of ethanol-mediated dopamine elevation by glycine receptors located on cholinergic interneurons in the nucleus accumbens.

Alcohol use disorder is one of the major psychiatric disorders worldwide, and there are many factors and effects contributing to the disorder, for example, the experience of ethanol reward. The rewarding and reinforcing properties of ethanol have been linked to activation of the mesolimbic dopamine system, an effect that appears to involve glycine receptors (GlyRs) in the nucleus accumbens. On which neuronal subtypes these receptors are located is, however, not known. The aim of this study was to explore the role of GlyRs on cholinergic interneurons (CIN) in sustaining extracellular dopamine levels and in ethanol-induced dopamine release. To this end, CIN were ablated by anti-choline acetyltransferase-saporin administered locally in the nucleus accumbens of male Wistar rats. Changes in dopamine levels induced by ablation, ethanol and/or a GlyR antagonist were monitored using in vivo microdialysis. The GlyRs antagonist strychnine depressed extracellular dopamine in a similar manner independent on local ablation, suggesting that GlyRs on CIN are not important for sustaining the extracellular dopamine tone. However, a low concentration of strychnine hampered ethanol-induced dopamine release in sham-treated animals, whilst no reduction was seen in ablated animals, suggesting that GlyRs located on CIN are involved in ethanol-induced dopamine release. Further, in ablated rats, ethanol-induced increases of the extracellular levels of the GlyR agonists glycine and taurine were attenuated. In conclusion, this study suggests that CIN are not important for GlyR-mediated regulation of basal dopamine output, but that CIN ablation blunts the ethanol-induced dopamine release, putatively by reducing the release of GlyR agonists.

Chemical constituents, pharmacological action, antitumor application, and toxicity of Strychnine Semen from Strychnons pierriana A.W.Hill.: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried and mature seeds of Strychnons pierriana A.W.Hill. have been called Strychnine Semen(S. Semen). It have been used in traditional Chinese medicine for nearly 400 years. In recent decades, scholars at home and abroad have widely used S. Semen in the treatment of tumor diseases, showing good anti-tumor effects. In this paper, the modern research achievements of S. Semen are reviewed, including traditional uses, phytochemistry, pharmacology, and toxicology. AIM OF THE STUDY: In recent years, the research on S. Semen has increased gradually, especially the research on its anti-tumor. This paper not only reviewed the traditional uses, chemical constituents and pharmacological activities of S. Semen, but also comprehensively listed the mechanisms of Strychnos in the treatment of different tumors, providing a review for further research and development of Strychnos resources. MATERIALS AND METHODS: A systematic review of the literature on Fuzi was performed using several resources, namely classic books on Chinese herbal medicine and various scientific databases, such as PubMed, the Web of Science, and the China Knowledge Resource Integrated databases. RESULTS: The main constituents of S. Semen include alkaloids, terpenoids, steroids, and their glycosides. Modern studies have proved that S. Semen has a wide range of pharmacological effects, including anti-inflammatory and analgesic, anti-thrombotic, myocardial cell protection, immune regulation, nerve excitation, and anti-tumor effects. Among them, the anti-tumor effect has been the focus of research in recent years. S. Semen have a certain therapeutic effect on many kinds of tumors, such as liver cancer, colon cancer, and stomach cancer in the digestive system, breast, cervical, and ovarian cancer in the reproductive system, myeloma and leukemia in the blood system, and those in the nervous system and the immune system. CONCLUSION: Strychnine has an inhibitory effect on a variety of tumors. However, modern studies of strychnine are incomplete, and more in-depth studies are needed on its stronger bioactive constituents and potential pharmacological effects. The antitumor effect of Strychnine is worth further exploration.

Promising anticonvulsant N-[(2,4-dichlorophenyl) methyl]-2-(2,4-dioxo-1H-quinazolin-3-yl) acetamide: dose-dependent study and evaluation of anticonvulsant action spectrum in vivo and in silico.

The anticonvulsant spectrum of the original promising anticonvulsant N-[(2,4-dichlorophenyl) methyl]-2-(2,4-dioxo-1H-quinazolin-3-yl) acetamide was studied. The compound had a pronounced anticonvulsant effect, significantly reducing the mortality of mice in models of seizures induced by pentylenetetrazole, picrotoxin, strychnine, and caffeine. In the thiosemicarbazideinduced seizure model, the test compound did not reduce mortality. The obtained results indicated that the mechanism of anticonvulsant action involved GABA-ergic (effective in models of pentylenetetrazole and picrotoxin-induced seizures), glycinergic (efficiency in the strychnine model of paroxysms), and adenosinergic (effectiveness in the model of caffeine induced seizures). Molecular docking of a promising anticonvulsant to anticonvulsant biotargets follow the mechanisms of chemo-induced seizures, namely GABA, glycine, and adenosine receptors type A2A, GABAAT, and BCAT enzymes. The conformity between in vivo and in silico studies results was revealed.

HPLC-MS/MS analysis of primary alkaloids in rat plasma after oral administration of "Nux vomica - Glycyrrhiza glabra decoction": A pharmacokinetic study.

ETHNOPHARMACOLOGICAL RELEVANCE: Decoction is the most common form of administering traditional Chinese medicine (TCM). During the preparation of decoction, the high temperature and complex chemical environment result in the formation of complex and multiple phases. The differences in drug components in different phases induce gastrointestinal absorption and physiological response. Nux vomica (Strychnos nux-vomica L) is a typical toxic TCM used in China, with remarkable pharmacological activity. In order to reduce its toxicity, nux vomica (NV) is often decocted with Glycyrrhiza glabra (GG) in clinic, and the detoxification mechanism has always been the focus of research interest. Most studies investigated the compatibility of NV-GG, but the in vivo behavior of individual constituents based on phase state has yet to be elucidated. AIM OF THE STUDY: To investigate the pharmacokinetic behavior of typical toxic components in different phase states of "NV-GG decoction" in rat plasma. MATERIALS AND METHODS: The sediment, suspension, colloid and true solution of "NV-GG decoction" was obtained via physical methods. The main components in different phase states were analyzed via reliable UFLC-Q-TOF-MS high-resolution mass spectrometry. A rapid and accurate HPLC-qqq-MS/MS method was established and validated for accurate determination of brucine and strychnine levels in plasma, followed by pharmacokinetic evaluation of different phase states of "NV-GG decoction" in rats. Kinetex F5 100A (50 mm × 3.0 mm, 2.6 µm) column was used for chromatographic separation. Aqueous solution containing acetonitrile and 0.1% formic acid was used as the mobile phase, followed by gradient elution at 0.4 mL/min. Mass spectra were detected by electrospray ionization (ESI) multiple reaction monitoring (MRM) in positive ion mode. RESULTS: Fifteen different alkaloids were detected in different phase states of "NV-GG decoction". Strychnine and brucine, which are toxic components with high content, were selected for quantitative analysis. The established UPLC-qqq-MS/MS method is accurate and reliable with a good linearity (R2 > 0.99) in the respective concentration range, satisfying the quantitative requirements. The pharmacokinetic parameters of different phase states of rats differed significantly after gavage. The deposition phase was the most prominent. The index components showed higher Cmax, AUC0 and Tmax, while the T1/2, MRT, V/F and CL/F were the smallest, with a relatively slow plasma clearance rate in rats. The true solution group showed the lowest Tmax and the fastest absorption. CONCLUSION: This method has been successfully utilized to study the pharmacokinetics of different phase states of "NV-GG decoction". Among the four phases, the deposition phase contributed to a large proportion of the in vivo kinetic behavior similar to that of sustained-release preparations, with slow absorption of toxic components and prolonged peak time. The pharmacokinetic parameters and plasma concentration-time curves of each phase can be used to study toxicity reduction of NV-GG and increase its biocompatibility.

Biosynthesis of strychnine.

Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.

Strychnine and its mono- and dimeric analogues: a pharmaco-chemical perspective.

Covering: up to November 2021Since its isolation in 1818, strychnine has attracted the attention of a plethora of chemists and pharmacologists who have established its structure, developed total syntheses, and examined its complex pharmacology. While numerous reviews on structure elucidation and total synthesis of strychnine are available, reports on structure-activity relationships (SARs) of this fascinating alkaloid are rare. In this review, we present and discuss structures, synthetic approaches, metabolic transformations, and the diverse pharmacological actions of strychnine and its mono- and dimeric analogues. Particular attention is given to its SARs at glycine receptors (GlyRs) in light of recently published high-resolution structures of strychnine-GlyR complexes. Other pharmacological actions of strychnine and its derivatives, such as their antagonistic properties at nicotinic acetylcholine receptors (nAChRs), allosteric modulation of muscarinic acetylcholine receptors as well as anti-cancer and anti-plasmodial effects are also critically reviewed, and possible future developments in the field are discussed.

Cross-Linked Poly-4-Acrylomorpholine: A Flexible and Reversibly Compressible Aligning Gel for Anisotropic NMR Analysis of Peptides and Small Molecules in Water.

Determination of the solution conformation of both small organic molecules and peptides in water remains a substantial hurdle in using NMR solution conformations to guide drug design due to the lack of easy to use alignment media. Herein we report the design of a flexible compressible chemically cross-linked poly-4-acrylomorpholine gel that can be used for the alignment of both small molecules and cyclic peptides in water. To test the new gel, residual dipolar couplings (RDCs) and J-coupling constants were used in the configurational analysis of strychnine hydrochloride, a molecule that has been studied extensively in organic solvents as well as a small cyclic peptide that is known to form an α-helix in water. The conformational ensembles for each molecule with the best fit to the data are reported. Identification of minor conformers in water that cannot easily be determined by conventional NOE measurements will facilitate the use of RDC experiments in structure-based drug design.

Quality by Design for Development, Optimization and Characterization of Brucine Ethosomal Gel for Skin Cancer Delivery.

Natural products have been extensively used for treating a wide variety of disorders. In recent times, Brucine (BRU) as one of the natural medications extracted from seeds of nux vomica, was investigated for its anticancer activity. As far as we know, this is the first study on BRU anticancer activity against skin cancer. Thus, the rational of this work was implemented to develop, optimize and characterize the anticancer activity of BRU loaded ethosomal gel. Basically, thin film hydration method was used to formulate BRU ethosomal preparations, by means of Central composite design (CCD), which were operated to construct (32) factorial design. Two independent variables were designated (phospholipid percentage and ethanol percentage) with three responses (vesicular size, encapsulation efficiency and flux). Based on the desirability function, one formula was selected and incorporated into HPMC gel base to develop BRU loaded ethosomal gel. The fabricated gel was assessed for all physical characterization. In-vitro release investigation, ex-vivo permeation and MTT calorimetric assay were performed. BRU loaded ethosomal gel exhibited acceptable values for the characterization parameters which stand proper for topical application. In-vitro release investigation was efficiently prolonged for 6 h. The flux from BRU loaded ethosome was enhanced screening optimum SSTF value. Finally, in-vitro cytotoxicity study proved that BRU loaded ethosomal gel significantly improved the anticancer activity of the drug against A375 human melanoma cell lines. Substantially, the investigation proposed a strong motivation for further study of the lately developed BRU loaded ethosomal gel as a prospective therapeutic strategy for melanoma treatment.

Brucine promotes apoptosis in cervical cancer cells (ME-180) via suppression of inflammation and cell proliferation by regulating PI3K/AKT/mTOR signaling pathway.

Brucine are the main constituents of Strychnos nux-vomica. Earlier reports have determined brucine shows anti-inflammatory, analgesic and excellent anti-tumor drug. Even though its anticervical cancer cells remains not clearly evaluated. So that, we hypothesized the anti-cervical cancer activity of brucine against the cervical (ME-180) cells. Brucine inhibited the inflammation, cell proliferation and promoted rate of apoptotic cell death ad reduced the mitochondrial potential, which is evidenced by respective (AO/EB, Rh-123, and PI) staining. Furthermore ELISA and real time PCR reaction determined that brucine were down regulated inflammatory (TNF-α, NF-kB, IL-6 & COX-2) cell proliferation (Cyclin D1) and apoptotic marker Bax, caspase-3, PI3K (phosphoinosital 3 kinase), AKT, mTOR (mammalian target of rapamycin) and over expression Bcl-2, associated death promoter. These findings were confirmed and finally suggested that brucine inhibited inflammation, cell proliferation and promoted the apoptosis through the down-regulation of PI3K/AKT/mTOR pathway. Taken together, these data were exhibited brucine as a good therapeutic agents for the prevention of anticancer cervical cancer drugs.

ATF3 contributes to brucine-triggered glioma cell ferroptosis via promotion of hydrogen peroxide and iron.

Ferroptotic cell death is characterized by iron-dependent lipid peroxidation that is initiated by ferrous iron and H2O2 via Fenton reaction, in which the role of activating transcription factor 3 (ATF3) remains elusive. Brucine is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica, which has shown potent antitumor activity against various tumors, including glioma. In this study, we showed that brucine inhibited glioma cell growth in vitro and in vivo, which was paralleled by nuclear translocation of ATF3, lipid peroxidation, and increases of iron and H2O2. Furthermore, brucine-induced lipid peroxidation was inhibited or exacerbated when intracellular iron was chelated by deferoxamine (500 µM) or improved by ferric ammonium citrate (500 µM). Suppression of lipid peroxidation with lipophilic antioxidants ferrostatin-1 (50 µM) or liproxstatin-1 (30 µM) rescued brucine-induced glioma cell death. Moreover, knockdown of ATF3 prevented brucine-induced accumulation of iron and H2O2 and glioma cell death. We revealed that brucine induced ATF3 upregulation and translocation into nuclei via activation of ER stress. ATF3 promoted brucine-induced H2O2 accumulation via upregulating NOX4 and SOD1 to generate H2O2 on one hand, and downregulating catalase and xCT to prevent H2O2 degradation on the other hand. H2O2 then contributed to brucine-triggered iron increase and transferrin receptor upregulation, as well as lipid peroxidation. This was further verified by treating glioma cells with exogenous H2O2 alone. Moreover, H2O2 reversely exacerbated brucine-induced ER stress. Taken together, ATF3 contributes to brucine-induced glioma cell ferroptosis via increasing H2O2 and iron.

Ethanolic Extract of the Red Algae Meristiella echinocarpa (Areschoug) Confers Neuroprotection in Mice / Extrato Etanólico da Alga Vermelha Meristiella echinocarpa (Areschoug) Confere Neuroproteção em Camundongos

Objectives: This study aimed to investigate the neuroprotective effects of the ethanolic extract obtained from red algae marine Meristiella echinocarpa (Areschougiaceae) ­ EEMe. Methods: EEMe was used in doses ranging from 10 to 40 mg/kg, administered intraperitoneally in mice. Behavioral tests were performed to assess locomotor activity (open field), anxiety (elevated plus maze), depression (tail suspension), and motor coordination (rota-rod). The anticonvulsant effect of the algae extract was evaluated in two models of seizures induced by strychnine and pentylenetetrazol. The level of oxidative stress was also evaluated in the following brain areas: the prefrontal cortex, hippocampus, and striatum. Statistical analysis was performed applying ANOVA followed by the Bonferroni test. Results: EEMe reduced significantly the number of crossing (36%) and rearing (54%) in the open field test and increased 1.3x the immobility time in the tail suspension test. In brain areas EEMe also reduced significantly malondialdehyde levels (striatum: 45%, hippocampus: 38%, prefrontal cortex: 37%) and nitrite levels (striatum: 72%, hippocampus: 79%, prefrontal cortex: 63%), and increased the reduced-glutathione levels (striatum: 72%, hippocampus: 73%, prefrontal cortex: 42%). In addition, the extract significantly prolonged the latency of seizures induced by strychnine (38%) or pentylenetetrazol (57%), and the latency of death induced by pentylenetetrazol (6.1x). Conclusion: EEMe exhibits antioxidant and anticonvulsant effects, probably involving GABAergic and glycinergic pathways.

Objetivos: este estudo teve como objetivo investigar os efeitos neuroprotetores do extrato etanólico da alga marinha vermelha Meristiella echinocarpa (Areschougiaceae) - EEMe. Métodos: EEMe foi utilizado em doses que variaram de 10 a 40 mg/kg, administrados via intraperitoneal em camundongos. Foram realizados testes comportamentais que avaliaram a atividade locomotora (campo aberto), a ansiedade (labirinto em cruz elevado), a depressão (suspensão em cauda) e a coordenação motora (rota-rod). O efeito anticonvulsivante do extrato da alga foi avaliado em dois modelos de convulsões por estricnina e pentilenotetrazol. Foi também realizada a avaliação do nível de estresse oxidativo nas seguintes áreas cerebrais: córtex pré-frontal, hipocampo e corpo estriado. A análise estatística foi realizada, aplicando a ANOVA seguida do teste de Bonferroni. Resultados: o EEMe reduziu, significativamente, o número de cruzamentos (36%) e o número de rearing (54%) no teste de campo aberto e aumentou, em 1,3x, o tempo de imobilidade no teste de suspensão pela cauda. Nas áreas cerebrais, o EEMe também reduziu, significativamente, os níveis de malondialdeído (estriado: 45%, hipocampo: 38%, córtex pré-frontal: 37%) e os níveis de nitrito (estriado: 72%, hipocampo: 79%, córtex pré-frontal: 63%) e aumentou a glutationa reduzida (estriado: 72%, hipocampo: 73%, córtex pré-frontal: 42%). Além disso, o EEMe prolongou, significativamente, a latência das convulsões induzidas por estricnina (38%) ou pentilenotetrazol (57%), e a latência da morte induzida por pentilenetetrazol (6,1x). Conclusão: o EEMe apresenta efeitos antioxidantes e anticonvulsivantes, provavelmente envolvendo as vias GABAérgica e glicinérgica.

Autophagy Impairment through Lysosome Dysfunction by Brucine Induces Immunogenic Cell Death (ICD).

Autophagy is an important tightly controlled cellular process that regulates cellular homeostasis and is involved in deciding cell fate such as cell survival and death. The role of autophagy in many intracellular signaling pathways explains its interaction with other different types of cell death, including apoptosis and immunogenic cell death (ICD). The reports showed the complex and intriguing relationship existing between autophagy and immune system signaling pathways. However, the role of autophagy in ICD remains to be clearly elucidated. In this study, we demonstrated that Brucine, a clinically-used small molecule in traditional Chinese medicine, elicited autophagy inhibition. Brucine also triggered cell stress and induced features of ICD, including calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) release in MDA-MB-231 and CT26 cancer cells. Brucine impaired autolysosomal degradation and exerted a feedback regulation of ERK1/2-mTOR-p70S6K signaling cascade. Brucine-elicited ICD was confirmed by the rejection of CT26 tumor cells, implanted in the mice after vaccination with Brucine-treated CT26 cells. The impaired autophagy contributed to Brucine-induced ICD, as knock-down of Atg5 significantly reduced Brucine-elicited CRT exposure and HMGB1 release. Our results revealed Brucine as a novel autophagy regulator, ICD inducer and hitherto undocumented role of autophagy in ICD. Thus, these results imply the importance of Brucine in cancer immunotherapy. Therefore, Brucine may be used as an ICD inducer and improve its application in cancer treatment with minimized toxicity.

Blocking glycine receptors reduces neuroinflammation and restores neurotransmission in cerebellum through ADAM17-TNFR1-NF-κß pathway.

BACKGROUND: Chronic hyperammonemia induces neuroinflammation in cerebellum, with glial activation and enhanced activation of the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway. Hyperammonemia also increases glycinergic neurotransmission. These alterations contribute to cognitive and motor impairment. Activation of glycine receptors is reduced by extracellular cGMP, which levels are reduced in cerebellum of hyperammonemic rats in vivo. We hypothesized that enhanced glycinergic neurotransmission in hyperammonemic rats (1) contributes to induce neuroinflammation and glutamatergic and GABAergic neurotransmission alterations; (2) is a consequence of the reduced extracellular cGMP levels. The aims were to assess, in cerebellum of hyperammonemic rats, (a) whether blocking glycine receptors with the antagonist strychnine reduces neuroinflammation; (b) the cellular localization of glycine receptor; (c) the effects of blocking glycine receptors on the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway and microglia activation; (d) whether adding extracellular cGMP reproduces the effects of strychnine. METHODS: We analyzed in freshly isolated cerebellar slices from control or hyperammonemic rats the effects of strychnine on activation of microglia and astrocytes, the content of TNFa and IL1b, the surface expression of ADAM17, TNFR1 and transporters, the phosphorylation levels of ERK, p38 and ADAM17. The cellular localization of glycine receptor was assessed by immunofluorescence. We analyzed the content of TNFa, IL1b, HMGB1, glutaminase, and the level of TNF-a mRNA and NF-κB in Purkinje neurons. Extracellular concentrations of glutamate and GABA were performed by in vivo microdialysis in cerebellum. We tested whether extracellular cGMP reproduces the effects of strychnine in ex vivo cerebellar slices. RESULTS: Glycine receptors are expressed mainly in Purkinje cells. In hyperammonemic rats, enhanced glycinergic neurotransmission leads to reduced membrane expression of ADAM17, resulting in increased surface expression and activation of TNFR1 and of the associated NF-kB pathway. This increases the expression in Purkinje neurons of TNFa, IL-1b, HMGB1, and glutaminase. Increased glutaminase activity leads to increased extracellular glutamate, which increases extracellular GABA. Increased extracellular glutamate and HMGB1 potentiate microglial activation. Blocking glycine receptors with strychnine or extracellular cGMP completely prevents the above pathway in hyperammonemic rats. CONCLUSIONS: Glycinergic neurotransmission modulates neuroinflammation. Enhanced glycinergic neurotransmission in hyperammonemia would be due to reduced extracellular cGMP. These results shed some light on possible new therapeutic target pathways for pathologies associated to neuroinflammation.

Effect of Baizhu (Rhizoma Atractylodis Macrocephalae) extract on intestinal absorption of brucine and strychnine in vitro and in situ.

OBJECTIVE: To investigate the antagonistic effect of the extract of Baizhu (Rhizoma Atractylodis Macrocephalae) (RAM) on the intestinal absorption of brucine and strychnine in Strychnos nux-vomica (NUX) and propose the mechanism of these effects. METHODS: The apparent permeability value (Papp) and absorption rate constant (Ka) were chosen as indices. The everted intestinal sac model and in situ single-pass intestinal perfusion model were used to study the effects of the RAM extract on the absorption of brucine and strychnine. To confirm the results, the brucine and strychnine concentrations in hepatic portal venous blood were determined. Western blotting was used to study P-glycoprotein (P-gp) expression in the Caco-2 cell line. RESULTS: Papp and Ka of brucine and strychnine were significantly increased in the presence of a P-gp inhibitor, but no significant increase was noted in the presence of a tight junction regulator. The RAM extract inhibited the absorption of brucine and strychnine and enhanced P-gp expression. CONCLUSION: The primary absorption mechanism for brucine and strychnine is passive transport, which is affected by P-gp.

Development, optimization, and evaluation of PEGylated brucine-loaded PLGA nanoparticles.

The application of nanotechnology to drug delivery systems for cancer therapy has progressively received great attention. The most heavily investigated approach is the development of nanoparticles (NPs) utilizing biodegradable and biocompatible polymers such as poly (lactic-co-glycolic acid) (PLGA). These NPs could be further improved by surface modification utilizing a hydrophilic biodegradable polymer such as polyethylene glycol (PEG) to achieve passive targeting. Modified NPs can deliver drugs such as brucine (BRU), which has shown its potential in cancer therapy. The objective of the current investigation was to develop and evaluate the passive targeting of long-circulating PLGA NPs loaded with BRU. NPs were characterized in terms of drug-excipient compatibility studies, including FTIR and DSC; physicochemical evaluations including particle size, zeta potential, morphological evaluation, entrapment efficiency and percentage yield; total serum protein adsorbed onto NP surfaces; and in vitro release of the loaded drug. Factorial design was employed to attain optimal PLGA-loaded NPs. Finally, the in vivo anti-tumor activity of BRU-loaded PLGA NPs was evaluated in tumor-bearing mice. The NPs obtained had smooth surfaces with particle sizes ranged from 94 ± 3.05 to 253 ± 8.7 nm with slightly positive surface charge ranged from 1.09 ± 0.15 to 3.71 ± 0.44 mV. Entrapment of BRU ranged between 37.5 ± 1.8% and 77 ± 1.3% with yields not less than 70.8%. Total protein adsorbed was less than 25.5 µg total protein/1 mg NP. In vitro drug release was less than 99.1% at 168 h. Finally, significant reductions in tumor growth rate and mortality rate were observed for PEG PLGA NP formulations compared to both BRU solution and naked NPs.