Advancing stroke therapy: A deep dive into early phase of ischemic stroke and recanalization.

Ischemic stroke, accounting for the majority of stroke events, significantly contributes to global morbidity and mortality. Vascular recanalization therapies, namely intravenous thrombolysis and mechanical thrombectomy, have emerged as critical interventions, yet their success hinges on timely application and patient-specific factors. This review focuses on the early phase pathophysiological mechanisms of ischemic stroke and the nuances of recanalization. It highlights the dual role of neutrophils in tissue damage and repair, and the critical involvement of the blood-brain barrier (BBB) in stroke outcomes. Special emphasis is placed on ischemia-reperfusion injury, characterized by oxidative stress, inflammation, and endothelial dysfunction, which paradoxically exacerbates cerebral damage post-revascularization. The review also explores the potential of targeting molecular pathways involved in BBB integrity and inflammation to enhance the efficacy of recanalization therapies. By synthesizing current research, this paper aims to provide insights into optimizing treatment protocols and developing adjuvant neuroprotective strategies, thereby advancing stroke therapy and improving patient outcomes.

An Unexpected Driving Force for Lipid Order Appears in Asymmetric Lipid Bilayers.

An ordered phase in one leaflet of an asymmetric bilayer can induce a precisely superimposed induced order domain in the apposed leaflet. Order is induced in such simple lipid compositions as dioleoylphosphatidylcholine/cholesterol (DOPC)/chol) which is expected to be a uniform and disordered lipid mixture. Dye partitioning can be used to label and identify coexisting liquid-disordered (Ld), liquid-ordered (Lo), or gel-ordered (Lß) molecules in a phase-separated leaflet. In the other leaflet of an asymmetric bilayer, dye partitioning also labels and identifies any induced order domains created by an Lo or gel phase domain in the apposed leaflet as well as the state of disorder of the lipid surrounding the induced ordered region. We explore a molecular level mechanism by which a disorder-prone uniform mixture of DOPC/chol = 0.8/0.2 would spontaneously separate into ordered regions coexisting with disordered regions. A redistribution of cholesterol seems to take place in the regions apposed to the ordered phase. The precision of the superposition of Lo or gel domains with their induced order domains implies a strong energy penalty that would be incurred if order/disorder interfaces were to form at the bilayer midplane. We conclude that the energy penalty for Lo/Ld or gel/Ld contact in the bilayer midplane is sufficient to drive disorderly DOPC/chol into an ordered state that reduces unfavorable order-disorder contacts at the bilayer midplane interface.

Psoralen induces liver injury and affects hepatic bile acids metabolism in female and male C57BL/6J mice.

Psoralen is a major component of Fructus Psoraleae that could induce liver injury. In this study, C57BL/6J mice were administered with psoralen at doses of 80 mg/kg for 3, 7 and 14 days. Blood and liver samples were collected for serum biochemistry and histopathology examinations, respectively. Psoralen led to liver injury with significantly increased liver weight and liver coefficient and up regulated serum ALT, AST and TG but down regulated serum TC and TP. The expression of bile acid-associated transporters and enzymes was detected by western blot, and the results showed that psoralen significantly down-regulates the expressions of CYP7A1, CYP27A1, BSEP and OSTα protein while up-regulates the expressions of HMGCR and FASN, resulting in the obstacles of bile acid efflux in the liver. The contents of 24 kinds of bile acids in the liver were measured by LC-MS/MS, and the results showed that psoralen led to the accumulation of unconjugated bile acids in the liver, such as ALCA and CA, which were more severe in male mice than female mice. It was indicated that psoralen may disrupt the balance of bile acid metabolism by inhibiting the expression of the efflux transporter, which then leads to liver damage.

Effects of Volatile Anesthetics versus Ketamine on Blood-Brain Barrier Permeability via Lipid-Mediated Alterations of Endothelial Cell Membranes.

The purpose of this study was to investigate the effects of the volatile anesthetic agents isoflurane and sevoflurane, at clinically relevant concentrations, on the fluidity of lipid membranes and permeability of the blood-brain barrier (BBB). We analyzed the in vitro effects of isoflurane or ketamine using erythrocyte ghosts (sodium fluorescein permeability), monolayers of brain microvascular endothelial cells ([13C]sucrose and fluorescein permeability), or liposomes (fluorescence anisotropy). Additionally, we determined the effects of 30-minute exposure of mice to isoflurane on the brain tight junction proteins. Finally, we investigated in vivo brain uptake of [13C]mannitol and [13C]sucrose after intravenous administration in mice under anesthesia with isoflurane, sevoflurane, or ketamine/xylazine in addition to the awake condition. Isoflurane at 1-mM and 5-mM concentrations increased fluorescein efflux from the erythrocyte ghosts in a concentration-dependent manner. Similarly, in endothelial cell monolayers exposed to 3% (v/v) isoflurane, permeability coefficients rose by about 25% for fluorescein and 40% for [13C]sucrose, whereas transendothelial resistance and cell viability remained unaffected. Although isoflurane caused a significant decrease in liposomes anisotropy values, ketamine/xylazine did not show any effects. Brain uptake clearance (apparent Kin) of the passive permeability markers in vivo in mice approximately doubled under isoflurane or sevoflurane anesthesia compared with either ketamine/xylazine anesthesia or the awake condition. In vivo exposure of mice to isoflurane did not change any of the brain tight junction proteins. Our data support membrane permeabilization rather than loosening of intercellular tight junctions as an underlying mechanism for increased permeability of the endothelial cell monolayers and the BBB in vivo. SIGNIFICANCE STATEMENT: The blood-brain barrier controls the entry of endogenous substances and xenobiotics from the circulation into the central nervous system. Volatile anesthetic agents like isoflurane alter the lipid structure of cell membranes, transiently facilitating the brain uptake of otherwise poorly permeable, hydrophilic small molecules. Clinical implications may arise when potentially neurotoxic drugs gain enhanced access to the central nervous system under inhalational anesthetics.

Accelerating therapeutic protein design with computational approaches toward the clinical stage.

Therapeutic protein, represented by antibodies, is of increasing interest in human medicine. However, clinical translation of therapeutic protein is still largely hindered by different aspects of developability, including affinity and selectivity, stability and aggregation prevention, solubility and viscosity reduction, and deimmunization. Conventional optimization of the developability with widely used methods, like display technologies and library screening approaches, is a time and cost-intensive endeavor, and the efficiency in finding suitable solutions is still not enough to meet clinical needs. In recent years, the accelerated advancement of computational methodologies has ushered in a transformative era in the field of therapeutic protein design. Owing to their remarkable capabilities in feature extraction and modeling, the integration of cutting-edge computational strategies with conventional techniques presents a promising avenue to accelerate the progression of therapeutic protein design and optimization toward clinical implementation. Here, we compared the differences between therapeutic protein and small molecules in developability and provided an overview of the computational approaches applicable to the design or optimization of therapeutic protein in several developability issues.

Harnessing Protein Corona for Biomimetic Nanomedicine Design.

Nanoparticles (NPs) are usually treated as multifunctional agents combining several therapeutical applications, like imaging and targeting delivery. However, clinical translation is still largely hindered by several factors, and the rapidly formed protein corona on the surface of NPs is one of them. The formation of protein corona is complicated and irreversible in the biological environment, and protein corona will redefine the "biological identity" of NPs, which will alter the following biological events and therapeutic efficacy. Current understanding of protein corona is still limited and incomplete, and in many cases, protein corona has adverse impacts on nanomedicine, for instance, losing targeting ability, activating the immune response, and rapid clearance. Due to the considerable role of protein corona in NPs' biological fate, harnessing protein corona to achieve some therapeutic effects through various methods like biomimetic approaches is now treated as a promising way to meet the current challenges in nanomedicine such as poor pharmacokinetic properties, off-target effect, and immunogenicity. This review will first introduce the current understanding of protein corona and summarize the investigation process and technologies. Second, the strategies of harnessing protein corona with biomimetic approaches for nanomedicine design are reviewed. Finally, we discuss the challenges and future outlooks of biomimetic approaches to tune protein corona in nanomedicine.

Astragalus saponins improves stroke by promoting the proliferation of neural stem cells through phosphorylation of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of major components of Buyang Huanwu decoction, Astragali Radix is broadly used for stroke treatment. Astragalus saponins (AST), the main active compound from Astragali Radix has the potentials for neuroprotection and improving spatial memory without clear pharmacological mechanism. AIM OF THE STUDY: The aim of this study was to investigate that pretreatment of AST is beneficial to protect against focal ischemic stroke in mouse model and its related underlying mechanism. MATERIALS AND METHODS: The neurological and motor function of MCAO mice were assessed by TTC staining and CatWalk gait analysis. The effect of AST on proliferation of NSCs was showed by the expression of Ki67 of MCAO mice and the number and size of primary neurospheres cultured from adult SVZ. The intersection of stroke-related targets, neurogenesis targets and drug-related targets were identified by the online website (https://www.omicstudio.cn/index). Then GO functional annotation and KEGG pathway enrichment analysis were performed. Candidate target Akt was confirmed to increase proliferation of cultured NSCs from adult SVZ by CCK8 assay and Western blot. RESULTS: We found that with the prolongation of administration time, AST improved neurological and motor function of MCAO mice, by promoting the proliferation of NSCs both in vivo and in vitro. Then, the primary network among drug, genes and biological pathway was established by using compound-target-disease & function-pathway analysis of astragalus membranaceus. PI3K/Akt which plays a key role in cell proliferation was among the top 10 most significant GO terms from above three aspects. Further analysis using cultured NSCs from adult SVZ confirmed that AST, astragaloside I (A1) and astragaloside III (A3) increased the proliferation of NSCs through targeting Akt. CONCLUSION: The present study elucidated that Astragalus saponins pretreatment could provide a protective effect on experimental stroke mainly by enhancing proliferation of NSCs through targeting Akt. The findings provided a basis for the development of novel strategies for the treatment of stroke.

Metal-Based Catalytic Drug Development for Next-Generation Cancer Therapy.

Considering the high increase in mortality caused by cancer in recent years, cancer drugs with novel mechanisms of anticancer action are urgently needed to overcome the drawbacks of platinum-based chemotherapeutics. Recently, in the area of metal-based cancer drug development research, the concept of catalytic cancer drugs has been introduced with organometallic RuII , OsII , RhIII and IrIII complexes. These complexes are reported as catalysts for many important biological transformations in cancer cells such as nicotinamide adenine dinucleotide (NAD(P)H) oxidation to NAD+ , reduction of NAD+ to NADH, and reduction of pyruvate to lactate. These unnatural intracellular transformations with catalytic and nontoxic doses of metal complexes are known to severely perturb several important biochemical pathways and could be the antecedent of next-generation catalytic cancer drug development. In this concept, we delineate the prospects of such recently reported organometallic RuII , OsII , RhIII and IrIII complexes as future catalytic cancer drugs. This new approach has the potential to deliver new cancer drug candidates.

Neoisoliquiritin exerts tumor suppressive effects on prostate cancer by repressing androgen receptor activity.

BACKGROUND: Prostate cancer (PCa) is a major cause of morbidity and mortality in men in both developed and developing countries. Androgens and the androgen receptor (AR) play predominant roles in the progression of PCa. Neoisoliquiritin (NEO) belongs to the class of licorice (Glycyrrhiza) flavonoids, which have a variety of biological activities including anti-depressant, anti-tumor-promoting, and anti-inflammation properties. Licorice root has cancer chemopreventive effects and has been given to PCa patients as an ingredient of PC-SPES, a commercially available combination of eight herbs. Therefore, we determined if NEO can suppress the proliferation of PCa cells. PURPOSE: We investigated whether and how NEO exerts its anti-neoplastic activity against PCa. METHODS: The Cell Counting Kit 8 and flow cytometry were used to evaluate the effects of NEO on the proliferation and cell cycle progression of AR-dependent (LNCaP) and AR-independent (PC3) PCa cells. RNA sequencing was employed to examine the genome-wide changes in responsiveness to NEO in LNCaP cells. Quantitative PCR, Western blotting, docking, chromatin immunoprecipitation, and dual-luciferase reporter assays were conducted to determine the mechanism of action of NEO and its potential cross-talk with AR. A LNCaP xenograft nude mouse model was used to determine the inhibitory effects of NEO on AR-dependent PCa tumors in vivo. RESULTS: NEO inhibited LNCaP cell proliferation in vitro by inducing G0/G1 phase cell cycle arrest. Conversely, NEO treatment had no effect on PC3 cells. Transcriptomic analysis indicated that AR signaling might be the key target of NEO in preventing PCa. NEO regulated AR-mediated cell growth suppression and AR-sensitized cell cycle arrest in LNCaP cells. NEO also blocked several key steps in the AR signaling pathway, including proposed targeting to the ligand binding pocket of AR by computer modeling, modulating AR-androgen response element DNA-binding activity, inhibiting the expression and transcriptional activity of AR, and suppressing downstream AR signaling. CONCLUSIONS: NEO negatively regulates AR expression and activity, thus supporting the tumor suppressive role for NEO in AR-dependent PCa.

Development of epimedin A complex drugs for treating the osteoporosis.

Osteoporosis is the most common disease involving bone degeneration. As the age of the population increases, the prevalence of the disease is expected to rise. However, current treatment methods do not provide a desirable solution for the restoration of the function of degenerated bones in patients with osteoporosis. This led to emergence of controlled delivery systems to increase drug bioavailability and efficacy specifically at the bone regeneration. In this study, an epimedin A (EA) complex drug system was prepared by solution blending method. In vitro cell-based experiments showed that the EA complex drug could significantly promote the differentiation and proliferation of osteoblasts and increase the alkaline phosphatase activity, calcium nodule formation, and the expression of osteogenesis-related genes and proteins. In vivo experiments further demonstrated that this novel drugs remarkably enhanced bone regeneration. These results suggest that EA may be used for the treatment of osteoporosis.

Effects of psoralen on hepatic bile acid transporters in rats.

Fructus Psoraleae (FP), widely used in traditional medicine, is increasingly reported to cause serious hepatotoxicity in recent years. However, the main toxic constituents responsible for hepatotoxicity and the underlying mechanisms are poorly understood. In the present study, psoralen, a main and quality-control constituent of FP, was intragastrically administered to Sprague-Dawley rats at a dose of 60 mg/kg for 1, 3 and 7 days. Blood and selected tissue samples were collected and analyzed for biochemistry and histopathology to evaluate hepatotoxicity. The results showed that psoralen could induce hepatotoxicity by enhanced liver-to-body weight ratio and alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total cholesterol after administration for 3 days. In addition, histopathological examinations also indicated the hepatotoxicity induced by psoralen. Furthermore, the mRNA and protein levels of hepatic bile acid transporters were significantly changed, in which MRP4, ABCG5 and ABCG8 were repressed, while the protein level of NTCP tended to increase in the rat liver. Taken together, psoralen caused liver injury possibly through affecting bile acid transporters, leading to the disorder of bile acid transport and accumulation in hepatocytes.

Isoflurane increases cell membrane fluidity significantly at clinical concentrations.

There is an on-going debate whether anesthetic drugs, such as isoflurane, can cause meaningful structural changes in cell membranes at clinical concentrations. In this study, the effects of isoflurane on lipid membrane fluidity were investigated using fluorescence anisotropy and spectroscopy. In order to get a complete picture, four very different membrane systems (erythrocyte ghosts, a 5-lipid mixture that mimics brain endothelial cell membrane, POPC/Chol, and pure DPPC) were selected for the study. In all four systems, we found that fluorescence anisotropies of DPH-PC, nile-red, and TMA-DPH decrease significantly at the isoflurane concentrations of 1 mM and 5 mM. Furthermore, the excimer/monomer (E/M) ratio of dipyrene-PC jumps immediately after the addition of isoflurane. We found that isoflurane is quite effective to loosen up highly ordered lipid domains with saturated lipids. Interestingly, 1 mM isoflurane causes a larger decrease of nile-red fluorescence anisotropy in erythrocyte ghosts than 52.2 mM of ethanol, which is three times the legal limit of blood alcohol level. Our results paint a consistent picture that isoflurane at clinical concentrations causes significant and immediate increase of membrane fluidity in a wide range of membrane systems.

Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats.

Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralen-treated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.

Sterol methyltransferase is required for optimal mitochondrial function and virulence in Leishmania major.

Limited knowledge on the exact functions of ergostane-based sterols has hampered the application of sterol synthesis inhibitors against trypanosomatid parasites. Sterol methyltransferase (SMT) is directly involved in the synthesis of parasite-specific C24-methylated sterols, including ergosterol and 5-dehydroepisterol. While pharmacological studies hint at its potential as a drug target against trypanosomatids, direct evidence for the cellular function and essentiality of SMT is lacking. Here, we characterized the SMT knockout mutants and their complemented strains in Leishmania major, the causative agent for cutaneous leishmaniasis. Deletion of SMT alleles led to a complete loss of C24-methylated sterols, which were replaced by cholestane-based sterols. SMT-null mutants were fully viable and replicative in culture but showed increased sensitivity to sphingolipid synthesis inhibition. They were not particularly vulnerable to heat, acidic pH, nitrosative or oxidative stress, yet exhibited high mitochondrial membrane potential and increased superoxide generation indicating altered physiology of the mitochondria. Despite possessing high levels of GPI-anchored glycoconjugates, SMT-null mutants showed significantly attenuated virulence in mice. In total, our study reveals that the biosynthesis of ergostane-based sterols is crucial for the proper function of mitochondria and the proliferation of Leishmania parasites in mammals.

Optimization of the Time Window of Interest in Ovariectomized Imprinting Control Region Mice for Antiosteoporosis Research.

This study was performed to determine the optimal window of time during which the properties of osteoporosis are obvious and to explore the best region of interest for microstructural evaluation in antiosteoporosis research in an ovariectomized mouse model by examining changes in micro-computed tomography parameters and serum indices. Ovariectomized mice and sham-operated mice were randomly divided into five groups. At the end of the 4th, 8th, 12th, 16th, and 20th weeks after ovariectomy, the microstructure of the proximal tibia and distal femur was scanned by micro-computed tomography and blood samples were collected to detect serum biochemical indicators including alkaline phosphatase, osteocalcin, N-terminal propeptide of type I procollagen (P1NP), and C-terminal telopeptide fragment of type I collagen (CTX1). The trabecular number and connectivity density decreased while the trabecular thickness and trabecular separation increased, indicating substantial changes in the trabecular microstructure of both the tibia and femur and significant changes in bone turnover after ovariectomy, as indicated by lower levels of serum alkaline phosphatase, osteocalcin, and P1NP and higher level of CTX1 in the ovariectomy than sham group. The proximal tibia from weeks 8 to 16 after ovariectomy was optimal for osteoporosis research in this model.

Reassessment of subacute MPTP-treated mice as animal model of Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model remains the most commonly used animal model of Parkinson's disease (PD). There are three MPTP-treatment schemes: acute, subacute and chronic. Considering the advantages of the period and similarity to PD, the subacute model was often chosen to assess the validity of new candidates, but the changes caused by the subacute MPTP treatment and the appropriate positive control for this model remain to be further confirmed. The aim of this study was: to estimate the value of the subacute MPTP mouse model in aspects of behavioral performance, biochemical changes and pathological abnormalities, and to find effective positive drugs. Male C57BL/6 mice were injected with MPTP (30 mg·kg-1·d-1, ip) for 5 consecutive days. Three days before MPTP injection, the mice were orally administered selegiline (3 mg·kg-1·d-1), pramipexole (3 mg·kg-1·d-1), or medopar (100 mg·kg-1·d-1) for 18 days. Behavioral performance was assessed in the open field test, pole test and rotarod test. Neurotransmitters in the striatum were detected using HPLC. Protein levels were measured by Western blot. Pathological characteristics were examined by immunohistochemistry. Ultrastructure changes were observed by electron microscopy. The subacute MPTP treatment did not induce evident motor defects despite severe injuries in the dopaminergic system. Additionally, MPTP significantly increased the α-synuclein levels and the number of astrocytes in the striatum, and destroyed the blood-brain barrier (BBB) in the substantia nigra pars compacta. Both selegiline and pramipexole were able to protect the mice against MPTP injuries. We conclude that the subacute MPTP mouse model does not show visible motor defects; it is not enough to evaluate the validity of a candidate just based on behavioral examination, much attention should also be paid to the alterations in neurotransmitters, astrocytes, α-synuclein and the BBB. In addition, selegiline or pramipexole is a better choice than medopar as an effective positive control for the subacute MPTP model.

Gramicidin Peptides Alter Global Lipid Compositions and Bilayer Thicknesses of Coexisting Liquid-Ordered and Liquid-Disordered Membrane Domains.

Effects of adding 1 mol % of gramicidin-A on the biochemical properties of coexisting liquid-ordered and liquid-disordered (Lo + Ld) membrane domains were investigated. Quaternary giant unilamellar vesicles (GUV) of di18:1PC(DOPC)/di18:0PC(DSPC)/cholesterol/gramicidin-A were prepared using our recently developed damp-film method. The phase boundary of Lo + Ld coexisting region was determined using video fluorescence microscopy. Through fitting Nile Red fluorescence emission spectra, the thermodynamic tie-lines in the Lo + Ld two-phase region were determined. We found that at 1 mol % (i.e., ∼7% of membrane area), gramicidin peptides alter the phase boundary and thermodynamic tie-lines. Gramicidin abolishes the coexisting phases at some lipid compositions but induces phase separation at others. Previous studies of gramicidin emphasize the local perturbation of bilayer thickness adjacent to the protein through the interaction of "hydrophobic mismatch". For the first time, it becomes clear that adding gramicidin produces significant long-range and global effects on the structure of membrane domains: it alters the overall lipid compositions and bilayer thicknesses of coexisting Lo and Ld domains. We also found that gramicidin partitions favorably into the Ld phase. Adding gramicidin decreases cholesterol in the Ld phase and increases cholesterol in the Lo phase. Those compositional changes broaden the bilayer thickness difference between Lo and Ld domains and facilitate preferential partition of gramicidin into thinner Ld domains. Our results demonstrate that membrane proteins play significant roles in determining lipid compositions and bilayer thicknesses of biomembrane domains.

Bibenzyl compound 20c protects against endoplasmic reticulum stress in tunicamycin-treated PC12 cells in vitro.

AIM: Accumulation of α-synuclein (α-syn) in the brain is a characteristic of Parkinson's disease (PD). In this study, we investigated whether treatment with tunicamycin, an endoplasmic reticulum (ER) stress inducer, led to the accumulation of α-syn in PC12 cells, and where α-syn protein was accumulated, and finally, whether bibenzyl compound 20c, a novel compound isolated from Gastrodia elata (Tian ma), could alleviate the accumulation of α-syn and ER stress activation in tunicamycin-treated PC12 cells. METHODS: PC12 cells were treated with tunicamycin for different time (6 h, 12 h, 24 h, 48 h). Cell viability was determined by a MTT assay. Subcellular fractions of ER and mitochondria were extracted with the Tissue Endoplasmic reticulum Isolation Kit. The levels of α-syn protein and ER-stress-associated downstream chaperones were detected using Western blots and immunofluorescence. RESULTS: Treatment of PC12 cells with tunicamycin (0.5-10 µg/mL) dose-dependently increased the accumulation of α-syn monomer (19 kDa) and oligomer (55 kDa), and decreased the cell viability. Accumulation of the two forms of α-syn was observed in both the ER and mitochondria with increasing treatment time. Co-treatment with 20c (10-5 mol/L) significantly increased the viability of tunicamycin-treated cells, reduced the level of α-syn protein and suppressed ER stress activation in the cells, evidenced by the reductions in phosphorylation of eIF2α and expression of spliced ATF6 and XBP1. CONCLUSION: Tunicamycin treatment caused accumulation of α-syn monomer and oligomer in PC12 cells. Bibenzyl compound 20c reduces the accumulation of α-syn and inhibits the activation of ER stress, which protected PC12 cells against the toxicity induced by tunicamycin.

20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway.

AIM: Our preliminary study shows that a bibenzyl compound isolated from Gastrodia elata, 2-[4-hydroxy-3-(4-hydroxybenzyl)benzyl]-4-(4-hydroxybenzyl)phenol (designated 20C), protects PC12 cells against H2O2-induced injury. In this study we investigated whether 20C exerted neuroprotective action in a cell model of Parkinson's disease. METHODS: A cell model of Parkinson's disease was established in PC12 cells by exposure to rotenone (4 µmol/L) for 48 h. Cell viability and apoptosis were assessed, and intracellular ROS level and the mitochondrial membrane potential (MMP) were detected. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome c, cleaved caspase-3, and oxidative stress-related proteins Nrf2, HO-1 and NQO1 were examined using Western blotting. The mRNA levels of HO-1 and NQO1 were determined with RT-PCR. The nuclear translocation of Nrf2 was observed with immunofluorescence staining. RESULTS: Treatment with rotenone significantly increased the number of apoptotic cells, accompanied by marked increases in the Bax/Bcl-2 ratio, cytochrome c release and caspase-3 activation. Rotenone also increased ROS accumulation, reduced MMP, and increased the nuclear translocation of Nrf2 as well as the mRNA and protein levels of the Nrf2 downstream target genes HO-1 and NQO1 in PC12 cells. Co-treatment with 20C (0.01-1 µmol/L) dose-dependently attenuated rotenone-induced apoptosis and oxidative stress in PC12 cells. Nrf2 knockdown by siRNA partially reversed the protective effects of 20C in rotenone-treated PC12 cells. CONCLUSION: The bibenzyl compound 20C protects PC12 cells from rotenone-induced apoptosis, at least in part, via activation of the Nrf2/ARE/HO-1 signaling pathway.

Claulansine F promoted the neuronal differentiation of neural stem and progenitor cells through Akt/GSK-3ß/ß-catenin pathway.

The persistence of neurogenesis raises the idea that neurons produced by the resident or transplanted neural stem cells could replace the neurons lost from brain injury or neurodegenerative disease. Therefore, compounds or methods for promoting neuronal differentiation become the focus of neurodegenerative disease therapy research. Claulansine F (Clau F), a newly discovered carbazole alkaloid, has been showed to induce neuritogenesis in PC12 cells. Herein, we studied the effect of Clau F on neuronal differentiation of neural stem/progenitor cells (NS/PCs). The current study demonstrated that Clau F initiated neuronal differentiation with a significant increase of TuJ1-positive cells and TuJ1 protein levels. We also found that Clau F promoted the maturity and sustainability of neurons by increasing MAP2-positive cells and MAP2 protein levels. At the same time, Clau F significantly inhibited the proliferation of NS/PCs. The underlying mechanism of Clau F was preliminary explored. Clau F treatment resulted in a profound increase of phosphorylation of Akt and GSK-3ß, which led to GSK-3ß inhibition and subsequently the nuclear accumulation of ß-catenin. Further, the interaction between ß-catenin and p300 in the nucleus was enhanced and the transcription of p300/ß-catenin responsive genes were increased significantly (c-jun, fra-1) by Clau F. Importantly, the positive effect of Clau F on neuronal differentiation was abolished by Akti-1/2, a specific inhibitor of Akt-1/2 kinase, which indicated the involvement of Akt/GSK-3ß in Clau F-mediated neuronal differentiation. In conclusion, these data suggested that Clau F promoted neuronal differentiation through Akt/GSK-3ß/ß-catenin signaling pathway in NS/PCs.