Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Neuron-targeted liposomal coenzyme Q10 attenuates neuronal ferroptosis after subarachnoid hemorrhage by activating the ferroptosis suppressor protein 1/coenzyme Q10 system.

Subarachnoid hemorrhage (SAH) is primarily attributed to the rupture of intracranial aneurysms and is associated with a high incidence of disability and mortality. SAH disrupts the blood‒brain barrier, leading to the release of iron ions from blood within the subarachnoid space, subsequently inducing neuronal ferroptosis. A recently discovered protein, known as ferroptosis suppressor protein 1 (FSP1), exerts anti-ferroptotic effects by facilitating the conversion of oxidative coenzyme Q 10 (CoQ10) to its reduced form, which effectively scavenges reactive oxygen radicals and mitigates iron-induced ferroptosis. In our investigation, we observed an increase in FSP1 levels following SAH. However, the depletion of CoQ10 caused by SAH hindered the biological function of FSP1. Therefore, we created neuron-targeted liposomal CoQ10 by introducing the neuron-targeting peptide Tet1 onto the surface of liposomal CoQ10. Our objective was to determine whether this formulation could activate the FSP1 system and subsequently inhibit neuronal ferroptosis. Our findings revealed that neuron-targeted liposomal CoQ10 effectively localized to neurons at the lesion site after SAH. Furthermore, it facilitated the upregulation of FSP1, reduced the accumulation of malondialdehyde and reactive oxygen species, inhibited neuronal ferroptosis, and exerted neuroprotective effects both in vitro and in vivo. Our study provides evidence that supplementation with CoQ10 can effectively activate the FSP1 system. Additionally, we developed a neuron-targeted liposomal CoQ10 formulation that can be selectively delivered to neurons at the site of SAH. This innovative approach represents a promising therapeutic strategy for neuronal ferroptosis following SAH. STATEMENT OF SIGNIFICANCE: Subarachnoid hemorrhage (SAH) is primarily attributed to the rupture of intracranial aneurysms and is associated with a high incidence of disability and mortality. Ferroptosis suppressor protein 1 (FSP1), exerts anti-ferroptotic effects by facilitating the conversion of oxidative coenzyme Q 10 (CoQ10) to its reduced form, which effectively scavenges reactive oxygen radicals and mitigates iron-induced ferroptosis. In our investigation, we observed an increase in FSP1 levels following SAH. However, the depletion of CoQ10 caused by SAH hindered the biological function of FSP1. Therefore, we created neuron-targeted liposomal CoQ10. We find that it effectively localized to neurons at the lesion site after SAH and activated the FSP1/CoQ10 system. This innovative approach represents a promising therapeutic strategy for neuronal ferroptosis following SAH and other central nervous system diseases characterized by disruption of the blood-brain barrier.

Ginsenoside Rg1 alleviates chronic inflammation-induced neuronal ferroptosis and cognitive impairments via regulation of AIM2 - Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng is a valuable herb in traditional Chinese medicine. Modern research has shown that it has various benefits, including tonifying vital energy, nourishing and strengthening the body, calming the mind, improving cognitive function, regulating fluids, and returning blood pressure, etc. Rg1 is a primary active component of ginseng. It protects hippocampal neurons, improves synaptic plasticity, enhances cognitive function, and boosts immunity. Furthermore, it exhibits anti-aging and anti-fatigue properties and holds great potential for preventing and managing neurodegenerative diseases (NDDs). AIM OF THE STUDY: The objective of this study was to examine the role of Rg1 in treating chronic inflammatory NDDs and its molecular mechanisms. MATERIALS AND METHODS: In vivo, we investigated the protective effects of Rg1 against chronic neuroinflammation and cognitive deficits in mice induced by 200 µg/kg lipopolysaccharide (LPS) for 21 days using behavioral tests, pathological sections, Western blot, qPCR and immunostaining. In vitro experiments involved the stimulation of HT22 cells with 10 µg/ml of LPS, verification of the therapeutic effect of Rg1, and elucidation of its potential mechanism of action using H2DCFDA staining, BODIPY™ 581/591 C11, JC-1 staining, Western blot, and immunostaining. RESULTS: Firstly, it was found that Rg1 significantly improved chronic LPS-induced behavioral and cognitive dysfunction in mice. Further studies showed that Rg1 significantly attenuated LPS-induced neuronal damage by reducing levels of IL-6, IL-1ß and ROS, and inhibiting AIM2 inflammasome. Furthermore, chronic LPS exposure induced the onset of neuronal ferroptosis by increasing the lipid peroxidation product MDA and regulating the ferroptosis-associated proteins Gpx4, xCT, FSP1, DMT1 and TfR, which were reversed by Rg1 treatment. Additionally, Rg1 was found to activate Nrf2 and its downstream antioxidant enzymes, such as HO1 and NQO1, both in vivo and in vitro. In vitro studies also showed that the Nrf2 inhibitor ML385 could inhibit the anti-inflammatory, antioxidant, and anti-ferroptosis effects of Rg1. CONCLUSIONS: This study demonstrated that Rg1 administration ameliorated chronic LPS-induced cognitive deficits and neuronal ferroptosis in mice by inhibiting neuroinflammation and oxidative stress. The underlying mechanisms may be related to the inhibition of AIM2 inflammasome and activation of Nrf2 signaling. These findings provide valuable insights into the treatment of chronic neuroinflammation and associated NDDs.

Leonurus japonicus Houtt. modulates neuronal apoptosis in intracerebral hemorrhage: Insights from network pharmacology and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houtt. (Labiatae), commonly known as Chinese motherwort, is a herbaceous flowering plant that is native to Asia. It is widely acknowledged in traditional medicine for its diuretic, hypoglycemic, antiepileptic properties and neuroprotection. Currently, Leonurus japonicus (Leo) is included in the Pharmacopoeia of the People's Republic of China. Traditional Chinese Medicine (TCM) recognizes Leo for its myriad pharmacological attributes, but its efficacy against ICH-induced neuronal apoptosis is unclear. AIMS OF THE STUDY: This study aimed to identify the potential targets and regulatory mechanisms of Leo in alleviating neuronal apoptosis after ICH. MATERIALS AND METHODS: The study employed network pharmacology, UPLC-Q-TOF-MS technique, molecular docking, pharmacodynamic studies, western blotting, and immunofluorescence techniques to explore its potential mechanisms. RESULTS: Leo was found to assist hematoma absorption, thus improving the neurological outlook in an ICH mouse model. Importantly, molecular docking highlighted JAK as Leo's potential therapeutic target in ICH scenarios. Further experimental evidence demonstrated that Leo adjusts JAK1 and STAT1 phosphorylation, curbing Bax while augmenting Bcl-2 expression. CONCLUSION: Leo showcases potential in mitigating neuronal apoptosis post-ICH, predominantly via the JAK/STAT mechanism.

Tetramethylpyrazine-loaded electroconductive hydrogels promote tissue repair after spinal cord injury by protecting the blood-spinal cord barrier and neurons.

Spinal cord injury (SCI) usually induces profound microvascular dysfunction. It disrupts the integrity of the blood-spinal cord barrier (BSCB), which could trigger a cascade of secondary pathological events that manifest as neuronal apoptosis and axonal demyelination. These events can further lead to irreversible neurological impairments. Thus, reducing the permeability of the BSCB and maintaining its substructural integrity are essential to promote neuronal survival following SCI. Tetramethylpyrazine (TMP) has emerged as a potential protective agent for treating the BSCB after SCI. However, its therapeutic potential is hindered by challenges in the administration route and suboptimal bioavailability, leading to attenuated clinical outcomes. To address this challenge, traditional Chinese medicine, TMP, was used in this study to construct a drug-loaded electroconductive hydrogel for synergistic treatment of SCI. A conductive hydrogel combined with TMP demonstrates good electrical and mechanical properties as well as superior biocompatibility. Furthermore, it also facilitates sustained local release of TMP at the implantation site. Furthermore, the TMP-loaded electroconductive hydrogel could suppress oxidative stress responses, thereby diminishing endothelial cell apoptosis and the breakdown of tight junction proteins. This concerted action repairs BSCB integrity. Concurrently, myelin-associated axons and neurons are protected against death, which meaningfully restore neurological functions post spinal cord injury. Hence, these findings indicate that combining the electroconductive hydrogel with TMP presents a promising avenue for potentiating drug efficacy and synergistic repair following SCI.

Celastrol inhibits oligodendrocyte and neuron ferroptosis to promote spinal cord injury recovery.

BACKGROUND: Spinal cord injury (SCI) is a traumatic injury to the central nervous system and can cause lipid peroxidation in the spinal cord. Ferroptosis, an iron-dependent programmed cell death, plays a key role in the pathophysiology progression of SCI. Celastrol, a widely used antioxidant drug, has potential therapeutic value for nervous system. PURPOSE: To investigate whether celastrol can be a reliable candidate for ferroptosis inhibitor and the molecular mechanism of celastrol in repairing SCI by inhibiting ferroptosis. METHODS: First, a rat SCI model was constructed, and the recovery of motor function was observed after treatment with celastrol. The regulatory effect of celastrol on ferroptosis pathway Nrf2-xCT-GPX4 was detected by Western blot and immunofluorescence. Finally, the ferroptosis model of neurons and oligodendrocytes was constructed in vitro to further verify the mechanism of inhibiting ferroptosis by celastrol. RESULTS: Our results demonstrated that celastrol promoted the recovery of spinal cord tissue and motor function in SCI rats. Further in vitro and in vivo studies showed that celastrol significantly inhibited ferroptosis in neurons and oligodendrocytes and reduced the accumulation of ROS. Finally, we found that celastrol could inhibit ferroptosis by up-regulating the Nrf2-xCT-GPX4 axis to repair SCI. CONCLUSION: Celastrol effectively inhibits ferroptosis after SCI by upregulating the Nrf2-xCT-GPX4 axis, reducing the production of lipid ROS, protecting the survival of neurons and oligodendrocytes, and improving the functional recovery.

Total paeony glycoside relieves neuroinflammation to exert antidepressant effect via the interplay between NLRP3 inflammasome, pyroptosis and autophagy.

BACKGROUND: Depression is a common mental illness characterised by abnormal and depressed emotions. Total paeony glycoside (TPG) is a naturally active saponin extracted from the traditional Chinese medicine Radix Paeoniae rubra. However, the antidepressant and neuroinflammatory effects of TPG have not been thoroughly studied. PURPOSE: To study the therapeutic potential of TGP in depression caused by neuronal injury and neuroinflammation and to explore the mechanism of TGP and the relationship between the NLRP3 inflammasome, pyroptosis, and autophagy. STUDY DESIGN: A chronic unpredictable mild stress (CUMS)-induced depression model and a cell model of corticosterone (CORT)-induced hippocampal neuron injury were established to evaluate the therapeutic effects of TPG. METHODS: The composition of TPG was analysed using high-performance liquid chromatography and mass spectrometry. The effects of TPG and fluoxetine on depression-like behaviour, neuronal injury, neuroinflammation, pyroptosis, and mitochondrial autophagy in the mice models were evaluated. RESULTS: TGP alleviated depression-like behaviours in mice and inhibited hippocampal neuronal apoptosis. The secretion of inflammatory cytokines was significantly reduced in CORT-induced hippocampal neuron cells and in the serum of a mouse model of CUMS-induced depression. In addition, TGP treatment reduced the levels of NLRP3 family pyrin structural domains, including NLRP3, pro-caspase-1, caspase-1, and IL-1ß, and the pyroptosis related proteins such as GSDMD-N. Importantly, TPG attenuated mitochondrial dysfunction, promoted the clearance of damaged mitochondria, and the activation of mitochondrial autophagy, which reduced ROS accumulation and NLRP3 inflammasome activation. An in-depth study observed that the regulatory effect of TPG on autophagy was attenuated by the autophagy inhibitor 3-methyladenine (3-MA) in vitro and in vivo. However, administration of the caspase-1 inhibitor Belnacasan (VX-765) successfully inhibited pyroptosis and showed a synergistic therapeutic effect with TPG. CONCLUSION: These results indicate that TPG can repair neuronal damage by activating autophagy, restoring mitochondrial function, and reducing inflammation-mediated pyroptosis, thereby playing an important role in the alleviation of neuroinflammation and depression. This study suggests new potential drugs and treatment strategies for neuroinflammation-related diseases and depression.

Yi-Qi-Huo-Xue decoction alleviates intracerebral hemorrhage injury through inhibiting neuronal autophagy of ipsilateral cortex via BDNF/TrkB pathway.

BACKGROUND: Yi-Qi-Huo-Xue Decoction (YQHXD), a traditional Chinese medicine formula, has demonstrated efficacy in the clinical treatment of intracerebral hemorrhage (ICH) for over a decade. Nevertheless, the precise pharmacotherapeutic compounds of YQHXD capable of penetrating into cerebral tissue and the pharmacological underpinnings of YQHXD remain ambiguous. METHODS: The active components of YQHXD in rat brains was analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The potential targets, pathways and biological progresses of YQHXD ameliorating ICH induced injury was predicted by network pharmacology. Moreover, collagenase-induced ICH rat model, primary cortex neurons exposed to hemin and molecular docking were applied to validate the molecular mechanisms of YQHXD. RESULTS: Eleven active components of YQHXD were identified within the brains. Employing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, our investigation concentrated on the roles of autophagy and the BDNF/TrkB signaling pathway in the pharmacological context. The pharmacological results revealed that YQHXD alleviated neurological dysfunction, brain water content, brain swelling, and pathological injury caused by ICH. Meanwhile, YQHXD inhibited autophagy influx and autophagosome in vivo, and regulated cortex neuronal autophagy and TrkB/BDNF pathway both in vivo and in vitro. Subsequently, N-acetyl serotonin (NAS), a selective TrkB agonist, was employed to corroborate the significance of the BDNF/TrkB pathway in this process. The combination of NAS and YQHXD did not further enhance the protective efficacy of YQHXD in ICH rats. Additionally, outcomes of molecular docking analysis revealed that nine compounds of YQHXD exhibited potential regulatory effects on TrkB. CONCLUSIONS: Ipsilateral neuronal autophagy and BDNF/TrkB pathway were activated 72 h after ICH. YQHXD effectively resisted injury induced by ICH, which was related with suppression of ipsilateral neuronal autophagy via BDNF/TrkB pathway. This study provides novel insights into the therapeutic mechanisms of traditional Chinese medicine in the context of ICH treatment.

Lesion of NPY Receptor-expressing Neurons in Perifornical Lateral Hypothalamus Attenuates Glucoprivic Feeding.

Glucoprivic feeding is one of several counterregulatory responses (CRRs) that facilitates restoration of euglycemia following acute glucose deficit (glucoprivation). Our previous work established that glucoprivic feeding requires ventrolateral medullary (VLM) catecholamine (CA) neurons that coexpress neuropeptide Y (NPY). However, the connections by which VLM CA/NPY neurons trigger increased feeding are uncertain. We have previously shown that glucoprivation, induced by an anti-glycolygic agent 2-deoxy-D-glucose (2DG), activates perifornical lateral hypothalamus (PeFLH) neurons and that expression of NPY in the VLM CA/NPY neurons is required for glucoprivic feeding. We therefore hypothesized that glucoprivic feeding and possibly other CRRs require NPY-sensitive PeFLH neurons. To test this, we used the ribosomal toxin conjugate NPY-saporin (NPY-SAP) to selectively lesion NPY receptor-expressing neurons in the PeFLH of male rats. We found that NPY-SAP destroyed a significant number of PeFLH neurons, including those expressing orexin, but not those expressing melanin-concentrating hormone. The PeFLH NPY-SAP lesions attenuated 2DG-induced feeding but did not affect 2DG-induced increase in locomotor activity, sympathoadrenal hyperglycemia, or corticosterone release. The 2DG-induced feeding response was also significantly attenuated in NPY-SAP-treated female rats. Interestingly, PeFLH NPY-SAP lesioned male rats had reduced body weights and decreased dark cycle feeding, but this effect was not seen in female rats. We conclude that a NPY projection to the PeFLH is necessary for glucoprivic feeding, but not locomotor activity, hyperglycemia, or corticosterone release, in both male and female rats.

Poria cocos (Schw.) Wolf, a Traditional Chinese Edible Medicinal Herb, Promotes Neuronal Differentiation, and the Morphological Maturation of Newborn Neurons in Neural Stem/Progenitor Cells.

Neurogenesis in the adult brain comprises the entire set of events of neuronal development. It begins with the division of precursor cells to form a mature, integrated, and functioning neuronal network. Adult neurogenesis is believed to play an important role in animals' cognitive abilities, including learning and memory. In the present study, significant neuronal differentiation-promoting activity of 80% (v/v) ethanol extract of P. cocos (EEPC) was found in Neuro-2a cells and mouse cortical neural stem/progenitor cells (NSPCs). Subsequently, a total of 97 compounds in EEPC were identified by UHPLC-Q-Exactive-MS/MS. Among them, four major compounds-Adenosine; Choline; Ethyl palmitoleate; and L-(-)-arabinitol-were further studied for their neuronal differentiation-promoting activity. Of which, choline has the most significant neuronal differentiation-promoting activity, indicating that choline, as the main bioactive compound in P. cocos, may have a positive effect on learning and memory functions. Compared with similar research literature, this is the first time that the neuronal differentiation-promoting effects of P. cocos extract have been studied.

Metabolic Fingerprinting of Different Sideritis Taxa Infusions and Their Neurogenic Activity.

Over the last years, Sideritis extracts were shown to improve memory. However, their potential to promote the generation of new neurons, starting with the neuronal differentiation of neural stem cells, remains unexplored. Therefore, the present study aimed to evaluate the neurogenic effects of different Sideritis infusions in neural stem and precursor cells and their impact on cell viability. Moreover, the metabolic fingerprints were recorded using LC-DAD, LC-HRESIMS, and GC-MS. The neurogenic potential of infusions of the eight Sideritis taxa tested was as potent as the classical neuronal inducer combination of retinoic acid and valproic acid. Further cytotoxicity assays revealed that the IC50 values of the extracts were between 163 and 322 µg/mL. Hierarchical cluster analyses of the metabolic fingerprints unveiled that the two Sideritis taxa with the lowest IC50 values were the most divergent in the analytical techniques used. As the analysis focused on polyphenols, it is reasonable to assume that these compounds are responsible for the effect on the cell viability of SH-SY5Y neuroblastoma cells. This study is the first report on the neurogenic potential of Sideritis taxa and might support the use of Sideritis herbal preparations in the context of neurodegenerative diseases.

Neuroprotective Effects of Ethanol Extract of Polyscias fruticosa (EEPF) against Glutamate-Mediated Neuronal Toxicity in HT22 Cells.

In traditional herbal medicine, the Polyscias fruticosa has been frequently used for the treatment of ischemia and inflammation. Oxidative stress mediated by elevated glutamate levels cause neuronal cell death in ischemia and various neurodegenerative diseases. However, so far, the neuroprotective effects of this plant extract against glutamate-mediated cell death have not been investigated in cell models. The current study investigates the neuroprotective effects of ethanol extracts of Polyscias fruticosa (EEPF) and elucidates the underlying molecular mechanisms of EEPFs relevant to neuroprotection against glutamate-mediated cell death. The oxidative stress-mediated cell death was induced by 5 mM glutamate treatment in HT22 cells. The cell viability was measured by a tetrazolium-based EZ-Cytox reagent and Calcein-AM fluorescent dye. Intracellular Ca2+ and ROS levels were measured by fluorescent dyes, fluo-3 AM and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Protein expressions of p-AKT, BDNF, p-CREB, Bax, Bcl-2, and apoptosis-inducing factor (AIF) were determined by western blot analysis. The apoptotic cell death was measured by flow cytometry. The in vivo efficacy of EEPF was evaluated using the Mongolian gerbil mouse by surgery-induced brain ischemia. EEPF treatment showed a neuroprotective effect against glutamate-induced cell death. The EEPF co-treatment reduced the intracellular Ca2+ and ROS and apoptotic cell death. Furthermore, it recovered the p-AKT, p-CREB, BDNF, and Bcl-2 levels decreased by glutamate. The EEPF co-treatment suppressed the activation of apoptotic Bax, the nuclear translocation of AIF, and mitogen-activated protein kinase (MAPK) pathway proteins (ERK1/2, p38, JNK). Further, EEPF treatment significantly rescued the degenerative neurons in the ischemia-induced Mongolian gerbil in vivo model. EEPF exhibited neuroprotective properties that suppress glutamate-mediated neurotoxicity. The underlying mechanism of EEPF is increasing the level of p-AKT, p-CREB, BDNF, and Bcl-2 associated with cell survival. It has therapeutic potential for the treatment of glutamate-mediated neuropathology.

SATB1/SLC7A11/HO-1 Axis Ameliorates Ferroptosis in Neuron Cells After Ischemic Stroke by Danhong Injection.

Neuronal damage after ischemic stroke (IS) is frequently due to ferroptosis, contributing significantly to ischemic injury. However, the mechanism against ferroptosis in IS remained unclear. The aim of this study was to investigate the potential mechanism of Danhong injection (DHI) and the critical transcription factor SATB1 in preventing neuronal ferroptosis after ischemic stroke in vivo and in vitro. The results showed that DHI treatment significantly reduced the infarct area and associated damage in the brains of the pMCAO mice, and enhanced the viability of OGD-injured neurons. And several characteristic indicators of ferroptosis, such as mitochondrial necrosis and iron accumulation, were regulated by DHI after IS. Importantly, we found that the expression and activity of SATB1 were decreased in the pMCAO mice, especially in neuron cells. Meanwhile, the SATB1/SLC7A11/HO-1 signaling pathway was activated after DHI treatment in ischemic stroke and was found to improve neuronal ferroptosis. Inhibition of SATB1 significantly reduced SLC7A11-HO-1 and significantly attenuated the anti-ferroptosis effects of DHI in the OGD model. These findings indicate that neuronal ferroptosis after IS can be alleviated by DHI through SATB1/SLC7A11/HO-1 pathway, and SATB1 may be an attractive therapeutic target for treating ischemic stroke.

Embryonic ethanol exposure induces ectopic Hcrt and MCH neurons outside hypothalamus in rats and zebrafish: Role in ethanol-induced behavioural disturbances.

Embryonic exposure to ethanol increases the risk for alcohol use disorder in humans and stimulates alcohol-related behaviours in different animal models. Evidence in rats and zebrafish suggests that this phenomenon induced by ethanol at low-moderate concentrations involves a stimulatory effect on neurogenesis and density of hypothalamic neurons expressing the peptides, hypocretin/orexin (Hcrt) and melanin-concentrating hormone (MCH), known to promote alcohol consumption. Building on our report in zebrafish showing that ethanol induces ectopic expression of Hcrt neurons outside the hypothalamus, we investigated here whether embryonic ethanol exposure also induces ectopic peptide neurons in rats similar to zebrafish and affects their morphological characteristics and if these ectopic neurons are functional and have a role in the ethanol-induced disturbances in behaviour. We demonstrate in rats that ethanol at a low-moderate dose, in addition to increasing Hcrt and MCH neurons in the lateral hypothalamus where they are normally concentrated, induces ectopic expression of these peptide neurons further anterior in the nucleus accumbens core and ventromedial caudate putamen where they have not been previously observed and causes morphological changes relative to normally located hypothalamic neurons. Similar to rats, embryonic ethanol exposure at a low-moderate dose in zebrafish induces ectopic Hcrt neurons anterior to the hypothalamus and alters their morphology. Notably, laser ablation of these ectopic Hcrt neurons blocks the behavioural effects induced by ethanol exposure, including increased anxiety and locomotor activity. These findings suggest that the ectopic peptide neurons are functional and contribute to the ethanol-induced behavioural disturbances related to the overconsumption of alcohol.

Mucuna pruriens seed protects the hippocampal neurons and abrogates seizure indices in chemically-convulsed mice: Evidence of the Nrf2 expression defense pathway.

The anti-convulsant mechanisms and neuroprotective effects of Mucuna pruriens (MP) seed in male BALB/c mice were evaluated. Ninety mice were kindled with picrotoxin, strychnine, or pilocarpine hydrochloride at the 30th minute of intraperitoneal injection (i.p) of normal saline (0.2 ml), MP (200, 100, 50 mg/kg), diazepam (7.5 mg/kg), or haloperidol (5 mg/kg). The onset of convulsion and percentage mortality was recorded. The histoarchitectural and immunohistochemical profiles of the brains were determined. Data were expressed as mean ± SEM with a one-way analysis of variance (ANOVA), while p < 0.05 was considered significant. There was a significant prolongation of the latency to first seizure across the treatment groups following picrotoxin, and pilocarpine-induced convulsion; a decrease in percentage mortality in the MP (50 mg/kg) treatment group, and an increase in the hippocampal nuclear factor erythroid 2-related factor 2 count, and Neu-N expression in the MP (200 mg/kg, and 100 mg/kg) treated mice. Treatment with MP seed may abolish seizure occurrence and consequential mortality; mechanisms traceable to its GABAergic expression and hippocampal NRF 2 and Neu N expression.

GP-14 protects against severe hypoxia-induced neuronal injury through the AKT and ERK pathways and its induced transcriptome profiling alteration.

Gypenosides are major bioactive ingredients of G. pentaphyllum. In our previous study, we found that gypenosides had neuroprotective effects against hypoxia-induced injury. In the current study, we focused on the protective effects of gypenoside-14 (GP-14), which is one of the newly identified bioactive components, on neuronal injury caused by severe hypoxia (0.3% O2). The results showed that GP-14 pretreatment alleviated the cell viability damage and apoptosis induced by hypoxia in PC12 cells. Moreover, GP-14 pretreatment also attenuated primary neuron injuries under hypoxic conditions. Additionally, GP-14 pretreatment significantly ameliorated neuronal damage in the hippocampal region induced by high-altitude cerebral edema (HACE). At the molecular level, GP-14 pretreatment reversed the decreased activities of the AKT and ERK signaling pathways caused by hypoxia in PC12 cells and primary neurons. To comprehensively explore the possible mechanisms, transcriptome sequencing was conducted, and these results indicated that GP-14 could alter the transcriptional profiles of primary neuron. Taken together, our results suggest that GP-14 acts as a neuroprotective agent to protect against neuronal damage induced by severe hypoxia and it is a promising compound for the development of neuroprotective drugs.

Phikud Navakot extract attenuates lipopolysaccharide-induced inflammatory responses through inhibition of ERK1/2 phosphorylation in a coculture system of microglia and neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Phikud Navakot (PN), a mixture of nine herbal plants, is an ancient Thai traditional medicine used for relieving circulatory disorders and dizziness. PN has also shown anti-inflammatory effects in rats with acute myocardial infarction. Moreover, phytochemical-inhibiting neuroinflammation, including gallic acid, vanillic acid, ferulic acid, and rutin were detected in PN extract; however, the anti-neuroinflammatory activity of PN extract and its components in a coculture system of microglia and neuronal cells is limited. OBJECTIVE: To investigate the anti-neuroinflammatory activities of PN on lipopolysaccharide (LPS)-induced inflammation in a coculture system of microglia and neuronal cells. METHODS: ELISA and qRT-PCR were used to assess cytokine expression. The phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by Western blotting. Microglia-mediated neuroinflammation was evaluated using a BV-2 microglia-N2a neuron transwell co-culture. RESULTS: PN extract and its component, gallic acid, decreased LPS-induced the mRNA expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as well as IL-6 protein levels in both microglial monoculture and coculture systems. This was accompanied by a reduction in neurodegeneration triggered by microglia in N2a neurons with increased neuronal integrity markers (ßIII tubulin and tyrosine hydroxylase (TH)). These effects were caused by the ability of PN extract to inhibit extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. CONCLUSION: This is the first study to show that PN extract inhibits neurodegeneration in LPS-activated BV-2 microglia by targeting ERK signaling activity.

Geraniin Protects against Cerebral Ischemia/Reperfusion Injury by Suppressing Oxidative Stress and Neuronal Apoptosis via Regulation of the Nrf2/HO-1 Pathway.

Geraniin, a polyphenol isolated from Phyllanthus amarus, possesses extensive biological and pharmaceutical activities. In this study, we investigated the protective effect against cerebral ischemia/reperfusion (I/R) injury of geraniin and explored its potential mechanism. Middle cerebral artery occlusion/reperfusion (MCAO/R) was used to simulate cerebral I/R injury in vivo, and oxygen-glucose deprivation/reoxygenation (OGD/R) was applied to establish an in vitro model of cerebral I/R injury. In this study, we performed TTC and HE staining and adopted a neurological score method to evaluate the neuroprotective effect of geraniin in vivo and used the CCK-8 assay to assess this effect in vitro. Indices of reactive oxidation capacity were measured in vivo and in vitro to verify the antioxidant capacity of geraniin. TUNEL staining and flow cytometry were applied to measure the apoptosis rate, and Western blotting was performed to assess the expression of apoptosis-related proteins. Finally, the expression of Nrf2 and HO-1 was evaluated in vivo and in vitro by Western blotting. Geraniin significantly reduced the infarct volume, decreased neurological deficit scores, alleviated pathological changes in neurons, and increased the cell survival rate. Geraniin increased the activity of superoxide dismutase (SOD) and decreased the activity of lactate dehydrogenase (LDH) and the contents of malondialdehyde (MDA), nitric oxide (NO), and neuronal nitric oxide synthase (nNOS) in vivo and in vitro. In addition, geraniin significantly reduced the apoptosis. Furthermore, geraniin also evidently increased Nrf2 (total and nuclear) and HO-1 protein expression in vivo and in vitro. Collectively, these results imply that geraniin may exert a protective effect against cerebral I/R injury by suppressing oxidative stress and neuronal apoptosis. The mechanism underlying the protective effect of geraniin is associated with activation of the Nrf2/HO-1 pathway. Our results indicate that geraniin may be a potential drug candidate for the treatment of ischemic stroke.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Label-Free Study of the Global Cell Behavior during Exposure to Environmental Radiofrequency Fields in the Presence or Absence of Pro-Apoptotic or Pro-Autophagic Treatments.

It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.