RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.
Assuntos
Medicamentos de Ervas Chinesas , Mitofagia , Farmacologia em Rede , Proteínas Quinases , Traumatismo por Reperfusão , Proteína Supressora de Tumor p53 , Animais , Masculino , Ratos , Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Mitofagia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células PC12 , Proteínas Quinases/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína LigasesRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Melastoma dodecandrum Lour. (MD), a traditional Chinese medicine used by the She ethnic group, has been used to treat cerebral ischemia-reperfusion (CIR) injury due to its efficacy in promoting blood circulation and removing blood stasiss; however, the therapeutic effects and mechanisms of MD in treating CIR injury remain unclear. AIM: To investigate the protective effects of MD on CIR injury, in addition to its impact on oxidative stress, endoplasmic reticulum (ER) stress, and cell apoptosis. MATERIALS AND METHODS: The research was conducted using both cell experiments and animal experiments. The CCK-8 method, immunofluorescence staining, and flow cytometry were used to analyze the effects of MD-containing serum on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cell viability, reactive oxygen species (ROS) clearance, anti-inflammatory, neuroprotection and inhibition of apoptosis. Furthermore, 2,3,5-Triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, Nissl staining, and immunohistochemistry were used to detect infarct size, pathological changes, Nissl corpuscula and neuronal protein expression in middle cerebral artery occlusion (MCAO) rats. Polymerase chain reaction and Western Blotting were conducted in cell and animal experiments to detect the expression levels of ER stress-related genes and proteins. RESULTS: The MD extract enhanced the viability of PC12 cells under OGD/R modeling, reduced ROS and IL-6 levels, increased MBP levels, and inhibited cell apoptosis. Furthermore, MD improved the infarct area in MCAO rats, increased the number of Nissl bodies, and regulated neuronal protein levels including Microtubule-Associated Protein 2 (MAP-2), Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP), and Neurofilament 200 (NF200). Additionally, MD could regulate the expression levels of oxidative stress proteins malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Both cell and animal experiments demonstrated that MD could inhibit ER stress-related proteins (GRP78, ATF4, ATF6, CHOP) and reduce cell apoptosis. CONCLUSION: This study confirmed that the therapeutic mechanism of the MD extract on CIR injury was via the inhibition of oxidative stress and the ER stress pathway, in addition to the inhibition of apoptosis.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Fármacos Neuroprotetores , Estresse Oxidativo , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Ratos , Células PC12 , Masculino , Fármacos Neuroprotetores/farmacologia , Apoptose/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêuticoRESUMO
The prevalence of Alzheimer's disease (AD) is increasing globally due to population aging. However, effective clinical treatment strategies for AD still remain elusive. The mechanisms underlying AD onset and the interplay between its pathological factors have so far been unclear. Evidence indicates that AD progression is ultimately driven by neuronal loss, which in turn is caused by neuroapoptosis and neuroinflammation. Therefore, the inhibition of neuroapoptosis and neuroinflammation could be a useful anti-AD strategy. Nonetheless, the delivery of active drug agents into the brain parenchyma is hindered by the blood-brain barrier (BBB). To address this challenge, we fabricated a black phosphorus nanosheet (BP)-based methylene blue (MB) delivery system (BP-MB) for AD therapy. After confirming the successful preparation of BP-MB, we proved that its BBB-crossing ability was enhanced under near-infrared light irradiation. In vitro pharmacodynamics analysis revealed that BP and MB could synergistically scavenge excessive reactive oxygen species (ROS) in okadaic acid (OA)-treated PC12 cells and lipopolysaccharide (LPS)-treated BV2 cells, thus efficiently reversing neuroapoptosis and neuroinflammation. To study in vivo pharmacodynamics, we established a mouse model of AD mice, and behavioral tests confirmed that BP-MB treatment could successfully improve cognitive function in these animals. Notably, the results of pathological evaluation were consistent with those of the in vitro assays. The findings demonstrated that BP-MB could scavenge excessive ROS and inhibit Tau hyperphosphorylation, thereby alleviating downstream neuroapoptosis and regulating the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Overall, this study highlights the therapeutic potential of a smart nanomedicine with the capability of reversing neuroapoptosis and neuroinflammation for AD treatment.
Assuntos
Doença de Alzheimer , Apoptose , Barreira Hematoencefálica , Azul de Metileno , Nanomedicina , Doenças Neuroinflamatórias , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Apoptose/efeitos dos fármacos , Células PC12 , Doenças Neuroinflamatórias/tratamento farmacológico , Ratos , Camundongos , Nanomedicina/métodos , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Masculino , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Introduction: Alzheimer's disease (AD), a neurodegenerative condition, stands as the most prevalent form of dementia. Its complex pathological mechanisms and the formidable blood-brain barrier (BBB) pose significant challenges to current treatment approaches. Oxidative stress is recognized as a central factor in AD, underscoring the importance of antioxidative strategies in its treatment. In this study, we developed a novel brain-targeted nanoparticle, Ce/Zr-MOF@Cur-Lf, for AD therapy. Methods: Layer-by-layer self-assembly technology was used to prepare Ce/Zr-MOF@Cur-Lf. In addition, the effect on the intracellular reactive oxygen species level, the uptake effect by PC12 and bEnd.3 cells and the in vitro BBB permeation effect were investigated. Finally, the mouse AD model was established by intrahippocampal injection of Aß1-42, and the in vivo biodistribution, AD therapeutic effect and biosafety of the nanoparticles were researched at the animal level. Results: As anticipated, Ce/Zr-MOF@Cur-Lf demonstrated efficient BBB penetration and uptake by PC12 cells, leading to attenuation of H2O2-induced oxidative damage. Moreover, intravenous administration of Ce/Zr-MOF@Cur-Lf resulted in rapid brain access and improvement of various pathological features of AD, including neuronal damage, amyloid-ß deposition, dysregulated central cholinergic system, oxidative stress, and neuroinflammation. Conclusion: Overall, Ce/Zr-MOF@Cur-Lf represents a promising approach for precise brain targeting and multi-target mechanisms in AD therapy, potentially serving as a viable option for future clinical treatment.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Cério , Curcumina , Estresse Oxidativo , Zircônio , Animais , Doença de Alzheimer/tratamento farmacológico , Células PC12 , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Zircônio/química , Zircônio/farmacocinética , Camundongos , Ratos , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacologia , Curcumina/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Cério/química , Cério/farmacocinética , Cério/farmacologia , Cério/administração & dosagem , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Distribuição Tecidual , Espécies Reativas de Oxigênio/metabolismo , Nanopartículas/química , Modelos Animais de Doenças , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacocinética , Estruturas Metalorgânicas/farmacologia , Masculino , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Humanos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismoRESUMO
BACKGROUND: Atrazine (ATR), a commonly used herbicide, is linked to dopaminergic neurotoxicity, which may cause symptoms resembling Parkinson's disease (PD). This study aims to reveal the molecular regulatory networks responsible for ATR exposure and its effects on dopaminergic neurotoxicity based on an integration strategy. METHODS: Our approach involved network toxicology, construction of protein-protein interaction (PPI) networks, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, as well as molecular docking techniques. Subsequently, we validated the predicted results in PC12 cells in vitro. RESULTS: An integrated analysis strategy indicating that 5 hub targets, including mitogen-activated protein kinase 3 (Mapk3), catalase (Cat), heme oxygenase 1 (Hmox1), tumor protein p53 (Tp53), and prostaglandin-endoperoxide synthase 2 (Ptgs2), may play a crucial role in ATR-induced dopaminergic injury. Molecular docking indicated that the 5 hub targets exhibited certain binding activity with ATR. Cell counting kit-8 (CCK8) results illustrated a dose-response relationship in PC12 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) displayed notable changes in the expression of hub targets mRNA levels, with the exception of Mapk3. Western blotting results suggested that ATR treatment in PC12 cells resulted in an upregulation of the Cat, Hmox1, and p-Mapk3 protein expression levels while causing a downregulation in Tp53, Ptgs2, and Mapk3. CONCLUSION: Our findings indicated that 5 hub targets identified could play a vital role in ATR-induced dopaminergic neurotoxicity in PC12 cells. These results provide preliminary support for further investigation into the molecular mechanism of ATR-induced toxicity.
Assuntos
Atrazina , Neurônios Dopaminérgicos , Herbicidas , Simulação de Acoplamento Molecular , Atrazina/toxicidade , Animais , Células PC12 , Ratos , Herbicidas/toxicidade , Neurônios Dopaminérgicos/efeitos dos fármacos , Mapas de Interação de Proteínas , Dopamina/metabolismoRESUMO
Depression is a prevalent mental disorder, but the side effects of antidepressants also make depressed patients resistant. Effective and safe antidepressants should be developed from traditional herbs, with the aim of reducing the side effects of antidepressants and improving the efficacy of drugs. In this study, the new macamide compound-4 (NMC-4) was synthesized for the first time, addressing the problem of difficult extraction, isolation, and low content of natural macamide. NMC-4 was characterized using mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy. The protective effect of NMC-4 against cell injury was demonstrated to be stronger than that of natural macamide (N-benzylhexadecanamide, XA) using a PC12 cell injury model. The study explored the effects of NMC-4 on chronic unpredictable mild stress (CUMS)-induced depressive symptoms. NMC-4 significantly improved depressive-like behaviors. NMC-4 ameliorated CUMS-induced depressive-like behaviors by mitigating neuroinflammation and modulating the NF-κB/Nrf2 and BDNF/PI3K/Akt pathways.
Assuntos
Antidepressivos , Depressão , Animais , Depressão/tratamento farmacológico , Ratos , Células PC12 , Masculino , Antidepressivos/farmacologia , Antidepressivos/química , Antidepressivos/uso terapêutico , Estresse Psicológico/tratamento farmacológico , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , NF-kappa B/metabolismo , Camundongos , Comportamento Animal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
In the present study, the PC12 cells as a bioassay system were used to screen the small molecules with nerve growth factor (NGF)- mimic effect from Lavandula angustifolia Mill. The ß-Cyclocitral (ß-cyc) as an active compound was discovered, and its chemical structure was also determined. Furthermore, we focused on the bioactive and action mechanism of this compound to do an intensive study with specific protein inhibitors and Western blotting analysis. The ß-cyc had novel NGF-mimic and NGF-enhancer effects on PC12 cells, while the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol 3 kinase, (PI3K)/serine/threonine-protein kinase (AKT), and glucocorticoid receptor (GR)/phospholipase C (PLC)/protein kinase C (PKC) signaling pathways were involved in the bioactivity of ß-cyc. In addition, the important role of the rat sarcoma (Ras)/protooncogene serine-threonine protein kinase (Raf) signaling pathway was observed, although it was independent of tyrosine kinase (Trk) receptors. Moreover, the non-label target protein discovery techniques, such as the cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS), were utilized to make predictions of its target protein. The stability of IGF-R and GR, proteins for temperature and protease, was dose-dependently increased after treatment of ß-cyc compared with control groups, respectively. These findings indicated that ß-cyc promoted the neuron differentiation of PC12 cells via targeting IGF-1R and GR and modification of downstream signaling pathways.
Assuntos
Fator de Crescimento Insulin-Like I , Lavandula , Fator de Crescimento Neural , Receptores de Glucocorticoides , Transdução de Sinais , Células PC12 , Ratos , Animais , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Lavandula/química , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/químicaRESUMO
By inhibiting acetylcholinesterase (AChE) activity, organophosphate compounds (OPs) can quickly cause severe injury to the nervous system and death, making it extremely difficult to rescue victims after OP exposure. However, it is quite challenging to construct scavengers that neutralize and eliminate these harmful chemical agents promptly in the blood circulation system. Herein, we report an enzyme-armed biomimetic nanoparticle that enables a 'targeted binding and catalytic degradation' action mechanism designed for highly efficient in vivo detoxification (denoted as 'Nanocleaner'). Specifically, the resulting Nanocleaner is fabricated with polymeric cores camouflaged with a modified red blood cell membrane (RBC membrane) that is inserted with the organophosphorus hydrolase (OPH) enzyme. In such a subtle construct, Nanocleaner inherits abundant acetylcholinesterase (AChE) on the surface of the RBC membrane, which can specifically lure and neutralize OPs through biological binding. The OPH enzyme on the membrane surface breaks down toxicants catalytically. The in vitro protective effects of Nanocleaner against methyl paraoxon (MPO)-induced inhibition of AChE activity were validated using both preincubation and competitive regimens. Furthermore, we selected the PC12 neuroendocrine cell line as an experimental model and confirmed the cytoprotective effects of Nanocleaner against MPO. In mice challenged with a lethal dose of MPO, Nanocleaner significantly reduces clinical signs of intoxication, rescues AChE activity and promotes the survival rate of mice challenged with lethal MPO. Overall, these results suggest considerable promise of enzyme-armed Nanocleaner for the highly efficient removal of OPs for clinical treatment.
Assuntos
Acetilcolinesterase , Inibidores da Colinesterase , Compostos Organofosforados , Animais , Acetilcolinesterase/metabolismo , Camundongos , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Ratos , Compostos Organofosforados/química , Membrana Eritrocítica , Células PC12 , Paraoxon/toxicidade , Paraoxon/análogos & derivados , Nanopartículas/química , Arildialquilfosfatase/metabolismo , Arildialquilfosfatase/química , Masculino , Intoxicação por Organofosfatos/tratamento farmacológicoRESUMO
BACKGROUND: Dysfunction of the cholinergic system and increased oxidative stress have a crucial role in cognitive disorders including Alzheimer's disease (AD). Here, we have investigated the protective effects of betanin, a novel acetylcholinesterase (AChE) inhibitor, on hydrogen peroxide (H2O2)-induced cell death in PC12 cells. METHODS AND RESULTS: The protective effects were assessed by measuring cell viability, the amount of reactive oxygen species (ROS) production, AChE activity, cell damage, and apoptosis using resazurin, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), Ellman method, lactate dehydrogenase (LDH) release, propidium iodide (PI) staining and flow cytometry, and Western blot analysis. H2O2 (150 µM) resulted in cell viability reduction and apoptosis induction while, pretreatment with the betanin (10, 20, and 50 µM) and N-Acetyl-L-cysteine (NAC) (2.5 and 5 mM) significantly increased the viability (P < 0.05, P < 0.01 and P < 0.001) and at 5-50 µM betanin decreased ROS amount (P < 0.05, P < 0.01 and P < 0.001). Whereas, pretreatment with the betanin (10, 20, and 50 µM) decreased AChE activity (P < 0.001), also at 20 and 50 µM betanin reduced the release of LDH (P < 0.001), and at 10-50 µM decreased the percentage of apoptotic cells (P < 0.001). Apoptosis biomarkers such as cleaved poly (ADP-ribose) polymerase (PARP) (P < 0.01 and P < 0.001) and cytochrome c (P < 0.05 and P < 0.001) were attenuated after pretreatment of PC12 cells with betanin at 10-20 µM and 10-50 µM respectively. Indeed, survivin (P < 0.001) increased after pretreatment of cells with betanin at 10-20 µM. CONCLUSIONS: Overall, betanin may use the potential to delay or prevent cell death caused by AD through decreasing the activity of AChE as well as attenuating the expression of proteins involved in the apoptosis pathway.
Assuntos
Acetilcolinesterase , Apoptose , Betacianinas , Sobrevivência Celular , Inibidores da Colinesterase , Peróxido de Hidrogênio , Estresse Oxidativo , Espécies Reativas de Oxigênio , Células PC12 , Animais , Ratos , Apoptose/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Betacianinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Fármacos Neuroprotetores/farmacologiaRESUMO
Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe disease with a poor prognosis, whose clinical treatment is still limited to therapeutic hypothermia with limited efficacy. Perillyl alcohol (POH), a natural monoterpene found in various plant essential oils, has shown neuroprotective properties, though its effects on HIE are not well understood. This study investigates the neuroprotective effects of POH on HIE both in vitro and in vivo. We established an in vitro model using glucose deprivation and hypoxia/reperfusion (OGD/R) in PC12 cells, alongside an in vivo model via the modified Rice-Vannucci method. Results indicated that POH acted as an indirect antioxidant, reducing inducible nitric oxide synthase and malondialdehyde production, maintaining content of antioxidant molecules and enzymes in OGD/R-induced PC12 cells. In vivo, POH remarkably lessened infarct volume, reduced cerebral edema, accelerated tissue regeneration, and blocked reactive astrogliosis after hypoxic-ischemic brain injury. POH exerted antiapoptotic activities through both the intrinsic and extrinsic apoptotic pathways. Mechanistically, POH activated Nrf2 and inactivated its negative regulator Keap1. The use of ML385, a Nrf2 inhibitor, reversed these effects. Overall, POH mitigates neuronal damage in HIE by combating oxidative stress, reducing inflammation, and inhibiting apoptosis via the Nrf2/Keap1 pathway, suggesting its potential for HIE treatment.
Assuntos
Animais Recém-Nascidos , Hipóxia-Isquemia Encefálica , Proteína 1 Associada a ECH Semelhante a Kelch , Monoterpenos , Fator 2 Relacionado a NF-E2 , Fármacos Neuroprotetores , Transdução de Sinais , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Monoterpenos/farmacologia , Monoterpenos/uso terapêutico , Células PC12 , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacosRESUMO
A bioactivity-guided separation strategy was used to identify novel antistroke compounds from Gymnadenia conopsea (L.) R. Br., a medicinal plant. As a result, 4 undescribed compounds (1-2, 13, and 17) and 13 known compounds, including 1 new natural product (3), were isolated from G. conopsea. The structures of these compounds were elucidated through comprehensive spectroscopic techniques, such as 1D/2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and quantum chemical calculations. An oxygen-glucose deprivation/reoxygenation (OGD/R)-injured rat pheochromocytoma (PC12) cell model was used to evaluate the antistroke effects of the isolates. Compounds 1-2, 10-11, 13-15, and 17 provided varying degrees of protection against OGD/R injury in the PC12 cells at concentrations of 12.5, 25, and 50 µM. Among the tested compounds, compound 17 demonstrated the most potent neuroprotective effect, which was equivalent to that of the positive control drug (edaravone). Then, transcriptomic and bioinformatics analyses were conducted to reveal the regulatory effect of compound 17 on gene expression. In addition, quantitative real-time PCR (qPCR) was performed to verify the results of the transcriptomic and bioinformatics analyses. These results suggest that the in vitro antistroke effect of compound 17 may be associated with the regulation of the Col27a1 gene. Thus, compound 17 is a promising candidate for the development of novel antistroke drugs derived from natural products, and this topic should be further studied.
Assuntos
Fármacos Neuroprotetores , Animais , Ratos , Células PC12 , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Acidente Vascular Cerebral/tratamento farmacológico , Orchidaceae/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Estrutura Molecular , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificaçãoRESUMO
BACKGROUND: Hypoxic-ischemic brain damage (HIBD) is a prevalent brain injury with high mortality and morbidity. It results from hypoxia and ischemia of the brain due to various perinatal factors. A previous study showed that knockdown of programmed cell death factor 4 (PDCD4) could reduce infarction injury resulting from ischemia/reperfusion injury. However, exact mechanism by which PDCD4 acts in HIBD is not yet understood. Our aim in present investigation was to investigate the function and mechanism of PDCD4 in alleviating HIBD. METHODS: An HIBD model was developed using neonatal rats. After 48 h of modeling, short-term neurological function was evaluated and the brain tissue removed for assessment of cerebral infarct volume and brain water content (BWC). A cell model of oxygen glucose deprivation/reoxygenation (OGD/R) was also constructed. Overexpression or knockdown of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) or PDCD4 was performed in pretreated cells. RESULTS: The geotaxis reflex time, cerebral infarct volume, and BWC all increased after HIBD in this neonatal rat model. Additionally, the levels of PDCD4 and of the N6-Methyladenosine (m6A) reader protein IGF2BP3 were increased in HIBD rats and OGD/R-stimulated pheochromocytoma (PC12) cells relative to controls. Moreover, OGD/R-stimulated pheochromocytoma PC12 cells showed decreased cell viability, increased apoptosis, and elevated Interleukin 6 (IL-6), Interleukin 1 ß (IL-1ß), and tumor necrosis factor-α (TNF-α) contents. These features were reversed after knocking down IGF2BP3. The interaction between IGF2BP3 protein and PDCD4 mRNA was confirmed by RNA immunoprecipitation and RNA pull-down assays. Furthermore, knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells reduced cell damage via down-regulation of PDCD4. Finally, the IGF2BP3/PDCD4 axis alleviated OGD/R-induced cell injury in primary cortical neurons (PCNs). CONCLUSIONS: PDCD4 and m6A reader protein IGF2BP3 were up-regulated in an HIBD neonatal rat model. Knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells or PCNs alleviated cell damage through reducing PDCD4.
Assuntos
Proteínas Reguladoras de Apoptose , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hipóxia-Isquemia Encefálica , Proteínas de Ligação a RNA , Ratos Sprague-Dawley , Animais , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/patologia , Ratos , Células PC12 , Animais Recém-Nascidos , Modelos Animais de Doenças , MasculinoRESUMO
Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.
Assuntos
Autofagossomos , Lisossomos , Metanfetamina , Metanfetamina/farmacologia , Animais , Células PC12 , Ratos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Catepsina D/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
BACKGROUND: Depression and anxiety disorders are prevalent psychiatric conditions, and currently utilized chemical drugs typically come with significant adverse effects. China boasts a wealth of medicinal and food herbs known for their safe and effective properties. PURPOSE: This study aimed to develop novel formulations with improved antidepressant and anxiolytic effects derived from medicinal and food herbs. STUDY DESIGN: Screening combinations with antidepressant and anxiolytic effects using techniques such as network pharmacology and validating their effects in vitro and in vivo experiments. METHODS: Utilizing network pharmacology and molecular docking, we identified the top ten medicinal herbs with anxiolytic and antidepressant potential. Herbs with cytoprotective effects and non-toxic characteristics were further screened to formulate the herbal blends. Subsequently, we established a PC12 cell injury model and a chronic unpredictable mild stress (CUMS) model in mice to assess the effects of our formulations. RESULTS: Ten medicinal herbs were initially screened, and six of them were deemed suitable for formulating the blend, namely Gancao, Dazao, Gouqizi, Sangye, Huangqi, and Jinyinhua (GDGSHJ). The GDGSHJ formulation reduced Lactate Dehydrogenase (LDH) leakage, decreased apoptosis, and demonstrated a favorable antidepressant and antianxiety effect in the CUMS mouse model. Besides, GDGSHJ led to the upregulation of serum 5-Hydroxytryptamine (5-HT) content and brain tissue 5-HT, Gamma-aminobutyric acid (GABA), and Dopamine (DA) levels. It also downregulated the expression of SLC6A4 and SLC6A3 genes in the mouse hippocampus while upregulating HTR1A, DRD1, DRD2, and GABRA1 genes. CONCLUSION: Our formulation exhibited robust antidepressant and antianxiety effects without inducing substantial toxicity. This efficacy appears to be mediated by the expression of relevant genes within the hippocampus of mice. The formulation achieved this effect by balancing 5-HT levels in the serum and DA, GABA, and 5-HT levels within brain tissue.
Assuntos
Ansiolíticos , Antidepressivos , Simulação de Acoplamento Molecular , Farmacologia em Rede , Animais , Antidepressivos/farmacologia , Ansiolíticos/farmacologia , Camundongos , Masculino , Células PC12 , Ratos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Depressão/tratamento farmacológico , Plantas Medicinais/química , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Modelos Animais de Doenças , Estresse Psicológico/tratamento farmacológico , Ansiedade/tratamento farmacológico , Serotonina/metabolismoRESUMO
Paeoniflorin (PF) is one of the active constituents of the traditional Chinese medicine Paeoniae Radix Rubra and has been actively explored in the pharmaceutical area due to its numerous pharmacological effects. However, severe difficulties such as limited bioavailability and low permeability limit its utilization. Therefore, this study developed and synthesized 25 derivatives of PF, characterized them by 1H NMR, 13C NMR, and HR-MS, and evaluated their antioxidant activity. Firstly, the antioxidant capacity of PF derivatives was investigated through DPPH radical scavenging experiment, ABTS radical scavenging experiment, reducing ability experiment, and O2 .- radical scavenging experiment. PC12 cells are routinely used to evaluate the antioxidant activity of medicines, therefore we utilize it to establish a cellular model of oxidative stress. Among all derivatives, compound 22 demonstrates high DPPH radical scavenging capacity, ABTS radical scavenging ability, reduction ability, and O2 .- radical scavenging ability. The results of cell tests reveal that compound 22 has a non-toxic effect on PC12 cells and a protective effect on H2O2-induced oxidative stress models. This might be due to the introduction of 2, 5-difluorobenzene sulfonate group in PF, which helps in scavenging free radicals under oxidative stress. Western blot and molecular docking indicated that compound 22 may exert antioxidant activity by activating Nrf2 protein expression. As noted in the study, compound 22 has the potential to be a novel antioxidant.
Assuntos
Antioxidantes , Glucosídeos , Simulação de Acoplamento Molecular , Monoterpenos , Glucosídeos/farmacologia , Glucosídeos/química , Glucosídeos/síntese química , Glucosídeos/metabolismo , Células PC12 , Monoterpenos/química , Monoterpenos/farmacologia , Monoterpenos/síntese química , Animais , Ratos , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/síntese química , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peróxido de Hidrogênio , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/síntese químicaRESUMO
The newly identified estrogen receptor, G protein-coupled receptor 30 (GPR30), is prevalent in the brain and has been shown to provide significant neuroprotection. Recent studies have linked ferroptosis, a newly characterized form of programmed cell death, closely with cerebral ischemia-reperfusion injury (CIRI), highlighting it as a major contributing factor. Consequently, our research aimed to explore the potential of GPR30 targeting in controlling neuronal ferroptosis and lessening CIRI impacts. Results indicated that GPR30 activation not only improved neurological outcomes and decreased infarct size in a mouse model but also lessened iron accumulation and malondialdehyde formation post-middle cerebral artery occlusion (MCAO). This protective effect extended to increased levels of Nrf2 and GPX4 proteins. Similar protective results were replicated in PC12 cells subjected to Oxygen Glucose Deprivation and Reoxygenation (OGD/R) using the GPR30-specific agonist G1. Importantly, inhibition of Nrf2 with ML385 curtailed the neuroprotective effects of GPR30 activation, suggesting that GPR30 mitigates CIRI primarily through inhibition of neuronal ferroptosis via upregulation of Nrf2 and GPX4.
Assuntos
Ferroptose , Infarto da Artéria Cerebral Média , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Transdução de Sinais/efeitos dos fármacos , Camundongos , Células PC12 , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Ratos , Masculino , Fármacos Neuroprotetores/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Modelos Animais de DoençasRESUMO
The therapy of large defects in peripheral nerve injury (PNI) suffers from several drawbacks, especially the lack of autologous nerve donors. Nerve conduits are considered as a solution for nerve injury treatment, but biocompatibility improvements is still required for conduits prepared with synthetic materials. Cell-derived extracellular matrix (ECM) has drawn attention due to its lower risk of immunogenic response and independence from donor availability. The goal of this study is to coat bone mesenchymal stem cell-derived ECMs on poly(lactic-co-glycolic) acid (PLGA) conduits to enhance their ability to support neural growth and neurite extensions. The ECM-coated conduits have better hydrophilic properties than the pure PLGA conduits. A marked increase on PC12 and RSC96 cells' viability, proliferation and dorsal root ganglion neurite extension was observed. Quantitative PCR analysis exhibited a significant increase in markers for cell proliferation (GAP43), neurite extension (NF-H, MAP2, andßIII-tubulin) and neural function (TREK-1). These results show the potential of ECM-coated PLGA conduits in PNI therapy.
Assuntos
Proliferação de Células , Sobrevivência Celular , Matriz Extracelular , Células-Tronco Mesenquimais , Regeneração Nervosa , Neuritos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Animais , Ratos , Neuritos/metabolismo , Células PC12 , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Regeneração Nervosa/efeitos dos fármacos , Alicerces Teciduais/química , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Gânglios Espinais , Traumatismos dos Nervos Periféricos/terapia , Engenharia Tecidual/métodos , Polímeros/química , Teste de MateriaisRESUMO
Providing antioxidants and targeting acetylcholinesterase (AChE) are key strategies in treating neurocognitive dysfunction. In this study, bioactive sturgeon (Acipenser schrenckii) spinal cord peptides (SSCPs) with antioxidant and AChE inhibitory potency were extracted and separated from sturgeon spinal cord by enzymatic hydrolysis and ultrafiltration, and targeted peptide PGGW was screened via computer simulated molecular docking. Further, the molecular dynamic interactions of the PGGW with superoxide dismutase (SOD) and AChE were analyzed, and the protective effect of PGGW on glutamate-induced PC12 cells in vitro was evaluated. The <3 kDa fraction of SSCPs displays the most potent antioxidative efficacy (1 mg/mL, DPPHâ¢: 89.07%, ABTS+: 76.35%). Molecular dynamics simulation showed that PGGW was stable within AChE and tightly bound to residues SER203, PHE295, ILE294 and TRP236. When combined with SOD, the indole group of PGGW was stuck inside SOD, but the tail chain PGG fluctuated greatly outside. Surface plasmon resonance demonstrated that PGGW has a high binding affinity for AChE (KD = 1.4 mM) and 0.01 mg/mL PGGW provided good protection against glutamate-induced apoptosis. The findings suggest a promising strategy for drug research on neurodegenerative diseases.
Assuntos
Acetilcolinesterase , Antioxidantes , Inibidores da Colinesterase , Proteínas de Peixes , Peixes , Simulação de Acoplamento Molecular , Peptídeos , Medula Espinal , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Ratos , Medula Espinal/química , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Células PC12 , Superóxido Dismutase/metabolismo , Superóxido Dismutase/químicaRESUMO
Controlling biomolecular-cell interactions is crucial for the design of scaffolds for tissue engineering (TE). Regenerated silk fibroin (RSF) has been extensively used as TE scaffolds, however, RSF showed poor attachment of neuronal cells, such as rat pheochromocytoma (PC12) cells. In this work, amphiphilic peptides containing a hydrophobic isoleucine tail (I3) and laminin or fibronectin derived peptides (IKVAV, PDSGR, YIGSR, RGDS and PHSRN) were designed for promoting scaffold-cell interaction. Three of them (I3KVAV, I3RGDS and I3YIGSR) can self-assemble into nanofibers, therefore, were used to enhance the application of RSF in neuron TE. Live / dead assays revealed that the peptides exhibited negligible cytotoxicity against PC12 cells. The specific interaction between PC12 cells and the peptides were investigate using atomic force microscopy (AFM). The results indicated a synergistic effect in the designed peptides, promoting cellular attachment, proliferation and morphology changes. In addition, AFM results showed that co-assembling peptides I3KVAV and I3YIGSR possesses the best regulation of proliferation and attachment of PC12 cells, consistent with immunofluorescence staining results. Moreover, cell culture with hydrogels revealed that a mixture of peptides I3KVAV and I3YIGSR can also promote 3D neurites outgrowth. The approach of combining two different self-assembling peptides shows great potential for nerve regeneration applications.
Assuntos
Nanofibras , Crescimento Neuronal , Peptídeos , Seda , Alicerces Teciduais , Animais , Células PC12 , Ratos , Nanofibras/química , Alicerces Teciduais/química , Crescimento Neuronal/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Seda/química , Proliferação de Células/efeitos dos fármacos , Fibroínas/química , Fibroínas/farmacologia , Engenharia Tecidual/métodos , Adesão Celular/efeitos dos fármacosRESUMO
Elemental analysis at the single-cell level is an emerging technique in the field of inductively coupled plasma mass spectrometry (ICP-MS). In comparison to the analysis of cell suspensions by fast time-resolved analysis, single-cell sampling by laser ablation (LA) allows the discriminatory analysis of single cells according to their size and morphology. In this study, we evaluated the changes in elemental contents in a single cell through differentiation of rat adrenal pheochromocytoma into neuron-like cells by LA-ICP-MS. The contents of seven essential minerals were increased about 2-3 times after the differentiation. In addition, we evaluated the degree of differentiation at the single-cell level by means of imaging cytometry after immunofluorescence staining of microtubule-associated protein 2 (Map2), a neuron-specific protein. The fluorescence intensities of Alexa Fluor 488-conjugated secondary antibody against the anti-Map2 primary antibody showed large variations among the cells after the onset of differentiation. Although the average fluorescence intensity was increased through the differentiation, there were still less-matured neuron-like cells that exhibited a lower fluorescence intensity after 5 days of differentiation. Since a positive correlation between the fluorescence intensity and the cell size in area was found, we separately measured the elemental contents in the less-matured smaller cells and well-matured larger cells by LA-ICP-MS. The larger cells had higher elemental contents than the smaller cells, indicating that the essential minerals are highly required at a later stage of differentiation.