RESUMO
P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X7 is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X7 activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X7 bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na+ ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.
Assuntos
Trifosfato de Adenosina , Microscopia Crioeletrônica , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Animais , Ratos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sítios de Ligação , Sódio/metabolismo , Humanos , Agonistas do Receptor Purinérgico P2X/farmacologia , Células HEK293 , Ligação Proteica , Modelos MolecularesRESUMO
Due to the widespread use of methamphetamine (METH) among reproductive-aged women, the effects of intrauterine exposure to METH need to be investigated, as previous studies on this topic have been limited. The goal of this study is to examine the influence of two regulatory genes (miRNA-151-3p and CACNA1C) on the intrauterine life of mice exposed to METH. Pregnant mice received doses of 2 and 5 mg/kg of METH and saline from day 10 of pregnancy until the end. Their offspring were then evaluated for miRNA-151-3p and CACNA1C gene expression levels using real-time PCR. The findings indicated that exposure to METH reduced the expression levels of both miRNA-151-3p and CACNA1C genes in offspring compared to the control group (p≤0.001). In conclusion, intrauterine exposure to METH leads to a decrease in expression levels of both miRNA-151-3p and CACNA1C genes, potentially disrupting regulatory pathways involving these genes and having an impact on male reproductive health.
Assuntos
Canais de Cálcio Tipo L , Regulação para Baixo , Metanfetamina , MicroRNAs , Efeitos Tardios da Exposição Pré-Natal , Testículo , Animais , Metanfetamina/toxicidade , MicroRNAs/genética , MicroRNAs/metabolismo , Feminino , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Testículo/efeitos dos fármacos , Testículo/metabolismo , Ratos , CamundongosRESUMO
The main objective of this work was to investigate the mechanism of Astragalus aqueous extract ulcer healing in diabetic foot model rats through the hypoxia-inducible factor 1-alpha (HIF-1É)/vascular endothelial growth factor (VEGF) signalling pathway. Fifty specific-pathogen-free male Sprague Dawley rats were divided into blank (A), model control (B), Astragalus extract (C) and mupirocin (D) treatment groups. Group A received a regular diet, whereas the other groups received a high-fat/high-sugar diet and intraperitoneal streptozotocin injections to induce diabetes. Diabetic foot ulcers were created via skin excision. Subsequently, ulcers were debrided daily. Groups B, C and D received wet saline gauze, wet gauze with Astragalus extract and gauze with mupirocin, respectively, on the affected area. Group A received no treatment. After 14 days, the rats were assessed for ulcer healing and general condition. Immunohistochemistry was used to detect HIF-1É and VEGF levels in the dorsalis pedis artery, and ELISA was used to determine serum IL-6 and CRP levels. The results revealed that Groups C and D had significantly faster ulcer healing compared with Group B (p < 0.01), and ulcer healing was faster in Group C than in Group D (p < 0.01). Group C exhibited notably higher HIF-1É and VEGF protein expression levels compared with Groups B and D (p < 0.01). IL-6 and CRP expression levels in Groups C and D were significantly lower than those in Group B (p < 0.01). In summary, Astragalus aqueous extract effectively treats diabetic foot ulcers by up-regulating HIF-1É and VEGF expression, activating the HIF-1É/VEGF pathway, improving local tissue ischaemia and hypoxia, promoting collateral circulation and enhancing dorsalis pedis artery formation, thereby accelerating ulcer repair in diabetic rats.
Assuntos
Astrágalo , Pé Diabético , Subunidade alfa do Fator 1 Induzível por Hipóxia , Extratos Vegetais , Ratos Sprague-Dawley , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Cicatrização , Animais , Pé Diabético/tratamento farmacológico , Pé Diabético/metabolismo , Masculino , Fator A de Crescimento do Endotélio Vascular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Astrágalo/química , Cicatrização/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicações , Ratos , Interleucina-6/metabolismo , Interleucina-6/sangue , Proteína C-Reativa/metabolismoRESUMO
AIMS: To explore the role of voltage-gated calcium channels (VGCC) in 5-HT2A/2C receptor agonist 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride's improvement of spinal cord injury (SCI) induced detrusor sphincter dyssynergia and the expressions of the 5-hydroxy tryptamine (5-HT) 2A receptors and VGCCs in lumbosacral cord after SCI. METHODS: Female Sprague-Dawley rats were randomized into normal control group and SCI group (N = 15 each). Cystometrogram (CMG), simultaneous CMG, and external urethral sphincter electromyography (EUS-EMG) were conducted in all groups under urethane anesthesia. Drugs were administered intrathecally during CMG and EUS-EMG. Rats were euthanized and L6-S1 spinal cord were acquired for immunofluorescence. RESULTS: In SCI rats, intrathecal administration of 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride or L-type VGCC blocker, nifedipine, could significantly increase voiding volume, voiding efficiency, and the number of high-frequency oscillations. They could also prolong EUS bursting activity duration on EUS-EMG. Moreover, the effect of 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride can be eliminated with the combined administration of L-type VGCC agonist, (±)-Bay K 8644. No significant differences were observed in CMG after intrathecal administration of T-type VGCC blocker TTA-P2. Additionally, immunofluorescence of the lumbosacral cord in control and SCI rats showed that the 5-HT2A receptor and Cav1.2 immunolabeling-positive neurons in the anterior horn of the lumbosacral cord were increased in SCI rats. CONCLUSIONS: Our study demonstrated that 5-HT2A/2C agonist 2,5-dimethoxy-4-iodophenyl-2-aminopropane hydrochloride may improve SCI-induced DSD by inhibiting the L-type voltage-gated calcium channel in lumbosacral cord motoneurons.
Assuntos
Canais de Cálcio Tipo L , Ratos Sprague-Dawley , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/complicações , Feminino , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Ratos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , AnfetaminasRESUMO
INTRODUCTION: Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. METHODS: We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. RESULTS: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. CONCLUSION: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.
Assuntos
Autofagia , Mitocôndrias , Proteínas Quinases , Receptores de Quinase C Ativada , Traumatismo por Reperfusão , Ubiquitina-Proteína Ligases , Animais , Humanos , Ratos , Apoptose/fisiologia , Autofagia/fisiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Linhagem Celular Tumoral , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias , Neuroproteção/fisiologia , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Ratos Sprague-Dawley , Receptores de Quinase C Ativada/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
AIMS: Cerebral aneurysm (CA) has been considered one of the most common cerebrovascular diseases, affecting millions of people worldwide. A therapeutic agent is currently missing for the treatment of CA. Nesfatin-1 (Nes-1) is an 82-amino acid adipokine which possesses a wide range of biological functions. However, the physiological function of Nes-1 in CA is still unknown. Here, we aimed to assess the preventive effects of Nes-1 in the pathological development of CA and elucidate the mechanisms behind this. METHODS: We used an elastase-induced CA model, accompanied by a high-salt diet to induce hypertension. Additionally, diverse experimental techniques, including Verhoeff-Van Gieson staining, real time PCR, enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence staining, were employed to assess CA formation, gene and protein expression, as well as the macrophage infiltration. RESULTS: Our results indicate that administration of Nes-1 significantly decreased the aneurysm size. Additionally, Nes-1 prevented inflammatory response by inhibiting the expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) at both the mRNA and protein levels in the Circle of Willis (COW) region. Also, the increased levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in the COW region were reduced by Nes-1. We found that Nes-1 administration suppressed the invasion of macrophages. Mechanistically, Nes-1 activated Nrf-2 by promoting its nuclear translocation but prevented the activation of the IκBα/NF-κB signaling pathway. CONCLUSION: These findings suggest that Nes-1 might be used as a promising agent for the prevention of CA.
Assuntos
Aneurisma Intracraniano , Fator 2 Relacionado a NF-E2 , NF-kappa B , Nucleobindinas , Transdução de Sinais , Animais , Masculino , Ratos , Aneurisma Intracraniano/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The cardioprotective effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) in adult hearts is mediated by nitric oxide (NO). During the early developmental period, rat hearts exhibit higher resistance to ischemia-reperfusion (I/R) injury, contain higher levels of serum nitrates, and their resistance cannot be further increased by IPC or IPoC. NOS blocker (L-NAME) lowers their high resistance. Wistar rat hearts (postnatal Days 1 and 10) were perfused according to Langendorff and exposed to 40 min of global ischemia followed by reperfusion with or without IPoC. NO and reactive oxygen species donors (DEA-NONO, SIN-1) and L-NAME were administered. Tolerance to ischemia decreased between Days 1 and 10. DEA-NONO (low concentrations) significantly increased tolerance to I/R injury on both Days 1 and 10. SIN-1 increased tolerance to I/R injury on Day 10, but not on Day 1. L-NAME significantly reduced resistance to I/R injury on Day 1, but actually increased resistance to I/R injury on Day 10. Cardioprotection by IPoC on Day 10 was not affected by either NO donors or L-NAME. It can be concluded that resistance of the neonatal heart to I/R injury is NO dependent, but unlike in adult hearts, cardioprotective interventions, such as IPoC, are most likely NO independent.
Assuntos
Animais Recém-Nascidos , Pós-Condicionamento Isquêmico , Traumatismo por Reperfusão Miocárdica , NG-Nitroarginina Metil Éster , Óxido Nítrico , Ratos Wistar , Animais , Óxido Nítrico/metabolismo , Pós-Condicionamento Isquêmico/métodos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , NG-Nitroarginina Metil Éster/farmacologia , Precondicionamento Isquêmico Miocárdico/métodos , Doadores de Óxido Nítrico/farmacologia , Masculino , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Molsidomina/farmacologia , Molsidomina/análogos & derivadosRESUMO
BACKGROUND: Myocardial infarction (MI) is a major disease with high morbidity and mortality worldwide. However, existing treatments are far from satisfactory, making the exploration of potent molecular targets more imperative. The E3 ubiquitin ligase RING finger protein 5 (RNF5) has been previously reported to be involved in several diseases by regulating ubiquitination-mediated protein degradation. Nevertheless, few reports have focused on its function in cardiovascular diseases, including MI. METHODS: In this study, we established RNF5 knockout mice through precise CRISPR-mediated genome editing and utilized left anterior descending coronary artery ligation in 9-11-week-old male C57BL/6 mice. Subsequently, serum biochemical analysis and histopathological examination of heart tissues were performed. Furthermore, we engineered adenoviruses for modulating RNF5 expression and subjected neonatal rat cardiomyocytes to oxygen-glucose deprivation (OGD) to mimic ischemic conditions, demonstrating the impact of RNF5 manipulation on cellular viability. Gene and protein expression analysis provided insights into the molecular mechanisms. Statistical methods were rigorously employed to assess the significance of experimental findings. RESULTS: We found RNF5 was downregulated in infarcted heart tissue of mice and NRCMs subjected to OGD treatment. RNF5 knockout in mice resulted in exacerbated heart dysfunction, more severe inflammatory responses, and increased apoptosis after MI surgery. In vitro, RNF5 knockdown exacerbated the OGD-induced decline in cell activity, increased apoptosis, while RNF5 overexpression had the opposite effect. Mechanistically, it was proven that the kinase cascade initiated by apoptosis signal-regulating kinase 1 (ASK1) activation was closely regulated by RNF5 and mediated RNF5's protective function during MI. CONCLUSIONS: We demonstrated the protective effect of RNF5 on myocardial infarction and its function was dependent on inhibiting the activation of ASK1, which adds a new regulatory component to the myocardial infarction associated network and promises to enable new therapeutic strategy.
Assuntos
Apoptose , Modelos Animais de Doenças , MAP Quinase Quinase Quinase 5 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio , Miócitos Cardíacos , Transdução de Sinais , Ubiquitina-Proteína Ligases , Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , MAP Quinase Quinase Quinase 5/metabolismo , MAP Quinase Quinase Quinase 5/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Masculino , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células Cultivadas , Camundongos , Função Ventricular Esquerda , Hipóxia Celular , RatosRESUMO
There is no effective and noninvasive solution for thrombolysis because the mechanism by which certain thrombi become tissue plasminogen activator (tPA)-resistant remains obscure. Endovascular thrombectomy is the last option for these tPA-resistant thrombi, thus a new noninvasive strategy is urgently needed. Through an examination of thrombi retrieved from stroke patients, we found that neutrophil extracellular traps (NETs), ε-(γ-glutamyl) lysine isopeptide bonds and fibrin scaffolds jointly comprise the key chain in tPA resistance. A theranostic platform is designed to combine sonodynamic and mechanical thrombolysis under the guidance of ultrasonic imaging. Breakdown of the key chain leads to a recanalization rate of more than 90% in male rat tPA-resistant occlusion model. Vascular reconstruction is observed one month after recanalization, during which there was no thrombosis recurrence. The system also demonstrates noninvasive theranostic capabilities in managing pigs' long thrombi (>8 mm) and in revascularizing thrombosis-susceptible tissue-engineered vascular grafts, indicating its potential for clinical application. Overall, this noninvasive theranostic platform provides a new strategy for treating tPA-resistant thrombi.
Assuntos
Terapia Trombolítica , Trombose , Ativador de Plasminogênio Tecidual , Animais , Ativador de Plasminogênio Tecidual/uso terapêutico , Humanos , Trombose/diagnóstico por imagem , Trombose/tratamento farmacológico , Masculino , Ratos , Terapia Trombolítica/métodos , Armadilhas Extracelulares/metabolismo , Suínos , Fibrinolíticos/uso terapêutico , Fibrinolíticos/farmacologia , Ratos Sprague-Dawley , Modelos Animais de Doenças , Fibrina/metabolismo , Nanomedicina Teranóstica/métodos , Resistência a Medicamentos , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/terapia , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Accumulated evidence suggested that gut microbial dysbiosis interplayed with progressive chronic kidney disease (CKD). However, no available therapy is effective in suppressing progressive CKD. Here, using microbiomics in 480 participants including healthy controls and patients with stage 1-5 CKD, we identified an elongation taxonomic chain Bacilli-Lactobacillales-Lactobacillaceae-Lactobacillus-Lactobacillus johnsonii correlated with patients with CKD progression, whose abundance strongly correlated with clinical kidney markers. L. johnsonii abundance reduced with progressive CKD in rats with adenine-induced CKD. L. johnsonii supplementation ameliorated kidney lesion. Serum indole-3-aldehyde (IAld), whose level strongly negatively correlated with creatinine level in CKD rats, decreased in serum of rats induced using unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (NX) as well as late CKD patients. Treatment with IAld dampened kidney lesion through suppressing aryl hydrocarbon receptor (AHR) signal in rats with CKD or UUO, and in cultured 1-hydroxypyrene-induced HK-2 cells. Renoprotective effect of IAld was partially diminished in AHR deficiency mice and HK-2 cells. Our further data showed that treatment with L. johnsonii attenuated kidney lesion by suppressing AHR signal via increasing serum IAld level. Taken together, targeting L. johnsonii might reverse patients with CKD. This study provides a deeper understanding of how microbial-produced tryptophan metabolism affects host disease and discovers potential pathways for prophylactic and therapeutic treatments for CKD patients.
Assuntos
Lactobacillus johnsonii , Insuficiência Renal Crônica , Insuficiência Renal Crônica/microbiologia , Insuficiência Renal Crônica/terapia , Insuficiência Renal Crônica/patologia , Animais , Ratos , Humanos , Camundongos , Masculino , Lactobacillus johnsonii/genética , Indóis , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Microbioma Gastrointestinal , FemininoRESUMO
Peripheral nerve defect are common clinical problem caused by trauma or other diseases, often leading to the loss of sensory and motor function in patients. Autologous nerve transplantation has been the gold standard for repairing peripheral nerve defects, but its clinical application is limited due to insufficient donor tissue. In recent years, the application of tissue engineering methods to synthesize nerve conduits for treating peripheral nerve defect has become a current research focus. This study introduces a novel approach for treating peripheral nerve defects using a tissue-engineered PLCL/SF/NGF@TA-PPy-RGD conduit. The conduit was fabricated by combining electrospun PLCL/SF with an NGF-loaded conductive TA-PPy-RGD gel. The gel, synthesized from RGD-modified tannic acid (TA) and polypyrrole (PPy), provides growth anchor points for nerve cells. In vitro results showed that this hybrid conduit could enhance PC12 cell proliferation, migration, and reduce apoptosis under oxidative stress. Furthermore, the conduit activated the PI3K/AKT signalling pathway in PC12 cells. In a rat model of sciatic nerve defect, the PLCL/SF/NGF@TA-PPy-RGD conduit significantly improved motor function, gastrocnemius muscle function, and myelin sheath axon thickness, comparable to autologous nerve transplantation. It also promoted angiogenesis around the nerve defect. This study suggests that PLCL/SF/NGF@TA-PPy-RGD conduits provide a conducive environment for nerve regeneration, offering a new strategy for peripheral nerve defect treatment, this study provided theoretical basis and new strategies for the research and treatment of peripheral nerve defect.
Assuntos
Hidrogéis , Fator de Crescimento Neural , Regeneração Nervosa , Oligopeptídeos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Nervo Isquiático , Transdução de Sinais , Animais , Regeneração Nervosa/efeitos dos fármacos , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células PC12 , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Oligopeptídeos/farmacologia , Oligopeptídeos/química , Hidrogéis/química , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Ratos Sprague-Dawley , Masculino , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Polímeros/químicaRESUMO
BACKGROUND: Rat models are valuable tools to study the lung microbiota in diseases. Yet the impacts of different lung parts, young and mature adult stages, and the different batches of the same conditions on the healthy rat lung microbiome have not been investigated. METHODS: The rat lung microbiome was analyzed to clarify the lung part-dependent and age-dependent differences and to evaluate the effects of several 'batch environmental factors' on normal rats, after eliminating potential contamination. RESULTS: The results showed that the contamination could be identified and excluded. The lung microbiome from left and right lung parts was very similar so one representative part could be used in the microbiome study. There were significantly different lung microbial communities between the young and mature adult groups, and also between the different feeding batches groups of the same repetitive feeding conditions, but a common lung microbiota characterized by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria as the most dominant phyla were present in all adult rats. It indicated that the experiment under the same condition of the same rats batch was needed to compare the difference in the lung microbiota and repeated experiments were necessary to confirm the results. CONCLUSION: These data represented that the lung bacterial communities were dynamic and rapidly susceptible to environmental influence, clustered strongly by age or different feeding batches but similar in the different lung tissue parts. This study improved the basic understanding of the potential effects on the lung microbiome of healthy rats.
Assuntos
Pulmão , Microbiota , Animais , Pulmão/microbiologia , Ratos/microbiologia , Masculino , Fatores Etários , Ratos Sprague-Dawley , Bactérias/classificação , Bactérias/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
Huanglian Jiedu decoction (HLJD) has been used to treat ischemic stroke in clinic. However, the detailed protective mechanisms of HLJD on ischemic stroke have yet to be elucidated. The aim of this study is to elucidate the underlying pharmacological mechanisms of HLJD based on the inhibition of neuroinflammation and the amelioration of nerve cell damage. A middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats and received HLJD treatment. Effects of HLJD on neurological function was assessed based on Bederson's score, postural reflex test and asymmetry score. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining, Hematein and eosin (HE) and Nissl staining were used to observe the pathological changes in brain. Then, transcriptomics was used to screen the differential genes in brain tissue in MCAO/R model rats following HLJD intervention. Subsequently, the effects of HLJD on neutrophil extracellular trap (NET) formation-related neuroinflammation, gamma-aminobutyric acid (GABA)ergic synapse activation, nerve cell damage and proliferation were validated using immunofluorescence, western blot and enzyme-linked immunosorbent assay (ELISA). Our results showed that HLJD intervention reduced the Bederson's score, postural reflex test score and asymmetry score in MCAO/R model rats. Pathological staining indicated that HLJD treatment decreased the cerebral infarction area, mitigated neuronal damage and increased the numbers of Nissl bodies. Transcriptomics suggested that HLJD affected 435 genes in MCAO/R rats. Among them, several genes involving in NET formation and GABAergic synapses pathways were dysregulated. Subsequent experimental validation showed that HLJD reduced the MPO+CitH3+ positive expression area, reduced the protein expression of PAD4, p-P38/P38, p-ERK/ERK and decreased the levels of IL-1ß, IL-6 and TNF-α, reversed the increase of Iba1+TLR4+, Iba1+p65+ and Iba1+NLRP3+ positive expression area in brain. Moreover, HLJD increased GABA levels, elevated the protein expression of GABRG1 and GAT3, decreased the TUNEL positive expression area and increased the Ki67 positive expression area in brain. HLJD intervention exerts a multifaceted positive impact on ischemia-induced cerebral injury in MCAO/R rats. This intervention effectively inhibits neuroinflammation by mitigating NET formation, and concurrently improves nerve cell damage and fosters nerve cell proliferation through activating GABAergic synapses.
Assuntos
Isquemia Encefálica , Medicamentos de Ervas Chinesas , Ratos Sprague-Dawley , Sinapses , Animais , Medicamentos de Ervas Chinesas/farmacologia , Ratos , Masculino , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamento farmacológico , Modelos Animais de Doenças , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Infarto da Artéria Cerebral Média/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/complicações , Fármacos Neuroprotetores/farmacologia , Encéfalo/patologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacosRESUMO
Background: Pentagalloyl glucose (PGG) is a polyphenol with vasoprotective properties. Targeted delivery of PGG reversed aortic aneurysm growth in several rodent models associated with decreased number of macrophages and transforming growth factor-ß (TGF-ß) expression. Thus, we sought to determine cellular mechanisms by which PGG reduces macrophage-induced aortic pathogenicity and its relationship to TGF-ß. Methods: Using THP-1 cells, primary human aortic cells, and explanted rat aortas, we assessed the anti-inflammatory effect of PGG. Expression of pro/anti-inflammatory macrophage markers was analyzed. Adhesion of monocytes as well as oxidative stress status, viability, and TGF-ß expression after primary aortic cell exposure to macrophage-conditioned medium with and without PGG were assessed. The release of TGF-ß was also examined in elastase-treated cultured rat aortas. Results: PGG pre-treatment of human aortic cell monolayers reduced the adhesion of THP-1 monocytes. PGG enhanced the expression of anti-inflammatory markers in THP-1-derived macrophages, and increased mitochondrial reactive oxygen species as well as mitochondrial polarization. Conditioned medium from THP-1-derived macrophages induced reactive oxygen species, cell death, and TGF-ß release from human aortic cells, which was suppressed by PGG. In explanted rat aortas, PGG reduced elastase mediated TGF-ß release. Conclusions: Combining anti-inflammatory, cytotoxic, and oxidative effects, PGG has high cardiovascular therapeutic potential. We confirmed previous in vivo observations whereby PGG suppressed TGF-ß response associated with disease resolution.
Assuntos
Anti-Inflamatórios , Aorta , Taninos Hidrolisáveis , Macrófagos , Fator de Crescimento Transformador beta , Taninos Hidrolisáveis/farmacologia , Humanos , Animais , Fator de Crescimento Transformador beta/metabolismo , Células THP-1 , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Anti-Inflamatórios/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Adesão Celular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacosRESUMO
The precise assessment of vascular heterogeneity in brain tumors is vital for diagnosing, grading, predicting progression, and guiding treatment decisions. However, currently, there is a significant shortage of high-resolution imaging approaches. Herein, we propose a contrast-enhanced susceptibility-weighted imaging (CE-SWI) utilizing the minimalist dextran-modified Fe3O4 nanoparticles (Dextran@Fe3O4 NPs) for ultrahigh-resolution mapping of vasculature in brain tumors. The Dextran@Fe3O4 NPs are prepared via a facile coprecipitation method under room temperature, and exhibit small hydrodynamic size (28 nm), good solubility, excellent biocompatibility, and high transverse relaxivity (r2*, 159.7 mM-1 s-1) under 9.4 T magnetic field. The Dextran@Fe3O4 NPs-enhanced SWI can increase the contrast-to-noise ratio (CNR) of cerebral vessels to 2.5 times that before injection and achieves ultrahigh-spatial-resolution visualization of microvessels as small as 0.1 mm in diameter. This advanced imaging capability not only allows for the detailed mapping of both enlarged peritumoral drainage vessels and the intratumoral microvessels, but also facilitates the sensitive imaging detection of vascular permeability deterioration in a C6 cells-bearing rat glioblastoma model. Our proposed Dextran@Fe3O4 NPs-enhanced SWI provides a powerful imaging technique with great clinical translation potential for the precise theranostics of brain tumors.
Assuntos
Neoplasias Encefálicas , Dextranos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Animais , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Dextranos/química , Ratos , Meios de Contraste/química , Humanos , Linhagem Celular Tumoral , Tamanho da PartículaRESUMO
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
Although minimally invasive interventional occluders can effectively seal heart defect tissue, they still have some limitations, including poor endothelial healing, intense inflammatory response, and thrombosis formation. Herein, a polyphenol-reinforced medicine/peptide glycocalyx-like coating was prepared on cardiac occluders. A coating consisting of carboxylated chitosan, epigallocatechin-3-gallate (EGCG), tanshinone IIA sulfonic sodium (TSS), and hyaluronic acid grafted with 3-aminophenylboronic acid was prepared. Subsequently, the mercaptopropionic acid-GGGGG-Arg-Glu-Asp-Val peptide was grafted by the thiol-ene "click" reaction. The coating showed good hydrophilicity and free radical-scavenging ability and could release EGCG-TSS. The results of biological experiments suggested that the coating could reduce thrombosis by promoting endothelialization, and promote myocardial repair by regulating the inflammatory response. The functions of regulating cardiomyocyte apoptosis and metabolism were confirmed, and the inflammatory regulatory functions of the coating were mainly dependent on the NF-kappa B and TNF signaling pathway.
Assuntos
Glicocálix , Hidrogéis , Polifenóis , Animais , Hidrogéis/química , Hidrogéis/farmacologia , Polifenóis/química , Polifenóis/farmacologia , Glicocálix/metabolismo , Glicocálix/química , Glicocálix/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Apoptose/efeitos dos fármacos , Camundongos , Miocárdio/metabolismo , Catequina/química , Catequina/análogos & derivados , Catequina/farmacologia , Ratos Sprague-Dawley , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , MasculinoRESUMO
Milk exosomes (mExos) have demonstrated significant promise as vehicles for the oral administration of protein and peptide drugs owing to their superior capacity to traverse epithelial barriers. Nevertheless, certain challenges persist due to their intrinsic characteristics, including suboptimal drug loading efficiency, inadequate mucus penetration capability, and susceptibility to membrane protein loss. Herein, a hybrid vesicle with self-adaptive surface properties (mExos@DSPE-Hyd-PMPC) was designed by fusing functionalized liposomes with natural mExos, aiming to overcome the limitations associated with mExos and unlock their full potential in oral peptide delivery. The surface property transformation of mExos@DSPE-Hyd-PMPC was achieved by introducing a pH-sensitive hydrazone bond between the highly hydrophilic zwitterionic polymer and the phospholipids, utilizing the pH microenvironment on the jejunum surface. In comparison to natural mExos, hybrid vesicles exhibited a 2.4-fold enhancement in the encapsulation efficiency of the semaglutide (SET). The hydrophilic and neutrally charged surfaces of mExos@DSPE-Hyd-PMPC in the jejunal lumen exhibited improved preservation of membrane proteins and efficient traversal of the mucus barrier. Upon reaching the surface of jejunal epithelial cells, the highly retained membrane proteins and positively charged surfaces of the hybrid vesicle efficiently overcame the apical barrier, the intracellular transport barrier, and the basolateral exocytosis barrier. The self-adaptive surface properties of the hybrid vesicle resulted in an oral bioavailability of 8.7% and notably enhanced the pharmacological therapeutic effects. This study successfully addresses some limitations of natural mExos and holds promise for overcoming the sequential absorption barriers associated with the oral delivery of peptides.
Assuntos
Exossomos , Lipossomos , Leite , Propriedades de Superfície , Animais , Administração Oral , Exossomos/química , Exossomos/metabolismo , Lipossomos/química , Leite/química , Peptídeos/química , Humanos , Sistemas de Liberação de Medicamentos , Camundongos , Ratos Sprague-Dawley , Ratos , MasculinoRESUMO
Purpose: Fabry disease is an X-linked lysosomal storage disorder that results in multi-systemic renal, cardiovascular, and neuropathological damage, including in the eyes. We evaluated anterior segment ocular abnormalities based on age, sex (male and female), and genotype (wild-type, knockout [KO] male, heterozygous [HET] female, and KO female) in a rat model of Fabry disease. Methods: The α-Gal A KO and WT rats were divided into young (6-24 weeks), adult (25-60 weeks), and aged (61+ weeks) groups. Intraocular pressure (IOP) was measured. Eyes were clinically scored for corneal and lens opacity as well as evaluated for corneal epithelial integrity and tear break-up time (TBUT). Anterior chamber depth (ACD) and central corneal thickness (CCT) using anterior segment-optical coherence tomography (AS-OCT). Results: The Fabry rats showed an age-dependent increase in IOP, predominantly in the male genotype. TBUT was decreased in both male and female groups with aging. Epithelial integrity was defective in KO males and HET females with age. However, it was highly compromised in KO females irrespective of age. Corneal and lens opacities were severely affected irrespective of sex or genotype in the aging Fabry rats. AS-OCT quantification of CCT and ACD also demonstrated age-dependent increases but were more pronounced in Fabry versus WT genotypes. Conclusions: Epithelial integrity, corneal, and lens opacities worsened in Fabry rats, whereas IOP and TBUT changes were age-dependent. Similarly, CCT and ACD were age-related but more pronounced in Fabry rats, providing newer insights into the anterior segment ocular abnormalities with age, sex, and genotype in a rat model of Fabry disease.
Assuntos
Segmento Anterior do Olho , Modelos Animais de Doenças , Doença de Fabry , Pressão Intraocular , Tomografia de Coerência Óptica , Animais , Doença de Fabry/genética , Doença de Fabry/patologia , Doença de Fabry/fisiopatologia , Feminino , Masculino , Ratos , Segmento Anterior do Olho/patologia , Segmento Anterior do Olho/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Pressão Intraocular/fisiologia , Fatores Sexuais , Envelhecimento/fisiologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , alfa-Galactosidase/genéticaRESUMO
The pathogenesis mechanism of acute gastric mucosal lesions (AGML) is still unclear; further exploration is urgently needed to find a new therapeutic target. This study aimed to investigate whether morphine might regulate the expression and function of transient receptor potential ankyrin 1 (TRPA1) through a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent pathway, thereby alleviating gastric mucosal lesions caused by water-immersion restraint stress (WIRS). Rats were administered with intrathecal morphine, TRPA1 antagonist (HC-030031), µ-opioid receptor antagonist, or protein kinase A inhibitor (H-89), respectively, before WIRS. After 6 hours of WIRS, microscopic lesions, hematoxylin and eosin staining, and transmission electron microscopy were applied to assess the damage of the gastric mucosa. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were conducted to detect the levels of TRPA1 and substance P (SP) in the dorsal root ganglia (DRG) and gastric tissues. In addition, immunofluorescence was used to explore the possible co-expression of TRPA1 and µ-opioid receptors in the DRG. The results indicated that WIRS upregulated TRPA1 and SP in gastric mucosa, and HC-030031 or H-89 could alleviate gastric mucosal lesions caused by WIRS (P < .0001). Morphine was found to suppress both WIRS-induced gastric mucosal lesions (P < .0001) and the upregulation of TRPA1 (P = .0086) and SP (P = .0013). Both TRPA1 and SP play important roles in the pathogenesis of WIRS-induced AGML. Exogenous gastroprotective strategies reduce elevated levels of TRPA1 via the cAMP/PKA-dependent pathway. Inhibition of TRPA1 upregulation in the DRG is critical for intrathecal morphine preconditioning-induced gastric protection.