RESUMO
Paracingulin (CGNL1) is recruited to tight junctions (TJs) by ZO-1 and to adherens junctions (AJs) by PLEKHA7. PLEKHA7 has been reported to bind to the microtubule minus-end-binding protein CAMSAP3, to tether microtubules to the AJs. Here, we show that knockout (KO) of CGNL1, but not of PLEKHA7, results in the loss of junctional CAMSAP3 and its redistribution into a cytoplasmic pool both in cultured epithelial cells in vitro and mouse intestinal epithelium in vivo. In agreement, GST pulldown analyses show that CGNL1, but not PLEKHA7, interacts strongly with CAMSAP3, and the interaction is mediated by their respective coiled-coil regions. Ultrastructure expansion microscopy shows that CAMSAP3-capped microtubules are tethered to junctions by the ZO-1-associated pool of CGNL1. The KO of CGNL1 results in disorganized cytoplasmic microtubules and irregular nuclei alignment in mouse intestinal epithelial cells, altered cyst morphogenesis in cultured kidney epithelial cells, and disrupted planar apical microtubules in mammary epithelial cells. Together, these results uncover new functions of CGNL1 in recruiting CAMSAP3 to junctions and regulating microtubule cytoskeleton organization and epithelial cell architecture.
Assuntos
Microtúbulos , Junções Íntimas , Camundongos , Animais , Junções Íntimas/metabolismo , Microtúbulos/metabolismo , Células Epiteliais/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoplasma/metabolismo , Junções Aderentes/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Exosomes are 30-120nm bio particles transferred from donor to recipient cells leading to modification in their regulatory mechanisms depending upon the coded message in the form of loaded biomolecule. Cancer cells derived exosomes the true representatives of the parent cells have been found to modify the tumor surrounding/distinct regions and participate in metastasis, angiogenesis and immune suppression. Tis study was aimed to study the effects of tumor mice derived exosomes on the normal mice spleen isolated T cells by using co-culture experiments and flow cytometer analysis. We mainly focused on some of the T cells population and cytokines including IFN-γ, FOXP3+ regulatory T (Treg) cells and KI67 (proliferation marker). Overall results indicated random changes in different set of experiments, where the cancer derived exosomes reduced the IFN-γ expression in both CD4 and CD8 T cells, similarly the Treg cells were also found decreased in the presence of cancer exosomes. No significant changes were observed on the Ki67 marker expression. Such studies are helpful in understanding the role of cancer exosomes in immune cells suppression in tumor microenvironment. Cancer exosomes will need to be validated in vivo and in vitro on a molecular scale in detail for clinical applications.
Os exossomos são biopartículas de 30-120 nm transferidas de células doadoras para células receptoras, levando à modificação em seus mecanismos reguladores, dependendo da mensagem codificada na forma de biomolécula carregada. Verificou-se que exossomos derivados de células cancerosas os verdadeiros representantes das células-mãe modificam as regiões circundantes / distintas do tumor e participam da metástase, angiogênese e imunossupressão. Este estudo teve como objetivo estudar os efeitos de exossomos derivados de camundongos com tumor nas células T isoladas de baço de camundongos normais, usando experimentos de cocultura e análise de citômetro de fluxo. Concentrou-se, principalmente, em algumas populações de células T e citocinas, incluindo IFN-γ, células T reguladoras FOXP3 + (Treg) e KI67 (marcador de proliferação). Os resultados gerais indicaram mudanças aleatórias em diferentes conjuntos de experimentos, em que os exossomos derivados de câncer reduziram a expressão de IFN-γ em células T CD4 e CD8, da mesma forma que as células Treg também foram encontradas diminuídas na presença de exossomos de câncer. Nenhuma mudança significativa foi observada na expressão do marcador Ki67. Esses dados são úteis para a compreensão do papel dos exossomos do câncer na supressão de células do sistema imunológico no microambiente tumoral. Exossomos de câncer precisarão ser validados in vivo e in vitro em escala molecular com detalhes para aplicações clínicas.
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Animais , Camundongos , Exossomos , Microambiente Tumoral , Sistema Imunitário , Metástase Neoplásica , NeoplasiasRESUMO
Introduction and objectives: Aging is an irreversible process associated with decreased biological functions that can lead to the reduction of reproductive organs capacities in males and females. Paternal age is a significant predictor of offspring health and development. So, the aim of this study was to evaluate the effects of vitamin C on histopathological and biochemical testicular changes following aging process with a focus on stereological methods. Material and methods: For this study, 48 adult male NMRI mice were divided into two control and experimental groups. Mice in experimental group were supplemented with vitamin C (150mg/kg) including 24-h interval by oral gavage for 33 weeks. Same regime was performed for animals in control group except that vitamin C was replaced by water. Then, right testes were extracted for stereological and left testes were used for molecular analyses on weeks 8, 12, and 33. (AU)
Introducción y objetivos: El envejecimiento es un proceso irreversible asociado a una disminución de las funciones biológicas que puede conducir a la reducción de la capacidad de los órganos reproductivos en hombres y mujeres. La edad paterna es un predictor significativo de la salud y el desarrollo de la descendencia. Por lo tanto, el objetivo de este estudio fue evaluar los efectos de la vitamina C sobre los cambios testiculares histopatológicos y bioquímicos posteriores al proceso de envejecimiento con un enfoque en los métodos estereológicos.Material y métodos: Para este estudio, 48 ratones NMRI machos adultos se dividieron en dos grupos de control y experimentales. Los ratones del grupo experimental se suplementaron con vitamina C (150mg/kg), incluido un intervalo de 24 horas mediante sonda oral durante 33 semanas. Se realizó el mismo régimen para los animales del grupo de control, excepto que se reemplazó la vitamina C por agua. Luego, se extrajeron los testículos derechos para estereología y los testículos izquierdos se utilizaron para análisis moleculares en las semanas 8, 12 y 33. (AU)
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Humanos , Animais , Camundongos , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Testículo , Análise do Sêmen , Testosterona , Túbulos Seminíferos/patologiaRESUMO
Purpose: The present study aimed to construct a co-loading platform encapsulating curcumin and paclitaxel at ratios of 2:1-80:1 (w/w) designated "CU-PTX-LNP" and explored the synergistic effects of CU-PTX at different composite proportions on liver cancer cells using the combination index (CI) method. Methods: The CU lipid nanoplatform (CU-LNP) formulation was optimized via single-factor and orthogonal experiments. Various concentrations of PTX were added to the optimal formulation of CU-LNP to generate CU-PTX-LNP and the nanoplatform characterized via differential scanning calorimetry (DSC), transmission electron microscope (TEM), X-ray diffraction (XRD), zeta potential, polydispersity index (PDI), and size analyses. The cumulative release, stability, and cytotoxicity of CU-PTX-LNP in LO2, HepG2, and SMMC-7221 cells were assessed in vitro, followed by safety investigation and pharmacokinetic studies in vivo. The anti-tumor activity of CU-PTX-LNP was also evaluated using nude mice. Results: CU-PTX-LNP formulations containing CU:PTX at a range of proportions (2:1-80:1; w/w) appeared as uniformly dispersed nanosized spherical particles with high entrapment efficiency (EE> 90%), sustained release and long-lasting stability. Data from in vitro cytotoxicity assays showed a decrease in the IC50 value of PTX of CU-PTX-LNP (by 5.47-332.7 times in HepG2 and 4.29-143.21 times in SMMC-7221 cells) compared to free PTX. In vivo, CU-PTX-LNP displayed excellent biosafety, significant anti-tumor benefits and enhanced pharmacokinetic behavior with longer mean residence time (MRT(0-t); CU: 4.31-fold, PTX: 4.61-fold) and half-life (t1/2z; CU: 1.83-fold, PTX: 2.28-fold) relative to free drugs. Conclusion: The newly designed CU-PTX-LNP platform may serve as a viable technological support system for the successful production of CU-PTX composite preparations.
Assuntos
Carcinoma Hepatocelular , Curcumina , Neoplasias Hepáticas , Camundongos , Animais , Paclitaxel/farmacocinética , Curcumina/farmacologia , Camundongos Nus , Cobre , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Lipídeos/químicaRESUMO
BACKGROUND: New drugs are urgently needed for the treatment of liver cancer, a feat that could be feasibly accomplished by finding new therapeutic purposes for marketed drugs to save time and costs. As a new class of national anti-infective drugs, carrimycin (CAM) has strong activity against gram-positive bacteria and no cross resistance with similar drugs. Studies have shown that the components of CAM have anticancer effects. AIM: To obtain a deeper understanding of CAM, its distribution, metabolism and anti-inflammatory effects were assessed in the organs of mice, and its mechanism of action against liver cancer was predicted by a network pharmacology method. METHODS: In this paper, the content of isovaleryl spiramycin III was used as an index to assess the distribution and metabolism of CAM and its effect on inflammatory factors in various mouse tissues and organs. Reverse molecular docking technology was utilized to determine the target of CAM, identify each target protein based on disease type, and establish a target protein-disease type network to ascertain the effect of CAM in liver cancer. Then, the key action targets of CAM in liver cancer were screened by a network pharmacology method, and the core targets were verified by molecular docking and visual analyses. RESULTS: The maximum CAM concentration was reached in the liver, kidney, lung and spleen 2.5 h after intragastric administration. In the intestine, the maximum drug concentration was reached 0.5 h after administration. In addition, CAM significantly reduced the interleukin-4 (IL-4) levels in the lung and kidney and especially the liver and spleen; moreover, CAM significantly reduced the IL-1ß levels in the spleen, liver, and kidney and particularly the small intestine and lung. CAM is predicted to regulate related pathways by acting on many targets, such as albumin, estrogen receptor 1, epidermal growth factor receptor and caspase 3, to treat cancer, inflammation and other diseases. CONCLUSION: We determined that CAM inhibited inflammation. We also predicted the complex multitargeted effects of CAM that involve multiple pathways and the diversity of these effects in the treatment of liver cancer, which provides a basis and direction for further clinical research.
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Medicamentos de Ervas Chinesas , Neoplasias Hepáticas , Animais , Camundongos , Simulação de Acoplamento Molecular , Neoplasias Hepáticas/tratamento farmacológico , Inflamação/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêuticoRESUMO
Kawasaki disease (KD) is a multisystem vasculitis that predominantly targets the coronary arteries in young children. Epidemiological data suggest both environmental and genetic factors contribute to the susceptibility and severity of the disease. Mercury (Hg) is a known environmental pollutant and a Ca2+ signaling modulator. Ca2+ signaling regulates the activation of NLRP3 inflammasome. Using the Lactobacillus casei cell wall extract (LCWE) induced coronary arteritis mouse model of KD; we studied the effect of mercury on inflammasome activation and its impact on the immunopathogenesis of KD. Mercury enhances the expression of inflammasome activation resulting in caspase-1 mediated secretion of IL-1ß and IL-18 cytokines. In vivo, the administration of mercury together with disease inducing LCWE exacerbates disease resulting in increased incidence and severity of coronary arteritis compared to LCWE alone. Mercury can act as a novel danger signal modulating Ca2+ signaling to increase IL-1ß and IL-18 secretion and intensifies coronary arteritis in an animal model of KD.
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Arterite , Doença da Artéria Coronariana , Lacticaseibacillus casei , Mercúrio , Síndrome de Linfonodos Mucocutâneos , Animais , Camundongos , Síndrome de Linfonodos Mucocutâneos/genética , Interleucina-18 , Inflamassomos/metabolismo , Modelos Animais de Doenças , Doença da Artéria Coronariana/genéticaRESUMO
Introduction: Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Methods: Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). Results: In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-ß1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Discussion: Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.
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Distrofia Muscular de Duchenne , Animais , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/tratamento farmacológico , Fator de Crescimento Insulin-Like I , Secretagogos , Camundongos Endogâmicos C57BL , Inflamação/tratamento farmacológico , Inflamação/genética , Fibrose , Hormônio do CrescimentoRESUMO
The JAK/STAT and NFκB signaling pathways are two major inflammatory signaling pathways that are usually activated simultaneously in the body's inflammatory response to bacterial or viral infections. Hyperactivation of these two prominent signaling pathways is associated with various immune-related diseases and mortality, pointing to an urgent need for drug development targeting JAK/STAT and/or NFκB signaling. In this study, we screened 18,840 compounds using our well-established dual STAT-NFκB driven luciferase reporter based high-throughput screening system and identified a bioactive compound C498-0670, which inhibits both JAK/STAT and NFκB signaling. C498-0670 inhibits the activation of STATs and p-IKKα/ß in both the immortalized cell lines and primary peritoneal macrophages, while suppressing the expression of LPS-induced inflammatory mediators in vitro. In addition, the overall anti-inflammatory effects of C498-0670 were investigated using transcriptome sequencing and bioinformatics approaches. C498-0670 was predicted to alleviate sepsis/septic shock by disease/function analysis using IPA software, which was further verified in the LPS-induced mouse sepsis model in vivo. C498 reduced LPS-induced liver and kidney damage, myeloid cell infiltration, and pro-inflammatory cytokine and chemokine production in vivo. Furthermore, the SPR-HPLC-MS-based target fishing approach was used to identify the putative drug targets, and the high affinities of JAK2 (JAK/STAT signaling), NFKBIA (NFκB signaling), and IL-1ß, NLRP1b (inflammasome signaling) for C498-0670 were verified by molecular docking approach. These results suggest that C498-0670 can be used as a dual-target inhibitor of JAK/STAT and NFκB signaling pathways for the treatment of various inflammatory diseases, especially septic shock.
Assuntos
Lipopolissacarídeos , Choque Séptico , Camundongos , Animais , Lipopolissacarídeos/farmacologia , Choque Séptico/induzido quimicamente , Choque Séptico/tratamento farmacológico , Simulação de Acoplamento Molecular , Janus Quinases/metabolismo , Transdução de Sinais , NF-kappa B/metabolismoRESUMO
Introduction: Macrophages are central cells in mediating the inflammatory response. Objective and Methods: We evaluated the effect of high glucose conditions on the inflammatory profile and the autophagy pathway in Bone-Marrow Derived Macrophages (BMDM) from diabetic (D-BMDM) (alloxan: 60mg/kg, i.v.) and non-diabetic (ND-BMDM) C57BL/6 mice. BMDM were cultured in medium with normal glucose (5.5 mM), or high glucose (25 mM) concentration and were primed with Nigericin (20µM) stimulated with LPS (100 ng/mL) at times of 30 minutes; 2; 4; 6 and 24 hours, with the measurement of IL-6, IL-1ß and TNF-α cytokines. Results: We have further identified changes in the secretion of pro-inflammatory cytokines IL-6, IL-1ß and TNF-α, where BMDM showed increased secretion of these cytokines after LPS + Nigericin stimulation. In addition, changes were observed in the autophagy pathway, where the increase in the autophagic protein LC3b and Beclin-1 occurred by macrophages of non-diabetic animals in hyperglycemic medium, without LPS stimulation. D-BMDM showed a reduction on the expression of LC3b and Beclin-1, suggesting an impaired autophagic process in these cells. Conclusion: The results suggest that hyperglycemia alters the inflammatory pathways in macrophages stimulated by LPS, playing an important role in the inflammatory response of diabetic individuals.
Assuntos
Interleucina-6 , Fator de Necrose Tumoral alfa , Camundongos , Animais , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Proteína Beclina-1/metabolismo , Nigericina/farmacologia , Camundongos Endogâmicos C57BL , Macrófagos/metabolismo , Citocinas/metabolismo , Autofagia , Glucose/metabolismoRESUMO
Introduction: The evolving tumor secretes various immunosuppressive factors that reprogram the tumor microenvironment (TME) to become immunologically cold. Consequently, various immunosuppressive cells like Tregs are recruited into the TME which in turn subverts the anti-tumor response of dendritic cells and T cells.Tumor immunotherapy is a popular means to rejuvenate the immunologically cold TME into hot. Mycobacterium indicus pranii (MIP) has shown strong immunomodulatory activity in different animal and human tumor models and has been approved for treatment of lung cancer (NSCLC) patients as an adjunct therapy. Previously, MIP has shown TLR2/9 mediated activation of antigen presenting cells/Th1 cells and their enhanced infiltration in mouse melanoma but the underlying mechanism by which it is modulating these immune cells is not yet known. Results: This study reports for the first time that MIP immunotherapy involves type 1 interferon (IFN) signaling as one of the major signaling pathways to mediate the antitumor responses. Further, it was observed that MIP therapy significantly influenced frequency and activation of different subsets of T cells like regulatory T cells (Tregs) and CD8+ T cells in the TME. It reduces the migration of Tregs into the TME by suppressing the expression of CCL22, a Treg recruiting chemokine on DCs and this process is dependent on type 1 IFN. Simultaneously, in a type 1 IFN dependent pathway, it enhances the activation and effector function of the immunosuppressive tumor resident DCs which in turn effectively induce the proliferation and effector function of the CD8+ T cells. Conclusion: This study also provides evidence that MIP induced pro-inflammatory responses including induction of effector function of conventional dendritic cells and CD8+ T cells along with reduction of intratumoral Treg frequency are essentially mediated in a type 1 IFN-dependent pathway.
Assuntos
Mycobacterium , Neoplasias , Animais , Camundongos , Humanos , Linfócitos T CD8-Positivos , Células Dendríticas , Interferons , Microambiente TumoralRESUMO
Introduction: Sepsis is a result of initial over-activation of the immune system in response to an infection or trauma that results in reduced blood flow and life-threatening end-organ damage, followed by suppression of the immune system that prevents proper clearance of the infection or trauma. Because of this, therapies that not only limit the activation of the immune system early on, but also improve blood flow to crucial organs and reactivate the immune system in late-stage sepsis, may be effective treatments. The tyrosine kinase FES may fulfill this role. FES is present in immune cells and serves to limit immune system activation. We hypothesize that by enhancing FES in early sepsis and inhibiting its effects in late sepsis, the severity and outcome of septic illness can be improved. Methods and analysis: In vitro and in vivo modeling will be performed to determine the degree of inflammatory signaling, cytokine production, and neutrophil extracellular trap (NET) formation that occurs in wild-type (WT) and FES knockout (FES-/- ) mice. Clinically available treatments known to enhance or inhibit FES expression (lorlatinib and decitabine, respectively), will be used to explore the impact of early vs. late FES modulation on outcomes in WT mice. Bioinformatic analysis will be performed to examine FES expression levels in RNA transcriptomic data from sepsis patient cohorts, and correlate FES expression data with clinical outcomes (diagnosis of sepsis, illness severity, hospital length-of-stay). Ethics and dissemination: Ethics approval pending from the Queen's University Health Sciences & Affiliated Teaching Hospitals Research Ethics Board. Results will be disseminated through scientific publications and through lay summaries to patients and families.
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Armadilhas Extracelulares , Sepse , Animais , Camundongos , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Sistema ImunitárioRESUMO
BACKGROUND: Ferroptosis is involved in developing inï¬ammatory diseases; yet, its role in acute hypertriglyceridemic pancreatitis (HTGP) remains unclear. AIM: To explore whether ferroptosis is involved in the process of HTGP and elucidate its potential mechanisms. METHODS: An HTGP mouse model was induced using intraperitoneal injection of P-407 and caerulein (CAE). Then, pancreatic tissues from the model animals were subjected to proteome sequencing analysis. The pathological changes and scores of the pancreas, lung, and kidney were determined using hematoxylin-eosin staining. The levels of serum amylase (AMY), triglyceride, and total cholesterol were measured with an automatic blood cell analyzer. Additionally, the serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß were determined by enzyme linked immunosorbent assay. Malonaldehyde (MDA), glutathione (GSH), and Fe2+ were detected in the pancreas. Finally, immunohistochemistry was performed to assess the expression of ferroptosis-related proteins. RESULTS: Proteome sequencing revealed that ferroptosis was involved in the process of HTGP and that NADPH oxidase (NOX) 2 may participate in ferroptosis regulation. Moreover, the levels of serum AMY, TNF-α, IL-6, and IL-1ß were significantly increased, MDA and Fe2+ were upregulated, GSH and ferroptosis-related proteins were reduced, and the injury of the pancreas, lung, and kidney were aggravated in the P407 + CAE group compared to CAE and wild type groups (all P < 0.05). Notably, the inhibition of ferroptosis and NOX2 attenuated the pathological damage and the release of TNF-α, IL-6, and IL-1ß in the serum of the mice. CONCLUSION: Ferroptosis was found to have an important role in HTGP and may be considered a potential target for clinical treatment.
Assuntos
Ferroptose , Pancreatite , Camundongos , Animais , Interleucina-6 , Fator de Necrose Tumoral alfa , Proteoma , Pancreatite/tratamento farmacológico , Doença AgudaRESUMO
Peripheral neurotoxicity injury caused by local anesthetics is a common complication of clinical anesthesia. The study of its mechanism is helpful to prevent and treat the neurotoxic injury of local anesthetics. Previous studies on peripheral neurotoxicity injury caused by local anesthetics have mainly focused on in vitro cell experiments. Due to the lack of an animal model of peripheral neurotoxicity damage caused by local anesthetics, there are few in vivo experimental studies regarding this topic. Herein, 1% ropivacaine hydrochloride was injected into the sciatic nerve by direct incision and exposure of the sciatic nerve to create a local anesthetic neurotoxic injury model. The results showed that 1% ropivacaine hydrochloride could reduce the lower limb motor score and mechanical paw withdrawal threshold in mice 48 hours after injection. Pathological sections showed that 48 hours after treatment with 1% ropivacaine hydrochloride, the sciatic nerve showed increased axonal edema and degeneration, edema between nerve fiber bundles, increased degeneration of axon and myelin sheath vacuoles, edema of nerve bundle membrane and local degeneration and necrosis, and a large number of inflammatory cells around the nerve adventitia were soaked. The above results show that under open vision, 1% ropivacaine hydrochloride can cause injury to the sciatic nerve after 48 h of treatment, which can simulate the neurotoxic damage of local anesthetics. This animal model provides a research tool for studying the mechanism of neurotoxic injury caused by local anesthetics.
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Anestésicos Locais , Síndromes Neurotóxicas , Animais , Camundongos , Anestésicos Locais/toxicidade , Ropivacaina/toxicidade , Nervo Isquiático/patologia , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/patologia , Modelos Animais , EdemaRESUMO
Objective: To investigate the role of the CXC chemokine receptor 1 (CXCR1)/CXC chemokine ligand 8 (CXCL8) axis in the abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis (PBC). Methods: 30 female C57BL/6 mice were randomly divided into the PBC model group (PBC group), reparixin intervention group (Rep group), and blank control group (Con group) in an in vivo experiment. PBC animal models were established after 12 weeks of intraperitoneal injection of 2-octanoic acid coupled to bovine serum albumin (2OA-BSA) combined with polyinosinic acid polycytidylic acid (polyI:C). After successful modelling, reparixin was injected subcutaneously into the Rep group (2.5 mg · kg(-1) · d(-1), 3 weeks). Hematoxylin-eosin staining was used to detect histological changes in the liver. An immunohistochemical method was used to detect the expression of cytokeratin 19 (CK-19). Tumor necrosis factor-α (TNF-α), γ-interferon (IFN-γ) and interleukin (IL)-6 mRNA expression were detected by qRT-PCR. Western blot was used to detect nuclear transcription factor-κB p65 (NF-κB p65), extracellularly regulated protein kinase 1/2 (ERK1/2), phosphorylated extracellularly regulated protein kinase 1/2 (p-ERK1/2), Bcl-2-related X protein (Bax), B lymphoma-2 (Bcl-2), and cysteine proteinase-3 (Caspase- 3) expression. Human intrahepatic bile duct epithelial cells were divided into an IL-8 intervention group (IL-8 group), an IL-8+Reparicin intervention group (Rep group), and a blank control group (Con group) in an in vitro experiment. The IL-8 group was cultured with 10 ng/ml human recombinant IL-8 protein, and the Rep group was cultured with 10 ng/ml human recombinant IL-8 protein, followed by 100 nmol/L Reparicin. Cell proliferation was detected by the EdU method. The expression of TNF-α, IFN-γ and IL-6 was detected by an enzyme-linked immunosorbent assay. The expression of CXCR1 mRNA was detected by qRT-PCR. The expression of NF-κB p65, ERK1/2 and p-ERK1/2 was detected by western blot. A one-way ANOVA was used for comparisons between data sets. Results: The results of in vivo experiments revealed that the proliferation of cholangiocytes, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the Con group compared with the PBC group. However, reparixin intervention reversed the aforementioned outcomes (P<0.05). In vitro experiments showed that the proliferation of human intrahepatic cholangiocyte epithelial cells, the expression of CXCR1 mRNA, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the IL-8 group compared with the Con group. Compared with the IL-8 group, the proliferation of human intrahepatic cholangiocyte epithelial cells, NF-κB and ERK pathway-related proteins, and inflammatory indicators were significantly reduced in the Rep group (P < 0.05). Conclusion: The CXCR1/CXCL8 axis can regulate the abnormal proliferation of bile duct epithelial cells in PBC, and its mechanism of action may be related to NF-κB and ERK pathways.
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Interleucina-8 , Cirrose Hepática Biliar , Animais , Camundongos , Feminino , Humanos , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Receptores de Interleucina-8A/metabolismo , Cirrose Hepática Biliar/patologia , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Ductos Biliares/patologia , Interleucina-6 , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Interferon gama/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismoRESUMO
Hepatitis B virus (HBV) infection is a global health problem. Animal models are important for the study of the HBV infection mechanism. In the study related to the mouse model of HBV infection, the researchers have established a variety of mouse models, including transgenic, plasmid hydrodynamic injection, virus vector transfection, cccDNA cycle simulation, human and mouse liver chimerism, and liver/immune dual humanization, according to the characteristics of HBV infection. Herein, the research progress of these models is summarized. Notably, the application of these models can further clarify the mechanism of HBV infection under the conditions of a specific immune response in vivo and lay the foundation for the development of new antiviral drugs and immunotherapy for HBV infection.
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Vírus da Hepatite B , Hepatite B , Humanos , Camundongos , Animais , Vírus da Hepatite B/genética , Replicação Viral , Hepatite B/tratamento farmacológico , Antivirais/uso terapêutico , Modelos Animais de Doenças , DNA Viral/genéticaAssuntos
Neuralgia , Animais , Camundongos , Humanos , Neuralgia/etiologia , Modelos Animais de Doenças , HiperalgesiaRESUMO
Background: Osteoporosis is a metabolic bone disease. Osteoclasts are significantly involved in the pathogenesis of osteoporosis. AS-605240 (AS) is a small molecule PI3K-γ inhibitor and is less toxic compared to pan-PI3K inhibitors. AS also exerts multiple biological effects including anti-inflammatory, anti-tumor, and myocardial remodeling promotion. However, the involvement of AS in the differentiation and functions of osteoclasts and the effect of AS in treating patients with osteoporosis is still unclear. Purpose: This study aimed to investigate if AS inhibits the differentiation of osteoclasts and resorption of the bones induced by M-CSF and RANKL. Next, we evaluated the therapeutic effects of AS on bone loss in ovariectomy (OVX)-induced osteoporosis mice models. Methods: We stimulated bone marrow-derived macrophages with an osteoclast differentiation medium containing different AS concentrations for 6 days or 5µM AS at different times. Next, we performed tartrate-resistant acid phosphatase (TRAP) staining, bone resorption assay, F-actin ring fluorescence, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting (WB). Next, MC3T3-E1s (pre-osteoblast cells) were differentiated to osteoblast by stimulating the cells with varying AS concentrations. Next, we performed alkaline phosphatase (ALP) staining, RT-qPCR, and WB on these cells. We established an OVX-induced osteoporosis mice model and treated the mice with 20mg/kg of AS. Finally, we extracted the femurs and performed micro-CT scanning, H&E, and TRAP staining. Results: AS inhibits the formation of osteoclasts and resorption of bone triggered by RANKL by inhibiting the PI3K/Akt signaling pathway. Furthermore, AS enhances the differentiation of osteoblasts and inhibits bone loss due to OVX in vivo. Conclusion: AS inhibits osteoclast production and enhances osteoblast differentiation in mice, thus providing a new therapeutic approach for treating patients with osteoporosis.
Assuntos
Reabsorção Óssea , Osteoporose , Feminino , Animais , Camundongos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Osteoclastos , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Osteogênese , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Diferenciação Celular , OvariectomiaRESUMO
Purpose: Our study aims to reveal the pharmacological mechanism of Astragaloside IV in the treatment of pulmonary fibrosis(PF) through network pharmacology and experimental validation. Methods: We first determined the in vivo anti-pulmonary fibrosis effect of Astragaloside IV by HE, MASSON staining, and lung coefficients, then used network pharmacology to predict the signaling pathways and molecularly docked key pathway proteins, and finally validated the results by in vivo and in vitro experiments. Results: In in vivo experiments, we found that Astragaloside IV improved body weight (P < 0.05), increased lung coefficients (P < 0.05), and reduced lung inflammation and collagen deposition in mice with pulmonary fibrosis. The network pharmacology results showed that Astragaloside IV had 104 cross-targets with idiopathic pulmonary fibrosis, and the results of KEGG enrichment analysis indicated that cellular senescence could be an important pathway for Astragaloside IV in the treatment of pulmonary fibrosis. Astragaloside IV also bound well to senescence-associated proteins, according to molecular docking results. The results of both in vivo and in vitro experiments showed that Astragaloside IV significantly inhibited senescence protein markers such as P53, P21, and P16 and delayed cellular senescence (P < 0.05). In in vivo experiments, we also found that Astragaloside IV reduced the production of SASPs (P < 0.05), and in in vitro experiments, Astragaloside IV also reduced the production of ROS. In addition, by detecting epithelial-mesenchymal transition(EMT)-related marker protein expression, we also found that Astragaloside IV significantly inhibited the development of EMT in both in vivo and in vitro experiments (P < 0.05). Conclusion: Our research found that Astragaloside IV could alleviate bleomycin-induced PF by preventing cellular senescence and EMT.
Assuntos
Bleomicina , Fibrose Pulmonar Idiopática , Camundongos , Animais , Simulação de Acoplamento Molecular , Farmacologia em Rede , Transição Epitelial-MesenquimalRESUMO
Background: Inflammation is closely associated with the pathogenesis of various ocular diseases. Uveitis is a condition characterized by the inflammation of the uvea and ocular tissues that causes extreme pain, decreases visual acuity, and may eventually lead to blindness. The pharmacological functions of morroniside, isolated from Cornus officinalis, are multifarious. Morroniside exerts various therapeutic effects, e.g., it ameliorates inflammation. However, the specific anti-inflammatory effect of morroniside on lipopolysaccharide-induced uveitis has not been reported widely. In this study, we investigated the anti-inflammatory effect of morroniside on uveitis in mice. Methods: An endotoxin-induced uveitis (EIU) mouse model was constructed and treated with morroniside. The inflammatory response was observed using slit lamp microscopy, and histopathological changes were observed by hematoxylin-eosin staining. The cell count in the aqueous humor was measured using a hemocytometer. The concentrations of TNF-α, IL-6, and IL-1ß in the ciliary body and retina were measured using ELISA kits. The expression of iNOS and Arg-1 in the ciliary body and retina was measured by immunofluorescence costaining, and western blotting was performed to measure the protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 in the ciliary body and retina. Results: Morroniside effectively ameliorated the inflammatory response in EIU mice. Furthermore, morroniside significantly reduced the concentrations of IL-1ß, IL-6, and TNF-α in the ciliary body and retina. Morroniside treatment significantly reduced the expression of iNOS in the ciliary body and retinal tissues. It also significantly inhibited p-JAK2 and p-STAT3 expression and promoted Arg-1 expression. In addition, morroniside boosted the effect of JAK inhibitors on the above indices. Conclusions: Collectively, these findings suggest that morroniside may protect against LPS-induced inflammation in uveitis by promoting M2 polarization through the inhibition of the JAK/STAT pathway.
Assuntos
Endotoxinas , Uveíte , Camundongos , Animais , Endotoxinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição STAT/metabolismo , Uveíte/induzido quimicamente , Uveíte/tratamento farmacológico , Uveíte/patologia , Corpo Ciliar/metabolismo , Corpo Ciliar/patologia , Lipopolissacarídeos/farmacologia , Inflamação/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Macrófagos/metabolismoRESUMO
How to achieve stable co-delivery of multiple phytochemicals is a common problem. This study focuses on the development, optimization and characterization of Huanglian-HouPo extract nanoemulsion (HLHPEN), with multiple components co-delivery, to enhance the anti-ulcerative colitis (UC) effects. The formulation of HLHPEN was optimized by pseudo-ternary phase diagram combined with Box-Behnken design. The physicochemical properties of HLHPEN were characterized, and its anti-UC activity was evaluated in DSS-induced UC mice model. Based on preparation process optimization, the herbal nanoemulsion HLHPEN was obtained, with the droplet size, PDI value, encapsulation efficiency (EE) for 6 phytochemicals (berberine, epiberberine, coptisine, bamatine, magnolol and honokiol) of 65.21 ± 0.82 nm, 0.182 ± 0.016, and 90.71 ± 0.21%, respectively. The TEM morphology of HLHPEN shows the nearly spheroidal shape of particles. The optimized HLHPEN showed a brownish yellow milky single-phase and optimal physical stability at 25 °C for 90 days. HLHPEN exhibited the good particle stability and gradual release of phytochemicals in SGF and SIF, to resist the destruction of simulated stomach and small intestine environment. Importantly, the oral administration of HLHPEN significantly restored the shrunk colon tissue length and reduced body weight, ameliorated DAI value and colon histological pathology, decreased the levels of inflammatory factors in DSS-induced UC mice model. These results demonstrated that HLHPEN had a significant therapeutic effect on DSS-induced UC mice, as a potential alternative UC therapeutic agent.
