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An efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is lacking. Here, we develop CoBATCH, combinatorial barcoding and targeted chromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions, and Tn5 generates indexed chromatin fragments ready for library preparation and sequencing. Importantly, this strategy enables not only low-input epigenomic profiling in intact tissues but also measures scalable up to tens of thousands of single cells per experiment under both native and cross-linked conditions. CoBATCH produces â¼12,000 reads/cell with extremely low background. Mapping of endothelial cell lineages from ten embryonic mouse organs through CoBATCH allows for efficient deciphering of epigenetic heterogeneity of cell populations and cis-regulatory mechanisms. Thus, obviating specialized devices, CoBATCH is broadly applicable and easily deployable for single-cell profiling of protein-DNA interactions.
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Cromatina/genética , Epigenoma , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Acetilação , Animais , Linhagem Celular , Cromatina/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.
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Ácidos Nucleicos Livres , Proteínas Proto-Oncogênicas B-raf , Padrões de Referência , Humanos , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/sangue , Proteínas Proto-Oncogênicas B-raf/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Classe I de Fosfatidilinositol 3-Quinases/genética , Receptores ErbB/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Biópsia Líquida/métodos , Biópsia Líquida/normas , Linhagem Celular Tumoral , Frequência do GeneRESUMO
Key features of Alzheimer's disease include neuronal loss, accumulation of beta-amyloid plaques, and formation of neurofibrillary tangles. These changes are due in part to abnormal protein metabolism, particularly the accumulation of amyloid beta. Mitochondria are the energy production centers within cells and are also the main source of oxidative stress. In AD, mitochondrial function is impaired, leading to increased oxidative stress and the production of more reactive oxidative substances, further damaging cells. Mitophagy is an important mechanism for maintaining mitochondrial health, helping to clear damaged mitochondria, prevent the spread of oxidative stress, and reduce abnormal protein aggregation. To this end, this article conducts an integrated analysis based on DNA methylation and transcriptome data of AD. After taking the intersection of the genes where the differential methylation sites are located and the differential genes, machine learning methods were used to build an AD diagnostic model. This article screened five diagnostic genes ATG12, CSNK2A2, CSNK2B, MFN1 and PGAM5 and conducted experimental verification. The diagnostic genes discovered and the diagnostic model constructed in this article can provide reference for the development of clinical diagnostic models for AD.
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Doença de Alzheimer , Autofagia , Metilação de DNA , Mitocôndrias , Doença de Alzheimer/genética , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Autofagia/genética , Metilação de DNA/genética , Biomarcadores/metabolismo , Mitofagia/genética , Transcriptoma/genética , Aprendizado de Máquina , MultiômicaRESUMO
Nucleic acid testing is a powerful tool for the detection of various pathogens. Respiratory syncytial virus (RSV) is a major cause of acute respiratory infection, especially in young children and infants. To improve the confidence and reliability of nucleic acid testing results for RSV, reference materials (RMs) of both type A and B of RSV were developed by the National Institute of Metrology, China, code numbers NIM-RM 4057 and 4058. The reference material was composed of in vitro transcribed RNA containing the nucleocapsid (N) gene, matrix (M) gene, and partial polymerase (L) gene of RSV. A duplex reverse transcription digital PCR method was established with limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) of 2, 5, and 23 copies per reaction for RSV-A and 4, 8, and 20 copies per reaction for RSV-B. The certified value and expanded uncertainty (U, k = 2) of the two RMs were determined to be (6.1 ± 1.4) × 104 copies/µL for RSV-A and (5.3 ± 1.2) × 104 copies/µL for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays.
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Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Lactente , Criança , Humanos , Pré-Escolar , Vírus Sincicial Respiratório Humano/genética , Infecções por Vírus Respiratório Sincicial/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/análiseRESUMO
This research aims to explore the influence of transient pressure fluctuation inside high-speed trains passing throught tunnels on the fetal growth of Sprague - Dawley (SD) rats. A pressure variation simulation system was designed and exposure experiments were performed on SD rats. Forty-eight SD rats are divided into two control groups and two experimental groups, and are then exposed to transient pressure alternation (-1200 Pa ~1200 Pa) from gestation day 0 to gestation day 5 (GD 0-5). Fetal growth and development indicators on GD12 and GD18 between experimental and control groups were compared. Statistical results showed that, compared to the control group, the key indicators in the experimental group, including placental weight, placental diameter, fetal weight, and crown-to-rump length have decreased by 4.77%, 3.38%, 6.20%, and 3.75% respectively on GD18. The findings imply that the pressure fluctuation environment of high-speed trains has potential effects on the fetal growth of SD rats.
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Exploration and dissection of potential actions and effects of long noncoding RNA (lncRNA) in animals remain challenging. Here, using multiple knockout mouse models and single cell RNA sequencing, we demonstrate that the divergent lncRNA Hand2os1/Uph has a key complex modulatory effect on the expression of its neighboring gene HAND2 and subsequently on heart development and function. Short deletion of the Hand2os1 promoter in mouse diminishes Hand2os1 transcription to â¼8-32%, but fails to affect HAND2 expression and yields no discernable heart phenotypes. Interestingly, full-length deletion of Hand2os1 in mouse causes moderate yet prevalent upregulation of HAND2 in hundreds of cardiac cells, leading to profound biological consequences, including dysregulated cardiac gene programs, congenital heart defects and perinatal lethality. We propose that the Hand2os1 locus dampens HAND2 expression to restrain cardiomyocyte proliferation, thereby orchestrating a balanced development of cardiac cell lineages. This study highlights the regulatory complexity of the lncRNA Hand2os1 on HAND2 expression, emphasizing the need for complementary genetic and single cell approaches to delineate the function and primary molecular effects of an lncRNA in animals.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Loci Gênicos/fisiologia , Coração/embriologia , Organogênese/genética , RNA Longo não Codificante/genética , Animais , Linhagem da Célula/genética , Proliferação de Células/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Cardiopatias Congênitas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/fisiologia , Gravidez , RNA Longo não Codificante/fisiologiaRESUMO
At present, there is not enough research about the application of liraglutide nano preparations in perioperative neurocognitive dysfunction. Therefore, the purpose of this study is the mechanism of the effect of liraglutide nano preparations on perioperative neurocognitive dysfunction in aged mice. In this study, 140 male SD rats aged 6-8 weeks were used as the research object, and were divided into 4 groups (n=24) according to the random number table method, which were group C (control group), group S (model group), and treatment. Group (low-dose liraglutide pretreated control group) and DS2 group (high-dose liraglutide pretreated control group) were treated with liraglutide anesthesia to establish a cognitive dysfunction model. Morris water maze experiment was conducted 4 days after anesthesia to compare the escape latency and the number of crossings of the original platform in each group; after 4 days of anesthesia, 18 old mice were randomly selected from each group for fluorescence quantitative polymerase chain reaction (RealTimePCR) and protein Western blotting (Western.Blot) was used to determine the mRNA and protein levels of Caspase-3, Bax and Bcl-2 in the hippocampus; the remaining 6 old mice in each group were taken to observe the pathological changes of the hippocampus neurons by transmission electron microscopy . Compared with saline-treated group, the levels of NF-KB, TNF-a and IL-1ß protein in mice treated with liraglutide decreased and IkB increased significantly (p<0.05). Liraglutide intervention may alleviate non-alcoholic fatty liver in diabetic mice by reducing the expression of inflammatory genes in liver tissue, thereby improving neurocognitive dysfunction in mice.
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Disfunção Cognitiva , Diabetes Mellitus Experimental , Animais , Disfunção Cognitiva/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Hipocampo/metabolismo , Liraglutida/metabolismo , Liraglutida/farmacologia , Liraglutida/uso terapêutico , Masculino , Camundongos , NF-kappa B/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Previous studies have evaluated the prognostic factors of patients with stage IIIB cervical cancer. However, there was only one study evaluating the relationship between LTI (lower third of vaginal invasion) and the prognosis of the patients with stage IIIB cervical cancer. Our research aimed to assess different therapeutic outcomes of the stage IIIB CCP (cervical cancer patients) with or without LTI. METHODS: From December 2007 to December 2014, patients with FIGO (International Federation of Gynecology and Obstetrics, 2009) stage IIIB cervical cancer admitted and treated in Zhejiang Cancer Hospital were enrolled and evaluated in this retrospective research. Different clinicopathological variables and treatment outcomes were analyzed by using multivariate and univariate Cox regression models and chi-square or Fisher's exact test. RESULTS: The number of enrolled patients was 622, among which 74 cases were with LTI and 548 without. The two- and five-year OS (overall survival) rates in non-LTI group were 79.9% and 58.9%, and the OS rates in LTI group were 68.9% and 38.8%, respectively (P = 0.001). The two- and five-year PFS (progression-free survival) rates in non-LTI group were 63.3% and 53.1%, and the PFS rates in LTI group were 45.9% and 37.0% respectively (P = 0.002). Multivariate Cox regression analysis indicated that histological type, total treatment time, hydronephrosis, and treatment protocol were factors significantly affecting the PFS rates in stage IIIB CCP, and OS rates were associated with histological type, hydronephrosis, treatment protocol, and LTI. CONCLUSIONS: Our study showed that stage IIIB CCP with LTI had worse prognosis than those without LTI.
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Neoplasias do Colo do Útero/patologia , Vagina/patologia , Braquiterapia , Feminino , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias do Colo do Útero/radioterapiaRESUMO
RATIONALE: We hypothesized that the differentiation processes of cardiac progenitor cell (CP) from first and second heart fields (FHF and SHF) may undergo the unique instructive gene regulatory networks or signaling pathways, and the precise SHF progression is contingent on the FHF signaling developmental cues. OBJECTIVE: We investigated how the intraorgan communications control sequential building of discrete anatomic regions of the heart at single-cell resolution. METHODS AND RESULTS: By single-cell transcriptomic analysis of Nkx2-5 (NK2 homeobox 5) and Isl1 (ISL LIM homeobox 1) lineages at embryonic day 7.75, embryonic day 8.25, embryonic day 8.75, and embryonic day 9.25, we present a panoramic view of distinct CP differentiation hierarchies. Computational identifications of FHF- and SHF-CP descendants revealed that SHF differentiation toward cardiomyocytes underwent numerous step-like transitions, whereas earlier FHF progressed toward cardiomyocytes in a wave-like manner. Importantly, single-cell pairing analysis demonstrated that SHF-CPs were attracted to and expanded FHF-populated heart tube region through interlineage communications mediated by the chemotactic guidance (MIF [macrophage migration inhibitory factor]-CXCR2 [C-X-C motif chemokine receptor 2]). This finding was verified by pharmacological blockade of this chemotaxis in embryos manifesting limited SHF cell migration and contribution to the growth of the outflow tract and right ventricle but undetectable effects on the left ventricle or heart tube initiation. Genetic loss-of-function assay of Cxcr2 showed that the expression domain of CXCR4 was expanded predominantly at SHF. Furthermore, double knockout of Cxcr2/Cxcr4 exhibited defective SHF development, corroborating the redundant function. Mechanistically, NKX2-5 directly bound the Cxcr2 and Cxcr4 genomic loci and activated their transcription in SHF. CONCLUSIONS: Collectively, we propose a model in which the chemotaxis-mediated intraorgan crosstalk spatiotemporally guides the successive process of positioning SHF-CP and promoting primary heart expansion and patterning upon FHF-derived heart tube initiation.
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Quimiotaxia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteína Homeobox Nkx-2.5/metabolismo , Transcriptoma , Animais , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/citologia , Proteína Homeobox Nkx-2.5/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Triple-negative breast cancer (TNBC) lacks major effective target molecules and chemotherapy remains the current main treatment. However, traditional chemotherapy drugs, such as doxorubicin (DOX), cause serious side effects and have a poor prognosis. Piperlongumine (PL), a natural alkaloid, has showed selective anticancer effects and is expected to become a new strategy against TNBC. In our research, cell viability, colony formation, flow cytometry, Western blot, and tumor xenograft model assays were established to evaluate the suppression effect of PL and DOX alone and in combination. Data showed that PL could effectively inhibit cell growth and induce apoptosis in two TNBC cell lines. We also demonstrated for the first time that the combination treatment of PL and DOX synergistically inhibited cell growth and induced apoptosis in TNBC cells. The suppression of STAT3 activation was indicated to be a mechanism of the anticancer effect. Moreover, the effectiveness of this combination was confirmed in a tumor xenograft model. These results revealed that inhibition of the JAK2-STAT3 pathway was a key anticancer mechanism when treated with PL alone or combined with DOX, suggesting that the combination of PL and chemotherapy drugs may be a potential strategy for the clinical treatment of TNBC.
Assuntos
Dioxolanos/farmacologia , Doxorrubicina/farmacologia , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this paper, we report a green and controllable synthetic method of gold nanoparticles (AuNPs) by directly ß-CD reduction under mild conditions. Analysis of UV-vis spectra, along with TEM was applied to study the effects of experimental parameters on morphologies of the gold nanoparticles. The corresponding formation mechanism of the nanoparticles was evaluated by redox potential. In particular, compared with the traditional method of sodium citrate or ascorbic reduction, this method can facilely realize multi-dimensional regulation. On this basis, we further studied the dispersion behavior of the as-prepared gold nanoparticles in oil/water mixed system that would provide a possible strategy for optical sensor.
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NADPH oxidase (NOX) is a major source of reactive oxygen species (ROS) in the body and it plays a key role in mediation of oxidative injury in the cardiovascular system. The purposes of this study are to evaluate the status of NOX in endothelial progenitor cells (EPCs) of hyperlipidemic rats and to determine whether NOX-derived ROS promotes the dysfunction of EPCs. The rats were fed on a high-fat diet for 8 weeks to establish a hyperlipidemic rat model, which showed the increased plasma lipids and the impaired functions of circulating EPCs (including the reduced abilities in migration and adhesion) accompanied by an increase in NOX activity and ROS production. Next, EPCs were isolated from normal rats and they were treated with oxidized low-density lipoprotein (ox-LDL) (100 µg/mL) for 24 h to induce a dysfunctional model in vitro. In agreement with our findings in vivo, ox-LDL treatment increased the dysfunctions of EPCs concomitant with an increase in NOX activity and ROS production; these phenomena were reversed by the NOX inhibitor. Based on these observations, we conclude that NOX-derived ROS involved in the dysfunctions of circulating EPCs in hyperlipidemic rats and inhibition of NOX might provide a novel strategy to improve EPC functions in hyperlipidemia.
Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Progenitoras Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Hiperlipidemias/induzido quimicamente , Lipoproteínas LDL/farmacologia , Masculino , NADPH Oxidases/antagonistas & inibidores , Fenótipo , Ratos , Ratos Sprague-DawleyRESUMO
Myeloperoxidase (MPO)-derived product hypochlorous acid (HOCl) is able to induce cellular senescence and MPO is also expressed in endothelial cells besides the well-recognized immune cells. This study aims to clarify the association of endothelium-derived MPO with endothelial senescence in hyperlipidemia. The rats were fed with high-fat diet for 8 weeks to establish a hyperlipidemic model, which showed an increase in plasma lipids, endothelium-derived MPO expression, endothelial senescence and endothelial dysfunction concomitant with a reduction in glycogen synthase kinase 3 beta (GSK-3ß) activity and phosphorylated ß-catenin (p-ß-catenin) level as well as an increase in ß-catenin and p53 levels within the endothelium. Next, human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low density lipoprotein (ox-LDL, 100 µg/ml) for 24 h to establish a senescent cell model in vitro. Consistent with the finding in vivo, ox-LDL-induced MPO expression and HUVECs senescence, accompanied by a decrease in GSK-3ß activity and p-ß-catenin level as well as an increase in HOCl content, ß-catenin and p53 levels; these phenomena were attenuated by MPO inhibitor. Replacement of ox-LDL with HOCl could also induce HUVECs senescence and activate the ß-catenin/p53 pathway. Based on these observations, we conclude that endothelium-derived MPO is upregulated in hyperlipidemic rats, which may contribute to the accelerated vascular endothelial senescence through a mechanism involving the ß-catenin/p53 pathway.
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Células Endoteliais/metabolismo , Hiperlipidemias/metabolismo , Ácido Hipocloroso/metabolismo , Lipoproteínas LDL/metabolismo , Peroxidase/metabolismo , beta Catenina/metabolismo , Animais , Senescência Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hiperlipidemias/patologia , Ácido Hipocloroso/farmacologia , Lipídeos/sangue , Lipoproteínas LDL/farmacologia , Masculino , Peroxidase/química , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.
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Miosinas Cardíacas/fisiologia , Glicoproteínas de Membrana/genética , Miocárdio/metabolismo , Cadeias Leves de Miosina/fisiologia , NADPH Oxidases/genética , Animais , Miosinas Cardíacas/análise , Núcleo Celular/química , Células Cultivadas , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Cadeias Leves de Miosina/análise , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , NADPH Oxidase 2 , Estresse Oxidativo , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Recent studies show that resveratrol exerts beneficial effects on prevention of pulmonary hypertension. This study is performed to explore the effects of trimethoxystilbene, a novel resveratrol analog, on rat pulmonary vascular remodeling and right ventricular hypertrophy in hypoxia-induced pulmonary arterial hypertension (PAH) and the underlying mechanisms. Sprague-Dawley rats were placed in a chamber and exposed to 10% O(2) continuously for 4 weeks to induce PAH. The effects of trimethoxystilbene (5 or 10 mg/kg per day, intragastric [i.g.]) and resveratrol (as a positive control, 25 mg/kg per day, i.g.) on hypoxia-induced PAH vascular remodeling and right ventricle hypertrophy were evaluated. At the end of experiments, the index for pulmonary vascular remodeling and right ventricle hypertrophy, inflammatory cell infiltration in lung tissue, the plasma levels and lung tissue contents of hydrogen peroxide (H(2)O(2)), the mRNA and protein levels for NADPH oxidases (NOX2, NOX4) and vascular peroxidase 1 (VPO1) in pulmonary artery or right ventricle were measured. The results showed that trimethoxystilbene treatment significantly attenuated hypoxia-induced pulmonary vascular remodeling (such as decrease in the ratio of wall thickness to vessel external diameter) and right ventricle hypertrophy (such as decrease in the ratio of right ventricle weight to the length of the tibia), accompanied by downregulation of NOX2, NOX4, and VPO1 expression in pulmonary artery or right ventricle, decrease in H(2)O(2) production and inflammatory cell infiltration in lung tissue. Trimethoxystilbene is able to prevent pulmonary vascular remodeling and right ventricle hypertrophy in hypoxia-induced rat model of PAH, which is related to inhibition of the NOX/VPO1 pathway-mediated oxidative stress and the inflammatory reaction.
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Hemeproteínas/antagonistas & inibidores , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , NADPH Oxidases/antagonistas & inibidores , Peroxidases/antagonistas & inibidores , Estilbenos/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Hemeproteínas/metabolismo , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Masculino , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estilbenos/química , Estilbenos/uso terapêutico , Remodelação Ventricular/fisiologiaRESUMO
BACKGROUND: High-grade serous ovarian cancer (HGSOC) is a common gynecologic malignancy with a poor prognosis. The traditional Chinese medicine formula Erzhimaoling decoction (EZMLD) has anticancer potential. This study aims to elucidate the anticancer effects of EZMLD on HGSOC in vitro and in vivo. MATERIALS AND METHODS: EZMLD-containing serum was prepared from Sprague-Dawley rats for treating SKOV3 ovarian cancer cells at varying concentrations for 24 h and 48 h to determine the IC50. Concentrations of 0%, 5%, and 10% for 24 h were chosen for subsequent in vitro experiments. The roles of METTL3 and METTL14 in SKOV3 cells were explored by overexpressing these genes and combining EZMLD with METTL3/14 knockdown. Investigations focused on cell viability and apoptosis, apoptosis-related protein expression, and KRT8 mRNA m6A modification. For in vivo studies, 36 BALB/c nude mice were divided into six groups involving EZMLD (6.75, 13.5, and 27 g/kg) and METTL3 or METTL14 knockdowns, with daily EZMLD gavage for two weeks. RESULTS: In vitro, EZMLD-containing serum had IC50 values of 8.29% at 24 h and 5.95% at 48 h in SKOV3 cells. EZMLD-containing serum decreased SKOV3 cell viability and increased apoptosis. EZMLD upregulated METTL3/14 and FAS-mediated apoptosis proteins, while downregulating Keratin 8 (KRT8). EZMLD increased KRT8 mRNA m6A methylation. METTL3/14 overexpression reduced SKOV3 cell viability and increased apoptosis, while METTL3/14 knockdown mitigated EZMLD's effects. In vivo, EZMLD suppressed SKOV3 xenografts growth, causing significant apoptosis and modulating protein expression. CONCLUSIONS: EZMLD has therapeutic potential for ovarian cancer and may be considered for other cancer types. Future research may explore its broader effects beyond cell apoptosis.
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Apoptose , Medicamentos de Ervas Chinesas , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas , Feminino , Animais , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/genética , Humanos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Camundongos , Apoptose/efeitos dos fármacos , Ratos , Proliferação de Células/efeitos dos fármacos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Cerebral ischemia-reperfusion injury involves a series of pathophysiological processes that occur when blood supply is restored after cerebral vascular obstruction, leading to neuronal damage. The AMPK/ERK1/2 signaling pathway has been identified as crucial in this process, although the exact mechanisms underlying the induction of ischemia-reperfusion injury remain unclear. In this study, we investigated the involvement of the AMPK/ERK1/2 signaling pathway in neuronal oxidative stress damage following cerebral ischemia-reperfusion by establishing animal and cell models. Our experimental results demonstrated that cerebral ischemia-reperfusion leads to oxidative stress damage, including cell apoptosis and mitochondrial dysfunction. Moreover, further experiments showed that inhibition of AMPK and ERK1/2 activity, using U0126 and Compound C respectively, could alleviate oxidative stress-induced cellular injury, improve mitochondrial morphology and function, reduce reactive oxygen species levels, increase superoxide dismutase levels, and suppress apoptosis. These findings clearly indicate the critical role of the AMPK/ERK1/2 signaling pathway in regulating oxidative stress damage and cerebral ischemia-reperfusion injury. The discoveries in this study provide a theoretical basis for further research and development of neuroprotective therapeutic strategies targeting the AMPK/ERK1/2 signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP , Isquemia Encefálica , Sistema de Sinalização das MAP Quinases , Neurônios , Estresse Oxidativo , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Apoptose , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Quinase 3 Ativada por Mitógeno/metabolismoRESUMO
The clinical application of doxorubicin (DOX) is mainly restricted by its serious side effects, poor drug delivery efficiency, and limited immunogenic death (ICD) effect. To improve DOX-based chemotherapy and ameliorate its adverse effects, we utilized 3LL cell-derived extracellular vesicles to encapsulate DOX and sodium nitroprusside (SNP) to obtain DOX/SNP@CM, which could effectively target the tumor site by harnessing the inherent homologous targeting property of tumor cell membranes. DOX performed its role on chemotherapy, and SNP successfully respond to the intracellular GSH to continuously generate nitric oxide (NO). The in situ-produced NO upregulated the Fas expression on the tumor cell surface, thereby sensitizing the Fas/FasL pathway-mediated tumor cell apoptosis of DOX. Furthermore, NO also boosted the intratumoral infiltration of cytotoxic T cells by promoted ICD effect towards tumor cells. Importantly, the anti-tumor immunity tightly cooperated with Fas/FasL mediated tumor cell apoptosis by NO-mediated manipulation on Fas/FasL interaction, collectively making DOX/SNP@CM exert significant tumor growth inhibition with low-dose DOX. Remarkably, DOX and SNP both are widely used clinical medicines, ensuring DOX/SNP@CM a potential opportunity for future practical applications.
Assuntos
Antibióticos Antineoplásicos , Apoptose , Doxorrubicina , Vesículas Extracelulares , Proteína Ligante Fas , Nitroprussiato , Receptor fas , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Proteína Ligante Fas/metabolismo , Receptor fas/metabolismo , Animais , Nitroprussiato/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Óxido Nítrico/metabolismo , Imunoterapia/métodos , Camundongos Endogâmicos C57BL , Feminino , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Neoplasias/imunologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Sistemas de Liberação de Medicamentos/métodosRESUMO
This study aimed to synchronously determine epitranscriptome-wide RNA N6-methyladenosine (m6A) modifications and mRNA expression profile in high grade serous ovarian cancer (HGSOC). The methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to comprehensively examine the m6A modification profile and the RNA-sequencing (RNA-seq) was performed to analyze the mRNA expression profile in HGSOC and normal fallopian tube (FT) tissues. Go and KEGG analyses were carried out in the enrichment of those differentially methylated and expressed genes. MeRIP-seq data showed 53,794 m6A methylated peaks related to 19,938 genes in the HGSOC group and 51,818 m6A peaks representing 19,681 genes in the FT group. RNA-seq results revealed 2321 upregulated and 2486 downregulated genes in HGSOC. Conjoint analysis of MeRIP-seq and RNA-seq data identified differentially expressed genes in which 659 were hypermethylated (330 up- and 329 down-regulated) and 897 were hypomethylated (475 up- and 422 down-regulated). Functional enrichment analysis indicated that these differentially modulated genes are involved in pathways related to cancer development. Among methylation regulators, the m6A eraser (FTO) expression was significantly lower, but the m6A readers (IGF2BP2 and IGF2BP3) were higher in HGSOC, which was validated by the subsequent real-time PCR assay. Exploration through public databases further corroborated their possible clinical application of certain methylation regulators and differentially expressed genes. For the first time, our study screens the epitranscriptome-wide m6A modification and expression profiles of their modulated genes and signaling pathways in HGSOC. Our findings provide an alternative direction in exploring the molecular mechanisms of ovarian pathogenesis and potential biomarkers in the diagnosis and predicting the prognosis of the disease.
Assuntos
Adenosina , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas , RNA Mensageiro , Humanos , Feminino , Adenosina/análogos & derivados , Adenosina/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Projetos Piloto , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Perfilação da Expressão Gênica , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/metabolismo , Gradação de Tumores , Pessoa de Meia-Idade , Transcriptoma , Metilação de DNARESUMO
Central nervous system lymphoma (CNSL) is a type of hematological tumor. Treatment of CNSL is difficult due to the existence of the blood-brain barrier (BBB). Here, we used exosomes (Exos), a type of extracellular vesicle, and iRGD to construct a new drug carrier system and use it to load doxorubicin (DOX). The results of in vitro and in vivo experiments showed that the iRGD-Exo-DOX system can efficiently and securely transport DOX through the BBB and target tumor cells. The results suggest that iRGD-Exo-DOX may cross the BBB through brain microvascular endothelial cell-mediated endocytosis. Together, our study indicates an impactful treatment of central nervous system tumors.