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The search for medications that can effectively reduce alveolar bone loss following tooth extraction is of great interest. This study aimed to observe the roles of 4-octyl itaconate (4-OI) in RANKL-induced osteoclastogenesis of bone marrow macrophages (BMMs) in vitro. Mandibular second molars were extracted to evaluate whether 4-OI could alleviate alveolar bone loss. 4-OI inhibited RANKL-induced osteoclastogenesis and promoted Nrf2 expression in bone marrow macrophages in vitro. Positive Nrf2 expressions were observed in inflammatory cells and osteoclasts in vivo. Treatment with 4-octyl itaconate increased Nrf2 expression, resulting in reduced inflammatory infiltration and osteoclastic activity after tooth extraction. Furthermore, increased expression of OCN and enhanced-alveolar bone healing of extraction socket were observed in the 4-OI group compared to the control group. Our results suggested that 4-OI could serve as a promising pharmacologic candidate for alveolar ridge preservation by alleviating alveolar bone loss following tooth extraction in rats.
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The roles and molecular mechanisms of Delta-like 1 (DLK1) in periodontitis remain largely unknown. Here, we investigated the expression of DLK1 and NF-κB p65 in Porphyromonas gingivalis (Pg.)-induced periodontitis in vivo. Periodontal inflammation and alveolar bone resorption were analyzed using western blotting, micro-computed tomography, TRAP staining, immunohistochemistry, and immunofluorescence. Raw246.7 cells were stimulated with 1 µg/ml Porphyromonas gingivalis lipopolysaccharide (Pg.LPS) to assess DLK1 expression in vitro. DLK1 overexpression was achieved, and transfection efficiency was confirmed using western blotting and immunofluorescence. The NF-κB and MAPK pathways were activated by treating cells with 1 µg/ml Pg.LPS to explore related mechanisms. Compared with normal tissues, both DLK1 and NF-κB p65 expression increased in periodontitis gingival tissues. DLK1-positive expression was observed in inflammatory infiltrating cells and osteoclasts in the marginal lacunae of the alveolar bone. DLK1 expression in CD68-positive macrophages was detected by immunofluorescence. However, DLK1 expression in Raw246.7 cells decreased after Pg.LPS stimulation and during osteoclast differentiation. DLK1 levels negatively correlated with TNF-α, IL-1ß, and NFATC1. Increased DLK1 in Raw246.7 cells further inhibited COX2 and iNOS expressions. Mechanistically, DLK1 overexpression down-regulated NF-κB p65 and JNK levels. In summary, these findings suggest that DLK1 overexpression inhibits periodontal inflammation through the NF-κB p65 and JNK pathways. Interventions targeting increased DLK1 levels may have therapeutic implications for periodontitis.
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BACKGROUND: Abnormalities in Ca2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive. METHODS: We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as, a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart. RESULTS: We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA. CONCLUSIONS: These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.
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Processamento Alternativo , Proteínas de Transporte , Insuficiência Cardíaca , Proteínas Musculares , RNA Longo não Codificante , Animais , Arritmias Cardíacas , Proteínas de Transporte/genética , Catecolaminas , Coração/fisiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Longo não Codificante/genéticaRESUMO
Dysregulation or aberrant signaling transduction contributes to tumorigenesis. Targeting these abnormal signaling pathways becomes an effective anticancer strategy. However, feedback activation or crosstalk between signaling pathways drives adaptive drug resistance which causes failure of cancer therapy. In this review article, we summarized treatments that cause feedback activation of AKT, ERK, STAT3, EGFR, FGFR, and HER2/3 signaling pathways and the combination therapy to enhance anti-tumor effect or to overcome drug resistance, to explore the underlying mechanisms that define the protein molecules participated or regulated the feedback activation. In addition, we reviewed clinical trials that employ combination treatments to suppress feedback activation and improve therapeutic efficacy of cancer treatments.
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Neoplasias , Transdução de Sinais , Humanos , Retroalimentação , Linhagem Celular Tumoral , Resistência a Medicamentos , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.
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Retículo Endoplasmático/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Família Multigênica , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Nó Sinoatrial/fisiologia , Nó Sinoatrial/fisiopatologiaRESUMO
BACKGROUND: Internet hospitals in China are in great demand due to limited and unevenly distributed health care resources, lack of family doctors, increased burdens of chronic diseases, and rapid growth of the aged population. The COVID-19 epidemic catalyzed the expansion of online health care services. In recent years, internet hospitals have been rapidly developed. Ping An Good Doctor is the largest, national online medical entry point in China and is a widely used platform providing online health care services. OBJECTIVE: This study aims to give a comprehensive description of the characteristics of the online consultations and inquisitions in Ping An Good Doctor. The analyses tried to answer the following questions: (1) What are the characteristics of the consultations in Ping An Good Doctor in terms of department and disease profiles? (2) Who uses the online health services most frequently? and (3) How is the user experience of the online consultations of Ping An Good Doctor? METHODS: A total of 35.3 million consultations and inquisitions over the course of 1 year were analyzed with respect to the distributions of departments and diseases, user profiles, and consulting behaviors. RESULTS: The geographical distribution of the usage of Ping An Good Doctor showed that Shandong (18.4%), Yunnan (15.6%), Shaanxi (7.2%), and Guangdong (5.5%) were the provinces that used it the most; they accounted for 46.6% of the total consultations and inquisitions. In terms of department distribution, we found that gynecology and obstetrics (19.2%), dermatology (17.0%), and pediatrics (14.4%) were the top three departments in Ping An Good Doctor. The disease distribution analysis showed that, except for nondisease-specific consultations, acute upper respiratory infection (AURI) (4.1%), pregnancy (2.8%), and dermatitis (2.4%) were the most frequently consulted diseases. In terms of user profiles, females (60.4%) from 19 to 35 years of age were most likely to seek consultations online, in general. The user behavior analyses showed that the peak times of day for online consultations occurred at 10 AM, 3 PM, and 9 PM. Regarding user experience, 93.0% of users gave full marks following their consultations. For some disease-related health problems, such as AURI, dermatitis, and eczema, the feedback scores were above average. CONCLUSIONS: The prevalence of internet hospitals, such as Ping An Good Doctor, illustrated the great demand for online health care services that can go beyond geographical limitations. Our analyses showed that nondisease-specific issues and moderate health problems were much more frequently consulted about than severe clinical conditions. This indicated that internet hospitals played the role of the family doctor, which helped to relieve the stress placed on offline hospitals and facilitated people's lives. In addition, good user experiences, especially regarding disease-related inquisitions, suggested that online health services can help solve health problems. With support from the government and acceptance by the public, online health care services could develop at a fast pace and greatly benefit people's daily lives.
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COVID-19/epidemiologia , Atenção à Saúde/métodos , Telemedicina/métodos , Adulto , China/epidemiologia , Estudos Transversais , Feminino , Humanos , Masculino , SARS-CoV-2/isolamento & purificação , Inquéritos e Questionários , Adulto JovemRESUMO
Dilated cardiomyopathy (DCM) is the most common cause of heart failure (HF) in children, resulting in high mortality and need for heart transplantation. The pathophysiology underlying pediatric DCM is largely unclear; however, there is emerging evidence that molecular adaptations and response to conventional HF medications differ between children and adults. To gain insight into alterations leading to systolic dysfunction in pediatric DCM, we measured cardiomyocyte contractile properties and sarcomeric protein phosphorylation in explanted pediatric DCM myocardium (N = 8 subjects) compared with nonfailing (NF) pediatric hearts (N = 8 subjects). Force-pCa curves were generated from skinned cardiomyocytes in the presence and absence of protein kinase A. Sarcomeric protein phosphorylation was quantified with Pro-Q Diamond staining after gel electrophoresis. Pediatric DCM cardiomyocytes demonstrate increased calcium sensitivity (pCa50 =5.70 ± 0.0291), with an associated decrease in troponin (Tn)I phosphorylation compared with NF pediatric cardiomyocytes (pCa50 =5.59 ± 0.0271, P = 0.0073). Myosin binding protein C and TnT phosphorylation are also lower in pediatric DCM, whereas desmin phosphorylation is increased. Pediatric DCM cardiomyocytes generate peak tension comparable to that of NF pediatric cardiomyocytes [DCM 29.7 mN/mm2, interquartile range (IQR) 21.5-49.2 vs. NF 32.8 mN/mm2, IQR 21.5-49.2 mN/mm2; P = 0.6125]. In addition, cooperativity is decreased in pediatric DCM compared with pediatric NF (Hill coefficient: DCM 1.56, IQR 1.31-1.94 vs. NF 1.94, IQR 1.36-2.86; P = 0.0425). Alterations in sarcomeric phosphorylation and cardiomyocyte contractile properties may represent an impaired compensatory response, contributing to the detrimental DCM phenotype in children.NEW & NOTEWORTHY Our study is the first to demonstrate that cardiomyocytes from infants and young children with dilated cardiomyopathy (DCM) exhibit increased calcium sensitivity (likely mediated by decreased troponin I phosphorylation) compared with nonfailing pediatric cardiomyocytes. Compared with published values in adult cardiomyocytes, pediatric cardiomyocytes have notably decreased cooperativity, with a further reduction in the setting of DCM. Distinct adaptations in cardiomyocyte contractile properties may contribute to a differential response to pharmacological therapies in the pediatric DCM population.
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Cálcio/metabolismo , Cardiomiopatia Dilatada/metabolismo , Miócitos Cardíacos/metabolismo , Troponina I/metabolismo , Cálcio/farmacologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Humanos , Masculino , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , FosforilaçãoRESUMO
Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo , human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro . Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.
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Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo, human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro. Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.
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Inhibition of SIRT3 exhibited potency in triggering leukemic cell differentiation. In discovery of potent SIRT3 inhibitors for cancer differentiation therapy, structural modification was performed on the previously developed lead compound P6. A total of 33 compounds were designed and synthesized. In the enzyme inhibitory assay, several molecules S18, S26, S27 and T5 showed potent SIRT3 inhibitory activity with IC50 value of 0.53, 1.86, 5.06, and 2.88 µM, respectively. Moreover, the tested compounds exhibited SIRT3 inhibitory selectivity over SIRT1 and SIRT2. Compounds S27 and T5 were potent in inhibition the growth of MM1.S and RPMI-8226 cells in the in vitro antiproliferative test. Significantly, representative compounds, especially S27 and T5, promoted differentiation of tested MM cells in the cellular morphological evaluation, accompanied by increasing the expression of differentiation antigen CD49e and human immunoglobulin light chain lambda and kappa. Additionally, molecule S18 without antiproliferative potency itself, showed significant inhibitory activity against growth factor IL-6 induced RPMI-8226 cell proliferation. Collectively, potent SIRT3 selective inhibitors with MM cell differentiation potency were developed for further discovery of anticancer drugs.
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Antineoplásicos , Diferenciação Celular , Proliferação de Células , Sirtuína 3 , Humanos , Sirtuína 3/metabolismo , Sirtuína 3/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Quinolinas/química , Quinolinas/farmacologia , Simulação de Acoplamento MolecularRESUMO
BACKGROUND: The roles and molecular mechanisms of tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) in periodontitis remain largely unknown. OBJECTIVE: This study aimed to determine the expression of TIPE2 and NF-κB p65 in rat Porphyromonas gingivalis-induced periodontics in vivo. METHODOLOGY: Periodontal inflammation and alveolar bone resorption were analyzed using western blotting, micro-computed tomography, TRAP staining, immunohistochemistry, and immunofluorescence. THP-1 monocytes were stimulated using 1 µg/ml Pg. lipopolysaccharide (Pg.LPS) to determine the expression of TIPE2 in vitro. TIPE2 mRNA was suppressed by siRNA transfection, and the transfection efficiency was proven using western blotting and real-time PCR. The NF-κB pathway was activated by treating the cells with 1 µg/ml Pg.LPS to explore related mechanisms. RESULTS: The expression of both TIPE2 and NF-κB p65 was increased in the gingival tissues of rat periodontitis compared with normal tissues. Positive expression of TIPE2 was distributed in inflammatory infiltrating cells and osteoclasts in the marginal lacunae of the alveolar bone. However, strong positive expression of TIPE2 in THP-1 was downregulated after Pg.LPS stimulation. TIPE2 levels negatively correlated with TNF-α and IL-1ß. Decreased TIPE2 in THP-1 further promoted NF-κB p65 phosphorylation. Mechanistically, TIPE2 knockdown upregulated NF-κB signaling pathway activity. CONCLUSIONS: Taken together, these findings demonstrate that TIPE2 knockdown aggravates periodontal inflammatory infiltration via NF-κB pathway. Interventions aimed at increasing TIPE2 may help in the therapeutic applications for periodontitis.
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NF-kappa B , Periodontite , Animais , Ratos , Inflamação , Lipopolissacarídeos/farmacologia , Fosforilação , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-XRESUMO
A number of protein signaling mechanisms are known to be involved in the progression of heart failure, yet the mechanism(s) by which the heart fails remains poorly understood. Therefore, we undertook a global approach to this question and used an antibody microarray to identify proteins differentially expressed in dysfunctional right ventricles in a bovine model of heart failure and the results were validated using cardiac tissue from both bovine and human heart failure. We found that protein disulfide isomerase 3, PDIA3, a protein that resides in the lumen of the endoplasmic reticulum, is significantly upregulated in both animal and human models of right and left heart failure. Altered expression of this protein has not previously been described in models of heart failure. In our initial microarray analysis, we found that CSK (c-Src kinase) was among the proteins upregulated in failing bovine ventricle. To further elucidate the role of CSK in heart failure, we studied the expression of its downstream target, Src, and found that Src expression and phosphorylation were markedly upregulated in failing ventricles. However, we also noted a smaller immunologically reactive protein that was only seen in experimental animals. In order to positively identify the smaller, Src-reactive protein, we used 2-dimensional gel electrophoresis and mass spectrophotometry. Surprisingly, we identified this protein as PDIA3, a protein that did not belong to the Src family of proteins. Upon sequence examination we found that PDIA3 contains a short C-terminal sequence with strong homology to Src and that it was this short sequence to which the antibody was generated. PDIA3 participates in MHC class I presentation and is implicated in the progression of valvular dysfunction in rheumatic heart disease, as well as calcium modulation in the sarcoplasmic reticulum. The molecule resides in the lumen of the endoplasmic reticulum and participates in disulfide bond formation during protein folding by interacting with calnexin and calreticulin. This interaction may indirectly effect SERCA (sarco/endoplasmic reticulum Ca(2+)-transport ATPase) activity and by extension contribute to the calcium dysregulation that characterizes progressive heart failure. Further studies are needed to elucidate the role that PDIA3 may play in the progression of heart failure.
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Insuficiência Cardíaca/enzimologia , Ventrículos do Coração/enzimologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Direita/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Apresentação de Antígeno , Bovinos , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/enzimologia , Insuficiência Cardíaca/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Análise Serial de Proteínas , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Quinases da Família src/imunologia , Quinases da Família src/metabolismoRESUMO
The goal of this work was to develop a sensitive and accurate method based on high performance liquid chromatography with tandem mass spectrometry (LC-MS) detection for determining the concentration of the Sinomenine derivative SWX in plasma and tissue of rat. Chromatographic separation was achieved on an Agilent SB-C18 (2.1*50â¯mm, 2.7⯵m) column. The mobile phase consisted of acetonitrile (solvent A) and 0.2â¯% formic acid in water (solvent B) with gradient elution as follows: 0-3â¯min, 50-90â¯% A, 3â¯min-3.01â¯min, 90-50â¯% A, 90â¯% A in 3.01â¯min-10â¯min. The flow rate was set to 0.3â¯mL/min. The column temperature was 40⯰C, and the sample injection volume was 10⯵L. The calibration curve had good linearity in the range of 10â¯ng/mL to 600â¯ng/mL. Biological samples were prepared by protein precipitation with acetonitrile. The area under the curve (AUC) and the peak drug concentration (Cmax) were linearly related to SWX dose. After oral administration of 25, 50 and 100 mg/kg SWX and intravenous administration of 0.5â¯mg/kg SWX, respectively, the absolute bioavailability of SWX was estimated as 12.4â¯%, 12.2â¯% and 10.6â¯%. SWX was widely distributed in heart, liver, spleen, lung, kidney, brain, breast, and fat, especially in lung, liver, and spleen. Distribution of SWX in brain suggested that it can pass the blood-brain barrier. The method established in this study has the advantages of high recovery and good reproducibility, and was suitable for the determination of the content of Sinomenine derivatives, providing a reliable scientific tool for carrying out pharmacokinetics research, providing a reliable scientific resource.
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Espectrometria de Massas em Tandem , Ratos , Animais , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodos , Acetonitrilas , SolventesRESUMO
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease.
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Cardiomiopatia Hipertrófica/fisiopatologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Linhagem Celular , Células Cultivadas , Meios de Cultura/química , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Modelos Biológicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/fisiologia , Fenilefrina/farmacologia , Sarcômeros/fisiologia , Análise de Sequência de RNA , Transdução de SinaisRESUMO
Whether external root resorption is associated with hypoxia in the periodontal ligaments of teeth with severe periodontitis remains unclear. Hypoxia inducible factor-1α (HIF-1α) expression and external resorption sites in the periodontal ligaments of these teeth were observed to elaborate upon the relationship between hypoxia and external root resorption in severe periodontitis. Histological analysis was performed to observe external root resorption. The expressions of HIF-1α and Nuclear factor-activated T cells c1 (NFATc1) in the periodontal ligaments were detected by immunofluorescence, western blotting and real-time PCR. Bone marrow macrophages (BMMs) were stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg.LPS) and cultured under hypoxia in vitro. High levels of HIF-1α and NFATc1 were detected in severe periodontitis. HIF-1α positive-cells were observed in the external resorption sites. Hypoxia promoted Pg.LPS-stimulated osteoclastogenesis of BMMs and bone resorption by the NFATc1 pathway. Increased HIF-1α in severe periodontitis are associated with external root resorption by the NFATc1 pathway.
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Suscetibilidade a Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição NFATC/metabolismo , Periodontite/etiologia , Periodontite/metabolismo , Transdução de Sinais , Adulto , Idoso , Animais , Biomarcadores , Diferenciação Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Periodontite/diagnóstico , Índice de Gravidade de DoençaRESUMO
Delayed resorption of crown-covered bone is a critical cause of delayed tooth eruption. Traditional herbal medicines may be good auxiliary treatments to promote the resorption of crown-covered bone. This study was carried out to analyse the effect of isorhamnetin 3-O-neohesperidoside on receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis in vitro and resorption of the crown-covered bone of the lower first molars in mice in vivo. Isorhamnetin 3-O-neohesperidoside promoted osteoclastogenesis and the bone resorption of mouse bone marrow macrophages (BMMs) and upregulated mRNA expression of the osteoclast-specific genes cathepsin K (CTSK), vacuolar-type H + -ATPase d2(V-ATPase d2), tartrate resistant acid phosphatase (TRAP) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). NFATc1, p38 and AKT signalling was obviously activated by isorhamnetin 3-O-neohesperidoside in osteoclastogenesis. Isorhamnetin 3-O-neohesperidoside aggravated resorption of crown-covered bone in vivo. In brief, isorhamnetin 3-O-neohesperidoside might be a candidate adjuvant therapy for delayed intraosseous eruption.
Assuntos
Osteogênese/fisiologia , Erupção Dentária/efeitos dos fármacos , Animais , Células da Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Flavonóis/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Quercetina/análogos & derivados , Quercetina/farmacologia , Ligante RANK/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Plenty of long non-coding RNAs (lncRNAs) play vital roles in the progression of atherosclerosis. Small nucleolar RNA host gene 6 (SNHG6) is a well known lncRNA that is aberrantly high expressed in atherosclerosis patients. However, its function and basic mechanism in atherosclerosis events have not been well clarified. METHODS: The expression patterns of SNHG6, miR-135a-5p, ROCK1 and ROCK2 in clinical samples and cells were detected by RT-qPCR assays. Cell Counting Kit-8 (CCK-8), flow cytometry assays, ELISA and reactive oxygen species (ROS) and malondialdehyde (MDA) detection, were performed to assess cell viability, apoptosis, inflammation and oxidative stress, respectively. Western blot analysis was carried out to examine the protein levels of Bax, Bcl-2, and SNHG6. Luciferase reporter and RIP assays were used to confirm the true interaction between SNHG6 and miR-135a-5p, or miR-135a-5p and ROCK. RESULTS: The levels of SNHG6, ROCK1 and ROCK2 were notably increased and miR-135a-5p was decreased in atherosclerosis patients and oxidized low-density lipoprotein (ox-LDL)-treated HUVECs. Knockdown of SNHG6 alleviated ox-LDL-induced injury of HUVECs, while this effect was partly reversed by miR-135a-5p inhibitor. Moreover, overexpression of ROCKs aggravated miR-135a-5p-alleviated atherosclerosis cell injury. SNHG6 contributed to ROCK expression through sequestering miR-135a-5p as a molecular sponge. CONCLUSION: SNHG6 functions as a promoter in atherosclerosis events by targeting miR-135a-5p/ROCK axis in ox-LDL-stimulated HUVECs. This finding will help to develop a novel therapeutic strategy for atherosclerosis.
RESUMO
The study is to verify whether Calcitonin gene related peptide (CGRP) gene-modified rat bone mesenchymal stem cells (BMSCs) are promising seed cells with great potential to repair bone defect. A recombinant lentiviral vector overexpressing CGRP peptide, pLenO-DCE-CGRP, was constructed and transfected into rBMSCs to obtain stable CGRP expression. Differently transfected rBMSCs: CGRP- modified rBMSCs (CGRP group), empty vector- modified rBMSCs (Vector group) and normal rBMSCs (Control group) were transferred to collagen scaffold to repair rat skull defects (n=6). Skull specimens were obtained at 4 and 8 weeks after operation. Expressions of osteogenesis-related indexes OCN (osteocalcin) and BMP-2 (Bone morphogenetic protein-2) were detected by immunohistochemistry and western blotting. Histological observation, Masson trichrome staining and Micro-computed tomography (Micro-CT) were applied to evaluate defect repair. Tartrate-resistant acid phosphatase (TRAP) was applied to observe bone remodeling at 4 weeks. PGP9.5 was detected to observe nerve fibers formation at 8 weeks. Osteogenic markers OCN and BMP-2 were significantly higher in CGRP -modified BMSCs at 4 weeks, compared with the vector and control groups. Few TRAP-positive expressions and scaffolds were observed in the CGRP group. More new bone formation and mineralization in defects were observed in the CGRP group at 4 and 8weeks. Many PGP9.5-positive expressions were found in the CGRP group. CGRP promoted osteogenic differentiation of BMSC and scaffold resorption in vivo. Repair effect of CGRP group was significantly better than the other two groups. CGRP gene-modified BMSCs are effective seed cells in tissue engineering to repair bone defects.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Osteogênese , Crânio , Engenharia Tecidual/métodos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , Peptídeo Relacionado com Gene de Calcitonina/genética , Células Cultivadas , Regeneração Tecidual Guiada , Osteocalcina/metabolismo , Osteogênese/fisiologia , Crânio/patologia , Fosfatase Ácida Resistente a Tartarato/metabolismo , Alicerces Teciduais , Transformação Genética , Microtomografia por Raio-XRESUMO
Osseous eruption is an important stage of tooth eruption process. The role of periodontal ligament-associated protein-1 (PLAP-1/asporin) in the development of osseous eruption canal remain undefined and were the focus of this study. C57BL/6 mice at postnatal days P11-13 and P 15-16 were chosen. The development of osseous eruption canal of lower first molar was observed and osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP). PLAP-1 expression in the process of osseous eruption (OE, P11-13) and post- osseous eruption (P-OE, P15-16) was assessed by immunohistochemistry, immunofluorescence and western blotting. Receptor activator of NF-κB ligand (RANKL) distribution in the process was also assessed by immunohistochemistry. A double immunofluorescence stain was used to reveal PLAP-1 in association with CD68 (osteoclast maker). Fresh occlusal tissues of erupting lower first molars at OE and P-OE were separated to detected RANKL/OPG ratio by western blotting to elucidate related mechanisms. At osseous eruption (OE), osseous and mucosal tissues could be observed on the occlusal side of lower first molar. Osseous eruption canal was developing. Many osteoclasts were found around occlusal alveolar bone in the development of osseous eruption canal. At post- osseous eruption (P-OE), osseous eruption canal had been built, only mucosal tissues were observed, and few osteoclasts were detected. More PLAP-1 expression was detected at OE, compared with that at P-OE. Similar distributions of PLAP-1 and RANKL in occlusal bone tissues of erupting lower first molars were detected at OE. Colocalization of PLAP-1 and CD68 revealed the positive relationship between PLAP-1 and osteoclasts in the development of osseous eruption canal. PLAP-1 positively correlated with RANKL and CD68+ osteoclasts, and areas of bone resorption. Higher RANKL/OPG ratio was detected at OE, compared with that at P-OE. PLAP-1 gets involved in the development of osseous eruption canal.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Erupção Dentária , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Reabsorção Óssea/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Minerais , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Irrigantes do Canal RadicularRESUMO
This study was undertaken to investigate the roles of importin 8 (IPO8) and infiltrating macrophages in human periodontitis. Gingival tissues were collected from 18 male patients with mild and severe periodontitis. IPO8 expression was observed by immunohistochemistry and western blotting. CD68 expression, a marker for macrophages, was observed by immunohistochemistry and immunofluorescence. IPO8 expression in mouse macrophage RAW264.7 cells stimulated by lipopolysaccharide (LPS) purified from Porphyromonas gingivalis was examined in vitro using small interfering RNA (siRNA). Phosphorylation of nuclear factor kappa B (NF)-κB pp65 was detected as an indicator of NF-κB activation by immunofluorescence and western blotting. IPO8 expression was increased in gingival tissues with periodontitis compared with healthy ones, and the elevation rose in proportion to the severity of periodontitis. Similarly, more CD68+ infiltrating macrophages were observed in severe periodontitis tissues. LPS stimulation promoted IPO8 expression in mouse macrophage cells. Knockdown of IPO8 by siRNA alleviated the LPS-induced increases in tumor necrosis factor-α and interleukin-6 gene expression, as well as NF-κB activation, by decreasing the expression of NF-κB pp 65 in the nuclei of cells in vitro. In conclusion, IOP8 plays a role in human periodontitis. Targeted inhibition of IOP8 expression could effectively alleviate periodontal inflammation.