RESUMEN
G-quadruplex structures within the nuclear genome (nG4) is an important regulatory factor, while the function of G4 in the mitochondrial genome (mtG4) still needs to be explored, especially in human sperms. To gain a better understanding of the relationship between mtG4 and mitochondrial function, it is crucial to develop excellent probes that can selectively visualize and track mtG4 in both somatic cells and sperms. Herein, based on our previous research on purine frameworks, we attempted for the first time to extend the conjugated structure from the C-8 site of purine skeleton and discovered that the purine derivative modified by the C-8 aldehyde group is an ideal platform for constructing near-infrared probes with extremely large Stokes shift (>220 nm). Compared with the compound substituted with methylpyridine (PAP), the molecule substituted with methylthiazole orange (PATO) showed better G4 recognition ability, including longer emission (â¼720 nm), more significant fluorescent enhancement (â¼67-fold), lower background, and excellent photostability. PATO exhibited a sensitive response to mtG4 variation in both somatic cells and human sperms. Most importantly, PATO helped us to discover that mtG4 was significantly increased in cells with mitochondrial respiratory chain damage caused by complex I inhibitors (6-OHDA and rotenone), as well as in human sperms that suffer from oxidative stress. Altogether, our study not only provides a novel ideal molecular platform for constructing high-performance probes but also develops an effective tool for studying the relationship between mtG4 and mitochondrial function in both somatic cells and human sperms.
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Colorantes Fluorescentes , Purinas , Humanos , Purinas/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Enfermedades Mitocondriales/metabolismo , Regulación hacia Arriba , Genoma Mitocondrial , G-Cuádruplex , Mitocondrias/metabolismo , Rayos Infrarrojos , Células HeLaRESUMEN
Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.
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Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Masculino , Microscopía por Crioelectrón , Sulfato de Deshidroepiandrosterona , Desoxicorticosterona , Ligandos , Receptores Acoplados a Proteínas G/químicaRESUMEN
Inflammation in the epididymis and testis contributes significantly to male infertility. Alternative therapeutic avenues treating epididymitis and orchitis are expected since current therapies using antibiotics have limitations associated to side effects and are commonly ineffective for inflammation due to nonbacterial causes. Here, we demonstrated that type 1 parathyroid hormone receptor (PTH1R) and its endogenous agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP), were mainly expressed in the Leydig cells of testis as well as epididymal epithelial cells. Screening the secretin family G protein-coupled receptor identified that PTH1R in the epididymis and testis was down-regulated in mumps virus (MuV)- or lipopolysaccharide (LPS)-induced inflammation. Remarkably, activation of PTH1R by abaloparatide (ABL), a Food and Drug Administration-approved treatment for postmenopausal osteoporosis, alleviated MuV- or LPS-induced inflammatory responses in both testis and epididymis and significantly improved sperm functions in both mouse model and human samples. The anti-inflammatory effects of ABL were shown to be regulated mainly through the Gq and ß-arrestin-1 pathway downstream of PTH1R as supported by the application of ABL in Gnaq± and Arrb1-/- mouse models. Taken together, our results identified an important immunoregulatory role for PTH1R signaling in the epididymis and testis. Targeting to PTH1R might have a therapeutic effect for the treatment of epididymitis and orchitis or other inflammatory disease in the male reproductive system.
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Epididimitis/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Orquitis/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , beta-Arrestina 1/metabolismo , Animales , Infertilidad Masculina/metabolismo , Infertilidad Masculina/virología , Lipopolisacáridos , Masculino , Ratones Endogámicos C57BL , Virus de la ParotiditisRESUMEN
BACKGROUND: Simulation is an increasingly used novel method for the education of medical professionals. This study aimed to systematically review the efficacy of high-fidelity (HF) simulation compared with low-fidelity (LF) simulation or no simulation in advanced life support (ALS) training. METHODS: A comprehensive search of the PubMed, Chinese Biomedicine Database, Embase, CENTRAL, ISI, and China Knowledge Resource Integrated Database was performed to identify randomized controlled trials (RCTs) that evaluated the use of HF simulation in ALS training. Quality assessment was based on the Cochrane Handbook for Systematic Reviews of Interventions version 5.0.1. The primary outcome was the improvement of knowledge and skill performance. The secondary outcomes included the participants' confidence and satisfaction at the course conclusion, skill performance at one year, skill performance in actual resuscitation, and patient outcomes. Data were synthesized using the RevMan 5.4 software. RESULTS: Altogether, 25 RCTs with a total of 1,987 trainees were included in the meta-analysis. In the intervention group, 998 participants used HF manikins, whereas 989 participants received LF simulation-based or traditional training (classical training without simulation). Pooled data from the RCTs demonstrated a benefit in improvement of knowledge [standardized mean difference (SMD) = 0.38; 95% confidence interval (CI): 0.18-0.59, P = 0.0003, I2 = 70%] and skill performance (SMD = 0.63; 95% CI: 0.21-1.04, P = 0.003, I2 = 92%) for HF simulation when compared with LF simulation and traditional training. The subgroup analysis revealed a greater benefit in knowledge with HF simulation compared with traditional training at the course conclusion (SMD = 0.51; 95% CI: 0.20-0.83, P = 0.003, I2 = 61%). Studies measuring knowledge at three months, skill performance at one year, teamwork behaviors, participants' satisfaction and confidence demonstrated no significant benefit for HF simulation. CONCLUSIONS: Learners using HF simulation more significantly benefited from the ALS training in terms of knowledge and skill performance at the course conclusion. However, further research is necessary to enhance long-term retention of knowledge and skill in actual resuscitation and patient's outcomes.
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Enseñanza Mediante Simulación de Alta Fidelidad , Humanos , Simulación por Computador , Escolaridad , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Preeclampsia gravely threatens the health of mothers and infants. At present, treatment based on the relevant mechanisms of pathogenesis is still not available, and there is no independent reliable clinical index for early prediction of preeclampsia. According to recent studies, analysis of the cell-free RNA in the peripheral blood of pregnant women has shown that testing certain cell-free RNA levels can help predict in advance the occurrence of preeclampsia before clinical symptoms appear. In this paper, we described the status of research and progress in using maternal cell-free RNA analysis in predicting preeclampsia. In addition, we stated that cell-free RNA in peripheral blood may become a promising, real-time and non-invasive monitoring method that can be used to explore the mechanisms of pathogenesis and pathophysiology of preeclampsia and to identify different subtypes of preeclampsia.
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Ácidos Nucleicos Libres de Células , Preeclampsia , Embarazo , Lactante , Femenino , Humanos , Mujeres Embarazadas , Preeclampsia/diagnóstico , FamiliaRESUMEN
Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Espermatogénesis/genética , Proteínas de Anclaje a la Quinasa A/genética , Animales , Femenino , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Motilidad Espermática/genética , Motilidad Espermática/fisiología , Cola del Espermatozoide/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
OBJECTIVE: To explore the association between polymorphisms of miRNA biogenesis related gene DICER,DROSHA,RAN and unexplained recurrent spontaneous abortion (URSA) in Chinese women. METHODS: We recruited 217 patients with URSA (URSA group) and 390 healthy controls who were fertile women with history of more than one successful pregnancy outcome (control group) from June 2013 to December 2015 in the West China Second University Hospital of Sichuan University. The control group was recruited from regular physical examination and prepregnancy check for women of childbearing age during the same period. A case-control study was performed to analyze polymorphism of miRNA machinery genes,including DICER rs3742330,DROSHA rs10719, RAN rs14035,by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. RESULTS: The frequencies of genotypes and alleles of DICER rs3742330,DROSHA rs10719,and RAN rs14035 showed no difference between URSA patients and control group (all P>0.05). DICER rs3742330/DROSHA rs10719 GG/TC+TT combinations were synergistically associated with increased URSA risk [odds ratio (OR)=1.657,95% confidence interval (CI)=1.006-2.731, P=0.047]. Although DICER rs3742330/RAN rs14035 GG / TT+TC combinations was significantly higher in the URSA group than in the control group,there was no statistical significance (OR=1.977,95%CI=0.956-4.087, P=0.066). However,DROSHA10719/RAN14035 TT+TC/TT+TC had no significant correlation with URSA (OR=0.958,95%CI=0.679-1.353, P=0.808). CONCLUSION: Our study demonstrated the relationship between URSA development and combined genotype of DICER rs3742330/DROSHA rs10719 GG/TC+TT.
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Aborto Habitual/genética , ARN Helicasas DEAD-box/genética , Predisposición Genética a la Enfermedad , Ribonucleasa III/genética , Proteína de Unión al GTP ran/genética , Estudios de Casos y Controles , China , Femenino , Frecuencia de los Genes , Humanos , MicroARNs , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , EmbarazoRESUMEN
Dicer, an RNAse III endonuclease, is critical for the biogenesis of small noncoding RNAs (microRNAs), including the biogenesis of microRNAs and small interfering RNAs, which transcriptionally and post-transcription ally regulate mRNA expression through binding to target mRNA and leading to subsequent mRNA degradation. Recent studies show that Dicer plays important roles in cell proliferation, differentiation and apoptosis. It has been attracted more and more attention in the reproductive field. In the male reproduction field, mouse model shows that Dicer is critical for the development of spermatogenic cell, sperm maturation, sperm motility and morphology. On the other hand, Dicer is broadly involved in not only follicular development, ovulation, luteinization, sex hormone synthesis, but also regulating the functions of the fallopian tube, endometrial receptivity in female reproduction. Since sperm and egg are the only two types of gametes for producing offspring, Dicer dysregulation may be the underlying cause of compromised embryo development through affecting the quantity or quality of sperm and eggs. Therefore, understanding the function of Dicer in reproduction of female and male is of great significance to study the pathogenetic mechanism related to dysfunctional reproduction, such as azoospermia and recurrent spontaneous abortion. We review the pivotal roles of Dicer in the male and female reproduction field in order to understand the relationship between Dicer and related disease.
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Reproducción/fisiología , Ribonucleasa III/fisiología , Animales , Desarrollo Embrionario , Femenino , Humanos , Masculino , Ratones , MicroARNs/biosíntesis , Ribonucleasa III/genética , EspermatogénesisRESUMEN
Due to the fact that Candida albicans colonizes in the upper respiratory tracts of healthy people, whether or not its isolation from airway secretions is sufficient to warrant treatment remains controversial. The animal models of immunosuppressive rats with pulmonary candidiasis were established by the intratracheal inoculating suspensions of C. albicans, and the animals were divided into the following three groups: (1) antifungal treatment group, (2) saline control group, and (3) blank control group. We noted the following in our studies: (1) The fungal load of the saline control group gradually increased such that it was higher than those of the antifungal treated group and was significant from the fourth day of treatment (P < 0.01). (2) The serum (1,3)-ß-D-glucan (BG) in the saline control group also gradually increased so that it was significantly higher than found with the treated group by the sixth day of treatment (P < 0.05), and in fact, the rank of pulmonary colony count and BG in the two groups at different time points showed an almost perfect linear correlation. (3) The median survival period of the rats in the antifungal treated group and saline control group was 15 and 8 days respectively, no rats died in the blank control group. (4) The lung lesions from the saline control group gradually became more aggravated than those in the antifungal treated group; no significant pathological changes were found in the blank control group. Antifungal treatment (micafungin) is capable of efficaciously decreasing the lung fungal burden, and continuous monitoring of BG is useful for the evaluation of therapeutic effect of antifungals. Infection of C. albicans with associated pathological damage implies the need for antifungal therapy.
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Antifúngicos/uso terapéutico , Candida albicans/química , Candidiasis/tratamiento farmacológico , Monitoreo de Drogas/métodos , Glucanos/sangre , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Suero/química , Animales , Candidiasis/microbiología , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Pulmón/patología , Masculino , Ratas Sprague-Dawley , Infecciones del Sistema Respiratorio/microbiología , Análisis de SupervivenciaRESUMEN
AIM: Carbonaceous dots (CDs), which have been used for diagnosis, drug delivery and gene delivery, are accumulated in heart at high concentrations. To improve their biocompatibility, polyethylene glycol-modified CDs (PEG-CDs) were prepared. In this study we compared the cardiac toxicity of CDs and PEG-CDs in mouse and zebrafish models. METHODS: Mice were intravenously treated with CDs (size: 4.9 nm, 5 mg·kg(-1)·d(-1)) or PEG-CDs (size: 8.3 nm, 5 mg·kg(-1)·d(-1)) for 21 d. Their blood biochemistry indices, ECG, and histological examination were examined for evaluation of cardiac toxicity. CDs or PEG-CDs was added in incubator of cmlc2 transgenic Zebrafish embryos at 6 hpf, and the shape and size of embryos' hearts were observed at 48 hpf using a fluorescent microscope. Furthermore, whole-mount in situ hybridization was used to examine the expression of early cardiac marker gene (clml2) at 48 hpf. RESULTS: Administration of CDs or PEG-CDs in mice caused mild, but statistically insignificant reduction in serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels detected at 7 d, which were returned to the respective control levels at 21 d. Neither CDs nor PEG-CDs caused significant changes in the morphology of heart cells. Administration of CDs, but not PEG-CDs, in mice caused marked increase of heart rate. Both CDs and PEG-CDs did not affect other ECG parameters. In the zebrafish embryos, addition of CDs (20 µg/mL) caused heart development delay, whereas addition of CDs (80 µg/mL) led to heart malformation. In contrast, PEG-CDs caused considerably small changes in heart development, which was consistent with the results from the in situ hybridization experiments. CONCLUSION: CDs causes greater cardiac toxicity, especially regarding heart development. Polyethylene glycol modification can attenuate the cardiac toxicity of CDs.
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Carbono/química , Carbono/toxicidad , Cardiotoxicidad/prevención & control , Corazón/efectos de los fármacos , Nanoestructuras/química , Nanoestructuras/toxicidad , Polietilenglicoles/química , Polietilenglicoles/farmacología , Animales , Animales Modificados Genéticamente , Carbono/administración & dosificación , Modelos Animales de Enfermedad , Corazón/embriología , Corazón/fisiología , Cardiopatías Congénitas/inducido químicamente , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratones , Nanoestructuras/administración & dosificación , Nanoestructuras/ultraestructura , Polietilenglicoles/administración & dosificación , Pez CebraRESUMEN
MicroRNAs (miRNAs) are noncoding RNAs with a short length of about 22 nucleotides. As major modulators participating in RNA interference, they affect cellular behaviors by regulating the expression of diverse genes at post-transcriptional levels. miR-15b is a member of the miR-15/16 family, which is broadly expressed in major tissues and specially enriched in the endovascular system of human beings. miR-15/16 affects cellular proliferation, apoptosis, invasion and angiogenesis. In this review, we summarize the role and the underlying mechanism of miR-15b as well as other miR-15/16 family members in different cells, especially in endothelial cells. We focus on the diverse roles of miR-15b in the occurrence, progression and prognosis of vascular diseases, with particular emphasis on preeclampsia, a hypertensive disorder related to endovascular dysfunction in the placenta.
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Células Endoteliales/fisiología , MicroARNs/fisiología , Enfermedades Vasculares/etiología , Animales , Ciclina D1/genética , Ciclina E/genética , Humanos , Neuropilina-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Aspergillus fumigatus is an intracellular opportunistic fungus causing invasive pulmonary mycosis, characterised by hyphal invasion and destruction of pulmonary tissue. Th1 cytokines could enhance fungicidal activity. The effects from the combination of interleukin-12 (IL-12) and IL-2 are rarely known in invasive pulmonary aspergillosis infection. To assess the cleaning of A. fumigatus infection in the pulmonary tissues by IL-12 and IL-2, interferon-γ (IFN-γ) was detected in the sera using ELISA, quantification of IFN-γ mRNA using real-time RT-PCR and lung Colony-forming unit was assayed by cultivation. Morphology was analysed by histopathological examination. Our results showed that IL-12 and/or IL-2 could enhance the IFN-γ expression in the pulmonary tissue, reduce the colony load in the pulmonary tissue and increase the survival rate of mouse. The combination of IL-12 and IL-2 could assist in increasing the IFN-γ expression in the pulmonary tissue, but neither reduce colony load in the pulmonary tissue nor increase the survival rate of mouse significantly. It was demonstrated that IL-12 and IL-2 were strong immunomodulatory cytokines as a prerequisite for protecting the host from infectious agents.
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Interleucina-12/inmunología , Interleucina-2/inmunología , Aspergilosis Pulmonar Invasiva/inmunología , Animales , Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/fisiología , Modelos Animales de Enfermedad , Humanos , Interferón gamma/sangre , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/sangre , Interleucina-2/sangre , Aspergilosis Pulmonar Invasiva/sangre , Aspergilosis Pulmonar Invasiva/genética , Aspergilosis Pulmonar Invasiva/microbiología , Pulmón/inmunología , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.
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Infertilidad Masculina , Mutación Missense , Humanos , Animales , Ratones , Masculino , Estudios Retrospectivos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Infertilidad Masculina/genética , Mutación , Conducto Deferente/anomalías , Espermatogénesis/genéticaRESUMEN
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased cyclooxygenase-2 (COX-2) expression and over-production of prostaglandin E(2) (PGE(2)) in human CF bronchial epithelia cell line (CFBE41o--) with elevated NF-κB activity compared to a wild-type airway epithelial cell line (16HBE14o--). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NF-κB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE(2) involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o-- cells were challenged with LPS as well as PGE(2), indicating possible involvement of CFTR in negative regulation of COX-2/PGE(2). In conclusion, CFTR is a negative regulator of PGE(2)-mediated inflammatory response, defect of which may result in excessive activation of NF-κB, leading to over production of PGE(2) as seen in inflammatory CF tissues.
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Ciclooxigenasa 2/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/enzimología , Dinoprostona/metabolismo , Neumonía/enzimología , Mucosa Respiratoria/enzimología , Infecciones del Sistema Respiratorio/enzimología , Animales , Línea Celular , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Humanos , Ratones , Ratones Endogámicos CFTR , FN-kappa B/metabolismo , Neumonía/genética , Neumonía/microbiología , Pseudomonas aeruginosa/patogenicidad , Mucosa Respiratoria/microbiología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/microbiología , Factores de Tiempo , TransfecciónRESUMEN
STUDY QUESTION: Does elevated temperature-induced cystic fibrosis transmembrane conductance regulator (CFTR) down-regulation in Sertoli cells in cryptorchid testis disrupt testicular tight junctions (TJs) through the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway? SUMMARY ANSWER: Our results suggest that CFTR may be involved in regulating testicular TJs and the blood-testis barrier (BTB) through its negative regulation of the NF-κB/COX-2/PGE(2) pathway in Sertoli cells, a defect of which may result in the spermatogenesis defect in cryptorchidism. WHAT IS KNOWN ALREADY: Cryptorchidism, or undescended testes, is known to result in defective spermatogenesis. Although an elevated testicular temperature is regarded as an important factor affecting spermatogenesis in cryptorchidism, the exact mechanism remains elusive. It is known that the expression of functional CFTR is temperature sensitive. Our previous study has demonstrated that CFTR negatively regulates NF-κB/COX-2/PGE(2) in bronchial epithelial cells. Disruption of TJs by COX-2/PGE(2) has been found in tumour cells. STUDY DESIGN AND METHODS: Expression of CFTR, NF-κB, COX-2 and TJ proteins was examined in the testes of a surgical-induced cryptorchidism mouse model and a testicular hyperthermia mouse model, as well as in control or CFTR-inhibited/knocked down primary rat Sertoli cells. PGE(2) production was measured by ELISA. Sertoli cell barrier function was determined by transepethelial resistance (TER) measurements in rat Sertoli cell primary cultures. BTB integrity in the cryptorchidism model was monitored by examining tracker dye injected into seminiferous tubules. MAIN RESULTS: Down-regulation of CFTR accompanied by activation of NF-κB, up-regulation of COX-2 and down-regulation of TJ proteins, including ZO-1 and occludin, was observed in a cryptorchidism mouse model. BTB leakage revealed impaired BTB integrity in cryptorchid testes, confirming the destruction of TJs. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and CFTR knockout mouse model. Culturing primary Sertoli cells at 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression and PGE(2) production compared with the culture at the physiological 32°C. Inhibition or knockdown of CFTR led to increased COX-2 but decreased ZO-1 and occludin expression in Sertoli cells, which could be mimicked by PGE(2), but reversed by NF-κB or COX-2 inhibitor, suggesting that the regulation of TJs by CFTR is mediated by a NF-κB/COX-2/PGE(2) pathway. Inhibition of CFTR or administration of PGE(2) significantly decreased Sertoli cell TER. LIMITATIONS: This study has tested only the CFTR/NF-κB/COX-2/PGE(2) pathway in mouse testes in vivo and in rat Sertoli cells in vitro, and thus, it has some limitations. Further investigations in other species, especially humans, are needed. WIDER IMPLICATIONS OF THE FINDINGS: Our study may shed more light on one of the aspects of the complicated underlying mechanisms of defective spermatogenesis induced by cryptorchidism.
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Criptorquidismo/metabolismo , Ciclooxigenasa 2/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Dinoprostona/biosíntesis , Regulación hacia Abajo , FN-kappa B/biosíntesis , Uniones Estrechas/patología , Animales , Barrera Hematotesticular , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Células de Sertoli/metabolismo , Temperatura , Regulación hacia ArribaRESUMEN
OBJECTIVE: To analyze the chemical constituents of petroleum fraction of Aconitum taipeicum. METHODS: The methanol extract of Aconitum taipeicum were extracted by petroleum and then analyzed by GC-MS. The compounds were quantitatively determined by normalization method. RESULTS: Twenty eight compounds were separated and identified. Most of them were alkane, fat acids and their esters and alkenes. The Nonacosane covered 13.057% of the total peaks, while 19-methyl-18,21-Hexatriacontanediether 8.180%, Ethylen eglycol monooctadecy ether 7.851%, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 7.805%, Metahyl Palmitate 6.676% and so on. CONCLUSION: This is the first report of constituents from the flower, stem and leaf of Aconitum taipeicum. The results will provide foundation for further exploitation and use of Aconitum taipeicum.
Asunto(s)
Aconitum/química , Alcanos/aislamiento & purificación , Ácidos Grasos/aislamiento & purificación , Componentes Aéreos de las Plantas/química , Plantas Medicinales/química , Alcanos/química , Ésteres/química , Ésteres/aislamiento & purificación , Ácidos Grasos/química , Alcoholes Grasos/química , Alcoholes Grasos/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Estructura Molecular , Terpenos/química , Terpenos/aislamiento & purificaciónRESUMEN
Objective: To analyze the mechanism of LINC00461 regulating the recurrence of diffuse large B cell lymphoma (DLBCL) through microRNA (miR)-411-5p/BCL2 interacting protein 3 (BNIP3) pathway. Methods: DLBCL samples in TCGA and GSE12453 were used for differential analysis to find long noncoding RNA (lncRNA) related to DLBCL recurrence. The 4 DLBCL data with the highest and lowest expression levels of LINC00461 in the TCGA database were selected for GSEA enrichment analysis. The targeting relationships of miR-411-5p with LINC00461 and BNIP3 were verified by the dual luciferase report. Blood samples from DLBCL patients were used to analyze the correlation between miR-411-5p and LINC00461 or BNIP3. LINC00461, miR-411-5p, or BNIP3 was overexpressed or silenced by transfection, and a tumor-bearing nude mice model was constructed to detect their effects on proliferation and apoptosis. Results: The level of LINC00461 in DLBCL was significantly higher than that in normal cases, and the level in recurrence DLBCL was significantly higher than that in nonrecurrence. The enrichment analysis results showed that the function of LINC00461 was closely related to apoptosis. The results shown that miR-411-5p bound to LINC00461 and BNIP3 and was negatively correlated with LINC00461 and BNIP3 mRNA in blood of DLBCL patients. Suppressing the level of LINC00461 inhibited cell proliferation and induced apoptosis. The inhibition of LINC00461 or overexpression of miR-411-5p reduced the expression of BNIP3 protein, thereby inducing apoptosis at the in vivo and in vitro levels. Conclusion: LINC00461 may induce miR-411-5p to "sponge," thereby increasing the expression of BNIP3 protein, and exerting the function of inhibiting apoptosis and promoting DLBCL recurrence.
RESUMEN
Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairmentinduced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygenglucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagyrelated proteins, including Beclin1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 α, and apoptosisrelated proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagyrelated proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNELpositive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.
Asunto(s)
Autofagia/fisiología , Isquemia Encefálica/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , China , Femenino , Glucosa/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Oxígeno/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel regulated by cAMP-dependent phosphorylation, which is expressed in epithelial cells of a wide variety of tissues including the reproductive tracts. Mutations in the gene encoding CFTR cause cystic fibrosis, a common genetic disease in Caucasian populations with a multitude of clinical manifestations including infertility/subfertility in both sexes. However, the physiological role of CFTR in reproduction and its involvement in the pathogenesis of reproductive diseases remain largely unknown. This review discusses the role of CFTR in regulating fluid volume and bicarbonate secretion in the reproductive tracts and their importance in various reproductive events. We also discuss the contribution of CFTR dysfunction to a number of pathological conditions. The evidence presented is consistent with an important role of CFTR in reproductive health and disease, suggesting that CFTR might be a potential target for the diagnosis and treatment of reproductive diseases including infertility.