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1.
Frontiers of Medicine ; (4): 302-312, 2021.
Article in English | WPRIM | ID: wpr-880973

ABSTRACT

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.


Subject(s)
Animals , Cullin Proteins/genetics , DNA-Binding Proteins/genetics , Factor VIII , Male , Mice , Mice, Knockout , Spermatogenesis/genetics , Ubiquitin-Protein Ligases
2.
Article in English | WPRIM | ID: wpr-878443

ABSTRACT

OBJECTIVES@#The effect of Vps4b gene mutation on the expressions of cytokeratin 14 (CK14) and proliferating cell nuclear antigen (PCNA) in the Hertwig's epithelial root sheath (HERS) is investigated.@*METHODS@#The bilateral mandibular tissues of mouse on postnatal days 5, 9, 11, 15, and 19 were removed. The mandibular first molar tissue sections were obtained after paraffin embedding. The CK14 and PCNA expressions in the epithelial root sheath of the normal mouse and Vps4b knockout mouse were compared through immunohistochemistry.@*RESULTS@#On postnatal day 5, the normal mouse began to form HERS and had a strong positive PCNA expression in the HERS cells; on postnatal day 9, the HERS structure was continuous, and PCNA was positive in the HERS cells; on postnatal day 11, a small portion of HERS began to break, and PCNA was weakly positive in the HERS cells; on postnatal day 15, HERS continued to fracture; PCNA was weakly and positively expressed in the HERS cells on the root surface; on postnatal day 19, the tooth root reached normal physiological length, and PCNA was positively expressed in the HERS cells of the terminal part. Similar to the normal mouse, the gene knockout mouse also formed a HERS structure on postnatal day 5. However, HERS began to break on postnatal day 9. On postnatal day 19, only a few fragments of HERS were found on the root surface, and the root development was immature. Moreover, the expression intensity of PCNA in the gene knockout mouse was decreased.@*CONCLUSIONS@#The Vps4b gene mutation may change the CK14 and PCNA expressions, leading to abnormal root development.


Subject(s)
ATPases Associated with Diverse Cellular Activities , Animals , Endosomal Sorting Complexes Required for Transport , Epithelial Cells , Keratin-14 , Mice , Mice, Knockout , Proliferating Cell Nuclear Antigen , Tooth Root
3.
Acta Physiologica Sinica ; (6): 42-50, 2021.
Article in Chinese | WPRIM | ID: wpr-878234

ABSTRACT

This study was designed to evaluate the role of short-chain fatty acid butyrate acid on intestinal morphology and function, and atherosclerotic plaque formation in apolipoprotein E-knockout (ApoE


Subject(s)
Animals , Apolipoproteins E/genetics , Atherosclerosis/prevention & control , Butyrates/pharmacology , Caco-2 Cells , Diet, High-Fat/adverse effects , Fatty Acids, Volatile , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Plaque, Atherosclerotic
4.
Rev. chil. cardiol ; 39(1): 24-33, abr. 2020. tab, graf
Article in Spanish | LILACS | ID: biblio-1115446

ABSTRACT

INTRODUCCIÓN: Los ratones SR-B1 KO/ApoER6 1h/h que son alimentados con una dieta rica en grasas saturadas, desarrollan enfermedad coronaria aterosclerótica severa, complicaciones isquémicas e insuficiencia cardíaca, con alta mortalidad. Los estudios con este modelo se han enfocado fundamentalmente en la enfermedad coronaria y menos en el remodelado cardíaco. El OBJETIVO del trabajo ha sido caracterizar el remodelado miocárdico, evaluar la evolución temporal de la función ventricular izquierda y la sobrevida asociada a enfermedad cardíaca por ateromatosis. MÉTODO: Ratones homocigotos SR-B1 KO/ApoER6 1h/h fueron alimentados por 8 semanas con dieta aterogénica o dieta normal y se comparó la sobrevida en ambos grupos. A las 4 semanas se realizó un ecocardiograma bidimensional. En los ratones eutanasiados se evaluó en la pared cardíaca fibrosis miocárdica y tamaño de los cardiomiocitos por morfometría, apoptosis con técnica de TUNEL e infiltración por células inflamatorias mononucleares (ED1) por inmunohistoquímica. RESULTADOS: En el grupo que recibió dieta aterogénica la sobrevida se redujo en 46,7% (p < 0.001), debido a muerte súbita y a falla cardíaca progresiva. En este grupo, a las 4 semanas se observó dilatación de cavidades izquierdas y disminución de la fracción de eyección del ventrículo izquierdo en comparación con el grupo control (79,3 ± 1,3% vs 66 ± 3,7%, p<0,01). También se observó aumento de la masa cardíaca relativa de 2.1 veces (p<0,001) y del peso pulmonar relativo en 80% (p<0,001), sin cambios en las dimensiones de los cardiomiocitos. En el miocardio de los ratones que recibieron dieta aterogénica hubo un aumento de la fibrosis cardíaca de 7.9 veces (p < 0.01) y del número de cardiomiocitos apoptóticos en 55.9 veces (p < 0.01), junto a un aumento del número de células inflamatorias mononucleares ED1. CONCLUSIONES: En el modelo de falla cardíaca severa de etiología isquémica con alta mortalidad en el ratón homocigoto SR-B1 KO/ApoER6 1h/h sometido a una dieta aterogénica, con falla cardíaca izquierda por disfunción sistólica, el remodelado patológico del miocardio está dado fundamentalmente por apoptosis y fibrosis. También se observa un aumento discreto de macrófagos en la pared cardíaca. Es posible que el edema parietal también pueda ser un mecanismo de remodelado relevante en este modelo.


Abstract: SR-B1 KO/ApoER6 1h/h mice fed a high saturated fat diet develop severe coronary atheromatosis, and cardiac failure with a high mortality rate. Cardiac remodeling under these conditions has not been well studied. AIM: To evaluate the time course of left ventricular function, cardiac remodeling and survival associated to the administration of an atherogenic diet. METHOD: Homozygote SR-B1 KO/ApoER6 1h/h mice received an atherogenic diet for 8 weeks. Mice receiving a normal diet served as controls. Survival rate, myocardial fibrosis, cardiomyocyte size, apoptosis and infiltration by inflammatory or mononuclear cells were compared between groups. A TUNEL technique was used to evaluate apoptosis. RESULTS: A 46.7% survival reduction compared to controls was observed in the experimental group (p<0.01), due to left ventricular and atrial dilatation associated to a decrease in ejection fraction (79,3 ± 1,3% vs 66 ± 3,7%, p<0,01, respectively). Also, an increased cardiac weight, 2.6 times greater was observed in the experimental group, compared to controls. Mice receiving the atherogenic diet showed an 80% increased lung weight. There was no evident change in cardiomyocytes, but there was more (7.9 times) cardiac fibrosis (p<0.01) and 55.9 times more apoptotic cells. (p<0.01), along with a greater number of inflammatory cells and ED1 mononuclear cells. CONCLUSION: Mice receiving an atherogenic diet develop heart failure and reduced survival rate. This is associated with cardiac remodeling with underlying apoptosis an ventricular wall fibrosis. It is posible that wall edema might contribute to the observed cardiac remodeling.


Subject(s)
Animals , Mice , Ventricular Remodeling , Diet, Atherogenic , Heart Failure/etiology , Hyperlipidemias/pathology , Ischemia/etiology , Fibrosis , Survival Analysis , Ventricular Function, Left , Apoptosis , Mice, Knockout , Ventricular Dysfunction , Disease Models, Animal , Heart Failure/physiopathology , Heart Failure/mortality , Heart Failure/pathology , Ischemia/physiopathology , Ischemia/mortality , Ischemia/pathology
6.
Arq. bras. cardiol ; 114(1): 100-105, Jan. 2020. tab, graf
Article in English | LILACS | ID: biblio-1055084

ABSTRACT

Abstract Background: The emergence of coronary heart disease is increased with menopause, physical inactivity and with dyslipidemia. Physical training is known to promote the improvement of cardiovascular functions. Objective: To investigate the effects of aerobic physical training on the left ventricle in ovariectomized LDL knockout mice. Methods: Thirty animals were divided into 6 groups (n = 5): Sedentary non-ovariectomized control; Sedentary ovariectomized control; Trained ovariectomized control; Sedentary non-ovariectomized LDL-knockout, sedentary ovariectomized LDL-knockout and trained ovariectomized LDL-knockout. We analyzed the average parameters of apparent density of collagen fibers types I and III, and metalloproteinase type 2 and type 9, were considered significant p < 0.05. Results: The results showed that the proposed exercise protocol altered the volume of type I collagen fibers, altered collagen remodeling parameters (MMP-2), and also reduced the 8-hydroxy-2'-deoxyguanosine (8OHdG) oxidative stress parameter. Conclusion: Moderate intensity aerobic training acts on collagen fiber volume, on collagen remodeling with the reduction of oxidative stress in the left ventricles of ovariectomized LDL-knockout mice.


Resumo Fundamento: O surgimento da doença cardíaca coronariana aumenta com a menopausa, inatividade física e dislipidemia. Sabe-se que o treinamento físico promove a melhora das funções cardiovasculares Objectivo: Investigar os efeitos do treinamento físico aeróbico sobre o ventrículo esquerdo em camundongos LDL knockout ovariectomizadas. Métodos: Trinta animais foram divididos em 6 grupos (n = 5): controle sedentário não ovariectomizado, controle sedentário ovariectomizado, controle treinado ovariectomizado, sedentário LDL-knockout não ovariectomizado, sedentário LDL-knockout ovariectomizado e treinado LDL-knockout ovariectomizado. Analisamos os parâmetros médios da densidade de volume de fibras colágenas tipo I e III, e metaloproteinases 2 e 9. Valores de p < 0,05 foram considerados significativos. Resultados: Os resultados mostram que o protocolo de exercício proposto alterou o volume de fibras colágenas do tipo I e os parâmetros de remodelamento do colágeno (MMP-2), e ainda reduziu o parâmetro de estresse oxidativo do 8-hidroxi-2'-deoxiganosina (8-OhdG). Conclusão: O treinamento aeróbico de intensidade moderada age sobre o volume das fibras colágenas e sobre o remodelamento de colágeno, com redução do estresse oxidativo em ventrículos esquerdos de camundongos ovariectomizados LDLr Knockout.


Subject(s)
Animals , Female , Rats , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Collagen Type I/metabolism , Collagen Type III/metabolism , Inflammation/physiopathology , Myocardium/metabolism , Physical Conditioning, Animal/physiology , Immunohistochemistry , Ovariectomy , Mice, Knockout , Oxidative Stress/physiology , Models, Animal
7.
Article in English | WPRIM | ID: wpr-828988

ABSTRACT

Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.


Subject(s)
Animals , Antibodies , Allergy and Immunology , Blotting, Western , Fluorescent Antibody Technique , Immunization , Immunohistochemistry , Mice , Mice, Knockout , PrPC Proteins , Allergy and Immunology , PrPSc Proteins , Allergy and Immunology , Prion Proteins , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology
8.
Article in Chinese | WPRIM | ID: wpr-828916

ABSTRACT

OBJECTIVE@#To construct a HIV-1 gp120 transgenic mouse model (gp120) with 7 nicotinic acetylcholine receptor (7nAChR) gene knockout.@*METHODS@#The 7nAChR gene knockout mice (7R) were crossed with HIV-1gp120 transgenic mice (gp120) to generate F1 generation mice. We selected the F1 mice with the genotype of 7R/gp120 to mate to obtain the F2 mice. The genotypes of the F3 mice were identified by PCR, and the protein expressions in the double transgenic animal model was analyzed by immunohistochemistry. BV2 cells were treated with gp120 protein and 7nAChR inhibitor, and the expressions of IL-1β and TNF- were detected using ELISA.@*RESULTS@#The results of PCR showed the bands of the expected size in F3 mice. Two F3 mice with successful double gene editing (7R/gp120) were obtained, and immunohistochemistry showed that the brain tissue of the mice did not express 7 nAChR but with high gp120 protein expression. In the cell experiment, treatment with gp120 promoted the secretion of IL-1β and TNF- in BV2 cells, while inhibition of 7nAChR significantly decreased the expression of IL-1β and TNF- ( < 0.001).@*CONCLUSIONS@#By mating gp120 Tg mice with 7R mice, we obtained gp120 transgenic mice with 7nAChR gene deletion, which serve as a new animal model for exploring the role of 7nAChR in gp120-induced neurotoxicity.


Subject(s)
Animals , Disease Models, Animal , Glycoproteins , Mice , Mice, Knockout , Mice, Transgenic , Tumor Necrosis Factor-alpha , alpha7 Nicotinic Acetylcholine Receptor , Metabolism
9.
Article in Chinese | WPRIM | ID: wpr-828105

ABSTRACT

OBJECTIVE@#To construct a HIV-1 gp120 transgenic mice (gp120 Tg) with vimentin (VIM) gene knockout.@*METHODS@#Female HIV-1 gp120 Tg mice were mated to VIM heterozygote mice (F0). All the offspring mice were derived from these original founders so that both genotypes had the same mixed genetic background. The F1 mice were bred to generate of VIM, VIM, VIM/gp120 Tg and VIM/gp120 Tg mice. PCR was performed for genotyping of the mice, and the expressions of VIM and gp120 in the brain tissues were examined using immunoblotting.@*RESULTS@#The results of PCR showed the presence of the target bands in VIM, VIM, VIM/gp120 Tg and VIM/gp120 Tg mice. In VIM/gp120 Tg mice, gp120 expression was detected throughout the brain regions while no VIM expression was detected.@*CONCLUSIONS@#We generated gp120 transgenic mouse models with VIM gene knockout, which facilitate the exploration of the role of VIM in gp120-induced neurotoxicity.


Subject(s)
Animals , Brain , Disease Models, Animal , Female , HIV Envelope Protein gp120 , HIV-1 , Mice , Mice, Knockout , Mice, Transgenic , Vimentin
10.
Acta Physiologica Sinica ; (6): 433-440, 2020.
Article in Chinese | WPRIM | ID: wpr-827044

ABSTRACT

The aim of the present study was to investigate the effect of zinc transporter Zip2 (SLC39A2) on mitochondrial respiration during myocardial ischemia/reperfusion (I/R) and the underlying mechanisms. An in vivo myocardial I/R model was established in mice by ligation of left anterior descending coronary artery. Cardiac zinc concentration was measured by inductively coupled plasma-optical emission spectrometer (ICP-OES), and the mitochondrial respiratory function and oxidative phosphorylation were determined by high-resolution respirometry (Oxygraph-2K). The phosphorylation levels of STAT3 and ERK in myocardial tissue were detected by Western blot. The results showed that, compared with the sham group, cardiac zinc concentration in myocardium was decreased in wild-type mice and further reduced in Zip2 knockout mice after I/R. Mitochondrial respiratory control rate (RCR) and oxidative phosphorylation were decreased in Zip2 knockout mice and worsened by I/R. Phosphorylation levels of STAT3 (Ser) and ERK were significantly decreased in Zip2 knockout mice after I/R. In I/R myocardial tissue, STAT3 overexpression significantly improved the mitochondrial respiratory function, while STAT3 dominant negative mutant (STAT3 S727A) inhibited mitochondrial respiratory function. Moreover, the impairment of mitochondrial function by Zip2 knockout was reversed by STAT3 overexpression. These results suggest that Zip2 regulates mitochondrial respiration via phosphorylation of STAT3 during myocardial I/R, which may represent the underlying mechanism of Zip2 cardioprotection against I/R injury.


Subject(s)
Animals , Carrier Proteins , Mice , Mice, Knockout , Mitochondria , Myocardial Reperfusion Injury , Myocardium , Myocytes, Cardiac
11.
Protein & Cell ; (12): 894-914, 2020.
Article in English | WPRIM | ID: wpr-880885

ABSTRACT

Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.


Subject(s)
A549 Cells , Animals , Apoptosis Regulatory Proteins/immunology , DEAD Box Protein 58/immunology , Dogs , HEK293 Cells , Humans , Influenza A Virus, H1N1 Subtype/immunology , Madin Darby Canine Kidney Cells , Mice , Mice, Knockout , Orthomyxoviridae Infections/pathology , Proteolysis , Signal Transduction/immunology , THP-1 Cells , TNF Receptor-Associated Factor 3/immunology , Ubiquitination/immunology , Viral Proteins/immunology
12.
Braz. j. med. biol. res ; 53(4): e9220, 2020. graf
Article in English | LILACS | ID: biblio-1089355

ABSTRACT

Rab7, an important member of the Rab family, is closely related to autophagy, endocytosis, apoptosis, and tumor suppression but few studies have described its association with renal fibrosis. In the early stage, our group studied the effects of Rab7 on production and degradation of extracellular matrix in hypoxic renal tubular epithelial cells. Because cell culture in vitro is different from the environment in vivo, it is urgent to understand the effects in vivo. In our current study, we established a renal fibrosis model in Rab7-knock-in mice (prepared by CRISPR/Cas9 technology) and wild type (WT) C57BL/6 mice using unilateral ureteral obstruction (UUO). Seven and 14 days after UUO, the expression of the Rab7 protein in WT mice, as well as the autophagic activity, renal function, and the degree of renal fibrosis in WT and Rab7-knock-in mice were examined by blood biochemical assay, hematoxylin-eosin and Masson staining, immunohistochemistry, and western blotting. We found that the Rab7 expression in WT mice increased over time. Furthermore, the autophagic activity constantly increased in both groups, although it was higher in the Rab7-knock-in mice than in the WT mice at the same time point. Seven days after UUO, the degree of renal fibrosis was milder in the Rab7-knock-in mice than in the WT mice, but it became more severe 14 days after surgery. Similar results were found for renal function. Therefore, Rab7 suppressed renal fibrosis in mice initially, but eventually it aggravated fibrosis with the activation of autophagy.


Subject(s)
Animals , Male , Female , Rabbits , Autophagy/physiology , Ureteral Obstruction/complications , rab GTP-Binding Proteins/genetics , Kidney/pathology , Kidney Diseases/etiology , Fibrosis , RNA/isolation & purification , Signal Transduction , Up-Regulation , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction , rab GTP-Binding Proteins/metabolism
13.
Arch. endocrinol. metab. (Online) ; 63(6): 557-567, Nov.-Dec. 2019. tab, graf
Article in English | LILACS | ID: biblio-1055021

ABSTRACT

ABSTRACT In order to provide new insights into the various activities of GH in specific tissues, recent advances have allowed for the generation of tissue-specific GHR knockout mice. To date, 21 distinct tissue-specific mouse lines have been created and reported in 28 publications. Targeted tissues include liver, muscle, fat, brain, bone, heart, intestine, macrophage, pancreatic beta cells, hematopoietic stem cells, and multi-tissue "global". In this review, we provide a brief history and description of the 21 tissue-specific GHR knockout mouse lines. Arch Endocrinol Metab. 2019;63(6):557-67


Subject(s)
Animals , Rats , Receptors, Somatotropin/physiology , Growth Hormone/physiology , Signal Transduction , Mice, Knockout , Models, Animal
14.
Acta cir. bras ; 34(10): e201901003, Oct. 2019. tab, graf
Article in English | LILACS | ID: biblio-1054672

ABSTRACT

Abstract Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.


Subject(s)
Animals , Reperfusion Injury/metabolism , Connexin 43/deficiency , Disease Models, Animal , Liver/blood supply , Aspartate Aminotransferases/analysis , Reference Values , Time Factors , Reperfusion Injury/pathology , Polymerase Chain Reaction , Mice, Knockout , Connexin 43/analysis , Alanine Transaminase/analysis , Genotyping Techniques , gamma-Glutamyltransferase/analysis , Liver/pathology , Necrosis
15.
Arq. bras. cardiol ; 112(2): 180-188, Feb. 2019. graf
Article in English | LILACS | ID: biblio-983821

ABSTRACT

Abstract Background: In menopause, there is greater cellular exposure to oxidative stress, related to the decreased antioxidative effects of estrogen. These metabolic changes favor the progression of cardiovascular diseases, such as atherosclerosis. Abnormal function of the aorta - the most important artery - is associated with many cardiovascular diseases. Collagen, especially types I and III, is one of the most important aortic wall components and it can be affected by many factors, including menopause. The 8-OHdG is one of the main markers of DNA oxidative damage induced by reactive oxygen species (ROS). Objective: We aimed to investigate effects of moderate aerobic training on the ascending aorta of LDL-knockout (LDL-KO) and ovariectomized female mice. Methods: A total of 15 C57BL/6 mice and 15 LDL-KO mice were divided into experimental groups. The thickness and volume density of types I and III collagen fibers were performed by morphoquantitative analysis, whereas the MMP-2 and MMP-9 and 8-OHdG were detected by immunohistochemistry and apoptosis was detected by the TUNEL assay. The significance level for all tests was p < 0.05. Results: Exercise causes an increase in the thickness of the aorta in LDL-KO groups, particularly accentuated in the ovariectomized groups. The type I collagen fibers showed an increase in volume density influenced by training in both Control groups and in the LDL-KO group. Type III collagen density decreased in both groups. The MMP-2 showed moderade immunostaining in the tunica media in LDL-KO groups, which did not occur in the control groups and the MMP-9 stained irregularly in all tissues. The marker 8-OhdG was stronger in the exercise training groups. Additionally, the ovariectomy, the exercise training and the LDL-KO treatments increased apoptosis. Conclusion: These results suggest that moderate-intensity aerobic exercise in ovariectomized mice associated to an increase in LDL rate possibly increases oxidative stress and apoptosis induction.


Resumo Fundamento: Na menopausa, há maior exposição celular ao estresse oxidativo, relacionada à diminuição dos efeitos antioxidantes do estrogênio. Essas alterações metabólicas favorecem a progressão das doenças cardiovasculares, como a aterosclerose. A função anormal da aorta - a artéria mais importante - está associada a muitas doenças cardiovasculares. O colágeno, especialmente os tipos I e III, é um dos mais importantes componentes da parede da aorta e pode ser afetado por muitos fatores, incluindo a menopausa. Por sua vez, 8-OHdG é um dos principais marcadores de danos oxidativos do DNA induzidos por espécies reativas de oxigênio (EROS). Objetivo: Investigar os efeitos do treinamento aeróbico moderado na aorta ascendente de camundongos fêmeas, nocaute para LDL (LDL-KO) e ovariectomizadas. Métodos: Um total de 15 animais C57BL/6 e 15 animais LDL-KO foram divididos em grupos experimentais. A espessura e a densidade de volume das fibras de colágeno tipos I e III foram realizadas por análise morfoquantitativa; MMP-2 e MMP-9 e 8-OHdG foram detectadas por imunohistoquímica; e a apoptose foi detectada pelo ensaio TUNEL. O nível de significância adotado para todos os testes realizados foi p < 0,05. Resultados: o exercício causa aumento da espessura da aorta em grupos LDL-KO, particularmente acentuada em grupos ovariectomizados. As fibras de colágeno de tipo I mostraram aumento da densidade de volume influenciado pelo treinamento em animais controle e LDL-KO. A densidade do colágeno tipo III diminuiu em ambos os grupos. A MMP-2 mostrou imunomarcação moderada na túnica média em animais LDL-KO; em grupos controle, a MMP-9 marcou irregularmente em todos os tecidos. O marcador 8-OHdG foi mais forte nos grupos de treinamento de exercícios. Além disso, a ovariectomia, o treinamento físico e os tratamentos de LDL-KO aumentaram a apoptose. Conclusão: Esses resultados sugerem que exercícios aeróbicos de intensidade moderada em camundongos ovariectomizados associados ao aumento da taxa de LDL, possivelmente, aumentam o estresse oxidativo e a indução da apoptose.


Subject(s)
Animals , Female , Rats , Aorta/metabolism , Physical Conditioning, Animal/physiology , Ovariectomy , Collagen/analysis , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 9/analysis , Aorta/pathology , Menopause/metabolism , Receptors, LDL/blood , Immunohistochemistry , Tunica Media/pathology , Apoptosis/physiology , Mice, Knockout , Oxidative Stress/physiology , In Situ Nick-End Labeling , Sedentary Behavior
16.
Article in English | WPRIM | ID: wpr-739532

ABSTRACT

14-3-3γ plays diverse roles in different aspects of cellular processes. Especially in the brain where 14-3-3γ is enriched, it has been reported to be involved in neurological and psychiatric diseases (e.g. Williams-Beuren syndrome and Creutzfeldt-Jakob disease). However, behavioral abnormalities related to 14-3-3γ deficiency are largely unknown. Here, by using 14-3-3γ deficient mice, we found that homozygous knockout mice were prenatally lethal, and heterozygous mice showed developmental delay relative to wild-type littermate mice. In addition, in behavioral analyses, we found that 14-3-3γ heterozygote mice display hyperactive and depressive-like behavior along with more sensitive responses to acute stress than littermate control mice. These results suggest that 14-3-3γ levels may be involved in the developmental manifestation of related neuropsychiatric diseases. In addition, 14-3-3γ heterozygote mice may be a potential model to study the molecular pathophysiology of neuropsychiatric symptoms.


Subject(s)
Animals , Anxiety , Brain , Heterozygote , Mice , Mice, Knockout , Williams Syndrome
17.
Article in Chinese | WPRIM | ID: wpr-777467

ABSTRACT

Curcumae Rhizoma is a Chinese medicinal herb that is contraindicated during pregnancy. Cold-congelation and blood-stasis are corresponding syndromes to Curcumae Rhizoma. Whether syndrome-based treatment is associated with developmental neurotoxicity of Curcumae Rhizoma remains to be unclear. To verify the theory of traditional Chinese medicine of "syndrome-based treatment during pregnancy", the present study induced the mice blood stasis model by immersing mice in ice water. Pregnant C57 BL/6 wild type(WT) mice and pregnant Nrf2 knock out(KO) mice were randomly divided into control groups and Rhizoma Curcumae exposure groups. The mice were exposed to Rhizoma Curcumae during day 5 to day 18 after pregnancy. The neurodevelopment was examined to evaluate the differences of developmental neurotoxicity between normal and blood-stasis pregnant mice exposed to Rhizoma Curcumae. caspase-3 and caspase-9 activity in brain of the offspring were measured by colorimetric assays. Bcl-2 mRNA and protein expression in brain of the offspring were examined by Real-time RT-PCR and Western blot, respectively. According to the findings, C57 BL/6 mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) had a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring, compared with the normal control group, but with no significant change in those of blood-stasis pregnant mice offspring. However, mice exposed to Rhizoma Curcumae(10.0 g·kg~(-1)) showed no change in Bcl-2 gene expression and p38 MAPK phosphorylation in brain of the offspring. Nrf2 gene knockout using CRISPR/Cas9 resulted in a longer positive occurring time of the surface righting reflex test of offspring and higher caspase-3 and caspase-9 activities in brain of offspring. In conclusion, developmental neurotoxicity of the blood-stasis pregnant mice exposed to Rhizoma Curcumae was weaker than that of the normal pregnant mice. Nrf2 activation involved in the phenomenon of Rhizoma Curcumae of "syndrome-based treatment during pregnancy", but the upstream signal pathway mechanism value shall be further investigated.


Subject(s)
Animals , Apoptosis , Brain , Caspases , Genetics , Curcuma , Chemistry , Drugs, Chinese Herbal , Pharmacology , Female , Maternal Exposure , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2 , Genetics , Pregnancy , Proto-Oncogene Proteins c-bcl-2 , Genetics , Random Allocation , Rhizome , Chemistry , Signal Transduction
18.
Acta Physiologica Sinica ; (6): 261-270, 2019.
Article in English | WPRIM | ID: wpr-777190

ABSTRACT

It was reported that α7 nicotinic acetylcholine receptor (α7-nAChR) knockout (α7 KO) mice showed few functional phenotypes. The purpose of this study was to investigate the effect of α7 KO on the electrophysiological characteristics of hippocampus in mice. The effect of α7 KO on hippocampal CA3-CA1 synaptic transmission in mice was evaluated by standard extracellular field potential recordings. The electrophysiological phenotype of γ-aminobutyrate A receptors (GABA-Rs) of single hippocampal neuron was detected by perforated patch-clamp recordings. The results showed that, the slope of field excitatory postsynaptic potential (fEPSP) and carbachol-induced theta oscillation were significantly decreased in the hippocampal CA1 neurons of α7 KO mice, compared with those of wild type mice. Under the treatment of GABA-R agonist muscimol, the I-V curves of both the hippocampal CA1 and CA3 neurons of α7 KO mice shifted towards depolarizing direction obviously, compared with those of wild type mice. These results suggest that the hippocampal CA3-CA1 synaptic transmission in α7 KO mice was significantly impaired and GABA-R maturation was significantly delayed, indicating that the deletion of α7-nAChR gene could significantly change the electrophysiological function of the hippocampus. The results may provide a new understanding of the role of α7-nAChR in hippocampal function and associated diseases.


Subject(s)
Animals , Hippocampus , Cell Biology , Mice , Mice, Knockout , Neurons , Physiology , Phenotype , Synaptic Transmission , alpha7 Nicotinic Acetylcholine Receptor , Physiology
19.
Acta Physiologica Sinica ; (6): 588-596, 2019.
Article in Chinese | WPRIM | ID: wpr-777152

ABSTRACT

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2 mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2 mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.


Subject(s)
Animals , CRISPR-Cas Systems , Female , Gene Knockout Techniques , Gene Targeting , Male , Mice , Mice, Knockout , RNA, Guide , Genetics
20.
Chinese Medical Journal ; (24): 690-698, 2019.
Article in English | WPRIM | ID: wpr-774807

ABSTRACT

BACKGROUND@#As a potent pro-inflammatory cytokine of the interleukin (IL)-1 family, IL-18 was elevated in early active and progressive plaque-type psoriatic lesions and that serum or plasma levels of IL-18 correlated with the Psoriasis Area and Severity Index (PASI). Although results from previous studies have established that IL-18 may aggravate psoriatic inflammation, the mechanisms of this process remain unknown. In this study, IL-18 knock out (KO) mice and wild-type (WT) mice were used to investigate the effects of IL-18 within a mouse model of psoriasis.@*METHODS@#WT and IL-18 KO mice were divided into four groups, including imiquimod (IMQ)-treated IL-18 KO group (n = 11) and WT group (n = 13) as well as their respectively gene-matched control mice (receiving vaseline; n = 12). PASI scores were used to evaluate psoriatic lesions in IMQ-treated mice. Pathological features and dermal cellular infiltration were investigated by hematoxylin and eosin staining. The levels of psoriasis-related cytokines including IL-23, IL-17, IL-12, IL-1β, IFNγ, IL-15, IL-27, and IL-4 were tested by real-time polymerase chain reaction (PCR). The protein level of IL-1β, IL-27, CXCL1, and Ly6 g were investigated by immunohistochemistry (IHC).@*RESULTS@#Acanthosis (98.46 ± 14.12 vs. 222.68 ± 71.10 μm, P < 0.01) and dermal cell infiltration (572.25 ± 47.45 vs. 762.47 ± 59.59 cells/field, P < 0.01) were significantly milder in IMQ-induced IL-18 KO mice compared with that in WT mice. IMQ-induced IL-18 KO mice manifested larger areas of Munro microabscesses (11,467.83 ± 5112.09 vs. 4093.19 ± 2591.88 μm, P < 0.01) and scales (100,935.24 ± 41,167.77 vs. 41,604.41 ± 14,184.10 μm, P < 0.01) as compared with WT mice. In skin lesions of IL-18 KO mice, the expressions of IL-1β, IL-4, and IL-27 were all significantly upregulated but IL-17 was decreased. Histologically, strong positive signals of Ly6g were observed within the epidermis of IL-18 KO mice but expressions of CXCL1 were decreased.@*CONCLUSIONS@#IL-18 may exacerbate prominent inflammation and influence pathological features in IMQ-induced mouse model of psoriasis. IL-18 may upregulate pro-inflammatory cytokines and reduce protective cytokines, thus aggravating psoriatic inflammation. In addition, IL-18 may be involved in the formation of Munro microabscesses and scales.


Subject(s)
Animals , Chemokine CXCL1 , Metabolism , Cytokines , Metabolism , Disease Models, Animal , Imiquimod , Toxicity , Interleukin-17 , Metabolism , Interleukin-18 , Metabolism , Mice , Mice, Knockout , Psoriasis , Genetics , Metabolism , Skin , Allergy and Immunology , Metabolism
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