In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Assessment of infection status in treponema pallidum antibody-reactive blood donors / 中国输血杂志

【Objective】 To evaluate the infection status and potential infectivity of Treponema pallidum specific antibody (anti-TP) reactive blood donors, and to provide reference for the key prevention and screening of TP under the current screening strategy. 【Methods】 From February to October 2021, 133 blood donors were tested reactive by two different anti-TP ELISA kits (77 cases were dual-reagent reactive and 56 cases were single-reagent reactive). Syphilis specific IgM antibody (TP-IgM) and IgG antibody (TP-IgG) were detected by Western blot (WB), and TRUST was conducted. The results were analyzed. 【Results】 Of the 133 samples, 24 (18.05%) were positive for TP-IgM, 40 (30.07%) were positive for TP-IgG, and 3 (2.26%) were positive for TRUST. Among them, 12 cases (15.58%) were TP-IgM positive and 40 cases (51.95%) were TP-IgG positive in 77 cases of double reagent reactivity, and 12 cases (21.43%) were TP-IgM positive and 0 was TP-IgG positive in 56 cases of single reagent reactivity. There was no significant difference in the positive rate of TP-IgM between the two groups (P>0.05), while the positive rate of TP-IgG in donors with double reagent reaction was higher than that in donors with single reagent reaction (P<0.05). In addition, among the 133 anti-TP-reactive blood donors, 15 cases were positive for single TP-IgM (11.28%, accounting for 62.50% of the total positive number of TP-IgM, a total of 12 cases of TP-IgM positive among the single reagent reactive patients, and all of them were TP-IgM positive and TP-IgG negative); 30 cases were positive for single TP-IgG (22.56%, accounting for 75.00% of the total positive number of TP-IgG). There were 55 cases (41.35%) who were negative for TP-IgM and TP-IgG, and 8 cases (6.02%) were both positive. 【Conclusion】 The TP-IgM positive donors in anti-TP reactive blood donors are infectious, but the positive rate is not high. Those with single reagent reactivity and single TP-IgM positive are prone to miss detection, which should be controlled. Those who were both TP-IgM and TP-IgG negative and those who were only TP-IgG positive may be false reactivity and the phenomenon of lifelong antibody expression. It is suggested to consider adding TP-IgM detection as a measurement index for permanent deferral of both reagents.

Effects of Chronic Restraint Stress on Apoptosis of Amygdala Cells in Rats / 法医学杂志

OBJECTIVES@#To explore the damage effects of chronic restraint stress (CRS) on amygdala cells through the rat CRS model.@*METHODS@#The rat CRS model was established, and the changes in body weight and adrenal mass in control group and CRS group were monitored at 1 d, 7 d, 14 d and 21 d. The behavior changes were evaluated by the percentage of retention time of open arms and open arm entries using the elevated plus maze (EPM). ELISA was used to detect the concentrations of rat's corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol. The changes of expression of glucocorticoid receptor (GR) and glial fibrillary acidic protein (GFAP) in amygdala were determined by immunohistochemistry and Western blotting. Ultrastructure changes of glial cell were observed by transmission electron microscopy. The apoptosis rate of amygdala was measured by flow cytometry.@*RESULTS@#Compared with the control group at the same time points, body weight of CRS 1 d, 7 d, 14 d and 21 d groups increased slowly, but adrenal mass increased significantly; the serum level of CRH, cortisol and ACTH increased significantly at 7 d, 14 d and 21 d respectively; the expression of GR in amygdala was increased while that of GFAP was decreased; EPM test suggested that the percentage of retention time of open arms and open arm entries decreased significantly after 14 d. The CRS group showed different degrees of glial cell damage in amygdala, and the apoptosis rate of glial cell was significantly increased in 21 d group.@*CONCLUSIONS@#This study successfully established a CRS model in rats, and anxiety-like behavioral changes in model rats may be caused by apoptosis of amygdala astrocytes.

Antitumor effect and mechanism of different extracts of cultivated Phellinus vaninii on H22 tumor bearing mice / 生物工程学报

In order to explore the antitumor effect and mechanism of different extracts of cultivated Phellinus vaninii fruit body on H22 tumor bearing mice, 150 ICR mice were randomly divided into blank group, model group, CTX group, P. vaninii water extract group, ethanol extract group, petroleum ether extract group and crude polysaccharide group. H22 liver cancer cells were used to establish a solid tumor model and the mice were sacrificed on the 10th day after administration. The spleen and thymus organ index and tumor inhibition rate were calculated, the serum levels of TNF-α, INF-γ, VEGF, and hematoxylin-eosin were detected, and the immunohistochemical staining method was used to observe the pathological changes of tumor tissues, while Western blotting was used to detect the expression of tumor-related proteins. The high-dose petroleum ether extract group showed the best tumor inhibition rate (73.21%), increased serum levels of TNF-α, IFN-γ, and VEGF, as well as significantly promoted tumor necrosis and ablation. The immunohistochemistry of the water extract group showed negative regulation, indicating an insignificant tumor suppression. Western blotting showed the apoptosis genes Caspase-3, Caspase-9 and pathway genes NF-κB and JAK were all highly expressed in each administration group compared with the model group, and their expression levels gradually decreased with increasing doses. In summary, the petroleum ether extract of P. vaninii fruit body showed a significant anti-tumor effect which is presumably mediated through the mitochondrial pathway. The metabolism of drug in the body induces activation of Caspase-3 and Caspase-9 apoptotic proteins by Bax, Bcl-2, and TNF, which further caused nuclear chromatin or DNA to condense or degrade, and subsequently destroy the normal proliferation of tumor cells, thereby inducing their apoptosis and inhibiting tumor growth.

Toxocara canis 30-35 kDa excretory-secretory antigen is an important marker in mice challenged by inocula containing different parasite load levels

ABSTRACT The Western-blotting technique was applied to identify antigenic fractions of excretory-secretory Toxocara canis antigen recognized by IgG antibodies throughout an experimental infection in mice challenged by different inocula. Mice were inoculated with 5, 50 and 500 embryonated eggs and serum samples were collected 15, 30, 60, 90 and 120 days post-infection. Serum samples were analyzed using an excretory-secretory Toxocara antigen. Antibodies recognized antigenic fractions from 30 to 90 kDa. The protein fraction of 30-35 kDa was the most frequently recognized regardless of the size of inoculum and the stage of infection represented by the different collection times, but the antigenic recognition was more evident in groups infected with 50 and 500 eggs. This study presents an antigenic panel of the excretory-secretory antigen of T. canis and suggests that the 30-35 kDa antigenic fraction is a promising marker of the infection and should be further explored in future studies on experimental toxocariasis.

Follow-up study of cases with indeterminate HIV antibody immunoblotting test results / 公共卫生与预防医学

Objective To study the characteristics and follow-up results of 94 people with indeterminate Western blot (WB) results of HIV antibody. Methods A retrospective analysis was conducted on the characteristics and outcome of 94 patients with indeterminate WB results of HIV antibody from AIDS confirmation laboratories in Jiading District from 2016 to 2019. Results The 94 subjects came mainly from clinical diagnostic tests, accounting for 68.09% of the total, followed by counseling tests and maternal examinations, accounting for 12.77% and 10.64%，respectively. Follow-up testing was performed on 48 patients with indeterminate HIV antibodies, with a follow-up detection rate of 51.06%. Among them, 25.00% turned positive during the follow-up, and 39.58% turned negative. There was no significant difference in the follow-up rate between different population groups and WB band-types. The positive conversion rate of counseling test samples was the highest, accounting for 55.56% of all positive conversion cases. There was a total of 14 WB band types, mainly in p24, gpl60 and p24pl60, accounting for 54.26%, 17.02% and 9.57%, respectively. There was a significant difference in the positive conversion rate of antibodies with different patterns of WB bands. The positive conversion rate of WB band type gp160gp41p24 was 100%, while the positive rate of gp160p24, gp160, and p24 was 66.67%, 37.5% and 11.54%, respectively. Conclusion The samples with indeterminate WB results of HIV antibody were mainly from medical institutions, and the follow-up retest rate was low. Follow-up tests of specific populations and samples with specific patterns of WB bands should be strengthened to confirm HIV infection status as soon as possible.

Analysis of allergens in children with common allergic diseases in Guangzhou / 公共卫生与预防医学

Objective To understand the distribution of allergens in children with common allergic diseases in Guangzhou, and to provide evidence for the prevention and treatment of environmental allergens in children with different allergic diseases. Methods This study enrolled a total of 6 869 children with allergic diseases, including wheezing diseases and asthma (1 013 cases), allergic rhinitis (660 cases), allergic conjunctivitis (422 cases), eczema (2 762 cases), atopic dermatitis (831 cases) and urticaria (1 181 cases). Western blotting was used to detect the serum allergen type E specific antibodies (sIgE) of the patients. The results were statistically analyzed. Results The total positive rate of sIgE in 6 869 children was 74.01%. The positive rate of male children (75.02%) was higher than that of female children (72.25%). The positive rate of inhalation allergens (53.47%) was higher than that of food allergens (57.39%). House dust mites and milk were the most common inhalation allergens and food allergens, respectively. Eczema was the most common allergic disease in infants and toddlers. Allergic rhinitis and urticaria were common in school-age children. Conclusion The main allergens causing allergic diseases in children in Guangzhou were dust mites and milk. Different allergic diseases had its specific allergens. It is necessary to take targeted prevention measures accordingly.

Caracterización del patrón proteico y perfil antigénico de larvas L 3 de anisákidos de importancia médica aisladas de peces comerciales de la costa peruana / Characterization of the protein pattern and antigen profile of L 3 larvae of medical importance in anisakids isolated from commercial fish of the peruvian coast

RESUMEN Introducción. La anisakidosis es una zoonosis causada por la ingestión accidental de larvas (L3) de anisákidos. Objetivo. Caracterizar el patrón proteico y perfil antigénico de la L3 de Anisakis simplex s.l. (tipo I), A. physeteris s.l. (tipo II) y Contracaecum osculatum s.l. aisladas de peces comerciales. Métodos. Se realizó una corrida electroforética en SDS-PAGE de los antígenos somáticos. Se inmunizó conejos experimentalmente y se evaluó por EITB. Resultados. El patrón proteico de Anisakis tipo I mostró 12 bandas, 18 Anisakis tipo II y C. osculatum 13, con las bandas 10 y 35 kDa específicas para Anisakis tipo II, 28 y 77 para C. osculatum no presentes en Anisakis tipo I. Conclusión. Se determinó bandas inmunogénicas específicas para Anisakis tipo I con las proteínas de peso molecular 11, 14, 25 y 40 kDa, para el tipo II de 9, 10, 12, 35 y 41 kDa, y C. osculatum 13, 15, 17, 30 y 47 kDa.

ABSTRACT Introduction. Anisakidosis is a zoonosis caused by accidental ingestion of anisakid larvae (L3). Objective. To characterize the protein pattern and antigenic profile of the L3 of Anisakis simplex s.l. (type I), A. physeteris s.l. (type II) and Contracaecum osculatum s.l. commercial3 fish isolated. Methods. An SDS-PAGE electrophoretic run of the somatic antigens was performed. Rabbits were immunized experimentally and evaluated by EITB. Results. The protein pattern of Anisakis type I showed 12 bands, 18 Anisakis type II and C. osculatum 13, with bands 10 and 35 kDa specific for Anisakis type II, 28 and 77 for C. osculatum, not present in Anisakis type I. Conclusion. Specific immunogenic bands were determined for Anisakis type I with the molecular weight proteins 11, 14, 25 and 40 kDa, for type II of 9, 10, 12, 35 and 41 kDa and C. osculatum 13, 15, 17, 30 and 47 kDa.

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21 / Comparação da progressão da senescência em células mesenquimais de parede de cordão umbilical humano medida por imunofluorescência e citometria de fluxo de p16 e p21

ABSTRACT Objective To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory. Methods Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence). Results A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence. Conclusion Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.

RESUMO Objetivo Acompanhar a expansão de células-tronco mesenquimais de cordão umbilical por dois marcadores clássicos de senescência, p16 (INK4A) e p21 (CDKN1A), usando métodos práticos, rápidos e com custo menor do que a técnica padrão-ouro de Western blotting, para avaliar sua aplicabilidade em laboratório. Métodos Células-tronco mesenquimais de cordão umbilical foram isoladas da geleia de Wharton e, após controle de qualidade e caracterização morfológica e imunofenotípica por citometria de fluxo, foram expandidas em cultura, até chegarem próximas à parada do ciclo celular (senescência replicativa). Resultados Foi feita a comparação entre células jovens, na passagem 5, e células pré-senescentes, na passagem 10, avaliando a expressão proteica dos marcadores clássicos de senescência celular p16 e p21, comparando os resultados obtidos por Western blotting com os obtidos por citometria de fluxo e imunofluorescência indireta. Conclusão O seguimento de culturas celulares, por meio da imunofluorescência indireta de p16, permite identificar as culturas de células-tronco mesenquimais de cordão umbilical em risco de atingirem a senescência replicativa.

Prokaryotic expression, purification and polyclonal antibody preparation of arginase in Agaricus bisporus / 国际药学研究杂志

Objective: To provide a method for preparing a specific polyclonal antibody of ARG. Methods: Firstly, the technology of polymerase chain reaction(PCR)was adopted to amplify the AbArg gene encoding arginase in Agaricus bisporus. Then the recombinant plasmid pET-28a(+)-Arg was constructed and transformed into E. coli BL21 for the expression of fusion protein. Afterwards, the purified protein was used to immunize New Zealand white rabbits, and the serum samples were collected. Results: The prokaryotic expression plasmid pET-28a(+)-Arg was constructed and the results of SDS-PAGE analysis showed that the fusion protein existing in form of inclusion body was successfully induced and expressed. After purification, the fusion protein with high purity (above 90%)was obtained. The immunity serum titer was 51 200 and Western blotting results showed that the polyclonal antibody could specifically recognize ARG protein in A. bisporus. Conclusion: Polyclonal antibody anti-ARG was prepared successfully, which lays a foundation for the further study on the mechanism of arginine metabolism.

Effects of hydrogen sulfide on pulmonary vascular remodeling and its inhibitors in rats with pulmonary hypertension / 解剖学报

Objective To explore the effects of hydrogen sulfide on pulmonary vascular remodeling and its inhibitors in rats with pulmonary hypertension ( PH). Methods Thirty male SD rats were randomly divided into control group ( 10 rats) , model group ( 10 rats) and H2S intervention group ( 10 rats) , PH model was induced by Lilium Wilfordii in model group, on the basis of model group, rats in H2S intervention group were injected with NaHS (56 u,mol/kg) intraperitoneally, while rats in control group were injected with normal saline at the same dose. Four weeks later, the hemodynamic parameters were measured, the right ventricular hypertrophy index (RVHI) was calculated, the pathological changes of pulmonary vessels were detected by HE staining, and the expressions of p38 and c-Jun N-terminal kinase( JNK) proteins in the mitogen-activated protein kinase (MAPK) family were detected by Western blotting and Real-time PCR. Results There were significant differences in hemodynamics, RVHI, wall thickness as a percentage of vessel diameter ( WT) % , pulmonary vessel wall area as a percentage of vascular cross-sectional area( WA) % , p38 and JNK in each group (P<0. 05). The expression levels of MSAP, MPAP, RVHI, WT%, WA%, p38 mRNA and JNK mRNA in the model group and H2S intervention group were significantly higher than those in the control group (P<0. 05) , while the levels of MSAP , MPAP , RVHI, WT% , WA% , p38 mRNA and JNK mRNA in H2S intervention group were significantly lower than those in the model group (P<0. 05). The pulmonary artery morphology showed that the wall thickness and lumen stenosis of the model group and the H2S intervention group increased compared with the control group, but the lumen thickness and lumen stenosis of the H2S intervention group were significantly reduced compared with the model group; Western blotting showed that the expressions of p38 and JNK in model group and H2S intervention group were higher than those in control group, while the expressions of p38 and JNK in H2S intervention group were lower than those in model group. Conclusion H2S can improve hemorheology, right ventricular hypertrophy index, alleviate pulmonary artery wall thickening and lumen stenosis, and inhibit pulmonary vascular remodeling in PH rats. Its mechanism may be related to the down-regulation of JNK and p38 protein expression in MAPK signaling pathway by H2S.

Polypeptide compound urantide inhibiting expression of type i collagen in rat heart of atherosclerosis / 解剖学报

Objective To observe the effect of the peptide compound urantide on the expression of type I collagen (Col I ) in the heart tissue of rats with atherosclerosis ( As) , and to explore its mechanism of prevention and treatment of heart damage in As rats. Methods Sixty healthy male 3-week-old SPF Wistar rats were selected. The As model was established by intraperitoneal injection of vitamin D3(VD3) to damage the arterial intima and high-fat diet. They were randomly divided into normal group, As model group, simvastatin group and urantide (3 days, 7 days, 14 days) groups. HE staining and Masson trichrome staining were used to observe the morphology and collagen fiber expression of rat hearts. Immunohistochemistry, Western blotting and Real-time PCR were used to detect the expression of Col I protein and gene in rat heart. Results Compared with the normal group, pathological phenomena such As myocardial cell degeneration, intercellular infiltration of a large number of neutrophils, scattered foam cells and hyperemia and hemorrhage were observed in the heart tissues of the As model group. Meanwhile, collagen fibers increased, and the gene and protein expression levels of Col I increased. Compared with the As model group, the cardiac pathological phenomena were effectively alleviated after the treatment with urantide. With the extension of the administration time, the collagen fibers decreased, and the gene and protein expression levels of Col I were gradually down-regulated, especially the effect was the best when the drug was given for 14 days. Conclusion Urantide can inhibit the expression of Col I in As heart to reduce myocardial interstitial damage, and has a protective effect on the heart of As rats.

Mechanism of microRAN-145 on proliferation and apoptosis of breast cancer cell line MCF-7 / 解剖学报

Objective To explore the mechanism of microRNA-145 (miR-145) involved in proliferation and apoptosis of breast cancer MCF-7 cells. Methods The immortalized breast cancer cell line MCF-7 cells were cultured in vitro and transfected with miR-145. Real-time PCR and Western blotting were used to detect mRNA and protein levels, respectively. The proliferation level of each group was detected by cell counting kit-8 (CCK-8) method , and the apoptosis level of MCF-7 cells in different treatment groups was detected by flow cytometer. Results Real-time PCR result showed that miR-145 did not affect Caspase-3, proliferating cell nuclear antigen (PCNA) and B cell lymphoma factor 2 (Bcl-2) mRNA levels in MCF-7 cells. Western blotting analysis showed that compared with the control group, transfection of miR-145 for 96 hours significantly increased the expression of Caspase-3 and inhibited the expression of PCNA and Bcl-2. The result of CCK-8 assay showed that the proliferation rate of MCF-7 cells was decreased after overexpression of miR-145 for 72 hours and 96 hours (P<0. 05). The result of flow cytometer showed that the apoptosis rate of MCF-7 cells in overexpression group was significantly higher than that in the control group (P<0. 05). Conclusion MiR-145 can inhibit the proliferation and promote the apoptosis of MCF-7 cells by down-regulating PCNA and Bcl-2 and up-regulating the expression of Caspase-3, which may be a new target for breast cancer treatment.

Gambogic acid suppresses invasion in human gastric carcinoma SGC-7901 cells by modulating inhibitor of nuclear factor kappa-B kinase α/p65 signaling pathway in vitro / 解剖学报

Objective To investigate the effect of gambogic acid (GA) on invasion in human gastric carcinoma SGC-7901 cells and its possible mechanism. Methods Cell counting kit-8(CCK-8) assay was performed to detect the effects of GA, inhibitor of nuclear factor kappa-B kinase( IKK) 16 and 5-fluorouracil (5-FU) on cell activity of GES-1 and SGC-7901 cells. Cell invasion was assessed with Transwell invasion assay. Western blotting was used to analyze the protein levels of vimentin, matrix metalloproteinase 2 ( MMP-2) and MMP-9 and protein phosphorylation of IKKα and p65. Results The cell activity was significantly decreased in SGC-7901 cells treated with GA in a dose-dependent manner with a half inhibiton concentration(IC50) value of 1. 89 μmol/L. But GA had no significant influence on cell viability of GES-1 cells. Meanwhile, 5-FU reduced the cell activity of GES-1 and SGC-7901 cells with IC50values of 7.36 μmol/L and 199.57 μmol/L respectively. Low-dose GA and IKK 16 impaired separately the ability of invasion in SGC-7901 cells, and down-regulated the protein levels of MMP-2, MMP-9 and vimentin, and inhibited phosphorylation of IKKot and p65, while a stronger inhibition was showed when the combination of GA and IKK16 was used. Conclusion Low-dose GA might inhibit invasion of SGC-7901 cells via IKKot/p65 signaling pathway.

Anti-apoptotic effect of human acute myeloid leukemia cell line HL-60 stimulated by interleukin-6 from bone marrow stromal cells HS-5 / 解剖学报

Objective To investigate the effect of interleukin( IL)-6 secreted by bone marrow stromal cells HS-5 on activity and apoptosis of human acute myeloid leukemia( AML) cells HL-60 and its possible mechanism. Methods HL-60 cells and HS-5 cells were cultured in vitro, and the co-culture system was established. Scanning electron microscope, ELISA, cell counting kit-8( CCK-8) , AnnexinV-FITC/PI, double-staining flow cytometry, Real-time PCR and Western blotting techniques were used to detect the changes of viability and apoptosis of HL-60 cells, respectively. HL-60 cells from different groups were inoculated into BALB/c nude mice to observe and record tumorigenesis. Results IL-6 secreted by bone marrow stromal cells HS-5 could enhance the viability and inhibit the apoptosis of HL-60 cells, and down-regulate the expression of pro-apoptotic gene Bax and and up-regulate the expression of anti-apoptotic gene Bcl-2 in HL-60 cells. HL-60 cells in co-culture group had the strongest tumorigenicity in BALB/c nude mice, while HL-60 cells alone had the weakest tumorigenicity. Conclusion Part of the mechanism by which bone marrow stromal cells HS-5 promote the proliferation of HL-60 cells and inhibit their apoptosis may be through the secretion of IL-6.

Enhancing effect of Lupeol on migration and invasion abilities of ST3Gaim-silenced MDA-MB-231 cells / 解剖学报

Objective In this study, we expored the enhancing effect of Lupeol on migration and invasion abilities of ST3Gal III-silenced MDA-MB-231 cells. Methods Human breast cancer cell line ST3Gal III -silenced MDA-MB-231 was cultured in vitro. The cell adhesion, Transwell and woud healing test were utilized to test the effect of Lupeol on ST3GalM-silenced MDA-MB-231 cells. The expression of matrix metalloproteinase( MMP)-2, -9, phosphatidylinositol 3-kinase/protein kinase B/nuclear factor κB ( PI3K/Akt/NF-KB) signaling pathway were examined by Western blotting. Results No significant influence of the decreased expression of ST3Gal III and Lupeol ( 5μmol/L) on proliferation and apoptosis of MDA-MB-231 cells were found. Lupeol inhibited the migration and invasion of ST3Gal III -silenced MDA-MB-231 cells in vilro( P<0. 05). Furthermore, the expression of NF-κB p65, p-Akt, Akt, p85, MMP-2 and MMP-9 levels were significantly down-regulated. Conclusion These observations suggest that Lupeol may inhibit the abilities of migration and invasion of ST3Gal III-silenced MDA-MB-231 cells in vitro by inhibiting the protein expression of MMP-2, -9 and effect the signaling pathway of PI3K/Akt/NF-κB.

Combining effects of aspirin and nimodipine on prognosis of cerebral ischemia reperfusion / 解剖学报

Objective To investigate the effects of the combination of aspirin and nimodipine preconditioning on the prognosis of cerebral ischemia-reperfusion. Methods Eighty healthy male SD rats were randomly divided into sham group, model group, aspirin preconditioning group and aspirin + nimodipine preconditioning group, with 20 rats in each group. The model of cerebral ischemia reperfusion was established. The rats in each group were given intragastric administration for 5 days before the model was established, and the drug was administered daily for 5 days. Sham group and model group were given normal saline; Aspirin preconditioning group was given 50 mg/kg aspirin; Aspirin + nimodipine preconditioning group was given 50 mg/kg aspirin and 10 mg/kg nimodipine. After 2 hours ischemia and 24 hours reperfusion, the animals were neurologically assessed, and then the volume of cerebral infarction was measured by TTC staining. The contents of superoxide dismutase (SOD), malondialdehyde (MDA), thromboxane B2, 6-keto-prostaglandin-la in brain tissue were determined by ELISA. The mRNA expression of Notch 1, Jagged 1 and Hesl in the brain tissues were detected by Real-time PCR, and the expressions of Jaggedl and Hesl, a downstream substance in Notch signaling pathway, were detected by Western blotting. Results The neurological deficit score of the aspirin+nimodipine pretreatment group was significantly lower than model group (P<0. 05) , and the cerebral infarction volume was significantly smaller than other groups. The SOD and 6-keto-prostaglandin 1 in the aspirin pretreatment group and the aspirin plus nimodipine pretreatment group were significantly higher than those in the model group, and the expressions of MDA, thromboxane B2 and thromboxane B2/6-keto-prostaglandin 1 were low. In the model group, the changes in the aspirin + nimodipine pretreatment group were more significant (P<0. 05). The expression levels of Notch 1 , Jagged 1 and Hesl mRNA and protein in the aspirin pretreatment group and aspirin + nimodipine pretreatment group were significantly lower than those in the model group (P<0. 05) , and the expression level of aspirin + nimodipine pretreatment group was lower than that of aspirin pretreatment group (P<0. 05). Conclusion The protective effect of aspirin plus nimodipine is superior to aspirin alone, which can significantly improve cerebral ischemia-reperfusion injury in rats, which may be exerted through influencing notch signaling pathway to achieve brain tissue protection.

Effect of silencing beclinl gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation / 解剖学报

Objective To investigate the effect of Beclinl gene on apoptosis of HT22 of mouse hippocampus neuron treated with oxygen and glucose deprivation/reoxygenation ( OGD/R). Methods HT22 cells in logarithmic growth phase were randomly divided into 4 groups; normal, model, beclin1 -/-and control. All groups were reoxygenated after 6 hours of oxygen and glucose deprivation except for normal group. The beclinl interference sequence was designed for mouse cDNA sequence using RNAi technology, and was transfected into HT22 cells by liposome Lipo2000. The transfection efficiency was observed under fluorescence microscope and Western blotting after 48 hours of transfection. Cell viability was detected by CCK-8 method. Cell damage was detected by lactate dehydrogenase(LDH) method , Bax and Bcl-2 were detected by immunofluorescence staining and the expression of LC3, P62 and Caspase-3 were tested by Western blotting after 24 hours of reoxygenation. SPSS 19. 0 statistical software was used for data analysis. Results Compared with the normal group, the cell viability and P62 expression decreased significantly (P<0. 01) , the LDH leakage rate, LC3 II/LC3 I , Caspase-3 expression and Bax/Bcl-2 increased significantly in model group (P<0. 01). Compared with the model group, the cell viability and LC3 II /LC3 I decreased significantly (P<0. 01) , the LDH leakage rate, Bax/Bcl-2,P62 and Caspase-3 expression increased significantly in beclin1-/-group (P<0.01). There was no difference between the control group and the model group. Conclusion Silencing beclinl inhibites autophagy, which aggravates the damage of OGD/R HT22 cells and further increases apoptosis.

Dact1 involvement in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation by regulating cx43

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Systemic injection of an H4 receptor agonist induces a decrease in CREB and pCREB levels in the cerebellar vermis and prefrontal cortex in mice

Studies have shown that an injection with the histamine H4 receptor agonist VUF-8430 modulates emotional memory processes. In the present study, the aim was to verify if intraperitoneal (ip) injection of VUF-8430 (500 ng/kg) in mice affects the synthesis of proteins required for memory consolidation processes by activating the phosphorylation of CREB (pCREB) in classical structures linked to emotional memory (prefrontal cortex, amygdala, and hippocampus) and the cerebellar vermis, a structure that has also been recently implicated in emotional memory. The results obtained using western blot analysis demonstrated that VUF-8430 induced a decrease in CREB and pCREB levels in the cerebellar vermis and prefrontal cortex, suggesting that this dose impaired the activation of cell signaling pathways in these structures. There was no change in protein expression in the amygdala and hippocampus. Our results are preliminary, and further investigations are needed to investigate the role of the H4 receptors in the central nervous system.