Effects of NFAT5 inhibition on proliferation, invasion, migration and apoptosis of lung adenocarcinoma cells / 国际肿瘤学杂志

Objective:To investigate the expressions of nuclear factor of activated T cell 5 (NFAT5) in lung adenocarcinoma tissues and cells, and the effects of NFAT5 on the proliferation, invasion, migration and apoptosis of lung adenocarcinoma cells.Methods:The clinical pathological specimens and paracancerous tissues of 61 patients with lung adenocarcinoma diagnosed and treated in 904th Hospital of Joint Logistic Support Force of People′s Liberation Army from June 2017 to June 2019 were collected. The expression levels of NFAT5 in lung adenocarcinoma tissues and paracancerous tissues were detected by quantitative real-time PCR (qRT-PCR), and the relationships between the expression of NFAT5 and clinicopathological features of patients were analyzed. H1975 cells were divided into control group (no treatment), NC group (transfecting siRNA-NC) and si-NFAT5 group (transfecting siRNA-NFAT5) . qRT-PCR was used to detect the expression level of NFAT5 in cell line. MTT and clone formation assay were used to detect cell proliferation. Transwell and scratch test were used to detect cell invasion and migration ability. Flow cytometry was used to detect cell apoptosis. The expressions of mitogen-activated protein kinase (MAPK) signaling pathway related proteins were detected by Western blotting.Results:The expression level of NFAT5 mRNA in lung adenocarcinoma (3.22±0.20) was significantly higher than that in paracancerous tissues (1.00±0.12), and there was a statistically significant difference ( t=75.662, P<0.001). The expression level of NFAT5 in lung adenocarcinoma tissue was associated with TNM stage ( χ2=10.357, P=0.001) and lymph node metastasis ( χ2=18.268, P<0.001). The expression levels of NFAT5 in the control group, NC group and si-NFAT5 group were 1.00±0.06, 1.01±0.05 and 0.31±0.06, and there was a statistically significant difference ( F=140.498, P<0.001). The absorbance ( A) values in the control group, NC group and si-NFAT5 group were 0.70±0.01, 0.55±0.01 and 0.35±0.01 at 24 h after transfection, 0.92±0.03, 0.87±0.06 and 0.57±0.06 at 48 h after transfection, 1.05±0.01, 0.90±0.01 and 0.66±0.01 at 72 h after transfection, and there were statistically significant differences ( F=9.815, P=0.013; F=45.977, P<0.001; F=129.494, P<0.001). Further pairwise comparison showed that the proliferation abilities of the si-NFAT5 group at 24, 48 and 72 h were significantly lower than those of the control group and NC group (all P<0.001). The cell clone numbers in the three groups were 452.33±31.50, 421.00±17.35 and 129.00±17.35 respectively, with a statistically significant difference ( F=128.200, P<0.001). The cell clone number in the si-NFAT5 group was significantly lower than that in the control group and NC group (both P<0.001). The invasion numbers of cells in the three groups were 262.67±28.02, 278.00±27.50 and 46.00±12.00 respectively, and there was a statistically significant difference ( F=89.896, P<0.001). The cell invasive ability in the si-NFAT5 group was significantly lower than that in the control group and NC group (both P<0.001). The relative scratch widths in the three groups were 0.28±0.04, 0.32±0.04 and 0.54±0.04 respectively, and there was a statistically significant difference ( F=42.889, P<0.001). The relative scratch width in the si-NFAT5 group was significantly increased than that in the control group and NC group (both P<0.001). The apoptosis rates in the three groups were (3.38±0.56)%, (3.14±0.62)% and (13.82±0.75)% respectively, and there was a statistically significant difference ( F=264.705, P<0.001). The apoptosis rate in the si-NFAT5 group was significantly higher than that in the control group and NC group (both P<0.001). The differences of protein expressions of NFAT5, p-P38/P38, p-ERK1/2/ERK1/2, p-JNK/JNK among the three groups were statistically significant ( F=91.245, P<0.001; F=132.896, P<0.001; F=243.332, P<0.001; F=118.358, P<0.001). The protein expressions of NFAT5, p-P38/P38, p-ERK1/2/ERK1/2, p-JNK/JNK in the si-NFAT5 group were all significantly lower than those in the control group and NC group, and there were statistically significant differences (all P<0.001). Conclusion:The expression of NFAT5 is increased in lung adenocarcinoma tissues and cells. Inhibition of NFAT5 can inhibit proliferation, invasion and migration of lung adenocarcinoma H1975 cells, and promote apoptosis of H1975 cells. The mechanism may be related to the inhibition of MAPK signal pathway by NFAT5.

Effect of rhizoma drynariae on osteoclast differentiation is related to the concentration of drug-containing serum / 中国组织工程研究

BACKGROUND: Total flavonoids of Rhizoma Drynariae can be used to prevent and treat osteoporosis, but its mechanism of action is mostly focused on osteoblasts rather than osteoclast differentiation. OBJECTIVE: To investigate the effect of Rhizoma Drynariae on osteoclast differentiation through T-cell nuclear factor 1 (NFATc1) signaling pathway. METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. Total flavonoids of Rhizoma Drynariae were intragastrically administered at 0, 11.6, 34.8 and 104.4 g/kg, respectively. Control serum and low-, middle-, high-concentration drug-containing sera were obtained. Bone marrow macrophages were isolated from 5-week-old rats and the effects of different drug-containing sera on macrophage activity were detected by cell counting kit-8 method. The macrophages were divided into low-, middle-and high-concentration groups, negative control group and blank group, with 5 multiple holes in each well. The low, middle and high concentration of drug-containing serum, negative control serum culture medium and normal culture medium were added respectively. All culture media were used to induce osteoclast differentiation with 20 μg/L macrophage colony-stimulating factor and 100 μg/L receptor activator of nuclear factor kappa B ligand. The number of osteoclasts and fusion index were detected by tartrate-resistant acid phosphatase staining 7 days after induction of differentiation. The expressions of c-Fos, Fra-1, Fra-2, NFATc1 and cathepsin K were detected by real-time fluorescence quantitative PCR and western blot at 7 days after differentiation. The number of bone resorption lacunae and the proportion of lacunae area in osteoclasts after 14 days of differentiation were detected by bone fragment absorption lacunae test. The study protocol was approved by the Animal Experiment Ethics Committee of Anyang District Hospital of Puyang City with approval No. PYSAYDQ-2019096. RESULTS AND CONCLUSION: Cell counting kit-8 results showed that there was no significant change in macrophage activity after intervention with different concentration of sera containing Rhizoma Drynariae. The morphology of macrophages was regular before differentiation. After differentiation, osteoclasts were identified by tartrate-resistant acid phosphatase staining. Compared with the blank group and negative control group, the number of osteoclasts, fusion index, number of bone resorption lacunae and proportion of lacunae area and the relative expressions of c-Fos, Fra-1, Fra-2, NFATc1, cathepsin K mRNA and protein in the low-, middle-and high-concentration groups decreased significantly (P middle-concentration group > high-concentration group, and there were significant differences between different concentration groups (P < 0.05). In conclusion, Rhizoma Drynariae may inhibit the differentiation of rat macrophages into osteoclasts through NFATc1 signaling pathway, and the degree of differentiation inhibition is related to the serum concentration of Rhizoma Drynariae.

East java extract propolis as potential intracanal medicament in experimentally induced chronic apical periodontitis

Introduction: A persistent infection after cleaning and shaping root canal is the main etiology of root canal treatment failure. Enterococcus faecalis has been considered as one of the most resistant species in root canal treatment. E. faecalis can stimulate receptor activator of nuclear factor-kappa B ligand (RANKL) which can increase nuclear factor of activated T-cell (NFATc1) in chronic apical periodontitis. East Java propolis has antibacterial effects and is biocompatible with in vitro effects. Aim: This study is aimed to analyze the East Java propolis extract as potential intracanal medicament in chronic apical periodontitis caused by E. faecalis bacterial infection. Materials and Methods: This study used 30 Wistar rats divided into three groups. In Group I, the first upper right molar tooth as healthy tooth was used for negative control group. In Group II, the first upper right molar tooth was used for a prepared root canal, and 10 ml brain heart infusion broth containing E. faecalis ATCC29212 106 CFU was injected into the canal and restored with glass-ionomer cement (GIC) for the experimentally induced chronic apical periodontitis group. In Group III, after root canal preparation, E. faecalis ATCC 29212 106 CFU was injected, and then, 10 μl propolis applied and tooth restored with GIC. It took 21 days for the periapical lesions to develop after pulp infection. The rats were then sacrificed to conduct immunohistochemical examinations in order to measure the expressions of RANKL and NFATc1. Results: The average of RANKL and NFATc1 expression in Group III was significantly lower than those in the experimentally induced chronic apical periodontitis group (P < 0.05). Conclusion: It can be concluded that East Java propolis extract is a potential intracanal medicament through the study of experimentally induced chronic apical periodontitis caused by E. faecalis infection in Wistar rats.

Mitochondrial aldehyde dehydrogenase 2 protects against high glucose-induced injury in neonatal rat cardiomyocytes by regulating CaN-NFAT3 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes.@*METHODS@#The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting.@*RESULTS@#Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05).@*CONCLUSIONS@#Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.

The expression of apoptosis associated protein 3 and nuclear factor 3 of activated T-cell in the tissue of epithelial ovarian tumors and its correlation with clinicopathological features / 中国基层医药

Objective To investigate the expressions of apoptosis associated protein 3 (APR3) and nuclear factor 3 of activated T-cell (NFAT3) in the tissue of epithelial ovarian tumors and its correlation with the clinicopathological features.Methods 92 patients with epithelial ovarian tumor were collected,23 cases with malignant tumor,24 cases with borderline tumor,45 cases with benign tumor.The expressions of APR3 and NFAT3 were detected by immunohistochemical methods,and the differences of different types of epithelial ovarian tumor were compared.The correlation of the expressions of APR3 and NFAT3 with the clinicopathological features of epithelial ovarian tumor was analyzed.The correlation of the expressions of APR3 with the expressions of NFAT3 in epithelial ovarian tumor was analyzed.Results The positive expression rate of APR3 in patients with malignant epithelial ovarian tumors (78.26％) was significantly higher than borderline tumors (41.67 ％) and benign tumors (22.22 ％),the differences were statistically significant (x2 =5.864,7.632,all P ＜ 0.05).The expression of APR3 in patients with malignant epithelial ovarian tumors was significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis and ascites (x2 =7.425,7.262,8.421,5.031,all P ＜ 0.05).The positive expression rate of NFAT3 in patients with malignant epithelial ovarian tumors (56.52％) was significantly higher than borderline tumors (29.17％) and benign tumors(17.78％),the differences were statistically significant (x2 =6.829,7.547,all P ＜0.05).The expression of NFAT3 in patients with malignant epithelial ovarian tumors was significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis (x2 =5.253,6.367,8.021,all P ＜ 0.05).The expressions of APR3 and NFAT3 in patients with malignant epithelial ovarian tumors were positively correlated (r =0.032,P ＜ 0.05).Conclusion The expressions of APR3 and NFAT3 in the tissue of malignant epithelial ovarian tumor obviously increase,are significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis and are positively correlated,and it may be correlated with the development and progression of malignant epithelial ovarian tumor.

Potential role of nuclear factor of activated T cells in cancer progression / 肿瘤

The nuclear factor of activated T cells (NFAT) - a group of transcription factors ubiquitously expressed in mammalian tissues, plays a critical role in orchestrating the intricate cellular interactions that characterize vertebrate development and morphogenesis. Recently, accumulated evidence points to an emerging role for NFAT transcription factors in cancer progression. Various NFAT isoforms are remarkably functional in tumor cells and multiple compartments in the tumor microenvironment, promoting carcinogenesis and cancer invasion. This review highlights the current knowledge about the role of NFAT in oncobiology, including tumor generation, growth, survival, malignant transformation, invasion, metastasis and angiogenesis. Clarifying the parts played by NFAT in tumor progression will help the development of effective therapeutics that target the NFAT pathway in neoplastic progression and metastasis.

EXPRESSION OF AN ACTIVATED T CELL ANTIGEN p140 IN ALLOGRAFT RENAL TRANSPLANTATION / 解放军医学杂志

To investigate expression of the novel membrane molecule p140 on activated T cell in patients after renal transplantation, im-munofluoescence staining and FCM analysis were utilized to monitor the expression of p140, and transplanted renal biopsy was employed to confirm acute allograft rejection. p140 is a transplantion antigen-induced molecule on activated T cells. It expresses weakly on T cells in patients after renal transplantation, but expresses remarkably during actue allograft rejection.

Study of the activated state of TH1/TH2 cytokines on ankylosing sondylitis / 中国免疫学杂志

Objective:To investigate the activated state of TH1/TH2 cytokines and T lymphocytes and to explore the pathogenesis of ankylosing sondylitis.Methods:Cytokine levels of Th1(IFN-?、TNF-?、IL-2)and Th2(IL-10、IL-5、IL-4) in plasma, percentages of CD3 +、CD4 +、CD8 + T cells、B cells(CD19 +) NK cells(CD16 +56 +)and CD3 +HLA-DR +、CD4 +HLA-DR +、CD8 +HLA-DR + T cells in peripheral blood lymphocytes were detected by Flow Cytometry.Results:In patients with AS ,plasma levels of TNF-?、IL-2 were significantly lower than that of healthy controls. IL-10 were significantly higher than that of healthy controls. In AS patients, percentages of CD3 +and CD8 + T cell from peripheral blood lymphocytes were significantly lower than that of healthy controls. Percentages of CD8 +HLA-DR + T cell were significantly lower than that of healthy controls. CD4 +HLA-DR +T cell was significantly highter than that of healthy controls.Conclusion:In patients with AS , lower levels of TNF-?、 IL-2 and higher level IL-10 at plasma indicate an inclination between TH1 and TH2, such as an impaired TH1 cytokine profile and an activated TH2 cytokine profile, especially in TNF-?.

Development of Monoclonal Antibodies Recognizing Human Peripheral Blood T Lymphocytes Cytoplasmic Proteins Induced upon Activation / 대한면역학회지

Antigen-specific T cell activation requires interaction of the T cell with specialized antigen-presenting cells. Signaling through the TCR is necessary but not sufficient to induce antigen-specific T cell activation and cytokine secretion. This first signal, termed signal 1, is both antigen-specific and MHC-restricted. Signal 2, which is neither antigen-specific nor MHC-restricted, is necessary to induce cytokine secretion, cellular proliferation, and effector function. Recently immunological studies in T cell activation area are mainly focused on biological and molecular biological characterization of TCR/CD3 complex and accessary molecules providing costimulatory signal (signal 2). If signal 2 is not delivered, T cell enter a state of long term un-responsiveness to specific antigen-termed anergy. Monoclonal antibody technique has been especially involved in recognizing novel inducible cell surface antigens on T cell activation. This study was aimed to develop monoclonal antibodies recognizing novel cytoplasmic proteins present in activated T cells. We make 6 monoclones involved in changing pattern of T cell activated cytoplasmic proteins. Using these 6 monoclonal antibodies analyze to find novel molecules involved in T cell activation associated response, apoptosis, and/or heat shock response of the T cells in early T cell activation.

Immunocytochemical Study on the Change of the Activated T Cells in Peripheral Blood of the Pulmonary Tuberculosis Patients / 결핵

BACKGROUND: It has been found that Helper T cells in the peripheral blood are decreased in the cell mediated immunity in the pulmonary tuberculosis But it has not been confirmed yet that only decrease in number of cells which has phenotype in the peripheral blood is defined to decrease in cell mediated immunity. The immunocytochemical study was performed to observe the change of the percentage of T-lymphocytes with their subsets and activated T cells in the peripheral blood of pulmonary tuberculosis and to know how many T cells would be activated, relative to resting cells in the peripheral blood. METHODS: The peripheral blood obtained from twenty two patients and ten healthy controls were smeared on the gelatin coated slide glass prepared for of mononuclear cells. The double bridge technique of alkaline phosphatase-antialkaline phosphatase(APAAP) method was used. As the primary antibodies, T1(anti-human T cell), T4(anti-human helper/inducer T cells) and T8(anti-human supressor/cytotoxic T cell) antibodies and interleukin-2 receptor (for early activated T cell),very late activation antigen (for activated cytotoxic T cell), T cell lineage specific activation antigen monoclonal actibodies were used. RESULTS: 1) There were significantly decrease in the absolute number of T4(+) cells but significantly increase of T8(+) cells in the peripheral blood of pulmonary tuberculosis (p<0.05). 2) The percentage of T4(+) cells showed significantly decrease in pulmonary tuberculosis but T8 (+)cells significantly increase(p<0.05). T4(+)/T8 (+) ratio showed significantly decrease in the peripheral blood of the pulmonary tuberculosis(p <0.05) 3) There were significantly increase in the absolute number of variable stages of activated T cells in the peripheral blood of the pulmonary tuberculosis(p<0.05). 4) The percentage of IL-2R, VLA-1, TLiSA were 6.45+1.56%, 7.64+1.34*, 10.45+1.16% in order which showed significantly increase in the peripheral blood of the pulmonary tuberculosis(p <0.05). CONCLUSION: We speculate that only a few percentage of T lymphocyte is activated in cell mediated immunity in pulmonary tuberculosis.