Role of CaN/NFATc4 signaling pathway in ventilator-induced lung injury / 中华麻醉学杂志

Objective To evaluate the role of calcineurin ( CaN)/nuclear factor of activated T cell cytoplasmic 4 protein ( NFATc4) signaling pathway in inflammatory responses in lung tissues of rats with ventilator-induced lung injury ( VILI) . Methods Twenty-four clean-grade healthy male Wistar rats, aged 5-8 weeks, weighing 220-250 g, were divided into 3 groups ( n=8 each) using a random number table method: control C (group C), VILI group and cyclosporine A plus VILI group (group CsA+VILI). The animals were anesthetized with pentobarbital and tracheostomized. The rats were mechanically ventilated for 6 h with the tidal volume set at 40 ml/kg and respiratory rate at 40 breaths/min to establish the model of VI-LI. The rats kept spontaneous breathing in group C. CaN specific inhibitor cyclosporine A 10 mg/kg was in-traperitoneally injected at 1 h before ventilation in group CsA+VILI. Rats were sacrificed immediately after ventilation, lung tissues were obtained and stained with hematoxylin and eosin to evaluate lung injury, broncho-alveolar lavage fluid was collected for determination of tumor necrosis factor-alpha ( TNF-α) , inter-leukin-1beta ( IL-1β) and IL-6 concentrations by enzyme-linked immunosorbent assay, and the lungs were removed for determination of the wet to dry weight ratio ( W/D ratio) , expression of intercellular adhesion molecule-1 ( ICAM-1) and vascular cell adhesion molecule-1 ( VCAM-1) ( by real-time polymerase chain reaction) , and expression of calcineurin and NFATc4 in lung tissues ( using Western blot ) . Results Compared with group C, the W/D ratio, lung injury scores and concentrations of IL-1β, IL-6 and TNF-αin BALF were significantly increased, and the expression of CaN, NFATc4, ICAM-1 mRNA and VCAM-1 mRNA was up-regulated in group VILI ( P＜0. 05) . Compared with group VILI, the W/D ratio, lung injury scores and concentrations of IL-1β, IL-6 and TNF-αin BALF were significantly decreased, and the expres-sion of CaN, NFATc4, ICAM-1 mRNA and VCAM-1 mRNA was down-regulated in group CsA+VILI ( P＜0. 05) . Conclusion CaN/NFATc4 signaling pathway mediates inflammatory responses in lung tissues of rats with VILI.

Expression and significance of nuclear factor of activated T-cells cytoplasmic 1 in hepatocellular carcinoma / 中华肝胆外科杂志

Objective To investigate the function of activated T cell nucleus factor 1 (NFATc1) in liver cancer tissues and the correlations between (NFATc1 and prognosis,and the effect of NFATc1 on liver cancer cell migration.Methods The hepatobiliary surgery department of Tianjin First Central Hospital collected 33 patients with hepatocellular carcinoma from January to June 2015,and studied the expression of NFATc1 in liver cancer and adjacent tissues and the prognosis of patients.HCC cell HepG2 were randomly divided into blank control group (without any intervention),interference group (infected with lentivirus interference sequence,down-regulated expression of NFATc1),and negative control group (infected with lentivirus negative control sequence).The relative expression of NFATc1 in the patients and the three groups were detected by real-time fluorescence quantitative polymerase chain reaction (rt-pcr),and the expressions of NFATc1,n-cadherin and e-cadherin in the three groups were detected by Western blot,and the migration of the cells was detected by Transwell.Results The mRNA level of NFATc1 in liver cancer tissues was higher than that in adjacent tissues,and the difference was statistically significant (P ＜ 0.05).According to the median expression of NFATc1,the 33 patients were divided into high-expression group (n =17) and low-expression group (n =16).The survival rate of the high-expression group was better than that of the low-expression group,and the difference was statistically significant (P ＜ 0.05).The mRNA level of NFATc1 in the interference group was lower than that in the negative control group and the blank control group,and the difference was statistically significant (P ＜0.05).The mRNA levels of NFATc1 and n-cadherin in the interference group were lower than those in the negative control group and the blank control group,while the mRNA levels of e-cadherin were higher than those in the negative control group and the blank control group,with statistically significant differences (P ＜ 0.05).The number of migrative cells in the interference group was (24.0 ± 5.6),which was lower than that in the blank control group [(69.0 ±4.0)] and the negative control group [(73.0 ±4.4)],and the difference was statistically significant (P ＜0.05).Conclusions The expression of NFATc1 is increased in liver cancer,and high level of NFATc1 indicated poor prognosis in liver cancer patients.Low expression of NFATc1 reduces the migration ability of liver cancer cells.

The expression of nuclear factor of activated T cells in hepatocellular carcinoma and its relationship with clinicopathologic factors / 中华肝胆外科杂志

Objective To analyze the expression of activated T cell nuclear factor (NFAT) in hepatoeellular carcinoma (HCC) tissues and its correlation with clinicopathological factors.Methods Data of 105 patients including 87 males and 18 females,aged 55.1 ± 10.8 years old,diagnosed with HCC who underwent hepatectomy in hepatobiliary surgery department of the first central hospital of Tianjin from September 2014 to December 2016 were retrospectively analyzed,Immunohistochemical staining was used to detect the expression of NFAT subtypes in HCC tissues and adjacent normal liver tissues,and the differences in expression of NFAT subtypes and related factors were analyzed.Results HCC tissues had higher expression of NFAT4 and lower expression of NFAT1 compared to adjacent tissues (P＜0.05).NFAT1 positive group had higher HBV infected rate (93.1％ vs.78.7％) and lower microvascular invasion rate than that in NFAT1 negative group (24.1％ vs.46.8％) (P＜ 0.05).NFAT3 positive group had more younger patients (≤ 60 years old) (80.0％ vs.60.0％) and higher microvascular invasion rate (46.2％ vs.15.0％) (P＜0.05).NFAT4 positive group had higher microvascular invasion rate (43.3％ vs.22.2％) (P＜0.05).Conclusion HCC tissues had different expressions of NFATs.The expressions of NFAT1,NFAT3 and NFAT4 are related to microvascular invasion.

Extracts of Flavoparmelia sp. Inhibit Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation

BACKGROUND: Osteoporosis is a geriatric disease with diminished bone density. The increase in the number of patients and medical expenses due to a global aging society are recognized as problems. Bone loss is the most common symptom of bone disease, not only osteoporosis but Paget's disease, rheumatoid arthritis, multiple myeloma, and other diseases. The main cause of this symptoms is excessive increase in the number and activity of osteoclasts. Osteoclasts are multinucleated giant cells that can resorb bone. They are differentiated and activation from monocytes/macrophages in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). METHODS: The effect of extract of Flavoparmelia sp. (EFV), a genus of lichenized fungi within the Parmeliaceae, on the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts was examined by phenotype assay and the cell cytotoxicity was evaluated by cell counting kit-8. The osteoclast differentiation-related genes and proteins were investigated by real-time polymerase chain reaction and immunoblotting. The functional activity of osteoclast in response to EFV treatment was evaluated by an Osteo Assay plate. RESULTS: In this study, we found that EFV, a genus of lichenized fungi within the Parmeliaceae, inhibited osteoclast formation. And we investigated its inhibitory mechanism. EFV reduced RANKL-mediated osteoclast formation and activation by inhibiting expression of nuclear factor of activated T cells 1, a key factor of osteoclastogenesis. CONCLUSIONS: Taken together, our results show that EFV is a promising candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.

Regulation of NFATc1 in Osteoclast Differentiation

Osteoclasts are unique cells that degrade the bone matrix. These large multinucleated cells differentiate from the monocyte/macrophage lineage upon stimulation by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL). Activation of transcription factors such as microphthalmia transcription factor (MITF), c-Fos, NF-kappaB, and nuclear factor-activated T cells c1 (NFATc1) is required for sufficient osteoclast differentiation. In particular, NFATc1 plays the role of a master transcription regulator of osteoclast differentiation. To date, several mechanisms, including transcription, methylation, ubiquitination, acetylation, and non-coding RNAs, have been shown to regulate expression and activation of NFATc1. In this review, we have summarized the various mechanisms that control NFATc1 regulation during osteoclast differentiation.

Recombinant mycobacterium tuberculosis heat shock protein 10 in human osteoclast differentiation / 中国组织工程研究

BACKGROUND:The mycobacterium tuberculosis heat shock protein 10 exerts effects on the osteoclasts by in vitro mouse cranium experiment, OBJECTIVE:To investigate the effect and mechanism of recombinant mycobacterium tuberculosis heat shock protein 10 (CPN10) on the differentiation of osteoclasts in the in vitro culture system that induces osteoclast differentiation. METHODHuman macrophage colony-stimulating factor-dependent adhesive blood mononuclear cells were divided into four groupreceptor activator for nuclear factor-κB ligand (RANKL)+CPN10 (1 mg/L), RANKL, CPN10 (1 mg/L), and negative control (complete culture medium). Monocytes were resuspended in a-MEM medium containing macrophage colony-stimulating factor, and were cultured in each group for 7, 14, 21 days. The morphology, quantity and bone resorption area of osteoclasts were examined by tartrate-resistant acid phosphatase (TRAP) staining. The expressions of NFATc1 and c-Fos gene and protein were also detected. RESULTS AND CONCLUSION:In negative control group, no TRAP-positive multinucleated osteoclasts generated, while in the other groups, TRAP-positive multinucleated osteoclasts differentiated and formed the lacunae in the smal bone grinding. The number of osteoclasts formation and resorption in CPN10 group were significantly lower than that in RANKL+CPN10 group. The expression of NFATc1 and c-Fos in the negative control group C was significantly lower than that of RANKL+CPN10 group and CPN10 group. However, CPN10 expressed NFATc1 and c-Fos protein, which was significantly lower than RANKL+CPN10 group. CPN10 is involved in the formation of osteoclasts, and the mechanism is related with the upregulation of NFATc1, c-Fos expression.

Nuclear factor of activated T cells negatively regulates expression of the tumor necrosis factor receptor-related 2 gene in T cells

Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.

Influence of coal-arsenic exposure on human T cells proliferation and its mechanism / 中国地方病学杂志

Objective To explore the influence of coal-arsenic exposure on human T cells proliferation and its mechanism.Methods Blood samples colleoted from individuals which lived in arsenism area of coal-burning type and non-arsenism area in Guizhou Province were divided into exposed group(17),mild(35),moderate(38) and severe arsenism group(19)and control group(35)according to Diagnosis Smndard for Endemic Arsenism (WS/T 211-2001).T cell stimulation index wag determined by methyl thiazolyl tetrazolium(MTT)colorimetric method.The intracellular Ca2+ exponential(IECa2+)in peripheral blood mononuclear cell(PBMC)was analyzed by Fho-3/AM dye and flow cytometry.DNA binding activity of actively T cells nuclear factor(NF-AT)in PBMC was evaluated by electrophoretie mobility shift assay(EMSA).Results Concanavalin A(ConA)stimulation decreased the T cells stimulation indexes in exposed group,mild,moderate and severe arsenism groups(1.315±0.962, 1.611±1.224,1.114±0.545,1.289±0.875)compared with control group(2.322±1.241),all the differences being statistically significant(P＜0.01).After stimulated by anti-CD3 monoclonal antibody(McAb),the T cells stimulation index in exposed group,mild,moderate and severe arsenism group(0.997±0.177,1.103±0.291,1.007±0.221, 0.957±0.205) were lower than that of control group(1.842±0.429,P ＜ 0.01 ). IECa2+ of PBMC after treated by anti-CD3 McAb in mild,moderate and severe arsenism group( 110.130±49.637,92.429±31.191,77.640± 35.372) were lower compared with control group(145.986±59.450,P ＜0.01 ). Moreover,IECa2+ in moderat and severe arsenism group were lower than exposed group(121.337±46.410,P ＜ 0.05). DNA binding activity of PBMC NF-AT in mild,moderate and severe arsenism group(1.354±0.446,1.290±0.291,1.159±0.411 ) were lowered than that of control group(1.722±0.291,P ＜ 0.01) and exposed group(1.611±0.294,P ＜ 0.05). Conclusions The coal-arsenic exposure can reduce the human T cells stimulation indexes,IECa2+ in PBMC and the DNA binding activity of NF-AT. It suggest that arsenic may suppress the proliferation ability of human T cells,which may be partly related to the influence of arsenic on T cell receptor(TCR)/CD3 signal transduetion pathway.