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Regulation of transcriptional factor NF-E2-related factor 2 by different doses of TNF-α

Jiaolin NING ; Liwen MO ; Zhengguo WANG ; Guocai TAO ; Xinan LAI

Chinese Journal of Pathophysiology.2010;26(4):791-796. doi:10.3969/j.issn.1000-4718.2010.04.032

AIM: To study the effects of tumor necrosis factor-α (TNF-α) on the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) in pulmonary microvascular endothelial cells. METHODS: Rat pulmonary micro-vascular endothelial cells (PMVECs) were cultured by lung tissue block pasted methods, and identified immunocytochemically using Ⅷ factor-related antigen. The cells were treated with different doses TNF-α (prepared in serum-free medium) for 4 h. Subcellular localization and levels of Nrf2 in PMVECs were observed with immunocytochemical methods. Nuclear extract were obtained to assayed transcriptional activity of Nrf2 with EMSA. Total RNA were isolated to assay the mRNA expression of Nrf2 by RT-PCR. RESULTS: The protein level of Nrf2 in the nuclei and transcriptional activity increased dose-dependently in PMVECs after treated with TNF-α at concentrations of 2.5, 5.0 or 10.0 μg/L. However, the protein level of Nrf2 in nuclei and transcriptional activity decreased dose-dependently in PMVECs after treated with TNF-α at concentrations of 20 or 40 μg/L. No different mRNA expression of Nrf2 in PMVECs treated with TNF-α at all concentration above was observed. CONCLUSION: Transcriptional activity of Nrf2 increases in PMVECs treated with low or moderate doses of TNF-α and decreases in PMVECs treated with high doses of TNF-α.

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Effect of epigenetic modification on Pdx-1 gene transcription in different mouse cells

Mingyue LI ; Xiaofang YU ; Shiyun BAO ; Baoheng LIN ; Chunyou WANG

Chinese Journal of Pathophysiology.2010;26(4):786-790. doi:10.3969/j.issn.1000-4718.2010.04.031

AIM: To investigate the role of epigenetic modification in Pdx-1 gene transcription and expression, and to compare the differences between epigenetic modifications of Pdx-1 gene promoter in various cell types of mice. METHODS: The promoter DNA methylation and histone modification status of Pdx-1 and MLH1 genes in NIT-1 cells, NIH3T3 cells and mouse embryonic stem cells were measured by chromatin immunoprecipitation-real time PCR method. The expression levels of these genes in the three cell lines were measured by real time RT-PCR. The relation between epigenetic modifications and gene expression was analyzed. RESULTS: (1) Compared to mES cells, there was lower DNA methylation and higher H3K4m3 modification levels in the promoter of Pdx-1 gene in NIT-1 cells (P<0.05). DNA methylation, H3 acetylation, H3K4m3 and H3K9m3 modification levels in the promoter of Pdx-1 gene in NIH3T3 cells were distinctly increased (P<0.05). (2) Pdx-1 gene transcription expressed only in NIT-1 cells. The Spearman's rho between Pdx-1 gene expression and DNA methylation (r=-0.802,P<0.01) was observed. The Pearson correlation between Pdx-1 gene expression and H3K4m3 modification (r=0.997,P<0.01) was also found. The Spearman's rho between Pdx-1 gene expression and H3K9m3 modification (r=-0.879,P<0.01) was observed. (3) No correlation between housekeeper MLH1 gene expression and epigenetic modification was found. CONCLUSION: DNA methylation, H3K4m3 and H3K9m3 modification coordinated participate to regulate and control the expression of Pdx-1 gene. It is of great significance to the differentiation of β cells from ES cells.

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SEB superantigen induces steroid resistance in PBMCs

Fengyu WANG ; Jianhua ZHOU ; Liru QIU

Chinese Journal of Pathophysiology.2010;26(4):781-785. doi:10.3969/j.issn.1000-4718.2010.04.030

AIM: To establish a model of staphylococcal enterotoxin B (SEB)-induced steroid resistance in human peripheral blood mononuclear cells (PBMCs), and to investigate the potential mechanism of SEB superantigen-induced steroid resistance in vitro. METHODS: PBMCs were isolated from normal children blood by Ficoll-Hypaque gradient centrifugation and stimulated with SEB at different concentrations. The proliferation rate of cells was measured by MTT assay. The subcellular localization of glucocorticoid receptor α (GRα) was examined by confocal microscopy. Protein phosphorylation was measured by means of Western blotting. RESULTS: SEB induced steroid resistance in a range of 10-500 μg/L and no significant difference among concentrations was observed. In SEB-stimulated PBMCs, the GRα did not translocate to the nuclear after dexamethasone treatment. ERK inhibitor U0126 significantly attenuated the inhibition of GRα nuclear translocation in SEB-stimulated PBMCs. SEB also induced more rapid and sustained phosphorylation of ERK1/2 in PBMCs. CONCLUSION: This study demonstrates that SEB may contribute to steroid resistance through ERK pathway and is associated with abrogation of GRα nuclear translocation.

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Expression of T cell-specific transcriptional factor Elf-1 in mononu-clear cells, CD4~+ T cells and CD8~+ T cells from umbilical cord blood

Chunlan LIN ; Shaohua CHEN ; Lijian YANG ; Haitao ZHENG ; Qi SHEN ; Yubin ZHOU ; Yangqiu LI

Chinese Journal of Pathophysiology.2010;26(4):777-780. doi:10.3969/j.issn.1000-4718.2010.04.029

AIM: To investigate the expression level of transcriptional factor Elf-1 in mononuclear cells, CD4~+ and CD8~+ T cells from umbilical cord blood (UCB). METHODS: Real-time PCR with SYBR green I technique was used for detecting the Elf-1 expression in mononuclear cells, sorted CD4~+ and CD8~+ T cells from 12 cases of umbilical cord blood. The relative mRNA expression level of Elf-1 was analyzed by a formula of 2~(-△Ct)×100%. The expression level of β_2-microglobulin gene (β_2M) was used as an endogenous reference. The peripheral blood from 10 cases of healthy adults was served as control. RESULTS: Elf-1 mRNA expressed in all blood samples collected from both UCB and healthy adults. The expression level showed apparent diversity in different individuals. The relative mRNA expression of Elf-1 in both mononuclear cells (18.55%±2.48%) and CD8~+ T cells (3.52%±0.45%) from UCB were significantly higher than those from healthy adults (9.16%±1.92%, 2.02%±0.27%, respectively, P<0.01, P<0.05). CONCLUSION: The results of Elf-1 expression level from umbilical cord blood indicate that the over-expression of Elf-1 gene in mononuclear cells and CD8~+ T cells might be one of the features of T cell immune state in umbilical cord blood.

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Antioxidant and immunoregulatory effects of Centella asiatica extracts

Huijuan HE ; Jing LI ; Weijie ZHU ; Lihua LU

Chinese Journal of Pathophysiology.2010;26(4):771-776. doi:10.3969/j.issn.1000-4718.2010.04.028

AIM: To determine the antioxidant and the immunoregulatory effects of Centella asiatica extracts. METHODS: Centella asiatica was extracted with alcohol and different organic solvent. The content of polyphenol was determined by Folin-ciocalteau method. The efficacy of the extracts to scavenge the hydroxy radical (OH·), 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH·) and intra-cellular reactive oxygen species (ROS) was measured. Lymphocyte proliferation was assessed to observe the influence of Centella asiatica extracts. RESULTS: (1) Centella asiatica was found to have abundant polyphenol extrated from different phases and in a descending order as follows: ethyl acetate extracts>n-butanol extracts>water extracts>ligarine extracts. (2) Extracts of Centella asiatica exhibited the scavenging efficacy of OH· and DPPH· free radicals, in which the acetic ether extracts showed the significant effect. (3) The acetic ether extracts had significant ability to inhibit the generation of ROS in stimulated lymphocytes. (4) The acetic ether extracts suppressed the lymphocyte proliferation. (5) The active ingredient was identified as flavone. CONCLUSION: Flavones in the Centella asiatica posseses antioxidant activity and effectively inhibits lymphocyte proliferation, showing ability of immunosuppre-ssion.

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siRNA knocks down Bax gene and inhibits TNF-α induced apoptosis in A549 cells

Gen HU ; Li LI ; Wenjie HUANG

Chinese Journal of Pathophysiology.2010;26(4):766-770. doi:10.3969/j.issn.1000-4718.2010.04.027

AIM: To investigate the influence of tumor necrosis factor-α (TNF-α) induced apoptosis through knocking down Bax gene by RNA interference (RNAi) in human alveolar type II epithelial cells (A549). METHODS: A549 cells were cultured in vitro and divided into 4 groups according to RNAi treatment: control group, TNF-α treated group, Bax siRNA group and control siRNA group. Chemically synthesized small interfering RNA (siRNA) directed against human Bax gene was transfected into A549 cells by cationic liposome. The effect of RNAi was investigated by reverse transcription PCR, Western blotting and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry. RESULTS: Bax gene was knocked down effectively in Bax siRNA group (P<0.05). Apoptosis was induced by TNF-α in TNF-α treated group and control siRNA group. However, it was abolished in Bax siRNA group by the downregulation of Bax gene compared to TNF-α treated group and control siRNA group (P<0.05). CONCLUSION: This result suggests that Bax gene has a significant role of pro-apoptosis in A549 cells and knocking down Bax gene by RNAi can effectively inhibit TNF-α-induced apoptosis in A549 cells.

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Expression of PPARγ, Nrf2 and γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid of guinea pigs with bronchial asthma

Jian CHEN ; Aiguo DAI ; Ruicheng HU ; Liming ZHU

Chinese Journal of Pathophysiology.2010;26(4):760-765. doi:10.3969/j.issn.1000-4718.2010.04.026

AIM: To investigate the expression of PPARγ and Nrf2/γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid(BALF) of guinea pig with bronchial asthma of acute episode, and to explore the roles of PPARγ on Nrf2/γ-GCS-h expression. METHODS: Forty adult male guinea pigs were randomly divided into 4 groups (10 guinea pigs in each group): control group (group A), asthmatic group (group B), dexamethasone treatment group (group C) and rogridone treatment group (group D). The asthmatic model was established by an ovalbumin challenge method. BALF was collected, and the total cell count and the proportion of the inflammatory cells were measured. After centrifugation, the concentrations of ROS and MDA in the clear supernatant were detected. The methods of in situ hybridization and immunohistochemistry were used for detecting the expression of PPARγ and Nrf2/γ-GCS-h at mRNA and protein levels. RESULTS: The proportion of eosinophils (EOS) in BALF in group B was significantly higher than that in groups A, C and D (P<0.01). The concentrations of ROS and MDA in BALF of group B was the highest. The difference of ROS and MDA was statistically significant (all P<0.05) as compared to the control. The results of immunohistochemistry and in situ hybridization indicated that the A value was the lowest in group B as compared to that in groups A, C and D (all P<0.01). In group B, the positive correlations were observed between PPARγ and Nrf2/γ-GCS-h, between γ-GCS-h and Nrf2. A negative correlation between the proportion of EOS in BALF and the expression of PPARγ and Nrf2/γ-GCS-h was also observed (all P<0.05). CONCLUSION: In acute asthmatic models induced by ovalbumin, the expression of PPARγ and Nrf2/γ-GCS-h is decreased, and PPARγ may up-regulate the expression of Nrf2/γ-GCS-h to inhibit the inflammatory and oxidative reactions, indicating a new way for prevention and treatment of bronchial asthma.

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Effect of high glucose toxicity on JNK pathway, cell viability and apoptosis in pancreatic β-cell line INS-1

Yan ZHANG ; Li YUAN ; Zhaosheng TANG

Chinese Journal of Pathophysiology.2010;26(4):755-759. doi:10.3969/j.issn.1000-4718.2010.04.025

AIM: To investigate the effect of high glucose toxicity on JNK pathway and cell function of INS-1 cells.METHODS: Cultured INS-1 cells with or without IGF-1 exposure, were treated with glucose at 3 concentrations (5.6 mmol/L, 11.2 mmol/L and 33.3mmol/L), respectively. MTT was used to measure the cell viability. Apoptosis was determined by immuno-fluorescence and flow-cytometry analysis. The serine 270 phosphorylation of IRS and phosphorylation of JNK in INS-1 cells were detected in the presence or absence of SP600125 treatment.RESULTS: The cell viability decreased and apoptosis increased with elevated glucose concentrations. The percentage of apoptosis cells was 11.3% in 5.6 G group, 12.7% in 11.2 G group and 28.2% in 33.3 G group. There was remarkable increase in apoptosis in 33.3 G group with a 2.49-fold increase to the cells in the basal 5.6 mmol/L glucose. High glucose activated the serine 270 phosphorylation of IRS correlates with JNK phosphorylation in INS-1 cells. Using Western blotting analysis, the levels of JNK phosphorylation were 3.33 fold increased and serine 270 phosphorylation of IRS was 1.17 fold increased in 33.3 G group compared to 11.2 G group (P<0.01). IGF-1 treatment inhibited phosphorylation of JNK and IRS. SP600125 treatment completely blocked JNK phosphorylation in 11.2 G group and reduced JNK phosphorylation by 90% in 33.3 G group. In addition, SP600125 treatment partly reduced serine 270 phosphorylation of IRS by 88.3% in 11.2 G group and 80% in 33.3 G group, the viability of INS-1 cells increased and the apoptosis decreased.CONCLUSION: The toxicity of chronic high glucose, which inhibits the cells viability and induces the cell apoptosis, might be related to suppress IRS signal by activating the JNK pathway. Blocking the JNK pathway might relieve the effect of glucose toxicity to the β cell function by improving the IRS signal pathway.

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Changes of PKAC-β, c-Fos and BDNF in cerebral cortex after intracerebral hemorrhage in rats treated with WIN55-212-2

Li ZHU ; Zhi DONG ; Guodong ZHANG

Chinese Journal of Pathophysiology.2010;26(9):1728-1733. doi:10.3969/j.issn.1000-4718.2010.09.014

AIM: To observe the effect of cannabinoid receptor (CB1R) agonist WIN55-212-2 on the expression of brain-derived neurotrophic factor (BDNF), c-Fos and protein kinase A beta-catalytic subunit (PKAC-β) in cerebrum cortex after intracerebral hemorrhage (ICH) in rats. METHODS: The intracerebral hemorrhage model of rat was made by the injection of collagenase Ⅶ, and WIN55-212-2 was intraperitoneally (ip) injected 30 min later. The rats were killed for sampling the brain tissues as specimens 24 h after ICH. The methods of immunohistochemical analysis and Western blotting were adopted to detect the expression of PKAC-β and BDNF. The mRNA expression of PKAC-β, c-Fos and BDNF was determined by RT-PCR. RESULTS: WIN55-212-2 obviously improved some nervous deficit symptoms and increased the expression of BDNF at mRNA and protein levels with upregulating the mRNA expression of c-Fos and downregulating the expression of PKA at mRNA and protein levels in the ipsilateral cerebral cortex. The proteins of PKAC-β, c-Fos and BDNF were expressed on the membrane or nucleus of the neuron or in the cytoplasm of glial cells, respectively. CONCLUSION: The expression of BDNF is induced not only by upregulation of c-Fos, but also by downregulation of PKA in WIN55-212-2 treated rats.

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Blunted response of L-type calcium current to simulated ischemia and reperfusion in ventricular myocytes from diabetic rabbits

Jian LI ; Qing LIU ; Guangping LI

Chinese Journal of Pathophysiology.2010;26(11):2155-2160. doi:10.3969/j.issn.1000-4718.2010.11.014

AIM: To evaluate the effects of simulated acute ischemia and reperfusion on L-type calcium current (ICa,L) in ventricular myocytes from diabetic and non-diabetic rabbits.METHODS: Using whole-cell patch clamp techniques, ICa,L was measured in left ventricular myocytes isolated from 6-week alloxan-induced diabetic rabbits and age-matched control ones at baseline, 5 min of simulated ischemia, and 5 min of reperfusion.RESULTS: There were no significant differences on baseline maximum ICa,L densities between diabetic and control ventricular myocytes. In control cells (n=11), maximal ICa,L densities of baseline, ischemia and reperfusion were (-8.36±1.63)pA/pF, (-5.90±1.75)pA/pF and (-4.22±1.02)pA/pF, respectively. The ICa,L of ischemia was less than that of baseline (P<0.01), while the ICa,L of reperfusion was less than those of baseline (P<0.01) and ischemia (P<0.05). In diabetic cells (n=9),the ICa,L of baseline, ischemia and reperfusion were (-7.55±1.62)pA/pF, (-6.05±1.58)pA/pF and (-5.12±1.13)pA/pF, respectively. Only ICa,L of reperfusion was less than that of baseline (P<0.01), while ICa,L of ischemia was not significantly different from that of baseline (P>0.05) or reperfusion (P>0.05).CONCLUSION: ICa,L in diabetic ventricular myocytes represents blunted response to acute ischemic injury, being decreased more slowly than that in control cells. Post-ischemic reperfusion is still a potent inhibitor against ICa,L in both diabetic and non-diabetic cells. This study may be indicative of the mechanism about ischemia-reperfusion injury to diabetic myocardium and the therapy for diabetic patients with ischemic heart disease.

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