Influence of coadministration of β-mercaptoethanol and nitroglycerin on cell viability of peripheral blood-derived endothelial progenitor cells in patients with coronary heart disease

AIM: To observe the effects and mechanisms of nitroglycerin (NTG) on cell viability and β-mercaptoethanol (β-ME) on ameliorating nitrate tolerance of peripheral blood-derived endothelial progenitor cells (EPCs) in coronary heart disease (CAD) patients.METHODS: We studied 75 patients with diagnosis of coronary artery disease who were assigned to control group and NTG group. EPCs were evaluated by flow cytometry. Vascular endothelial growth factor-A (VEGF-A) and peroxynitrite anion (ONOO^-) production were measured by ELISA. EPCs were cultured in vitro with NTG and β-ME stimulation. The cell viability was determined by MTT assay. The levels of VEGF-A, ONOO^- and reactive oxygen species (ROS) were measured by ELISA and DCFH-DA assay. The protein levels of Akt,p-Akt,endothelial nitric oxide synthase (eNOS) and p-eNOS were determined by Western blot. RESULTS: Compared with the control group, the circulating EPCs levels were significantly lowered, plasma ONOO^- production was vitally increased, but there was a markedly decrease of VEGF-A production in the patients treated with excess NTG(P<0.05). Moderate dose of NTG increased the viability of EPCs, VEGF-A production, and phosphorylated protein levels of Akt and eNOS. Excess NTG was shown to reverse the effect of moderate dose of NTG, but β-ME improved the adverse effect of excess NTG. CONCLUSION: Moderate dose of NTG effectively promotes EPCs viability by PI3K/Akt/eNOS signaling pathway and β-ME improves NTG-induced tolerance by reducing oxidative stress and up-regulating the PI3K/Akt/eNOS signaling pathway.     

Overexpression of HBXIP in HepG2 cells induces cell migration and regulates expression of β-catenin

AIM: To investigate the effects of hepatitis B virus X-interacting protein(HBXIP) in hepatic cancer cells on the cell migration and expression of β-catenin. METHODS: Transwell assay was used to assess the cell migration. Gelatin zymography was used to observe the activity of matrix metalloproteinase 9 (MMP-9). The expression of MMP-9, glycogen synthase kinase 3β(GSK-3β), p-GSK3β, β-catenin and p-β-catenin in HepG2 cells was determined by Western blotting. RESULTS: HepG2 cells which stably overexpressed HBXIP (HepG2-HBXIP) exhibited higher migration ability than the control cells. The results of the gelatin zymography assay showed that HBXIP overexpression increased the activity of MMP-9 in HepG2 cells. The results of Western blotting indicated that HBXIP increased the expression of MMP-9 and β-catenin, inhibited the phosphorylation of β-catenin and promoted the phosphorylation of GSK-3β (Ser9). CONCLUSION: HBXIP regulates the GSK-3β/β-catenin signaling pathway, resulting in a significant improvement of hepatocellular carcinoma cell migration.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Effect of mutant β-catenin gene on the proliferation of hepatocytes

AIM:The β-catenin is a key molecule in the Wnt signal pathway, which plays a critical role in normal development and tumorigenesis. However, the mechanisms of the β-catenin on the cell growth control are still not completely defined. The aim of this study was to test the hypothesis that the mutant β-catenin may regulate the hepatocyte proliferation. METHODS: The immortalized murine hepatocyte cell line, AML12, was used for this study. A plasmid that contain mutant β-catenin S33Y was transfected into the AML12 cells and a stable cell line AML12S33Y was established. The cell growth property of this cell line and the parental cell were compared by flow cytometry analysis and direct cell count. The cells were also tested for the ability to form soft agar colonies, and the ability to form tumors in the severe immune deficient mice (SCID). RESULTS:1. The mutant β-catenin containing cell line AML12S33Y has higher proliferating index compared with the parental AML12 cells (P<0.01), suggesting that mutant β-catenin promotes cell growth. 2. The mutant β-catenin cells formed small colonies in soft agar after 4 weeks of culture, but did not generate tumor in SCID mice. CONCLUSION:The mutant β-catenin promotes liver cell growth.     

β-estradiol promotes invasion and migration of lung cancer A549 cells via ERβ-mediated ERK1/2 membrane-initiated steroid signaling

AIM: To investigate the regulatory mechanism of β-estradiol in the invasion and migration of lung cancer A549 cells. METHODS: Breast cancer MCF-7 cells and lung cancer A549 cells were cultured in vitro. The MCF-7 cells were used as the estrogen receptor (ER) positive expression cell model. Real-time PCR and immunofluorescence were employed to measure the expression level and the localization of ER in A549 cells. The phosphorylation of ERK1/2 upon β-estradiol stimulation was quantified by Western blot. The invasion and migration abilities of A549 cells upon β-estradiol stimulation with or without ERK1/2 inhibitor PD98059 were measured by Transwell and Cell-IQ assays. RESULTS: ERβ was the dominant ER subtype in the A549 cells and primarily comprised of ERβ2 and ERβ5. Immunofluorescence revealed that ERβ expression was mainly localized in the cytoplasm. β-estradiol induced phosphorylation of ERK1/2 and promoted the invasion and migration of the cells. Inhibition of ERK1/2 signaling reversed β-estradiol-promoted invasion and migration of A549 cells. CONCLUSION: ERβ-mediated membrane-initiated steroid signaling is involved in the process of β-estradiol-promoted invasion and migration of A549 cells, through which ERK1/2 signaling plays a pivotal role.     

Effect of endothelin-1 on inflammation and oxidative stress in COPD

AIM:To investigate the effect of endothelin-1 on inflammation and oxidative stress in chronic obstructive pulmonary disease (COPD). METHODS:Healthy non-smokers (30 cases), healthy smokers (30 cases) and COPD patients (29 cases) were collected and induced to produce sputum. The concentration of endothelin-1 in the induced sputum was detected. The model of emphysema was established by cigarette smoke extract to stimulate SD rats. Endothelin A receptor antagonist BQ123 and non-selective endothelin receptor antagonist bosentan were used to intervene with the model rats. The experiment was divided into control group, cigarette-treated group, selective antagonist group and non-selective antagonist group. The protein levels of cleaved caspase-3 in the lung tissue were determined by Western blot. Gelatin zymography was used to analyze the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the lung tissue. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The bioantioxidant power (BAP) was detected by BAP assay kit. RESULTS:The concentrations of endothelin-1 in induced sputum of healthy smokers and COPD patients were significantly higher than that of healthy non-smokers (P<0.05), and the level of endothelin-1 in COPD patients was significantly higher than that in healthy smokers (P<0.05). The levels of cleaved caspase-3, MMP-2, MMP-9, TNF-α and IL-1β in the lung tissues from cigarette-treated group were significantly higher than those in control group (P<0.05). The endothelin A receptor antagonist significantly inhibited the levels of cleaved caspase-3, MMP-2, MMP-9, TNF-α and IL-1β (P<0.05). The serum BAP in cigarette-treated group was significantly lower than that in control group (P<0.05). However, endothelin A receptor antagonist significantly increased serum BAP (P<0.05). CONCLUSION:Endothelin-1 may play an important role in the development and progression of COPD through regulating apoptosis, matrix metalloproteinase activity, inflammation and oxidative stress.     

Palmitic acid inhibits proliferation of pancreatic β cell line INS-1 via up-regulating expression of receptors Frizzled-2 and Ror-2

AIM:To investigate the mechanism of palmitic acid inhibiting the proliferation of rat pancreatic β cell line INS-1. METHODS:INS-1 cells were treated with palmitic acid in time or concentration gradient experiments. The expression of Frizzled-2 (Fzd-2) and receptor tyrosine kinase-like orphan receptor 2 (Ror-2) at mRNA and protein levels was detected by RT-qPCR and Western blot. Interaction of Wnt5a with Fzd-2 or Ror-2 in INS-1 cells treated with palmitic acid was determined by the method of direct co-immunoprecipitation. The proliferation rates of the cells were measured by EdU assay after transfected with siRNA-Fzd-2 or siRNA-Ror-2. RESULTS:Fzd-2 and Ror-2 were up-regulated in INS-1 cells stimulated with palmitic acid at both mRNA and protein levels. Palmitic acid promoted Wnt5a to recruit receptors Fzd-2 and Ror-2. Silencing the expression of Fzd-2 or Ror-2 weakened the inhibitory effect of palmitic acid on INS-1 cell proliferation (P<0.05). CONCLUSION:Palmitic acid inhibits the proliferation of INS-1 cells via up-regulating the expression of Fzd-2 and Ror-2, as well as promoting Wnt5a to recruit these receptors.     

Effects of different β-adrenergic receptors on cardiac systolic and diastolic functions in hypoxic stress rats

AIM:To explore the effects of different β-adrenergic receptors (β-AR) on the left and right ventricular systolic and diastolic functions in rats under acute hypoxic stress. METHODS:The healthy male SD rats were randomly divided into 4 groups (n=7):control group, non-selected β-AR blocker propranolol group, selected β₁-AR blocker atenolol group and selected β₂-AR blocker ICI 118,551 group, and then the rats were exposed to normoxia (20.9% O₂, 79.1% N₂) and hypoxia (15.0% O₂, 85.0% N₂) condition respectively at the altitude of 2 260 m (Xining, China). The heart rate (HR), the left ventricular systolic pressure (LVSP), the right ventricular systolic pressure (RVSP), and the maximum raise/decline rate of left and right ventricular pressure (±dp/dt_max) were monitored, and the arterial blood gas in normoxia and hypoxia condition were compared to explore the effect of β-AR on the left and right ventricular systolic and diastolic functions in acute hypoxic stress rats. RESULTS:Under normoxia condition, the LVSP, ±dp/dt_max of left ventricular were decreased in propranolol group, atenolol group and ICI 118,551 group, the RVSP and ±dp/dt_max of right ventricle were decreased in propranolol group and atenolol group (P<0.05). Under hypoxia condition, the PaO₂, LVSP, ±dp/dt_max of left ventricle were decreased in all groups compared with the normoxia group, and the ±dp/dt_max of right ventricle was increased in all groups (P<0.05), also the degree of index change in control group was more obvious than that in propranolol group and atenolol group. CONCLUSION:The activation of β₁-AR is an important compensatory regulation for heart function during hypoxic stress. However, the compensatory enhancement of right heart function under acute hypoxia condition which through tonogenic dilation is more significant for maintaining the normal circulating blood flow.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling

AIM: To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS: The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured. The protein expression of β-catenin, glycogen synthase kinase-3β (GSK-3β), c-Myc and cyclin D1 in the ASMC was determined by Western blot. After depressing the interaction between β-catenin and p300/CBP, the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry. Meanwhile, the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS: The protein levels of β-catenin, c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P<0.05). After depressing the interaction between β-catenin and p300/CBP, the cell activity of ASMC was decreased in asthma group compared with control group (P<0.05), and the change of the cell cycle distribution in asthma group was also more obvious (P<0.05). After inhibiting P38 MAPK activity, the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P<0.05).CONCLUSION: Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1, reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.     

Effect of ischemic post-conditioning on Egr-1 and IL-1β expression in ischemia-reperfusion injured lung in rats

AIM: To investigate the protective effects of ischemic post-conditioning on the expression of early growth response factor 1 (Egr-1) and interleukin-1β(IL-1β) in ischemia-reperfusion injured lung in rats. METHODS: The model of lung ischemia-reperfusion injury was established in 24 rats and the rats were randomly allocated to 3 different groups (n=8 in each group): (1) sham group: only sham operation (thoracotomy) and no ischemia for 3 h; (2)ischemia-reperfusion group (I/R group): interruption of pulmonary perfusion and ventilation for 1 h followed by reperfusion for 2 h; (3) ischemic post-conditioning group (IPostC group): ischemic post-conditioning (5 min of reperfusion and 5 min of ischemia for 3 times) between the end of ischemia and the beginning of the reperfusion followed by reperfusion for 1.5 h. The lung tissues (prepared to small pieces of about 20 mg) were collected and homogenized at the end of the experiment. The concentration of myeloperoxidase (MPO) in the homogenate was determined. The wet to dry weight ratio (W/D) of the lung tissues was also measured at the end of reperfusion. The pathological changes of the lung tissues were observed under light microscope after reperfusion. The mRNA expression of Egr-1 and IL-1β in the lung tissues was detected by RT-PCR. RESULTS: Compared with sham group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D were significantly increased in I/R group (P<0.05). The inflammatory responses of the lungs in I/R group were significantly severer than those in sham group. Compared with I/R group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D in IPostC group were significantly decreased (P<0.05). The inflammatory responses of the lungs in IPostC group were also significantly attenuated. CONCLUSION: Ischemic post-conditioning significantly reduces ischemic reperfusion injury of the lung by inhibiting the expression of Egr-1 and IL-1β.     

Promoting cell viability and migration of gastric cancer cells by PRRX2 via activation of Wnt/β-catenin signaling pathway

AIM: To study the effect of paired-related homeobox 2 (PRRX2) gene on the viability and migration ability of gastric cancer cells, and to analyze the underlying mechanism of regulating Wnt/β-catenin signaling pathway.METHODS: The expression of PRRX2 in gastric cancer and normal gastric tissue and the correlation between PRRX2 expression in gastric cancer tissues with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The small interfering RNA (siRNA) and over-expressed plasmids of PRRX2 were transfected into gastric cancer cells MGC-803 and SGC-7901, respectively. MTT assay and Transwell assay were used to detect the viability and migration ability of gastric cancer cells. Western blot and TOPflash/FOPflash dual-luciferase reporter gene assay were used to detect the activity of Wnt/β-catenin signaling pathway. Co-immunoprecipitation was used to detected the interaction between PRRX2 and β-catenin proteins.RESULTS: Knockdown of PRRX2 attenuated the viability and migration ability of gastric cancer cell line MGC-803 (P<0.05). Over-expression of PRRX2 enhanced the viability and migration ability of SGC-7901 cells (P<0.05), increased the protein levels of β-catenin, c-Myc and cyclin D1 (P<0.05) and the activity of TOPflash/FOPflash dual-luciferase reporter gene (P<0.05). PRRX2 interacted with β-catenin protein in gastric cancer cells.CONCLUSION: PRRX2 promotes the viability and migration ability of gastric cancer cells, which may be related to Wnt/β-catenin signaling pathway.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

Effect of cigarette smoking condensate on HDAC2 and inflammatory mediators in mouse myoblast C2C12 cells

AIM: To investigate the effect of cigarette smoking condensate on histone deacetylase 2 (HDAC2) and inflammatory mediators in mouse myoblast C2C12 cells. METHODS: C2C12 cells were treated with cigarette smoke extract (CSE). HDAC2 siRNA was transfected into the cells using Lipofectamine^TM 2000. The levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in the culture supernatants were measured by ELISA, and the expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blotting. RESULTS: The expression of HDAC2 at mRNA and protein levels in CSE group was lower than that in control group (P<0.05). The supernatant levels of IL-8 and TNF-α in CSE group were significantly higher than those in control group (P<0.05). When the cells were transfected with HDAC2 siRNA followed by CSE stimulation, the expression of HDAC2 at mRNA and protein levels was decreased, and the supernatant levels of IL-8 and TNF-α were significantly increased as compared with CSE group and control group (P<0.05).CONCLUSION: Under the oxidative stress condition, C2C12 cells generate high levels of IL-8 and TNF-α by down-regulating the expression of HDAC2.