Effects of resveratrol on TNF-α-induced injury and expression of MCP-1 in isolated rat pulmonary artery endothelial cells

AIM: To investigate the possible mechanism of resveratrol (Res) on tumor necrosis factor-α (TNF-α)-induced monocyte chemoattractant protein-1 (MCP-1) expression in primary rat pulmonary artery endothelial cells (RPAECs).METHODS: RPAECs were randomly divided into 4 groups:control group, solvent (1% DMSO) group, TNF-α group and Res group. Each group was divided into 1 h, 4 h and 8 h subgroups (n=6 per time point). The TNF-α+C1142 (a rodent chimeric mAb that neutralizes rat MCP-1) group was set up at the 8 h time point. At each time point, the protein and mRNA expression of MCP-1 was measured by Western blot and real-time PCR.RESULTS: Pretreatment of the RPAECs with C1142 significantly down-regulated the expression of MCP-1 (P<0.05). The protein and mRNA expression of MCP-1 was markedly increased in TNF-α group (P<0.05). Notably, incubation with Res down-re-gulated the protein and mRNA expression of MCP-1, which was significantly lower than that in TNF-α group (P<0.05).CONCLUSION: MCP-1 was involved in the process of TNF-α-induced injury of RPAECs. Res down-regulates the expression of MCP-1 in RPAECs, thus attenuating cell injury.     

TNF-α induces PIP3-mediated necroptosis in MLO-Y4 cells

AIM: To explore whether tumor necrosis factor-α (TNF-α) induces necroptosis in murine long bone osteocyte-like cell line MLO-Y4 and the possible mechanism. METHODS: The MLO-Y4 cells were divided into control group, TNF-α group, TNF-α+necrostatin-1 (Nec-1) group, TNF-α+Z-VAD group and TNF-α+receptor-interacting protein 3 (RIP3)-siRNA group. The death rate of MLO-Y4 cells was assessed by flow cytometry with Annexin V-FITC/PI staining. The morphological features of the cells were observed under transmission electron microscope (TEM). The protein levels of RIP1, RIP3 and cleaved caspase-3 were determined by Western blot. Finally, the numbers of total cells and RIP1-RIP3-positive cells were observed under laser scanning confocal microscope. The production of reactive oxygen species (ROS) in the cells was measured by DCFH-DA staining. RESULTS: Compared with control group, the apoptotic or necroptotic rate of the cells induced by TNF-α was increased significantly (P<0.01). The increased apoptotic or necroptotic rate was dramatically reduced by treating with Nec-1, Z-VAD or RIP3-siRNA transfection (P<0.01). In TNF-α group and TNF-α+Z-VAD group, a lot of MLO-Y4 cells with typical necroptotic morphological features were observed under TEM. However, obvious necroptotic cells were not found in Nec-1 or RIP3-siRNA treatment group. The protein level of RIP1 in the cells treated with Nec-1 was sharply lower than that in TNF-α group (P<0.01). However, Z-VAD did not reduce the elevated levels of RIP1 and RIP3. RIP3-siRNA effectively down-regulated the protein level of RIP3 compared with TNF-α group (P<0.01). Nec-1 effectively down-regulated the protein levels of RIP1 colocalized with RIP3 compared with TNF-α group (P<0.01). However, Z-VAD did not reduce the levels of RIP1 colocalized with RIP3. Nec-1, Z-VAD and RIP3 siRNA significantly decreased the ROS levels (P<0.01). CONCLUSION: TNF-α induces the necroptosis of MLO-Y4 cells. RIP3 play vital roles in the cell necroptotic signal pathway. ROS may be the executor of necroptosis of MLO-Y4 cells.     

Inhibitory effect of proanthocyanidins B1 and B2 on inflammation of BV-2 cells induced by lipopolysaccharide

AIM: To investigate the different inhibitory effects of proanthocyanidins B1 and B2, which are isomers, on the inflammatory response of BV-2 cells induced by lipopolysaccharide (LPS). METHODS: MTT assay was used to detect the effects of proanthocyanidins B1 and B2 on the viability of BV-2 cells. LPS (1 mg/L) was used to promote BV-2 cells to secrete inflammatory factors. ELISA, chemotaxis assay and Western blot were used to detect the influence of proanthocyanidins B1 and B2 on the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cell chemotaxis and phosphorylation of NF-κB. RESULTS: Proanthocyanidins B1 and B2 did not show cytotoxicity effect on BV-2 cells. Proanthocyanidin B1 and B2 inhibited the cell chemotaxis, phosphorylation of NF-κB, and releases of TNF-α and IL-1β. CONCLUSION: Proanthocyanidins B1 and B2 inhibit the inflammatory response of BV-2 cells induced by LPS, and their action intensity didn't show significant difference.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

Effect of oleanolic acid on expression of TNF-α and collagen in silicotic rats in vivo

AIM: To observe the effect of oleanolic acid (OA) on the expression of Tumor necrosis factor-α (TNF-α) and collagen in silicotic rats in vivo and its possible mechanism. METHODS: Male Wistar rats were divided into 4 groups according to the randomized block design: control group, model group, OA group and solvent control group (20 rats in each group). Except control group, the rats in other groups were induced by intratracheal instillation of silicon di-oxide (SiO₂; 250 mg/kg). The rats in OA group were intragastrically administered with OA (60 mg/kg) from the second day of giving SiO₂. The rats in solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose solution (10 mL/kg). The rats in control group were given normal saline under the same condition for 56 consecutive days. All rats were killed at the 7th, 14th, 28th and 56th days. The lung coefficient was detected and the morphological changes were observed. The serum contents of TNF-α were detected by ELISA. The content of total collagen in the lung tissue was measured. The protein level of nuclear factor-κB (NF-κB) in the lung tissue was determined by immunohistochemical method. RESULTS: (1) According to the morphological changes, the silicosis model was successfully established. Compared with control group, the lung coefficient and total collagen increased obviously in model group and solvent control group. The lung coefficient and total collagen content in OA group at each time point reduced compared with those in model group and solvent group, and increased compared with those in control group at the corresponding time points. (2) The serum contents of TNF-α in model group and solvent control group significantly increased, peaking at the 14th day, slightly decreasing afterward, and showing statistically significant difference at each time point compared with those in control group. No significant difference between model group and solvent group at different time points was observed. OA had inhibitory effect on the contents of TNF-α compared with model group and solvent group at the corresponding time points. (3) NF-κB in model group and solvent control group significantly increased, peaking at the 28th day, and showing statistically significant difference at each time point compared with those in control group. The NF-κB expression in OA group was similar to model group, but significantly decreased compared with control group at each time point. CONCLUSION: OA inhibits the expression of TNF-α and collagen and attenuates the silicosis fibrosis, which may be related to the NF-κB pathway.     

Total flavonoids of onion attenuate hydrogen peroxide-induced oxidative damage in retinal pigment epithelial cells

AIM: To investigate the effects of total flavonoids of onion (FO) on hydrogen peroxide (H₂O₂)-induced oxidative damage in retinal pigment epithelial cells. METHODS: The retinal pigment epithelium ARPE-19 cells were divided into 5 groups:control group, H₂O₂ group (treated with H₂O₂), FO-L+H₂O₂ group (treated with H₂O₂ and low concentration of FO), FO-M+H₂O₂ group (treated with H₂O₂ and medium concentration of FO) and FO-H+H₂O₂ group (treated with H₂O₂ and high concentration of FO). The cell viability was measured by MTT assay. Apoptosis was analyzed by flow cytometry. DCFH-DA staining was used to detect reactive oxygen species (ROS) level in the cells. WST assay was used to detect superoxide dismutase (SOD) activity. The content of malonaldehyde (MDA) was measured by TBA method. Mitochondrial membrane potential was analyzed by JC-1 staining. The protein levels of cytochrome C (Cyt C) in the cytoplasm, and cleaved caspase-3 and cleaved caspase-9 in the cells were determined by Western blot. RESULTS: Treatment with H₂O₂ decreased ARPE-19 cell viability, increased the apoptotic rate and the level of ROS in the cells, decreased SOD activity, increased the content of MDA, decreased mitochondrial membrane potential, and increased the protein levels of Cyt C in the cytoplasm and cleaved caspase-3 and cleaved caspase-9 in the cells (P<0.05). Compared with H₂O₂ group, the cell viability in FO-L+H₂O₂ group, FO-M+H₂O₂ group and FO-H+H₂O₂ group was increased, the apoptotic rates were decreased, the levels of ROS were decreased, SOD activity was increased, the content of MDA was decreased, mitochondrial membrane potential was increased, the protein level of Cyt C was decreased in the cytoplasm, and the protein levels of cleaved caspase-3 and cleaved caspase-9 protein in the cells were decreased gradually (P<0.05). CONCLUSION: Total flavonoids of onion reduce H₂O₂-induced oxidative damage in retinal pigment epithelial cells.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Lipopolysaccharide stimulates TNF-α and endothelin-1secretion from cultured rat Kupffer cells

AIM: To investigate lipopolysaccharide (LPS) stimulated cytokine secretion from normal rat Kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured. Tumor necrosis factor -α (TNF-α) and endothelin-1 (ET-1) secreted by LPS stimulated Kupffer cells were detected. RESULTS: LPS had an stimulative effect on Kupffer cell activity. LPS in definite concentrations promoted Kupffer cell secretion. CONCLUSION: LPS promotes Kupffer cell secretion, which may be associated with liver injury induced by LPS.     

Effect of Egr-1 gene transfection on renal inflammation in diabetic mice

AIM: To observe the effects of Egr-1 gene transfection on the expression of tumor necrosis factor-α(TNF-α) and intercellular adhesion molecule-1(ICAM-1), and to investigate the role of Egr-1 in the pathogenesis of diabetic nephropathy.METHODS: The diabetic mouse model was established. Ten mice were randomly selected as the diabetic group. The remaining 40 mice were injected with empty plasmid, Egr-1 expression plasmid or Egr-1 siRNA plasmid via the tail vein once a week. The normal control group was also set up. The animals were sacrificed at the end of the 4th week. The renal tissues were harvested. The expressions of Egr-1, TNF-α and ICAM-1 were detected by immunohistochemistry and Western blot. The pathological changes were observed under electron microscope.RESULTS: In diabetic mouse kidney, the expression of Egr-1, TNF-α and ICAM-1 was increased, and irregular thickening of glomerular basement membrane, mesangial expansion and fusion of foot were observed. The change trend was more significant in Egr-1 gene transfection group, and these changes in siRNA plasmid transfection group were obviously reduced compared with diabetes group.CONCLUSION: Egr-1 up-regulates the expression of TNF-α and ICAM-1, and induces mesangial cell proliferation and mesangial extracellular matrix accumulation, which is probably one of the mechanisms of accelerating glomerulosclerosis.     

Expression and function of TNF-α in dorsal root ganglion of rats with chronically compressed dorsal root ganglion

AIM: To investigate the role of TNF-α and NF-κB in the mechanism of neuropathic pain due to chronically compressed dorsal root ganglion (CCD).METHODS: Based on the CCD model, von Frey filaments were used to quantify behavior test. The expression changes of TNF-α and NF-κB were determined by Western blotting, and the correlation between the expression of TNF-α and the 50% paw withdrawal threshold was also analyzed. Moreover, the location of TNF-α in dorsal root ganglion (DRG) was observed with immunofluorescence double staining.RESULTS: We found 50% paw withdrawal threshold of CCD decreased at the first day after operation. The mechanical allodynia was the most obvious at postoperative 7~14 d and lasted longer than 35 d. The expression of TNF-α and NF-κB increased significantly in DRG after operation (P<0.01), especially at 7~14 d, and then restored gradually. Moreover, there was a correlation between the protein expression of TNF-α and the changes of neuropathic behavior (P<0.05).CONCLUSION: TNF-α and NF-κB are involved in the mechanism of mechanical allodynia after chronically compressed DRG.     

Effects of Ku70 on protein expression of HTLV-1 in HTLV-1 positive T cells

AIM:To explore the effects of Ku70 on the protein expression of human T-lymphotrophic virus 1 (HTLV-1) in HTLV-1 positive T cells. METHODS:The expression level of Ku70 in HTLV-1 positive T cells was exa-mined by Western blot. The siRNA targeting Ku70 was constructed and the effect of the siRNA on knockdown of Ku70 expression was determined by Western blot. After knockdown of Ku70 expression in the HTLV-1 positive T cells by siRNA, the expression of HTLV-1-related proteins at mRNA and protein levels was examined by real-time PCR and Western blot, and the expression levels of interferons and proinflammatory cytokines were examined by real-time PCR. RESULTS:The HTLV-1 positive T cells, including MT2, MT4 and C8199 cells, displayed a higher expression level of Ku70. The protein expression of HTLV-1 was increased in Ku70-silencing MT2 cells and MT4 cells. After knockdown of Ku70 expression in the MT2 cells and MT4 cells, the production of interferon (IFN)-α, IFN-γ and tumor necrosis factor-α was reduced.CONCLUSION:The HTLV-1 positive T cells have a higher expression level of Ku70. In HTLV-1 positive T cells, Ku70 promotes the production of interferons and proinflammatory cytokines and inhibits HTLV-1-related protein expression.     

Role of endogenous nitric oxide in apoptosis of alveolar epithelial cells in the development of pulmonary fibrosis in rats

AIM: To study the role of high level of endogenous nitric oxide (NO) in apoptosis of alveolar epithelial cells in the development of pulmonary fibrosis in rats. METHODS: The content of nitrite/nitrate (NO₂^-/NO₃^-) in out-flowing pulmonary blood (OPB) was assayed by nitric acid reduction method. The apoptosis of alveolar epithelial cells was observed by TdT-mediated dUTP nick-end labeling (TUNEL) and electron microscopy, respectively. The above indices were observed on the day 14 and the day 30 after intratracheal administration of BLMA₅ alone or along with blockade of iNOS by aminoguanidine (AG) in rats. RESULTS: (1) Both the content of NO₂^-/NO₃^- in OPB and the number of apoptotic alveolar epithelial cells in lung were increased in BLMA₅ 14 d group, compared with normal control group and BLMA₅ 30 d group, respectively (P<0.05). The high level of NO₂^-/NO₃^- in OPB and the apoptosis of alveolar epithelial cells were ameliorated by AG. CONCLUSION: The apoptosis of alveolar epithelial cell is induced by high level of endogenous NO in the development of pulmonary fibrosis.     

Effects of TRPC1 on TGF-β1-induced epithelial-mesenchymal transition of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the epithelial-mesenchymal transition (EMT) of human bronchial epithelial (HBE) cells induced by transforming growth factor-β1 (TGF-β1). METHODS: EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluorescence and Western blotting. Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells. The influence of SKF96365 (a TRPC1 blocker) and siRNA-mediated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting. RESULTS: Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells. Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells. Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive. The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 (P<0.05). The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group. The protein expression of E-cadherin and α-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05). CONCLUSION: TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.     

Inhibitory effect of IGF-1 overexpression on oxidative damage in umbilical cord mesenchymal stem cells

AIM: To analyze the inhibitory effect of insulin-like growth factor-1 (IGF-1) overexpression in umbilical cord mesenchymal stem cells (UC-MSCs) on oxidative damage and to develop new application model for UC-MSCs. METHODS: UC-MSCs were isolated from human umbilical cord with enzymatic digestion, and further characte-rized with flow cytometry. IGF-1-overexpressing UC-MSCs (UC-MSCs-IGF-1) were established by retrovirus infection. IGF-1 expression of UC-MSCs-IGF-1 was evaluated by real-time quantitative PCR and flow cytometry, and its surface markers, as well as osteogenic and adipogenic differentiation ability, were further analyzed. The proliferation, anti-oxidative damage and anti-apoptosis abilities of UC-MSCs-IGF-1 were evaluated when treated with H₂O₂ at different concentrations (0 μmol/L, 10 μmol/L, 50 μmol/L and 100 μmol/L). RESULTS: UC-MSCs showed positive expression of CD29, CD90 and CD105, but negative expression of CD34, which coincided with the normal phenotype of mesenchymal stem cells. UC-MSCs-IGF-1 established with retrovirus infection showed much higher expression of IGF-1 compared with normal UC-MSCs, and expressed the same surface markers as UC-MSCs.The osteogenic and adipogenic differentiation abilities were also observed. With the oxidative damage by H₂O₂ treatment, UC-MSCs-IGF-1 showed more strong proliferation, anti-oxidative damage and anti-apoptosis abilities as compared with normal UC-MSCs. In addition, the activity of SOD in UC-MSCs-IGF-1 was a little higher than that in control group. CONCLUSION: IGF-1 overexpression in UC-MSCs inhibits oxidative damage and cell apoptosis. UC-MSCs-IGF-1 may have more advantagies in clinical application.     

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.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Influences of bypass-activated complement and TNF-α on endothelial cells to express ICAM-1 and PMN-EC adhesion

AIM:To investigate the influences of bypass-activated complement and TNF-α on intercellular adhesion molecule 1(ICAM-1) expression in endothelial cells, and polymorphonuclear neutrophil-endothelial cell (PMN-EC) adhesion.METHODS:In vitro, zymosan-activated human serum(ZAHS)and/or TNF-Adirectly stim-ulated the HUVECS monolayers.PMN-EC adhesion percentage was measured, and modified whole-cell ELISA was used for measurement of ICAM-1.RESULTS:ZAHS alone resulted in no significant change in the degree of PMN adhesion and the level of ICAM-1. Activation of HUVECS with TNF-α followed by ZAHS stimulation resulted in greater increase in PMN-EC adhesion and ICAM-1 expression, as compared with the activation with TNF-α alone.CONCLUSION:Bypass-activated complement can promote ICAM-1 expression and neutrophil -endothelium adhesion induced by TNF-α, and so it is effective for promoting inflammation.     

Pretreatment with external trigeminal nerve electrostimulation inhibits seizures and decreases expression of IL-1β and TNF-α in hippocampus with status epilepticus induced by pentylenetetrazol in rats

AIM:To observe the effect of pretreatment with external trigeminal nerve electrostimulation (eTNS) on behavioral changes and the expression of interleukin-1β (IL-1β) and 　tumor necrosis factor α (TNF-α) in hippocampus of pentylenetetrazol (PTZ)-treated rats. METHODS:The rats were randomly divided into control group, PTZ group and eTNS group, and kindled by PTZ after administered 7 d, 14 d and 28 d of consecutive fake electrostimulation or eTNS. Subsequently, the severity and duration of seizure were quantitatively evaluated. The concentrations of IL-1β and TNF-α in hippocampus were detected by the methods of ELISA and immunohistochemisty. RESULTS:Compared with PTZ group, treatment with eTNS significantly inhibited the severity and duration of seizure (P<0.05), and significantly reduced the content of IL-1β and TNF-α in the hippocampus after status epilepticus (P<0.05 or P<0.01). CONCLUSION:Pretreatment with eTNS may provide a new approach for prevention and treatment of epileptogenesis.     

TNF-α exacerbates cholesterol accumulation via down-regulating LXRα promoter activity in HepG2 cells

AIM: To investigate the exacerbating effect of tumor necrosis factor alpha (TNF-α) on lipid accumulation in HepG2 cells by inhibiting liver X receptor alpha (LXRα) signaling pathway. METHODS: Luciferase reporter plasmid driven by the LXRα promoter (pGL3-Basic-LXRα-P) was constructed and transfected into HepG2 cells to detect the LXRα promoter activity. HepG2 cells were incubated with serum-free medium (control), 20 μg/L TNF-α (TNF-α), 100 mg/L LDL (LDL) and 20 μg/L TNF-α plus 100 mg/L LDL (LDL+TNF-α), respectively. The effects of TNF-α on cholesterol accumulation were examined by oil red O staining and quantitative intracellular cholesterol assay. The expression of LXRα, ABCA1 and ABCG1 at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS: The pGL3-Basic-LXRα-P was constructed successfully. TNF-α decreased the activity of LXRα promoter in the absence or presence of LDL. Inflammatory stress inhibited the expression of LXRα, ABCA1and ABCG1 at mRNA and protein levels. The cholesterol efflux was increased after loading of LDL, while TNF-α decreased intracellular cholesterol efflux. The results of oil red O staining and quantitative intracellular cholesterol assay demonstrated that inflammatory stress increased cholesterol levels in HepG2 cells. CONCLUSION: TNF-α exacerbates the cholesterol accumulation in hepatic cells via inhibiting LXRα promoter activity and gene expression.