Toll-like receptor 4 expression on mast cells in human gingival tissues with chronic periodontitis

AIM: To observe the expression of Toll-like receptor 4 (TLR4) on mast cells in human gingival tissues with chronic periodontitis. METHODS: A total of 68 volunteers, including 23 cases of mild chronic periodontitis, 25 cases of severe chronic periodontitis and 20 healthy controls, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining. The expression of TLR4 in gingival tissues was detected by immunohistochemical staining, and TLR4 expression on mast cells was detected by immunofluorescence double staining. RESULTS: The expression of TLR4 in gingival tissues and on mast cells in chronic periodontitis groups was significantly higher than that in normal control group (P<005), and that in severe chronic periodontitis group was significantly higher than that in mild chronic periodontitis group (P<005). CONCLUSION: The expression of TLR4 in gingival tissues and on mast cells is increased with the severity of chronic periodontitis, suggesting that TLR4, especially TLR4 on mast cells, may play an important role in human chronic periodontitis.     

Effect of Toll-like receptor 4 signal transduction on airway inflammation in infant asthmatic rat

AIM: To study the change and regulatory mechanism of Toll-like receptor 4 (TLR4) on eosinophil (EOS) apoptosis. METHODS: Twenty-seven SD rats were randomly divided into control group (A), asthma group (B) and dexamethasone group (D). Asthmatic model rats were sensitized and repeatedly exposed to aerosolized ovalbumin. Pulmonary tissues were observed under light microscope (LM). The inflammatory cells in BALF were counted. The levels of IL-10 in serum were measured by ELISA. Expressions of TLR4 mRNA were tested by hybridization. The apoptotic EOS was detected by TUNEL.RESULTS: (1) LM showed that inflammatory cells infiltrated around the bronchus, airway mucous plug in group B, obviously lightened in group D. (2) Inflammatory cells count in BALF: the total cellular score, EOS absolute count and EOS% in group B were significantly increased (P<0.01). Compared to group B, a significant decrease in group D was observed (P<0.01). (3) The level of IL-10 in group B was significantly higher than that in group A and in group D (P<0.01). (4) No significant difference (P>0.05) of TLR4 mRNA expression was observed between group A and group B. However, that in group D were significantly increased (P< 0.01). (5) Percentages of apoptotic EOS in group B were significantly lower than those in group A (P<0.01), those in group D were significantly increased (P<0.01). A significant correlation between TLR4 mRNA and apoptotic EOS (r=0.612, P<0.01) was observed. CONCLUSION: Dexamethasone can increase IL-10 secretion, induce EOS apoptosis, which may correlate with TLR4 signal transduction.     

Effects of angiopoietin 4 on lipopolysaccharide-induced injury of human umbilical vein endothelial cells

AIM:To observe the effects of angiopoietin 4 (Ang-4) on lipopolysaccharide (LPS)-induced injury of human umbilical vein endothelial cells (HUVECs). METHODS:The EnVision immunohistochemical method was used to identify the HUVECs. After pre-treated with different doses of Ang-4 for 0.5 h, HUVECs was exposed to LPS at concentration of 10 mg/L for 24 h. The cell viability was evaluated by MTT assay. The content of tumor necrosis factor-alpha (TNF-α) in the supernatant and the concentrations of intracellular and supernatant von Willebrand factor (vWF) were detected by ELISA. The mRNA levels of Toll-like receptor 4 (TLR4), NF-κB p65 and TNF-α were determined by real-time PCR. RESULTS:Factor Ⅷ in the cytoplasm was positive in the HUVECs.Compared with normal group, LPS reduced the cell viability (P<0.01), and significantly increased the secretion of TNF-α and vWF (P<0.01). The mRNA expression of TLR4, NF-κB p65 and TNF-α also increased (P<0.01). Ang-4 at concentration of 100 μg/L enhanced the cell viability (P<0.01), reduced the content of vWF and TNF-α, and inhibited the LPS-induced increases in the mRNA levels of TLR4, NF-κB p65 and TNF-α (P<0.01). CONCLUSION: Ang-4 antagonizes LPS-induced damage in HUVECs by inhibiting TLR4-NF-κB p65-TNF-α signaling pathways.     

Correlation between Toll-like receptor 4 on peripheral blood monocytes and serum IL-6 in patients with chronic severe hepatitis B

AIM:To study the change of Toll like receptor 4(TLR4) on peripheral blood monocytes (PBMCs) and its role in the pathogenesis of chronic severe hepatitis B.METHODS:The expression of TLR4 on CD14+ PBMCs was determined by flow cytometry in 30 healthy control,31 patients with chronic hepatitis B and 30 patients with chronic severe hepatitis B. The level of serum interleukin-6 (IL-6) was detected by ELISA. RESULTS:The expressions of TLR4 on PBMCs and serum IL-6 in the groups of healthy control,patients with chronic hepatitis B and patients with chronic severe hepatitis B were 2.3±1.1,3.7±2.3,6.9±4.1 mean fluorescence intensity (MFI) and （11.5±7.2) ng/L,(40.8±31.2) ng/L,(77.6±33.3） ng/L. The TLR4 value in the group of patients with chronic severe hepatitis B was significant higher than that in the group of healthy control and the group of patients with chronic hepatitis B (P<0.05). However,there was no significant difference between the group of patients with chronic hepatitis B and the group of healthy control (P>0.05). Serum IL-6 increased gradually and significantly between the group of healthy control and the groups of patients with chronic hepatitis B and patients with chronic severe hepatitis B. There was a significant positive correlation between the expression of TLR4 and the content of serum IL-6 in the group of chronic severe hepatitis B. CONCLUSION:TLR4 may play a role in the pathogenesis of chronic severe hepatitis B.     

Effect of Chinese propolis on PC-PLC activity and TLR4 expression in LPS-treated vascular endothelial cells

AIM: To investigate the effect of Chinese propolis on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and the expression of Toll-like receptor 4 (TLR4) in LPS-treated vascular endothelial cells (VECs). METHODS: Confluent VECs were stimulated with LPS at the concentration of 100 μg/L in the presence of 0.5% fetal bovine serum. The cells were treated with Chinese propolis at the concentration of 12.5 mg/L for 12 h and 24 h. The viability of VECs and the level of nitric oxide (NO) were detected by sulforhodamine B (SRB) assay and chemical method, respectively. The activity of PC-PLC was measured using L-α-phosphatidylcholine as substrate. The protein levels of TLR4, nuclear factor-κB p65 (NF-κB p65) and p53 were determined by Western blotting. The level of intracellular reactive oxygen species (ROS) was examined using a fluorescent probe, 2,7-dichlorodihydrofluorescin (DCHF). For the measurement of mitochondrial membrane potential, the fluorescent dye JC-1 was used. RESULTS: Treatment with Chinese propolis for 24 h had no effect on the viability of VECs. However, the levels of NO and ROS were significantly decreased by Chinese propolis. PC-PLC activity and NF-κB p65 expression were significantly depressed by Chinese propolis treated for 12 h, and the expression of TLR4 and p53 were dramatically decreased by Chinese propolis treated for 12 and 24 h. No effect of Chinese propolis on mitochondrial membrane potential was observed. CONCLUSION: Chinese propolis depresses the activity of PC-PLC and the expression of TLR4, and then inhibits the downstream signal molecules such as NF-κB p65, p53, ROS and NO in VECs.     

Protective effect of capsaicin on lipopolysaccharide-induced activation of vascular endothelial cells

AIM:To investigate the effect of capsaicin on lipopolysaccharide (LPS)-induced activation of cultured endothelial cells of mouse aorta in vitro. METHODS:The endothelial cells were isolated from mouse aorta and cultured in vitro, and the specific cell markers of the cells were identified by immunofluorescence staining. The cells were stimulated with LPS (100 μg/L) combined with or without capsaicin, and the cells and supernatant were collected at 12 h, 24 h and 48 h. The levels of soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1) and soluble P-selectin (sP-selectin) in the supernatant were measured by ELISA. The levels of nuclear NF-κB p65 and cytopasmic p-IκBα and IκBα were detected by Western blotting. RESULTS:Compared with control group, the levels of sP-selectin, sICAM-1 and sVCAM-1 in LPS group were significantly increased (P<0.05), and LPS promoted the expression of sICAM-1 and sVCAM-1 in a time-dependent manner. Compared with LPS group at the same time point, capsaicin inhibited the expression of sP-selectin, sICAM-1 and sVCAM-1 in a dose-dependent manner. Compared with control group, the protein levels of NF-κB p65 and p-IκBα in LPS group at 24 h were significantly increased (P<0.05), while the protein level of IκBα in LPS group at 24 h were significantly decreased (P<0.05). Compared with LPS group, capsaicin decreased the protein levels of NF-κB p65 and p-IκBα and increased the protein level of IκBα in a dose-dependent manner. CONCLUSION: Capsaicin has a protective effect on LPS-induced vascular endothelial cell activation, which potentially contributes to the suppression of IκBα degradation and NF-κB p65 nuclear translocation.     

Effects of interleukin -13 on interleukin-12 production in mesangial cells induced by LPS

AIM: To investigate the effect of interleukin-13 (IL-13) on interleukin-12 (IL-12) production in mesangial cells.METHODS: The protein synthesis of IL-12 in mesangial cells was measured by ELISA.The expression of IL-12 mRNA in mesangial cells was evaluated by RT-PCR.RESULTS: The production of IL-12 in mesangial cells stimulated by lipopolysaccharide(LPS) was significantly increased (P<0.01).IL-13 (1-100 μg/L) inhibited the protein and mRNA expression of IL-12 in a dose-dependent manner (P<0.05 or P<0.01).CONCLUSION: IL-13 inhibits IL-12 expression induced by LPS in mesangial cells.IL-13 may regulate immune responses by balancing Th1/Th2 in glomerulonephritis.     

Knock-down of TLR4 expression reduces secretion of inflammatory factors in LPS-induced pancreatic acinar AR42J cells

AIM:To study the effect of Toll-like receptor 4 (TLR4) on the secretion of inflammatory factors in the pancreatic acinar AR42J cells induced by lipopolysaccharides (LPS). METHODS:The rat pancreatic acinar AR42J cells were treated with LPS. The expression of TLR4 at mRNA and protein levels was determined by real-time PCR and Western blot. The lentivirus carrying TLR4 small interfering RNA (siRNA) was used to infect the AR42J cells. Under LPS stimulation, the interference efficacy was measured by real-time PCR and Western blot. The cell viability was measured by MTT assay, and the leakage rate of lactate dehydrogenase (LDH) was examined by dinitrophenylhydrazine method. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture medium were detected by ELISA, and the malondialdehyed (MDA) content in supernatant was measured by thiobarbituric acid method. The activity of superoxide dismutase (SOD) in the supernatant was determined by xanthine oxidation, and the activity of glutathione peroxidase (GSH-Px) and catalase (CAT) was detected by colorimetry. RESULTS:The expression of TLR4 at mRNA and protein levels in LPS-treated AR42J cells was significantly increased (P<0.05). Infection with TLR4 siRNA-carrying lentivirus significantly inhibited the expression of TLR4 at mRNA and protein levels in the AR42J cells under LPS stimulation(P<0.05). The viability of AR42J cells was decreased after LPS treatment. The leakage rate of LDH was increased, the levels of IL-1β and TNF-α secreted by the AR42J cells were increased, the content of MDA was increased in the supernatant, and the activity of SOD, GSH-Px and CAT was reduced (P<0.05). After knock-down of TLR4 expression, the viability of AR42J cells was increased under LPS stimulation, the LDH leakage rate, secreted levels of IL-1β and TNF-α, and the content of MDA in cell culture medium were decreased, and the SOD, GSH-Px and CAT levels were increased (P<0.05). CONCLUSION:LPS induces the expression of TLR4 in the pancreatic acinar AR42J cells. Knock-down of TLR4 expression reduces the secretion of inflammatory factors IL-1β and TNF-α, and attenuates the oxidative damage in pancreatic acinar AR42J cells induced by LPS.     

Lipoxin A4 reduces lipopolysaccharide-induced expression of cyclooxygenase 2 and prostaglandin E2 in human bronchial epithelial cells

AIM: To explore the effects of lipoxin A₄ on the expression of cyclooxygenase 2 (COX-2) in human bronchial epithelial cells (HBECs). METHODS: HBECs were incubated with various concentrations (0.1, 1 and 10 mg/L) of lipopolysaccharide(LPS) for 9 h, or 1 mg/L LPS for different time (3 h, 6 h and 9 h). The levels of COX-2 mRNA in HBECs and prostaglandin E₂ (PGE₂) in the culture supernatant were measured. In addition, the HBECs were exposed to lipoxin A₄ at concentration of 0, 100 and 400 μmol/L after stimulated with LPS at concentration of 1 mg/L for 9 h, and the supernatant of the culture cells was collected for determining the content of PGE₂ by ELISA. The cells were also harvested, and the mRNA and protein levels of COX-2 were analyzed by RT-PCR and Western blotting, respectively. RESULTS: LPS increased the mRNA expression of COX-2 and production of PGE₂ in a dose and time dependent manners in HBECs. Induction of COX-2 mRNA and protein by LPS were inhibited by lipoxin A₄ in a dose-dependent manner. Lipoxin A₄ also significantly decreased LPS-induced production of PGE₂. CONCLUSION: Lipoxin A₄ down-regulates LPS-induced expression of COX-2 and consequently inhibits the production of PGE₂ in HBECs.     

Inhibitory effect of NF-κB decoy ODN on expressions of TLR4 and IL-8 in LPS- induced intestinal epithelial cells

AIM: To study the effect of NF-κB decoy oligodeoxynucleotides(ODNs) on TLR4 and IL-8 expression in LPS-induced SW480 cells. METHODS: SW480 cells were cultured in vitro and stimulated for 3 h with LPS (10 μg/L). NF-κB decoy oligodeoxynucleotides mediated by lipofectin 2000 were added into the cell culture for 6 h. The supernatants were collected and messured for IL-8 by ELISA. TLR4 mRNA and IL-8 mRNA were examined by RT-PCR, respectively. The results were compared with control group, Scrambled ODNs group and lipofectin 2000 group. RESULTS: After SW480 cells were stimulated by LPS, TLR4 mRNA, IL-8 mRNA and IL-8 expressions were significantly increased, and the difference compared with control group was obvious. After treated with NF-κB decoy oligodeoxynucleotides, TLR4 mRNA, IL-8 mRNA and IL-8 expressions were significantly inhibited. The Scrambled ODNs group and lipofectin 2000 group had no effect on them. CONCLUSION: NF-κB decoy ODNs will become a new gene drug for treating inflammatory bowel disease(IBD).     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

TGF β1 inhibits the maturation of dendritic cells and down-regulates TLR4 expression

AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF β-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD80, CD86, I-Ab and CD40 in TGF β-DC were lower. The TGF β-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF β-DC was also found. CONCLUSION: TGF-β1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.     

Effect of homocysteine on expression of interleukin-8 mRNA and protein in THP-1 macrophages

AIM: To investigate the effect of homocysteine (Hcy) on expression of interleukin-8 (IL-8) mRNA and protein in THP-1-derived macrophages (THP-1 macrophages). METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 μmol/L PMA treatment for 72 hours, then the differentiated THP-1 macrophages were incubated with homocysteine (0.01 mmol/L-0.20 mmol/L) for 24 hours, or with 0.10 mmol/L Hcy for various time up to 48 hours. IL-8 protein in THP-1 supernatants was measured by ELISA, and IL-8 mRNA expression was detected by semiquantitive RT-PCR. RESULTS: Compared with control, Hcy significantly increased the expression of IL-8 protein in a concentration-dependent manner. 0.05 mmol/L, 0.10 mmol/L and 0.20 mmol/L Hcy increased IL-8 production by 1.28 fold, 1.32 fold and 1.55 fold, respectively (P＜0.01). IL-8 production were elevated significantly 3 h after treatment with 0.10 mmol/L Hcy. In addition, Hcy also increased IL-8 mRNA expression in a concentration-and time-dependent manner. CONCLUSION: Hcy may contribute to atherogenesis by inducing IL-8 expression and secretion in THP-1 macrophages.     

Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway

AIM: To determine whether laminar shear stress regulates nitric oxide (NO) production in vascular endothelial cells through Pim1/endothelial nitric oxide synthase (eNOS) signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to laminar shear stress using a parallel-plate flow system. NO production is evaluated by NO assay kit. Pim1 protein expression and eNOS phosphorylation were determined by Western blot. A specific small interfering RNA was used to knock down Pim1 gene expression, and then the changes of above indicators were detected. RESULTS: After 15-min exposure of HUVECs to laminar shear stress (15 dyn/cm²), rapid increases in Pim1 protein expression and NO production were observed (P < 0.05). Shear stress also caused time-dependent stimulation of eNOS phosphorylation (P < 0.05). The shear-induced Pim1 expression and NO production were abrogated in the HUVECs transfected with siPim1 (P < 0.05). Pim1 silencing also prevented shear-induced rise of eNOS-Ser1177 phosphorylation (P < 0.05). CONCLUSION: Pim1 may account for shear-induced NO production in endothelial cells due to phosphorylation activation of eNOS.