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.     

Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

AIM: To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on the apoptosis, oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation (A/R). METHODS: The H9c2 cells were used to establish a model of A/R. The H9c2 cells were transfected with PTEN small interfering RNA (siRNA) and negative control. After A/R, the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Xanthine oxidase method was used to determine superoxide dismutase (SOD) activity. The content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The lactate dehydrogenase (LDH) activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in culture supernatant were examined by ELISA. The protein levels of cleaved caspase-3, Bax and FasL in the cells were determined by Western blot. RESULTS: After A/R, the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells (P<0.05). The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA (P<0.05). The viability of H9c2 cells was decreased after A/R, while the apoptotic rate was increased. The protein levels of cleaved caspase-3, Bax and FasL were increased in the cells. The MDA level was elevated, the activity of SOD was decreased, and the levels of LDH, TNF-α, IL-1β and IL-6 in the culture supernatant were increased (P<0.05). Down-regulation of PTEN partly antagonized the effects of A/R on the viability, apoptotic rate, MDA content, SOD activity, and the levels of LDH, TNF-α, IL-1β and IL-6 in culture supernatant. CONCLUSION: Down-regulation of PTEN attenuates oxidative damage induced by A/R, reduces apoptosis and secretion levels of TNF-α, IL-1β and IL-6 in the H9c2 cells.     

Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.     

Artemisinin attenuates intestinal epithelial barrier damage induced by LPS

AIM: To investigate the effect of artemisinin on lipopolysaccharide(LPS)-induced intestinal epithelial barrier damage in IEC-6 cells and its molecular mechanism. METHODS: Cultured IEC-6 cells were divided to 5 groups: control group, LPS(100 mg/L) group and LPS+Artemisinin(30, 50 and 100 μmol/L) groups. The cytotoxicity was detected by MTT assay. The releases of TNF-α, IL-1β and IL-6 in the IEC-6 cells were measured by ELISA. The transepithelial electrical resistance(TER) was detected by electrical resistance tester, and the horseradish peroxidase(HRP) flux permeability were analyzed by a microplate reader. The expression of tight junction proteins, ZO-1, claudin-1 and occludin, and the expression of TLR4/MyD88/NF-κB at mRNA and protein levels were determined by RT-qPCR and Western blot. RESULTS: Artemisinin alone(up to 100 μmol/L) or in combination with LPS(100 mg/L) was not toxic to IEC-6 cells. Compared with control group, the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells significantly increased after treatment with LPS. The expression of TLR4/MyD88/NF-κB was activated by LPS. LPS down-regulated the protein expression of ZO-1, claudin-1 and occludin. However, artemisinin treatment decreased the releases of TNF-α, IL-1β and IL-6 in the culture supernatant of IEC-6 cells. The expression of TLR4/MyD88/NF-κB at mRNA and protein levels was gradually reduced after treatment with artemisinin. In addition, artemisinin upregulated the protein expression of ZO-1, claudin-1 and occludin significantly(P<0.01) in a dose-dependent manner. CONCLUSION: Artemisinin attenuates LPS-induced intestinal epithelial barrier damage by inhibiting TLR4/MyD88/NF-κB activation in the IEC-6 cells.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.     

Effect of netrin-1 on high glucose induced injury of renal tubular epithe-lial cells

AIM:To study the effect of netrin-1 on the damage of renal tubular epithelial cells induced by high glucose. METHODS:Human renal tubular epithelial HK-2 cells were treated with high glucose. Real-time PCR and Western blot were used to detect the expression level of netrin-1 in the cells. HK-2 cells were infected with netrin-1-over-expressing lentivirus, and the effect of netrin-1 over-expression on the HK-2 cells treated with high glucose was observed. The apoptosis rate was analyzed by flow cytometry. The protein level of cleaved caspase-3 was determined by Western blot. lactate dehydrogenase (LDH) activity in the culture medium was measured by 2,4-binitrobenzene hydrazine method. The content of malondialdehyde (MDA) in the culture medium was detected by thiobarbituric acid method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the culture medium were measured by ELISA. RESULTS:The expression of netrin-1 at mRNA and protein levels in the HK-2 cells after high glucose treatment was significantly lower than that in the control cells (P<0.05). Infection with netrin-1-over-expressing lentivirus up-regulated the expression of netrin-1 in the HK-2 cells treated with high glucose. High glucose promoted the secretion of IL-1β and TNF-α, decreased the levels of LDH and MDA in the cell culture supernatant, and induced apoptosis and activation of caspase-3 in renal tubular epithelial cells (P<0.05). After the HK-2 cells with up-regulation of netrin-1 were induced by high glucose, the IL-1β and TNF-α secretion, the levels of LDH and MDA in the culture medium, the apoptosis, and the level of activated caspase-3 protein in the cells were all decreased, as compared with the control cells (P<0.05). CONCLUSION:Up-regulation of netrin-1 expression attenuates oxidative damage and inflammatory injury, and reduces apoptosis induced by high glucose in renal tubular epithelial cells.     

Observation of cardiomyocytes stimulated by TNF-α, IL-1β, LPS in vitro

AIM: To observe the changes of cardiomyocytes after stimulation by TNF-α, IL-1β, LPS.METHODS: Cardiac ventricular myocytes were cultured in vitro. Different doses of TNF-α, IL-1β, LPS were added to stimulate the cardiomyocytes, the hypertrophy of cardiomyocytes 8 h, 24 h, and 48 h after stimulation was determined and the apoptosis were also observed 24 h, 48 h, 72 h after stimulation. RESULTS: Compared to the normal myocytes, the cardiomyocytes were hypertrophied after stimulation by 10 μg/L, 15 μg/L of TNF-α, 20 μg/L, 100 μg/L of IL-1β and 10 mg/L, 15 mg/L, 20 mg/L of LPS, and the effect was dose-dependent, the strongest effect was showed in 24 h. Moreover, 20 μg/L of TNF-α, 100 μg/L of IL-1β and 30 mg/L of LPS caused cardiomyocyte apoptosis, especially in 72h. CONCLUSION: TNF-α, IL-1β, LPS induced the cardiomyocyte hypertrophy and apoptosis, suggesting the inflammation may be the main cause of cardiovascular disease.     

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.     

Experimental study on anti-endotoxin activity of a tetrahydropyrimidine derivative,ZL-5015

AIM: To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS: Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice. Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model. The levels of interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to evaluate the mRNA expression of the cytokines. RESULTS: Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1β and TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group. The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40 μmol/L) inhibited the expression of IL-1β and TNF-α at both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels. CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1β and TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.     

Role of p38MAPK in production of pro-inflammatory cytokines in Kupffer cells from severe acute pancreatitis rats

AIM:To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in the Kupffer cells (KCs) production of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β), in severe acute pancreatitis (SAP) rats.METHODS:Sprague-Dwaley rats were randomized into three groups:①sham operation rats, ②SAP rats, ③SAP rats given the p38 MAPK inhibitor CNI-1493(10 mg/kg, iv). The SAP model was induced by the bili-pancreatic duct infusion with 5% sterile soduim taurocholate solution. Rats from each group were killed at 12 h after sham operation or SAP and Kupffer cells (KCs) were isolated. The mRNA expressions of TNF-α and IL-1β (by quantitative real-time RT-PCR) and p38 MAPK activity (by Western blot analysis) in KCs were examined. The levels of TNF-α and IL-1β in plasma were determined by ELISA.RESULTS:There was a significant acvitation of p38 MAPK in KCs harvested from SAP rats than those from sham operation rats. SAP also promoted the mRNA expressions of TNF-α and IL-1β in KCs and the plasma levels of TNF-α and IL-1β. These events were significantly inhibited by treatment with CNI-1493.CONCLUSIONS:p38 MAPK activation is one important aspect of the signaling events that may mediate the KCs production of pro-inflammatory cytokines, TNF-α and IL-1β, in SAP rats. The inhibition of the p38 MAPK may be a potential target in the prevention and treatment of SAP.     

Minocycline inhibits BV-2 cell activation by regulating P2X7 receptor

AIM：To explore the role of P2X7 receptor in inhibition of lipopolysaccharide (LPS)-stimulated BV-2 cell activation by minocycline. METHODS：BV-2 cells were divided into 5 groups: control group, LPS group, LPS+0.1 μmol/L Mino group, LPS+1 μmol/L Mino group and LPS+10 μmol/L Mino group. The expression of P2X7 receptor was determined by real-time PCR and Western blotting. The levels of TNF-α and IL-1β in the microglia culture supernatants were measured by ELISA. The morphological changes of the cells were also observed. RESULTS：After exposed to LPS, the expression of P2X7 receptor increased in BV-2 cells at mRNA and protein levels. The concentrations of TNF-α and IL-1β in the microglia culture supernatants also increased. Meanwhile, 0.1~10 μmol/L minocycline inhibited those changes in a dose-dependent manner. CONCLUSION：Minocycline inhibits the activation of microglia. The mechanism may be related to the P2X7 receptor.     

Opening of ATP-sensitive K+ channels protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting TLR4/NF-κB pathway

AIM: To investigate whether the opening of ATP-sensitive K⁺(K_ATP) channels protects H9c2 cardiac cells against high glucose(HG)-induced injury and inflammation by inhibiting the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) pathway. METHODS: The protein levels of TLR4 and NF-κB p65 were determined by Western blot. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The cell viability was measured by CCK-8 assay. Mitochondrial membrane potential(MMP) was examined by rhodamine 123(Rh 123) staining followed by photofluorography. The intracellular levels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein- diacetate(DCFH-DA) staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG(35 mmol/L glucose) for 24 h, the protein levels of TLR4 and phosphorylated NF-κB p65(p-NF-κB p65) were significantly increased. Pretreatment of the cells with 100 μmol/L diazoxide(DZ, a K_ATP channel opener) for 30 min before exposure to HG considerably blocked the up-regulation of the TLR4 and p-NF-κB protein levels induced by HG. Moreover, co-treatment of the cells with 30 μmol/L TAK-242(an inhibitor of TLR4) obviously inhibited the HG-induced up-regulation of the p-NF-κB p65 protein level. On the other hand, pretreatment of the cells with 100 μmol/L DZ had a clear myocardial protection effect, which attenuated the HG-induced cytotoxicity, inflammatory response, mitochondrial damage, oxidative stress and apoptosis, evidenced by an increase in the cell viability, and decreases in the levels of IL-1β and TNF-α, MMP loss, ROS generation and the number of apoptotic cells. Similarly, co-treatment of H9c2 cardiac cells with 30 μmol/L TAK-242 or 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries and inflammation induced by HG.CONCLUSION: The opening of K_ATP channels protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the TLR4/NF-κB pathway.     

Effect of recombinant rat CC16 protein on LPS-induced expression of TNF-α, IL-6 and IL-8 in rat tracheal epithelial cells

AIM: To explore the effect of recombinamt rat CC16 protein (rCC16) on LPS-induced expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-8 in the rat tracheal epithelial (RTE) cells.METHODS: The RTE cells were incubated with rCC16 at concentrations of 0.5, 1.0 and 2.0 mg/L in serum-free media for 2 h prior to LPS (0.1 mg/L) treatment for further 24 h. The cells were harvested for assessing the mRNA levels of TNF-α, IL-6 and IL-8 by RT-qPCR. The cell culture supernatants were collected for analyzing the protein levels of TNF-α, IL-6 and IL-8 by ELISA. In addition, the nuclear translocation of nuclear factor-κB (NF-κB) p65 was tested by Western blot.RESULTS: rCC16 inhibited LPS-induced IL-6 and IL-8 expression at both mRNA and protein levels in the RTE cells in a concentration-dependent (0~2 mg/L) manner, as demonstrated by RT-qPCR and ELISA. However, no concentration-dependent manner between the dose of rCC16 and TNF-α expression was observed, and rCC16 inhibited LPS-induced TNF-α expression at lower concentration (0.5 mg/L). rCC16 concentration-dependently inhibited the effects of LPS on the level of nuclear translocation of NF-κB p65.CONCLUSION: rCC16 suppresses LPS-mediated TNF-α, IL-6 and IL-8 production through inactivation of NF-κB activity in RTE cells.[KEY WORDS] CC16 protein; Airway inflammation; LPS; Inflammatory mediators; Nuclear factor-κB     

Homocysteine thiolactone damages cultured endothelial cells

AIM: To approach the mechanisms of homocysteine thiolactone (HTL)-induced damage in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with HTL. The concentrations of soluble intercellular adhesion molecule (sICAM)-1 and TNF-α in the conditioned medium were measured by ELISA. The activity of NF-κB and the level of ROS were determined by fluorescence microscopy. Cell viability,activity of lactate dehydrogenase (LDH) and content of nitric oxide (NO) in the medium were also detected. RESULTS: Exposure of HUVECs to HTL at concentration of 1 mmol/L for 3 h potentiated the activity of NF-κB and increased the level of ROS. Incubation of HUVECs with HTL (1 mmol/ L for 24 h) markedly decreased the cell viability and NO content, and increased the level of LDH, sICAM-1 and TNF-α in the culture medium. Pretreatment with NAC, apocynin or PDTC markedly inhibited the increased activity of NF-κB and decreased the levels of ROS, TNF-α, sICAM-1, NO and LDH in a dose-dependent manner. CONCLUSION: The dysfunction of endothelial cells induced by homocysteine thiolactone in vitro may be related to the oxidative stress and the activation of NF-κB.     

α7nAChR is involved in anti-inflammation of physiological concentrations of glucocorticoids

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR) in anti-inflammation of glucocorticoids (GCs) at physiological concentrations. METHODS: MTT assay was used to measure the viability of BV-2 cells, which were processed by hydrocortisone at different concentrations. On the basis of inflammatory model induced by LPS in BV-2 cells, experimental groups were divided as follows: (1) control; (2) LPS; (3) GCs＋LPS; (4) methyllycaconitine (MLA)＋GCs＋LPS. The levels of TNF-α and IL-1β in the cell supernatants were detected by ELISA. RESULTS: Hydrocortisone at concentrations of 2 000 and 1 000 nmol/L decreased the cell viability to (76.9±5.5)% and (90.8±7.3)%, respectively, indicating the cellular injury by GCs at over-physiological doses. LPS significantly induced the releases of TNF-α and IL-1β in a time- and dose-dependent manner in BV-2 cells. Hydrocortisone at physiological concentrations (500 and 250 nmol/L) reduced the releases of TNF-α and IL-1β in BV-2 cells stimulated by LPS, and MLA at concentration of 10 nmol/L antagonized the anti-inflammatory effect of GCs. CONCLUSION: α7nAChR is involved in the anti-inflammatory effect of the physiological concentrations of GCs.