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.     

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.     

Inhibitory effect of madecassoside on LPS-stimulated microglia

AIM: To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to investigate its possible mechanism. METHODS: Microglia cells of neonatal Sprague-Dawley (SD) rats were cultured, isolated and purified. Microglia cells were activated with lipopolysaccharide (LPS). The inhibitory effect of madecassoside on microglia was measured by MTT assay. Tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β) were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry. The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR. RESULTS: LPS induced the proliferation of microglia and release inflammatory cytokines significantly. Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner. The IC₅₀ value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h. Madecassoside also decreased the levels of TNF-α and IL-6, increased the ratios of microglia at the G₂ phase and the apoptotic rate, decreased the expression of TLR4 and NF-κB significantly (P<0.05). CONCLUSION: Madecassoside has inhibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.     

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.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

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.     

Effects of tripterygium glycosides combined with dexamethasone on TLRs/NF-κB signaling pathway in EAE rats

AIM: To investigate the role of TLRs/NF-κB pathway in experimental allergic encephalomyelitis (EAE) rats treated with tripterygium glycosides (TG) + dexamethasone (DX). METHODS: Lewis rats were used in the study and divided into control group, EAE model group, therapy 1 group (EAE rats treated with DX) and therapy 2 group (EAE rats treated with DX+TG). The mean clinical score of the rats was determined. The expression of TLR4 and TLR9 at mRNA and protein levels was detected by the methods of real-time quantitative RT-PCR and immunohistochemistry. The protein level of NF-κB p65 was also measured. The levels of TNF-α, IL-1β and IL-6 were assayed by ELISA. RESULTS: The mean clinical scores at 5th, 16th and 20th day were lower in therapy 1 group and therapy 2 group than that in EAE model group. The mean clinical score in therapy 2 group was even lower than that in therapy 1 group. At the 16th day (the peaking period), the mRNA expression of TLR4 and TLR9 in therapy 1 group and therapy 2 group were obviously lower than that in EAE model group. The protein levels of TLR4, TLR9 and NF-κB p65 were also significantly lower in therapy 1 group and therapy 2 group than those in EAE model group at peak stage of EAE. The levels of TNF-α, IL-1β and IL-6 were lower in therapy1 group and therapy2 group than those in EAE model group. The significant differences of the mean clinical score, the mRNA expression of TLR4 and TLR9, the positive ratio of NF-κB p65 and the levels of TNF-α, IL-1β and IL-6 between therapy 1 group and therapy 2 group were found. The result of orthogonal factorial analysis of variance indicated that the difference of therapeutic effect between DX and DX+TG was significant (F=75.749, P<0.01). CONCLUSION: The TLRs/NF-κB pathway takes part in the pathological process of EAE. TG combined with DX alleviates the symptoms of EAE by suppressing inflammatory and immunological reactions of EAE.     

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.     

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.     

Mechanism of systemic inflammatory response syndrome induced by EC DNA

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-α and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-α and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-JB was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-α and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-α peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-α and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NFκB. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, andinduces serum TNF- αand IL- 6 level to increase in rats and ANA- 1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF- κB may be its importantmolecular mechanism, but other pathway probably exists to play an important role.     

miR-451 inhibits inflammatory responses in glomerular mesangial cells by targeting Psmb8 in diabetic nephropathy mice

AIM: To investigate the effect of microRNA (miR)-451 by targeting proteasome subunit β type 8 (Psmb8) on the inflammatory responses in mouse glomerular mesangial cells (MCs) under high-and low-glucose conditions. METHODS: The expression levels of miR-451, IL-18 mRNA and TNF-α mRNA were detected by qPCR. The protein expression levels of IL-18, TNF-α and Psmb8 were determined by Western blot when miR-451 was over-expressed and down-expressed in the MCs. Moreover, the expression of IL-18 and TNF-α was detected when Psmb8 was silenced by si-Psmb8 in MCs. RESULTS: The expression of miR-451 was significantly decreased in the MCs treated with high glucose compared with low glucose group (P<0.01). However, the expression of Psmb8 was increased in high glucose group as compared with low glucose group (P<0.01). Moreover, the expression levels of Psmb8, IL-18 and TNF-α were significantly decreased when miR-451 was over-expressed in high glucose group (P<0.01). Additionally, the expression levels of IL-18 and TNF-α were significantly reduced when Psmb8 was silenced in the MCs under high glucose condition. CONCLUSION: miR-451 reduces the expression of inflammatory factors via targeting Psmb8 in the MCs under high glucose condition. Therefore, miR-451 may play a role in inflammation of diabetic nephropathy.     

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.     

Effects of miR-155 over-expression on expression of inflammatory factors and indolamine 2, 3-dioxygenase in microglial BV-2 cells

AIM:To explore the effect of microRNA-155(miR-155)over-expression on the expression of inflammatory factors and indolamine 2, 3- dioxygenase (IDO) in the microglial BV-2 cells. METHODS:For over-expression of miR-155, the BV-2 cells were transfected with lentiviral vector carrying mmu-miR-155. The expression of inflammatory factors was detected by cytometric bead array system (CBA). The mRNA expression of inflammatory factors and IDO was analyzed by real-time PCR. The protein levels of suppressor of cytokine signalling 1 (SOCS1), p-p38 MAPK and IDO were determined by Western blot. RESULTS:The expression of miR-155 was up-regulated in the BV-2 cells transfected with lentiviral vector carrying mmu-miR-155 compared with LPS treatment group (P<0.01). The miR-155 over-expression promoted the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and IL-10, and inhibited the secretion of IL-12. The miR-155 over-expression increased the mRNA expression of IL-6, TNF-α, IL-10 and IDO, also increased the protein levels of IDO and p-p38 MAPK, but decreased the protein expression of SOCS1 (P<0.01). LPS promoted the secretion of IL-6, TNF-α, MCP-1 and IL-12, also increased the mRNA expression of IL-6, TNF-α and IDO, meanwhile, increased the protein levels of IDO, p-p38 MAPK and SOCS1 (P<0.01). CONCLUSION:Over-expression of miR-155 promotes the secretion of related imflammatory factors and protein expression of IDO in microglial BV-2 cells mediated with SOCS1 and p38 MAPK signaling pathway.     

Effect of high-fructose diet on adipose tissue renin-angiotensin system in rats

AIM: To observe the effects of high-fructose diet on adipose tissue inflammation and renin-angiotensin system (RAS), and to reveal the role of Toll-like receptor 2 (TLR2) in this process.METHODS: Male SD rats (n=16) were randomly divided into control group, high fructose group, high fructose+siRNA negative control group, and high fructose+TLR2-siRNA group. The rats in control group were fed with a standard chow diet. The rats in high fructose group were fed with a diet with 60% fructose, and the rats in high fructose+TLR2-siRNA group and high fructose+siRNA negative control group were transfected with TLR2 siRNA and scrambled siRNA, respectively. Serum uric acid was measured and visceral adipose tissue was weighed at the 14th week. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), angiotensinogen (AGT), and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. Infiltrating macrophages in the adipose tissues were measured with immunohistochemistry. The mRNA expression of IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), AGT, angiotensin-converting enzyme 1 (ACE1), angiotensin Ⅱ type 1 receptor (AT1R), and angiotensin Ⅱ type 2 receptor (AT2R) was detected by RT-qPCR. The protein level of TLR2 was determined by Western blot.RESULTS: High fructose-fed rats showed elevated serum uric acid, raising fat content, higher serum concentrations of IL-6, TNF-α, AGT and AngⅡ, and more infiltrating macrophages in the adipose tissues (P<0.05). Moreover, the mRNA levels of IL-6, TNF-α,MCP-1, AGT, ACE1, AT1R and AT2R in the adipose tissues were increased (P<0.05). When high fructose-fed rats were transfected with TLR2-siRNA, the dramatic decreases in TLR2 protein level and number of infiltrating macrophages in the adipose tissues were found. Both in serum and adipose tissues, the mRNA levels of inflammatory cytokines and RAS components were all significantly decreased (P<0.05).CONCLUSION: High-fructose diet up-regulates RAS in adipose tissues via activation of TLR2 inflammation signaling 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     

Norepinephrine attenuates injury of vascular endothelial cells induced by LPS

AIM: To investigate the effects of norepinephrine (NE) on vascular endothelial cell damage induced by lipopolysaccharides (LPS). METHODS: Human umbilical vein endothelial cells (HUVEC-12) were cultured with LPS at 100 mg/L to establish the cell damage model. Real-time PCR and Western blot were used to determine the expressions of VE-cadherin at mRNA and protein levels. The levels of TNF-α, IL-1β, IL-2 and IL-10 in culture supernatant were measured by ELISA. The reactive oxygen species (ROS) production in the endothelial cells was detected by ROS assay kit. RESULTS: LPS decreased both mRNA and protein levels of VE-cadherin accompanied by increased levels of TNF-α, IL-1β, IL-2 and intracellular ROS, and decreased level of IL-10 in the endothelial cells. NE reversed the expression of VE-cadherin at mRNA and protein levels under the condition of LPS treatment in a dose-dependent manner, and also alleviated the intracellular oxidative stress. CONCLUSION: NE reverses the endothelial damage induced by LPS, which may be related to the up-regulation of VE-cadherin level and the decreases in oxidative stress and inflamatory mediators.