Inhibition of HMGB1 expression and release by nicotine in RAW264.7 cells

AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α7 subunit-containing nicotinic receptor (α7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α7nAch receptor expression.     

Effect of TNF-α on the cartilage endplate chondrocytes of New Zealand white rabbits

AIM:To evaluate the biological roles of TNF-α on the cartilage endplate cells (chondrocytes). METHODS:The chondrocytes were isolated and harvested from the cartilage endplate of New Zealand rabbits and then the biological characteristics of cells were identified by methods such as toluidine blue staining for type Ⅱ collagen. After different concentrations of TNF-α were added to culture medium respectively, the rate of the proliferation of chondrocytes in different time was measured with MTT. The protein expressions of Bax, Bcl-2, Fas and caspase-3 were measured by immunocytochemistry. The changes of the mRNA of aggrecan and type Ⅱ collagen were measured by RT-PCR. RESULTS:The TNF-α at concentration of 50 μg/L and 100 μg/L decreased the rate of the proliferation on chondrocytes. Though TNF-α at concentrations of 10 μg/L and 50 μg/L increased the level of Bax, Fas and caspase-3, only 50 μg/L TNF-α decreased the level of Bcl-2. TNF-α at concentrations of 10 μg/L and 50 μg/L decreased the level of collagen IIa mRNA and only 50 μg/L TNF-α decreased the level of aggrecan. CONCLUSION:TNF-α not only inhibits the proliferation and the matrix synthesis in chondrocytes, but also increases the expression of pro-apoptotic factors in chondrocytes.     

The mechanism of lipopolysaccharide-activated rat alveolar macrophages producing tumor necrosis factor alpha

AIM: To investigate the expression of hypoxia inducible factor-1alpha (HIF-1α) and the role of HIF-1α in tumor necrosis factor alpha (TNF-α) production in rat alveolar macrophages activated by lipopolysaccharide (LPS). METHODS: HIF-1α function was inhibited by using the method of HIF-1α decoy. Western blotting and semiquantitative RT-PCR were applied to determine the expression of HIF-1α protein and mRNA, respectively. The production of TNF-α was determined with ELISA. RESULTS: The content of HIF-1α protein in LPS group (1.95±0.57) and HIF-1α decoy group (1.89±0.59) were 4.8 times and 4.6 times higher than that in control group (0.41±0.14), respectively. The expression of HIF-1α mRNA showed no difference among three groups (F=3.14，P>0.05). The production of TNF-α in LPS group was higher than that in control group (61 ng/L vs 156 ng/L, q=5.12, P<0.05) and HIF-1α decoy group (90 ng/L vs 156 ng/L, q=4.63, P<0.05), respectively. However, the content of TNF-α in HIF-1α decoy group was still higher than that in control group (61 ng/L vs 94 ng/L, q=4.47, P<0.05). CONCLUSION: The enhanced stability of HIF-1α protein results in the marked upregulation of its protein and HIF-1α is contributed to the production of TNF-α in LPS-stimulating rat alveolar macrophages. It is indicated that HIF-1α plays important role in the pathogenesis of chronic inflammation involved in diseases such as COPD.     

The effect of nucleolin on interleukin-1β secretion induced by lipopolysaccharide

AIM: To explore the expression of nucleolin in lipopolysaccharide(LPS)-mediated inflammatory models, and further investigate the role of nucleolin in expression and secretion of LPS-induced interleukin-1β(IL-1β). METHODS: To establish inflammatory models, mice suffered intraperitoneal injection of LPS(15 mg/kg)and RAW264.7 cells were treated with LPS(500 μg/L).Western blotting were applied to identify the expression of nucleolin in these inflammatory models. After over-expression of nucleolin by transient pcDNA3.1-C23 transfection and down-regulation by transient transfection of nucleolin antisense oligonucleotides, the secretion of IL-1β were examined by enzyme-linked immunosorbent assay (ELISA) in LPS-stimulated RAW264.7 cells. RESULTS: Westem blotting assays showed that the 110 kD nucleolin increased in RAW264.7 cells treated with LPS (500 μg/L) and the lung tissues of the mice treated with LPS (15 mg/kg), while the 80 kD component of nucleolin decreased. ELISA showed that LPS-induced IL-1β release in RAW264.7 cells transfected with pcDNA3.1-C23 was higher than that in pcDNA3.1 empty vector transfected cells. LPS-induced IL-1β release in RAW264.7 cells transfected with C23 antisense oligonucleotide was lower than that in normal cells and scramble oligonucleotide transfected cells. CONCLUSION: In LPS-mediated mouse endotoxemia model and LPS-mediated RAW264.7 cell inflammatory model, the expression of 110 kD nucleolin was up-regulated, but 80 kD nucleolin fragment decreased. Nucleolin promoted secretion of LPS-induced IL-1β.     

Effects of TNF-α-mediated insulin resistance on INSIG2 and SCAP expressions in vivo

AIM: To investigate the effects of TNF-α induced insulin resistance (IR) on INSIG1, INSIG2, SCAP and SREBP expressions in mice. METHODS: Male C57BL/6J mice were randomly divided into 4 groups. The mice were given an intraperitoneal injection of TNF-α (6 μg·kg-1·d-1; 3 μg·kg-1·d-1 and 1 μg·kg-1·d-1) and saline (NC group) twice daily for 7 d. The insulin sensitivity and glucose metabolism in awaken mice were evaluated by intravenous glucose tolerance test (IVGTT). The mRNA expression and protein levels of gene were measured by RT-PCR and Western blotting. RESULTS: After TNF-α treatment, fasting blood glucose (FBG), plasma insulin and free fatty acids (FFA) were significantly elevated in TNF-α (6 μg·kg-1·d-1) group compared to NC, TNF-α (1 μg·kg-1·d-1) and TNF-α (3 μg·kg-1·d-1) groups (P<0.01 and P<0.05, respectively). There was a lower glucose tolerance in TNF-α (6 μg·kg-1·d-1) group than that in other three groups during IVGTT. In TNF-α (6 μg·kg-1·d-1) group, the insulin release of glucose-stimulation was higher than that in NC and TNF-α (1 μg·kg-1·d-1) groups (P<0.01 and P<0.05). The INSIG2 mRNA expression of adipose tissues in TNF-α (6 μg·kg-1·d-1) group was significantly increased compared with NC group (P<0.01), and INSIG2 protein levels were also increased (P<0.05). In TNF-α treatment mice, SCAP mRNA level in adipose tissues was significantly up-regulated than that in the controls (P<0.05). The mRNA expressions of INSIG1 and SREBP1 in two groups were not significantly changed (P>0.05). CONCLUSION: In TNF-α induced insulin resistance, INSIG2 and SCAP may be involved in the pathways of lipid metabolism.     

CO2 与养分交互作用对番茄幼苗养分利用的影响

以番茄(Lycopersicon esculentumMill.)‘合作906’为材料进行溶液培养试验,设2个因子:CO2和营养液浓度;CO2浓度设正常(360μL/L)和倍增(720μL/L)2个水平;营养液浓度设基本营养液(日本山崎番茄营养液),微量元素采用阿农营养液配方的1/2、1/4、1/8、1/164个水平,完全试验方案8个处理,3次重复。pH为6·0±0·2,3d更换1次营养液。移植到1·2L盆(2株/盒)中,植株在CO2生长箱(VS-3DMC)中培养,全天施放CO2,白天25℃,晚上15℃,光照为14h/d,光照强度11000lx,相对湿度60%。46d时收获,根、茎、叶经蒸馏水冲洗吸干水分后,放入纸袋105℃杀青,75…     

Expression of IGFBP2 and IGFBP4 in hepatocytes with experimental injury

AIM:To explore the mechanism of IGFBP2 and IGFBP4 in hepatocytes with injury induced by TNF-α and TGF-β1. METHODS:Human hepatocyte line (HL-7702) was cultured in vitro and treated with TNF-α or TGF-β1 for 24 h. The expression of IGFBP2 and IGFBP4 was detected by immunochemistry staining. The inhibition ratio of hepatocytes was detected by MTT assay. HL-7702 was treated with TNF-α at concentration of 20 μg/L for 48 h, then the apoptosis of hepatocytes was detected by both Annexin-V /PI and TUNEL assay. RESULTS:The expression of IGFBP2 and IGFBP4 in TNF-α or TGF-β1 treated groups was significantly higher than that in control group (P<0.05). The positive staining of IGFBP2 and IGFBP4 in TNF-α (20 μg/L) treated groups or TGF-β1 (4 μg/L) treated groups was the strongest among all groups. A positive correlation was found between IGFBP2 or IGFBP4 and inhibitory ratio of hepatocytes (P<0.05 or P<0.01). Compared with normal control group, the percentage of apoptosis markedly increased in TNF-α (20 μg/L) treated group (P<0.01). CONCLUSION:IGFBP2 and IGFBP4 involved in the injury process in hepatocytes, indicating an important role in injury of hepatocytes induced by TNF-α or TGF-β1.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

IL-17A reduces lipid accumulation by up-regulation of ABCA1 expression in Raw264.7 macrophages

AIM:To investigate the effects of interleukin-17A (IL-17A) on the expression of adenosine triphosphate binding cassette transporter A1 (ABCA1) in RAW264.7 macrophages. METHODS:Mouse RAW264.7 macrophages were treated with IL-17A at different concentrations for 6 or 24 h, or treated with IL-17A at the same concentration for different time. The expression of ABCA1 at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Cholesterol efflux to apolipoprotein A1 (ApoA-1) was evaluated by NBD-cholesterol method. Lipid accumulation in the cells was evaluated by Oil Red O staining. RESULTS:Compared with control group, IL-17A increased the expression of ABCA1 at protein level in the RAW264.7 cells significantly (P<0.05), but had no effect on the mRNA expression of ABCA1. In addition, cholesterol efflux to ApoA-1 was increased and lipid accumulation in the RAW264.7 cells was decreased obviously after treatment with IL-17A. CONCLUSION:IL-17A increases the protein expression of ABCA1 but not at mRNA level in the RAW264.7 macrophages, which may be correlated with its anti-atherosclerosis effect.     

Corticosterone inhibits LPS-induced expression of NLRP3 in mouse macrophages and its relation with XO

AIM: To investigate the inhibitory effect of corticosterone (CORT) on lipopolysaccharide (LPS)-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and its relation with xanthine oxidase (XO). METHODS: An inflammatory model of mouse macrophage RAW 264.7 was established by stimulating with LPS. Total cellular protein was extracted after the macrophages were treated with CORT at different concentrations (0~900 μg/L). The protein levels of NLRP3 and caspase-1 were determined by Western blot. According to the treatments, the macrophages were divided into control group, LPS group, LPS+CORT group and LPS+allopurinol group. Cell components were extracted at 0, 0.5, 1, 1.5 and 2 h. The protein levels of NLRP3 and XO were determined by Western blot,and the mRNA expression of NLRP3 and XO was detected by real-time PCR. RESULTS: CORT at 700 μg/L and above significantly inhibited the expression of NLRP3 and the activation of caspase-1 in the macrophages induced by LPS (P<0.05). Compared with LPS group, the expression of NLRP3 and XO in LPS+CORT group was inhibited (P<0.05), and the expression of NLRP3 in LPS+allopurinol group was also reduced (P<0.05).CONCLUSION: High concentration of CORT inhibits the expression of NLRP3 in LPS-induced mouse macrophages, which is associated with XO. The inhibitory effect of CORT may be related to the reduction of XO expression.     

Effect of high mobility group box protein 1 on expression of STAT/SOCS/cyclin D1 in rat class fibroblast-like synovial cells

AIM: To investigate the regulatory effect of high mobility group box protein 1 (HMGB1) on STAT/SOCS signal transduction in RSC-364 synoviocytes. METHODS: RSC-364 cells induced by 10 μg/L human recombinant HMGB1 were collected at 6 h, 12 h or 24 h after treatment, respectively. The cells without any treatment were also collected at same time points for normal control. The mRNA expression of STAT 1/3 was analyzed by RT-PCR. The phospho-STAT 1/3 (p-STAT 1/3) and STAT 1/3 proteins were detected by Western blotting. The flow cytometric analysis (FCM) was applied to detect the protein expression of p-STAT 1/3, SOCS 1/3 and cyclin D1. RESULTS: HMGB1 at concentration of 10 μg/L significantly up-regulated the mRNA and protein levels of STAT1 expression, as well as cyclin D1 protein, and markedly increased the protein expression of p-STAT1 (P<0.01). Compared to control group, no significant difference of STAT3 and p-STAT3 expression was observed. HMGB1 elevated the protein expression of SOCS1, while the protein level of SOCS3 was only up-regulated at 6 h. CONCLUSION: HMGB1 may activate the process of STAT1 signal transduction and up-regulate the gene expression of cyclin D1, suggesting the role of HMGB1 in promoting the proliferation of synoviocytes.     

Effects of ouercetin on lipopolysaccharide induced hepatocyte injury in vitro

AIM: To study the effects of quercetin on lipopolysaccharide (LPS) induced hepatocyte injury and the expression of TNF-α in vitro. METHODS: Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion. LPS at concentration of 40 mg/L was used to induce injury to the cultured cells, and 0.5-10 μmol/L quercetin was added at the same time. After 24 h of incubation, the cell apoptosis rates were detected by MTT and PI-AnnexinV. LDH and TNF-α were measured by kits. RESULTS: 40 mg/L LPS caused a 27% growth inhibition. The apoptosis rate was 30.2%. LDH leakage was 20 folds higher than normal. TNF-α expression significantly increased. Treated with quercetin at doses of 0.5-10 μmol/L, the apoptosis rate, LDH leakage and TNF-α expression in hepatocytes were attenuated in a dose dependent manner. CONCLUSION: 0.5-10 μmol/L of quercetin protects hepatocytes from injury induced by LPS, which is associated with suppression of the inflammatory cytokine TNF-α.     

Effect of Kupffer cell blockade on activation of mitogen-activated protein kinases in response to lipopolysaccharide

AIM: Using the mouse model of lipopolysaccharide(LPS) attack,we study the effect of Kupffer cell (KC) blockade on the activation of mitogen-activated protein kinases(MAPKs) signal transduction pathway induced by LPS.METHODS: GdCl₃ (10 mg/kg) or the same volume of NS was continually injected intravenously at 48 h and 24 h before LPS (5 mg/kg) was injected into the male mice of Kunming species.The liver was then took out and KCs were isolated 30 minute after LPS was injected.The KCs isolated from the mice were cultured,and pretreated with GdCl₃ (100 μmol/L) for 1 h.The culture medium containing LPS (100 μg/L) was added and continuously incubated for 30 minute.The protein expression and phosphorylation level of ERK1/2 and p38MAPK in liver or KCs were assayed in vivo and in vitro,and effect of GdCl₃ on the phagocytosis function was observed,respectively.RESULTS: LPS induced the protein phosphorylation of ERK1/2 and p38MAPK in KCs or liver,no effect on the protein expression was observed.GdCl₃ treatment inhibited LPS-induced KCs activation and secretion of TNF-α,however,it had no effect on ERK1/2 and p38MAPK in KCs or liver,neither at the protein expression nor the phosphorylation.KCs secreted a few TNF-α with short time treatment with GdCl₃ alone in vitro.CONCLUSION: KC blockade with GdCl₃ alleviates LPS-induced KCs activation and the release of TNF-α not through modulating intracellular ERK1/2 or p38MAPK signal transduction pathways.We presume that GdCl₃ might reduce liver injury through cross talk of other intracellular signal transduction pathways (JNK,NF-кB,GPCR,etc).     

Role of p38 MAPK in cyclic mechanical stretch induced HMGB1 expression in alveolar macrophages

AIM: To investigate the role of p38 mitogen-activated protein kinase (MAPK) in cyclic mechanical stretch induced the expression of high mobility group box 1 protein (HMGB1) in alveolar macrophages (AMs). METHODS: AMs were cultured and seeded at 1×108 cells/L in 6-well Bioflex cell culture plates. Subsequently, the cells were exposed to cyclic mechanical stretch at 20% (group B) elongation using Flexercell 4000T cell stretching unit. In group C, cells were pre-treated with SB203580 (40 μmol/L) for 2 h before mechanical stretch. Group A served as control. The expression of HMGB1 mRNA in alveolar macrophages was detected by RT-PCR. p38 MAPK activity and the expression of HMGB1 protein were measured by Western blotting analysis. RESULTS: The expression of HMGB1 mRNA and protein, and the activity of p38 MAPK in AMs were significantly increased in group B than those in group A (P<0.05). SB203580, an inhibitor of p38 MAPK, significantly inhibited the inducing effect of mechanical stretch (P<0.05). CONCLUSION: Mechanical stretch regulates the expression of HMGB1 mRNA and protein in alveolar macrophages by activating p38 MAPK signal pathway.     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.