uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Effect of recombinant MIF on lung fibroblast proliferation and collagen synthesis in vitro

AIM: To investigate the influence and mechanism of recombinant macrophage migration inhibitory factor (rMIF) on fibroblasts. METHODS: MRC-5 fibroblasts were divided into two groups: the treated group was treated with rMIF (25-100 μg/L, 12 h, 24 h or 48 h) and the control was non-rMIF treatment. The activity of proliferation in both groups was investigated and compared by CCK-8 means. Synthesis of collagen in the culture supernatants was detected by the hydroxyproline. The expression of collagen type I mRNA was examined using RT-PCR analysis. The level of collagen type I protein induced by rMIF was quantified by Western blotting. RESULTS: The production of proliferation ratio of fibroblasts treated with 50 μg/L and 100 μg/L rMIF at 24 h or 48 h were increased obviously (P<0.05 or P<0.01). The collagen synthesis significantly increased after stimulation with 100 μg/L rMIF for 48 h (P<0.01). rMIF significantly increased the expression of collagen type I mRNA and protein in a dose dependent manner compared with control (P<0.05 or P<0.01). CONCLUSION: rMIF upregulates the proliferation of fibroblasts and has an effect on the production of collagen. These results suggest that MIF may play important roles in the pathogenesis of airway remodeling.     

Effect of acyl coenzyme A: cholesteryl acyltransferase 1 antisense oligonucleotides on the formation of foam cells

AIM: To study the effect of acyl coenzyme A: cholesteryl acyltransferase 1 (ACAT1) antisense oligonucleotides on the formation of foam cells (FC). METHODS: THP-1 cells were cultured and differentiated into macrophages (MP) by phorbol myristate acetate (PMA). Over-expressing ACAT1 gene THP-1 cells were constructed. The ACAT1 antisense and missense oligonucleotides conducted by LipofectamineTM 2000 were incubated with above cells. Ac-LDL was added 6 h later and incubated for 24 h. The expression of ACAT1 protein was detected by Western blotting. The ACAT activity was measured by quantifying the incorporation of ［1-14C］ oleoyl CoA into cholesteryl esters. The formation of foam cells was detected by oil red O staining. RESULTS: The ACAT1 antisense oligonucleotides inhibited the activity of ACAT in macrophages and over-expressing ACAT1 gene THP-1 cells. It also inhibited the formation of foam cell in macrophages and over-expressing ACAT1 gene THP-1 cells with lipid loading. The missense oligonucleotides did not show the inhibitory effects. CONCLUSION: The ACAT1 antisense oligonucleotides inhibit the activity of ACAT and the formation of foam cells.     

Role of 17β-estradiol in regulation of MIF expression in endometrial stromal cells of endometriosis

AIM: To investigate the effects of 17β-estradiol on the expression of macrophage migration inhibitory factor (MIF) in cultured endometrial stromal cells from endometriosis. METHODS: Immunohistochemistry was used to identify the endometrial stromal cells. The expression of MIF at mRNA and protein levels was assessed by RT-PCR and Western blotting. RESULTS: Elevated expression of MIF at mRNA and protein levels was observed in the cultured endometrial stromal cells treated with 17β-estradiol. In endometrial stromal cells from the women with endometriosis, the level of MIF up-regulation by 17β-estradiol was significantly higher than that in the cells from the women without endometriosis. CONCLUSION: Endometrial stromal cells from endometriosis are more sensitive to 17β-estradiol, which up-regulates the expression of MIF and contribute to the pathogenesis and progression of endometriosis.     

Oxidized low density lipoprotein induces the expression of macrophage migration inhibitory factor

AIM: To examine expression of macrophage migration inhibitroy factor (MIF) gene and protein in macrophages induced by oxidized low density lipoprotein (ox-LDL). METHODS: Macrophages were incubated with ox-LDL at the concentration of 150 mg/L for time course (0-36 h) and with ox-LDL at the different concentrations (0-300 mg/L) for 24 h, expression of MIF mRNA and protein were detected by RT-PCR and ELISA. RESULTS: The results showed that ox-LDL increased MIF gene and protein expression in macrophages in a dose and time-dependent manner. After the exposure of macrophage to ox-LDL, the expression of MIF mRNA level increased consistently with protein. CONCLUSION: MIF may play an important role in atherosclerosis.     

Macrophage migration inhibitory factor gene expression is suppressed by using antisense RNA mediated by expression vector

AIM:To study the potential of using antisense RNA mediated by expression vector to suppress MIF expression. METHODS:MIF gene was sub-cloned into plasmid pcDNA3 to construct MIF antisense RNA expression vector, pcDNA3-antiMIF, which was identified by restriction enzyme digestion and DNA sequencing. By using lipofectamine 2000, plasmid pcDNA3 and pcDNA3-antiMIF were transfected into MIF expression cells, 293-MIF, separately. 60 h later, the 293-MIF cells were collected and used to determine the MIF mRNA expression by real-time quantitive PCR. Plasmid pcDNA3-antiMIF was transformed into HUVECs, named HUVECs-antiMIF, to express MIF antisense RNA. HUVECs-antiMIF was screened by sulfate G418 and identified by PCR and RT-PCR analysis. Then the MIF expression vector, pSecTag-MIF, was transfected into HUVECs-antiMIF, and the MIF mRNA expression in HUVECs-antiMIF was determined by quantitative PCR. RESULTS:Restriction enzyme digestion and DNA sequencing analysis showed that MIF antisense RNA expression vector, pcDNA3-antiMIF, was constructed correctly. The results of quantitative PCR showed that MIF mRNA expression was suppressed by MIF antisense RNA at level about 32% (P<0.05) in 293-MIF cells. The HUVECs-antiMIF, which expressed MIF antisense RNA, was obtained and identified by PCR and RT-PCR assay. The results of quantitative PCR revealed that MIF mRNA expression was also down-regulated by about 40% (P<0.05) in HUVECs-antiMIF cells. CONCLUSION:MIF expression was suppressed efficiently by MIF antisense RNA mediated by expression vector, and the HUVECs-antiMIF was established to express MIF antisense RNA.     

Change of ICAM-1 expression in intestine tissue of mice induced by LPS and role of p38 MAPK in its expression

AIM: To study the change of intercellular adhesion molecule-1(ICAM-1) expression in intestine tissues of mice induced by LPS and regulatory effect of p38 mitogen-activated protein kinase(p38 MAPK) on ICAM-1 expression. METHODS: Protein and mRNA of ICAM-1 were measured using Western blotting and RT-PCR respectively in intestine tissue of BALB/c mice treated by lipopolysaccharide(LPS) or LPS plus SB203580, a specific inhibitor of p38 MAPK. RESULTS: Compared with control group, the expression of ICAM-1 protein and mRNA was increased significantly by LPS stimulation in dose- and time-dependent manner. ICAM-1 expression reached peak value at 12-36 h after LPS stimulation. 20.0 mg/kg of LPS could induce the maximum of ICAM-1 expression. Pretreatment of mice with SB203580 for 30 min could inhibit significantly LPS-induced expression of ICAM-1 protein and mRNA expression in mouse intestine tissues. CONCLUSIONS: These data highlight that LPS could up-regulate ICAM-1 protein and mRNA expression in intestine tissue of mice in dose- and time-dependent manner, and p38 MAPK signal pathway plays an important role in ICAM-1 expression induced by LPS. It suggests that inhibition of p38 MAPK might be a useful principle for the prevention and treatment of intestine damage of endotoxic shock.     

LPS and cerulein induce isolated rat pancreatic tissue injury and inhibit expression of HSP60 protein

AIM: To investigate the effects of lipopolysaccharides (LPS) or cerulein on the expression of HSP60 in isolated rat pancreatic tissues. METHODS: The tissue of rat pancreas was isolated by surgical operation and prepared into tissue snips. The isolated pancreatic tissue was cultured, and stimulated with low- and high-concentrations of cerulein (Cer, 10-11 mol/L, 10-5 mol/L) or lipopolysaccharides (LPS, 10 mg/L, 20 mg/L). Normal saline (NS) was used as control reagent. Before stimulation and 1 h or 4 h after stimulation, the following parameters for evaluating injury were detected: the viability and the level of trypsinogen activation peptide (TAP) in the pancreatic tissues, and the level of interleukin 6 (IL-6) in the culture supernatants. Meanwhile, real-time PCR and Western blotting were used to determine the HSP60 expression at mRNA and protein levels respectively. RESULTS: Under the stimulation with LPS or cerulein, the viability of the pancreatic tissues decreased slightly at 1 h and became much lower with prolonged treatment for 4 h (P<0.05). The TAP level in the pancreatic tissues increased obviously at both 1 h and 4 h (P<0.05) after treatment, except LPS at lower dose. The IL-6 level in the culture supernatant showed no significant change at 1 h stimulation, then increased remarkably at 4 h after stimulation, especially under the conditions of stimulating with LPS or cerulein at higher doses (P<0.05). The expression of HSP60 at mRNA and protein levels decreased sharply with the increase in the concentration of LPS and the prolonged stimulation time; In cerulein stimulation group, the expression of HSP60 mRNA showed an obvious increase (P<0.01), whereas HSP60 protein was reduced remarkably, especially stimulated with cerulein at higher dose and with longer stimulation time (P<0.05). CONCLUSION: LPS or cerulein induce injuries in isolated pancreatic tissues in dose and time dependent manners. Meanwhile, the protein expression of HSP60 is reduced significantly, indicating that the decrease in the cellular protection of HSP60 may involve in the injury of pancreatic tissues.     

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.     

Role of Akt/NF-κB pathway in immune-complexes-induced MCP-1 and CSF-1 expression in murine glomerular mesangial cells

AIM: To explore the role of Akt/NF-κB pathway in immune-complexes-induced monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor-1 (CSF-1) expression in Mesangial Cells. METHODS: Primary murine glomerular mesangial cells were cultured in vitro and divided into control group, stimulation group and antisense, sense and mismatched oligodeoxynucleotide group. In control group, the cells were stimulated with monomeric IgG after treatment with 0.5% lipofectin for 8 h. In stimulation group, the cells, which had been treated with 0.5% lipofectin for 8 h, were stimulated with aggregated IgG. In antisense, sense and mismatched oligodeoxynucleotide group, being transduced antisense, sense and mismatched oligodeoxynucleotide respectively with 0.5% lipofectin 8 h, the cells were stimulated with AIgG. MCP-1 and CSF-1 in supernatant were deteced with ELISA. In addition, RT-PCR was used to determine MCP-1 and CSF-1 mRNA expression, and EMSA to investigated the activation of NF-κB. RESULTS: Mesangial cells cultured in vitro had a low level NF-κB activation and a low level constitutive expression of MCP-1 and CSF-1. Stimulated with AIgG, activation of NF-κB was markedly increased(0.35±0.06 vs 0.75±0.16, P＜0.01), expression of MCP-1 and CSF-1 mRNA (0.48±0.03 vs 0.72±0.02, P＜0.05; 0.44±0.01 vs 0.59±0.02, P＜0.05), MCP-1 and CSF-1 levels in supernatant(15.52±1.81 vs 43.05±3.18, P＜0.05; 389.06±13.75 vs 764.22±31.78, P＜0.05) were markedly increased. Akt1 antisense oligodeoxynucleotide markedly inhibited immune-complexes-induced NF-κB activation, MCP-1 and CSF-1 mRNA and protein expression. CONCLUSION: Akt/NF-κB pathway mediates immune-complexes-induced MCP-1 and CSF-1 expression in mesangial cells. It suggests that Akt/NF-κB pathway may be a new therapy target for macrophage recruitment and activation in immune complexes nephritis.     

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.     

Regulation of ATP-binding acssette transporter A1 on apolipoprotein E secretion from macrophages

AIM: To investigate the regulatory effect of the adenosine triphosphate binding cassette transporter A1 (ABCA1) on apolipoprotein E secretion from human THP1 macrophages.METHODS: Differentiation of THP1 macrophages from monocytes was stimulated with phorbol 12-myristate 13-acetate. The macrophages then were incubated with factors which regulate ABCA1 expression. After periods of incubation, apo E secreted in the medium and synthesized in the cell was determined with ELISA, and apo E mRNA espression was detected with Northern blot.RESULTS: An increase in apo E secretion from THP1 macrophages was observed by 8 h of incubation with 8-Br-cAMP, an activator of ABCA1 expression (P＜0.05). Glyburide, a putative ABCA1 inhibitor, and antisense oligonucleotides specifically against ABCA1 mRNA remarkably decreased apo E secretion from both THP1 macrophages and macrophage foam cells (P＜0.01,respectively). Neither apo E mRNA expression nor intracellular apo E level in THP1 macrophages was changed by inhibition of ABCA1.CONCLUSION: ABCA1 may promote the secretion of apo E from macrophages and macrophage foam cells and the effect may occur at the level of post-translation. The present results reveal a new aspect underlying antiatherogenic properties of ABCA1.     

Changes of pulmonary apoptosis and NOS mRNA in the lipopolysaccharide-induced acute lung injury

AIM: To observe the chronological changes of pulmonary apoptosis and the expression of iNOS mRNA,nNOS mRNA and eNOS mRNA in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to investigate the mechanisms of ALI.METHODS: Rats were randomly divided into 2 groups: control group and LPS treated group.The rats were injected with either saline or LPS and killed at 1,3,6,9 and 12 h after LPS injection.The expressions of iNOS mRNA,nNOS mRNA and eNOS mRNA in the lung tissue were respectively measured with RT-PCR methods.Apoptosis and expressions of Bcl-2 and Bax were respectively determined by flow cytometry (FCM) and immunohistochemistry (IHC).The pathological changes of lung tissue were observed under light and electron microscope.RESULTS: Compared with that in control group,the expression of iNOS mRNA was significantly increased at 3,6,9 and 12 h after administration of LPS (P<0.05).The eNOS mRNA was significantly decreased at 3,6,9 and 12 h after administration of LPS (P<0.05).The nNOS mRNA had no significant change during the 12 h in LPS group.Degree of ALI was gradually worsened after administration of LPS.Apoptosis of pulmonary cells was significantly increased,and reached the top level at 9 h after administration of LPS (P<0.01).The expression of Bcl-2 was markedly decreased and the expression of Bax was significantly enhanced in alveolar and airway epithelial cells in LPS treated group.CONCLUSION: The expressions of iNOS mRNA,eNOS mRNA and nNOS mRNA are not identical in LPS-induced acute lung injury.NOS regulates the apoptosis of pulmonary cells through affecting the balance of Bcl-2 and Bax.     

Influence of siRNA-mediated MIF knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids

AIM：To investigate the influence of siRNA-mediated macrophage migration inhibitory factor (MIF) knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids.
METHODS：Mouse macrophage cell line RAW2647 was transiently transfected with MIF siRNA and control siRNA by liposome method. The transfection efficiency was assessed by immunofluorescence technique. The expression of MIF mRNA and protein was examined by RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production in cell culture supernatants was measured by ELISA, and the protein expression of Annexin 1, cytosolic phospholipase A2α (cPLA2α) and phospho-cPLA2α were evaluated by Western blotting.
RESULTS：MIF siRNA significantly inhibited MIF expression both at mRNA and protein levels in RAW2647 cells and subsequently enhanced the inhibitory effect of dexamethasone (Dex) on PGE2 and LTB4 production. MIF siRNA also increased Annexin 1 expression becreased by Dex, and strengthened the inhibitory effect of Dex on the phosphorylation of cPLA2α.
CONCLUSION：MIF siRNA enhances the inhibitory effect of Dex on PGE2 and LTB4 production from RAW2647 cells partly via increasing Annexin 1 expression and inhibiting cPLA2α phosphorylation. Intracellular MIF knockdown mediated by siRNA may enhance the sensitivity of RAW2647 cells to the anti-inflammatory effect of glucocorticoids.     

The effect of antisense bcr-abl oligonucleotides on the growth of K562 cells

AIM:To study the effect of bcr- abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chrenic myelogeneous leukemia (CML) gene therapy.METHODS:Cells were exposed to oligomeis, observed by inverted microscope.Cells inhibitory rate were determined by 0.4 trypan blue exclusion . CFU-K562 were cultured in 0.8 % methylcellulose . P210 was measured by flow cytomety RESULTS: K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than Spznol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h . There was signifi-cant inhibition of cell proliferation in a rang‘cells number from 1×10⁴/mL to 5×10⁴/mL after treatment with 10unol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA.CONCLUSION: bcr-abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.