Objective To observe the expression of GdCl3 on Toll-like receptors （TLRs） of RAW264.7 from murine macrophage cell line induced by lipopolysaccharide (LPS) stimulation. Methods Cells were divided into 3 groups: blank group, LPS group and GdCl3 group. And these cells dyed by goat anti-mouse TLR2/4 poly-antibody and anti-goat IgG labelled with fluorescein isothiocyanate (FITC). The synthesis of TLR2/4 protein were determined by flow cytometry (FCM) analysis and reverse transcription polymerase chain reaction (RT-PCR) analyzed their gene expression. Cell supernatants were taken to measure TNF-α production following the ELISA (enzyme-linked immunosorbent assay) protocol. Results The expressions of TLR2/4 protein and mRNA in GdCl3 group under action of different concentration of GdCl3〔TLR2/4 protein, 200 μmol/L: (70.2±1.28）％/(66.7±2.59)％, 400 μmol/L: (64.9±1.43)％/(60.4±1.25)％, 2 000 μmol/L: (47.4±0.98)％/(32.1±0.74)％; TLR2/4 mRNA (the value of absorbance), 200 μmol/L: (76.42±2.76)/(101.72±3.14), 400 μmol/L: (75.60±3.76)/(89.65±5.17), 2 000 μmol/L: (64.22±4.67)/(78.44±4.88)〕 were significantly lower than those of in LPS group 〔TLR2/4 protein: (94.4±1.76)％/(95.7±0.87)％, P＜0.01; TLR2/4 mRNA: (127.64±3.25)/(119.82±5.59), P＜0.05, P＜0.01〕. The expression of TNF-α in GdCl3 group under action of different concentration of GdCl3〔200 μmol/L: (2 540±77) pg/ml, 400 μmol/L: (2 041±106) pg/ml, 2 000 μmol/L: (1 020±220) pg/ml〕 was also significantly lower that that of in LPS group 〔(4 688±127) pg/ml, P＜0.01)〕. Conclusion GdCl3 significantly inhibits TLR expression and secretion of TNF-α under the condition of LPS stimulation in vivo.
Objective To study the effect of pravastatin on the survival of islet xenografts.MethodsPigtomouse islet transplantation was performed. The models were divided into 4 groups: group A (control); group B, treated with CsA; group C, treated with pravastatin; group D, treatment with combined CsA with pravastatin. The survival time (ST) of the grafts in each group were recorded. Histological examination was used to detect the inflammation and islet cells in the graft. The infiltrated cells were detected by immunohistochemistry with CD4+, CD8+ and CD68 monoclonal antibody. The serum NO was measured. RTPCR was used in the test of IFNγ mRNA.ResultsThe ST of group A,B,C,D was (6.2±0.82) d, (9.2±1.92) d, (7.2±1.30) d, (11.2±1.76) d respectively, the ST of group D was much longer than that of the other groups (P＜0.05).Compared to that in other groups, less infiltrated cell in group D was found. On the 4th postoperative day, the serum NO in group A was (105.0±19.3) mmol/L,significantly higher than that in group B 〔(88.20±21.04) mmol/L〕, in group C 〔(70.7±17.8) mmol/L)〕 and in group D 〔(56.30±16.4) mmol/L〕. When rejection occurred, the serum NO in group C and D was (83.7±10.6) mmol/L and (71.3±13.8) mmol/L, also lower than that in group A (P＜0.05), the serum NO in group B was (104.7±16.3) mmol/L, compared that in group A, no significance was present (Pgt;0.05）. On the 4th postoperative day, the serum expression of IFNγ mRNA in group D was 23.5±4.6, lower than that in group A (28.8±4.8), and no significance was present compared with that in group B and C. ConclusionPravastatin can abate the role of macrophages, especially combined with Cyclosporine, and can prolong the survival of islet xenograft.
ObjectiveTo investigate changes of lipopolysaccharidebinding protein (LBP) and its clinical significance in activation of Kupffer cells (KCs) during endotoxemia.MethodsWistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111∶B4) via the tail vein of rats, then sacrificed 1, 3, 6 and 12 hour respectively. Hepatic tissue was collected to measure LBP mRNA expression by reverse transcritasepolymerase chain reaction (RTPCR). The levels of plasma endotoxins, LBP, TNFα and IL6 were determined. The pathological changes of hepatic tissue were observed under electron microscope.ResultsWhen the levels of plasma LPS elevated, expression of LBP mRNA in hepatic tissue were ber than that in control rats. The levels of plasma LBP, TNFα and IL6 were increased markedly also in rat with endotoxemia when compared with that in control groups (P＜0.01). KCs were seen to be enlarged in size, their surface projections were increased in number, and their cytoplasm was full of phagocytic vacuoles or electron dense phagosomes which indicated active phagocytosis.ConclusionLPS can markedly upregulate LBP mRNA expression in hepatic tissue, the levels of plasma LBP also increased. LBP may be a critical factor of LPS which stimulates KCs to produce and release different proinflammary mediators.
63 normal human gallbladders (non-stone group) and 47 inflammed cholesterol stone gallbladders(stone group) were assayed for the amount of macrophages(ΜΦ),the levels of tumor necro-sis factor (TNF) and interleukin 1(1L-1).It was found that in stone group,the amount of ΜΦ was significantly higher than in non-stone group(ΜΦ4101.90±295.72 vs 572.13±30.07AU,Plt;0.01).The levels of TNF and 1L-1 released mainly from the MΦ in stone group were also significantly increased in comparison with those in non-stone group(TNF 18.12±2.03 vs 4.45±0.39ng/mg,Plt;0.001;1L-1 102.42±7.84 vs 66.75±9.50u/mg protein,Plt;0.05).These results suggest that the activited ΜΦ and increases of TNF,1L-1 may be closely related to the inflammatory reaction in gallbladders and the formation of cholesterol gallstones.
Objectives To explore the expression of macrophage inflammatory protein-1beta (MIP-1β) in patients with none-small cell lung cancer (NSCLC) of different pathological types and its association with cancer clinical stages and metastasis of lymph nodes.Methods MIP-1β mRNA from fresh lung tissue of 38 NSCLC patients was amplified by RT-PCR and half-quantified.Immunohistochemical technique was performed to find out the expression of MIP-1β in paraffin-embedded lung tissue from 66 patients with NSCLC.The area and degree of stain were evaluated to determine the positive rate，which was compared between with or without metastasis of lymph nodes，different pathological types and TNM clinical stages.Results MIP-1β protein was found in cytoplasm of malignant cells of squama cell cancer and adenocarcinoma without significant difference between them，while not found in bronchus-alveolus cell cancer.The MIP-1β mRNA expression in squama cell cancer and adenocarcinoma were significant higher than which in bronchus-alveolus cell cancer without significant difference between each other.The positive rates of MIP-1β in lung cancer of Ⅰ，Ⅱ and Ⅲ stages were 74.2%，29.4% and 85.7% respectively，which of Ⅰ and Ⅲ stages cancer were significant higher than Ⅱ stage without significant difference between each other.The positive rates of MIP-1β in lung cancer with or without metastasis of lymph nodes were 45.8% and 76.3% respectively with significant difference between them.Conclusion MIP-1β is expressed in lung cancer cells and relates to the pathological type，TNM stage and the metastasis of lymph nodes.
Objective To investigate the effects of mechanical ventilation on lung pathology and concentration of proinflammatory cytokines in rats.Methods Forty-five healthy Sprague-Dawley rats were randomly divided into three groups with 15 rats each group，ie.a control group(No mechanical ventilation)，a high tidal volume group(VT 34 mL/kg，RR 30 bpm) and low tidal volume group(VT 8 mL/kg，RR 60 bpm).Results There were enlarged alveolar spaces，interalveolar septum collapse，swollen and spotty hemorrhages，inflammatory cell infiltration in the ventilation groups，which was more serious in the high tidal volume group.Lung wet-to-dry weight ratio of high tidal volume group was significantly higher than that of other groups（both Plt;0.05）.The concentrations in both macrophage-inflammatory protein-2（MIP-2）and tumor necrosis factor-α(TNF-α) of bronchoalveolar lavage fluid(BALF) and blood samples was significantly higher in two ventilation groups than the control group（Plt;0.05），which were higer in the high tidal volume group than in the low tidal volume group（Plt;0.05）.Conclusions These results indicate that mechanical ventilation can cause lung injury，which is more serious in high tidal volume ventilation.And mechanical ventilation-induced lung injury can cause proinflammatory cytokines release，with different levels in bronchoalveolar lavage fluid and blood samples.
Objective To study the role of E-selection and macrophage inflammatory protein-2（MIP-2） in the airway inflammation in a rat model of chronic obstructive pulmonary disease（COPD）.Methods Twenty-four male SD rats were randomly divided into a normal control group and a COPD group.The rat model of COPD was established by exposure to cigarette smoking.Lung function，pathologic features of lung tissues and inflammatory cell differentials in bronchoalveolar lavage fluid（BALF） were investigated.Immunohistochemistry was employed to examine the expression of E-selection in lung tissue.The levels of MIP-2 in BALF，serum and lung tissues were measured with ELISA.Results The changes in histopathology and pathophysiology in the rat COPD model were similar to those in COPD patients.The expression of E-selection on bronchopulmonary vein endothelial cells of the COPD group significantly increased as compared with the normal control group（Plt;0.05） and was positively correlated with the neutrophil counts in BALF in the COPD group（ｒ=0.809，Plt;0.01）.The levels of MIP-2 in BALF and lung tissues in the rats with COPD were signicantly higher than those in normal rats，both of which were positively correlated with the neutrophil counts in BALF in the COPD group（r=0.893，Plt;0.01；r=0.716，Plt;0.05）.Conclusion Both E-selection and MIP-2 may be involved in the process of airway inflammation in COPD.
Objective To observe the effects of mechanical stretch on cytokines release from alveolar macrophages( AMs) and the expression of macrophage inflammatory protein-2( MIP-2) induced by lipopolysaccharide( LPS) . Methods AMs were divided into the following groups: ①AMs were subjected to 20% elongation by Flexercell 4000T cell stress system for 24 hours and the supernatant was collected to detect the levels of TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, macrophage inflammatory protein-1α( MIP-1α) , MIP-2, monocyte chemoattractant protein-1( MCP-1) , granulocyte /macrophage colony stimulating factors( GM-CSF) , interferon inducible protein-10( IP-10) , regulated on activation in normal T-cell expressed and secreted( Rantes) and keratinocyte chemoattractant( KC) , by using LiquiChip system. ② AMs were subjected to 5% , 10% , 15% and 20% elongation for 24 hours and the supernatant was collected to detect the levels of MIP-2. ③AMs were subjected to 20% elongation and MIP-2 in supernatant was detected 1, 3,6, 12, and 24 hours later. ④ AMs were subjected to 20% elongation and/ or LPS at a concentration of 10 ng/mL, and MIP-2 in supernatant was detected 24 hours later. Unstretched AMs were used as control in all kind of test. Results ①The levels of IL-1β, IL-6,MIP-2, MCP-1, IFN-γand IP-10 secreted by stretched AMs were 8. 7, 4. 3, 38. 6, 4. 8, 14. 2 and 5. 0 times those of the control group( all P lt; 0. 001) . ② The levels of MIP-2 secreted by AMs subjected to 10% , 15% and 20% elongation were ( 480. 5 ±93. 1) pg /mL,( 806. 3 ±225. 9) pg/mL and ( 1335. 7 ±18. 5) pg/mL respectively, all significantly higher than those oft he control group [ ( 34. 6 ±11. 4) pg/mL, all P lt;0. 001] . ③ Three hours after the stimulation of stretch the level of MIP-2 began to increase gradually. And 6, 12, and 24 hours after the stimulation the levels of MIP-2 secreted by the AMs were ( 819. 4 ±147. 5) pg/mL, ( 1287. 6 ±380 ±3 ) pg/mL and ( 1455. 9 ±436. 7) pg/mLrespectively, all significantly higher than those of the control group[ ( 33. 4 ±10. 2) pg/mL, all P lt; 0. 001] . ④When the AMs were stimulated individually by LPS( 10 ng /mL) or mechanical stretch ( 20% ) , the levels of MIP-2 increased to ( 1026. 3 ±339. 5 ) pg/mL and ( 1335. 7 ±318. 5 ) pg/mL respectively( both P lt; 0. 001) . When the AMs were costimulated by LPS and mechanical stretch, the level of MIP-2 increased to ( 2275. 3 ±492. 1) pg/mL, implicating a synergistic effect between mechanical stretch and LPS ( F = 121. 983, P lt; 0. 001) . Conclusions Mechanical stretch activates AMs to produce multiple inflammatory cytokines and induce AMs to secret MIP-2 in a strength- and time-dependent manner.Mechanical stretch also has synergistic effect with LPS in inducing MIP-2 release, which might play an important role in the development of ventilator-induced lung injury.
Objective To explore the role of macrophage-stimulating protein ( MSP) and receptor tyrosine kinase RON in the airway inflammation of chronic obstructive pulmonary disease( COPD) , and investigate its possible mechanism. Methods The rat COPDmodel was established by exposing the rats to cigarette smoke daily for three months. Rat alveolar macrophages ( AMs) were isolated in vivo and cultured,and then challenged with different concentrations of MSP for 24 hours. The concentrations of MSP in broncho-alveolar lavage fluid ( BALF) and serum, and the levels of IL-1β, TNF-α, IL-8, and IL-10 in the supernatants were measured by ELISA. The expression of RONmRNA in lung tissue was assessed by reverse transcription-polymerase chain reaction. The levels of RON protein in the lung tissue and AMs cultured in vitro were observed by immunohistochemistry. The activity of superoxide dismutase ( SOD) and malondialdehyde ( MDA) content in the culture solution were measured with chromatometry method. Results Compared with the control group, the concentrations of MSP in serum and BALF of the COPD rats were significantly higher ( P lt;0. 01) . The levels of RONmRNA and RON protein in the COPD rats were also upregulated significantly ( P lt; 0. 01) . MSP evoked the AMs isolated from the normal and COPD rats to generate more content of MDA and caused a reduction in activity of SOD. In addition, MSP stimulated TNF-α, IL-8, IL-1βand IL-10 release fromAMs of the normal and COPD rats dose-dependently. The levels of TNF-α, IL-8, and IL-1βwere higher, while the level of IL-10 and the SOD activity were lower in AMs of the COPD group than those of the control group in the same dose of MSP ( P lt;0. 01) . The more significant increase in the levels of TNF-α, IL-8, IL-1β, and the more notable decrease in the activity of SOD was found in the COPD group compared with the control group. But the degree of increasing MDA and IL-10 in the AMs of the COPD group was lower than that in the control group. Linear correlation analysis showed that the MSP concentration and the RON protein level in the COPD rats were positively associated with the total cellcounts and AM counts in BALF, and were related to the indexes for pulmonary emphysema. Conclusions There is a close correlation between the MSP and receptor tyrosine kinase RON with the airway inflammation of COPD. The mechanism might be that MSP promote the macrophages release inflammatory factors and increase the production of oxygen free radicals.
Objective To explore the effects of several immunosuppressants on the cell numbers of cultured rat macrophages and Schwann’s cells. Methods The macrophages and Schwann’s cells were cultured from the newborn Wistar rats. Different concentrations of methylprednisolone(10-3, 10－4,10－6 and 10－8 mol/L), CsA(10－5, 10－6, 10－7 and 10－8 mol/L) and FK506(10-6, 10－7, 10－8 and 10－9 mol/L) were administrated to the cells, while control group was given no drugs. Twentyfour, 48 and 72 hours after administration, the cells from different concentrations were measured with MTT methods respectively. Theresults were compared and analyzed statistically. Results Only high concentration methylprednisolone (10-4 mol/L) and a certain range of concentrations of CsA (10-6，10-7 and 10-8 mol/L) and FK506 (10-7,10-8 and 10-9 mol/L) can provide protection to culturedrat macrophages. Under most concentrations, CsA and FK506 had no effects onthe cell number of cultured rat Schwann’s cell. Only with high concentration CsA (10-5 mol/L) and methylprednisolone (10-3 mol/L) could significantly decreased the cell number of Schwann’s cell. Long time (72 hours) and low dosage (10-8 mol/L) administration of methylprednisolone could significantlyprotect Schwann’s cell. Conclusion High concentration methylprednisolone and some certain concentration CsA and FK506 can protect cultured rat macrophages. But high concentration CsA and methylprednisolone prohibit the proliferation of Schwann’s cells. Only long time and low dosage methylprednisolonecan protect cultured rat Schwann’s cells.