Objective To investigate the transduction pathway of TREM-1 during endotoxininduced acute lung injury ( ALI) in mice through the specific activating or blocking TREM-1.Methods 40 mice were randomly divided into a saline control group, an ALI group, an antibody group, and a LP17 group ( 3.5 mg/kg) . All mice except the control group were intraperitoneally injected with lipopolysaccharide ( LPS) to establish mouse model of ALI. Two hours after LPS injection, anti-TREM-1mAb ( 250 μg/kg) was intraperitoneally injected in the antibody group to activation TREM-1, and synthetic peptide LP17 was injected via tail vein in the LP17 group to blocking TREM-1. After 6,12,24, 48 hours, 3 mice in each group were sacrificed for sampling. The expression of NF-κB in lung tissue was determined by immunohistochemistry. The levels of TNF-α, IL-10, TREM-1, and soluble TREM-1 ( sTREM-1) in lung tissue and serumwere measured by ELISA. Pathology changes of lung were observed under light microscope, and Smith’s score of pathology was compared. Results Administration of anti-TREM-1mAb after ALI modeling significantly increased the NF-κB expression in lung tissue at 48h, resulting in a large number of pro-inflammatory cytokines releasing in the lung tissue and serumand lung pathology Smith score increasing. Administration of LP17 after modeling significantly down-regulated the expressions of NF-κB and pro-inflammatory cytokines, while led to a slight increase of anti-inflammatory cytokines and a decline of lung pathology Smith’s score.Conclusion TREM-1 may involve in inflammatory response by promoting the generation of inflammatory factors via NF-κB pathway, thus lead to lung pathological changes in ALI.
【Abstract】 Objective To study the effects of ischemic preconditioning (IP) on the activity of nuclear factor-κB （NF-κB） and the expressions of TNF-α and intercellular adhesion molecule-1 (ICAM-1) during early reperfusion following liver transplantation in rats. Methods The models of rat orthotopic liver transplantation were established. The donor livers were stored for 2 hours in Ringers solution at 4 ℃ before transplantation. All rats were randomly divided into sham operation group （SO group）， control group and IP group. IP group was achieved by clamping the portal vein and hepatic artery of donor liver for 10 minutes followed by reperfusion for 10 minutes before harvesting. The activity of NF-κB and expressions of TNF-α and ICAM-1 at 1 h, 2 h, 4 h and 6 h after reperfusion were measured. Serum ALT, LDH were also determined. Results The liver function of recipients with IP were significantly improved. Compared with SO group， the graft NF-κB activity increased after transplantation in control group and IP group (P＜0.05), while compared with control group that was significantly attenuated at 1 h and 2 h in IP group. Similarly, hepatic levels of TNF-α and ICAM-1 were significantly elevated in control group and were reduced in IP group. Conclusion IP might down-regulated TNF-α and ICAM-1 expression in the grafts after orthotopic liver transplantation through depressed NF-κB activation， and attenuate neutrophil infiltration in the grafts after reperfusion.
Objective To investigate the role of alveolar macrophages ( AMs ) in airway inflammation of smoke-induced COPD rat model and its possible regulating mechanism. Methods Twelve Wistar rats were randomly divided into a COPD group and a control group. The rat model of COPD was established with smoke exposure and LPS intrathacheal instillation. Bronchoalveolar lavage fluid ( BALF)was collected for measurement of total and differential cell counts. Then AMs were isolated and identified byimmunofluorescence. Western blot was employed to analyze the cytoplasmic and nuclear NF-κB p65 expression of AMs. The concentrations of TNF-α,macrophage inflammatory protein 2 ( MIP-2) and IL-10 in cell culture supernatantwere assayed by ELISA.Results The scores of bronchitis and mean liner intercepts in the COPD group were significantly higher than those in the control group [ 4. 33 ±1. 16 vs. 1. 33 ±0. 58,P =0. 016; ( 168. 77 ±11. 35) μm vs. ( 93. 61 ±4. 16) μm, P = 0. 000) ] . The total cell count in BALF of the COPD group was significantly higher than that in the control group ( P lt; 0. 05) , and the AMs and neutrophils were predominant [ ( 72. 00 ±2. 22) % and ( 18. 29 ±8. 34) % ] . The cytoplasmic NF-κB p65 expression of AMs in the COPD group was significantly lower , while the nuclear NF-κB p65 expression was significantly higher ( P lt; 0. 05) compared with the control group. The ELISA results showed that the concentrations of TNF-αand MIP-2 in culture supernatant of AMs in the COPD group were significantly higher than those in the control group ( P lt;0. 05) , while the concentration of IL-10 was not significantly different between the two groups ( P gt;0. 05) . Conclusions COPD rat model was established successfully with smoke exposure and LPS intratracheal instillation with a profile of macrophage-based chronic inflammation and increased secretion of TNF-αand MIP-2. The mechanismis closely related to activation of NF-κB.
ObjectiveTo explore the protective mechanism and effect of the resveratrol for kidney injury of obstructive jaundice. MethodsThe rats were randomly divided into three groups: sham operation group receiving laparotomy without bile duct ligation (BDL), the obstructive jaundice group with BDL, and the obstructive jaundice + resveratrol group given resveratrol following BDL. The levels of total bilirubin (TBIL), direct bilirubin (DBIL), blood urea nitrogen (BUN), and creatinine (Cr) in the serum were tested. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione (GSH) level in the renal tissues were detected. The expressions of the silent information regulator 1 (SIRT1) and nuclear factor-κB (NF-κB) proteins were tested by Western blot. The expression of SIRT1 mRNA was detected by RT-PCR and the renal cell apoptosis was examined by TUNEL staining. Results①Compared with the sham operation group, the levels of serum TBIL, DBIL, BUN and Cr were significantly higher (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were signi-ficantly lower (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly higher (P < 0.05) in the obstructive jaundice group.②Compared with the obstructive jaundice group, the levels of serum TBIL, DBIL, BUN and Cr were significantly lower (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were significantly higher (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly lower (P < 0.05) in the obstructive jaundice+resveratrol group. ConclusionThe resveratrol could alleviate renal damage and play a beneficial role to resist inflammation, oxidation, and apoptosis by activating the SIRT1 which probably inhibits the expression of NF-κB protein and promotes the activity of SOD in cholestatic kidney injury.
Objective To construct the mouse NF-κB P65 subunit expression plasmid, and identify its biological activity. Methods NF-κB P65 siRNA retrovirus expression vectors were reconstructed by molecular clone technology. Recombinant vectors were transfected into 293E package cells and virus suspension was collected. RT-PCR was used to detect the expression level of NF-κB P65 mRNA and TNF-α mRNA at different time-point of LPS stimulation. Western blot was performed to analyze the protein level of NF-κB P65. ELISA was applied to detect the expression level of TNF-α released by LPS-stimulated J774A.1. Results NF-κB P65 siRNA retrovirus expression vectors of mouse were successfully constructed. From2 hours after the stimulation of LPS, the expression level of NF-κB P65 mRNA of the siRNA group was obviously lower than the scramble control group ( 0.91 ±0.03 vs. 1.02 ±0.02, Plt;0.01) . At24,36, 48 and 72 hours after the LPS stimulation, the expression level of NF-κB P65 protein of the siRNA group was significantly decreased compared with the scramble control group ( 0.97 ±0.02 vs. 1.01 ±0.01, 0.94 ± 0.01 vs. 1.02 ±0. 01,0.94 ±0.02 vs. 1.02 ±0.01, 0.93 ±0.01 vs. 1.00 ±0.02, Plt;0. 05) . At 2, 6, 12, 24 hours after the LPS stimulation, both the expression level of TNF-α mRNA and the content of TNF-α in the culture medium supernatant of the siRNA group were lower than the scramble control group ( Plt;0. 01) . Conclusions The construction of NF-κB P65 siRNA retrovirus expression vectors is feasible. Inflammation factors in mouse monocyte-macrophages are significantly inhibited after NF-κB expression is depressed by RNA interference technology, which may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.
Objective To investigate the expressions of ubiquitin-proteasome markers，including E2-14K，MAFbx，MuRF-1，and nuclear factor-κB（NF- κB） p50，in diaphragm of COPD rats，and their relationship with IL-17 level in diaphragm and serum in order to elucidate the potential mechanism of diaphragm atrophy. Methods Thirty healthy adult male SD rats were randomly divided into a model group （n=18） and a normal control group （n=12）. The COPD rat model was established by instillation of lipopolysaccharide （LPS） and exposure to cigarette smoke for 28 days. The protein levels of E2-14K，MAFbx，MuRF-1，and NF-κB p50 in diaphragm were measured by Western blot. The concentration of IL-17 in serum and diaphragm was measured by ELISA. Results Western blot showed that the protein expressions of E2-14K，MAFbx，MuRF-1，and NF-κB p50 in diaphragm increased significantly in the COPD model group compared with the normal control group （0.96±0.12 vs. 0.53±0.09，0.99±0.10 vs. 0.53±0.08，0.95±0.08 vs. 0.51±0.16，1.11±0.10 vs. 0.64±0.50，respectively，Plt;0.01）. The IL-17 level in serum and diaphragm was significantly higher in the COPD group than the control group. The expression of NF-κB p50 was positively correlated with E2-14K，MAFbx，and MuRF-1 expressions （r=0.82，0.92，0.86，respectively，Plt;0.01）. Both in serum and diaphragm，IL-17 level was positively correlated with the percentage of neutrophils，levels of NF-κB p50，E2-14K，MAFbx，and MuRF-1 expressions（all Plt;0.01）. The IL-17 levels in serum and diaphragm were also positively correlated each other （r=0.84，Plt;0.01）. Conclusions The results show that the ubiquitin-proteasom pathway，the NF-κB pathway and IL-17 are up-regulated in diaphragm of COPD rats .These alterations may contribute to diaphragm atrophy in COPD.
【 Abstract 】 Objective To investigate the protective effect of peroxisome proliferator-activated receptor γ (PPAR γ ) activator 15-deoxyprostaglandin J2 (15d-PGJ2) in rat hepatic ischemia-reperfusion injury and its mechanism. Methods The models of 70% warm ischemia-reperfusion injury were established in SD rats, rats were randomly divided into 4 groups: sham operation group, ischemia-reperfusion group, 15d-PGJ2 group and 15d-PGJ2+GW9662 group. After reperfusion, serum AST and ALT levels were determined; the liver tissues were removed for measurement of activity of NF-κB and myeloperoxidase (MPO), TNF-α content and expression of ICAM-1. Results Compared with sham operation group, the serum levels of ALT and AST, and the activities of MPO and NF- κ B, TNF- α content and expression of ICAM-1 in ischemia-reperfusion group, 15d-PGJ2 group and 15d-PGJ2+GW9662 group were greatly improved (P ＜ 0.05). Compared with ischemia-reperfusion group, the serum levels of ALT and AST and the activities of MPO and NF- κ B, TNF- α content and expression of ICAM-1 in 15d-PGJ2 group were significantly decreased (P ＜ 0.05). Compared with 15d-PGJ2 group, the serum levels of ALT and AST, and the activities of MPO and NF- κ B, TNF- α content and the expression of ICAM-1 in 15d-PGJ2+GW9662 group were obviously increased (P ＜ 0.05). Conclusion PPAR γ activator 15d-PGJ2 could protect against ischemia-reperfusion injury in rats, with its possible mechanism of inhibiting NF-κB activation and down-regulating TNF-α content and ICAM-1 expression in a PPARγ dependent fashion.
ObjectiveTo explore the role of osteopontin (OPN) in hyperoxia-induced acute lung injury and its relationship with nuclear factor-κB (NF-κB),matrix metalloproteinase 2 and 9 (MMP-2,MMP-9). MethodsNinety-six mice were randomly divided into a phosphate buffer solution intranasal inhalation group (PBS group) and a recombinant OPN intranasal inhalation group. The mice were exposed in sealed cages >95% oxygen for 24-72 hours to induce lung injury or room air as control. The severity of lung injury was evaluated. The expression of NF-κB,MMP-2,MMP-9,TIMP-1 and TIMP-2 mRNA in lung tissue at 24,48 and 72 hours under hyperoxia were examined by reverse transcript-polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was performed for detection of NF-κB protein in lung tissue. ResultsPBS group mice developed more severe acute lung injury at 72 hours under hyperoxia.TIMP-1 and TIMP-2 mRNA expressions were significantly increased in r-OPN group than their matched PBS group when exposed to hyperoxia. IHC study showed higher expression of NF-κB protein in lung tissue of PBS group at 72 hours of hyperoxia. ConclusionOPN can protect against hyperoxia-induced lung injury by inhibiting the expressions of NF-κB,MMP-2 and MMP-9.
ObjectiveTo explore the mechanism of lung injury in Sprague-Dawley (SD) rats induced by acute organic phosphorus pesticides (AOPP) by observing the changes of the blood serum nuclear factor (NF)-κB consistence, NF-κB level of lung tissue and lung coefficient. MethodNinety-six healthy male SD rats (six weeks old) were randomly divided into group A (control, n=48) and group B (poison, n=48). The rats of group B were given omethoate by gavage (45 mg/kg), and the rats of group A accepted normal saline. Then the rats were killed at designated observing points (30 minutes; 3, 6, 12, 24, and 48 hours), and the lung coefficient, blood serum NF-κB consistence and NF-κB level of lung tissue were measured. At the same time, we observed the pathological changes of the rats' lung tissue. ResultsCompared with group A, blood serum NF-κB consistence, NF-κB level of lung tissue and the level of lung coefficient in group B were significantly higher (P<0.01). The lung tissues of group A were normal at each time point, but in group B, the lung pathological changes gradually appeared 30 minutes later with pulmonary interstitial engorging, alveolar septum widening and some alveolus being full of red blood cells, and this situation reached its peak at hour 12. Then it gradually mitigated from 24 to 48 hours. ConclusionThere are significant increases in blood serum NF-κB consistence and NF-κB level in lung tissues in rats with lung injury induced by omethoate poisoning. The NF-κB may play a role in the process of lung injury induced by organophosphorus pesticide.
Objective To observe the effects of exogenous pulmonary surfactant (PS) on ventilation-induced lung injury (VILI) in rats, and to investigate its possible mechanisms. Methods A total of 40 Wistar rats were divided into 4 groups with randomized blocks method: control group, high tidal volume (HV) group, VILI group, and PS group, with 10 rats in each group. The control group was subjected to identical surgical procedure but was never ventilated. After 30 min of mechanical ventilation (MV) with Vt 45 ml/kg, the rats in HV group were killed immediately; rats in the VILI group were continually ventilated for up to 150 min with Vt 16 ml/kg; in the PS group, 100 mg/kg of PS administered intratracheally and with the same settings as VILI group. Mean artery pressure (MAP), blood gas analysis, lung wet to dry weight ratios (W/D), thorax-lung compliance, and cell counts in bronchoalveolar lavage fluid (BALF) were determined. Nuclear factor-κB(NF-κB) activity in lungs was measured by enzyme-linked immunosorbent assay (ELISA), interleukin-8(IL-8) in serum and BALF was determined by radioimmunoassay (RIA). Pathological examination of the lung was performed. Results Injurious ventilation significantly decreased MAP and PaO2/FiO2, but increased NF-κB activity and W/D. MAP and PaO2/FiO2 improved, but NF-κB activity, IL-8 in serum and BALF, and cell counts in BALF reduced significantly in PS group compared with those in VILI group. Histological studies showed reduced pulmonary edema and atelectasis in the PS group. Conclusion PS administered intratracheally can suppress the increased activity of NF-κB induced by VILI, exogenous PS can be used to treat VILI.