Objective To investigate the protective effects of antitumor necrosis factor-α antibody （TNF-αAb） on lung injury after cardiopulmonary bypass （CPB） and their mechanisms. Methods Forty healthy New Zealand white rabbits，weighting 2.0-2.5 kg，male or female，were randomly divided into 4 groups with 10 rabbits in each group. In groupⅠ，the rabbits received CPB and pulmonary arterial perfusion. In group Ⅱ，the rabbits received CPB and pulmonary arterial perfusion with TNF-αAb. In group Ⅲ，the rabbits received CPB only. In group Ⅳ，the rabbits only received sham surgery. Neutrophils count，TNF-α and malondialdehyde （MDA） concentrations of the blood samples from the left and right atrium as well as oxygenation index were examined before and after CPB in the 4 groups. Pathological and ultrastructural changes of the lung tissues were observed under light and electron microscopes. Lung water content，TNF-α mRNA and apoptoticindex of the lung tissues were measured at different time points. Results Compared with group Ⅳ，after CPB，the rabbitsin group Ⅰ to group Ⅲ showed significantly higher blood levels of neutrophils count，TNF-α and MDA（P＜0.05），higherTNF-α mRNA expression，apoptosis index and water content of the lung tissues （P＜0.05），and significantly lower oxyg-enation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with group Ⅱ，after CPB，the rabbits in groups Ⅰ and Ⅲ had significantly higher blood concentrations of TNF-α （5 minutes after aortic declamping，220.43±16.44 pg/ml vs.185.27±11.78 pg/ml，P＜0.05；249.99±14.09 pg/ml vs.185.27±11.78 pg/ml，P＜0.05），significantly higher apoptosis index （at the time of CPB termination，60.7‰±13.09‰ vs. 37.9‰±7.78‰，P＜0.05；59.6‰±7.74‰ vs. 37.9‰±7.78‰，P＜0.05），significantly higher blood levels of neutrophils count and MDA （P＜0.05），significantly higher TNF-α mRNA expression and water content of the lung tissues （P＜0.05），and significantly loweroxygenation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with groupⅠ，rabbits in group Ⅲ had significantly higher above parameters （P＜0.05） but lower oxygenation index （P＜0.05） only at 30 minutes after the start of CPB. Conclusion Pulmonary artery perfusion with TNF-αAb can significantly attenuate inflammatory lung injury and apoptosis of the lung tissues during CPB.
ObjectiveTo establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene, and explore the mechanism of GSTM5 nuclear translocation. MethodsRecombinant lentiviral expression vector containing GSTM5 gene was constructed and lentivirus was produced. After lentivirus infection of 16HBE cells, 16HBE-GSTM5 cell lines were obtained by screening with puromycin. Expression of GSTM5 in different cells was examined by RT-qPCR and Western blot. The nuclear translocation of GSTM5 was observed by confocal laser scanning microscope, after the 16HBE-GSTM5 cell lines were treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) for 0.5 hour. ResultsLentiviral expression plasmids, PLVX-puro-3*flag-SBP-GSTM5-C and PLVX-puro-GSTM5-SBP-3*flag-N, were constructed and lentiviral particles were successfully packed. After infected with lentivirus and screened by puromycin, two cell lines, 16HBE-GSTM5-SBP-3*flag-N and 16HBE-3*flag-SBP-GSTM5-C, were obtained. GSTM5 expression in these two cell lines was significantly higher compared with the control group and parental cells. After treated with TNF-α for 0.5 hour, the nuclear translocation of GSTM5 in 16HBE-GSTM5-SBP-3*flag-N was much more obviously than that in 16HBE-3*flag-SBP-GSTM5-C. ConclusionThe N-terminal region of GSTM5 is critical for nuclear translocation induced by TNF-α, which is mediated by a novel and non-classical nuclear localization signal.
【Abstract】ObjectiveTo explore the changes of colon motility of the rats in multiple organ dysfunction syndrome （MODS） induced bacterial peritonitis and the effects of IL6, TNFα and induce nitricoxide synthase (iNOS) on colon motility. MethodsWistar rats were divided into two groups, which were the control group and the MODS group. The number of stool, the amplitude changes of circular smooth muscle strip, the length of smooth muscle cell, and the changes of serum NO in two groups were observed. The expressions of IL6, TNFα and iNOS protein and IL6 mRNA, TNFα mRNA and iNOS mRNA in distal colon were investigated by using immunohistochemical methods and RTPCR. ResultsThe numbers of stool and the amplitude in the MODS group were lower than those of the control group （P＜0.05）. The expressions of IL6, TNFα and iNOS were negative in the control group, while they were positive in the MODS group. IL6 mRNA,TNFα mRNA and iNOS mRNA were negative expression in the control group, but they were positive expression in the MODS group. The concentration of serum NO and the length of smooth muscle cells in the MODS group were higher than those of the control group (P＜0.01). ConclusionColon motor dysfunction of the rats is related to the iNOS, IL6 and TNFα.
Objective To investigate the protective effects of endotoxin pretreatment on lung injury of rats with endotoxemia. Methods The rat model of acute endotoxemia was established by injecting lipopolysaccharide (LPS) intraperitoneally. Seventy-two male Wistar rats were randomly divided into three groups, ie. a saline control group (N, n=24) , a LPS-treated group (L, n=24) , and a LPS pretreated group ( P, n=24) . Each group was divided into 2 h, 4 h, 6 h, and 12 h subgroups. The rats in group P were firstly administered with introperitoneal injection of 0.25 mg/kg LPS. After 24 hours, they were subjected to the injection of 0.5 mg/kg LPS. The rats in group N and L received injection of equivalent amount of saline. After 72 hours, the rats in group L and P were challenged with intravenous injection of 10 mg/kg LPS, otherwise saline in group N. Six rats were killed at 2, 4, 6 and 12 hours respectively after injection of LPS in group L and P. The lungs were removed for detecting intercellular adhesion molecule-1 ( ICAM-1) , superoxide dismutase ( SOD) , and malondialdehyde (MDA) . Meanwhile the level of tumor necrosis factoralpha ( TNF-α) in serum was measured, and the pathological changes of lung were also examined. Results The contents of ICAM-1, MDA and TNF-α in the LPS-treated 4 h group were 75.07 ±0. 53, ( 3.93 ± 0.42) μmol/g, and (478.62 ±45.58) pg/mL respectively, significantly higher than those in the saline control group. The endotoxin pretreatment reduced the above indexes to 42.40 ±0.44, ( 2.89 ±0.49) μmol / g and ( 376.76 ±43.67) pg/mL respectively (Plt;0.05) . The content of SOD in the LPS-treated 4 h group was ( 6.26 ±0.31) U/mg, significantly lower than that in the saline control group. The endotoxin pretreatment increased SOD to ( 8.79 ±0.35) U/mg. Conclusion Endotoxin pretreatment can suppress the progress of lung injury in rats with endotoxemia and protect the lung tissue by down-regulating the inflammatory response and oxygen free radical production.
Objective To investigate the effects of ginkgo biloba extract （GBE） on expressions of IL-1β, IL-6,and TNF-α in the pancreas and brain tissues of rats with severe acute pancreatitis （SAP）, and further to explore the pathogenesis of SAP and the efficacy of GBE on brain injury. Methods Fifty-four Winstar rats were randomly divided into normal control group, model group, and treatment group, with 18 rats for each group. For rats in the normal control group, only conversion of pancreas was performed by abdomen opening , followed by wound closure immediately. For rats in the model group and treatment group, 5% sodium taurocholate hydrate were injected under pancreatic capsule to establish SAP model, and then GBE and normal saline were infected into intra-abdomen repeatedly every 8 hours, respectively. At 6 h, 12 h, and 24 h after the model establishment, experimental samples were extracted and serum amylase was detected. Pathogenic scoring for pancreas tissues was performed under light microscopy, and immunohistochemistry method was employed to detect the expression levels of IL-1β, IL-6, and TNF-α in pancreas and brain tissues. Results For the treatment group, both serum amylase and pancreas scoring were significantly lower than those of the model group （P＜0.01）. At 24 h after model establishment, the expressions of IL-1β, IL-6, and TNF-α of pancreas tissues in model group were significantly higher than those at 6 h and 12 h （P＜0.05 or P＜0.01）, but no significant differences wereobserved in treatment group （P＞0.05）. The expressions of IL-1β, IL-6, and TNF-α of brain tissues in model group were significantly higher than those at 6 h and 12 h （P＜0.05 or P＜0.01）, but in treatment group decreased （P＜0.05 or P＜0.01）. The expressions of IL-1β, IL-6, and TNF-α in the treatment group were significantly lower than those of the model group at same time （P＜0.01）. Conclusions During SAP, the expressions of IL-1β, IL-6 and TNF-α in pancreas and brain tissues increased obviously. GBE showed suppressing and scavenging effects on IL-1β, IL-6 and TNF-α in pancreas and brain tissues.
【Abstract】ObjectiveTo investigate the effect of Salvia Miltiorrhiza (SM) and Shengmai injection （SI） in treating systemic inflammatory response syndrome (SIRS) and their mechanism. Methods The animal model of SIRS was established by injectinglipopolysaccharide(LPS, 1 mg/kg)intraperitoneally. Forty Wistar rats were randomly divided into four groups: control group, SM group, SI group and combined treatment group (SM+SI group), which were treated with normal saline（5 ml/kg） plus LPS（1 mg/kg）, SM（5 ml/kg）plus LPSKG4（1 mg/kg）, SI（5 ml/kg）plus LPS（1 mg/kg）, SM（2.5 ml/kg） plus SI（2.5 ml/kg） and LPS（1 mg/kg） respectively. Six rats of each group were sacrificed for sample collection of blood, liver, lung and kidney 8 hours after LPS injection. Blood routine, serum TNF-α and IL-6 were measured. Specimen of organs were fixed in formalin and sent for routine pathological examination. The survival of other 4 rats of each group were observed untill 48 hours after LPS injection. SPSS 10.0 was used in statistical analysis. Results Two rats in control group died 13 hours and 22 hours after LPS injection respectively, the remaining 2 rats in this group and the rats in other 3 groups survived 48 hours after LPS injection. The white blood cell count of control group was significantly higher than that of other groups. The serum TNF-α and IL-6 of control group were significantly more than those of other groups. Pathological damages were found in all groups, and the most severe ones were in control group. SM and SI could decrease the level of serum TNF-α and IL-6 in the process of LPS-stimulated SIRS, down-regulate the severe inflammatory response, attenuate organ damages of the liver, lung and kidney, and increase forty-eihgt-hour survival rate obviously. Conclusion The experiment provides a theoretical base for clinical use of SM and SI in treatment of SIRS.