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.
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.
The synthesis and secretion of inflammatory cytokines in the monocytes of 68 cases of multiple system organ failure (MSOF) patients was investigated by the method of MTT stained in cytokines dependent defferential cell strain. The data showed that the serum levels of tumor necrosis factor, interleukine 1 and interleukine 6 were increased (Ｐ＜0.01) in the monocytes of MSOF patients. The synthesis and secretion of these inflammatory cytokines gradually increased in the monocytes after onset of MSOF. After 5 days of treatment with antibiotics and electrolytes intravenous infusion, the secretion of TNF, IL-1 and IL-6 were decreased respectively. These results suggested that the TNF, IL-1 and IL-6 are integrated into system inflammatory responese and caused the injury to the tissues and organs. The production levels of these cytokines can be regarded as the index of MSOF and its severity.
Objective To investigate the changes of expression of zonula occludens-1(ZO-1) in rats with severe acute pancreatitis (SAP), and to study the relationship between the ZO-1 protein and microvascular injury in rats with SAP. Methods Forty-eight Wistar rats were randomly divided into sham-operation (SO) group and SAP group, each group enrolled 24 rats. Pancreas of rats in SO group were flipped only after laparotomies, but rats of SAP group were injected with 5% sodium taurocholate by retrograding cholangiopancreatography micro pump to produce the SAP model. At 6, 12, and 24 hours after operation, 8 rats were sacrificed to get abdominal aortic blood for testing the levels of peripheral blood amylase, trypsin, interleukin-8(IL-8), tumor necrosis factor-α(TNF-α), and ZO-1 protein. At the same time, pancreatic tissues were got to perform HE staining and immunohistochemical staining for observation of the pathological changes and the expression of ZO-1 protein respectively. Results Compared with SO group at the same time, the levels of peripheral blood amylase, trypsin, IL-8, TNF-α, and ZO-1 protein were all higher in SAP group (P < 0.05). The level of amylase in SAP-24 hours group was higher than those of 6 hours group and 12 hours group(P < 0.05), the levels of trypsin, IL-8, and ZO-1 protein in SAP group increased over time (P < 0.05), but levels of TNF-αin 3 time points of SAP group did not differ with each other significantly(P > 0.05). Results of regression showed that in the SAP group, the level of ZO-1 protein in serum was significantly positive correlated with pathological score of pancreatic tissue(b=0.96, P < 0.05), levels of serum amylase(b=0.87, P < 0.05), trypsin(b=0.72, P < 0.05), and serum IL-8 (b=0.69, P < 0.05), but was not significantly correlated with level of TNF-α(P > 0.05). HE staining results showed that damage of pancreatic tissues became worse over time in SAP group, and the pathological score of SAP-6 hours group was lower than those of 12 hours group and 24 hours group (P < 0.05). Immunohistochemical staining results showed that, in SAP group, with the extension of time, the number of ZO-1 protein granules in pancreatic acinar cells and capillary wall reduced, and expressed in capillaries discontinuously. Conclusion During the course of SAP, the concentration of serum ZO-1 protein increase, but its expression in the pancreatic tissue degrade, which is closely associated with microvascular injury and progression of pancreatic tissues.
ObjectiveTo investigate the role of Wnt5a in the mechanism of radiculopathy and the relation between Wnt5a and tumor necrosis factorα(TNF-α) by observing the change of the expression of Wnt5a in the rat model of chronic compression of dorsal root ganglia (CCD). MethodsA total of 192 adult male Sprague Dawley rats were allocated into 4 groups: shame group (group A, n=48), CCD group (group B, n=48), CCD+sal ine group (group C, n=48), and CCD+etanercept group (group D, n=48). An L-shaped needle (about 3.5 mm in length, 0.6 mm in diameter) was inserted into the L5 intervertebral foramen, and the dorsal root ganglia (DRG) was compressed by the needle to prepare the CCD model in groups B, C, and D, and then normal sal ine (5.5 mg/kg) or etanercept was injected intraperitoneally in groups C and D. The intervertebral foramen was exposed in group A. The mechanical pain threshold of the posterior paw was tested by the von Frey filaments at 1, 3, 5, and 7 days after operation; the expressions of Wnt5a protein and mRNA were detected at 3 and 7 days after operation by immunohistochemical staining and RT-PCR, respectively. ResultsThe mechanical pain threshold of groups B and C was significantly lower than that of groups A and D, and in group D than in group A (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The Wnt5a positive cells and the mRNA expression of Wnt5a at 7 days were significantly more than those at 3 days in groups B, C, and D (P < 0.05). The Wnt5a positive cells and the mRNA expression of Wnt5a in groups B and C were significantly more than in groups A and D, and in group D than in group A (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05). ConclusionThe expression of Wnt5a in the DRG is increased after CCD. The expression of Wnt5a in the DRG is decreased after the administration of the inhibitor of TNF-α.
Objective To investigate the role of vascular endothelial growth factor ( VEGF) in the pathogenesis of emphysema and its relationship with tumor necrosis factor alpha ( TNF-α) . Methods 48 rats were randomly divided into four groups, ie. a normal control group, an emphysema group, a rhTNFR∶Fc intervention group, and a sham intervention group. The rats in the emphysema group, the rhTNFR: Fc intervention group, and the shamintervention group were exposed to cigarette smoking for 80 days. After 30 days of exposure, rhTNFR: Fc hypodermic injection was administered in the rhTNFR: Fc intervention group while placebo was injected in the sham intervention group as control. Lung tissue sections were stained by hematoxylin and eosin. Mean linear intercept ( MLI) and mean alveolar numbers ( MAN) were measured to estimate the extent of emphysema. The level of TNF-αin serumand BALF, and the level of VEGF in BALF were measured with ELISA. Results In the emphysema group, MLI was higher and MAN was lower than those in the normal control group. Moreover, the levels of TNF-αin serum and BALF were higher, and thelevel of VEGF in BALF was lower significantly ( P lt;0. 05) . After the intervention with rhTNFR∶Fc, MAN increased and the serum TNF-αdecreased significantly compared with the emphysema group ( P lt; 0. 05) .However there were no significant differences in MLI, VEGF, and TNF-α in BALF ( P gt; 0. 05 ) . No correlation was found between the level of TNF-αand VEGF in BALF in the emphysema group. Conclusion VEGF and TNF-αare related to the pathogenesis of emphysema of smoking rats, and may contribute to the development of emphysema in different pathways.
Objectve To measure the serum levels of human IL-32 and explore the clincal implication in patients with chronic obstructive pulmonary disease( COPD) at acute exacerbation or stable stage. Methods 120 patients with COPD were recruited, including 60 patients with acute exacerbation COPD and 60 patients with stable COPD from October 2010 to May 2011. Thirty healthy nonsmoking volunteers were included as controls. The concentrations of interleukin-8 ( IL-8) , tumor necrosis factor alpha ( TNF-α) , and IL-32 in serum were measured by enzyme-linked immunosorbent assay ( ELISA) . The correlations among IL-32, IL-8, TNF-αand lung functions were investigated. The datas were analyzed using a statistical software package ( SPSS13. 0) . Variables were compared with one-way ANOVA . The correlations between variables were analyzed using Pearson’s correlation coefficient or Spearman correlation coefficient. Results SerumIL-32 level was significantly higher in AECOPD patients [ ( 174. 56 ±88. 15) ng/L] than that in healthy subjects [ ( 59. 41 ±20. 98) ng/L] and in stable COPD patients [ ( 89. 40 ±33. 84) ng/L]( P lt;0. 05) while serum IL-32 level was also significantly higher in stable COPD patients than in healthy subjects( P lt;0. 05) . The serumIL-32 1evel in patients with acute exacerbation COPD and stable COPD was positively correlated with the serumIL-8 level, TNF-αlevel ( respectively P lt;0. 01) . The serumIL-32 level was negatively correlated with FEV1 /predicted value, FEV1 /FVC and PaO2 ( respectively, P lt;0. 01) . There was no statistical significance of the serum IL-32, IL-8 or TNF-α levels in COPD patients with different severity ( all P gt;0. 05) . Conclusion The serumlevel of IL-32, a newpro-inflammatory cytokine is elevated in COPD patients, which may be involved in the pathogenesis of inflammation in COPD.
Objective To examine the levels of interferon-gamma; (INF-gamma;), tumor necrosis factor-alpha; (TNF-alpha;) and interleukin-6(IL-6) in serum of patients with acute uveitis before and after treatment, and to explore the possible roles of those cytokines in the initiation and progression of the uveitis. Methods A series of 75 patients with acute uveitis,and 30 healthy persons from our hospital were investigated. The levels of INF-gamma;, TNF-alpha; and IL-6 in acute phase and convalescent phase were measured by the enzymelinked immunosorbent assay. Result The serum levels of INF-gamma;, TNF-alpha; and IL-6 in acute phase were significantly higher than that of the convalescent phase and the healthy controls (F=65.805/50.418/155.381, P=0.000). A significant negative correlation was found between the serum levels of INF-gamma;, TNF-alpha; and IL-6 in acute phase with their initial visual acuity(r=-0.656, -0.592 and -0.653, Plt;0.01). There was also a positive correlation among the serum levels of INF-gamma;, TNF-alpha; and IL-6(r=0.340, 0.467 and 0.338, Plt;0.05). Conclusions There are high serum levels of INF-gamma;, TNF-alpha; and IL-6 in patients with acute uveitis, and the cytokines levels were decreased after the treatment. The results suggested that the INF-gamma;, TNF-alpha; and IL-6 involved in initiation and progression of uveitis.