Objective To detect the effects of cytokines on the expression of early growth response gene-1 (Egr-1) in cultured human retinal pigment epithelial (RPE) cells. Methods Immunofluorescence staining, Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were used to detect and quantitatively analyze the expression of Egr-1 protein and mRNA in cultured human RPE cells which were exposed to stimulants, including 20 mu;g/ml lipopolysaccharide (LPS), 40 ng/ml tumor necrosis factor (TNF)-alpha;, 10 U/ml interferon (IFN)gamma;, 30% supernatant of monocyte/macrophage strain (THP1 cells) and the vitreous humor from healthy human eyeballs, for 0, 10, 20, 30, 40 and 60 minutes, respectively. Results The RPE cells stimulated for 0 minute revealed faint green fluorescence of Egr-1 in the cytoplasm. With exposure to the stimulants, the expressionof Egr-1 increased obviously and b green fluorescence was found in cytoplasm in some nuclei of RPE cells. Compared with the untreated RPE cells, after stimulated by 20 mu;g/ml LPS, 40 ng/ml TNFalpha;, 10 U/ml IFNgamma;, 30% supernatant of THP-1 cells and the vitreous humor, the approximate ultimate amplitudes of Egr-1 mRNA enhanced 1.9, 1.3, 14, 1.2, and 1.4 times, respectively; the greatest amplitudes of Egr-1 protein increased 3.4, 1.2, 1.7, 32, and 1.3 times, respectively. Conclusion LPS, TNF-alpha;, IFN-gamma;, supernatant of THP-1 cells and the vitreous humor can upregulate the expression of Egr-1 mRNA and protein in cultured human RPE cells, and induce its nuclear transposition, which suggests the activation of Egr-1.
Objective To investigate whether ADAM33 ( A disintegrin and metalloproteinase 33) gene polymorphismhas effect on the airway inflammation of COPD. Methods A total of 312 COPD patients were recruited for this study. Four polymorphic loci ( T2, T1, S2, and Q-1) of ADAM33 were selected for genotyping by using the polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP) method. Total and differential cell counts, contents of TNF-α, IL-6, IL-8, and VEGF in induced sputumwere detected. The relationship between genotypes and inflammatory reaction was analyzed. Results On locus T2, the cell counts and content of TNF-αin induced sputum increased significantly in the carriers with GG genotype than those with AA and AG genotypes ( Plt;0.01 and Plt;0.05) . On locus T1, the lymphocyte counts in induced sputumincreased significantly in the carriers with GG genotype than those with AA and AG genotypes ( Plt;0.05) ; but the content of IL-8 in induced sputumwas higher in AA and AG genotypes ( Plt;0.05) . On locus Q-1, the contents of VEGF and IL-8 in induced sputum increased significantly in the carriers with GG genotype than those with AA and AG genotypes (Plt;0.05) . On locus S2, the total cell counts in induced sputumincreased significantly in the carriers with GG genotype than those with CC and CG genotypes ( Plt;0.05) , and the content of IL-8 in induced sputum increased significantly in GG genotype ( Plt;0.01 ) . Conclusion These results suggest that ADAM33 polymorphism may participate the pathogenesis of COPD by promoting airway inflammation.
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.
ObjectiveTo investigate the effects of transforming growth factor-β (TGF-β) in choroidal neovascularization (CNV) induced by laser in mice. Methods Eighty male C57BL/6J mice at the age of 6-8 weeks old were randomly divided into the normal control, photocoagulation model, photocoagulation with phosphate buffered saline (PBS control group) and photocoagulation with TGF-β receptor inhibitor groups (TGF-β receptor inhibitor group), twenty mice of each group. Fundus argon laser photocoagulation was performed in the photocoagulation model group, PBS control group and TGF-β receptor inhibitor group to induce CNV. One week, two, three and four weeks after the laser procedure, fundus fluorescein angiography (FFA) was carried out in the normal control or photocoagulation model groups to observe CNV formation dynamically. Western blot was used to analyze the expressions of TGF-β in the retina from the mice of normal control or photocoagulation model groups, and VEGF or TNF-α in the retina of normal control, PBS control or TGF-β receptor inhibitor groups. The CNV areas of each group were evaluated by using fluorescein stain on retinal pigment epithelium (RPE)/choroid flat mounts after two weeks of photocoagulation. ResultsThe FFA results showed the retinal vessels centered on the optic disc and arranged radially, while the choroidal vascular present network distribution in the normal control mice. Significant leakage of fluorescein showed discoid strong fluorophore in photocoagulation sites of retina at one week after photocoagulation. The quantitative analysis results of Western blot demonstrated that the TGF-β protein expression levels in retina of photocoagulation model mice gradually increased with time passing. The protein expression levels of TGF-β were significant differences in the photocoagulation model group comparing with the normal control group (F=13.042, P < 0.05). The protein expression levels of TNF-α (F=14.721, 17.509) and VEGF (F=18.890, 11.251) increased significantly in retina of PBS control or TGF-β receptor inhibitor groups when compared with that of normal control group at one week, two, three and four weeks after photocoagulation, and the differences were both statistically significant (P < 0.05). Compared with PBS control group, the protein levels of TNF-α and VEGF in retina from TGF-β receptor inhibitor group were significantly reduced, the differences was statistically significant (F=21.321, 16.160, P < 0.05). Two weeks after laser photocoagulation, a distinct reduction in CNV lesion size in the TGF-β receptor inhibitor group mice when compared to PBS or normal control groups, the differences was statically significant (F=4.482, P < 0.05). ConclusionTGF-β may promote CNV formation by up-regulating both TNF-α and VEGF protein expressions, the application of its specific inhibitor is able to reduce CNV progression.
ObjectiveTo observe the expression of vascular endothelial growth inhibitor (VEGI, TL1A), vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in diabetes rats' serum, vitreous and retina, and discuss the role of VEGI in the pathogenesis of diabetic retinopathy (DR). MethodsA total of p70 adult male Wistar rats were randomly divided into 4 groups, the control group (10 rats), the diabetes mellitus (DM) 1 month group (20 rats), the DM 3 month group (20 rats) and the DM 6 month group (20 rats). Cytokines of serum and vitreous were determined by enzyme-linked immunosorbent assay (ELISA), and the concentrations of the cytokines in the retina were determined by immunohistochemistry on paraffin retinal sections. Hematoxylin-eosin (HE) staining of retina was used to estimate the pathological change of DR. The results were analyzed by one-way analysis of variances, independent samples t-test and LSD test. ResultsThe serum TL1A levels of the control group, the DM 1 month group, the DM 3 month group and the DM 6 month group rats were (92.09±2.05), (118.36±8.30), (85.90±7.51) and (78.90±4.88) ng/L respectively, the level of TL1A in serum of the DM 1 month group, the DM 3 month group and the DM 6 month group were significantly lower than that of the control group (F=77.405, P < 0.05). The concentration of serum TNF-α and IL-1β increased after DM model was established (F=3.508, 15.416; P < 0.05); the VEGF level in serum showed no difference between the groups (F=1.242, P > 0.05). The vitreous TL1A levels of the control group, the DM 1 month group, the DM 3 month group and the DM 6 month group were (91.50±8.18), (67.03±6.74), (47.44±4.92) and (46.01±4.62) ng/L respectively, every DM groups showed significant difference with the control group (F=114.777, P < 0.05); VEGF level in vitreous increased from 1 month after DM model was established (F=8.816, P < 0.05); TNF-α and IL-1β level in vitreous also showed an upward tendency (F=4.392, 3.635; P < 0.05). Paraffin section immunohistochemistry showed that the absorbance (also called optical density) of TL1A of the DM 1 month group and the DM 3 month group were significantly lower than that of the control group (t=6.851, 6.066; P < 0.05), but the DM 6 month group showed no difference with the control group (t=1.401, P > 0.05); the level of VEGF and TNF-α in DM groups were higher than that of the control group (tVEGF=-4.709, -16.406, -9.228; tTNF-α=-4.703, -6.583, -17.762; P < 0.05); the level of IL-1β were significantly higher in the DM 1 month group and the DM 6 month group (t=-4.108, -3.495; P > 0.05); but the DM 3 month showed no difference with the control group (t=-0.997, P > 0.05). HE staining of retina showed that the retina of the control group and the DM 1 month group had normal retinal structures, the DM 3 month group had retinal edema and disorganization, the DM 6 month group had severe retinal edema, deep stain of ganglion cells, and more neovascularization in inner plexiform layer. ConclusionVEGI is involved in the pathogenesis of DR, and it might interacts with VEGF, TNF-α and IL-1β to affect the development of DR.
Uveitis is the most common extra-articular manifestation of juvenile idiopathic arthritis, typically as chronic anterior uveitis with insidious onset. Delayed and inadequate treatment may result in loss of patients' vision and even blindness. For refractory or severe uveitis related to juvenile idiopathic arthritis, systemic immunosuppressive agents should be used as early as possible. With the advantage of controlling ocular inflammation, avoiding ocular complications and reducing the use of traditional immunosuppressant drugs and glucocorticoid, tumor necrosis factor-α inhibitors have been new therapeutic options for uveitis associated with juvenile idiopathic arthritis, although methotrexate is known as the first-line approach. However, there are no internationally unified guidelines for clinical issues regarding the timing of application, reduction and withdrawal of tumor necrosis factor-α inhibitors, and no agreement on the application of tumor necrosis factor-α inhibitors in the management of ocular complications either. An in-depth understanding of the application status and progress of tumor necrosis factor alpha inhibitors in the treatment of juvenile idiopathic arthritis-associated uveitis has important clinical significance.
Objective To investigate the effect of aerosolized perfluorocarbon (PFC) (FC77) on gas exchange，histopathological changes of lung in acute lung injury and pulmonary expression of tumor necrosis factor-α (TNF-α) mRNA.Methods After acute lung injury (ALI) was induced by oleic acid (OA)，16 rabbits were assigned randomly into 2 groups,ie.aerosolized perfluorocarbon group (PFC group) and conventional mechanical ventilation group (CMV group).Gas exchange parameters were measured before and after ALI，at 1，2，3，4 h after treatment.Histological sections taken from 6 different parts of lung were stained by hematoxylin and eosion.The express of TNF-α mRNA in the 2 different parts of lung were detected by in situ hybridization (ISH).Results Compared with CMV group，the PaO2 and static lung compliance (CLst) were significantly increased (Plt;0.05)，the histopathological lesions of lung were attenuated，and the TNF-α mRNA expression was decreased significantly in PFC group (all Plt;0.05).There was more expression of TNF-α mRNA in backside than that in foreside of lung in two groups (Plt;0.05).Conclusion Aerosolized perfluorocarbon (PFC) can decrease expression of tumor necrosis factor-α mRNA in the lung，and improve the CLst and oxygenation during acute lung injury.
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.
【Abstract】Objective To investigate the potential role of tumor necrosis factoralpha (TNFα) in apoptosis after combined liver and kidney transplantation in rats. MethodsEighty rats which had combined liver and kidney transplantation were randomly paired, were divided into study group (n=20) and control group (n=20). 40 ml of 4 ℃ sodium chloride and antiTNFα monoclonal antibody (30 ml was infused from portal veins to donated livers and 10 ml from renal arteries to donated kidneys) were infused to the study group (0.1 mg/kg weight),and the same quantity of 4 ℃ sodium chloride was infused the control group. Venous blood was drew at different phases after the transplantations to detect the function of kidney and liver. The level of TNFα and the cell apoptosis were detected in the transplanted tissues of liver and kidney by ELISA and terminal deoxynucleotidy transferase mediated dTUPbiotin nickend labeling (TUNEL). ResultsThe levels of AST, ACT, Cr and BUN in the study group were significantly lower than those of the control group at the same phases (P＜0.05). The level of TNFα in the transplanted tissues of kidney and liver was also significantly lower as compared with those of control group. The cell apoptosis index of the transplanted tissues of kidney and liver was significantly smaller in the study group （P＜0.05）. There was no dramatically pathological change in the tissues of transplanted kidney and liver, which were treated with antiTNFα monoclonal antibody, and the structures are almost normal. ConclusionAntiTNFα monoclonal antibody may reduce cell apoptosis and accelerate the restoration of function of liver and kidney after combined liver and kidney transplantation.
The exact pathophysiological mechanisms of retinopathy of prematurity (ROP) remain elusive. The risk factors of ROP include excessive oxygen therapy, malnutrition, infection and inflammation. Among the factors above, the effect of inflammation on ROP has received more attention. TNF-α is a biological active protein which is involved in neovascularization and inflammation. It may play a role in the development of ROP. This review summarized the studies on the association between TNF-α and ROP in recent years, so as to provide a new way to further study the pathogenesis and treatment methods of ROP.