ObjectiveTo dynamically observe the effect of N-acetylserotonin (NAS) on the expression of tumor necrosis factor-α (TNF-α) protein in retina of retinal ischemia reperfusion injury (RIRI) rats, and to explore the mechanism.MethodsBy using random number table method, 90 healthy male Sprague-Dawley rats were divided into sham operation group (n=10), RIRI group (n=40), and NAS group (n=40). The right eye was as the experimental eye. In the RIRI group and NAS group, the anterior chamber high intraocular pressure method was used to establish the RIRI model. In the NAS group, 10 mg/kg NAS was injected intraperitoneally before modeling and 30 minutes after modeling. At 6, 12, 24, 72 h after modeling, hematoxylin-eosin staining was used to observe the pathological changes of the retina, and the retinal ganglion cells (RGC) were counted. Each group was detected by immunohistochemical staining and Western blot about the relative expression of TNF-α, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein in the rat retina. One-way analysis of variance was used for differences between groups. The general linear regression method was used to analyze the correlation between the relative expression changes of TNF-α protein and the changes of Nrf2 and HO-1 protein expression after NAS intervention.ResultsOptical microscope observation revealed that the retinal edema of rats in the RIRI group was observed at 6, 12, and 24 h after modeling; the thickness of the retina in the NAS group was significantly thinner than that in the RIRI group, and the difference was statistically significant (F=9.645, 477.150, 2.432; P＜0.01). At 6, 12, 24, and 72 h after modeling, the retinal RGC counts in the NAS group were significantly higher than those in the RIRI group, and the difference was statistically significant (F=12.225, 12.848, 117.655, 306.394; P＜0.05). The results of immunohistochemical staining and Western blot showed that 6 h after modeling, the relative expression of TNF-α protein in the retina of the RIRI group increased significantly compared with that in the sham operation group, reaching a higher level at 12 h, and decreased at 24 and 72 h. But all were significantly higher than the sham operation group, the difference was statistically significant (immunohistochemical staining: F=105.893, 1 356.076, 434.026, 337.351; P＜0.01; Western blot: F=92.906, 534.948, 327.600, 385.324; P＜0.01). At different time points after modeling, the relative expression of TNF-α protein in the retina of the NAS group was significantly lower than that of the RIRI group (immunohistochemical staining: F=15.408, 570.482, 21.070, 13.767; P＜0.05; Western blot: F=12.618, 115.735, 13.176, 111.108; P＜0.05), but still higher than the sham operation group (immunohistochemical staining: F=40.709, 151.032, 156.321, 216.035; P＜0.01; Western blot: F=33.943, 79.729, 74.057, 64.488; P＜0.01), the difference was statistically significant; 12 h after modeling, Nrf2 in the retina of the NAS group (immunohistochemical staining: F=51.122, P＜0.05; Western blot: F=33.972, P＜0.05), HO-1 (immunohistochemical staining: F=30.750, P＜0.05; Western blot: F=18.283, P＜0.05) protein relative expression was significantly higher than that of RIRI group, and the differences were statistically significant. The results of linear regression analysis showed that the difference in the number of TNF-α+ cells in the RIRI group and the NAS group was negatively correlated with the difference in the number of Nrf2+ and HO-1+ cells (r2=0.923, 0.936; P＜0.01).ConclusionsNAS can inhibit the expression of TNF-α protein in the retina of RIRI rats and reduce RIRI. The mechanism may be related to the Nrf2/HO-1 pathway.
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.
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.
【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.
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.
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.
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 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.
Vogt-Koyanagi-Harada syndrome (VKH) is an autoimmune disorder primarily characterized by bilateral granulomatous uveitis, which can lead to severe visual impairment and related complications. Traditional treatment typically involves glucocorticoid combined with immunosuppressants, but these therapies are associated with significant side effects, limited efficacy, and poor long-term prognosis. In recent years, biologic agents have emerged as a promising treatment for refractory VKH due to their targeted action, high efficacy, and low toxicity. Tumor necrosis factor-alpha (TNF-α) inhibitors, such as infliximab and adalimumab, have shown significant benefits in controlling inflammation, improving vision, and reducing steroid dependence, making them a key option for difficult-to-treat VKH. Among interleukin (IL) blockers, tocilizumab has demonstrated potential in patients who do not respond to traditional treatments. Rituximab, a B-cell targeting agent, has shown good efficacy and safety in patients resistant to TNF-α inhibitors. Additionally, research into novel biologics targeting the IL-23/IL-17 axis and IL-33 offers new directions for VKH therapy. While biologics provide clear advantages in VKH treatment, further research is needed to explore their long-term safety, cost-effectiveness, and optimal treatment regimens. Large-scale randomized controlled trials are required to validate their efficacy and identify personalized treatment strategies to improve long-term patient outcomes.
Objective To investigate the mechanism of dexamethasone in the treatment of acute necrotizing pancreatitis (ANP). Methods The ANP of 48 SD rats were induced by retrograde infusion of sodium taurocholate through biliopancreatic duct.After 30 minutes,the therapy group was administrated with dexamethasone at a dose of 0.2 mg/100 g alone. The control group was administrated with the same amount of 0.9% saline solution.At fourth hour and twelfth hour,8 rats of each group were sacrificed to examine the levels of serum tumor necrosis factor-alpha(TNFα) and serum amylase,to score the degree of pancreatic necrosis and to evaluate acinar cell apoptosis by in situ hybridization by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL). The survial period of 8 rats in each group were observed. Results In therapy group, the level of TNFα was (17.8±2.7) pg/ml and (8.5±1.6) pg/ml,the apoptosis index was （36.94±4.12）% and （ 32.79±3.31）%,the survival period was (33.4±21.5) h.While the control group with the indexes mentioned above were as follows: (53.6±18.7) pg/ml and (37.2±11.1) pg/ml ( P＜0.01),（4.37±1.24）% and （5.12±2.11）% （P＜0.01）,(14.6±5.7) h (P＜0.01) ,the histologic scoring for ANP between therapy group and control group was a significantly distinct (P＜0.01). Conclusion Dexamethasone can induce pancreatic acinar cell apoptosis in this model. Proper leves of TNFα may play an important role in regulating the apoptosis.Apoptosis can protect pancreas from necrosis in ANP.
