ObjectiveTo study the inhibitory effects of pigment epithelium derived factor (PEDF) on oxygen-induced retinal neovascularization in mice, and to investigate the possible involvement of interleukin-1β (IL-1β) in the neovascular-inhibitory function of PEDF. Methods A total of 140 postnatal day (P)7 C57BL/6 mice were randomly divided into normal control group, oxygen-induced retinopathy (OIR) model group, PEDF treatment group and PBS treatment control group. All mice except normal control group with their mothers were exposed to (75±2)% oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model. Mice in normal control group were kept in room air only. At P12 and P14, respectively, mice in PEDF treatment group received intravitreous injections of 1 μl PEDF (2 μg/μl), while PBS treatment control group received the same volume of PBS (10 mmol/L, pH7.4).All mice were euthanized at P17 and eyes were isolated. The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy. Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR). ResultsChanges of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina, the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group (t=15.02, P < 0.01), and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group (t=5.96, P < 0.01). Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer (OPL) to ganglion cell layer (GCL) of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group. The specific expression levels of IL-1β protein in retinas of OIR mice by Western-blot analysis were higher than those of normal control group(t=3.35, P < 0.05), While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.764, P > 0.05). Similarly, expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group(t=4.43, P < 0.01). After treated with PEDF, expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.15, P > 0.05). ConclusionsPEDF can inhibit oxygen-induced retinal neovascularization. The mechanism may be related to that PEDF can downregulate the expression of IL-1β in retina.
Objective To observe the influence of interleukin-1beta; (IL-1beta;) on the expression of phosphorylated signal transducers and activators of transcription 3 (pSTAT 3) in rat retinal Muuml;ller cells.Methods For in vitro study cultured Muuml;ller cells were treated with IL-1beta; of different concentrations (0, 0.1, 1, 5 and 10 ng/ml) for 24 hours. For in vivo study, 32 Sprague-Dawley（SD）rats were divided into 4 groups randomly (control group，100，500 and 1000 ng/ml group) with 8 rats in each group. After 24 hours of injection with phosphate buffered solution (PBS), or 100,500,1000 ng/ml IL-1beta; into the vitreous cavities of the above rats, retinas were harvested. The expressions of pSTAT3 in cultured Muuml;ller cells or treated retinas were evaluated by indirect immunofluorescence and western blotting.Results After 24 hours of incubation without IL-1beta;, pSTAT3 has little expression in cultured Muuml;ller cells, but was upregulated by 1 ng/ml or higher IL-1beta; in a dosagedependent manner (F=46.64, 43.78;P＜0.01). pSTAT3 was not expressed in adult rat retina, but was upregulated by vitreous injection of 100 ng/ml or higher IL-1beta; in a dosagedependent manner (F=73.53,43.70;P＜0.01).pSTAT3 expressed mainly in inner nuclear layer and ganglion cell layer. Doublelabeling showed that there was no costaining of pSTAT3 and glial fibrillary acidic protein (GFAP) in retina of control group, but there were many costained Muuml;ller cells in retinas treated with IL-1beta;.Conclusions Expression of pSTAT3 in Muuml;ller cells could be activated by IL-1beta; which may represent one pathway link to reactive gliosis.
Objective To explore the effect of IL-10 on the inhibition of early proinflammatory cytokine release in intraabdominal infection and early sepsis. Methods Forty eight SD rats were randomized into 4 groups, 12 in each group, ①sham operation group, ②control group, ③prophylactic group, ④therapeutic group. Group 1 underwent laparotomy only, group 2 received laparotomy and cecal ligation plus punctures (CLP) with saline injected once every 3 hrs, group 3 underwent CLP and IL-10 injection intraperitoneally 1 hr before surgery and once every 3 hrs following operation, group 4 received CLP and IL-10 injection once every 3 hrs after operation. At 3 and 9 hr points, rats were sacrificed and blood samples were taken for measurement of inflammatory cytokines. Results Almost no inflammatory cytokines were detected in sham group, CLP produced a significant rise in serum TNF-α (tumor necrosis α), IL-1, IL-6 (interleukin 1,6) in control group, IL-10 reduced the rise of inflammatory cytokines significantly. Conclusion IL-10 could inhibit the early inflammatory cytokine release in rat model of sepsis. Suggesting it may attenuate the severity of inflammation.
Objective To explore whether interleukin-10 ( IL-10 ) gene single nucleotide polymorphisms are associated with asthma. Methods The frequency of three single nucleotide polymorphisms ( rs1800896, rs3024492, and rs3024496) and haplotypes of IL-10 gene were analysed in 302 adult asthmatic subjects and 275 controls of Han Chinese in Guangzhou using MALDI-TOF-MS and MassARRAY-IPLEX. The genotype and allele frequencies were analyzed by Chi-square test in both groups.Logistic regression analysis with adjustment for age and sex was conducted. Odds ratio ( OR) and 95%confidence interval ( 95% CI) were calculated to analyze the associations between the susceptibility of asthma and genotypes. Results ①Three genotypes GG, GA, and AA of rs1800896 were found in Han Chinese inGuangzhou. The frequencies of GG, GA, and AA genotypes were 2. 12% , 39. 65% , and 58. 23% ,respectively. The relative risk of developing asthma in the carriers of GA was significantly higher than that in the carriers of AA ( OR=4. 827, P lt;0. 001) . ②Two genotypes AA and AT of rs3024492 were found in Han Chinese in Guangzhou. The frequencies of AA and AT genotypes were 1. 22% and 98. 78% , respectively.The rs3024492 polymorphism was not related to susceptibility in asthma. ③Two genotypes TT and CT of rs3024496 were found in Han Chinese in Guangzhou. The frequencies of TT and CT genotypes were 90. 59% and 9. 41% , respectively. The rs3024496 polymorphism was not related to susceptibility in asthma. Conclusion In IL-10 gene single nucleotide polymorphisms, rs1800896 but not rs3024492 and rs3024496 isstatistically related with the development of asthma.
Objective To observe the histopathologic features and expression patterns of tumor necrosis factor-alpha; （TNF-alpha;）, interleukin-1beta;（IL-1beta;） and Escherichia coli lipopolysaccharide (LPS) in the rat vitreous with LPS inducedendophthalmitis. Methods Wistar rats were randomly divided into saline control group (SC,136 rats）,endophthalmitis group （EO, 168 rats）and blank control group （BC,12 rats）.EO group received an intravitreal injection of 5 mu;l LPS; SC group received 5 mu;l sterile saline and no intervention for BC group.Six,12,24,48, and 72 hours,5 and 7 days after injection, intraocular inflammation were observed and the eyes and vitreous were collected for histopathological examination and measurement of TNF-alpha;, IL-1beta; and LPS expression. Results Severe inflammatory responses in the eyes were observed in EO group between six and 72 hours after LPS injection，ocular inflammation subsided seven days after LPS injection. In the vitreous, a peak neutrophil count was observed at 24 hours （1224.64plusmn;132.2） cells/eye that rapidly declined at 72 hours （342.25plusmn;47.7） cells/eye. The levels of TNF-alpha; and IL-1beta; in EO group were peaked at 24 hours with （996.18plusmn;89.45） and（5556plusmn;1440）pg/ L, respectively；Persisted at 48 hours and began to decline rapidly thereafter. Seven days after LPS injection, levels of TNF-alpha; and IL-1beta; returned to baseline with （22.16plusmn;5.84）and （73.7plusmn;18.7） pg/L, respectively. LPS concentration in EO group decrease rapidly at 72 hours with （11.03plusmn;3.41） ng and disappear on days 7 with (0.22plusmn;0.08) ng after LPS injection.Conclusions Massive neutrophils infiltration, high levels expression of TNF-alpha; and IL-1beta; and spontaneous elimination of bacterial elements in vitreous cavity were major pathologic characteristics in this experimental model. The expression patterns of TNF-alpha;,IL-1beta; were in accord with LPS clearance process.
ObjectiveTo address the effect and mechanism of interleukin 17 (IL-17) on the proliferation, migration and apoptosis of human retinal vascular endothelial cells (HREC). MethodsIL-17 receptor (IL-17R) mRNA and protein expression in human retinal vascular endothelial cells (HREC) were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation of HREC was examined using CCK-8 assay in the presence of different concentrations of IL-17. Cell migration of HREC was detected using wound scratch assay. Flow cytometry was used to test the effect of IL-17 on the apoptosis of HREC. The effects of IL-17 on HREC expression of basic fibroblast growth factor (bFGF), Caspase-3 and thrombospondin-1 (TSP-1) were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). The effect of IL-17 on HREC expression of Caspase-3 was examined using Western blot. ResultsIL-17 receptor (IL-17R) expressed in HREC as quantified by RT-PCR and Western blot. The proliferation of HREC in the presence of IL-17 was promoted in a dosage-dependent manner (t=-3.235, -6.276;P=0.032, 0.000). Wound scratch assay showed a significant increase in the migrated distance of HREC with IL-17 stimulation under the concentration of 100μg/L(t=-3.551, -2.849; P=0.006, 0.019), 200μg/L(t=-10.347, -4.519; P=0.000, 0.001) and 500μg/L (t=-3.541, -2.607; P=0.008, 0.036). The intervention of 200μg/L IL-17 can effectively inhibit the apoptosis of HREC, compared with the control group using flow cytometry (t=5.682, P=0.047). RT-PCR results showed that IL-17 can promote the expression of bFGF and inhibit the expression of Caspase-3 and TSP-1. Western blot result also showed that IL-17 can suppress the protein expression of Caspase-3. ConclusionThe mechanism of IL-17 promoting proliferation, migration but suppress apoptosis of HREC may via regulating the expression of bFGF and Caspase-3.