ObjectiveTo observe the effect of interleukin (IL) 10 modified endothelial progenitor cells (EPC) in diabetic retinopathy (DR).
MethodsEPC cells were collected and cultivated from the bone marrow of rats and identified by immuno-fluorescence staining. EPC cells were infected with lentivirus (LV) of EPC-LV-IL10-GFP (EPC-LV-IL10-GFP group) or EPC-LV-NC-GFP (GFP group). EPC cells without lentivirus infection was the EPC group. Enzyme-linked immuno sorbent assay (ELISA) was used to measure the concentrations of tumor necrosis factor (TNF)-α, IL10, IL8 and vascular endothelial growth factor (VEGF) in the supernatant of these three groups. 168 male Wistar rats were divided into normal control group (28 rats), diabetes mellitus (DM) group (28 rats), DM-blank control group (56 rats) and DM-intervention group (56 rats). DM was introduced in the latter 3 groups by streptozotocin intravenous injection. Three months later, the rats in the DM-blank control group and DM-intervention group were injected with EPC-LV-NC-GFP or EPC-LV-IL10-GFP by tail vein, respectively. Immunohistochemistry was used to observe the GFP expression in rat retinas. The blood-retinal barrier breakdown was detected by Evans blue (EB) dye. The retinal histopathologic changes were observed by transmission electron microscope. The mRNA level of VEGF, matrix metallproteinases-9 (MMP-9), angiopoietin-1 (Ang-1), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in retina were measured by reverse transcription-polymerase chain reaction (RT-PCR).
ResultsELISA showed that the levels of TNF-αand IL8 in the supernatant significantly decreased, while the levels of IL10 and VEGF increased (P < 0.05) in EPC-LV-IL10-GFP group. GFP expressed in the retina of blank control group and intervention group, mainly in the ganglion cell layer, inner nuclear layer and outer plexiform layer. The retinal blood vessel pathological change and EB permeability significantly decreased in intervention group compared with DM group (P < 0.05), and blank control group (P < 0.05). RT-PCR revealed that the mRNA level of VEGF, MMP-9 and Ang-1 significantly increased, and eNOS decreased in DM group compared to the normal control group (P < 0.05). The mRNA level of VEGF and iNOS decreased, eNOS increased while Ang-1 and MMP-9 had not changed in DM-blank control group and DM-intervention group compared with DM group (P < 0.05).
ConclusionsIL10 modified EPC can improve the inflammative microenvironment and suppressed the pathogenesis of DR. Furthermore, EPC transplantation can increase the number of EPC and exerted their effect.
Objective
To determine the concentration of int erleukin-12(IL-12),interleukin-2(IL-2) and tumor necrosis factor(TNF)and the irpossible role in the pathogenesis of proliferative vitreoretinopathy(PVR) ．
Methods
Patients were divided into 3 groups:18 with PVR,7 with simples retinal detachment caused by macular hole and 4 samples from normal eyes were used as control.Sample s of vitreous were obtained by aspiration through pars plana before cryotherapy ,vitrectomy and gas injection and stored in liquid nitrogen at －70℃ within 30 minites for ELISA.
Results
①The levels of IL-12,IL-2，and TNF in the vitreous of PVR were positively correlated with the degree of severity of disease.②The levels of IL-12, IL-2,and TNF in the PVR were higher than those in simple retinal detachment caused by macular hole and those in control group(Plt;0.01 ).③The levels of IL-12,IL-2，and TNF in retinal detachment caused by macular hole were also higher than those in the control group(Plt;0.01).
Conclusion
IL-12,IL-2，and TNF may play a role at lease to some extent in the pathogenesis of PVR.
(Chin J Ocul Fundus Dis,1999,15:75-77)
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.
Interleukin-18 is an inactive precursor which lacks a signal peptide, it has a role in regulating retinal pathological angiogenesis. It also inhibits experimental choroidal neovascularization (CNV) via interferon-γand thrombospondin-1. Currently little is known about its mechanisms of inhibition for CNV, may be speculated to be due to effects of anti-angiogenesis, down-regulates vascular permeability and lower vascular endothelial growth factor (VEGF) levels via directly acting on the vascular endothelial cell and epithelial cells. Exogenous administration of mature recombinant interleukin-18 has no adverse effect on retinal pigment epithelial cell viability. In addition, the anti-VEGF role of interleukin-18 is tested to be safe and effective for humans. Interleukin-18 alone or in combination with anti-VEGF shows to be a good prospect for improving the prognosis of experimental CNV. However, more large clinical studies are required to confirm the exact efficacy of interleukin-18 for CNV.
Objective
To investigate the changes and clinical significance of cytokines and inflammatory species in exhaled breath condensate (EBC) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD).
Methods
Thirty AECOPD patients admitted in the Department of Respiratory Medicine from March 2015 to August 2016 (smokers and passive smokers) and 21 healthy volunteers (non-smokers) were recruited in this prospective study. General information and EBC were collected from each subject. The concentrations of interleukin-17 (IL-17), IL-10, and 8-isoprestane (8-iso-PG) in EBC were measured by enzyme-linked immunosorbent assay, meanwhile lung function test was performed in the AECOPD patients.
Results
Both IL-17 (ng/L) and 8-iso-PG (ng/L) levels increased significantly in the AECOPD patients before and after treatment compared with the healthy controls (10.74±1.02 and 5.65±0.88 vs. 3.36±0.61, 12.35±2.25 and 9.65±1.22 vs. 6.93±1.15, P<0.05). However, IL-10 level significantly decreased in the AECOPD patients before and after treatment compared with the healthy controls (1.68±0.17 and 2.59±0.31 vs. 2.85±0.43, P<0.05). Both IL-17 and 8-iso-PG levels in the AECOPD patients were significantly lower after treatment than those before treatment (5.65±0.88 vs. 10.74±1.02, 9.65±1.22 vs. 12.35±2.25, P<0.05), but IL-10 level were significantly higher aftertreatment than those before treatment (2.59±0.31 vs. 1.68±0.17, P<0.05). FEV1, FVC, and FEV1%pred improved significantly after treatment (P<0.01). FEV1, FEV1/FVC and FEV1%pred were not significantly correlated with IL-17, IL-10 or 8-is-PG levels.
Conclusion
IL-17, IL-10 and 8-iso-PG may be involved in the pathogenesis of COPD, and may be important biomarkers in monitoring airway inflammation and oxide stress during the treatment of AECOPD patients.
ObjectiveTo investigate the expressions of IL-10,tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum and lung tissue of COPD rats in order to elucidate the potential mechanism of airway inflammation.
MethodsForty-five healthy adult male SD rats were randomly divided into a COPD model group (n=30) and a normal control group (n=15). The COPD rat model was established by intratracheal instillation of lipopolysaccharide (LPS) and exposure to cigarette smoke for 28 days. The concentrations of IL-10,TNF-α and IFN-γ in serum and lung tissue were measured by ELISA.
ResultsTNF-α level of serum and lung tissue in the COPD model group increased significantly compared with the control group(P<0.05),while the levels of IFN-γ and IL-10 decreased significantly[serum:(44.68±8.67) ng/L vs. (75.96±10.59) ng/L;lung tissue:(64.55±9.03) ng/L vs. (94.06±8.71) ng/L,P<0.01]. The level of IL-10 in serum and lung tissue was negatively correlated with TNF-α (serum:r=-0.67,lung tissue:r=-0.80,P<0.01). The level of IL-10 in serum and lung tissue was positively correlated with IFN-γ (serum:r=0.64,lung tissue:r=0.72,P<0.01). The level of IL-10 in serum and lung tissue was negatively correlated with the percentage of neutrophils(serum:r=-0.70,lung tissue:r=-0.67,P<0.01).
ConclusionIn COPD rats,down regulation of IL-10 plays an important role in regulation of airway inflammation.
Objective To investigate the response of retinal ganglion cells （RGC）in detached and reattached retina in adult rats, and the effect of IL-1beta antibody and IL-1Ra on the loss of RGC. Methods A total of 73 Sprague-Dawley (SD) rats were subretinally injected with healon GV（1.4% hyaluronate）and retrograde labeled with fluorogold (FG), and 10 ng IL-1 Ra and 500 ng IL-1beta antibody were injected into the subretinal space combined with healon GV. The retinal flakes were observed under the fluoroscope and the number of RGC was counted 2 hours, 1, 3, 5, 7, 10, 20, 50, and 90 days after deta chment; 10 days after detachment and 30 days after reattachement; 90 days after detachment and 20 days after reattachement, and 1 and 10 days after injection with IL-1beta antibody and IL-1Ra,respectively. And the control group was only developed an intraocular injection of the same valume of healon GV. Result Two hours after detachment, the RGC loss was found, reached the peak at first day, and decreased gradually. RGC loss was also found in the non-detached area. The reattachment 10 days after detachment (early reattachment) stopped the loss of RGC, and the reattachment 90 days after detachment (late reattachment) promoted the loss, which rested on a certain level. Subretinal space injection of IL-1Ra and IL-1beta antibody decreased the loss of RGCs in the detached retina. Conclusion The RGCs loss were found both in the detached and attached retina. Early reattachment may stop the loss of RGC, and late reattachment may promote the loss. Both IL-1beta antibody and IL-1Ra have neuro protective effect on RGC. (Chin J Ocul Fundus Dis，2004，20：233-236)
OBJECTIVE: To study the nerve growth factor (NGF) expression and the influence of IL-1 alpha or IL-1 beta on NGF secretion in newborn rat astrocytes. METHODS: Astrocytes obtained from the brain cortex of newborn rats were cultured and purified, and they were divided into three groups, experimental, control and blank groups. IL-1 alpha or IL-1 beta were added into the experimental group with 25, 50 and 100 U/ml, each group was cultured for 24, 48 or 72 hours, and then the NGF contents in cultured astrocytes suspension media were measured by a two-cite enzymelinked immunoserbent assay (ELISA). RESULTS: Astrocytes could secret NGF by themselves and each concentration of IL-1 alpha or IL-1 beta media at any testing time could enhance NGF secreting in newborn rat astrocytes in certain degrees. The effects of IL-1 beta were ber than IL-1 alpha, the best effect in the unit time was observed in IL-1 beta with 50 U/ml for 24 hours. CONCLUSION: Astrocytes can express NGF, and IL-1 alpha or IL-1 beta can enhance the NGF expression in newborn rat astrocytes.
ObjectiveTo investigate the expression of interleukin-18（IL-18）and signal transducers and activators of transcription 5（STAT5）in retina of 4-24-week-old diabetic rats, and explore the potential molecular mechanisms involved in diabetic retinopathy (DR).MethodsRetinal gene expression profile of healthy and 8-week-old diabetic rats was established with restriction fragment differential displaypolymerase chained reaction (RFDD-PCR), and the differences was analyzed by bioinformatics. IL-18 and STAT5 were filtrated as the candidate genes of DR. The expression of IL-18 and STAT5 in retina of diabetic rats with the age of 4, 8, and 24 weeks was observed by semi-quantitative reverse transcriptase-polymerase chain reaction（RT-PCR）.ResultsThe result of RFDD-PCR showed:expression of IL-18 was higher in healthy retina than that in diabetic one; expression of STAT5 was not found in healthy rats but in diabetic ones. The result of RT-PCR showed：compared with the normal, high expression of IL-18 was found in 4-week diabetic retina, reduced in 8-week one, and decreased to the lowest in 24-week one. The expression of STAT5 was not observed in healthy or 4week diabetic retina, but occurred in 8-week one, and increased in 24-week one. ConclusionThe expression of IL-18 and the activation of STAT5 may relate to the occurrance of DR. The expression of IL-18 doesn′t depend on the activation of STAT5. (Chin J Ocul Fundus Dis, 2005,21:258-260)
Objective To explore the effects of asiaticoside on the activation of nuclear factor kappa B ( NF-κB) and cytokines expression in RAW264. 7 cells induced by lipopolysaccharide ( LPS) . Methods RAW264. 7 cells were allocated to 5 groups, ie. a blank group, a model group stimulated by LPS at dose of 10 μg/mL, and three asiaticoside treatment groups stimulated by LPS and different doses of asiaticoside simultaneously. The effects of asiaticoside ( 10 - 7 , 10 - 6 , 10 - 5 mol /mL) on the proliferation of cells were examined by MTT assay. The activation of NF-κB was detected and analyzed by the laser scanning confocal microscope( LSCM) ,meanwhile the concentrations of TNF-α, IL-1, and IL-10 in supernatants were quantified by ELISA. Results MTT assay showed that asiaticoside ( 10 - 7 , 10 - 6 ,10 - 5 mol /mL) had no effects on the proliferation of RAW264. 7 cells. Asiaticoside significantly decreased the activation of NF-κB, downregulated the secretion of TNF-αand IL-1, and upregulated IL-10 secretion in a dose dependent manner. According to LSCM, the ratio of NF-κB activation was ( 3. 5 ±1. 5) % , ( 75. 7 ±9. 1) % , ( 66. 8 ±7. 1) % , ( 58. 9 ±9. 0) % , and ( 40. 1 ±8. 8) % in the blank, model, and asiaticoside( 10 - 7 , 10 - 6 , 10 - 5 mol /mL) treatment groups respectively. The contents of TNF-α in supernatants were ( 171. 12 ±35. 42, 1775. 45 ±193. 97,1284. 63 ±162. 13,1035. 22 ±187. 97, 598. 90 ±107. 73) pg/mL respectively and IL-1 were ( 5. 66 ±0. 98,26. 93 ±3. 48,22. 41 ±2. 84, 17. 05 ±1. 70, 10. 64 ±1. 29) ng/mL respectively, while IL-10 were ( 25. 23 ±2. 17,71. 75 ±8. 31, 82. 82 ±6. 00, 98. 70 ±8. 84, 119. 97 ±9. 13) pg/mL respectively. Conclusion The antiinflammation mechanism of asiaticoside may be mediated by downregulating inflammatory factors throughNF-κB signal pathway and keeping the balance between proinflammatory and antiinflammatory system.
