RESUMEN
Background Interleukin-1β (IL-1β) is an important inflammation-related factor in the initial stage of proliferative vitreoretinopathy (PVR).The previous research showed that curcumin can inhibit IL-1 β-induced proliferation of rabbit retinal pigment epithelium (RPE) cells,but the anti-inflammatory mechanism and effect of curcumin are still undefined.Objective This study was to observe the migration of IL-1β-induced rabbit RPE cells,and evaluate the function and mechanism of inhibition of curcumin on IL-1β-induced inflammation of RPE cells.Methods Cultured rabbit RPE cells of generation 4 were used in this experiment.The cells were cultured in serum-free DMEM and 0,0.1,1.0 and 10.0 μg/L IL-1β were separately added in the medium for 24 hours.The expressions of cyclooxygenase-2 (COX-2) protein and mRNA in the cells were detected by Western blot and reverse transcription PCR to determine the optimal concentration of IL-1β.The cells were divided into IL-1β group and curcumin+IL-1β group,and 1.0 μg/L IL-1 or 1.0 μμg/L IL-1 β combined with 10 μg/ml curcumin was respectively added into the medium for 24,48 and 72 hours.The cells cultured by only serum-free medium served as the control group.Hematoxylin and eosin staining was conducted for the cells to count the number of cells migrating into the injured area under the optical microscope.The relative expression levels of COX-2 protein and mRNA in the cells were detected by Western blot and reverse transcription PCR,and the relative expression levels of nuclear factor (NF)-κBp65 and inhibitor of NF-κB-α (IκB-α) protein were also detected by Western blot assay.The expression intensity and location of NF-κBp65,IκB-α and COX-2 in the cells were detected by immunochemistry.Results RPE cells just isolated from the rabbit eyes were in round shape and abundant in melanin.The melanin significantly decreased in the fourth generations of RPE cells.The shape of cells became long and narrow,and net shaped distribution.Immunochemistry demonstrated the strong positive response of RPE cells for keratin (AE1/AE3).There were (31.93 ±1.21),(36.27±2.50) and (38.33±2.40) migratory cells in the control group after 24,48 and 72 hours respectively.The number of migratory cells increased to 45.73 ± 2.30,71.13 ± 1.92 and 80.60 ± 1.71 in the IL-13 group,but obviously decreased to 13.13 ± 2.20,14.93 ± 1.10 and 12.60 ± 1.51 in the curcumin + IL-1β group.A Significant increase in the migrating cell number was found in the IL-1 β group compared with the control group and the curcumin+IL-1β group in various time points (all at P＜0.05).The relative expression levels of COX-2 protein and mRNA peaked in the 1.0 μg/L IL-1β group,so 1.0 μg/L of IL-1β was determined as the optimal concentration in the experiment.In 24,48 and 72 hours after culture,the expression levels of COX-2 protein and mRNA in the cells were significantly lower in the curcumin + IL-1β group than those in the control group (all at P＜0.05).The relative expression level reached peak in NF-κBp65 protein and lowed bottom in IκB-α proteins at 48 hours after cultured in the IL-1β group,and the reverse trend was seen in the curcumin+IL-1β group,with the significant differences between the two groups (both at P＜0.05).Immunochemistry showed that NF-κBp65 was expressed strongly in the cell nuclei and cytoplasm in the IL-1 β group and presented the weaker expression in the control group and the curcumin+IL-1 β group.Compared with the control group,the expression was weaker in IκB-α and stronger in COX-2 in the IL-1β group.In addition,the expression of IκB-α was enhanced and that of COX-2 was attenuated in the curcumin+IL-1β group in comparison with the IL-1β group.Conclusions Curcumin inhibits the movement of rabbit RPE cells induced by IL-1β.IL-1β up-regulates the expression of COX-2 by activating NF-κB signal pathway,and curcumin plays an anti-inflammatory role by blocking this pathway.
RESUMEN
Background Neovascular glaucoma (NVG) is a serious eye disease with a variety of causes.It results from the secretion of growth factors by hypoxic retinal tissue,such as vascular endothelial growth factor (VEGF),but anti-VEGF treatment alone dose not completely inhibit neovascularization.Our previous study found that platelet-derived growth factor-c (PDGF-C) is also an important factor in pathological angiogenesis.However,the role of PDGF-C in the development of NVG remains unknown.Objective This study was to quantitatively detect the levels of VEGF and PDGF-C in the aqueous humor of patients with NVG and provide a basis for the anti-angiogenesis therapy.Methods A prospective cohort study was carried out.Sixty-two eyes of 62 patients with advanced NVG and 11 control subjects with age related cataract were included in Xijing Hospital of the Fourth Military Medical University from January 2014 to August 2015.Ten of 62 NVG eyes received retinal photocoagulation and/or cryotherapy,and 16 eyes resulted from central retinal vein occlusion,20 eyes from diabetic retinopathy,5 eyes from postopeartion of retinal detachment,4 eyes from Eales disease and 7 eyes from unknown cause.Iris neovascularization of grade Ⅱ was found in 13 eyes,grade Ⅲ in 29 eyes and grade Ⅳ in 10 eyes.Aqueous humor specimen was collected for 0.1-0.2 ml during the surgery,and the contents of VEGF and PDGF-C in the aqueous humor were detected by ELISA assay.Written informed consent was obtained from each subject prior to any medical procedure.Results The VEGF and PDGF-C concentrations in the aqueous humor were (1 138.17±69.31) ng/L and (29.80±1.64) ng/L in the NVG group,respectively,which were significantly higher than (679.54±49.81) ng/L and (18.60±1.85)ng/L in the control subjects (t=20.95,20.49,both at P＜0.01).The VEGF and PDGF-C concentrations in aqueous humor were (1 095.99±52.71) ng/L and (28.55±0.94) ng/L in the retinal photocoagulation group,showing significantly decreases in comparison with (1 146.28±69.57) ng/L and (30.04± 1.64) ng/L in the untreatment group (t =-2.160,P =0.034;t =-2.760,P =0.008).The VEGF content in aqueous humor was positively correlated with PDGF-C content in the patients with NVG (r=0.346,P=0.006).However,the aqueous levels of VEGF and PDGF-C were not significantly different among various primary disease groups and different iris neovascularitation gradings (all at P＞0.05).Conclusions Both VEGF and PDGF-C contents in the aqueous humour are considerably elevated in NVG patients.Retinal photocoagulation and/or cryotherapy can inhibit the release of VEGF and PDGF-C in the aqueous humor.PDGF-C may be a target for the treatment of NVG.