ABSTRACT
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.
ABSTRACT
Background The primary pathologic mechanism of posterior capsular opacification (PCO) is proliferation,migration and epithelial-mesenchymal transformation of residuary lens epithelial cells (LECs) following cataract surgery.Matrix metalloproteinases (MMPs) play a role during the migration of LECs.Researches showed that GM6001,a broad inhibitor of MMPs,can arrest the migration of LECs,but as specific inhibitors of MMPs,the efficacy and safety of MMP-2/9 inhibitor Ⅰ and Ⅱ on LECs migration remain unclear.Objective This study was to determine and compare the inhibitory efficacy among GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ on human LECs and search the clinical medication to prevent PCO.Methods Human LECs were cultured and passaged in vitro,and the cells of 3-4 generation were incubated in 6-well plates.Then the cells of 70％ confluent monolayer were cultured in DMEM without fetal bovine serum for 12 hours.GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ at different concentrations (0.25,0.50,1.00,2.00,4.00,8.00,16.00,32.00,64.00,128.00 μmol/L) were added into the culture medium for 24 hours separately,and regularly cultured cells served as the control group.A bare area was made by a 200 μl sterile spear on the cell layer,and the migrated distance and inhibitory rate were calculated.The second or third generation of cells were incubated in 96-well plates at a density of 5×105/ml (200 μl/well).GM6001 (128.00 μmol/L),MMP-2/9 inhibitor Ⅰ (64.00 μmol/L) and Ⅱ (32.00 μmol/L) were added into the culture medium for 24 hours,and the cell viability was assayed by using MTT assay.Results Cultured cells grew well with irregular arrangement and presented the polygon in shape.The migrated distance was gradually reduced as the increase of concentrations of GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ,showing significant differences among the various concentration groups (GM6001:F=248.647,P＜0.05;MMP-2/9 inhibitor Ⅰ:F=357.125,P＜0.05;MP2/9 inhibitor Ⅱ:F=396.374,P＜ 0.05).The cell migrated distance in the control group was set to 1,the relative migrated distances were 0.478 ± 0.091,0.294±0.088 and 0.191 ±0.081 in the GM6001 group,MMP-2/9 inhibitor Ⅰ group and MMP-2/9 inhibitor Ⅱ group at the concentrations of 32.00 μmol/L,respectively,showing a significant difference among the groups (F =116.031,P＜0.01),and cell migrated distance was obviously shorter in the MMP-2/9 inhibitor Ⅱ group than that in the GM6001 group or MMP-2/9 inhibitor Ⅰ group (all at P＜0.01).The A values were 0.607±0.016,0.567±0.015,0.583±0.010 and 0.595 ±0.0138 in the control group,GM6001 group (128.00 μmol/L),MMP-2/9 inhibitor Ⅰ group (64.00 μmol/L) and MMP-2/9 inhibitor Ⅱ group (32.00 μmol/L),respectively,without significant difference among the groups (F=1.403,P＞0.05).Conclusions GM6001,MMP-2/9 inhibitor Ⅰ and Ⅱ reduce the mobility of human LECs effectively but do not affect the viability of the cells in vitro.MMP-2/9 inhibitor Ⅱ appears to be most dominant in inhibiting migration of human LECs.