Authors: Liu Liya, Ma Jingxue, Liu Danyan, An Jianbin, Zhou Nalei, Ma Yuelei
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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-1β-induced proliferation of rabbit retinal pigment epithelium (RPE) cells, but the anti-inflammatory mechanism and effect of curcumin are still undefined.
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.
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 μg/L IL-1β 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.
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-1β 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-1β 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-1β group and presented the weaker expression in the control group and the curcumin+ IL-1β 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.
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.