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 Normal ultrastructure is the anatomical basis of retinal pigment epithelial(RPE) cells to perform normal physiological function.At present the precipitation method is often used to detect the ultrastructure of RPE cells with transmission electron microscopy(TEM).Objective The aim of this study was to explore a simple and feasible approach to examine the ultrastructure of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells.Methods hESCs were induced and differentiated into RPE cells by the spontaneous differentiation method,and the expressions of microphthalmia associated transcription factor MITF and paired-box gene 6 (PAX6),specific protein of RPE cells,in the cells were detected by immunofluorescence assay.hESC-RPE cells were inoculated into Transwell filter,and the ultrastructure of the cell sheet was examined under the TEM.Then the ultrastructure of the cell sheet specimens was compared with those of hESC-RPE cells from cell precipitation and RPE cell specimens of 90-day-old Long Evans rats.Results MITF and PAX6 were positively expressed in hESC-RPE cells.The normal ultrastructure were visible in the RPE cells of rats under the TEM,including apical microvilli,polarized melanin granules,cellular nucleus,basement membrane and intercellular junctions,and the ultrastructure of hESC-RPE cell sheet on Transwell was similar to the RPE cells in rats.However,only scatter melanin granules,nonpolar nucleus and scanty microvilli were observed under the TEM in the hESC-RPE cells by cell precipitation method.Conclusions Without digestion process,hESC-RPE cell sheet on Transwell can retain the normal ultrastructure of hESC-RPE cells under the TEM,with a more simple and reliable advantage.
ABSTRACT
Objective To observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice.Methods A total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group,OIR model group and OIR model cell transplanted group,10 mice in each group.The OIR model was induced in mice of OIR model group and OIR model cell transplanted group.The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group.At 20 days after the injection,the RPE thickness was evaluated by fluorescence microscope.The expression of RPE65,Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR).Results The thickness of RPE in OIR model mice was thinner than that in normal mice;the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice.The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597,18.864,25.691) and mRNA expression (F=39.458,11.461,34.796) of RPE65,Bestrophin,ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P＜0.05).Conclusions Subretinal injection of RPE cells can promote RPE thickening.RPE65 and Bestrophin protein relative expression levels increased,ZO-1 protein relative expression levels reduced;mRNA expression levels of RPE65,Bestrophin and ZO-1 genes increased.
ABSTRACT
Background The imbalance of cell cycle regulation results in proliferative vitreoretinopathy (PVR).Studing the effects of cyclin dependent kinase (CDK) and CDK inhibitor (CKI) on the cell cycle regulation of retinal pigment epithelial (RPE) cells and fibroblasts in PVR formation is of important significance.Objective This study was to investigate the expressing trend of p21 ,p27 and CDK inhibitors in retinas of different ages of rabbits and explore the relationship between p21 or p27 and cell growth.Methods Nine clean New Zealand rabbits were assigned to 10-week group,20-week group and 30-week group according to the age and 3 rabbits for each.The eyeballs were enucleated binocularly after the animals were sacrificed and retinas were isolated.Real-time PCR and Western blot were employed to detect the expressions of p21 and p27 mRNA and their proteins in retinas of the rabbits.The use and care of the animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The relative expressing levels of p21 mRNA were 1.631±0.063,1.506±0.012 and 1.585 ±0.015, and those of p27 m RNA were 1.581 ± 0.048,1.470 ± 0.012 and 1.490 ±0.013 in the 10-week group,20-week group and 30-week group, respectively, showing significant differences among the groups (p21 mRNA: F=9.311,P=0.014;p27 mRNA: F=12.360, P=0.007) , and the p21 and p27 mRNA expressing levels were significantly higher in the 10-week group and 30-week group than those in the 20-week group (all at P＜ 0.05).The expressing levels of p21 protein were 0.675 ± 0.061,0.089 ±0.001 and 0.200 ± 0.007, and those of p27 protein were 0.928±0.019,0.183±0.005 and 0.576±0.089 in the 10-week group,20-week group and 30-week group, respectively, with remarkable differences among the groups (p21 : F =228.905, P＜0.001;p27 : F =148.957,P＜0.001), and the expressions were significantly raised in the 10-week group and 30-week group in comparison with the 20-week group (all at P＜0.01).Significantly positive correlations were found in the expressing levels of p21 and p27 both in transcriptional and protein levels (mRNA : r =0.906, P＜0.01;protein : r =0.913, P＜0.01).Conclusions The expressions of p21 and p27 up-regulate in the retinas of developing stage of rabbits but gradually reduce with adultness.However, p21 and p27 levels appear to be increasingly raised with aging of the rabbits.It is implied that p21 and p27 play a balancing role in the process of cycle regulation in retina cells.