The Inhibition Effect And Molecular Mechanism Of Crocetin On Development Of Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy（PVR）occurs after rhegmatogenous retinal detachment（RD）or major ocular trauma and its surgical repair and is characterized by the formation of epiretinal,subretinal and intravitreal fbrotic membranes that can contract and lead to tractional retinal（re-）detachment.It is estimated that PVR develops in approximately 5-10%of all RD cases and is still the main cause of failure of RD surgery.Although most PVR cases can be surgically repaired with modern surgical techniques,recurrent detachment and PVR itself often result in limited visual recovery and irreversible damage.The major cell type in the fibrotic membrane is retinal pigment epithelial（RPE）cells.RPE cells undergo epithelial-mesenchymal transition（EMT）through which the detached polarized cells transdifferentiate into fibroblast-or myofibroblast-like cells and acquire several abilities,including enhanced migratory capacity,invasiveness,resistance to apoptosis,extracellular matrix production and fibrotic membrane formation.Eventually,the contraction of the fibrotic membrane leads to fixed folds and redetachment of the retina.Crocetin is one of the major components of saffron that is derived from the dried stigma of the Crocus sativus L.Crocetin is a low-molecular weight carotenoid compound consisting of a C-20 carbon chain with multiple double bonds,and a carboxylic acid group at each end of the molecule.This compound possesses various biological activities including anticancer,anti-oxidation,anti-inflammatory,hepatoprotective,and neuroprotective activities.Previousreportshaveshownthatcrocetincaninhibit ischemia/reperfusion induced retinal damage,suppress the VEGF-induced angiogenesis,and demonstrate the antifibrotic effects in scleroderma fibroblasts.However,the interaction between crocetin and the proliferation,migration,and EMT of RPE cells associated with PVR has not been studied.In the present study,we investigated the effect of crocetin on the proliferation,migration,and transforming growth factor-β₂（TGF-β₂）induced EMT of RPE cells.Further we evaluated the safety,pharmacokinetics,and prevention effect in proliferative vitreoretinopathy of intraocular crocetin.Part one Crocetin inhibits the proliferation and migration of ARPE-19cellsObjective:To investigate the potential impact of crocetin on the proliferation and migration of cultured ARPE-19 cells.Methods:The cells were treated with different concentrations of crocetin（0,50,100,and 200μM）at various time intervals（0,24,48 and 72 h）for cell proliferation and migration assays.Cell proliferation was examined using the CCK-8 assay.Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining.The expression levels of proliferating cell nuclear antigen（PCNA）,p21 and p53 were examined by Western blot analysis.Cell migration was assessed by in vitro scratch and Transwell assays.Results:Crocetin treatment significantly inhibited cell proliferation in a dose-and time-dependent manner.Crocetin demonstrated a dose-dependent increase in the number of cells in G1 phase and decrease in the number of cells in S phase after 24 h of treatment.Treatment with crocetin especially 200μM steadily reduced PCNA protein expression and increased p21 and p53accumulation.As shown by the scratch assay,untreated and 50μM crocetin treated cells displayed enhanced migratory ability and almost covered the scratched area after 72 h,whereas the migratory ability of cells treated with high concentrations of crocetin was significantly reduced at 24,48,and 72 h,particularly in the group treated with 100μM or 200μM crocetin.Similar results were obtained in Transwell assay,where crystal violet staining showed that much fewer cells had migrated through the membrane in the high-concentration crocetin-treated groups than the untreated and 50μM crocetin-treated groups after 20 h of incubation.Conclusions:Treatment of ARPE-19 cells with crocetin（50-200μM）significantly inhibited their proliferation and migration in a concentration-and time-dependent manner.Crocetin induced G1 arrest,reduced PCNA protein expression and increased the p21 and p53 accumulation in ARPE-19 cells.Part two Crocetin inhibits the TGF-β₂-induced epithelial-mesenchymaltransition of ARPE-19 cells via suppression phosphorylation of p38Objective:To investigate the potential impact of crocetin on the TGF-β₂-induced epithelial-mesenchymal transition（EMT）of cultured ARPE-19 cells.Methods:After 1 h of pretreatment with various concentrations of crocetin（0,50,100,and 200μM）,the cells used for the EMT assay were stimulated with or without recombinant human 10 ng/ml TGF-β₂.EMT markers,such as E-cadherin（epithelial marker）,ZO-1（epithelial marker）,vimentin（mesenchymal marker）andα-SMA（mesenchymal marker）were examined using RT-qPCR,Western blot and immunofluorescence staining.At the end of the experiments,the cells were preincubated with p38 MAPK inhibitor（SB203580,10μM）for 30 min before treatment with or without exogenous TGF-β₂ to test whether crocetin work through surpressing the activation of p38 MAPK.Results:Treatment of ARPE-19 cells with 10 ng/ml TGF-β₂ induced a significant change in cell morphology from a cuboidal to an elongated spindle-like shape,reduced the expression of E-cadherin and ZO-1,and increased the expression of vimentin andα-SMA at both mRNA and protein levels.On the other hand,cell elongation induced by TGF-β₂ in ARPE-19 cells was blocked by co-treatment with 200μM crocetin.The TGF-β₂-induced decrease in E-cadherin and ZO-1,and increase in vimentin andα-SMA could be inhibited by 100μM and 200μM crocetin.Immunofluorescence staining validated the effect of crocetin on the expression of E-cadherin andα-SMA in the cells treated with TGF-β₂.Preincubation of ARPE-19 cells with 10μM SB203580 blocked the TGF-β₂-induced activation of p38,and inhibited TGF-β₂-induced expression ofα-SMA.In addition,crocetin prevented the TGF-β₂-induced activation of p38 in a dose-dependent manner for 24 h.Conclusions:Crocetin inhibits TGF-β₂-induced EMT of ARPE-19 cells through the p38 MAPK pathway.Part three The safety of intravitreous injection of crocetin in rabbitsObjective:To determine the safety of intraocular crocetin in rabbits.Methods:The right eyes of eight rabbits were injected with 0.2μmol（four eyes）and 0.4μmol（four eyes）crocetin suspended in 0.1 ml phosphate buffered saline（PBS）.The left eyes were injected with 0.1 ml PBS containing the same concentration of only DMSO.The animals underwent eye examinations before injection and at 1,3,5,7,and 14 days through such methods as slit-lamp biomicroscopy and indirect ophthalmoscopy.Fundus photography,optical coherence tomography（OCT）,and full-field electroretinogram（ffERG）were obtained at baseline 7 days and 14 days.Afterward,the eyes were enucleated for histopathological analysis.Results:One left eye and one right eye were excluded from the study for vitreous hemorrhage and cataract formation,respectively.Indirect ophthalmoscopy and OCT demonstrated no signs of retinal hemorrhage,edema,or optic nerve pallor.Histological examination in H&E staining showed complete preservation of all retinal layers in both the experimental eyes and the control eyes.There were no signs of atrophy or necrosis in the inner and outer retina.Under scotopic testing conditions,there were no significant differences in a-wave and b-wave amplitudes between crocetin-injected right eyes and vehicle-injected left eyes at any time point.Under photopic testing conditions,there was a decrease in the a-wave amplitude at 1 week in 0.4μmol crocetin-injected right eyes,but the decrease had no difference compared with the left eyes and returned to normal at 2weeks.No difference in a-wave and b-wave amplitudes were found between crocetin-injected right eyes and vehicle-injected left eyes at any time point.Conclusions:A single intraocular injection of crocetin（under 0.4μmol）appears to be nontoxic to the retina.Part four The pharmacokinetics of intravitreous injection of crocetin innormal rabbitsObjective:To observe the pharmacokinetic properties of intravitreally injected crocetin in normal rabbits.Methods:A high-performance liquid chromatography（HPLC）method was developed by setting chromatographic conditions,building vitreous samples processing methods and standard curve,testing the recovery and precision.Both eyes of fourteen pigmented rabbits received an intravitreous injection of 0.4μmol crocetin.The entire vitreous were obtained immediately and at 1,3,7,12,24,48,and 72 hours after injection,and processed according to the methods built before.Then concentrations of crocetin at each time point were collected detected by HPLC technology.Pharmacokinetic parameters were caculated with DAS 3.0 pharmacokinetic software.Results:The retention time of crocetin and curcumin was 8.30 min and 4.79min separately with no interference from vitreous endogenous substances.Standard curve equation for vitreous supernatant was y=0.0686x-0.0021,R2=0.9996.Standard curve equation for vitreous pellet was y=0.0069x-0.0015,R2=0.9996.The recovery and precision of these methods were fit for the experimental requirements.The mean crocetin concentrations and insoluble crocetin amount in the vitreous at specific time points after intravitreal injection with 0.4μmol crocetin were caculated according to the standard curve equations.The highest concentration of crocetin in the vitreous was 36.77±3.39μg/ml（111.98±10.33μM）with the half-time 4.23±1.31h calculated using a non-compartmental model.Conclusions:The HPLC method developed in the research was specific and sensitive for pharmacokinetic analysis of crocetin in vitreous cavity.Pharmacokinetic parameters of 0.4μmol crocetin in vitreous included Cmax36.77±3.39μg/ml（111.98±10.33μM）,volume of distribution 3±0.11 ml,clearance 0.1±0.02 ml/h,half-life 4.23±1.31h.Part five Prevention effect in proliferative vitreoretinopathy ofintraocular crocetinObjective:To examine whether crocetin can inhibit the development of proliferative vitreoretinopathy（PVR）in a rabbit model.Methods:PVR was induced in the right eye of twenty pigmented rabbits.Ten eyes were later injected with 0.4μmol crocetin,and the other 10 eyes（as controls）received 0.1 ml PBS.Slit-lamp biomicroscopy,indirect ophthalmoscopy,a digital fundus camera,optical coherence tomography（OCT）and full-field electroretinogram（ffERG）were performed at days 3 and7 and weekly for a total of 4 weeks after injection.Afterward,the eyes were enucleated and subjected to histological analysis（hematoxilin-eosine,Masson’s trichrome for collagen and immunohistochemical staining forα-SMA）.The stage of PVR was graded according to Fastenberg classification from 0 through 5 at days 7,14,and 28 after injection.Results:The Mann-Whitney U Test for nonparametric data showed statistically significant differences（P<0.05）at days 14,and 28 between the experimental and control groups.We showed the representative baseline,1-and 4-week scotopic and photopic ffERG waveforms.In the group injected with ARPE-19 cells+PBS,there was a significant reduction in a-wave amplitudes and b-wave amplitudes at 4 weeks under both scotopic and photopic conditions.The group injected with ARPE-19 cells+0.4μmol crocetin showed a slight reduction in a-and b-wave amplitudes at 1 and 4weeks in both scotopic and photopic 3.0 ERG.Histological findings and OCT confirmed the formation of epiretinal fibrotic membranes in two groups in different severity.In the eyes injected with ARPE-19 cells+PBS,fibrotic membranes with dense collagen fibers and cells were drawn up through the retina and made it convoluted.In contrast,the eyes injected with ARPE-19cells+0.4μmol crocetin showed a mild traction with formation of loose epiretinal membranes.Immunohistochemistry results and Masson’s trichrome stain showed a more prominentα-SMA expression and larger amounts of collagen deposition in the eyes injected with ARPE-19 cells+PBS than those injected with ARPE-19 cells+0.4μmol crocetin.Conclusions:Intraocular crocetin significantly inhibits the development of PVR in a rabbit model.