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

Proliferative vitreoretinopathy(PVR)can complicated with rhegmatogenous retinal detachment(RD)or dramatic ocular trauma.The incidence of PVR is estimated around 5%-10% in all RD cases.The main cell involoved in the fibrotic membrane is retinal pigment epithelium(RPE).RPE cell plays an important role in the physiology of the retina and also the maintenance of the eye development and neural retina.It is a highly differentiated monolayer cell and an atomically located between the Bruch` s membrane and neurosensory retina.RPE allows transportation of nutrients from the choroid to the photoreceptors,phagocytosis of the metabolites of outer segment of the photoreceptors,maintaining the blood-retinal barrier el al.In the process of retinal detachment,the blood-retinal barrier is affected and RPE cells are directly exposed to serum exudates and vitreous.A variety of cytokines in vitreous promote migration of RPE into vitreous cavity,which can further proliferate and transform into a proliferative membrane.In addition,previous studies have shown that apoptosis co-exists in the vitreoretinal stretch membrane of PVR,which plays an important role in the development of the pathogenesis of PVR related ophthalmic diseases.Among the growth factors,there are four PDGF isoforms(PDGF-A,-B,-C,and-D)that form homodimers or heterodimers(PDGF-AA,-BB,-AB,-CC,and-DD)through disulfide bonds.PDGF is involved in many important biological activities such as cell differentiation,proliferation,apoptosis and migration.In addition,studies have shown that PDGF enhances the contraction of fibroblasts and RPE cells.Various in vivo studies found that PDGF content increased in both the vitreous of PVR patients and animal models.In the retinal detachment patients after laser surgery and the RPE cells of PVR mouse models,PDGF expression was shown to be improved.PDGF-BB showed extensive proliferation and migration onvascular cells,RPE cells and glial cells,and PDGF-AA acts on non-vascular cells.Crocetin,an antioxidant carotenoid,is abundant in saffron-has been shown multiple pharmacologicalactions on many cell types,including inhibiting ischemia/reperfusion induced retinal damage,antifibrotic effects in scleroderma fibroblasts,anti-oxidation,anti-inflammatoryand neuroprotective activities.In our previous studies,crocetin has been shown to inhibit the proliferation,migration and interstitial transformation of ARPE-19 cells.In this study,we further investigate intoitspathological activity for ARPE-19 cells,and studied the effect and mechanism of crocetin on ARPE-19 cells induced by PDGF-BB,and proposed to take rabbit eye PVR model as the research object and study it in vivo and in vitro.Part one Potential impact and mechanism of crocetin on proliferation,migration and apoptosis of PDGF-BB-induced ARPE-19 cellsObjective: To investigate the potential impact and mechanism of crocetin on proliferation,migration and apoptosis of PDGF-BB-induced ARPE-19 cells.Methhods: Effects of different concentrations of crocetin(0 ?M,100 ?M,200 ?M,400 ?M)and PDGF-BB on the proliferation of ARPE-19 cells were determined by Cell Counting Kit-8(CCK-8).The inhibitory effect of crocetin was further demonstrated with 5-ethynyl-2-deoxyuridine(Ed U)assays.The effect of crocetin on migration of PDGF-BB-induced ARPE-19 cells was verified by Transwell migration assay and an in vitro scratch assay.Flow cytometric analysis was used to detect the effect of crocetin on apoptosis of PDGF-BB-induced ARPE-19 cells.Western blot was used to detect the effects of different concentrations of crocetin on the expression of Bcl-2 family.Results: The cell proliferation inhibition rate of low concentration crocetin(100? M)was significantly increased at 72 h.However,the cell proliferation inhibition rate increased in a concentration-and time-dependent manner after the intervention of medium concentration(200 ?M)and high concentration(400 ?M)of crocetin.The Ed U transfection probability of the cells treated with crocetin was lower than that of the control group in a concentration dependent manner,and the results were consistent with CCK-8 assay.In the in vitro scratch assay,the migration of cells was significantly inhibited by various concentrations of drugs at 48 and 72 hours after treatment.The results of Transwell migration assay showed that the inhibitory effect of crocetin on cell migration was concentration dependent.Flow cytometric analysis detection revealed that crocetin induced apoptosis of ARPE-19 cells induced by PDGF-BB,and the ability of promoting apoptosis increased with the increase of concentration.Western blot analysis showed that crocetin significantly inhibited the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl(P<0.01),and increased the expression of pro-apoptotic proteins Bax and Bak(P<0.01)in a concentration-dependent manner.Summary: Crocetin inhibites the proliferation and migration of PDGF-BB-induced ARPE-19 cells and induces apoptosis of PDGF-BBinduced ARPE-19 cells.Part two Crocetin inhibited PDGF-BB-induced phosphorylation of platelet derived factor receptor(PDGFR)and downstream activation of the PI3k/Akt and MAPK signaling pathwaysObjective: To verify the possible involvement of crocetin in the signaling pathways.Methods: Western blot analysis was performed to determine the effects of different concentrations of crocetin(0 ?M,100 ?M,200 ?M,400 ?M)on PDGF-BB-induced PDGFR phosphorylation and downstream PI3k/Akt and MAPK signaling pathway activation.Results: Different concentrations of crocetin can inhibit the phosphorylation of PDGFR and significantly inhibit the activation of the downstream PI3k/Akt and MAPK signaling pathways.Summary: Crocetin inhibites PDGF-BB-induced phosphorylation of PDGFR and downstream activation of the PI3k/Akt and MAPK signaling pathways.Part three Experimental study on the treatment of proliferative vitreoretinopathy with crocetin.Objective: To further investigate the effect of crocetin on proliferative vitreoretinopathy in rabbit eyes.Methods: Twenty adult pigmented rabbits were randomly divided into experimental group and control group,and 10 rabbits in each group were intravitreal injected with ARPE-19 cells,2.5?l PDGF-BB,0.4 ?mol of crocetin and ARPE-19 cells,2.5?l PDGF-BB,0.1ml PBS(containing 2% DMSO).Slit-lamp biomicroscopy and indirect ophthalmoscopy were performed on 3,7,14 and 28 days.Electroretinograms(ERG)and optical coherence tomography(OCT)examinations were performed at 7,14 and 28 days.On the 28 th day,the experimental rabbits were sacrificed and their eyeball specimens were collected for immunohistochemical staining.The detection indexes were p-Akt,p-p38,p-PI3 k,p-ERK and p-JNK.Results: The fundus conditions of the experimental animals in each group were statistically analyzed at different time points according to the Fastenberg classification.ERG waveformons shown that on day 7,there was no significant change in the amplitude of b wave in the eyes after injection compared with the contralateral eyes.on day 14 and 28,the amplitude of b wave in the eyes after injection slightly decreased compared with the contralateral eyes.HE staining and OCT showed that the whole retinal structure of the experimental group was clear with normal morphology,and the optic ganglion cell layer,inner and outer nuclear layer cells were arranged neatly.In the control group,ganglion cell layer edema,inner and outer nuclear layer cells were disordered,and extracellular ganglion of photoreceptor was lost.Immunohistochemical staining results showed a more prominent p-Akt,p-ERK,p-PI3 k,p-p38 and p-JNK expression in the eyes injected with ARPE-19 cells +PBS than those injected with ARPE-19 cells +crocetin.Summary: Crocetin inhibites the pathogenesis of rabbit PVR,has a protective effect on visual function,and decreases p-Akt,p-PI3 K,p-p38,p-ERK and p-JNK expression.Conclusions:1.Crocetin inhibites the proliferation and migration of PDGFBB-induced ARPE-19 cells and induces apoptosis of PDGF-BB-induced ARPE-19 cells.2.Crocetin inhibites PDGF-BB-induced phosphorylation of PDGFR and downstream activation of the PI3k/Akt and MAPK signaling pathways.3.Crocetin inhibites the pathogenesis of rabbit PVR,has a protective effect on visual function,and decreases p-Akt,p-PI3 K,p-p38,p-ERK and p-JNK expression.