Curcumin And Retinoic Acid Suppress The Migration Of Human Retinal Pigment Epithelial Cells And Remodel The Extracellular Matrix Of Human Retinal Pigment Epithelial Cells

Proliferative vitreoretinopathy (PVR) is characterized as fibrocellularepiretinal membrane at the vitreoretinal interface formation, contraction andtraction, which lead to retinal detachment. PVR is considered to be one of themost severe complications of rhegmatogenous retinal detachment (RRD)including early cell migration, cell proliferation, excellullar matrix (ECM)remodeling, fibrocellular epiretinal membrane formation and contraction. Theformation and gradual contraction of epiretinal membranes at the vitreoretinalinterface cause a marked distortion of the retinal architecture and result inretinal reattachment surgery failure and eventually lead to severe vision loss orblindness. Moreover, PVR is also the most serious and common complicationoccurred in traumatic and retinal vascular disease. Retinal pigment epithelium(RPE) cell is the most important factor which triggers and is involed in thedevelopment and progression of PVR. Three obvious pathologic stages runthrough the formation and development of PVR. The first one involves amigration process of RPE cells into the preretinal and subretinal places, wherethe cells begin to proliferate. The next includes several cells, such as RPE cells,continually proliferation and the ECM remodeling. The final stage results inRD following fibrocellular membrane formation and contraction. Overgrowthof the fibrocellular membrane is known as a process of wound healingassociated with the interactions between RPE cells and ECM remodeling.Once RPE cells detach from the basement membrane site (the Brunchmembrane) in the RRD cases, the properties of them become more active likefribroblast in the cultures.In clinics, the most effective management for PVR is the surgery on thevitreoretinal interface and the rapid development of technical operation is always helpful for the retina to be reattached at the time of treatment. However,retinal reattachment surgery failure is inevitable because of recurrent PVR,and results in the deterioration of visual acuity and blindness. Therefore, it isan impending and promising mission to select some safe and effectivepharmaceutical precautions for early prevention and treatment of PVR, whichare being highlighted in clinics and in the animal experiments. Now, numerousof western medicines have been tried in the project for PVR prevention. Butthe western medicines have not been applied in practice, though their potenteffects on anti-proliferation in vitro, because some disadvantages of thememerge such as cytotoxicity and simple function. However, accumulatingevidences have demonstrated that the traditional Chinese medicine display atheoretically promising prospect because of abundant sources in nature,multiple-function and low toxicity. Then, the traditional Chinese medicine,Curcumin, which was separated and extracted from natural plants, enters intoour sight. Curcumin, or diferuloylmethane, is a major chemical component ofturmeric (Curcuma longa). Literatures have demonstrated that a promisingapplication of Curcumin to exhibit several properties such as anticarcinogenic,anti-inflammatory, antiangiogenic, antioxidant, antirheumatic, andantimetastatic activities. Curcumin have been extensively put into use ininternal medicine disease and anti-cancer, but rarely in ocular diseases.Meanwhile, the previous experimental researches were only foucused on theproliferation activities of RPE cells. However, the mechanism contributing toimbalance in the microenviroment-ECM remains elusive or has been rarelyreported.So we selected Curcumin, a traditional medicine, which wereinvestigated in the experiments in vitro and in vivo to prevent PVR formationand development. And Retinoic acid (RA), which had been identified as aneffective medicine in PVR treatment, was the control one in this experiment.On the view of ECM, we investigated whether Curcumin and RA treatmentsmay affect RPE cell behaviors other than cell proliferation. The results wouldprovide a new sight. Part Ⅰ Curcumin and Retinoic acid suppress the migration of humanRPE cells in vitro and in vivoObjective：The study was designed to better understand how differentconcentrations of Curcumin and Retinoic acid (RA) influenced the migrationand invasion abilities of human RPE cells in vitro and to select the optimalconcentration. Meanwhile, the identified optimal concentration of Curcuminand RA were intravitreally injected into the rabbit PVR models to investigatethe invasion activities of human RPE cells in vivo.Methods：RPE cells were isolated by trypsinization in human eye cups,and the harvested RPE cells were incubated in Ham’s F12culture. The purityand characterization were demonstrated by using the immunocytochemicalmethods. Early passages (from the forth to the sixth) of cultured human RPEcells were used in the present study. The effects of several concentrations ofCurcumin and RA on the viability of human RPE cells were determined usingthe methyl-thiazolyl tetrazolium bromide assay (MTT). The differentconcentrations of Curcumin (0、0.25×10-5、0.5×10-5、1.0×10-5、2.0×10-5、4.0×10-5M）and different concentrations of RA（0、10-6、10-5、10-4、10-3M）stimulated proliferation of RPE cells after48h and the inhibition effectswere recorded. The half maximal inhibitory concentrations (IC50) of Curcuminand RA at the time of48h were calculated. And choosing the adoptconcentrations of Curcumin and RA were applied in the next steps, such aswound healing assay, transwell invasion assay,3D collagen gel contractionassay in vitro and RPE ivasion assay in vivo.Results：RPE cells showed in round shape and were abundant in melaninwhen they were just isolated from the eye cups. The adherence time ofprimary RPE cells happened from48h to72h in the cultures. After one week’sincubation, the cells became nearly confluence. The melanin were nearlydisappeared until the forth generations of RPE cells. The purity andcharacterization were demonstrated by using the immunocytochemicalmethods where the RPE cell was positive stained in both cytokeratin AE1/AE3and S-100. And the IC-550of Curcumin and RA were1.2×10M and10-2.77M, respectively.In the wound healing assay: the different concentrations of Curcumin (0、0.25×10-5、0.5×10-5、1.0×10-5M) and different concentrations of RA (0、10-6、10-5�'�10-4M) treated the human RPE cells, respectively. Curcuminand RA showed the significant and dose-dependant effects on the migration ofRPE cells. In transwell cell invasion assay: the different concentrations ofCurcumin (0、0.25×10-5、0.5×10-5M) and different concentrations of RA (0、10-6、10-5M) treated the human RPE cells for48h, respectively. Curcumin andRA showed the significant and dose-dependant suppression effects on theinvasion of RPE cells. In3D collagen gel contraction assay: the differentconcentrations of Curcumin (0、0.25×10-5、0.5×10-5M) and differentconcentrations of RA (0、10-6、10-5M) treated the polymers including humanRPE cells (2×105) for96h, respectively. Curcumin and RA showed thesignificant and dose-dependant suppression effects on the contraction ofcollagen gel. In RPE cells invasion assay in vivo, a1:1suspension in200μl of2×1066thRPE cells and medicine (0.5×10-5M Curcumin or10-5M RA) wereinjected into vitreous cavities of rabbit PVR models. The control groupunderwent PVR induction by being injected with the same amount of cells andsaline instead of medicine. PVR formation was monitored weekly till the endof the4thweek. Curcumin and RA significantly suppressed the invasionacitivites of RPE cell in vivo.Conclusion：Lots of cultured human RPE cells can provide stabilized cellsources for researches in vitro and in vivo. Associated with MTT assay, woundhealing assay, transwell invasion assay in vitro,3D collagen gel contractionassay and invasion assay in vivo,0.5×10-5M Curcumin and10-5M RAexhibited the significantly inhibition effects on the migration and invasionactivities of human RPE cells. And the observation time point of48h wasidentified and would be applied in the next experiments.Part Ⅱ Curcumin and RA regulate the mRNA levels of human retinalpigment epithelium cells’ ECM and adhesion molecules in vitroObjective：After identifying the inhibition effects of Curcumin and RA on the migratory and invasion behaviors of human RPE cells in vitro and invivo, we investigated whether Curcumin and RA could selectively modulatethe mRNA expressions of84known human RPE cell’ ECM and adhesionmolecules by microarray and real-time PCR.Methods：The differential ECM and adhesion molecule gene expressionprofiles were evoked by the exponential phase9thpassage of RPE cellstreatment with Curcumin (0.5×10-5M), with RA (10-5M) or without any drugsusing the human ECM and adhesion molecules microarray and RT-PCRmethods.Results：To substantiate the results of the microarray studies, RT-PCRwas performed to assess the84known mRNA expressions. In Curcumingroup, there were17regulated genes for down-regulation and10forup-regulation compared to the control with the statistically significant (P <0.05). And similarly,24regulated genes in RA group were recorded (20fordown-regulation and4for up-regulation) compared to the control with thestatistically significant (P <0.05).Conclusion：Curcumin and RA could have significantly and selectivelyinfluenced on the mRNA levels of human RPE cells’ ECM and adhesionmolecules. They had the common characters to block numerous genesexpression, such as MMP1, MMP2, MMP9, most collagen proteins,Fribronectin-1and laminins, and to increase the mRNA levels of severalintegrins and MMP3. Compared with each other, Curcumin showed thestronger and more system effects on the regulation of the mRNA expression ofMMP3, MMP9and tissue inhibitor of metalloproteinase1(TIMP1).Part ⅢCurcumin and RA regulate the protein levels of human retinalpigment epithelium cells’ ECM and adhesion molecules in vitroObjective：After identifying the inhibition the effects of Curcumin andRA on the migratory and invasion behaviors of human RPE cells in vitro andin vivo, we investigated whether Curcumin and RA could selectivelymodulate the partial ECM protein expressions of RPE cell by westernblotting. And we wanted to identify whether there existed the coherence between the genes and the proteins expression.Methods： The differential ECM and adhesion molecule proteinexpressions were evoked by the exponential phase9thpassage of RPE cellstreatment with Curcumin (0.5×10-5M), with RA (10-5M) or without any drugsusing western blotting method.Results：Compared to the control, Curcumin (0.5×10-5M) significantlyinhibited several proteins expression, such as Fribronectin-1, MMP1, MMP2and MMP9. Meanwhile, Curcumin increased the MMP3protein expression.Similarly, RA (10-5M) significantly inhibited several proteins expression,such as Fribronectin-1, MMP1, MMP2, MMP9, MMP14, integrin AM andintegrin B2. Meanwhile, RA increased the MMP3and E-cadherin expression.By comparision, Curcumin had the stronger effect on augmenting the proteinlevel of MMP3.Conclusion：Curcumin and RA could have significantly influenced onthe protein levels of human RPE cells’ ECM and adhesion molecules. Theyhad the common characters to block numerous protein expressions, such asMMP1, MMP2, MMP9, Fribronectin-1, and to increase the protein levels ofMMP3. Compared with the results of genes expression, Curcumin and RA hadthe coherent effects on protein expression.Part Ⅳ Curcumin regulate the intercellular calcium signal expression ofhuman retinal pigment epithelium cells in vitroObjective：Observing the expressions of the intracellular calcium signalof human RPE cells in vitro， we investigated the correlated signaltransduction mechanism between the human RPE cells and their ECM afterCurcumin treatment at different concentrations.Methods： Every group of6thpassage of human RPE cells was treatedby5μM Fluo-3for30min. After this treatment，Curcumin at0、0.25×10-5、0.5×10-5、1.0×10-5、2.0×10-5、4.0×10-5M concentrations affected everygroup respectively. And intracellular calcium fluorescence changes of RPEcells were measured by laser scanning confocal microscope（LSCM）.Results： The data showed that the calcium fluorescences were appeared in the empty control group and Curcumin groups (0.25×10-5、0.5×10-5、1.0×10-5、2.0×10-5、4.0×10-5M). However，the expression of fluorescence inthe Curcumin groups was significantly greater than that in the empty controlgroups. And there was positive correlationship between the fluorescencedegree of intracellular calcium and the different Curcumin concentrations（r=0.922, P＜0.05）.Conclusion： The calcium fluorescences were significantly increasedinside the cultured human RPE cells induced by different concentrationCurcumin groups compared with the control group. Above all the data, theintracellular calcium concentrations were immediately increased afterCurcumin external stimulus.Then the signal transduction may be responsiblefor the biological activities of cells by their ECM remodelling.