| Proliferative vitreoretinopathy(PVR)is a common eye disease that seriously threatens vision.It occurs in 5-10%of patients with rhegmatogenous retinal detachment(RD).The disease is characterized by the formation of the epiretinal and/or subretinal membranes,which may distort the tissue structure of the retina and lead to re-detachment.The proliferation of retinal pigment epithelium(RPE)cells is the initial stage of PVR.The formation process of the epiretinal membranes are similar to the wound healing reaction of RPE cell proliferation.The cells undergo epithelial-mesenchymal transformation and migration.Triggering factors of PVR proliferative membranes formation are thought to be related to damage to the tight junctions of the blood-retinal barrier and the release of growth factors,chemotactic mediators and inflammatory cytokines.Areas of retinal tears and detachment may cause fluid to leak from the vitreous into the subretinal space and create conditions for RPE cells to migrate to the inner surface of the retina.Among many growth factors,fibroblast growth factor-2(FGF-2)plays an important role in the proliferative process at the vitreoretinal interface,stimulating migration and proliferation of RPE cells,fibroblasts and vascular endothelial cells,and inducing experimental PVR.Therefore,we used FGF-2 to induce ARPE-19cells,modeling to more closely resemble the pathological environment of PVR in vivo.At present,there is no clinically approved drug treatment for PVR,and surgery is still the main method.Surgical treatment includes removal of the fibrous membranes and,in some cases,removal of part of the retina.Drugs that treat or prevent the formation of PVR may improve surgical success and protect vision after retinal detachment.Certain drugs that act on different processes in the pathological process of PVR,including cell proliferation,migration,and contraction,will be a potential therapeutic approach to treat or prevent the formation of PVR.Our team has conducted studies on curcumin,plumbagin,and crocetin in vitro and in vivo.Studies have shown that these three drugs have inhibitory effects on PVR with small side effects.In order to find an effective and safe drug,the effect of these three drugs was compared and the mechanism of action of the best drug was studied,which provided a new idea for the prevention and treatment of PVR.Part one:Experimental study on inhibition of FGF-2-Induced human retinal pigment epithelial cell Proliferation in vitro by curcumin,plumbagin,and crocetinObjective:In this study,different concentrations of FGF-2 were used to act on human retinal pigment epithelial cells to observe its pro-proliferation effect and to select the best concentration of FGF-2 to induce cells.To explore the inhibitory effects of curcumin,plumbagin,and crocetin on proliferation of human retinal pigment epithelial cells induced by FGF-2 and to evaluate the best anti-proliferative drug.Methods:1.ARPE-19 cells were cultured in vitro.The effects of different concentrations of FGF-2(1,5,10,20,40 ng/ml)on proliferation of ARPE-19cells at different time(24 h,48 h,72 h)were detected by CCK-8 assay.2.CCK-8 assay is used to detect cell proliferation.ARPE-19 cells are treated with or without FGF-2 and different concentrations of curcumin(0,1,3,10,20,30,40,50,100?M),plumbagin(0,0.3,0.9,2.7,5,8,10?M),crocetin(0,30,50,100,200,300,400,500?M)for 24 h.Compare the effect of these three drugs.Results:1.At the three time points of 24 h,48 h and 72 h,FGF-2 concentrations(1,5,10,20,40 ng/ml)could induce proliferation of cells in all groups(P<0.05),and OD values increased gradually with the extension of cell incubation time.In each time point and concentration group,10 ng/ml FGF-2 had the strongest effect on promoting proliferation.2.The results of CCK-8 assay showed that all three drugs could inhibit proliferation of FGF-2-induced ARPE-19 cells(P<0.05).Among them,IC50value of plumbagin was the smallest among three drugs,suggesting that it had the best therapeutic effect and showed obvious inhibitory effect on cells at low concentration.Conclusion:1.The optimal concentration of FGF-2 on ARPE-19 cells was 10 ng/ml.2.Different concentrations of curcumin,plumbagin,and crocetin could inhibit the proliferation of FGF-2-induced ARPE-19 cells.The most effective drug to inhibit cell proliferation is plumbagin.Part Two:Experimental study on inhibition of FGF-2-induced proliferation,migration,and invasion of human retinal pigment epithelial cells in vitroObjective:On the basis of the findings in the first part that plumbagin has the strongest inhibitory effect on cell proliferation,to further explore the effect of plumbagin on proliferation,migration,and invasion of FGF-2-induced ARPE-19 cells and its regulatory mechanism.Methods:1.CCK-8 and EdU assays were used to detect the proliferation of ARPE-19 cells.Western blot was used to detect the changes of PCNA protein content in ARPE-19 cells treated with different concentrations of plumbagin.2.The migration and invasion of ARPE-19 cells were detected by cell scratch assay and Transwell assay.3.The mRNA and protein expressions of MMP-2,MMP-9,Collagen I,? and ? in ARPE-19 cells were detected by RT-PCR and western blot.4.Phosphorylation of FGFR-1/2,ERK,p38 and JNK in ARPE-19 cells was detected by Western blot.Results:1.The CCK-8 and EdU assays were used to detect cell proliferation.The results showed that 1.25 and 2.5?M of plumbagin had no significant effect on cell viability,while 5 and 7.5?M of plumbagin significantly reduced cell viability.The results of EdU assay showed that FGF-2 could promote proliferation of ARPE-19 cells.Western blot was used to detect the expression level of PCNA protein,and it was found that the expression level of PCNA protein decreased gradually with the increase of the concentration of plumbagin(P<0.05)in a dose-dependent manner.Therefore,we demonstrate that plumbagin inhibits FGF-2-induced proliferation of ARPE-19 cells.2.The migration and invasion of ARPE-19 cells were detected by cell scratch assay and Transwell assay.The cell migration was significantly inhibited in the cell scratch assay(P<0.05).The cell invasion was significantly inhibited in Transwell assay(P<0.05).3.The expression levels of MMP-2 and MMP-9 in ARPE-19 cells were detected by RT-PCR and western blot.The results showed that the mRNA and protein expressions of MMP-2 and MMP-9 were significantly inhibited by plumbagin(P<0.05)in a dose-dependent manner.RT-PCR and western blotting were used to evaluate the expression levels of collagen ?,?,and ? in FGF-2-induced ARPE-19 cells.The results showed that FGF-2 could promote the mRNA and protein expression of collagen ?,?,and ?(P<0.05).The mRNA and protein expression of collagen ?,?,and ? was inhibited in a dose-dependent manner.4.The phosphorylation of FGFR-1/-2,ERK,p38,and JNK were detected by RT-PCR and western blot.The results showed that the intracellular phosphorylation of FGFR-1 and FGFR-2 was significantly increased after FGF-2 treatment,while the phosphorylation of FGFR-1 and FGFR-2 was significantly decreased by plumbagin.FGF-2-induced phosphorylation of ERK,p38,and JNK was significantly increased.However,the phosphorylation levels of ERK,p38,and JNK were significantly decreased with the gradual increase of the concentration of plumbagin(P<0.05).Conclusion:1.Plumbagin inhibited proliferation,migration,and invasion of FGF-2-induced ARPE-19 cells.2.The inhibition of proliferation,migration,and invasion of ARPE-19cells may be related to the inhibition of mRNA and protein expressions of MMP-2/-9,collagen ?,?,and ?.3.The inhibition of proliferation,migration,and invasion of ARPE-19cells may be achieved by inhibiting the phosphorylation of FGFR-1/-2,ERK,p38,and JNK.Part three:Experimental study on prevention and treatment of proliferative vitreoretinopathy of rabbits in vivoObjective:In the second part of this study,it was found that plumbagin could inhibit proliferation,migration,and invasion of ARPE-19 cells in vitro.In this part of the experiments,we used FGF-2 combined with ARPE-19 cells to model rabbit eyes,and further explored whether plumbagin had inhibitory effect on the development of PVR in rabbit eyes.Methods:1.The pigment rabbits were randomly divided into 2 groups with 5 rabbits in each group.The rabbits in control group were injected with FGF-2(50 ng),ARPE-19 cells and phosphate buffer(PBS)in vitreous cavity.The rabbits in the experimental group were injected with FGF-2(50 ng),ARPE-19 cells and plumbagin in vitreous cavity.Fundus photography,B-ultrasound and OCT were used to examine the eyes of rabbits before modeling and after modeling3,7,14,21,28 days.According to the examination results,the PVR level of each group was divided according to Fastenberg classification.2.On the 28th day after modeling,the rabbit eye tissues were removed and the pathological changes of retinal tissues in each group were observed by HE staining.The expression of?-SMA in retina of each group was observed by immunohistochemistry.Results:1.3th days after establish the experimental model:In the control group,1eye was PVR?level.In the experimental group,5 eyes were PVR 0.The7th days after establish the experimental model:In the control group,1 eye was PVR?level,2 eyes were PVR?level.In the experimental group,1eye was PVR?level.The 14th day after establish the experimental model:In the control group,2 eyes were PVR?level,2 eyes were PVR?level,1eye was PVR?level.In the experimental group,1 eye was PVR?level,3 eyes were PVR?level.21th days after establish the experimental model:In the control group,2 eyes were PVR?level,3 eyes were PVR?level.In the experimental group,2 eyes were PVR?level,2 eyes were PVR?level.28th days after establish the experimental model:In the control group,2eyes were PVR?level,3 eyes were PVR?level.In the experimental group,2 eyes were PVR?level,2 eyes were PVR?level.The incidence of retinal detachment in the control group was 60%at 14th days and 100%at21th days after modeling.The incidence of retinal detachment in the experimental group at 28th days after modeling was 40%.2.HE staining showed clear structure of normal retinal layers.In the control group,the retinal ganglion cell layer was edema,the internal and external nuclear layer cells were disordered,and the extracellular segment of the photoreceptor was lost.In the experimental group,the retinal structure was orderly,and the extracellular segment of the photoreceptor was also lost.Immunohistochemical staining showed that there was no positive staining of?-SMA in the retina of normal pigmented rabbits,while the retina of the control group showed strong staining of brown-yellow,and the retina of the experimental group showed weak staining of light brown.Conclusion:Plumbagin can inhibit formation of epiretinal membranes and delay the progression of PVR in rabbits. |
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