The Control Effect And Mechanism Research Of Plumbagin In Proliferative Vitreoretinopathy

The main character of proliferative vitreoretinopathy(PVR)is cell proliferation and membrane contraction on retinal surface or in the vitreous cavity,which leads to retinal detachment and visual impairment.It remains a significant obstacle to successful retinal reattachment surgery,and the common complication of severe ocular trauma,vascular retinopathy and so on.The retinal pigment epithelial(RPE)cells are considered as the most important cell type in PVR pathogenesis,which dedifferentiate and migrate through a retinal break and then proliferate on the retinal layers and vitrea,resulting in formation of epi-retinal membranes with other cells and cytokines.At present,more attempts have been made to develop nonsurgical therapies for PVR,especially pharmacological strategies.Plumbagin(PLB;5-hydroxy-2-methyl-1,4-naphthoquinone)is a kind of Chinese natural naphthoquinone component isolated from the root of Plumbago zeylanica L,which is abundant,with less side effect and has a extensive range of effects including anti-inflammatory and cancer.In the present study,we aimed to investigate whether PLB can effectively inhibit proliferation,migration and epithelial-to-mesenchymal transition(EMT)of RPE cells in vitro and in vivo,trying to find out the underlying mechanism and provide a new way to prevention and control of PVR.Part one The effects of plumbagin(PLB)on ARPE-19 cells proliferation and the underlying mechanismPurpose: This study aimed to explore the effects of a series concentrations of plumbagin(PLB)on ARPE-19 cells proliferation and underlying mechanism in order to assess its effectiveness.Methods: ARPE-19 cells were cultured with incremental concentrations(0,5,15,and 25 ?M)of PLB for 24 hours or with 15 ?M PLB for 12,24 and 48 hours.Then cell viability was evaluated by MTT assay and DAPI staining,while apoptosis and cell cycle progression of ARPE-19 cells were assessed by flow cytometric analysis.Furthermore,the level of Bcl-2 family regulatory proteins was examined by Western blotting.Results: Typical apoptosis morphological changes could be observed after ARPE-19 cell treated with plumbagin for 24 hours,such as nucleus pycnosis and nuclear cracking to pieces.As the drug concentration increased,the cell number decreased.MTT results showed plumbagin has obvious inhibitory effect on cell proliferation activity.Flow cytometry testing indicated plumbagin could significantly promote the ARPE-19 apoptosis through cell G2 / M phase retardation.Western Blotting detected plumbagin raised the expression of pro-apoptotic protein and inhibited the expression of anti-apoptotic proteins,and this pro-apoptosis effect was dose and time dependent.Conclusions: Plumbagin could inhibit ARPE-19 cell proliferation and promote apoptosis dose and time dependently.Plumbagin caused cell G2 / M phase retardation,then induced apoptosis and inhibited proliferation by regulating the biological effects of Bcl-2 family.Part two The effects of plumbagin on ARPE-19 cells epithelial-tomesenchymal transition and the underlying mechanismPurpose: This study aimed to explore the effects of a series concentrations of plumbagin on ARPE-19 cells migration and epithelial-tomesenchymal transition in order to assess its effectiveness.Methods: ARPE-19 cells were cultured with incremental concentrations of PLB.Then migration and invasion of PLB-treated cells were determined using Transwell chamber assays and scratch wound assay.The contractile ability was assessed by contraction assay.Expression of matrix metalloproteinases(MMPs)and epithelial-mesenchymal transition(EMT)markers were assessed by Western blotting.Results: With the increase of drug concentration,the scratches became wider in scratch experiment,and the number of cells through the artificial basement membrane decreased in invasive experiment showing that plumbagin could inhibit ARPE-19 cell migration and invasion.Meanwhile,it was found that the higher the concentration of plumbagin,the more obviously inhibition of collagen gel contraction.These above three kinds of abilities were concentration-dependent.Western blotting detected plumbagin depressed the secretion of MMP-1,MMP-2 in ARPE-19 cells so that weaken the migration and invasion abilities.At the same time plumbagin reduced the level of alpha-SMA and had a protective effect on ZO-1,which could inhibit the EMT of ARPE-19 cell.These effects on MMP-1,MMP-2,?-SMA were concentration and time-dependent.Conclusions: Plumbagin could inhibit ARPE-19 cell migration and invasion ability dose-dependently.Plumbagin could also reduce the EMT of ARPE-19 cells,and inhibit their contraction and fibrosis.Part three The effects of plumbagin on signaling pathways of human retinal pigment epithelial cellsPurpose: This study aimed to explore the specific effects of plumbagin on the different signaling pathways of ARPE-19 cells.Methods: ARPE-19 cells were cultured with incremental concentrations of PLB for 24 hours or with 15 ?M PLB for 12,24 and 48 hours.P38 MARK,PI3 K,?-catenin and Notch-1 were chosen as the markers of four common signal pathways in life activities: MARK,PI3K-AKT-m TOR,Wnt/?-catenin and Notch.The expression of m RNA was tested by Real-Time PCR and the level of relative proteins was examinated by Western blotting.Results: Compared to the control group,plumbagin of middle and high concentrations could significantly inhibit the m RNA expression of p38 MARK and PI3 K in a dose and time dependent manner.But there was no statistical difference in ?-catenin and Notch-1 m RNA expression quantity changes.Meanwhile,low,middle and high concentration of plumbagin couldsignificantly suppress the expression of p38 MARK,PI3 K protein;although the content ?-catenin protein decreased with increase of plumbagin concentration,only high concentration group had statistical significance;however,the protein content of Notch-1 had no statistical difference in any group.Conclusions: PLB inhibited p38 MARK,PI3 K signal pathways of ARPE-19-19 cells significantly in a concentration-and time-dependent manner.But Wnt/?-catenin,Notch pathways were not the main approach which plumbagin worked.Part four In vivo study of plumbagin on proliferative vitreoretinopathy in rabbit eyesPurpose: This study aimed to explore the effects of plumbagin in proliferative vitreoretinopathy rabbit models as well as assess the efficacy and safety of plumbagin intraocular injection.Methods: New Zealand albino rabbits were divided into three groups: 25 ?M PLB,50 ?M PLB and DMSO only as the control group.Bilateral ERG were operated on the day before the injection,on days 3,7,14 and 21 after the injection.Three weeks later,the eyeballs were taken out to receive histological testing.Moreover,plumbagin was injected in rabbit eyes along with RPE cells and plasma.After 1,7,14 and 21 days,the degrees of PVR were assessed using indirect ophthalmoscopy,and optical coherence tomography(OCT),ultrasound images,electroretinograms(ERG)as well as histopathology were used to evaluate efficacy.Results: After plumbagin injection,the rabbit eyes were in a stable condition.The b wave amplitudes were relatively stable in low concentration of PLB and control group,while b wave amplitude of high concentration group is mild lower,but there was no significant difference.Histology detection showed the rabbit retina in each group had clear structures and normal form.Furthermore,the eyes injected with PLB presented lower PVR severity than the untreated eyes in rabbit models.PLB exhibited a wide safetymargin,indicating no evidence of causing retinal toxicity.Conclusion: PLB effectively inhibited the rabbit PVR in the experimental PVR models without obvious toxic and side effect.