The Experimental Study Of Delphinidin To Prevent Age-related Macular Degeneration In Vitro

BACKGROUNDWith the aging of the population,age-related macular degeneration (ARMD) is becoming a public health concern and there will be more elderly persons who blind because of ARMD than glaucoma and diabetic retinopathy.The excitement associated with the advances in antiangiogenic therapy for patients with ARMD has been tempered by the clinical findings that after successful regression of subretinal neovascularization,visual improvement has been limited.In both the TAP (Treatment of ARMD with Photodynamic Therapy) and the VIP (Verteporfin in Photodynamic Therapy) studies,only 5%and 8%of patients,respectively,gained vision compared with control subjects after the treatment of subretinal neovascularization.As a result,it is becoming increasingly clear that new approaches for treatment should be focused on both preventing the initial insults that lead to disease progression and rescuing the retinal pigment epithelium(ARPE-19) and photoreceptor cells that have been damaged.Nowdays medicine of food has become more and more popular in prevention and treatment of many diseases.Of the 520 new drugs approved between 1983 and 1994,39%were natural products,of which 60—80%were antibiotics and anti-cancer drugs from natural products.Of the 20 best-selling non-protein drugs in 1999,9 were derived or developed as the result of leads generated by natural products.Some medicine have come into being such as ocuvite for preventing ARMD,but the expensive price and mainly function for anti-oxidant has limit it's applying.Delphinidin as one part of anthocyanidin,has many effect for anti-oxidant,anti-angiogenicanti-carcinosis and anti-inflammatory properties which may be helpful for the prevention and treatment of many diseases,but has not been elucidate could the effect to antioxidant on RPE cells and inhibited the angiogenesis on RF/6A(rhesus choroidsretina endothelial cell line)cells.We think delphinidin have these functions on RPE and RF/6A cells,then we can find an effective prevention for ARMD.OBJECTIVE1)To investigate the effect of delphinidin on anti-oxidative stress injury and the relative mechanisms in ARPE-19.2) To investigate the efect of delphinidin on expression of VEGF (vascular endothelial growth factor)and the relative mechanisms in RPE and RF/6A cells induced by hypoxia mimic cobalt chloride.3) To investigate the effect of delphinidin on angiogenesis and the relative mechanisms in cultured RF/6A cells.METHODS1) MTT was used to determine the effect of delphinidin on ARPE-19 cell survival and proliferation at a series of concentration.Cells was pretreated with delphinidin(1,5,10,20μM ) for 24 hours then under H₂O₂ induced-stress-injury.The morphodifferentiation of apoptotic nuclei was analyzed by Hoechst-PI fluorescence staining.The concentration of ROS and JC-1 was measured by ROS and JC-1 kit.The mRNA expression of bcl-2 was measured by RT-PCR.2) The primary cultured RF/6A cells was identified by immunohistochemical method.MTT was used to determine the effect of delphinidin on RF/6A cell survival and proliferation at a series of concentration.The RPE and RF/6A cells was pretreated with delphinidin(1,5,10,20μM )for 24 hours then under COCl₂ induced-hypoxic condition.The expression of VEGF,HIF-1αand HIF-2αprotein were measured by Western-blot.3) Cells was pretreated with delphinidin(1,5,10,20μM ) for 24 hours then under VEGF-stimulating.The cell proliferation was assessed using MTT after 24 hours.Cell migration was investigated by transwell chamber and tube formation on matrigel by endothelial cells was also analyzed.The protein expression of p-FAK and cell apoptosis was measured by western-blot and caspase-3 immunohistochemical method. manner between the concentration of 1μM～20μM.As compared with negativ group,bcl-2 mRNA were significantly up-regulated in 5μM～20μM delphinidin pretreated group(P＜0.01),while in 1μM delphinidin pretreated group(P＜0.05).2) Immunohistochemical method identificated RF/6A cell was endothelial cell.Delphinidin at the concentration of 10^-6M～10^-1M didn't inhibit the proliferation of RF/6A cells in vitro.While delphinidin inhibits VEGF and HIF-1αprotein in a dose-dependent manner.As compared with negativ group,VEGF and HIF-1αprotein were significantly down-regulated in 5μM～20μM delphinidin pretreated group(P＜0.01) and in 1μM delphinidin pretreated group(P＜0.05),while HIF-2αprotein didn't change compared with negativ group(P＞0.05).3) Delphinidin inhibits VRGF-stimuated RA/6A cell proliferation, migration,tube formation and protein expression of p-FAK in a dose-dependent manner.As compared with negativ group,cell proliferation,migration,tube formation and protein expression of p-FAK were significantly down-regulated in 5μM～20μM delphinidin pretreated group(P＜0.01),while in 1μM delphinidin pretreated group(P＜0.05). Caspase-3 immunohistochemical show delphinidin inhibit cell proliferation was not through apoptosis.CONCLUSION1) Delphinidin exerts a potent anti-oxidative effect in vitro and the relative mechanism may be associated with the inhibition of the function of mitochondrion.2) Delphinidin could inhibit the expression of VEGF under hypoxic condition,and the relative mechanism may be associated with the inhibition of HIF-1α,while not associated with HIF-2α.3) Delphinidin exerts a potent antiangiogenic effect in vitro and the relative mechanism may be associated with the inhibition of p-FAK.