| Objective: To observe and discuss the effect of lipid oxidative damage induced by ox-LDL on(non)exudative Age-Related Macular Degeneration disease.Meanwhile demonstrate inhibiting effect of Salvianolic acid A(Sal A)on Retial Pigment Epithelium(RPE)cells inflammation and Choroidal Neovascularization(CNV)progression.Method: 1.Establish animal models of high lipid oxidative damage: Male SD mice were randomized into four groups: control,ox-LDL and ox-LDL + Sal A group.Each group undergone injections of ox-LDL(4mg/kg)or PBS intravenously,with(without)Sal A(5 mg/kg or 10mg/kg)intraperitoneal injection once per day for 1 week.C57bl6/J mouse were randomized into control,ox-LDL,Sal A and ox-LDL + Sal A group.Each group undergone injections of ox-LDL(4mg/kg)or PBS intravenously,with(without)Sal A(10mg/kg)intraperitoneally once per day for 1 week.2.According to former method,SD mice were sacrificed and eyes were enucleated 7 days after injection.Paraffin sections were used to detect ox-LDL lipidosis and apoptosis in retina by immunofluorescence and TUNEL analysis;Toluidine blue staining was used to assess retinal photoreceptor cells apoptosis.3.Establish choroidal neovascularization model in C57 mouse.After continuous ox-LDL and Sal A injection for 7 days.C57 mouse were anesthesized and CNV model was induced by 530 nm laser photocoagulation.4.7 days after fundus laser,C57 mouse were anesthesized and FFA examination was used to evaluate fluorescence leakage grade and CNV area.Then eyes were enucleated,choroid flatmouts were produced to analysis CNV volume.Pathological sections were used to analyse CNV width and length.5.Induce chronic lipid oxidative damage to ARPE-19 cells:ARPE-19 cells were randomized into Control(serum free medium),ox-LDL(100mg/L)group and ox-LDL plus Sal A(5uM/50uM)group and cultured for 24 hours.Cells were pre-administrated with Sal A 3 hours before ox-LDL stimulation.IL-1?/18 and TNF-? levels,MAPK and P2X7R-PKR-NLRP3 pathway were evaluated;Nrf2 protein and PI3K/AKT/mTOR pathway were also assessed when studying anti-oxidation and anti-inflammation effect of Sal A.6.Induce chronic lipid oxidative damage to ARPE-19 and HUVECs cells.Angiogenesis and anti-angiogenesis factors,ZO-1 and CYLD protein expression were detected 48 hours after ox-LDL and Sal A stimulation.CYLD Function in modulating angiogenesis process was also studied.Results: 1.7 days after intravenous injection of ox-LDL in animals increased serum total and ox-LDL cholesterol concentrations.Immunofluorescence experiment demonstrated obvious lipidosis and apoptosis in retinal.Sal A pre-treatment prohibited ox-LDL induced high concentrations of serum lipid proteins and protected retina from ox-LDL induced lipidosis and apoptosis.2.Ox-LDL injection increased fluorescence leakage grade and areas compared with PBS group 7 days after laser.Pathological sections and choroid flatmounts revealed that CNV volume was larger in ox-LDL group than control;Sal A pre-treatment significantly reduced CNV volume and fluorescence leakage severity.3.Ox-LDL increased IL-1?/18 and TNF-? levels,meanwhile activated MAPK and P2X7R-PKR-NLRP3 signal in ARPE-19 cells after 24 hours;Sal A pre-treatment exerted anti-oxidation and anti-inflammation functions by promoting Nrf2 expression and activating PI3K/AKT/mTOR pathway.4.Ox-LDL up-regulated VEGF/PDGF levels in ARPE-19 cells after 48 hours,meanwhile disrupted ZO-1 integrity and promoted tube formation in HUVECs cells.Sal A pre-treatment inhibited VEGF/PDGF expression and promoted Anti-angiostatin expression,maintained RPE cells junction integrity and inhibited tube formation in HUVECs.5.Ox-LDL induced angiogenesis via activating CYLD function.Sal A prohibited angiogenesis by down-regulating CYLD and inducing P62-CYLD-TRAF6 interaction.Conclusion: 1.Intravenous injection of ox-LDL established animal model of hyperlipemia and is used to study machenisms of oxidative damage and chronic inflammation in RPE layer of dry-AMD patients.2.Chronic lipid oxidation damage mediates chronic inflammation in RPE layer,Sal A protected RPE from inflammation oxidation damage.3.High circulating level of ox-LDL promotes exacerbate CNV.Sal A pretreatment inhibited CNV progression.4.Lipid oxidation damage promotes angiogenesis process via activating CYLD.Sal A inactivates CYLD function and promotes P62-CYLD-TRAF6 interaction,which restrained angiogenesis process.5.Salvianolic acid A could be a potential therapeutic medicine in treating AMD clinically... |
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