Ethanol Extract Of Fructus Lycii And Its Constituents Lutein/zeaxanthin Protect Against Age-related Macular Degeneration In Vivo And In Vitro

Objective The aim of this study was to investigate the inhabiting effect of lyceum barbarum extracts on sub-RPE deposit formation in high-fat diet and oral hydroquinone (HQ) model mice in vivo, and the effect of an extract of Fructus lycii (gou qi zi) on Cathepsin B (Cat B)、Cystatin C (Cys C) expression in model mice in vivo, And to investigate the effect of Lutein and Zeaxanthin to hydrogen peroxide on MMP-2, TIMP-2proteasome expression induced by oxidative stress in ARPE-19cells in vitro.Methods In vivo:100eight-month-old C57BL/6female mice, Then mice were divided randomly into aging control model aging control, control (20mice) and3treatment groups (60mice,20mice each group).20were fed regular diet as aging control,80were fed a high-fat diet for6months followed by HQ (0.8%) in the drinking water for3months. Ethanol extract of Fructus lycii were given once-daily through oral administration in different dose (high:3.75g/kg/d, middle:2.50g/kg/d, low:1.25g/kg/d) for3months. At the end of the experimental period, the mice were killed and the eyes immediately removed. Transmission electron microscopy was used to observe sub-RPE deposit formation and Bruch membrane (BrM) thickness, semiquanttative grading of deposit severity was performed, The expression and location of Cat B and Cys C were examed by immunohistochemistry, The mRNA expression Cat B and Cys C were detected by real-time PCR and and the protein expression Cat B and Cys C were detected by Western blot, respectively. In vitro:Methylthiazolyltetrazolium was used to detect the effects of different concentrations of Hydrogen peroxide and Lutein and Zeaxanthin for ARPE-19cells, then the expression and distribution of MMP-2, TIMP-2proteasome in cells were detected by Western blot.Results In vivo:sub-RPE deposit formation:Compared to the aging control group, the sub-RPE deposit in the model control group mice significantly higher (P＜0.01), but Fructus lycii ethanol extract decreased the score in a dose-dependent fashion and at high and middle dose also led to a significant effect, at low dose led to a significant effect compared to the model mice (P＜0.05). The Bruch membrane of the model control was significantly thicker than that of aging control mice (P<0.01), Fructus lycii ethanol extract led to significantly reduced the thickness of Bruch membrane in a dose-dependent manner compared to the model mice (all P<0.01). Compared to the aging control, the mRNA expression of Cat B and Cys C were significantly higher in the model control mice (P<0.01), the mRNA expression of Cat B and Cys C were down-regulated by the extract of Fructus lycii (P<0.01). Compared to the aging control, the protein expression of Cat B and Cys C were significantly higher in the model control mice (P<0.05), At middle and high dose led to a significant effect compared to the model mice (P＜0.05), But at low dosen’t led to a significant effect compared to the model mice (P＞0.05). In vitro:cell viability assay showed that H2O2reduced ARPE-19cell proliferation dose-dependently and at1、10、100μM have no ignificant effect compared to the control cells (P>0.05), and produced a significant effect at200μM compared to the control cells (P<0.05), and also produced a significant effect at300、400、500μM compared to the control cells (P<0.01). Lutein/zeaxanthin at30、50、70μM produced a significant effect compared to the cells treated with H2O2alone (P<0.05). Compared to the control, the protein expression of MMP-2and TIMP-2were significantly higher in the model control (P<0.05).The protein expression of MMP-2also produced a significant effect at30、50、70μM compared to the model control(P<0.05at30、50, P＜0.01at70μM). The protein expression of TIMP-2at30μM led to no significant effect compared to the model mice (P>0.05), and have significant effect at50、70μM(P<0.05at50μM, P<0.01at70μM).Conclusion Fructus lycii ethanol extract could reduce RPE sediment and restore Bruch membrane in vivo leading to the improvement in AMD histopathology, and could down-regulated the expression of Cat B and Cys C in high-fat diet and oral HQ model mice. Lutein and Zeaxanthin could down-regulated the expression of MMP-2, TIMP-2proteasome in ARPE-19cells which treated by hydrogen peroxide first.