Inhibtiory Effects Of Grape Seed Extract On Inflammatory Cytokines In RPE Cells

Age-related macular degeneration?AMD?is an eye disease that occurs in the elderly?aged 50 years and older?.Symptoms of AMD often manifest as blindness caused by impaired central vision.Macular area in the eyes can provide a clear central vision,and retina is the"light receiver"of the eye.AMD has a serious impact on both macular area and retina,and can even lead to blindness.Drusen,with?-amyloid as the main components,will be gradually accumulated in human eyes with the increase of age,which can activate self-protective responses such as oxidative stress and endoplasmic reticulum stress,thus causing oxidative damage or other chronic diseases in the body.In addition to medical treatment,dietary supplementation of antioxidants has become an important approach of preventing or alleviating age-related diseases.Studies have shown that grape seed extract?GSE?are rich in polyphenolic compounds and are highly effective antioxidants and anti-inflammatory agents.Therefore,the use of dietary intake of GSE to interfere with oxidative damage and to delay the aging of human body has practical research value.This paper constructed a A?_?1-40?-induced oxidative damaged animal model and a A?_?1-40?-induced cell injury model to explore the endoplasmic reticulum stress in vivo and vitro.Whether GESs have a regulatory effect on ER-stress,as well as on inflammation response,was studied in animal and cell models.The main research results include:1.Construction of A?_?1-40?-induced retina-injured mice model.AFM images showed that when the polymerization time was 72h,A?_?1-40?formed a fibrillar structure in addition to the granular structure.When the polymerization time was 96h,A?_?1-40?is completely fibrous.The oligo-A?_?1-40?was intravitreally injected into the mouse eyes to construct an AMD animal model.2.Grape seed extract had a protective effect on the retina of the mouse model induced by A?_?1-40?.TUNEL showed that A?_?1-40?can induce apoptosis of retinal pigment epithelium cells while the apoptosis of PRE cells significantly reduced in mice treated with GSE gavage.H&E staining indicated that A?_?1-40?induced cell dysfunction in the ganglion cell layer?GCL?,inner nuclear layer?IN?and outer nuclear layer?OL?,with an increased thickness of cell layer.Besides,the cell structure of the RPE layer was lost,with a decreased thickness of cell layer.The above changes were all inhibited after administration of GSE.GSE inhibits the overexpression of inflammatory factors in PRE-choroid layer induced by A?_?1-40?.Real-time PCR showed that A?_?1-40?induced an increase in inflammatory factors IL-12,IL-1?,IL-6 and IL-18in RPE-choroid complexes compared with non-A?_?1-40?group,which were4.6,2.2,3.6 and 2.6 times higher than those in sham operation group.The upregulation was significantly down-regulated after GSE administration by32.6%,22.7%,30.5%and 26.9%in low GSE gavage group,39.1%,18.2%,39.8%and 30.8%in high GSE gavage group.3.GSE inhibits A?_?1-40?-induced endoplasmic reticulum stress in APRE-19 cells,thereby reducing the expression of inflammatory cytokines.GSE inhibits the expression of inflammatory factors in APRE-19 cells induced by A?_?1-40?.The levels of IL-12,IL-1beta,IL-6,IL-18 and IL-8 in APRE-19 cells were 1.98,3.31,2.89,3.45 and 2.55 times higher than those in the control group,which were all down-regulated after further administration of GSE dose-dependently.When the concentration of GSE was 10 ug/mL,the levels of five inflammatory factors were 52.6%,48.4%,55.7%,78.6%and 43.9%lower than those of the model group,respectively.Western Blotting showed that the activation of ATF6,the phosphorylation of IRE1 and the splicing level of XBP1 decreased in the cells treated with appropriate concentration of GSE.Furthermore,GSE up-regulates the expression of GRP78,a key molecule chaperone of ER stress-UPR,suggesting that GSE may inhibit ER stress by up-regulating GRP78 expression,thus inhibiting the over-expression of inflammatory factors in RPE cells.In summary,GSE can inhibit the expression of inflammatory cytokines by regulating the IRE1-XBP1 signaling pathway of endoplasmic reticulum stress and increasing the expression level of molecular chaperone GRP78,thus inhibiting cellular inflammatory response.