Protective Effect Of Curcumin On H₂O₂-induced Oxidative Stress In ARPE-19 And Its Mechanisms

Age-related macular degeneration（AMD） is a class of age-related macular diseases, which involved the retinal pigment epithelial and choroid, leads to visual loss, especially progressive loss of central vision, and is more prevalent among seniors. AMD is the leading cause of severe visual loss in developed countries. Although the pathogenesis of AMD consists of aging process, oxidative stress, inflammation, autoimmune and imbalance of growth factors, oxidative damage is the most important one. As retinal pigment epithelial（RPE） phagocytize photoreceptor outer-segment discs constantly, and produce large amount of oxidized lipids and H₂O₂ exposure to longperiod photo oxidation, RPE runs high risk of oxidative damage and its damage can compromise the function of photoreceptors. The aging process and compromised antioxidant capacity could make ocular tissues more vulnerable to oxidative stress. The surge of oxidative stress and compromised antioxidant capacity leads to abnormal RPE structure and RPE dysfunction, which is the well-known pathogenesis of AMD.Curcumin（diferuloylmethane）, a relatively low-molecular weight polyphenol, is the active essential of Curcuma longa. Curcumin is well-documented for its anti-oxidative, antimicrobial, anti-proliferation and anticancer potentials. Curcumin exerts its effects by modulation of gene expression, nuclear factors, cytokines, micro RNA profiles and enzymes. Micro RNA and nuclear factors play important roles in anti-oxidative mechanisms of curcumin.To investigate the protective effect of curcumin on oxidative stress in RPEs, we established oxidative stress model in ARPE-19 cell line by exposure to hydrogen peroxide, and investigated whether mi R199a/Nrf2 was involved in this mechanism. Then we observed the changes of Nrf2 target genes’ products, such as HO-1, γ-GCS, SOD and NQO1 in the protective effect curcumin against oxidative stress. To elucidate the role of mi R199 a in this protective mechanism, we mimicked mi R199 a and counteracted mi R199 a by transfection ARPE-19 cells with mi R199a-mimic sequence and anti-mi R199 a sequence respectively. Role of p38 MAPK in this anti-oxidative mechanism was further investigated.The elucidation of curcumin protective effect on H₂O₂-induced oxidative stress in ARPE-19 can provide new perspectives and ground breaking lights in prevention and treatment of AMD. Part ICurcumin can alleviate HⅠ2O2-induced oxidative stress in ARPE-19cellsObjectives: To establish oxidative stress model in ARPE-19 cells by exposure to series concentration of H₂O₂, to determine the appropriate intervention levels of curcumin, and to investigate whether curcumin can protect ARPE-19 cells from oxidative damage induced by H₂O₂.Methods: We investigated the cell viability with Cell Titer-Blue and changes of MDA, SOD, GSG-Px induced by exposure ARPE-19 cells of 5th-7th generation to series concentration of H₂O₂（10μmol/L, 50μmol/L, 100μmol/L and 500μmol/L） for 18 h, then the model of oxidative stress was established. We quantified the cell viability of ARPE-19 cells after exposure to series concentration of curcumin（5μmol/L, 10μmol/L, 20μmol/L and 30μmol/L） for 6h to select the appropriate intervention levels of curcumin, and then investigated the protective effect of curcumin on oxidative stress in ARPE-19 cells induced by H₂O₂.Results: Exposure to 100μmol/L H₂O₂ for 18 h was efficient to decrease ARPE-19 viability, upregulate MDA level and compromise viability of SOD and GSH-Px, while pretreatment with 10μmol/L curcumin for 6 h can alleviate all these stresses.Conclusions: H₂O₂ can induce oxidative stress in ARPE-19 cells, as indicated by the increase of MDA and decrease of SOD and GSH-Px capacity, and compromise the viability of ARPE-19 cells. Curcumin pretreatment can alleviate H₂O₂-induced oxidative stress in ARPE-19. Part IIRole of mi R199a/Nrf2 pathway in the alleviation of HⅡ2O2-inducedoxidative stress in ARPE-19 by curcuminObjectives: To determine the expression of Nrf2, HO-1, γ-GCS and NQO1 at both m RNA and protein levels, and the expression of mi R199 a in ARPE-19 after exposure to H₂O₂ and/or curcumin.Methods: After exposure to 0μmol/L curcumin for 6h plus 0μmol/L H₂O₂ for 18 h, 10μmol/L curcumin for 6h plus 0μmol/L H₂O₂ for 18 h, 0μmol/L curcumin for 6h plus 100μmol/L H₂O₂ for 18 h,or 10μmol/L curcumin for 6h plus 100μmol/L H₂O₂ for 18 h, expression of Nrf2, HO-1, γ-GCS and NQO1 at both m RNA and protein levels, and expression of mi R199 a were determined in each condition.Results: Curcumin alleviated the oxidative stress in ARPE-19 cells by counteracting the upregulation of mi R199 a, and downregulation of Nrf2 and its target genes at both m RNA and protein levels caused by H₂O₂.Conclusions: Curcumin exerted its protective effect against H₂O₂-induced oxidative stress in ARPE-19 cells by inhibiting mi R199 a expression, upregulating Nrf2, HO-1, γ-GCS and NQO1 at both m RNA and protein levels. Part ⅢModulation of Nrf2 and target genes of Nrf2 by mi R199 a inARPE-19 cellsObjectives: To investigate the effects of mi R199 a on Nrf2 and target genes of Nrf2 in ARPE-19 cells by construction and transfection mi R199 a mimic RNA sequence and anti-mi R199 a RNA sequence.Methods: We constructed mi R199 a mimic RNA sequence and anti-mi R199 a RNA sequence to mimic and inhibit mi R199 a expression respectively. After transfection ARPE-19 cells with mi R199a-mimic or anti-mi R199 a, the expression of Nrf2, HO-1, γ-GCS and NQO1 at both m RNA and protein levels were measured retrospectively.Results: Transfection with mi R199a-mimic can represent the effect of mi R199 a and inhibit the expression of Nrf2 and its target genes at both m RNA and protein levels, while transfection with anti-mi R199 a can increase the expression of Nrf2, HO-1, γ-GCS and NQO1 at both levels, and mimic the effect of curcumin.Conclusions: Transfection with mi R199a-mimic represented the upregulation of mi R199 a, and transfection with anti-199 a mimicked the effect of curcumin. Modulation of Nrf2 and target genes of Nrf2 by curcumin was mediated by mi R199 a, at least partially. Part ⅣEffect of curcumin on nuclear translocation of Nrf2 and role ofp38MAPK in the modulation of Nrf2 by curcuminObjectives: To elucidate the effect of curcumin on distribution and nuclear translocation of Nrf2, and to determine the role of p38 MAPK in the modulation of Nrf2 and its target genes by curcumin.Methods: Nuc-Nrf2 and T-Nrf2 were determined after exposure to 0μmol/L, 10μmol/L, 20μmol/L or 30μmol/L curcumin in ARPE-19 cells. Expression of Nrf2, HO-1, γ-GCS, and NQO1 were measured after pretreatment with 0μmol/L or 20μmol/L SB203580 for 2 h and the subsequent culture with 0μmol/L or 10μmol/L curcumin for 6h.Results: Curcumin increased the expression of Nrf2 and enhanced the nuclear translocation of Nrf2 concurrently. SB203580, a specific inhibitor of p38 MAPK, inhibited the phosphorylation of p38 MAPK, and the effect of curcumin on Nrf2 and its target genes.Conclusions: Curcumin increased the expression of Nrf2 and enhanced the nuclear translocation of Nrf2. p38 MAPK could be involved in the Nrf2-mediated antioxidant effect of curcumin.