| Our experiments firstly investigated that hesperetin exhibited antioxidant andanti-inflammatory activities through using t-BHP-induced oxidative stress andLPS-stimulated inflammation in RAW264.7cells. Then, we employed H2O2-inducedARPE-19damage to further investigate the protective effect of hesperetin on oxidative stressand inflammatory damage and its pharmacological mechanism, and also tested the effect ofhesperetin in primary cultured RPE cells. It will provide a new strategy for the prevention andtreatment of AMD. In present study, depending on by the use of technique of enzyme-linkedimmunosorbent (ELISA), Western Blot, reverse transcription polymerase chain reaction(RT-PCR), flow cytometry (FCM) and Co-Immunoprecipitation (Co-IP) from the cell，molecular level and signal transduction level, we investigated the mechanism of action ofhesperetin.The following is the study outcomes:(1) In this study, hesperetin markedly protected against t-BHP-induced oxidativecytotoxicity and ROS production via activating the Keap1/Nrf2/ARE signialling pathway andupregulating antioxidative enzymes including HO-1, NQO1and GST expression in RAW264.7cells. In addition, hesperetin significantly inhibited LPS-induced TNF-α, IL-6, IL-1β,iNOS and COX-2genes expression in RAW264.7cells, which suppressed the activation ofMAPK and NF-κB signaling pathway.(2) RPE cells subjected to oxidative stress and inflammatory damage may be a key factorresulting in the development and progression of AMD. Hence, based on above outcome, weused H2O2-induced ARPE-19damage to further investigate the protective effect of hesperetinon antioxidative stress and anti-inflammatory damage. These results suggested that hesperetindisplayed the action of antioxidant stress by upregulating antioxidative enzymes HO-1, NQO1and GST expression, reducing H2O2-induced SOD and GSH depletion as well as MDAproduction in ARPE-19cells. In addition, hesperetin exhibited the role of anti-inflammatory by inhibiting H2O2-induced TNF-α, IL-1β， IL-18and IL-6genes expression, whichsuppressed the activation of MAPK and NF-κB signaling pathway. Therefore, the protectionof effect of hesperetin on ARPE-19cells damage is the effective inhibition of H2O2-inducedcell apoptosis, ROS generation and cell viability. However, hesperetin markedly inhibitedH2O2-induced cell viability and ROS production, blocked cytokines production, which werereversed by SnPP treatment in ARPE-19cells.(3) Our results indicated that hesperetin significantly upregulated Nrf2-mediated HO-1protein expression via activating the Keap1/Nrf2/ARE signialling pathway in ARPE-19cells.Herein, hesperetin evidently suppressed H2O2-induced Txnip, IL-1β and IL-18genesexpression, but it has no effect on NLRP3,Caspase-1and ASC protein expression when H2O2inactivated HO-1and Nrf2protein expression and activated Txnip, NLRP3and Caspase-1protein expression. In addition, Hesperetin evidently blocked a H2O2-induced significantincrease in ASC and Caspase-1interacted with NLRP3, and Txnip associated with NLRP3aswell as enhanced a H2O2-induced significant decrease in Txnip interacted with Trx, whichwere eliminated by SnPP treatment. In last, our results suggested that hesperetin dramaticallyupregulated Nrf2/HO-1protein expression and decreased H2O2-induced cell apoptosis anddeath in primary cultured RPE cells.Together, this study first demonstrated that hesperetin protected from H2O2-inducedARPE-19cells damage through decreasing H2O2-induced cell viability, ROS generation andcytokines production, which activated Nrf2-mediated HO-1protein expression to inhibit theactivation of Txnip and the association of TXNIP with NLRP3, which triggers the subsequentactivation of the NLRP3inflammasome. Therefore, to activate the Nrf2/HO-1as a target,hesperetin exert its antioxidant and anti-inflammatory activity, which is expected to become anew way of prevention and treatment of AMD. |
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