Catalpol Protects ARPE-19 Cells against Oxidative Stress

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Catalpol Protects ARPE-19 Cells against Oxidative Stress ( catalpol-protects-arpe-19-cells-against-oxidative-stress )

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Cells 2021, 10, 2635 2 of 24 of the hallmarks of early AMD [9,10]. A growing corpus of evidence suggests that antiox- idants, by inhibiting the production of ROS, protect RPE cells from oxidative stress, thereby playing a key therapeutic role in early AMD [11]. Therefore, investigations on the antioxidant pathways involved in retinopathy are crucial for the development of new therapies for AMD. Nrf2-mediated activation of the antioxidant pathway is one of the key endogenous defense mechanisms for maintaining cellular redox balance [12]. Overproduction of intra- cellular ROS stimulates the dissociation of Nrf2 from Keap1 and its translocation to the nucleus, where Nrf2 binds to its downstream antioxidant response elements (AREs) to trigger the transcriptional activation of many phase II detoxifying and antioxidant en- zymes, such as HO-1, glutaredoxin 1 (Grx1), CAT, and NQO1, thereby mitigating cellular redox imbalance caused by oxidative stress [13–16]. On the other hand, the uncontrolled and continuous generation of ROS further leads to DNA damage and mitochondrial dys- function, thereby activating downstream caspase-dependent apoptotic pathway [17,18]. In addition, it was confirmed that activation of Nrf2 signaling pathway could inhibit ROS- mediated apoptosis in RPE cells. [19]. Therefore, the development of novel antioxidant drugs targeting the activation of the Keap1/Nrf2/ARE antioxidant pathway is considered a promising approach to the treatment of AMD and other degenerative diseases involving a delicate intracellular oxidant–antioxidant imbalance. Catalpol, an iridoid glucoside extracted from the dry roots of Rehmannia glutinosa Libosch, has been widely used to treat AMD and other degenerative diseases [20,21]. Re- cent studies indicate that catalpol exerts various biological properties, such as antioxidant, anti-apoptosis, neuroprotective, and anti-inflammation effects [22–24]. Although the mechanism involved in the treatment of certain diseases with catalpol has been gradually understood in the past few years, the potential of catalpol in the treatment of retinal dis- eases is still unknown. The purpose of this study was to investigate whether catalpol could protect RPE cells from oxidative stress damage and to further elucidate its role in the an- tioxidant defense mechanism. This study provides a novel therapeutic strategy for treat- ing AMD or slowing down its progression. 2. Materials and Methods 2.1. Reagents and Chemicals Catalpol (purity > 98:72%) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), 0.05% trypsin, Dulbecco’s Modified Ea- gle Medium: Nutrient Mixture F-12 (DMEM/F-12), and penicillin/streptomycin solutions were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), N-Acetylcysteine (NAC) and hydrogen peroxide (H2O2) were bought from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and 3-(4,5- dime- thyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Beijing Solarbio Science and Technology Co. Ltd. (Beijing, China). ROS, MMP, LDH, TUNEL, Apoptosis and Cell Cycle detection kits were purchased from Beyotime Biotechnology (Shanghai, China). 2.2. Cell Cultures and Treatment Human retinal pigment epithelial cells (ARPE-19) were purchased as frozen vials from BeNa Culture Collection (Beijing, China), and all cell experiments were performed between the third and fifth generations. The cells were cultured in DMEM/F-12 (supple- mented with 10% FBS, 1% streptomycin/penicillin) at 37 °C in a humidified atmosphere containing with 95% air and 5% CO2. Cells at 80-90% confluence were selected for subcul- ture and subsequent experimentation. For H2O2-induced apoptotic studies, the cells were cultured overnight in DMEM/F12 containing 2% FBS and then incubated in serum-free medium for 30 min, followed by exposure to H2O2 (400 μM) for 6 h. Catalpol was dis- solved in DMSO and stored at 4 °C. Under the experimental conditions, the concentration

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