Journal of Functional Foods 40 (2018) 785–797 Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff The isoflavones and extracts from Maclura tricuspidata fruit protect against T neuronal cell death in ischemic injury via induction of Nox4-targeting miRNA-25, miRNA-92a, and miRNA-146a Sungeun Honga, Jaeyoung Kwonb,c, Nguyen Tuan Hiepb,d, Su Jin Sime, Nahyun Kime, ⁎ ⁎ Kyeong Ho Kimf, Dongho Leeb, , Woongchon Mara, a Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea b Department of Biosystems and Biotechnology, Korea University, Seoul 02841, Republic of Korea c Natural Constituents Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute, Gangneung 25451, Republic of Korea d Department of Extraction Technology, Vietnam National Institute of Medicinal Materials, 3B Quang Trung, Hoan Kiem, Hanoi, Viet Nam e ForestMedicinal Resources Research Center, Korea Forest Research Institute, Yeongju-si 36040, Republic of Korea f Collegeof Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea ARTICLE INFO ABSTRACT Keywords: This study evaluated the neuroprotective effects 50% EtOH extract of Maclura tricuspidata fruits (FME50) and Maclura tricuspidata isolated compounds, cudraisoflavone I (CFI), and cudraisoflavone H (CFH), on ischemic damage in in vitro model MiroRNAs and in vivo model of cerebral ischemia. FME50, CFI, and CFH inhibited OGD/R + glucose-induced neuronal cell Oxygen-glucose deprivation/ death, ROS generation, and Nox4 expression via induction of Nox4-targeting miRNA-25, miRNA-92a, and reoxygenation + glucose miRNA-146a in SH-SY5Y cells. Also, FME50 suppressed OGD/R + glucose-induced activation of ASK1-JNK1/ Middle cerebral artery occlusion/reperfusion p38 MAPK signal cascade. Furthermore, FME50 significantly reduced the MCAO/R-induced brain infarct, Nox4 NADPH oxidase MAPkinase expression via induction of Nox4-targeting three miRNAs. Additionally, FME50 suppressed MCAO/R-induced MAPK signal pathway. These results demonstrate that FME50, CFI, and CFH exert neuroprotective effects via Nox4 inhibition by the induction of Nox4-targeting miRNAs and inhibition of MAPK signal cascade, suggesting that they might be possible candidates for the treatment of cerebral ischemia. 1. Introduction against 6-hydroxydopamine-induced cell death in SH-SY5Y cells (Hiep et al., 2015). Maclura tricuspidata Carrière (formerly known as Cudrania tricuspi- Cerebral reperfusion injury initially caused by ischemia is due to data, Moraceae) is a tree widespread in Korea, China, and Japan, and insufficient blood supply to the brain, leading to poor oxygen supply the ripe fruit can be eaten fresh or processed into pies, sherbets, tarts and eventually neuronal cell death. After the ischemic period, re- and wines. M. tricuspidata contains prenylated isoflavonoids, benzylated perfusion or reoxygenation is essential for survival of neuronal cells. flavonoids, xanthones, quercetin and kaempferol (Han et al., 2009; However, the restoration of blood circulation results in the over- Hiep et al., 2015; Kim et al., 2009; Lee et al., 1996; Lee et al., 1995), production of mitochondrial reactive oxygen species (ROS) in neuronal and it has been shown to have anti-atherosclerotic, anti-inflammatory cells and thereby, causes the cellular damage observed in neurological (Park et al., 2006), hepatoprotective (Tian, Kim, Cui, & Kim, 2005), diseases, such as stroke (Chouchani et al., 2014). Mitochondrial ROS neuroprotective (Jeong et al., 2010), antihypertensive (Kang et al., have been reported to be able to be formed by xanthine oxidase, cy- 2002), and antiallergic activity (Lee, Kwon, Lee, & Mar, 2015), as well tochrome P450, nitric oxide synthase, and nicotinamide adenine di- as pancreatic lipase inhibitory activity (Jeong et al., 2014). Cudraiso- nucleotide phosphate oxidase (Nox). The human genome encodes flavone I (CFI) and cudraisoflavone H (CFH) from the fruit of M. tri- seven members of the Nox family (Nox1 - Nox5, Duox1 and Duox2), cuspidata have previously been reported to have neuroprotective effects and Nox is a membrane-bound enzyme complex composed of six Abbreviations: FME50, 50% EtOH extract of Maclura tricuspidata fruits; CFI, cudraisoflavone I; CFH, cudraisoflavone H; Nox, NADPH oxidase; OGD/R + glucose, oxygen-glucose deprivation/reoxygenation + glucose; MCAO/R, middle cerebral artery occlusion/reperfusion; MAPK, mitogen-activated protein kinases; ASK1, apoptosis signal-regulating kinase 1; JNK1, c-Jun N-terminal kinases1; ROS, reactive oxygen species ⁎ Corresponding authors. E-mail addresses: [email protected] (D. Lee), [email protected] (W. Mar). https://doi.org/10.1016/j.jff.2017.12.011 Received 28 March 2017; Received in revised form 30 November 2017; Accepted 5 December 2017 1756-4646/ © 2017 Elsevier Ltd. All rights reserved. S. Hong et al. Journal of Functional Foods 40 (2018) 785–797 subunits with different patterns of expression (Sumimoto, Minakami, & (Cat#SC-47778) primary antibodies and secondary antibodies were pur- Miyano, 2011). When Nox4 expression is stimulated by external stress chased from Santa Cruz Biotechnology, Inc. (CA, USA). The miRNA mi- or brain injury, the integral membrane protein p22phox forms a het- mics and inhibitors (miRNA-25(Cat#4426961), miRNA-92a erodimeric functional complex with Nox4 to generate ROS (Bedard & (Cat#4426961), miRNA-146a(Cat#4448892)) were purchased from Krause, 2007), suggesting that ROS generated by the Nox4 activation Applied Biosystems (Foster City, CA, USA). plays a major role in cerebral ischemia. Recent studies have reported that Nox activity is connected with expression of Nox-targeting miRNAs 2.2. UPLC chromatogram of the 50% EtOH extract from the Maclura (Liu et al., 2016). MiRNAs are small noncoding RNAs with 19–25 nu- tricuspidata fruit (FME50) cleotides and regulate basic cellular functions such as proliferation, differentiation, and death (Hwang & Mendell, 2006). MiRNAs are All samples were analyzed using an Acquity UPLC system (Waters, known to play an important role in many pathological processes in the Millford, MA, USA) equipped with an Acquity BEH C18 column brain (Adlakha & Saini, 2014), and some miRNAs are expressed in (2.1 mm × 150 mm i.d., 1.7 μm). The mobile phases consisted of 0.05% neuronal cells. Among them, three different miRNAs (miRNA-25, formic acid in water (A) and acetonitrile (B), with a flow rate of 0.3 ml/ miRNA-92a, and miRNA-146a) are highly related in the regulation of min. The initial eluent was 35% B, and its proportion was increased Nox4 mRNA expression (Fu et al., 2010; Tsao, Behren, Cebon, & linearly to 95% B for 10 min, held constant at 100% for 11.5 min, re- Christophi, 2015; Wang et al., 2014). These miRNAs modulate protein turned to the initial composition for 10.5 min, and then held constant expression by binding the complementary target mRNA, initiating de- for 1.5 min to re-equilibrate the column. The sample injection volume gradation or translational inhibition of the target mRNA (Jeyaseelan, was 4 μl for extracts and 2 μl for compounds. The column and sample Lim, & Armugam, 2008). Silencing of Nox4 mRNA expression has been managers were set at 35 and 15 °C, respectively, and the UV wavelength reported to inhibit ROS production (Wang et al., 2014) and inhibit was monitored at 265 nm. hypoxia-induced activation of apoptotic signals such as ASK1, JNK1, and p38 (Mkaddem et al., 2010). Ischemia-induced ROS are in part 2.3. Plant material and sample preparation attributable to the activation of mitogen-activated protein kinases (MAPKs). Apoptosis signal-regulating kinase 1 (ASK1) is an upstream The fruits of Maclura tricuspidata was collected from the Korea regulator of the MAPK cascade and activated by various stresses in- Forest Research Institute, Southern Forest Research Center (Jinju, cluding oxidative stress, ER stress, calcium influx, receptor-mediated Korea). A voucher specimen (accession No. KH1-5-090904) was de- inflammatory signals and ischemic injuries (Ichijo, Fujisawa, & Takeda, posited at the Department of Biosystems and Biotechnology, Korea 2007). ASK1 activates c-Jun N-terminal kinase 1 (JNK1), known as a University (Seoul, Korea). The dried fruits of M. tricuspidata (3.44 kg, mediator of neuronal degeneration in response to stress, and phos- yield 18.88%, w/w) were ground and sifted through a 120-mesh sieve. phorylates p38 mitogen-activated protein kinase, one of the apoptosis A portion of the samples (7.0 g) was refluxed three times by means of a factors related to MAPK (Irving & Bamford, 2002). JNK1 is known to heating mantle with 250 ml of each 0, 30, 50 70, and 100% ethanol for bind and phosphorylate the DNA-binding protein c-Jun and elevate its 1 h. Each extract were combined, filtered, and concentrated in vacuo to transcriptional activity, and p38 kinase is expressed in most cell types obtain various samples (2.85 g, 2.71 g, 2.58 g, 2.67 g, and 2.15 g, re- and regulates the activity of many cytokines. Inhibition of these pro- spectively). The chemical investigation led to the isolation of nine teins induces cell survival; therefore, proteins in the MAPK pathway are isoflavones, namely, cudraisoflavone I (1, 0.0007%), cudraisoflavone H targets for drug development (Johnson & Lapadat, 2002). (2, 0.0013%), gancaonin A (3, 0.0086%), 6,8-diprenylorobol (4, Previously, we reported that isoflavones and extracts from the fruit 0.0015%), alpinumisoflavone (5, 0.0762%), 4′-O-methylalpinumiso-