antioxidants Article Inhibitory Effect of Nelumbo nucifera Leaf Extract on 2-Acetylaminofluorene-induced Hepatocarcinogenesis Through Enhancing Antioxidative Potential and Alleviating Inflammation in Rats Mon-Yuan Yang 1, Tung-Wei Hung 2,3, Chau-Jong Wang 4,5,* and Tsui-Hwa Tseng 6,7,* 1 Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung 40201, Taiwan 2 Division of Nephrology, Department of Medicine, Chung Shan Medical University Hospital, Taichung 40201, Taiwan 3 School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan 4 Department of Health Diet and Industry Management, Chung Shan Medical University, Taichung 40201, Taiwan 5 Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan 6 Department of Medical Applied Chemistry, Chung Shan Medical University, Taichung 40201, Taiwan 7 Department of Medical Education, Chung Shan Medical University Hospital, Taichung 40201, Taiwan * Correspondence: [email protected] (C.-J.W.); [email protected] (T.-H.T.); Tel.: +886-4-24730022 (ext. 11670) (C.-J.W.); +886-4-24730022 (ext. 12230) (T.-H.T.) Received: 8 July 2019; Accepted: 19 August 2019; Published: 22 August 2019 Abstract: Leaf extract of Nelumbo nucifera (NLE) has been demonstrated to possess anti-atherosclerosis, improve alcohol-induced steatohepatitis, prevent high-fat diet-induced obesity, and inhibit the proliferation and metastasis of human breast cancer cells. This study determines the chemopreventive role of NLE against 2-acetylaminofluorene (AAF)-induced hepatocellular carcinoma (HCC) in rats. AAF was used to induce hepatocarcinogenesis in rats through genetic and nongenetic effects. After administration for 12 weeks, NLE (0.5–2%) supplementation orally inhibited AAF (0.03%)-induced hepatic fibrosis which appears during the development of premalignant lesions in rats. After the 6-month experiment, NLE supplementation resulted in decreasing AAF-induced serum parameters of hepatic injury, including the level of triglycerides, total cholesterol, alpha-fetoprotein (AFP), and inflammatory mediator interleukin (IL)-6 and tumor necrosis factor (TNF)-α as well as the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γGT). NLE supplementation also reduced AAF-induced lipid peroxidation and 8-hydroxy-20-deoxyguanosine (8-OHdG) formation in the rat liver. Hepatic histopathological investigation revealed that NLE supplementation attenuated the AAF-induced HCC and glutathione S-transferase-Pi (GST-Pi) expression. Furthermore, NLE supplementation increased the expression of transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream targets, including catalase, glutathion peroxidase (GPx), and superoxide dismutase 1 (SOD-1) in the rat liver. Our findings indicate that NLE supplementation inhibited AAF-induced hepatocarcinogenesis by enhancing antioxidative potential and alleviating inflammation in rats. Keywords: 2-acetylaminofluorene; antioxidant enzymes; hepatocellular carcinoma; inflammation; Nelumbo nucifera leaf extract; oxidative stress Antioxidants 2019, 8, 329; doi:10.3390/antiox8090329 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 329 2 of 15 1. Introduction Hepatocellular carcinoma (HCC) is a common malignant tumor and has a high mortality rate worldwide. Because most HCC patients are frequently diagnosed at an advanced stage, the overall 5-year survival rate of HCC is estimated to be below 20% [1]. In addition, the recurrence rate can be as high as 50%. The development of HCC is a complex process involving the accumulation of genetic and epigenetic alteration, which passes through states of initiation, promotion, and progression, with both viral and chemical carcinogens involved in the multistate process. The onset of HCC is usually late in patients with chronic liver diseases such as hepatitis B and C infections and dietary carcinogen exposure. Accordingly, hepatic fibrosis occurs when liver is repeatedly and continuously injured. Damage to hepatocytes triggers the release of reactive oxygen species (ROS) and inflammatory mediators, which would stimulate carcinogenic process [2]. Untreated fibrosis may progress to liver cirrhosis, leading to organ failure and hepatoma [3,4]. Therefore, approaches on antioxidant, anti-inflammation, and anti-fibrosis are considered the effective strategy for lowering the morbidity and mortality of HCC. Nelumbo nucifera Gaertn (Nelumbonaceae), commonly referred to as lotus, is a widely distributed crop in central and southern Taiwan. In Southeastern Asia, lotus seed and rhizomes are usually considered popular dietary staples, and lotus leaf is also used as a common food item in Taiwan. Additionally, all parts of N. nucifera, including the leaves, flowers, stamens, embryos, and rhizomes, have been used as medicinal herbs in Eastern Asia and exhibit pharmacological properties such as antipyretic, antidiabetic, antioxidative and antiobesity effects [5–10]. In addition, Nelumbo nucifera leaf extract (NLE) was demonstrated to have remarkable effectiveness in reducing the development of atherosclerosis [10], preventing high-fat diet-induced obesity and body fat accumulation [11], improving alcohol-induced steatohepatitis [12], and inhibiting the proliferation and metastasis of human breast cancer cells [13,14]. Moreover, our study found that NLE alleviated high-fat diet-induced hepatic injuries in hamsters [15]. Our previous study demonstrated that NLE contains phenolic compounds including phenolic acid and flavonoids [12,13]. In related studies, gallic acid induces HepG2 cell apoptosis [16] and protects against diethylnitrosamine-induced rat hepatocellular carcinoma [17]. Catechin prevents the development of hepatic neoplasma by modulating several signal transduction and metabolic pathways [18]. Rutin suppresses epidermal growth factor receptor (EGFR) kinase activity [19] and inhibits N-nitrosodiethylamine-induced HCC in Wistar rats [20]. Isoquercitrin inhibits the growth of HepG2 cells and xenograft tumor growth in nude mice [21]. Miquelianin promots doxorubicin-induced apoptosis under endoplasmic reticulum (ER) stress in HCC cells [22]. Astragalin reduces the proliferation of HCC cells and inhibits the growth of xenograft tumors in vivo [23]. These reports imply that NLE may exert antihepatoma potential. However, the protective effects of NLE on oxidative stress-triggering hepatocarcinogenesis remain unclear. Therefore, we investigated the effects of NLE on hepatocarcinogenesis by 2-acetylaminofluorene AAF, which can induce liver cancer through genotoxic and nongenotoxic process associated with promoting oxidative stress in rats [24,25]. 2. Materials and Methods 2.1. Chemicals and Reagents The leaves of N. nucifera Gaertn. were purchased from Paiho Farmers’ Association Organization in Tainan County, Taiwan. Formic acid (reagent grade, 96%) was purchased from Tedia (Fairfield, OH, USA). Acetonitrile and methanol (high-performance liquid chromatography-grade, Darmstadt, Germany). The liquid chromatography grade polyphenol standards including isoquercitrin, peltatoside, and miquelianin were obtained from Extrasynthese (Genay, France). AAF, bovine serum albumin, dithiothreitol, ethylenediaminetetraacetic acid, formaldehyde, N-(2-hydroxyethyl) piperazine-N’-ethanesulfonic acid, phenylmethylsulfonyl fluoride, and sodium phosphate were purchased from Sigma (St. Louis, MO, USA). Antibodies against catalase, glutathion peroxidase (GPx), superoxide dismutase 1 (SOD-1), and nuclear factor erythroid 2-related factor 2 (Nrf2) were purchased Antioxidants 2019, 8, 329 3 of 15 from Cell Signaling Technology (Beverly, MA, USA), and β-actin as internal control was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated antibodies against rabbit IgG or mouse IgG were purchased from Sigma. 2.2. Preparation of NLE and HPLC Analysis NLE was prepared using a method similar to that in our previous paper and analyzed using HPLC [12,13]. Briefly, dried N. nucifera leave (20 g) was resuspended in 100 mL of distilled water at 4 ◦C for 12–16 h. The supernatant was filtered and concentrated under reduced pressure. Finally, the concentrated solution was lyophilized as NLE. HPLC analysis were using a Waters system which consisted of a HPLC controller and pump (Waters 600), a detector (2998 PDA) and an in-line degasser AF. A systemic procedure for analyzing the polyphenols contained a Mightysil RP-18 GP 250 column (Kanto, Tokyo, Japan), mobile phase solvent A (formic acid in water, pH = 2.5), solution B (acetonitrile) and solution C (methanol). HPLC method was flowing with gradient program: 0 min, 100% A; 5 min, 90% A, 0% B; 15 min, 85% A, 3% B; 20 min, 85% A, 5% B; 30 min, 85% A, 8% B; 50 min, 85% A, 15% B; 65 min, 75% A, 25% B; 70 min, 70% A, 30% B; 80 min, 50% A, 50% B; 85 min, 100% B; 100 min, 100% B. Flow rate was 1.0 mL/min and injection volume was 20 µL. The UV detection range was on 210–400 nm. 2.3. Animals Maintenance and Treatment Male Wistar rats, aged 4–5 weeks and weighing 140–160 g, were purchased from the National Laboratory Animal Center (Taipei, Taiwan), kept at a constant temperature between 22 and 24 ◦C with a 12 h light/12 h dark cycle. All protocols were approved by the Institutional Animal Care and Use Committee of Chung Shan Medical University (IACUC, CSMU, approval no. 1070). Rats were fed with Laboratory Rodent Diet 5001 (PMI Nutrition International) containing 23.0% crude protein, 4.5% crude fat, 6.0%