Torularhodin Ameliorates Oxidative Activity in Vitro and D-galactose

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Bioactive Constituents, Metabolites, and Functions

Torularhodin Ameliorates Oxidative Activity in Vitro and D-galactoseinduced Liver Injury via the Nrf2/HO-1 Signaling Pathway in Vivo Chang Liu, Yan Cui, Fuwei Pi, Yahui Guo, Yuliang Cheng, and He Qian J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03847 • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Journal of Agricultural and Food Chemistry

Graphical Abstract

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Torularhodin Ameliorates Oxidative Activity in Vitro and D-galactose-induced Liver

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Injury via the Nrf2/HO-1 Signaling Pathway in Vivo

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Chang Liu, a, b Yan Cui, c Fuwei Pi, a, b Yahui Guo, a, b Yuliang Cheng, *, a, b and He

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Qian, *, a, b

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a

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Technology, Jiangnan University, Wuxi 214122, China.

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b

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.

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c

Institute of Agricultural Products Processing, Key Laboratory of Preservation

State Key Laboratory of Food Science and Technology, School of Food Science and

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Engineering of Agricultural Products, Ningbo Academy of Agricultural Sciences,

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Ningbo 315040, China.

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*Correspondence:

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He Qian ([email protected]) and Yuliang Cheng ([email protected])

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Address: Jiangnan University 1800 Lihu Avenue Wuxi, Jiangsu Province, P. R. China

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Abstract

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Torularhodin is a natural product, extracted from Sporidiobolus pararoseus, and has

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the similar chemical structure to beta-carotene. The antioxidative effects of torularhodin

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were investigated using DPPH, ABTS, and a cell oxidative damage model in vitro, and

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a D-galactose-induced liver-injured mouse model in vivo. Cell experiments

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demonstrated torularhodin had a powerful effect on oxidative damage caused by H2O2

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to AML12 cells. Torularhodin significantly reduced inflammatory cytokines and

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increased the activity of antioxidant enzymes both in mouse serum and liver. The

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inhibition of D-galactose-induced oxidative damage in the liver was correlated with the

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torularhodin-mediated effects on improving the activity of Nrf2/HO-1, reducing the

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expression of Bax and NF-κB p65 by western blot analysis. RT-PCR results

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demonstrated torularhodin upregulated the antioxidative mRNA expression of Nrf2,

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NQO1, and HO-1 in the liver. In summary, torularhodin significantly scavenged free

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radicals and prevented oxidative damage in vitro and reduced D-galactose-induced liver

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oxidation via promotion of the Nrf2/HO-1 pathways in vivo.

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Keywords: Torularhodin; Sporidiobolus pararoseus; antioxidation in vitro and in vivo; liver injury; Nrf2/HO-1 pathways

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INTRODUCTION

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Currently, there is growing interest in the pharmacological potential of natural products

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such as microbial fermentation products.1 Sporidiobolus pararoseus is a facultative

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aerobic yeast and is widely distributed in nature owing to its high adaptability. S.

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pararoseus is widely used in the food processing and production owing to its ability to

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produce many functional metabolites such as extracellular polysaccharides, ergosterol,

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unsaturated fatty acids, and, in particular, carotenoids.2-4 S. pararoseus JD-2 was

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isolated and purified from pepper sauce. Torularhodin is the main carotenoid produced

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by S. pararoseus JD-2, and the differences between its structure and that of β-carotene

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is an extra carboxyl group (Figure 1 A).5-6 Our previous research has shown that

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torularhodin has significant anti-cancer effects both in vitro and in vivo,7-8 and that

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carotenoids of S. pararoseus have the ability to reducing blood fat in mice fed a high-

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fat diet.8-10 However, its biological activity, nutritional function, and, in particular, its

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antioxidant effects in vitro and in vivo have not been well studied.

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Liver disease is one of the deadliest diseases and is commonly caused by a variety

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of factors worldwide. Increasing evidence shows that oxidative damage, which is

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characterized by an imbalance between the reactive oxygen species (ROS) production

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system and the radicals scavenging system, is the leading cause of chronic liver injury.11

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Under this imbalance, oxygen free radicals cannot be scavenged in time and, as a result,

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the ROS content increases excessively, leading to cell dysfunction and damage.12-13 It

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has been shown that an overload of D-galactose can increase the overproduction of

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ROS, resulting in mitochondrial dysfunction, cell dysfunction, hepatocyte apoptosis, 3


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and, ultimately, oxidative and inflammatory damage in mouse livers.14-15 More

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importantly, many studies have shown that the antioxidant properties of natural

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products such as carotenoids can alleviate organ damage caused by D-galactose.16 This

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also indicates that liver damage induced by D-galactose can be alleviated by

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antioxidants.

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Besides, many researches have confirmed that Nrf2 is one of the critical

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transcription factors involved in the regulation of multiple antioxidant mechanisms in

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the antioxidant defense system.17 When the cells are in equilibrium, transcription

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dependent on Nrf2 is inhibited by negative regulatory factor Keap1. However, once the

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cells are exposed to ROS, the Nrf2-Keap1-related signaling pathways are activated.

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Nrf2 is then separated from Keap1 and transferred to the nucleus, where it binds to the

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antioxidant response elements (AREs) in the promoter regions. As a result, a number

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of antioxidative enzymes and phase 2 detoxifying enzymes are activated, such as NQO1

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and HO-1, and it then plays anti-inflammatory, antioxidative, and anti-apoptotic roles.18

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Therefore, the purpose of the present study was to investigate the antioxidative

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effects of torularhodin produced by S. pararoseus against DPPH, ABTS, a cell

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oxidative damage model in vitro, and D-galactose-induced oxidative damage in a

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mouse liver model in vivo. Additionally, the antioxidative mechanisms of torularhodin

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were investigated in D-galactose-induced mouse liver. The results of this study will

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promote a better understanding of the potential health-promoting roles of torularhodin,

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especially in liver health and other oxidation-related chronic diseases.

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MATERIALS AND METHODS

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Materials. S. pararoseus JD-2 (save number: CCTCC M2010326). β-carotene

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standard (95% pure, stored at -80 ℃) was purchased from Sigma (Shanghai, China).

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Corn oil was obtained from Golden dragon fish (Wuxi City, Jiangsu China). Alpha

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mouseliver 12 (AML12) cells were purchased from the cell resource center of the

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Shanghai Institute of Biotechnology (Shanghai, China). Cell cytotoxicity, Proliferation

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Assay and Electrochemical luminescence (ECL) Detection Kit were purchased from

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Beyotime Biotechnology (Haimen City, Jiangsu, China). Commercial kits for catalase

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(CAT), reduced glutathione (GSH), malondialdehyde (MDA), and total antioxidant

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capacity (T-AOC) were obtained from Nanjing Jiancheng Technology Co. (Nanjing

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City, Jiangsu, China); Antibodies, including HO-1, Nrf2, and Bax, were purchased

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from Proteintech (Wuhan City, Hubei, China). Antibodies NF-κB p65 and GAPDH

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were purchased from Abcam (Cambridge, USA). ELISA kits for interleukin 1β (IL-1β)

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and tumor necrosis factor-α (TNF-α) were obtained from Nanjing SenBeiJia Biological

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Technology Co (Nanjing City, Jiangsu, China). Other analytical-grade chemicals were

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purchased from local companies.

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Sample preparation. Torularhodin was obtained from the extract of S. pararoseus

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JD-2 according to our previously published method2-3, 19. First, S. pararoseus JD-2

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stored at -80 ℃ was grown in culture media with horizontal rocking (28 ℃, 24 h, 100

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r/min). S. pararoseus JD-2 in liquid media was incubated and cultured in Petri dishes

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(28 ℃, 48 h). Then, the red colonies were transferred to fresh fermentation media and

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incubated for 72 h (28 ℃, 100 r/min). Second, crude torularhodin in the fermentation 5


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media was extracted and enriched using high-pressure homogenization (80 MPa).

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Finally, torularhodin was isolated and purified using a silica gel column and TLC,

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HPLC, and LC-MS. Torularhodin (purity > 96%) was obtained and identified by HPLC

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method.

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ABTS* radical scavenging activity. Referring to a previously reported method with

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appropriate modification.20 ABTS was dissolved in pure water at a concentration of 7

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mM. ABTS radical cation (ABTS*) was obtained by mixing 7 mM ABTS solution with

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an same amount of potassium persulfate, it was then kept in the dark (4 ℃, 15 h). The

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ABTS* solution was diluted with 100 % ethyl alcohol and the absorbance is 0.70 (±

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0.02) at 734 nm at 30 ℃. Then, 90 % diluted ABTS* solution was added to 10 % of

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sample, which was diluted in ethyl acetate. In the end, reacting for 10 minutes and

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measuring absorbance at 30 ℃. The formula is as follows:

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ABTS* scavenging activity (%) = 100 - [(As - Ab) × 100/Ac]

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As = absorbance of the sample; Ab = absorbance of the blank; Ac = absorbance of the

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control. All experiments were done in triplicate.

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DPPH radical scavenging activity. Referring to a previously reported method with

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appropriate modification.20 Mixing samples diluted in methanol at different

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concentrations with DPPH diluted in methanol (0.04 mg/mL). Reacting for 30 minutes

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and measuring absorbance at 517 nm in the dark at room temperature. The formula is

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as follows:

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DPPH scavenging activity (%) = 100 - [(As - Ab) × 100/Ac]

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As = absorbance of the sample; Ab = absorbance of the DPPH solution, and Ac = 6



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absorbance of the methanol. All experiments were done in triplicate.

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AML12 cell viability. The viability of AML12 was measured by a WST-1 Cell

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Cytotoxicity and Proliferation Assay kit. Torularhodin was diluted with medium (89%

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dulbecco's modified eagle medium + 10% fetal calf serum + 1% Penicillin-

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Streptomycin Solution) prior to the experiment.21 AML12 cells were pretreated with or

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without torularhodin for 18 h in 96-well plates at a density of 1 × 106 cells/mL. Next,

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cells were incubated with H2O2 for 9 h. The plates were incubated (37 °C, 3 h), and the

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absorbance was measured at 450 nm after adding 10 μL of the WST-1 solution to each

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plate.22-23 All the cells were maintained at 37 °C with 5% CO2, passaged every 4 to 6

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days when the cells reached approximately 80% to 90% confluence, and the medium

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was changed twice times each week.

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Animal Experiments. (Figure 1, B) In total, 48 healthy ICR mice (8 weeks old, male,

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20 ± 2 g) [SPF, SCXK (Hu) 2013001823759] were provided by the SiLaiKe Laboratory

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Animal Company (Shanghai, China). Approval was obtained from the Committee of

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Jiangnan University [No. JN. No 20170601-20171121 (68)] to use the animals for the

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following experiments. The mice were placed at a twelve-hours light/dark schedule

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under invariant conditions (23 ± 1 ℃ and 60% humidity) and free to common food and

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water. 48 ICR mice were randomly assigned to six groups, with eight mice in each

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group:

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Control group: Corn oil; saline; Model group: Corn oil; D-galactose dissolved in

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saline; Low-dose group: Torularhodin (0.2 mg/kg) dissolved in corn oil; D-galactose

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dissolved in saline; Medium-dose group: Torularhodin (0.4 mg/kg) dissolved in corn 7


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oil; D-galactose dissolved in saline; High-dose group: Torularhodin (0.8 mg/kg)

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dissolved in corn oil; D-galactose dissolved in saline; and Positive group: Vitamin E

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(VE) (150 mg/kg) dissolved in corn oil; D-galactose dissolved in saline. All the groups

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except the control group were intraperitoneally administered D-galactose (200 mg/kg)

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dissolved in saline (0.9%, w/v) daily for 8 weeks.14, 24 Meanwhile, the mice were fed

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daily with VE or torularhodin dissolved in corn oil before the D-galactose. In the control

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group, all mice were treated with saline solution or corn oil in the same day and manner.

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Determination of body weight and liver indices. The body weights of mice were

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recorded once a week at the same time. At 12 h after the last treatment, all the mice

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were anesthetized by injecting 1% Pentobarbital sodium intraperitoneally at the dose of

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40mg/kg and killed, and blood (from the inferior vena cava) and livers were collected.

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The serum was produced by centrifugation on the speed of 4000 rpm at 4 ℃ for 15 min

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with blood plasma. The liver was weighed immediately, and the liver indices was

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calculated based on the following formula: Liver coefficient (g/g) = organ weight (mg)

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× 100%/body weight (g).

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Determination of biochemical indices in serum and liver. Serum biochemical

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indices detections of TBIL, ALT, AST, and ALP were carried out in the Fourth People’s

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Hospital of Wuxi City (Wuxi, China) on a Murray Biochemical Analyzer 800. The

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levels of IL-6, IL-1β, and SOD in serum and liver were assessed using an ELISA assay

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kit in accordance with the specification. Liver tissues were homogenized in the PBS to

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analyze the BCA, MDA, CAT, and T-AOC levels in accordance with the specification.

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Determination of liver histology. Fresh liver tissues were fixed in 4% 8



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paraformaldehyde solution for 48 h, which was dissolved in PBS buffer, and then

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embedded in hot paraffin. Paraffin, which contained liver tissues, were cut into slices

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with the thickness of 5 μm and stained with hematoxylin and eosin in accordance with

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the standard protocol. The images of liver pathology were observed by an inverted

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microscope (Zeiss, Germany).

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Determination of western blotting. The mouse livers were removed from the -80 ℃

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refrigerators and weighed. Then, RIPA buffer containing the protease inhibitor and a

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phosphatase inhibitor were added to make a homogenate and centrifuged. The

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manufacturer’s protocols were followed, using a BCA Kit (Nanjing, China) to

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determine the protein concentration. The total protein samples were separated on a 10%

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SDS-polyacrylamide gel, and then transferred to a PVDF membrane. After blocking at

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room temperature for 2 h with blocking solution (3% BSA in TBST), membranes were

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incubated with an appropriate dilution of the primary polyclone rabbit antibodies Nrf2,

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HO-1, Bax, NF-κB, and GAPDH overnight at 4 ℃. After 14 h, membranes were washed

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six times with TBST solution and further incubated with secondary antibody for 2 h.

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Next, the membranes were washed six times with TBST solution again. Finally, the

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membranes were stained for ECL detection and observed by chemiluminescence

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analyzer (Tanon-5200, Shanghai, China).

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Determination of real-time quantitative PCR. Total RNA in the mouse liver was

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extracted using the RNA Isolation Kit (Generay Biotech Co., Ltd, Shanghai, China)

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following the specification. Then, the cDNA was generated and reverse-transcribed

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from RNA using the reverse-transcription kit according to the standard protocols of the 9


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RT-PCR kit (Generay Biotech Co., Ltd, Shanghai, China). Detailed steps of

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transcription and extension for PCR response were carried out as published previously8.

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Sequences of qPCR primers used in this study are listed in Table 2.

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Statistical analysis. All data were analyzed using one-way analysis of variance

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(ANOVA) (Tukey’s test). The statistical results are expressed as the means ± standard

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deviation (SD). Data were processed using GraphPad Prism software version 7, and p

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< 0.05 was recognized significant.

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RESULTS AND DISCUSSION

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Influence on antioxidant activities in vitro. It has commonly been assumed

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that the structure of a compound is closely related to its biological activity and

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pharmacological action. Torularhodin belongs to the carotenoids, and the differences

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between its structure and that of β-carotene is an extra carboxyl group (Figure 2). It has

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been confirmed that phytochemical carotenoids have significant antioxidant functions.5,

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25

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scavenging free radicals,26 and torularhodin can be classified as a provitamin A

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carotenoid.27 Thus, the antioxidant function of torularhodin is related to the theoretical

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basis of its chemical structure. However, no relevant research has focused on the

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antioxidant activities of torularhodin in vitro.

Recent studies have shown that vitamin A has a significant antioxidant function in

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DPPH method has been commonly used to test the antioxidant capacity of bioactive

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compounds., due to its hydrogen-donating ability.28 ABTS can be catalyzed to generate

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a stable green free radical ABTS•+ in the presence of potassium sulfate. ABTS method 10



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has been widely used to determine the antioxidant activities by evaluation of ABTS•+

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radical scavenging ability.29 Figure 2 A, B show the results of ABTS and DPPH

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methods.

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The torularhodin exhibited higher radical scavenging activity with the lower IC50

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value of 2.22 μM in the ABTS and 9.61 μM in the DPPH compared with β-carotene,

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which has the IC50 value of 2.82 μM and 10.09 μM. This may mean torularhodin

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exhibited better antioxidant activity than β-carotene. The intercorrelations between the

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radical scavenging activity of the DPPH and ABTS methods are also shown in Figure

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2 A, B.

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The correlation between torularhodin and ABTS was positive with the correlation

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coefficient of 0.9869. Similarly, the correlation between torularhodin and DPPH was

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also positive with the correlation coefficient of 0.9835. The results indicating that the

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radical scavenging ability is related to the dose of torularhodin. These results suggest

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that torularhodin was responsible for the free radical scavenging and antioxidant

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activity. Recent advances in techniques suggested the antioxidant activity of

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carotenoids, which were extracted from different sources of microorganisms, that were

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also correlated with the content of carotenoids.30 This is in line with the results of our

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present study.

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Influence on the viability of AML12 cells exposed to H2O2. In particular,

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a large number of cellular model in vitro studies have helped to establish a connection

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between carotenoids and oxidative damage.31 H2O2 is widely used as an oxidizing

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reagent to trigger oxidative stress. H2O2 administration caused the generation of ROS, 11


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modulation of cellular redox status, and decreasing of cell viability.32 As in the animal

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experiments, D-galactose induced release of free radical production and pro-

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inflammatory cytokines. Thus, the effects of torularhodin on AML12 cells damaged by

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H2O2 were investigated to simulate D-galactose-exposure mice in this study. In our

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study, H2O2 was used to induce oxidative stress at various concentrations for 9 h, and

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cell viability was determined after exposure. In Figure 2 (C), the effect of torularhodin

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at the 6-μmol is similar to that of β-carotene at an 8-μmol concentration on increasing

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AML12 cell viability. The improvement levels of torularhodin were highest at the 8-

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μmol concentrations, respectively (p < 0.001). These results demonstrated that

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torularhodin exerts a dose-dependent antioxidation on AML12 cells exposed to H2O2,

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suggesting that torularhodin is a potential extract in preventing diseases due to oxidative

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damage.

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Influence on body weight, liver indices, and serum biochemical

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functions. The liver is involved in almost all important biological processes,

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including detoxification, the immune response, and bacterial defense. The liver indices

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and serum enzymes are the main indices used to evaluate liver health.33-34 During the

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8-week feeding period, the body weight of each group of mice increased steadily.

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However, it was clear that the control group had a significantly higher weight gain as

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compared with the model group, torularhodin, and VE groups (Figure 1 C). They

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showed the different pattern in the comparison of the liver indices, especially the

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torularhodin (0.8 mg/kg) group (p < 0.001) (Table 1). Other authors have also reported

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that D-galactose triggers a remarkable decrease both in weight and organ indices 12



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compared with normal mice.34 Our results indicated that torularhodin exerted a

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protective effect against D-galactose-induced weight loss in mice.

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The extent of hepatic injury can be evaluated by measuring the serum activities or

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levels of ALT, AST, ALP, SOD, IL-6, and IL-1β.35 Compared with the control group,

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the levels of ALT, AST, ALP, IL-6, and IL-1β in the liver injury mice dramatically

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increased, whereas the level of SOD decreased (p < 0.05, Figure 3). Torularhodin

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decreased the levels of ALT, AST, ALP, IL-6, and IL-1β and increased the level of

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SOD in the serum to varying degrees relative to the control mice. However,

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torularhodin treatment at a dose of 0.8 mg/kg significantly suppressed the release of

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these serum liver function indices as compared to the model group. These results

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suggested that the torularhodin needs to reach a certain dose to have a strong antioxidant

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effect. IL-1β and IL-6 have important regulatory effects in the immune system; they are

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mediators of the acute phase response and regulate hepatic cell interaction and crosstalk

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of the various inflammatory pathways.36 Torularhodin could alleviate the inflammatory

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response in the serum in a manner similar to that of VE because the inflammatory

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cytokines in the serum were significantly inhibited in the torularhodin treatment groups.

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This could explain why torularhodin had a hepatoprotective effect against D-galactose-

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induced liver injury.

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Influence on liver biochemical function. Oxidative stress is the one of the

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key mechanisms of liver injury, and is mainly characterized by the excessive production

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of MDA and the decrease in antioxidant activities of CAT, SOD, and T-AOC.37-39 SOD

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is the major antioxidant enzyme in the antioxidant system, and decomposes to oxygen 13


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and hydrogen peroxide by catalyzing the decomposition of superoxide anions.40 Next,

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the products are further metabolized by CAT.41 T-AOC represents the overall

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antioxidant capacity in the body.42 In Figure 4, compared to the model group, CAT,

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SOD, and T-AOC content of the control group decreased noticeably (p < 0.05), whereas

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the levels of inflammatory cytokines IL-6 and IL-1β increased significantly (p < 0.01).

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However, torularhodin ameliorated strikingly these enzymatic antioxidant activities

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and inflammatory cytokines in the liver by treatment in a dose-dependent manner,

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which was similar to the results of the Vitamin E. The metabolism of D-galactose

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causes oxidative stress accompanied by MDA, which is the common product of

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oxidative stress.43 Compared with the control group, it is important to emphasize that

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D-galactose substantially increased the MDA content in the liver of the model group (p

301

< 0.001) but decreased the MDA content under treatment with torularhodin.

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Interestingly, the effect of torularhodin (0.8 mg/kg) was superior to that of the Vitamin

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E. Our results confirmed that torularhodin can increase the activity of antioxidant

304

enzymes and reduce the production of MDA and inflammatory cytokines in the liver.

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Therefore, torularhodin has potential as a natural product to prevent liver injury caused

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by D-galactose.

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Influence on liver histology. Histological changes in the D-galactose-induced

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mouse livers showed hepatocyte apoptosis, sinusoidal dilatation, necrocytosis, cell

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infiltration, and nucleation.34 As shown in Figure 5, the structure of the control group

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(a) hepatic lobule was normal, with uniformly sized hepatic cell, and the hepatocyte

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section showed visible central thin sinusoids and veins. Compared with the treated 14



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groups, the number of binuclear hepatocytes in the model group was significantly

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increased (b), the liver cells were arranged in disorder, the size of the nuclei varied, and

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some of the nuclei were dissolved. Local hepatocyte swelling resulted in inflammatory

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cell infiltration. In the VE group (c) and TT-low group (d), the swelling of the liver

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cells was alleviated, the nucleus was of a different size, and inflammatory cell

317

infiltration could be seen. However, torularhodin treatment significantly ameliorated

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the hepatic pathological alterations, and the protective effect of torularhodin at 0.8

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mg/kg was the greatest, with minor morphological and structural changes and no

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obvious inflammatory cell infiltration. Taken together, these results proved that

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torularhodin can efficiently improve D-galactose-induced oxidation damage in the liver.

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Influence on the Expression of Nrf2, HO-1 NQO1, NF-𝜅B, and Bax.

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Accumulating studies have shown that an overload of D-galactose can increase the

324

overproduction of ROS, resulting in mitochondrial dysfunction, cell dysfunction,

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hepatocyte apoptosis, and, ultimately, oxidative and inflammatory damage in mouse

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livers.16 In addition, researches have demonstrated that Nrf2 is one of the key

327

transcription factors involved in the regulation of multiple antioxidant mechanisms.17

328

In Figure 7, when the cells are in equilibrium, transcription dependent on Nrf2 is

329

inhibited by negative regulatory factor Keap1. Once the cells are exposed to ROS, the

330

Nrf2 related signaling pathway is activated. Nrf2 is then separated from Keap1 and

331

transferred to the nucleus, where it binds to AREs in the promoter regions. As a result,

332

antioxidative enzymes like NQO1 and HO-1, and it then plays anti-inflammatory,

333

antioxidative, and anti-apoptotic roles.18 15


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Nrf2 is the main redox-sensitive transcription factor, which can upregulate the

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expression of cell protective genes in the oxidative stress response.44 In general, low

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dose of oxidant can induce the expression of Nrf2 genes, whereas high concentrations

337

can decrease the expression of these genes.45 HO-1 plays a key antioxidant role, and

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the expression and activity of HO-1 are low in normal, whereas HO-1 has obvious

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antioxidation effects through the expression of Nrf2 under stress conditions such as

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high/low oxygen.46 Except HO-1, NQO1 is another downstream gene of Nrf2 and a

341

reductase that protects cells from quinone and oxidative stress. However, different

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concentrations of oxidants may further metabolize reduction products of NQO1 and

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show different antioxidant effects.47

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In Figure 6 (D) and (E), our results clearly showed that torularhodin significantly

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increased Nrf2 and HO-1 levels in D-galactose-treated mice livers. In Figure 6 (F),

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expression of Nrf2, NQO1, and HO-1 mRNA in D-galactose-treated mice significantly

347

decreased as compared to the control mice (p < 0.05). Our results observed that mRNA

348

expression of Nrf2, NQO1, and HO-1 markedly increased in the livers of the

349

torularhodin and VE groups compared with the model group, especially the

350

torularhodin (0.8 mg/kg) group (p < 0.01). However, there was no noticeable result in

351

the Nrf2 mRNA expression of the TT-low group relative to the model group. Therefore,

352

the results emphasized that torularhodin can activate Nrf2 from Keap1 and transfer it

353

from the cytoplasm to the nucleus and can increase the expression of HO-1 and NQO1,

354

resulting in a decrease in the production of ROS and the alleviation of oxidative damage

355

in the liver. 16



356

Studies have shown that inflammation is another form of oxidative stress, and the

357

activation of the Nrf2 signaling pathway inhibits the activation of NF-κB and the

358

production of inflammatory cytokines.48 Our study explored the expression of

359

inflammatory cytokines in the liver. Like Nrf2, NF-κB is a fast-inducing transcription

360

factor in many intracellular signaling pathways involved in the stress response, and it

361

is highly sensitive to changes in cell oxidative status, cell transformation, and

362

apoptosis.49

363

In Figure 6 (B), the protein levels and the mean optical densities of NF-𝜅B were

364

higher in the model mice as compared to the control mice (p < 0.01). Torularhodin and

365

VE treatments decreased the protein levels and the mean densities of NF-𝜅B was dose-

366

dependent in D-galactose-induced liver injury in mice (p < 0.05) compared with the

367

model group. By triggering the release of proinflammatory mediators, the activation of

368

NF-κB plays a significant role in the process of liver oxidative stress.50 In this study,

369

D-galactose caused the increased expression of NF-κB p65 protein in the mouse liver.

370

Treatment with torularhodin inhibited the expression of p65 protein in mouse liver,

371

which indicated that torularhodin could inhibit inflammatory responses against D-

372

galactose-induced liver injury via the NF-κB pathway.

373

NF-κB is closely related to inflammatory cytokines. ROS promote the rapid entry of

374

activated NF-κB protein from the cytoplasm to the nucleus, which released

375

inflammatory cytokines including IL-1β and IL-6.51 In present study, D-galactose was

376

injected subcutaneously into mice daily for 8 weeks, resulting in markedly increased

377

concentrations of IL-6 and IL-1β. However, torularhodin inhibited the production of 17


Page 18 of 36

Page 19 of 36


378

inflammatory cytokines and NF-κB in the mouse liver. This is consistent with the

379

results of related studies. Bax is a pro-apoptotic protein that mainly exists in the

380

cytoplasm. Bax migrates to the outer membrane of mitochondria under oxidative

381

stimulation, which results in the permeability of mitochondria and cell apoptosis.35 Our

382

results indicated that torularhodin decreased the protein levels of Bax in a dose-

383

dependent manner in D-galactose-induced liver injury in mice. That suggests that the

384

protective effects of torularhodin might also be the result of inhibition of apoptosis. The

385

deeper mechanisms deserve further study in the future.

386 387

In summary, our study showed that torularhodin produced by S. pararoseus protected

388

against oxidation in DPPH and ABTS assays and cell oxidative damage models in vitro

389

as well as ameliorated oxidative damage in vivo, i.e., in D-galactose-induced liver

390

injury in mice. It demonstrated that torularhodin has the ability to scavenge free radicals

391

along with an antioxidative effect on AML12 cells exposed to H2O2. Furthermore, our

392

study demonstrated that treatment with torularhodin effectively improved the levels of

393

biomarkers in a dose-dependent manner, which suggests that the antioxidative effects

394

of torularhodin on D-galactose-induced liver injury are achieved by promoting the

395

expression of Nrf2, HO-I, NQO1, SOD, and CAT and inhibiting the expression of NF-

396

κB, Bax, IL-1β, and IL-6. Thus, torularhodin effectively promoted antioxidant gene

397

expression and inhibited the expression of pro-inflammatory cytokines, effectively

398

ameliorating mice liver injury induced by D-galactose.

399 18



Page 20 of 36

400 401

Abbreviations: VE Vitamin E, ALT alanine aminotransferase, AST aspartate

402

aminotransferase, ALP alkaline phosphatase, SOD superoxide dismutase, IL-1β

403

interleukin-1 beta, IL-6 interleukin-6, MDA malondialdehyde, CAT activity of

404

catalase, SOD superoxide dismutase, T-AOC total antioxidant capacity, H&E

405

hematoxylin and eosin staining, Nrf2 nuclear factor erythroid 2-related factor 2, HO-1

406

heme oxygenase-1, NF-κB nuclear factor-kappa B, NQO1 quinine oxidoreductase 1,

407

RT-PCR real-time quantitative PCR, ROS reactive oxygen species, TLC thin-layer

408

chromatography, HPLC high performance liquid chromatography, LC-MS liquid

409

chromatography - mass spectrometry, ABTS 2,2′-Azino-bis-3-ethylbenzthiazoline-6-

410

sulphonic acid, DPPH 2,2-Diphenyl-1-picryl-hydrazyl-hydrate, ICR Institute of

411

Cancer Research, RIPA Radio-immunoprecipitation assay buffer, BCA Bicinchoninic

412

acid, PVDF Polyvinylidene fluoride, BSA bovine serum albumin, TBST Tris Buffered

413

Saline Tween, ECL electrochemical luminescence

414 415

Acknowledgments

416

This work is supported by the National Key Research and Development Program of

417

China

418

2017YFC1601806), National Natural Science Foundation of China (No. 31601552,

419

21603087), Natural Science Foundation of Jiangsu Province (No. BK20160178)

420

Forestry Science and Technology Innovation and Extension Project of Jiangsu Province

421

(No. LYKJ [2017] 26) and the National First-class Discipline Program of Food Science

(No.

2017YFC1601704,

2018YFC1604202,

19


2018YFC160015,

Page 21 of 36

422


and Technology (JUFSTR20180509).

423 424

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Pathways in Mice. Oxid. Med. Cell. Longev. 2018, 2018, 8284107.

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27


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586 587

Figure 1. Chemical structures of β-carotene (C40H56) and torularhodin (C40H52O2) (A);

588

Design diagram of mice experiment (B); Effects of torularhodin on the changes in body

589

weight of mice in eight weeks (C).

28



590 591

Figure 2. Effects of torularhodin on DPPH, ABTS, cell oxidative damage model

592

experiments in vitro. *p < 0.05, **p < 0.01, ***p < 0.001 vs model group. 29


Page 30 of 36

Page 31 of 36


593 594

Figure 3. Effects of torularhodin on serum biochemical function. The serum activities

595

or levels of ALT (A), AST (B), ALP (C), SOD (D), IL-1β (E), and IL-6 (F) in mice

596

with D-galactose-induced liver injury. *p < 0.05, **p < 0.01, ***p < 0.001 vs model

597

group.

30



598 599

Figure 4. Effects of torularhodin on liver biochemical function. Hepatic activities or

600

levels of CAT (A), SOD (B), T-AOC (C), MDA (D), IL-1β (E), and IL-6 (F) in mice

601

with D-galactose-induced liver injury. *p < 0.05, **p < 0.01, ***p < 0.001 vs. model

602

group.

31


Page 32 of 36

Page 33 of 36


603 604

Figure 5. Effects of torularhodin on liver HE staining of D-galactose-induced liver

605

injury in mice. Con group (a); Model group (b); Torularhodin (0.2 mg/kg) group (c);

606

Torularhodin (0.4 mg/kg) group (d); Torularhodin (0.8 mg/kg) group (e); Vitamin E

607

(150 mg/kg) group (f). Red arrow indicates inflammatory infiltration; Blue arrow

608

indicates binucleate hepatocytes (original magnification, 200×).

32



609 610

Figure 6. Effects of torularhodin on the expression of Nrf2, HO-1, NQO-1, Bax, and

611

NF-κB in mice with D-galactose-induced liver injury. Representative western blotting 33


Page 34 of 36

Page 35 of 36


612

of Nrf2, HO-1, Bax, and NF-κB in the liver (A); Quantification of NF-κB/GAPDH ratio

613

(B); Bax/GAPDH ratio (C); Nrf2/GAPDH ratio (D); and HO-1/GAPDH ratio (E) in the

614

liver; mRNA expression of Nrf2, NQO1, and HO-1 (F). *p < 0.05, **p < 0.01, ***p

Torularhodin Ameliorates Oxidative Activity in Vitro and D-galactose

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