Comparison of the hepatoprotective effects of the three main stilbenes

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Bioactive Constituents, Metabolites, and Functions

Comparison of the hepatoprotective effects of the three main stilbenes from mulberry twigs Jia Ya Nan, Peng Ya Lin, Zhao Yi Ping, Cheng Xi Fei, Zhou You, Chai Chun Li, Zeng Lin Shu, Pan Min Hui, and Li Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07245 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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Journal of Agricultural and Food Chemistry

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Comparison of the hepatoprotective effects of the three main stilbenes from mulberry

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twigs

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Ya-Nan Jia1, Ya-Lin Peng1, Yi-Ping Zhao1, Xi-Fei Cheng1, You Zhou1, Chun-Li Chai1, Ling-Shu

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Zeng2, Min-Hui Pan1, Li Xu1,*

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1College

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of Biotechnology, Southwest University, No. 2 Tiansheng Road, BeiBei District, Chongqing 400715, China;

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Chongqing sericulture science and technology research institute, No. 1 Shangba Road, Dongyang

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Street, Beibei District, Chongqing 400700, China;

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* Li Xu: Tel: 86-13996005452; Fax: 86-23-68250191; Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: The purpose of this study was to compare the hepatoprotective effects of Oxy

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(oxyresveratrol), Res (resveratrol), and MulA (mulberroside A) (80 mg/ kg body weight/d, i.g.) on

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acute liver injury (ALI) induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN) in mice.

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After 7 h of LPS (50 μg/kg body weight, i.p.) and D-GalN (500 mg/kg body weight, i.p.) exposure,

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the activities of serum transaminases and antioxidant enzymes were determined. The expressions of

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Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signal

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pathway, nuclear factor-kappa B (NF-κB) signal pathway and Mitogen-activated protein kinase

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(MAPK) signal pathway related proteins were evaluated by western blot assays. Histopathological

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analysis was performed by hematoxylin-eosin (H&E) staining on the separated livers of mice. The

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results showed that treatment with Oxy, Res, and MulA could significantly decrease the levels of

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alanine transaminase (ALT) and aspartate transaminase (AST) (P < 0.01). MulA was the most

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effective ingredient among the three and the ALT and AST levels were reduced at 90.3 ± 1.3% and

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93.9 ± 1.1% compared with LPS/D-GalN treated group (P < 0.01). Meanwhile, the stilbenes curbed

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the expression of inflammatory factors, NF-κB pathway activation and MAPKs phosphorylation, and

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upregulated antioxidant enzymes, Nrf2, NAD (P) H:quinone oxidoreductase (NQO1) and heme

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oxygenase-1 (HO-1) expression levels. Stilbenes might protect the ALI caused by LPS/D-GalN

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through inhibiting the negative effectiveness of oxidation stress and inflammation. The protective

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performance of MulA was better than those of Oxy and Res, and we hypothesize that which might be

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due to the mediation of specific metabolic pathway of the MulA in vivo. All of these results implied

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that stilbenes in mulberry twigs might be promising as natural additives.

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KEYWORDS: mulberry, hepatoprotective, resveratrol, oxyresveratrol, mulberroside A

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INTRODUCTION

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Mulberry (Moraceae, Morus alba L.), a traditional industrial tree, has been cultivated

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widespread in China. Mulberry twigs have been recorded in the Chinese ancient pharmacopoeia for

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their application in various diseases. Many researchers have indicated that mulberry twigs contain

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abundant secondary metabolites, which exhibit many bioactivities (1-3). However, due to the

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insufficient development of mulberry twigs, they are generally treated as agricultural waste or used

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as fuel, resulting in a great waste of resources. Recently, there has been interest in stilbenes, and

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mulberry twigs are commonly studied due to their rich stilbenes (4).

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Res (trans-3,4′,5-trihydroxystilbene, Fig. 1A) is a natural polyhydroxy distyrene compound

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found in more than 70 plant species, including Morus alba L., Arachis hypogaea L., Vitis vinifera,

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and Polygonum cuspidatum (5-7). Res has become known for its antioxidant properties,

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anti-inflammatory properties, and its neuroprotective and general health-improving abilities in

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mammals (8-10). Oxy，trans-2',3,4',5-tetramethoxystilbene, Fig. 1B, generally speaking, is primarily

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distributed in plants, such as Artocarpus and Moraceae in the Moraceae (11). Oxy is the major

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stilbene in mulberry, which is a natural structural analog of Res with one more phenolic hydroxyl

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group than Res at the 2' position. The biological activities of Oxy include antioxidant,

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anti-inflammatory,

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(2,5'-dihydroxy-4,3'-bis(beta-D-glucopyranosyloxy)-trans-stilbene, Fig. 1C) is the Oxy diglycoside,

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in which 3′-OH and 4-OH are replaced by glucose molecules, which have many biological activities

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and pharmacological functions within antioxidant, anti-inflammatory, antivirus, and anti-tyrosinase

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activities (4, 16-18).

antiviral,

and

cell

protection


activities

(12-15).

MulA


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ALI is an intricate inflammatory disease caused by multi-factor, including microbial infections

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and alcohol or drug-induced liver injuries (19). The sudden loss of the metabolic and immune

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function of the liver could lead to symptoms, such as jaundice, hepatic encephalopathy,

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coagulopathy, and multiple organ failure. Due to significant mortality, researchers have begun to

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examine liver diseases more closely, and these diseases have become a serious worldwide public

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health problem.

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To the best of our knowledge, there are no reports comparing the hepatoprotective actions that

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Oxy, Res, and MulA have against LPS/D-GalN-induced ALI. In consequence, this research aims to

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examine the potential protective actions that Oxy, Res, and MulA have on LPS/D-GalN-induced

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hepatic damage through exploring hepatic antioxidant, anti-inflammatory, and histopathologic events

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in a mouse model.

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MATERIALS AND METHODS

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Chemicals

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Res standard (CAS 501-36-0, >99.0% purity) was introduced from Chengdu Must

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Biotechnology Co., Ltd. (Chengdu, China). Standards of Oxy (CAS 29700-22-9, >99.0% purity) and

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MulA (CAS 102841-42-9, >99.0% purity) were purchased from Shanghai Yuanye Biotechnology

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Co., Ltd. (Shanghai, China). D-GalN was got from Aladdin Reagent Database, Inc. (Shanghai,

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China). LPS was provided by Sigma-Aldrich (Shanghai, China). Silymarin (Sily, 75.0%~80.9%

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purity) was provided by MADAUS AG Co. (Chongqing, China).

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Materials

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The kit of protein extraction and RBITC-conjugated Goat anti-Rabbit IgG were purchased from


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Sangon Biotech Co., Ltd (Shanghai, China). The primary antibodies of Toll-like receptor 4 (TLR4),

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myeloid differentiating factor 88 (MyD88), IκB kinases β (IKKβ), inhibitor kappa B α (IκBα), p65,

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interleukin-1β (IL-1β), interleukin-6 (IL-6), Keap1, Nrf2, NQO1, HO-1, c-Jun N-terminal kinase

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(JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, β-actin and Lamin B were supplied

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by Proteintech Group, Inc (Wuhan, China). The primary antibodies of p-IKKβ, p-IκBα, p-ERK,

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p-JNK, and p-p38 were procured from Cell Signaling Technology (Beverly, MA, USA). Diagnostic

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kits for ALT, AST, catalase (CAT), malondialdehyde (MDA), and superoxide dismutase (SOD) were

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provided by Nanjing Jiancheng Technology Co., Ltd. (Nanjing, China).

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Animals and ethical statement

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The male Kunming mice (20 ± 2 g) obtained from Sbf (Beijing) Biotechnology Co., Ltd.

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(SCXK(Jing)2014-0006) (Beijing, China). The mice were got under favorable humidity degree

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(50%) and temperature (25 °C), easily to get food and sterile water. All of the experimental

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guidelines of animals were approved by the Committee of Southwest University Laboratory Animal

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Management and animal handling followed the dictates of the National Animal Welfare Law of

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China.

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Mice treatments

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Mice treatments were performed as described previously with some adjustments (20). The mice,

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after 7 days of adaptation to the environment around, were in random assigned in six groups (n = 10

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per group). The mice in control group and LPS/D-GalN group were intragastrically (i.g.) pretreated

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with normal saline. The Oxy group, Res group, and MulA group were administered with Oxy, Res,

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and MulA (80.0 mg/kg body weight/d, dissolved in normal saline, i.g.) for 6 days, respectively. The



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Sily group pretreated with Sily (54.0 mg/kg body weight/d, dissolved in normal saline, i.g.). After 6

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h of the sixth treatment, the mice of the LPS/D-GalN group, stilbene groups and Sily group were

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attacked by intraperitoneal (i.p.) injection of LPS (50.0 μg/kg body weight) and D-GalN (500 mg/kg

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body weight). An experimental evaluation was made based on the sacrifice of relevant animals with

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anesthesia after 7 h’s intraperitoneal injection of the LPS/D-GalN.

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Serum determinations

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Following the commercial kits instructions (Nanjing Jiancheng Biotechnology Institute,

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Nanjing, China) the serum levels of ALT, AST, CAT, SOD, and MDA were determined.

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Histopathological analysis

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Histopathological analysis was performed as the previous description (20). Liver tissues from

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several mice per group were fixed in 10% formaldehyde solution, embedded in paraffin, cut into 5

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μm-thick continuous sections, and stained with H&E. The tissue damage was assessed by a

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microscope (Nikon, Tokyo, Japan).

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Western Blotting

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Western blotting analysis was performed as the previous description (20). The proteins of liver

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tissue were extracted according to the manufacturer’s instructions (Sangon Biotech Co. Ltd.,

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Shanghai, China), separated by 12% SDS-PAGE and transferred to PVDF membranes. The primary

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and secondary antibodies were incubated at 4 ºC overnight. The blots were visualized using ECL

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reagents and quantitated by Image Jet (1.43) software.

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Statistical analysis


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All the data are revealed being the mean ± S.D. It was determined using ANOVA in SPSS 19.0.

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The research of one-way variance (ANOVA) was followed by Least Significant Difference (LSD)

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test, Duncan’s Multiple Range (DMR) test, and Student Newman-Keuls (SNK) test. P < 0.05 was

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considered statistically significant.

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RESULTS

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Effects of Oxy, Res and MulA on the growth of mice

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As shown in Table 1, the liver weight (LW) and the liver index (liver weight/body weight;

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LW/BW) of each group were not of significant difference (P > 0.05), indicating that the

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pre-administration with Oxy, Res, and MulA did not affect the normal growth of the mice.

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Effects of Oxy, Res and MulA on liver histopathological changes in mice

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According to the H&E staining section of the mouse liver tissue in Fig. 2, the liver tissue of the

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control group was intact. The structure of the hepatic lobule was normal. There was no swelling,

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edema, or steatosis, and the hepatocytes were arranged in a strip-like shape centering on the central

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vein. The hepatocyte boundaries were obvious. The nucleus was round and clear. The cytoplasm was

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rich, and there was no inflammatory cell infiltration in the hepatic lobule or the portal. The structure

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of the liver tissue revealed in LPS/D-GalN group disappeared. The hepatocytes were obviously

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swollen, edematous, and degenerated, and there were large amounts of inflammatory cell infiltration

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in the hepatic lobules and portals. The degree of hepatocyte degeneration and necrosis was

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significantly reduced by the drug treatments. The inflammatory cell infiltration was significantly

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reduced. The liver tissue structure was basically intact, and the hepatic lobule structure was clear.

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The differences in the liver tissue morphology between the MulA, the Res, and the Oxy mice were

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not significant, and further experimental validation is required.



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Effects of Oxy, Res and MulA on mice’s liver function

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AST and ALT are important indicators for detecting liver function in mice. As observed in Fig.

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3, ALT and AST levels in LPS/D-GalN group were significantly higher than those in control group

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(**P < 0.01), showing that the ALI model in mice was successfully constructed. The ALT and AST

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levels in the stilbene groups were significantly less than the LPS/D-GalN group (##P

Comparison of the hepatoprotective effects of the three main stilbenes

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