Arch. Pharm. Res. DOI 10.1007/s12272-014-0370-0

RESEARCH ARTICLE

Aloin protects against chronic alcoholic liver injury via attenuating lipid accumulation, oxidative stress and inflammation in mice Yan Cui • Qing Ye • Heya Wang • Yingchao Li Xiuhua Xia • Weirong Yao • He Qian

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Received: 26 September 2013 / Accepted: 13 March 2014 Ó The Pharmaceutical Society of Korea 2014

Abstract The present study was designed to investigate the protective effect of aloin against alcoholic liver disease in a chronic alcohol feeding mouse model. Mice were given alcohol twice a day by intragastric administration for 11 weeks (4.0, 4.7, 5.5 g/kg bw/day for the first 3 weeks respectively, 6.3 g/kg bw/day for the following 8 weeks). Aloin (10, 30 mg/kg bw) or vehicle was given by gavage to mice after each alcohol administration. Alcohol elevated the serum transaminases alanine aminotransferase, aspartate aminotransferase, total cholesterol and triglyceride levels which were significantly attenuated by the co-administration of aloin (p \ 0.05). Histopathological observations were consistent with these indices. Co-administration of aloin significantly suppressed the alcohol-dependent induction of sterol regulatory element-binding protein-1c expression (p \ 0.01) and remarkably up-regulated the mRNA levels of AMP-activated protein kinase-a2 (p \ 0.001). Furthermore, aloin supplementation significantly inhibited the alcoholdependent elevation of malondialdehyde and cytochrome P4502E1 expression (p \ 0.05), and significantly elevated superoxide dismutase activity (p \ 0.01). The up-regulation of serum lipopolysaccharide (LPS), hepatic nitric oxide, tumor necrosis factor a, toll-like receptor-4, and myeloid differentiation primary response gene 88 were also markedly suppressed by the co-administration of aloin (p \ 0.05) in alcohol-treated mice. These results suggest that aloin may represent a novel, protective strategy against chronic alcoholic liver injury by attenuating lipid accumulation, oxidative stress and LPS-induced inflammatory response.

Y. Cui  Q. Ye  H. Wang  Y. Li  X. Xia  W. Yao  H. Qian (&) School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, China e-mail: [email protected]

Keywords Aloin  Chronic alcoholic liver injury  Hepatoprotection  Lipid accumulation  Oxidative stress  Inflammation

Introduction Chronic alcohol consumption, the major cause of alcoholic liver disease (ALD), results in steatosis, alcoholic hepatitis and fibrosis/cirrhosis (Albano 2008; Tuma 2002). Due to its role in increasing morbidity and mortality, ALD has already become a significant health and economic problem all over the world. Although the underlying mechanisms are still not well understood, increasing evidence indicates the involvement of lipid accumulation, oxidative stress, and endotoxin lipopolysaccharide (LPS)-induced inflammatory injury in the development of ALD (An et al. 2012; Ding et al. 2012; Noh et al. 2011). Therefore, treatments with antioxidants and anti-inflammatory agents such as vitamin C, vitamin E, and bicyclol which enhance hepatic antioxidant and anti-inflammatory capacities have been tried in an attempt to improve alcoholic liver injury (Hu et al. 2009; Yanardag et al. 2007; Zhao et al. 2008). Recently, agents developed from natural products and traditional medicinal plants which possess hepatoprotective effects have become increasingly attractive in the prevention and therapy of ALD (Ding et al. 2012; Kanuri et al. 2009; Rejitha et al. 2012). Aloe is a classical traditional medicinal plant which contains multiple bioactive components. Aloin, also known as barbaloin, is the major anthraquinone obtained from the leaf exudates of Aloe ferox Mill. and Aloe vera L. (Groom and Reynolds 1987; Patel et al. 2012; van Wyk et al. 1995), and is a natural C-glucoside of aloe-emodin anthrone (10glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)-

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Fig. 1 Chemical structure of aloin

anthracenone, Fig. 1). A study conducted by Park et al. (2009a) on the intestinal absorption of active aloe components in vitro showed that aloin has low bioavailability, and only about 5.5 % is absorbed. As it is well known, aloin has an excellent laxative effect, and participates in many important physiological functions, including anti-oxidation and anti-inflammation (Beppu et al. 2003; Esmat et al. 2012; Park et al. 2009b, 2011, 2012). However, the application of aloin is strictly limited due to its possible adverse effects including laxative property, cytotoxicity and possible carcinogenesis (Avila et al. 1997; Bottenberg et al. 2007; Esmat et al. 2006; Koch, 1996). Recently, Chandan et al. (2007) showed that the aqueous extract of Aloe vera L., which contained aloin, exerted a potential hepatoprotective effect against carbon tetrachloride-induced liver injury, possibly through its antioxidant activity. In addition, previous research also proved that aloin had a potential effect on accelerating the rate of alcohol oxidation (Chung et al. 1996). However, to the best of our knowledge, few studies have explored the protective effect of aloin against ALD and its underlying mechanisms. In our previous studies, aloin showed significant protective effects against acute alcohol-induced liver injury, and effectively alleviated hangover and inhibited alcohol absorption in mice (Ye et al. 2012). In the present study, we investigate the possible protective effect of aloin against ALD in a mouse model of chronic alcoholic hepatotoxicity. The potential underlying mechanisms such as lipid accumulation, oxidative stress, LPS-induced Kupffer cell activation, and the signal pathways involved are also examined.

Materials and methods Materials and chemicals Aloin (purity [98 %) extracted from Aloe ferox Mill. leaves was purchased from Chengdu Herbpurify CO., Ltd

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(Chengdu, China) and verified by HPLC. Anhydrous alcohol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Compound Biejiaruangan Troche (CBT), a product of Inner Mongolia Furui Medical Science Co., Ltd (China), was procured from a local pharmacy (Wuxi, China). Superoxide dismutase (SOD), reduced glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). LPS, interleukin-1b (IL-1b), interleukin-10 (IL-10), and tumor necrosis factor a (TNF-a) enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, USA). All other chemicals used were of analytical grade. Animals and experimental design Kunming mice (male, 18–22 g body weight, bw) obtained from SLAC Laboratory Animal (Shanghai, China) were used for all experiments. The mice were housed in standard cages under set temperature (23 ± 2 °C) and humidity (60 ± 5 %) with a 12-h light/dark cycle and fed with commercial standard chow (SLAC Laboratory Animal, Shanghai, China) and water ad libitum. After 1 week of acclimatization, 60 mice were randomly divided into five experimental groups (n = 12): Group I (Control): vehicle-treated mice; Group II (Alcohol): alcohol-treated mice; Group III (Positive control, PC): alcohol-treated mice receiving CBT (500 mg/kg bw, dissolved in corn oil) intragastrically; Group IV (Alcohol ? aloin at low dose, AAL): alcoholtreated mice receiving low-dose aloin (10 mg/kg bw, dissolved in corn oil) intragastrically; Group V (Alcohol ? aloin at high dose, AAH): alcoholtreated mice receiving high-dose aloin (30 mg/kg bw, dissolved in corn oil) intragastrically. During experiments, alcohol (50 %, v/v, in water) was administered intragastrically by gavage twice daily as the previous studies (Zhang et al. 2009; Forsyth et al. 2009). The amount of the 50 % alcohol was initially 10 mL/ kg bw/day (4.0 g/kg bw/day) and gradually increased as tolerance developed during the first 3 weeks to a maintenance dose of 16 mL/kg bw/day (6.3 g/kg bw/day) that was continued for 8 more weeks: Week 1: 5 mL/kg bw 2 9 a day (corresponding alcohol was 4.0 g/kg bw/day); Week 2: 6 mL/kg bw 2 9 a day (corresponding alcohol was 4.7 g/kg bw/day); Week 3: 7 mL/kg bw 2 9 a day (corresponding alcohol was 5.5 g/kg bw/day);

Aloin protects against alcoholic liver injury

Week 4–11: 8 mL/kg bw 2 9 a day (corresponding alcohol was 6.3 g/kg bw/day); Control mice received the corresponding vehicle (water) twice daily, also by gavage. After each alcohol administration, the mice in the experimental groups received an oral dose of the corresponding tested substance, respectively, according to the doses listed above. The Control and Alcohol group received an equal volume of vehicle (corn oil) to aloin-treated groups. At the end of the 11-week treatment, the mice were anesthetized and sacrificed after 12 h of fasting. Blood samples were collected and the serum was separated after clot formation by centrifugation (3,000 rpm, 10 min, 4 °C) for the determination of serum marker enzymes and lipids levels. The excised livers were washed with ice-cold physiological saline (0.9 % NaCl solution) and weighed. Two small sections of liver from the same lobe in each animal were fixed for pathological examination. The remaining liver tissues were frozen in liquid nitrogen and stored at -80 °C until analysis. All experimental procedures involving animals were developed in compliance with the animal care guidelines of China, which conform to internationally accepted principles in the care and use of experimental animals. The approval of this experiment was obtained from the Institutional Animal Ethics Committee of Jiangnan University (Wuxi, China). All animal protocols were reviewed and approved by the Animal Care and Use Committee of our institute.

For the hepatic biochemical assays, 10 % (w/v) homogenate of liver was prepared using ice-cold physiological saline (0.9 % NaCl solution). After centrifugation, supernatants were isolated and kept at -80 °C during the analysis. Colorimetric estimations of hepatic MDA, GSH, SOD, and NO levels were performed using commercially available kits according to the manufacturer’s instructions. Briefly, MDA was measured by estimating the color production generated with thiobarbituric acid treatment at 532 nm. GSH, an important peptide that detoxifies and scavenges free radicals, was measured as non-protein thiols according to the method described by Ellman (1959). Liver homogenate (0.1 mL) was mixed with 0.1 mL of the precipitating solution. After centrifugation (4,000 rpm at 4 °C for 10 min), the free thiol groups in the supernatant were determined based on its reaction with 5,50 -dithiobis-2-nitrobenzoic acid to generate 5-thio-2-nitrobenzoic acid, which was measured at 405 nm. The activity of SOD was obtained by the method of McCord (1994). NO was determined on the basis of the Griess reaction (Ding et al. 1988), which generates a purple-azo dye product that can be monitored using a spectrophotometer at 540 nm. The corresponding protein content was determined by the Bradford method (Bradford 1976) using bovine serum albumin as a standard. Endotoxin assay Serum endotoxin (LPS) levels were determined using a commercially available ELISA kit (R&D Systems, Minneapolis, USA) following the manufacturer’s instructions.

Clinical chemistry and pathological evaluation Hepatic TNF-a, IL-1b, and IL-10 assays Serum marker enzymes, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured using commercially available kits (Roche, Basle, Switzerland) according to the manufacturer’s instructions using a Cobas C501 automatic biochemical analyzer (Roche, Basle, Switzerland). For the pathological evaluation, the fixed liver tissues were embedded in paraffin. Tissue sections (5 lm) were cut and stained with hematoxylin-eosin (H&E). The slides were observed and photographed using a light microscope (Leica, Solms, Germany).

Serum and hepatic biochemical assays Serum lipids including total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), and high density lipoprotein (HDL) were assayed on a Cobas C501 automatic biochemical analyzer (Roche, Basle, Switzerland) with commercially available kits (Roche, Basle, Switzerland).

Ten percent (w/v) liver homogenates were obtained as described above. The levels of the hepatic inflammatory cytokines TNF-a, IL-1b, and IL-10 were determined with commercially available ELISA kits (R&D Systems, Minneapolis, USA), according to the manufacturer’s protocol. The levels of TNF-a, IL-1b, and IL-10 were normalized against total protein content. RNA isolation and real-time PCR Total RNA was extracted from the stored frozen liver tissues with Trizol reagent using a commercial RNA kit (Shanghai Generay Biotech Co., Ltd, Shanghai, China). The expression of target mRNAs, including cytochrome P4502E1 (CYP2E1), sterol regulatory element-binding protein-1c (SREBP-1c), AMP-activated protein kinase-a2 (AMPK-a2), toll-like receptor-4 (TLR-4), and myeloid differentiation primary response gene 88 (MyD88), was measured by real-time PCR following reverse-transcription. The single-stranded cDNA

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Y. Cui et al. Table 1 Primers used for quantitative real-time PCR Target gene

Forward/reverse primers

GADPH

Forward: 50 -CTTTGGCATTGTGGAAGGGCTC-30 Reverse: 50 -GCAGGGATGATGTTCTGGGCAG-30

CYP2E1

Forward: 50 -GTTTTCCCTAAGTATCCTCCGTGAC-30 Reverse: 50 -GAAGCGTTTGTTGAAGAGAATATCC-30

AMPK-a2

Forward: 50 -GCTACCTATTTCCTGAAGACCCCTC-30 Reverse: 50 -CTTGGTTCATTATTCTCCGATTGTC-30

SREBP1c

Forward: 50 -TAGAGCGAGCGTTGAACTGTATTG-30 Reverse: 50 -CCATGCTGGAGCTGACAGAGAA-30

TLR-4

Forward: 50 -CTGTATTCCCTCAGCACTCTTGATT-30 Reverse: 50 -TGCTTCTGTTCCTTGACCCACT-30

MyD88

Forward: 50 -ATGGTGGTGGTTGTTTCTGACG-30 Reverse: 50 -GTCGCATATAGTGATGAACCGCA-30

was synthesized from 1.0 lg of total RNA using RevertAidTM M-MuLV Reverse Transcriptase, following the manufacturer’s instructions (Thermo Scientific, Rockford, USA). For each target mRNA, 2 lL of cDNA was mixed with SYBR GreenÒ PCR Premix (TaKaRa, Dalian, China) and 0.8 lL of each specific forward/reverse primer (Table 1) in a final volume of 20 lL. The PCR amplification was monitored in real time using the ABI 7900 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, USA) with an initial hold step (95 °C for 30 s) and 40 cycles of a 2-step RCR (95 °C for 3 s, 60 °C for 30 s). The levels of target genes were determined by the comparative CT method by normalizing to an internal reference (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) and relative to a calibrator (2-44Ct). The purity of the PCR product was verified by melting curve analysis. Statistical analyses Data were expressed as the mean ± standard deviation (SD). Statistics were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test using the Statistical Package for Social Studies software. A difference of p \ 0.05 was considered to be statistically significant.

Results Aloin prevents chronic alcohol-induced hepatotoxicity To evaluate liver injury, serum ALT and AST activities were measured as markers. As shown in Fig. 2a, alcohol group revealed the abnormal higher levels of serum ALT (p \ 0.001) and AST (p \ 0.05) than control group. However, the levels of these marker enzymes were

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significantly decreased when aloin or CBT was coadministered with alcohol (p \ 0.05). Alcohol caused significantly higher serum TC, TG, and LDL levels than those observed in the control group (p \ 0.05, p \ 0.001 and p \ 0.01, respectively) (Fig. 2b). Co-administration of aloin with alcohol significantly suppressed the elevation of serum TC (30 mg/kg bw, p \ 0.05) and TG (10 and 30 mg/kg bw, p \ 0.05 and p \ 0.01, respectively), however, there was no significant decrease in serum LDL levels when compared with the alcohol group. Moreover, aloin treatment also showed a protective effect by increasing the serum HDL level, and statistical significance was observed in the high-dose group (30 mg/kg bw, p \ 0.05). Co-administration of CBT with alcohol caused significant reduction in the alcohol-dependent elevation of TC (p \ 0.05), TG (p \ 0.001) and LDL (p \ 0.05) levels, but had no significant effect on serum HDL level. Representative H&E staining photomicrographs of liver histology are shown in Fig. 2c. Daily alcohol intake induced greater steatosis (fat accumulation) and inflammatory injury (Kupffer cell activation) in mouse liver, compared to the control animals. However, co-administration of aloin or CBT suppressed the deleterious effects of chronic alcohol intake in the liver, as indicated by diminished Kupffer cells and fatty infiltration of hepatocytes, which was consistent with biochemical analyses. In addition, the body weight was not affected regardless of treatment. No mortality or diarrhea was observed during the entire study. The colon and rectum of the aloin-treated mice were all looked normal as the control group. Aloin suppresses chronic alcohol-induced lipid peroxidation and oxidative stress Effect of aloin on chronic alcohol-induced lipid peroxidation and oxidative stress is shown in Fig. 3. The hepatic MDA, a marker of lipid peroxidation, was significantly elevated by 1.5-fold after chronic alcohol feeding compared with the control group (p \ 0.05). However, this elevation was markedly attenuated when aloin was co-administered with alcohol (10 and 30 mg/kg bw, p \ 0.05 and p \ 0.001, respectively). Chronic ingestion of alcohol significantly decreased the total hepatic GSH concentration (p \ 0.01) and SOD activity (p \ 0.05), and dramatically elevated the expression of CYP2E1 (p \ 0.001) as compared to the control group. With the co-administration of aloin, SOD activity was significantly increased (10 and 30 mg/kg bw, p \ 0.01 and p \ 0.001, respectively) and the over-expression of CYP2E1 was significantly inhibited (30 mg/kg bw, p \ 0.05). However, aloin had no significant effect on the alcohol-dependent GSH depletion. Co-administration of CBT with alcohol markedly attenuated alcohol-dependent GSH depletion (p \ 0.05) and over-expression of CYP2E1 (p \ 0.001), but

Aloin protects against alcoholic liver injury

Fig. 2 Effect of aloin supplementation on a serum marker enzymes and b lipids. c Representative photomicrographs of H&E staining of liver sections (9400). Alcohol treatment induced activation of Kupffer cells (KC) and fatty infiltration of hepatocytes (arrows); central vein (CV). Data are expressed as mean ± SD for 12 mice in

each group. One-way ANOVA followed by Duncan’s multiple range test was used to calculate statistical significance. *p \ 0.05, **p \ 0.01, ***p \ 0.001 vs. control group; #p \ 0.05, ##p \ 0.01, ### p \ 0.001 compared with the alcohol group

had no significant effect on SOD and MDA levels when compared with the alcohol group.

significantly decreased in the alcohol group when compared with the control group (p \ 0.01) (Fig. 4b). However, highdose aloin (30 mg/kg bw, p \ 0.05) or CBT (p \ 0.05) supplementation significantly inhibited its reduction. In addition, the serum endotoxin (LPS) level was markedly elevated in the alcohol-treated mice relative to the control value (p \ 0.01), and it was significantly decreased by the coadministration of aloin (30 mg/kg bw, p \ 0.01) or CBT (p \ 0.01; Fig. 4c).

Aloin inhibits chronic alcohol-induced inflammation Chronic alcohol administration for 11 weeks resulted in a significant development of inflammatory injury in the livers of the alcohol group when compared with the control mice (Fig. 4). The hepatic contents of NO and TNF-a were significantly elevated by alcohol treatment (p \ 0.05), but they were all significantly decreased when aloin was co-administered with alcohol (p \ 0.05; Fig. 4a, b). The IL-1b level was dramatically increased in alcohol-treated mice (p \ 0.05), but no significant effect was found with the co-treatment of aloin or CBT. The anti-inflammatory cytokine IL-10 was

Aloin prevents SREBP-1c but activates AMPK-a2 expression in liver To identify the mechanism underlying the protective role of aloin on chronic alcohol-induced steatosis in mice, we

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Fig. 3 Effect of aloin supplementation on the levels of a MDA, b GSH, c SOD, and d the relative mRNA level of CYP2E1. Data are expressed as mean ± SD for 12 mice in each group. One-way ANOVA followed by Duncan’s multiple range test was used to

calculate statistical significance. *p \ 0.05, **p \ 0.01, ***p \ 0.001 versus control group; #p \ 0.05, ##p \ 0.01, ### p \ 0.001 compared with the alcohol group

profiled the expression of a group of lipid metabolismrelated genes. As shown in Fig. 5, alcohol administration significantly decreased the mRNA level of AMPK-a2 as compared to the control group (p \ 0.05). With the coadministration of aloin, the mRNA expression of AMPKa2 was dramatically elevated as compared with the alcohol group (10 and 30 mg/kg bw, p \ 0.001 and p \ 0.001, respectively). The mRNA level of SREBP-1c was dramatically increased by 13.0 fold by chronic alcohol ingestion (p \ 0.001). Interestingly, its over-expression in alcoholic liver was markedly inhibited by the co-administration of aloin (10 and 30 mg/kg bw, p \ 0.01 and p \ 0.001, respectively) or CBT (p \ 0.001).

(p \ 0.001), while the mRNA levels of MyD88 were dramatically decreased (p \ 0.01).

Aloin blocks hepatic induction of TLR and MyD88 The expression of hepatic TLR-4, MyD88, was quantitated by real-time PCR. Chronic alcohol intake dramatically elevated TLR-4 (p \ 0.001) and MyD88 mRNA expression (p \ 0.001) as compared with the control group (Fig. 6). However, aloin or CBT co-administered during alcohol consumption significantly reduced the expression of TLR-4 when compared with the alcohol group

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Discussion ALD is a syndrome of progressive inflammatory liver injury associated with chronic intake of alcohol (An et al. 2012). It progresses through different stages ranging from a fatty liver with minimal injury to more advanced liver damage, including alcoholic hepatitis and fibrosis/cirrhosis (Albano 2008; O’Shea et al. 2010). It has been shown that ALD is associated with a multifactorial pathological progress involving an increased NADH/NAD? ratio and endotoxin translocation. This results in lipid accumulation, and induces oxidative stress and inflammatory injury, resulting in concurrent changes with elevation of the activity of serum enzymes (An et al. 2012; Stewart et al. 2001). Therefore, great efforts are needed to provide effective protection from these damaging agents. Recent studies showed that aloin had a substantial antioxidant ability against lipid peroxidation and free radicals, and also exerted a potential effect on alleviating inflammatory

Aloin protects against alcoholic liver injury

Fig. 5 Effect of aloin on the expression of hepatic lipid metabolismrelated genes. Data are expressed as mean ± SD for 10-12 mice in each group. One-way ANOVA followed by Duncan’s multiple range test was used to calculate statistical significance. *p \ 0.05, ***p \ 0.001 versus control group; ##p \ 0.01, ###p \ 0.001 compared with the alcohol group

Fig. 6 Effect of aloin on the mRNA levels of TLR-4 and MyD88 in mouse liver. Data are expressed as mean ± SD for 10–12 mice in each group. One-way ANOVA followed by Duncan’s multiple range test was used to calculate statistical significance. ***p \ 0.001 versus control group; ##p \ 0.01, ###p \ 0.001 compared with the alcohol group Fig. 4 Effect of aloin supplementation on a NO, b cytokines and c LPS levels. Data are expressed as mean ± SD for 12 mice in each group. One-way ANOVA followed by Duncan’s multiple range test was used to calculate statistical significance. *p \ 0.05, **p \ 0.01 versus control group; #p \ 0.05, ##p \ 0.01 compared with the alcohol group

injury (Asamenew et al. 2011; Beppu et al. 2003; Esmat et al. 2012; Park et al. 2009b, 2011). This prompted us to investigate the possible hepatoprotective effect of aloin against chronic alcohol-induced liver injury. Since aloin is known to exert diarrhea, we also conducted an in vivo study to determine the safe intake level and found that

aloin at the doses of 10 and 30 mg/kg bw did not cause diarrhea. The results of the present study demonstrated that chronic alcohol consumption caused liver injury as evidenced by elevation of serum ALT and AST activity, serum TC, TG, and LDL levels, as well as enlargement and fatty infiltration of hepatocytes, all of which reflected early biochemical and pathological changes in ALD. With the co-administration of aloin or CBT, the levels of these

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serum marker enzymes and lipids were near normal or only slightly elevated, showing its potential to maintain the normal functional status of the liver. In addition, histopathological liver changes induced by alcohol were also remarkably attenuated by aloin or CBT co-treatment. Results indicated that aloin was able to suppress the hepatotoxicity of chronic alcohol and the hepatoprotective effect was comparable to the conventionally reputed CBT (a commercially available hepatoprotective compound in China). Fatty liver is the initial and most common response of the liver to alcohol consumption, and is characterized by an accumulation of cholesterol and TG (Wang et al. 2009). In the present study, we found that co-administration of aloin with alcohol significantly attenuated the alcohol-dependent elevation in serum TC and TG levels, increased the HDL level, and reduced the fatty infiltration of hepatocytes in tissues, suggesting that the protective effect of aloin on ALD was partly due to its ability to suppress hepatic lipid accumulation. Multiple mechanisms are involved in alcohol-induced hepatic lipid accumulation, including enhanced lipogenesis and the inhibition of fatty acid oxidation (You et al. 2002). AMPK, which may be regulated by TNF-a, is a key modulator of lipid metabolism in the liver (An et al. 2012; You and Crabb 2004b). It can lead to a decrease in the mRNA and protein expression of SREBP-1c regulating genes and thus a reduction in lipid synthesis, and an increase in ATP-generating catabolic pathways such as lipolysis and fatty acid oxidation (An et al. 2012; Yamauchi et al. 2002; Zang et al. 2004). Our results and other published research (You and Crabb 2004b; You et al. 2004; Zeng et al. 2012) showed that alcohol consumption increased SREBP-1c levels, associated with decreased AMPK activity. However, aloin co-administrated during alcohol consumption clearly lowered the hepatic mRNA expression of SREBP-1c and dramatically elevated the expression of AMPK-a2 as compared to the alcohol group, which could explain the lower serum TC, TG levels and alleviated histopathological liver changes in the alointreated mice. These results demonstrated that aloin suppressed alcohol-dependent lipid accumulation in mice presumably through restoring the balance between lipid synthesis and fatty acid oxidation/lipolysis, which may be mediated at least in part by AMPK-a2 activation and down-regulation of SREBP-1c expression. Recent studies have also shown that an elevation of TNF-a level was related to increasing rates of fatty acid synthesis and esterification (You and Crabb 2004a; You et al. 2002). The results of the present study showed that aloin inhibited the over-production of TNF-a caused by chronic alcohol ingestion, suggesting that the ability of aloin to inhibit alcohol-induced lipid accumulation may also be associated with its suppression of TNF-a production.

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Oxidative stress is a feature of alcohol hepatotoxicity and plays an important role in the development of ALD (Yurt and Celik 2011). Many pathways, including increased lipid peroxidation, damage to the mitochondria, free radical generation, and decrease of hepatic antioxidant defense, have been proposed to be involved (Albano 2006; Noh et al. 2011; Zeng et al. 2012). Our results confirmed the involvement of oxidative stress in chronic alcoholinduced liver injury and showed a significant hepatoprotective effect of aloin, as evidenced by the significant alleviation of the lipid peroxidation marker MDA. In addition, the enzymatic antioxidant SOD was markedly elevated by aloin co-treatment. Aloin co-treatment also showed a significant reduction on the hepatic mRNA expression of CYP2E1 as compared to the alcohol group, which resulted fewer free-radical production, thus alleviating oxidative stress and decreasing the development of alcoholic fatty liver. In combination with previous reports concerning its capacity to inhibit lipid peroxidation and scavenge free radicals in vitro and in vivo (Asamenew et al. 2011; Esmat et al. 2012), it can be concluded that the attenuation of chronic alcohol-induced oxidative stress by aloin was most likely due to its anti-oxidative effects against lipid peroxidation and free radicals. Increasing evidence shows that intestinal permeation and translocation of bacterial endotoxins followed by the induction of TLR-4-dependent signaling pathway in the liver are key mechanisms in the development of ALD (An et al. 2012; Mandrekar and Szabo 2009; Nagy 2003). Alcohol consumption causes increased intestinal leakage, which leads to the translocation of endotoxins into circulation. These endotoxins then induce TLR-4 expression followed by the activation of Kupffer cells which leads to the release of pro-inflammatory cytokines (TNF-a, IL-1b) and NO, and ultimately results in hepatocellular necrosis (An et al. 2012; Hu et al. 2009). Previous work showed that aloin exerted anti-inflammatory activity both in a rat colitis model and murine macrophages (Park et al. 2009b, 2011). In this chronic alcohol-induced liver injury model, we confirmed the anti-inflammatory property of aloin. Aloin not only decreased the accumulation of LPS, but also significantly inhibited the elevation of hepatic TNF-a and NO levels induced by alcohol administration. Moreover, aloin significantly suppressed the alcohol-dependent reduction in anti-inflammatory cytokine IL-10. In an effort to understand the mechanism by which aloin attenuates alcohol-induced inflammation, the effects of aloin on the alcohol-dependent induction of mRNA expression of TLR4, and MyD88 in mouse livers were also investigated. Similar to the results of previous studies (An et al. 2012), over-expression of hepatic TLR-4 and MyD88 was observed after 11 weeks of alcohol consumption. Cotreatment with aloin during alcohol consumption almost

Aloin protects against alcoholic liver injury

completely protected mice from chronic alcohol-induced elevation of TLR-4 expression and significantly inhibited the activation of MyD88. The above results suggest that aloin supplementation can alleviate liver inflammatory injury in chronic alcohol-treated mice via the decrease of endotoxin accumulation and the down-regulation of TLR-4 and MyD88 gene expression. In summary, the results of the present study showed that aloin had a significant protective effect against chronic alcohol-induced liver injury in mice. The hepatoprotective action of aloin was most likely mediated by its ability to suppress lipid accumulation through regulation of lipid metabolism, depress oxidative stress, as well as inhibit LPS-induced inflammatory injury by decreasing gene expression of TLR-4 and MyD88. Although further detailed studies are required to establish its clinical application, our results suggest that aloin may be a good candidate for the development of novel protective strategies against alcohol-induced liver injury. Acknowledgments This work was supported by the National Science and Technology Support Program in the 12th 5 year Plan of China (No. 2011BAZ02169), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD), and the Fundamental Research Funds for the Central Universities of China (No. JUDCF10057, JUSRP11121). Conflict of interest of interest.

The authors declare that there are no conflicts

References Albano, E. 2006. Alcohol, oxidative stress and free radical damage. Proceedings of the Nutrition Society 65: 278–290. Albano, E. 2008. Oxidative mechanisms in the pathogenesis of alcoholic liver disease. Molecular Aspects of Medicine 29: 9–16. An, L., X. Wang, and A.I. Cederbaum. 2012. Cytokines in alcoholic liver disease. Archives of Toxicology 86: 1337–1348. Asamenew, G., D. Bisrat, A. Mazumder, and K. Asres. 2011. In vitro antimicrobial and antioxidant activities of anthrone and chromone from the latex of Aloe harlana reynolds. Phytotherapy Research: PTR 25: 1756–1760. Avila, H., J. Rivero, F. Herrera, and G. Fraile. 1997. Cytotoxicity of a low molecular weight fraction from Aloe vera (Aloe barbadensis Miller) gel. Toxicon 35: 1423–1430. Beppu, H., T. Koike, K. Shimpo, T. Chihara, M. Hoshino, C. Ida, and H. Kuzuya. 2003. Radical-scavenging effects of Aloe arborescens Miller on prevention of pancreatic islet B-cell destruction in rats. Journal of Ethnopharmacology 89: 37–45. Bottenberg, M.M., G.C. Wall, R.L. Harvey, and S. Habib. 2007. Oral aloe vera-induced hepatitis. Annals of Pharmacotherapy 41: 1740–1743. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248–254. Chandan, B.K., A.K. Saxena, S. Shukla, N. Sharma, D.K. Gupta, K.A. Suri, J. Suri, M. Bhadauria, and B. Singh. 2007. Hepatoprotective potential of Aloe barbadensis Mill. against carbon

tetrachloride induced hepatotoxicity. Journal of Ethnopharmacology 111: 560–566. Chung, J.H., J.C. Cheong, J.Y. Lee, H.K. Roh, and Y.N. Cha. 1996. Acceleration of the alcohol oxidation rate in rats with aloin, a quinone derivative of Aloe. Biochemical Pharmacology 52: 1461–1468. Ding, A.H., C.F. Nathan, and D.J. Stuehr. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. Journal of Immunology 141: 2407–2412. Ding, R.B., K. Tian, L.L. Huang, C.W. He, Y. Jiang, Y.T. Wang, and J.B. Wan. 2012. Herbal medicines for the prevention of alcoholic liver disease: A review. Journal of Ethnopharmacology 144: 457–465. Ellman, G.L. 1959. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82: 70–77. Esmat, A.Y., M.M. Said, G.M. Hamdy, A.A. Soliman, and S.A. Khalil. 2012. In vivo and in vitro studies on the antioxidant activity of aloin compared to doxorubicin in rats. Drug Development Research 73: 154–165. Esmat, A.Y., C. Tomasetto, and M.C. Rio. 2006. Cytotoxicity of a natural anthraquinone (aloin) against human breast cancer cell lines with and without erbB-2-topoisomerase II alpha coamplification. Cancer Biology and Therapy 5: 97–103. Forsyth, C.B., A. Farhadia, S.M. Jakate, Y.M. Tang, M. Shaikh, and A. Keshavarzian. 2009. Lactobacillus GG treatment ameliorates alcohol-induced intestinal oxidative stress, gut leakiness, and liver injury in a rat model of alcoholic steatohepatitis. Alcohol 43: 163–172. Groom, Q.J., and T. Reynolds. 1987. Barbaloin in aloe species. Planta Medica 53: 345–348. Hu, S., S. Yin, X. Jiang, D. Huang, and G. Shen. 2009. Melatonin protects against alcoholic liver injury by attenuating oxidative stress, inflammatory response, and apoptosis. European Journal of Pharmacology 616: 287–292. Kanuri, G., S. Weber, V. Volynets, A. Spruss, S.C. Bischoff, and I. Bergheim. 2009. Cinnamon extract protects against acute alcohol-induced liver steatosis in mice. The Journal of nutrition 139: 482–487. Koch, A. 1996. Metabolism of aloin-The influence of nutrition. Journal of Pharmaceutical and Biomedical Analysis 14: 1335–1338. Mandrekar, P., and G. Szabo. 2009. Signalling pathways in alcoholinduced liver inflammation. Journal of Hepatology 50: 1258–1266. Mccord, J.M. 1994. Mutant mice, Cu, Zn superoxide dismutase, and motor neuron degeneration. Science 266: 1586–1587. Nagy, L.E. 2003. Recent insights into the role of the innate immune system in the development of alcoholic liver disease. Experimental Biology and Medicine 228: 882–890. Noh, J.R., Y.H. Kim, G.T. Gang, J.H. Hwang, H.S. Lee, S.Y. Ly, W.K. Oh, K.S. Song, and C.H. Lee. 2011. Hepatoprotective effects of chestnut (Castanea crenata) inner shell extract against chronic ethanol-induced oxidative stress in C57BL/6 mice. Food and Chemical Toxicology 49: 1537–1543. O’shea, R.S., S. Dasarathy, A.J. Mccullough, A.a.S.L. Dis, and A.C. Gastroenterology. 2010. Alcoholic liver disease. Hepatology 51: 307–328. Park, M.Y., H.J. Kwon, and M.K. Sung. 2009a. Intestinal absorption of aloin, aloe-emodin, and aloesin; A comparative study using two in vitro absorption models. Nutrition Research and Practice 3: 9–14. Park, M.Y., H.J. Kwon, and M.K. Sung. 2009b. Evaluation of aloin and aloe-emodin as anti-inflammatory agents in aloe by using murine macrophages. Bioscience, Biotechnology, and Biochemistry 73: 828–832.

123

Y. Cui et al. Park, M.Y., H.J. Kwon, and M.K. Sung. 2011. Dietary aloin, aloesin, or aloe-gel exerts anti-inflammatory activity in a rat colitis model. Life Sciences 88: 486–492. Patel, D.K., K. Patel, and V. Tahilyani. 2012. Barbaloin: A concise report of its pharmacological and analytical aspects. Asian Pacific Journal of Tropical Biomedicine 2: 835–838. Rejitha, S., P. Prathibha, and M. Indira. 2012. Amelioration of alcohol-induced hepatotoxicity by the administration of ethanolic extract of Sida cordifolia Linn. The British Journal of Nutrition 108: 1256–1263. Stewart, S., D. Jones, and C.P. Day. 2001. Alcoholic liver disease: New insights into mechanisms and preventative strategies. Trends in Molecular Medicine 7: 408–413. Tuma, D.J. 2002. Role of malondialdehyde-acetaldehyde adducts in liver injury. Free Radical Biology and Medicine 32: 303–308. Van Wyk, B.E., M.C. Van Rheede Van Oudtshoorn, and G.F. Smith. 1995. Geographical variation in the major compounds of Aloe ferox leaf exudate. Planta Medica 61: 250–253. Wang, Y., G. Millonig, J. Nair, E. Patsenker, F. Stickel, S. Mueller, H. Bartsch, and H.K. Seitz. 2009. Ethanol-induced cytochrome P4502E1 causes carcinogenic etheno-DNA lesions in alcoholic liver disease. Hepatology 50: 453–461. Yamauchi, T., J. Kamon, Y. Minokoshi, Y. Ito, H. Waki, S. Uchida, S. Yamashita, M. Noda, S. Kita, K. Ueki, K. Eto, Y. Akanuma, P. Froguel, F. Foufelle, P. Ferre, D. Carling, S. Kimura, R. Nagai, B.B. Kahn, and T. Kadowaki. 2002. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nature Medicine 8: 1288–1295. Yanardag, R., O. Ozsoy-Sacan, S. Ozdil, and S. Bolkent. 2007. Combined effects of vitamin C, vitamin E, and sodium selenate supplementation on absolute ethanol-induced injury in various organs of rats. International Journal of Toxicology 26: 513–523. Ye, Q., H.Y. Wang, and H. Qian. 2012. Protective effects of crude aloe polysaccharides and aloin on mice with acute liver injury induced by alcohol. Science and Technology of Food Industry 33: 355–358. You, M., and D.W. Crabb. 2004a. Molecular mechanisms of alcoholic fatty liver: Role of sterol regulatory element-binding proteins. Alcohol 34: 39–43.

123

You, M., and D.W. Crabb. 2004b. Recent advances in alcoholic liver disease II. Minireview: Molecular mechanisms of alcoholic fatty liver. American journal of physiology. Gastrointestinal and liver physiology 287: G1–G6. You, M., M. Fischer, M.A. Deeg, and D.W. Crabb. 2002. Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP). The Journal of biological chemistry 277: 29342–29347. You, M., M. Matsumoto, C.M. Pacold, W.K. Cho, and D.W. Crabb. 2004. The role of AMP-activated protein kinase in the action of ethanol in the liver. Gastroenterology 127: 1798–1808. Yurt, B., and I. Celik. 2011. Hepatoprotective effect and antioxidant role of sun, sulphited-dried apricot (Prunus armeniaca L.) and its kernel against ethanol-induced oxidative stress in rats. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 49: 508–513. Zang, M.W., A. Zuccollo, X.Y. Hou, D. Nagata, K. Walsh, H. Herscovitz, P. Brecher, N.B. Ruderman, and R.A. Cohen. 2004. AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells. Journal of Biological Chemistry 279: 47898–47905. Zeng, T., C.L. Zhang, F.Y. Song, X.L. Zhao, and K.Q. Xie. 2012. Garlic oil alleviated ethanol-induced fat accumulation via modulation of SREBP-1, PPAR-alpha, and CYP2E1. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 50: 485–491. Zhang, R.R., Y.Y. Hu, J.Y. Yuan, and D.Z. Wu. 2009. Effects of Puerariae radix extract on the increasing intestinal permeability in rat with alcohol-induced liver injury. Journal of Ethnopharmacology 126: 207–214. Zhao, J., H. Chen, and Y. Li. 2008. Protective effect of bicyclol on acute alcohol-induced liver injury in mice. European Journal of Pharmacology 586: 322–331.