Gynura procumbens Reverses Acute and Chronic Ethanol-Induced

Article pubs.acs.org/JAFC

Gynura procumbens Reverses Acute and Chronic Ethanol-Induced Liver Steatosis through MAPK/SREBP-1c-Dependent and -Independent Pathways Xiao-Jun Li,‡,# Yun-Mei Mu,‡,# Ting-Ting Li,‡ Yan-Ling Yang,‡ Mei-Tuo Zhang,‡ Yu-Sang Li,‡ Wei Kevin Zhang,‡ He-Bin Tang,*,‡,§ and Hong-Cai Shang*,§ ‡

Department of Pharmacology, College of Pharmacy, South-Central University for Nationalities, No. 182, Minyuan Road, 430074 Wuhan, China § Key Laboratory of Chinese Internal Medicine of MOE and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, 100700 Beijing, China ABSTRACT: The present study aimed to evaluate the hepatoprotective effect and mechanism of action of Gynura procumbens on acute and chronic ethanol-induced liver injuries. Ethanol extract from G. procumbens stems (EEGS) attenuated acute ethanolinduced serum alanine aminotransferase levels and hepatic lipid accumulation. Therefore, EEGS was successively extracted by petroleum, ethyl acetate, and n-butyl alcohol. The results showed that the n-butyl alcohol extract was the active fraction of EEGS, and hence it was further fractionated on a polyamide glass column. The 60% ethanol-eluted fraction that contained 13.6% chlorogenic acid was the most active fraction, and its effect was further evaluated using a chronic model. Both the n-butyl alcohol extract and the 60% ethanol-eluted fraction inhibited chronic ethanol-induced hepatic lipid accumulation by modulating lipid metabolism-related regulators through MAPK/SREBP-1c-dependent and -independent signaling pathways and ameliorated liver steatosis. Our findings suggest that EEGS and one of its active ingredients, chlorogenic acid, may be developed as potential effective agents for ethanol-induced liver injury. KEYWORDS: Gynura procumbens, chlorogenic acid, alcoholic liver disease, steatosis, MAPK, SREBP-1c

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INTRODUCTION Alcohol has long been identified as a major risk factor for liver diseases. Sustained excessive drinking of alcohol can lead to the development of alcoholic liver disease (ALD), which refers to a broad range of liver injury, including steatosis, alcoholic hepatitis, fibrosis, and cirrhosis.1 Globally, approximately 70% of alcohol-related mortalities are directly attributed to hepatic disease,2 and 4% of human deaths are related to ALD, which seriously affects patients’ quality of life and places a huge burden on health care systems.3,4 The development of ALD is a complex process that involves a multitude of signal pathways, and the mechanism behind ALD is still not well understood. Although multiple attempts have been made to improve patient outcome, we have yet to find a reliable treatment, except for alcohol abstinence.1 During the past decades, less toxic multitargeting herbs have attracted considerable attention as potential therapeutic candidates against ALD.5 Gynura procumbens (Lour.) Merr., a traditional food and herb, enthusiastically used in Southeast Asia, possesses a wide range of pharmacological properties, such as reducing blood glucose and lipids levels,6 possessing anti-liver cancer activity,7 and relieving hepatotoxicity and other ALD-associated symptoms.8,9 As a new food material recommended by the National Health and Family Planning Commission of China in 2012, G. procumbens became a popular vegetable and folk medicine. People in southern and central China like planting G. procumbens in their yards. However, until now, very little was known about its active ingredients or pharmacologic mechanisms. In the current study, we performed in vivo experiments © XXXX American Chemical Society

to screen the active fraction(s) from G. procumbens for protective effects against ethanol-induced liver steatosis and further elucidate its probable mechanisms. The progression of ethanol-induced liver steatosis, an early stage in the development of ALD, is a multi-factorial and multistep process that involves multiple metabolic pathways. Acetylcoenzyme A carboxylase (ACC), which converts acetyl-CoA to malonyl-CoA, is the committed step of the fatty acid synthesis.10 Fatty acid synthase (FAS) is another key enzyme that catalyzes fatty acid synthesis. The expression of genes required for fatty acid and lipid production, including ACC and Fasn,11,12 are positively regulated by a master regulator of lipid homeostasis, sterol regulatory element binding protein 1c (SREBP-1c). Previous studies have proven that SREBP-1c activation in ALD is directly influenced by AMP-activated protein kinase (AMPK)13 and mitogen-activated protein kinases (MAPKs).1,14,15 AMPK is a protein kinase that inhibits lipid synthesis through phosphorylation and inactivates key lipogenic genes such as SREBP-1 and ACC.13 Meanwhile, MAPKs are also the protein kinases that inhibit SREBP-1c through phosphorylation.15 Because G. procumbens affects lipids metabolism and SREBP-1c plays a predominant role in alcoholinduced hepatic steatosis,16 we focused on the pathways upstream and downstream of SREBP-1c to elucidate the Received: July 20, 2015 Revised: September 7, 2015 Accepted: September 7, 2015

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DOI: 10.1021/acs.jafc.5b03504 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Schematic diagram of the bioguided fractions of G. procumbens on alcohol-induced liver injury. and identified in EEGS in our previous study. HPLC analysis was carried out with a U3000-Dionex instrument with a 5 μm Acclaim C18 column (4.6 × 250 mm, Thermo Fisher Scientific, Inc., Waltham, MA). Detection was carried out at 326 nm with a 70 min gradient. Solvent A was acetonitrile, and solvent B was a 0.1% aqueous phosphoric acid solution. The gradient system was A-B (v/v) = 19/81 (0 min) → 27/73 (65 min) → 19/81 (70 min). The flow rate of the mobile phase was 1 mL/min (Figure 2). The contents of neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in EEGS as calculated by external standard method were 0.01%, 0.27%, 0.28%, 0.051%, and 0.12%, respectively. The contents of chlorogenic acid in petroleum extract, ethyl acetate extract, n-butyl alcohol extract, water extract, fraction 1, fraction 2, and fraction 3 were 0.0078%, 0.38%, 1.0%, 0.03%, 0% (not detectable), 13.6%, and 42.0%, respectively. Screening of the Active Fraction(s) of EEGS in the Acute Alcohol Exposure Model. The acute alcohol exposure model designed by Carson and Pruett17 is particularly valuable when used to screen the efficacy of agents that may offer clinical benefits and for the mechanistic and predictive analysis of therapeutic compounds.18 In the current study, male Kunming mice (body weight 18−22 g) were used, and all experiments followed the WHO Guidance of Humane Care and Use of Laboratory Animals. Ethanol (5 g/kg) was given orally (by gavage) every 12 h for a total of three doses to induce liver injury in the acute alcohol exposure model. EEGS and its fractions were suspended in the ethanol solution so that the mice received ethanol and EEGS or its fractions at the same time. The mice were sacrificed 4 h after the last ethanol treatment, and their blood samples and whole livers were immediately collected. Blood samples were used to test for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity. Serum levels of ALT and AST were determined using commercial spectrophotometric kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. Liver samples were used for RTPCR, Western blot, triglyceride (TG) content analysis, and histological examination. We performed three rounds of screening with the acute ALD model to evaluate the active fraction(s) of G. procumbens. One round of screening was to determine the optimal EEGS dose, and the other two were to screen the active fraction(s) of the EEGS and nbutyl alcohol extract. Estimation of the Effect of EEGS and Fraction 2 in the Chronic Alcohol Exposure. Because acute alcohol exposure in mice induces hepatic steatosis in a manner similar to chronic ethanol administration, the acute model is useful as a screening tool and/or a mechanistic and predictive analysis for agents against liver disease induced by chronic alcohol intake.18 However, negative health consequences of alcohol use vary according to the drinking pattern, such as acute or chronic.1 The acute model with three binge-drinking episodes did not evoke the full range of symptoms of chronic alcohol consumption in the liver.19

molecular mechanisms underlying the protective effects of G. procumbens against ALD.

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MATERIALS AND METHODS

Reagents. G. procumbens was produced in Shantou, Guangdong, China (identified by Prof. Bingkun Zhang, Wuhan Botany, Chinese Academy of Sciences). P44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), acetyl-CoA carboxylase, phospho-acetyl-CoA carboxylase (Ser79) antibodies were purchased from Cell Signaling Technology (Beverly, MA). SREBP-1 antibody was purchased from Abcam (Cambridge, UK). AMPK1/2 and phospho-AMPK1/2 (Thr172) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The GAPDH antibody was purchased from AB CLONAL Biotechnology (Wuhan, Hubei, China). Chlorogenic acid and silymarin were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals purchased were of the purest form commercially available. Animals, Grouping, Treatment, and Sampling. Preparation of the Ethanol Extract from G. procumbens Stems (EEGS) and Its Subfractions. Fresh stems were minced, dried in the sun, and powdered for the experiments. The extract was prepared from 1000 g of powder extracted by heat reflux in 10 L of 80% ethanol at 85 °C for 1 h and filtered. The extraction was repeated three times. The filters were combined and evaporated under reduced pressure at 50 °C to remove the solvent. Then, the extract was lyophilized to obtain EEGS with a yield of 18.5%. The yield in the present study is always calculated using the following formula: yield = (weight of the dried extract/weight of sample used for extraction) × 100%. EEGS was further fractionated according to the schematic diagram shown in Figure 1. EEGS was dissolved in 500 mL of water, and the solution was successively extracted by the same volumes of petroleum, ethyl acetate, and water-saturated n-butyl alcohol. Each solvent extraction was repeated three times. The extracts of each solvent were combined and then evaporated under reduced pressure at 50 °C. The crude extracts of the four fractions were classed, yielding petroleum, ethyl acetate, n-butyl alcohol, and water at 5.4%, 14.2%, 13.3%, and 67.0%, respectively. The n-butyl alcohol extract was further fractionated on a polyamide glass column (custom-made, inner diameter 2 cm, packing height 40 cm). Elution was started with distilled water followed by 60% ethanol and ending with 95% ethanol, 375 mL each, 2.5 mL/min. The eluted fractions were evaporated under reduced pressure at 50 °C to give fraction 1 (yield 92.74%), fraction 2 (yield 6.49%), and fraction 3 (yield 0.76%). Analysis of Chlorogenic Acids by HPLC-UV. Five chlorogenic acidsneochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid Chave been isolated B


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Figure 2. Representative HPLC-UV chromatograms of EEGS and its subfractions. HPLC analysis was carried out with a U3000-Dionex instrument with a 5 μm Acclaim C-18 column (4.6 × 250 mm) (Thermo Fisher Scientific, Inc. Waltham, MA). Detection was carried out at 326 nm with a 70 min gradient. Solvent A was acetonitrile, and solvent B was 0.1% aqueous phosphoric acid solution. The gradient system was A-B (v/v) = 19/81 (0 min) → 27/73 (65 min) → 19/81 (70 min). The flow rate of the mobile phase was 1 mL/min. Peaks: 1, neochlorogenic acid; 2, chlorogenic acid; 3, isochlorogenic acid B; 4, isochlorogenic acid A; and 5, isochlorogenic acid C. Thus, we also evaluated the protective effect of G. procubens on chronic ethanol-induced liver steatosis. Liquid diets were based on the Lieber−DeCarli formulation (Dyets, Inc., Bethlehem, PA). Each mouse was housed in an individual cage and allowed ad libitum access to ethanol-containing diet. Control animals were pair-fed the same diet but with maltodextrin isocalorically substituted for ethanol.20 Either the ethanol-containing diet or the isocaloric maltose-dextrin (control) diet was fed to the animals for 8 weeks. The ethanol content began at 2.5% and was increased in a stepwise manner 0.5% every 2 days until the end of the first week and then 0.5% every 4 days until 5% was achieved. This level was maintained until the end of the experiment. Silymarin, a mixture of flavonolignans with anti-oxidant and antiinflammatory features, is extracted from the seed of Silybum marianum and protects against both acute21 and chronic22 ethanol-induced liver injury.2 In view of its nontoxic nature, easy availability, and wellstudied quality standard, silymarin was used as the positive control in the present experiment. Blood ethanol concentrations were measured using gas chromatography as previously described.23,24 Blood alcohol levels ranged between 66 and 186 mg/dL and were similar among the groups. Histological Assay. Liver tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. Three-micrometer sections were stained with hematoxylin and eosin (H&E) by standard methods and

then studied by light microscopy. Hepatic lipids were determined by staining 16 μm thick frozen liver sections with oil red O. Multispectral imaging was performed using the Nuance Multispectral Imaging System (Cambridge Research and Instrumentation Inc., Woburn, MA) as described in our previous study.25 Briefly, spectral optical density data were automatically acquired from 420 to 720 nm in 10 nm increments. Spectral unmixing was accomplished using Nuance software v1.42 with pure spectral libraries of oil red O (slide stained with only oil red O). For the quantification, three equal-sized fields of each photograph were randomly chosen for each experiment. Measurement of Liver TG Content. Hepatic levels of TG in mice were measured using a commercially available Tissue Triglyceride Assay kit (Applygen Technologies Inc., Beijing, China). A 20-fold volume of lysate was added to the 40 mg liver preparations. Supernatants were collected after centrifugation at 2000 rpm for 5 min and analyzed for TG content according to the manufacturer’s protocol. The TG concentrations were normalized to protein concentrations and expressed as mg of TG/g of protein. Real-Time PCR Assay. Total RNA was extracted from the stored frozen liver tissues using RNAiso Plus (TaKaRa Bio, Dalian, Liaoning, China) according to the manufacturer’s protocol. The isolated RNA was converted into complementary DNA using Advantage RT-forPCR Kit (TaKaRa Bio, Dalian, Liaoning, China). RT-PCR was C


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Journal of Agricultural and Food Chemistry Table 1. Primers Used for Quantitative PCR gene

forward primer (5′ to 3′)

reverse primer (5′ to 3′)

CPTIA Fabp1 Fabp4 PPAR-α Fasn SREBP-1 PPAR-γ ApoB MTTP CD68 IL-1β IL-6 CD163 Arginase TGF-β F4/80 MCP-1 TNF-α GAPDH

CTCAGTGGGAGCGACTCTTCA AAACTCACCATCACCTATGGAC GATGTGCGAACTGGACACAG TATTCGGCTGAAGCTGGTGTAC TCCTGGGAGGAATGTAAACAGC GATGTGCGAACTGGACACAG CGCTGATGCACTGCCTATGA AAGCACCTCCGAAAGTACGTG ATACAAGCTCACGTACTCCACT GTGTCTGATCTTGCTAGGACC TGGTGTGTGACGTTCCCATT CCACTTCACAAGTCGGAGGCTTA TCCACACGTCCAGAACAGTC ATGGAAGAGACCTTCAGCTAC GTGTGGAGCAACATGTGGAACTCT CTTTGGCTATGGGCTTCCAGTC CCAGCCTACTCATTGGGATCA ACCCTCACACTCAGATCATCTTC TGTGTCCGTCGTGGATCTGA

GGCCTCTGTGGTACACGACAA ATTGAGTTCAGTCACGGACTTT CATAGGGGGCGTCAAACAG CTGGCATTTGTTCCGGTTCT CACAAATTCATTCACTGCAGCC CATAGGGGGCGTCAAACAG AGAGGTCCACAGAGCTGATTCC CTCCAGCTCTACCTTACAGTTGA TCCACAGTAACACAACGTCCA TGTGCTTTCTGTGGCTGTAG CAGCACGAGGCTTTTTTGTTG CCAGTTTGGTAGCATCCATCATTTC CCTTGGAAACAGAGACAGGC GCTGTCTTCCCAAGAGTTGGG ACGCTGAATCGAAAGCCCTGTA GCAAGGAGGACAGAGTTTATCGTG CTTCTGGGCCTGCTGTTCA TGGTGGTTTGCTACGACGT TTGCTGTTGAAGTCGCAGGAG

performed on the Thermal Cycler Dice TP800 system (TaKaRa Bio, Otsu, Shiga Prefecture, Japan) using SYBR Premix Ex TaqII (TaKaRa Bio, Dalian, Liaoning, China) with 30−40 cycles of denaturation at 95 °C for 5 s, annealing, and extension at 60 °C for 30 s. GAPDH was used as an internal standard, and the mRNA expression levels of the target genes were normalized to that of GAPDH. The sequences of both forward and reverse primers are listed in Table 1. Western Blotting Assay. Total proteins were extracted from liver using Cell Lysis Buffer for Western and IP (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) containing 1% phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) and 1% phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentrations were determined by the Lowry method.26 Twenty micrograms of protein was separated by electrophoresis on a 6%, 8%, or 12% sodium dodecyl sulfate-polyacrylamide gel, and electrophoretically transferred to polyvinylidene difluoride membranes. The blots were incubated with specific primary antibodies for 1 h at room temperature: rabbit anti-AMPKα1/2 antibodies, rabbit anti-phosphop44/42 MAPK, rabbit p44/42 MAPK, rabbit anti-phospho-p38 MAPK, and rabbit p38 MAPK antibodies (1:200 dilution); mouse anti-SREBP-1 and rabbit anti-phospho-AMPKα1/2 (1:750 dilution); rabbit anti-acetyl-CoA carboxylase and rabbit anti-phospho-acetyl-CoA carboxylase antibodies (1:2000 dilution); and mouse anti-GAPDH antibody (1:10000 dilution) overnight at 4 °C. They were then incubated with a secondary antibody: rabbit polyclonal antiimmunoglobulin G (IgG), or mouse or rabbit monoclonal anti-IgG (1:2000 dilution). Antibody binding was detected using an enhanced chemiluminescence kit with hyper-enhanced chemiluminescence film. Statistical Analysis. All results were expressed as the mean ± standard deviation (SD). Statistical comparisons were performed using one way analysis of variance (ANOVA) with Dunnett’s multiple comparison test or unpaired Student’s t test. The analyses were performed with SPSS software (version 16.0, SPSS Inc., Chicago, IL). A p value less than 0.05 was considered to be statistically significant. The Western blot and optical histological results were obtained from at least three independent experiments, and the analysis was carried out with triplicate samples.

As shown in Figure 3A, acute alcohol gavage significantly increased serum activities of ALT (37.9 ± 6.0 U/L vs 30.5 ± 4.2 U/L); this elevation was significantly diminished by treatment with EEGS at a dose of 50 mg/kg. EEGS at a dose of 25 mg/kg also prevented the increase in ALT activity to a certain extent but with no significant difference compared with Group IB. As shown in Figure 3B, acute alcohol gavage increased serum activities of AST. This elevation was significantly diminished by treatment with 50 mg/kg EEGS. EEGS at a dose of 25 mg/kg also slightly prevented the increase in AST activity, but with no significant difference compared with Group IB. According to the above ALT and AST results, the suppressive effect on ethanol-induced injury occurred in a dose-dependent manner. To assess the effect of EEGS on hepatic steatosis induced by acute ethanol intake, hepatic lipid accumulation was qualitatively examined by oil red O staining and quantitatively determined by a TG quantification kit. After three doses of ethanol administration via oral gavage, mice from both the ethanol group and ethanol plus EEGS groups exhibited obvious accumulation of neutral lipid droplets in their livers compared with the control group, as illustrated by oil red O staining (Figure 3D,E). The lipid droplets in the livers of the EEGStreated groups were much fewer than those in the model group. Quantitative analysis (Figure 3F) confirmed the histological results by demonstrating that acute ethanol gavage dramatically increased the hepatic TG content in mice, and this elevation was significantly diminished by treatment of EEGS at doses of 12.5 and 50 mg/kg. These data clearly indicated that EEGS could effectively reverse acute ethanol-induced hepatic lipid accumulation. Although the data from the H&E staining and oil red O staining (Figure 3C−E) indicated the treatment with EEGS at dose of 25 mg/kg exerted a protective effect, the data in Figure 3F showed a tendency without statistical significance. In view of significant effect of both 12.5 and 50 mg/kg EEGS, the medium dose of 25 mg/kg was considered to be a turning point, and EEGS exerted bidirectional regulation in a dosedependent manner, which usually indicates the involvement of complex active ingredients and multiple pathways. Therefore,

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RESULTS EEGS Attenuated Acute Ethanol-Induced Lipid Accumulation in the Liver. ALT is released from the cytoplasm of impaired hepatocytes. Its serum activity is most commonly used in clinical practice as a reliable primary indicator for ALD.5,27 D


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Figure 3. EEGS attenuated acute ethanol-induced liver lipid accumulation. Animals were randomly divided into six groups: Group IA, control (normal saline treated); Group IB received EEGS at a dose of 50 mg/kg only; Groups IC−IF were treated with 5 g/kg ethanol orally every 12 h for a total of three doses; and Groups ID, IE, and IF were treated with EEGS at doses of 12.5, 25, and 50 mg/kg, respectively. The mice were sacrificed 4 h after the last ethanol treatment. (A) Serum alanine aminotransferase (ALT) activity. (B) Serum aspartate aminotransferase (AST) activity. (C) Representative photomicrographs of H&E staining of liver sections. Arrow indicates vacuolated hepatocytes. (D) Representative photomicrographs of oil red O staining of liver sections and (E) densitometric analysis of staining. Red is positive oil red O staining, and blue is hematoxylin counterstain. Top panel in (D): RGB images. Bottom panels: Unmixed oil red O images from the top panel. (F) Quantitation of hepatic triglyceride (TG) content. Data are expressed as the mean ± SD, n = 15; ***P < 0.001 vs control; ##P < 0.001 vs model. Scale bar = 100 μm.

the medium dose of 25 mg/kg was selected as an optimal dose for further study as specified below. The n-Butanol Fraction Extracted from EEGS Was the Active Fraction of EEGS. Serum ALT activity and hepatic lipid accumulation were used as assessment criteria to screen the active fraction(s) of EEGS. Hepatic lipid accumulation was evaluated by vacuolation in H&E stained sections and quantitatively determined by a TG quantification kit. As shown in Figure 4B, acute alcohol gavage increased serum ALT activity to a certain extent, and this elevation was significantly diminished by treatment with the n-butyl alcohol fraction from EEGS. In addition, the n-butyl alcohol fraction significantly reduced the ethanol-induced hepatic lipid accumulation as shown in Figure 4A,C. Again, treatment with EEGS only showed a tendency without statistical significance (Figure 4C), which was consistent with the data in Figure 3F. Moreover, these data provide a further evidence for the existence of bidirectional regulation in protective effect of EEGS against ethanol-induced liver steatosis. Together, the n-

butanol fraction was determined to be the most active fraction of EEGS and was selected for further fractionation by the polyamide glass column. Fraction 2 Was the Active Fraction of the n-Butyl Alcohol Extract. As shown in Figure 5B, acute alcohol gavage significantly increased serum ALT activity, and this elevation was diminished by treatment of fraction 1, 2, or 3 to a certain extent. In addition, fractions 1 and 2 significantly reduced the ethanol-induced hepatic lipid accumulation as shown in Figure 5A,C. Thus, fractions 1 and 2 were determined to be the active fractions of the n-butyl alcohol extract according to the screening criteria mentioned above. Compared with fraction 1, fraction 2 had fewer compounds (Figure 2) and required a lower dose to achieve similar activity. Thus, fraction 2 was selected for further study. EEGS, Fraction 2, and Chlorogenic Acid Attenuated Chronic Ethanol-Induced Hepatic Lipid Accumulation. As shown in Figure 6D, chronic alcohol treatment increased serum ALT activity, and this elevation was significantly E


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Figure 4. The n-butanol fraction was the active fraction of EEGS. Animals were randomly divided into seven groups: Group IIA, control (normal saline treated); Group IIB was treated with 5 g/kg ethanol only; Groups IIC, IID, IIE, IIF, and IIG received oral administration with 5 g/kg ethanol plus EEGS (25 mg/kg), petroleum ether fraction (1.35 mg/kg), ethyl acetate fraction (3.55 mg/kg), n-butanol fraction (3.33 mg/kg), or water fraction (16.75 mg/kg), respectively, every 12 h for a total of three doses. (A) Representative photomicrographs of H&E staining of liver sections. The arrow indicates vacuolated hepatocytes. (B) Serum alanine aminotransferase (ALT) activity. (C) Quantitation of hepatic triglyceride (TG) content. Data are expressed as the mean ± SD, n = 12; **P < 0.01 vs control; #P < 0.05 and ##P < 0.01 vs model. Scale bar = 100 μm.

Figure 5. Fraction 2 was the active fraction of the n-butyl alcohol extract. Animals were randomly divided into six groups with 6−9 mice per group: Group IIIA, control (normal saline treated); Group IIIB, treated with 5 g/kg ethanol only; Groups IIIC, IIID, IIIE, and IIIF were orally treated with 5 g/kg ethanol plus the n-butanol fraction (3.33 mg/kg), fraction 1 (3.10 mg/kg), fraction 2 (0.22 mg/kg), or fraction 3 (0.03 mg/kg), respectively, every 12 h for a total of three doses. (A) Representative photomicrographs of H&E staining of liver sections. The arrow indicates vacuolated hepatocytes. (B) Serum alanine aminotransferase (ALT) activity. (C) Quantitation of hepatic triglyceride (TG) content. Data are expressed as the mean ± SD, n = 6−9; **P < 0.01 vs control; #P < 0.05 and ##P < 0.01 vs model. Scale bar = 100 μm.

2, chlorogenic acid, and silymarin, attenuated chronic ethanolinduced liver lipid accumulation. EEGS and fraction 2 were more potent than chlorogenic acid and silymarin. Fraction 2 had similar effects as EEGS but was purer and more powerful (10 mg/kg/day vs 75 mg/kg/day). EEGS Alleviated the Ethanol-Induced Expression of Genes Involved in Lipid Metabolism and Inflammation

diminished by treatment with fraction 2. EEGS, chlorogenic acid, and silymarin also prevented the increase in ALT activity to a certain extent, but were not significantly different from Group IVB. Chronic ethanol-induced vacuolation in H&E staining and red staining in the oil red O staining of liver sections were far more serious than that of the acute sections (Figure 6A−C). All of the treatments, including EEGS, fraction F


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Figure 6. EEGS, fraction 2, and chlorogenic acid attenuated chronic ethanol-induced lipid accumulation in liver. Mice were divided into eight groups with 6−10 mice per group: Group IVA received a standard solid diet and water ad libitum; Group IVB, control (received an isocaloric maltodextrincontaining diet in a pair-fed fashion); Group IVC received an isocaloric maltodextrin-containing diet and was orally treated with EEGS (75 mg/kg/ day) for the last 2 weeks; Groups IVD−IVH were treated with an ethanol-containing diet; Groups IVE, IVF, IVG, and IVH were orally treated with EEGS (75 mg/kg/day), fraction 2 (10 mg/kg/day), chlorogenic acid (10 mg/kg/day), or silymarin (100 mg/kg/day), respectively, for the last 2 weeks. Either the ethanol-containing diet or the isocaloric maltodextrin (control) diet was fed to the animals for 8 weeks. (A) Representative photomicrographs of oil red O staining of liver sections and (C) densitometric analysis of staining. Red is positive oil red O staining, and blue is hematoxylin counterstain. Top panels in (A): RGB images. Bottom panels: unmixed oil red O images of the up panel. (B) Representative photomicrographs of H&E staining of liver sections. Arrow indicates vacuolated hepatocytes. (D) Serum alanine aminotransferase (ALT) activity. Data are expressed as the mean ± SD, n = 6−10; ***P < 0.001 vs pair-fed control; #P < 0.05 and ###P < 0.001 vs model. Scale bar for (A) = 100 μm. Scale bar for (B) = 50 μm.

in an Acute Model. Ethanol significantly induced the expression of fatty acid-uptake genes, including Fabp1 and Fabp4, and fatty acid-synthesis genes, including Fasn and SREBP-1, in the liver (Figure 7A). Intriguingly, EEGS treatment reduced the expression levels of Fabp1, Fabp4, Fasn, and SREBP-1. The genes involved in fatty acid βoxidation (CPT1A) and TG-secretion (MTTP) were also enhanced by ethanol exposure. However, EEGS significantly reduced the mRNA expression of CPT1A and MTTP. Moreover, ethanol did not show any significant effects on PPAR-α, PPAR-γ, or ApoB mRNA expression when compared to normal controls. PPAR-γ and ApoB mRNA expressions were upregulated by EEGS in the presence or absence of ethanol. We also analyzed inflammation markers including CD68, IL1β, IL-6, CD163, Arginase, TGF-β, F4/80, MCP-1, and TNF-α (Figure 7B). We found that ethanol exposure elevated the expression of almost all of the above genes except for F4/80 and TNF-α. However, EEGS treatment reversed these ethanolinduced mRNA changes. Even F4/80 and TNF-α mRNA expressions were reduced by EEGS treatment. These data indicated that EEGS might relieve ethanol-induced liver damage by regulating of lipid metabolism and reducing inflammatory responses in the liver. Administration of EEGS, Fraction 2, or Chlorogenic Acid Alleviated Hepatic Steatosis through MAPK/ SREBP-1c-Dependent and -Independent Pathways. Premature SREBP-1c (pSREBP-1c) is cleaved and activated in response to ethanol feeding, which is closely associated with an increased expression of hepatic lipogenic genes and the accumulation of TG in the liver.28 To investigate the possible underlying mechanisms of EEGS-mediated hepatic improve-

ments, we measured the changes of SREBP-1c and its upstream regulators (MAPK and AMPK) and downstream effector (ACC) in the chronic model. There are two obvious bands of SREBP-1c in Figure 8A, the top one is so-called pSREBP-1c, and the bottom one is mSREBP-1c. The pSREBP-1c band in the present study represented both normal and phosphorylated form of premature SREBP-1c. Both the premature and mature forms of SREBP-1c were significantly elevated by chronic ethanol administration. After treatment with EEGS or fraction 2, the influence of ethanol on mSREBP-1c was abolished. Consequently, we measured the changes of key MAPK members, which suppress SREBP-1c activity by phosphorylation.15,29 The administration of ethanol significantly decreased the phosphorylation of both p38 MAPK and p44/ 42 MAPK (Figure 8B). After treatment with EEGS or fraction 2, the influence of ethanol on the phosphorylation of both p38 MAPK and p44/42 MAPK proteins was abolished. In addition, ethanol treatments decreased the total expression of p38 MAPK but not p44/42 MAPK. Moreover, the effect of ethanol on SREBP-regulated promoter activation was mediated, at least in part, through inhibition of AMPK.30 However, neither the ethanol nor the EEGS treatments demonstrated any effects on the expression of the total or phosphorylated form of AMPK (Figure 8C). These data indicated that AMPK was not involved in the SREBP-1c pathway in the present study. The first committed step in fatty acid biosynthesis is carried out by ACC.10 The regulation of ACC occurs at multiple levels. The first level is regulated by phosphorylation/dephosphorylation, and AMPK phosphorylates and inhibits ACC enzymatic activity.10,31 The second level is regulated by G


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Figure 7. Hepatic expression levels of lipid metabolism-related and inflammation marker genes in mice. Group IA, control (normal saline treated); Group IB received EEGS at a dose of 50 mg/kg only; Groups IC−IF were treated with ethanol orally every 12 h for a total of three doses; Groups ID, IE, and IF were treated with EEGS at a dose of 12.5, 25, and 50 mg/kg, respectively. The mice were sacrificed 4 h after the last ethanol treatment. The mRNA expression of lipid metabolism-related genes (A) and the mRNA expression of inflammation markers (B) were evaluated by quantitative real-time PCR analysis and normalized to the housekeeping gene GAPDH. Data are expressed as the mean ± SD of 15 mice; *P < 0.05 and **P < 0.01 vs control; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs model.

transcription, and SREBP-1c increases ACC expression.11,12 In the present study, total ACC and its phosphorylated form were evaluated during chronic ethanol administration (Figure 8C). However, the ratio between the phosphorylated and total ACC forms was progressively decreased by chronic ethanol administration, and after treatment with EEGS or fraction 2, the influence of ethanol on the ratio was abolished. The administration of EEGS and fraction 2 abolished the influence of ethanol on SREBP-1c activation, its upstream regulators (phosphorylation of both p38 MAPK and p44/42 MAPK) and downstream effectors, such as ACC. Thus, the MAPK/SREBP-1c-dependent pathway participated in the protective effect of EEGS and fraction 2 against hepatic steatosis. As one of the major active ingredients of fraction 2, chlorogenic acid canceled the negative effect of ethanol on ACC expression and activation. However, unlike fraction 2, chlorogenic acid did not change the MAPK or SREBP-1c activity in the current study. This result indicated that chlorogenic acid inhibited ACC in a MAPK/SREBP-1cindependent manner and that there are other ingredient(s) in fraction 2 that impact the MAPK/SREBP-1c pathway.

protective effects against ethanol-induced liver injury. A previous review has collected 34 herbal medicines and/or active compounds specifically used for that purpose.27 As summarized by Ding et al. in that review, the underlying mechanisms and active ingredients have not been sufficiently elucidated. In the present study, the capability of EEGS and its fractions to protect against ethanol-induced liver injury were investigated. Emphasis was placed upon the underlying molecular mechanisms and the active ingredient and its contributions to the protective effect. The ability to prevent increased serum ALT activity and hepatic lipid accumulation in acute ethanol-induced liver steatosis was used as an assessment criterion to screen the active fraction(s) from EEGS. The n-butyl alcohol extract was the active fraction of EEGS and was selected for further fractionation by the polyamide glass column. The 60% ethanoleluted fraction that contained 13.6% chlorogenic acid was the most active fraction, and its effects were further evaluated in the chronic model. Data in the chronic model demonstrated that treatment with the n-butyl alcohol extract, 60% ethanol-eluted fraction, or chlorogenic acid protected against ethanol-induced liver steatosis, indicating that chlorogenic acid, at least in part, contributed to the beneficial effect of G. procumbens on alcoholic fatty liver. Compared with the doses of well-known commercial products, such as Panax notoginseng saponins

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DISCUSSION Over the past decade, dozens of herbs and individual compounds isolated from herbs have been shown to possess H


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Figure 8. Effect of EEGS, fraction 2, or chlorogenic acid on the protein expression of the MAPK/SREBP-1c/ACC pathway. Mice were divided into eight groups with 6−10 mice per group: Group IVA received a standard solid diet and water ad libitum; Group IVB, control (received an isocaloric maltodextrin-containing diet in a pair-fed fashion); Group IVC received an isocaloric maltodextrin-containing diet and was orally treated with EEGS (75 mg/kg/day) for the last 2 weeks; Groups IVD−IVH were treated with an ethanol-containing diet; Groups IVE, IVF, IVG, and IVH were orally treated with EEGS (75 mg/kg/day), fraction 2 (10 mg/kg/day), chlorogenic acid (10 mg/kg/day), or silymarin (100 mg/kg/day), respectively, for the last 2 weeks. Either the ethanol-containing diet or the isocaloric maltose-dextrin (control) diet was fed to the animals for 8 weeks. Western blot analysis using a specific antibody was used to examine the expression of the proteins in the mouse liver homogenates. Bands densities were determined using ImageJ, normalized to GAPDH, and expressed as a percentage of the standard solid diet control. Representative photographs of Western blots of SREBP-1c (A), p44/42 MAPK and p38 MAPK (B), and ACC and AMPK (C), with quantitative analysis of blots given below each set of photographs, where each bar represents the mean ± SD of the results obtained from three mice. *P < 0.05, **P < 0.01, and ***P < 0.001 vs pair-fed control; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs model.

(100−300 mg/kg/day),5 silymarin (100−200 mg/kg/day),21 or most of the above-mentioned herbal medicines, the dose of fraction 2 and chlorogenic acid used in the present study was much lower, just 10 mg/kg/day. In addition, in the present study, fraction 2 showed better potential than chlorogenic acid and silymarin to prevent ethanol-induced liver steatosis. The mechanism by which ethanol causes fatty liver and liver injury is complicated. Anti-oxidative stress, anti-inflammation,

and lipid metabolism regulation are three major mechanisms that are involved in the protective effect of herbal medicines against ALD.1,27 Alcoholic fatty liver is the earliest and most common response of the liver to alcohol and may be a precursor of more severe forms of liver injury. SREBP-1c is a master regulator of lipid homeostasis, and SREBP-1 knockout mice are completely protected from ALD, indicating a causal involvement of SREBP-1 in ALD.16 SREBP-1c positively I


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In summary, our data show that G. procumbens protected against ethanol-induced liver steatosis, possibly through ameliorating hepatic lipid accumulation by modulating lipid metabolism-related genes through MAPK/SREBP-1c-dependent and -independent pathways. Our findings also suggest that G. procumbens and one of its active ingredients, chlorogenic acid, could potentially be developed as effective agents for acute or chronic ethanol-induced liver injury.

regulates the expression of genes encoding lipogenic enzymes, including ACC and FAS.11,12 The most important function of ACC is to provide the malonyl-CoA substrate for the synthesis of fatty acids, and the main function of FAS is to catalyze the synthesis of palmitate. The present study indicated that ethanol induced expression levels of ACC and FAS, which is in accordance with a previous report.28 The effect of ethanol on SREBP-regulated promoter activation was mediated, at least in part, through inhibition of AMPK and MAPK.30 In several studies, AMPK phosphorylation is increased,32,33 whereas in other studies, AMPK phosphorylation is decreased.34,35 Ethanol (amount and feeding duration) and fat (type and dose) used in these studies might contribute to the discrepancies.36 Unlike AMPK, the changes of MAPK were in accordance with SREBP-1c, indicating a possible link between MAPK and SREBP-1c and its downstream genes in the present study. The administration of EEGS and fraction 2 abolished the influence of ethanol on SREBP-1c activation and its upstream regulators and downstream effectors. Thus, the MAPK/SREBP-1c-dependent pathway contributes to the mechanism of the beneficial effect of EEGS and fraction 2 on alcoholic fatty liver. As one of the major active ingredients of fraction 2, chlorogenic acid was chosen to further investigate the underlying mechanism. Chlorogenic acid has many biological properties, including anti-bacterial, anti-oxidant, and anticarcinogenic activities. Recently, the roles and applications of chlorogenic acid in relation to hepatic steatosis have been highlighted.37,38 Chlorogenic acid inhibits the MAPK pathway39,40 to protect drug-induced liver injury while activating AMPK to improve lipid metabolism in non-alcoholic fatty liver disease.37 However, unlike fraction 2, chlorogenic acid did not change MAPK or SREBP-1c activity in the current study, which indicated that chlorogenic acid inhibited ACC in a MAPK/ SREBP-1c-independent pathway and that there was an addtional ingredient(s) in fraction 2 that impacted the MAPK/SREBP-1c pathway. In addition to AMPK, protein kinase A also has the ability to phosphorylate ACC. Other kinases are also suspected to be important in this regulation, because many other possible phosphorylation sites exist on ACC.41 Both MAPK/SREBP-1c-dependent and -independent pathways contributed to the regulation of G. procumbens on lipid metabolism-related genes, which exerted subsequent protective effects against ethanol-induced hepatic lipid accumulation. ALD is a complex process. In addition to lipid accumulation, ethanol-induced liver injury is highly linked to inflammatory and oxidative stress.1 Inflammation and oxidative stress are two etiological factors that have been suggested to play important roles in the development of ethanol-induced liver injury. Silymarin, the positive control in the present study, is a good example of an agent that protects against alcohol-induced liver disease via anti-inflammatory and anti-oxidative features.21 Increased pro-inflammatory cytokine levels have been well documented in ALD.42 EEGS treatment alleviated the ethanolinduced upregulation of cytokine levels, which should have resulted in a corresponding attenuation of ethanol-induced liver injury. In addition, because chlorogenic acid is an anti-oxidant and ethanol treatment enhances oxidative stress in the liver,43 we could assume that the reduction of oxidative stress may also partially contribute to the prevention of liver injury in the present study, but more precise experiments are needed to further confirm that hypothesis.

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AUTHOR INFORMATION

Corresponding Authors

*(H.-B.T.) Tel/fax: +86 27 6784 2332. E-mail: hbtang2006@ mail.scuec.edu.cn. *(H.-C.S.) Tel/fax: +86 10 8401 2510. E-mail: shanghongcai@ 126.com. Author Contributions #

X.-J.L. and Y.-M.M. contributed equally to this work.

Author Contributions

Y.-M.M., T.-T.L., M.-T.Z., Y.-L.Y., and X.-J.L. carried out the experiments. X.-J.L., H.-B.T., and H.-C.S. proposed and designed the research, performed the data analysis, and wrote the paper. Y.-S.L. and W.K.Z. assisted in the data analysis and interpretation. Funding

This study was supported by grants from the National Natural Science Foundation of China (81403188 and 81373842), the Modernization Engineering Technology Research Center of Ethnic Minority Medicine of Hubei province (2015ZY002), and the Natural Science Foundation of China Hubei (2013CFB451). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EEGS, ethanol extract from Gynura procumbens stems; MAPK, mitogen-activated protein kinase; SREBP-1c, sterol regulatory element binding protein 1c; ALT, alanine transaminase; AST, aspartate transaminase; ALD, alcoholic liver disease; AMPK, 5′adenosine monophosphate-activated protein kinase; TG, triglyceride; H&E, hematoxylin and eosin; RT-PCR, real-time polymerase chain reaction; IgG, immunoglobulin G; ACC, acetyl-coenzyme A carboxylase; CPT1A, carnitine palmitoyltransferase 1A; Fabp1, fatty acid binding protein 1; Fabp4, fatty acid binding protein 4; PPAR-α, peroxisome proliferatoractivated receptor-α; Fas, fatty acid synthase; PPAR-γ, peroxisome proliferator-activated receptor-γ; ApoB, apolipoprotein B; MTTP, microsomal triglyceride transfer protein; CD68, cluster of differentiation 68; IL-1β, interleukin-1β; IL-6, interleukin-6; CD163, cluster of differentiation 163; TGF-β, transforming growth factor-β; F4/80, EGF-like modulecontaining mucin-like hormone receptor-like 1; MCP-1, monocyte chemotactic protein 1; TNF-α, tumor necrosis factor-α

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Gynura procumbens Reverses Acute and Chronic Ethanol-Induced

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