Prevention of Free Fatty Acid–Induced HepaticLipotoxicity by 18�-Glycyrrhetinic Acid Through

Lysosomal and Mitochondrial Pathways

Xudong Wu,1,2 Luyong Zhang,1 Emily Gurley,2 Elaine Studer,2 Jing Shang,1 Tao Wang,1 Cuifen Wang,1 Ming Yan,1

Zhenzhou Jiang,1 Phillip B. Hylemon,2,3 Arun J. Sanyal,3 William M. Pandak, Jr,3 and Huiping Zhou1-3

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and affectsmillions of people worldwide. Despite the increasing prevalence of NAFLD, the exactmolecular/cellular mechanisms remain obscure and effective therapeutic strategies arestill limited. It is well-accepted that free fatty acid (FFA)-induced lipotoxicity plays apivotal role in the pathogenesis of NAFLD. Inhibition of FFA-associated hepatic toxicityrepresents a potential therapeutic strategy. Glycyrrhizin (GL), the major bioactive com-ponent of licorice root extract, has a variety of pharmacological properties includinganti-inflammatory, antioxidant, and immune-modulating activities. GL has been used totreat hepatitis to reduce liver inflammation and hepatic injury; however, the mechanismunderlying the antihepatic injury property of GL is still poorly understood. In thisreport, we provide evidence that 18 �-glycyrrhetinic acid (GA), the biologically activemetabolite of GL, prevented FFA-induced lipid accumulation and cell apoptosis in invitro HepG2 (human liver cell line) NAFLD models. GA also prevented high fat diet(HFD)-induced hepatic lipotoxicity and liver injury in in vivo rat NAFLD models. GAwas found to stabilize lysosomal membranes, inhibit cathepsin B expression and enzymeactivity, inhibit mitochondrial cytochrome c release, and reduce FFA-induced oxidativestress. These characteristics may represent major cellular mechanisms, which account forits protective effects on FFA/HFD-induced hepatic lipotoxicity. Conclusion: GA signif-icantly reduced FFA/HFD-induced hepatic lipotoxicity by stabilizing the integrity oflysosomes and mitochondria and inhibiting cathepsin B expression and enzyme activity.(HEPATOLOGY 2008;47:1905-1915.)

Abbreviations: AO, acridine orange; DCF, 2,7-dichlorofluorescein; DCFH, 2,7-dichlorofluorescin; DCFH-DA, 2,7-dichlorofluorescein diacetate; FFA, free fatty acid;FITC, fluorescein isothiocyanate; GA, 18�-glycyrrhetinic acid; GL, glycyrrhizin; HFD, high fat diet; IC50, median inhibitory concentration; JC-1, 5,5�,6,6�-tetrachloro-1,1�,3,3� tetraethyl benzimidazolylcarbocyanine iodide; JNK, c-jun N-terminal kinase; Me, methyl ester; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholicsteatohepatitis; PA, palmitate; PBS, phosphate-buffered saline; PI, propidium iodide; ROS, reactive oxygen species; SA, stearate; SEM, standard error of the mean; TG,triglyceride (or triacylglycerol); TNF, tumor necrosis factor

From the 1Jiangsu Center for Drug Screening, Jiangsu Center for Pharmacodynamic Research and Evaluation, China Pharmaceutical University, Nanjing, Jiangsu,People’s Republic of China; and Departments of 2Microbiology and Immunology and 3Internal Medicine/Gastrointestinal Division, Virginia Commonwealth University,Richmond, VA.

Received November 9, 2007; accepted January 16, 2008.Supported by the Megaproject of Science Research for the 10th Five-Year Plan (no. 2004AA2Z3785) of the Ministry of Science and Technology of the People’s Republic

of China; the 333 Key Program of Jiangsu Province R&D Human Resource (2005-2006) of the People’s Republic of China; the Program for New Century Excellent Talentsin University of the Ministry of Education of the People’s Republic of China (grant NCET-04-0507, 2004-2007); National Institutes of Health grants R21 AI068432 andR01 AT004148 to H.Z. and grants R01 AI057189 and P01 DK38030 to P.B.H; the GlaxoSmithKline Research Fund; and the A.D. Williams Fund.

Address reprint requests to: Huiping Zhou, Ph.D., Department of Microbiology & Immunology, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA23298-0678. E-mail: hzhou@vcu.edu; fax: 804-828-0676; or Lu-yong Zhang, Ph.D., China Pharmaceutical University, Nanjing, Jiangsu, 210009. E-mail:lyzhang@cpu.edu.cn.

Copyright © 2008 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.22239Potential conflict of interest: Nothing to report.Supplementary material for this article can be found on the HEPATOLOGY Web site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

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Nonalcoholic fatty liver disease (NAFLD) is themost common liver disease in the United Statesand worldwide, and has emerged as a major

public health concern. NAFLD has two stages: a fattyliver and nonalcoholic steatohepatitis (NASH), the mostsevere form of NAFLD, and end-stage liver disease.1 Cur-rently, the cellular mechanisms of NAFLD developmentand disease progression remain undefined and the thera-peutic strategies are still limited. Numerous studies sug-gest that obesity, diabetes, and the metabolic syndromeare closely associated with the disease progression ofNAFLD.2,3

Free fatty acids (FFAs) are the important mediators oflipotoxicity. Circulating FFAs are the primary contribu-tor to the liver triacylglycerol (TG) content, and plasmaFFAs levels are correlated with disease severity in NAFLDpatients.4 Although the mechanism by which FFAs in-duce lipoapoptosis is still not fully identified, most recentstudies indicate that multiple mechanisms might be in-volved in FFA-induced hepatic lipotoxicity. FFAs notonly induce c-jun N-terminal kinase (JNK)-dependentactivation of apoptotic Bcl-2 proteins Bim and BCL-2-associated X protein, which trigger the mitochondrial ap-optotic pathway,2 but also induce tumor necrosis factor(TNF)-� expression through a lysosomal pathway.5 Ac-cumulation of intracellular FFAs results in lysosomal per-meabilization and release of cathepsin B into the cytosol,which further promotes mitochondrial reactive oxygenspecies (ROS) production and release of cytochrome c.6,7

Both lysosomal and mitochondrial apoptotic pathwaysplay pivotal roles in TNF-�-induced hepatocyte apopto-sis and liver injury.6,8

Cathepsin B is the best characterized mammalian cys-teine peptidase and is ubiquitously expressed.9 Intracellu-lar cathepsin B is localized in the lysosomes. It has beenshown that hepatic steatosis is associated with lysosomalpermeabilization.10,11 Disruption of the lysosomes andsubsequent release of lysosomal proteases into cytosol hasbeen implicated in cellular injury. Recent studies havedemonstrated that inactivation of cathepsin B attenuateshepatocyte apoptosis and liver damage induced byTNF-�, cholestasis, and cold ischemia–warm reperfu-sion.10-12 These findings suggest that cathepsin B plays aprominent role in apoptosis and tissue injury. CathepsinB inhibition may have a potential therapeutic effect intreating liver diseases.

Licorice is one of the most ancient medicinal plants; ithas long been used as a conditioning and flavoring agentand in traditional Chinese medicine for the treatment ofvarious inflammatory diseases.13 Glycyrrhizin (GL) is amajor bioactive triterpene glycoside of licorice root ex-tract and has a variety of pharmacological properties. GL

is one of the leading natural compounds currently usedclinically for treatment of chronic hepatitis C and humanimmunodeficiency virus infections.14,15 Pharmacokineticstudies have shown that GL exhibits its pharmacologicalfunctions through its biologically active metabolite, 18�-glycyrrhetinic acid (GA), which is formed by presystemichydrolysis (Fig. 1).15 Although it has been shown that GAhas a protective effect on hepatic injury, the underlyingmechanism by which GA improves liver biochemistry andhistology remains unknown. Whether GA has a protec-tive effect on FFA-induced lipotoxicity in liver has notbeen explored so far. The primary objective of the currentstudy was to examine the effects of GA on FFA-inducedlipotoxicity and the potential underlying mechanisms.

Materials and Methods

Cell Culture Treatment. Human HepG2 cells werecultured in modified Eagle’s medium with 10% fetal bo-vine serum. FFAs (oleate/palmitate [PA], 2:1) were mixedwith FFA-free bovine serum albumin and the mixture wasadded to medium to a final concentration of 1 mM. GAwas dissolved in dimethylsulfoxide.

Fig. 1. Biotransformation of GL to GA.

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Isolation and Culture of Primary Hepatocytes.Primary hepatocytes were isolated from male Sprague-Dawley rats (250 to 300 g) and cultured as described.16

Animal Studies. To examine the effect of high fatdiet (HFD)-induced hepatic injury, male Sprague-Daw-ley rats (180 to 200 g) were randomly assigned to fourgroups (n � 8). The first control group was fed a normaldiet; the three HFD groups were fed HFD containing 2%cholesterol and 10% lard for 4, 6, and 8 weeks, respec-tively. To examine the protective effect of GA on HFD-induced lipotoxicity in liver, rats were randomly assignedto the following four groups: (I) normal control; (II)HFD group; (III) HFD plus GA 25 mg/kg; and (IV)HFD plus GA 50 mg/kg. Rats in the normal controlgroup (I) were fed a standard diet; the rats in the otherthree groups (II-IV) were fed HFD. The rats were gavagedwith GA or control solution every day for 8 weeks. All ratswere housed under identical conditions in an aseptic fa-cility and given free access to water and food. The ratswere weighed once a week to adjust drug intake. At theend of each time period, rats were fasted for 16 hours andblood samples were collected. Serum total cholesterol,free cholesterol, TG, low density lipoprotein-cholesterol,high density lipoprotein-cholesterol, alanine aminotrans-ferase, and aspartate aminotransferase were measured us-ing standard enzymatic techniques. All studies wereapproved by the Animal Study Committee of ChinaPharmaceutical University.

Histopathology Analysis. For each rat, three speci-mens from different regions of the liver were collected andfixed in 4% paraformaldehyde in 0.1 M phosphate bufferat room temperature overnight. The regions of the spec-imens were standardized for all rats. The paraffin-embed-ded tissue sections (4 �m) were stained with hematoxylinand eosin according to standard techniques. The sampleswere examined blindly by a professional pathologist toevaluate the presence of ballooning, steatosis, inflamma-tion, and fibrosis. Lesions were evaluated semiquantita-tively on a four-point scale (1 � absent, 2 � mild, 3 �moderate, and 4 � intense) for each damage as describedby Brunt et al.17 The extent of lesions in each rat wasexpressed as the average score of three separate specimens.

Electron Microscopy. The HepG2 cells were col-lected and fixed with 4% glutaraldehyde in 0.1 M phos-phate buffer, pH 7.2, at 4°C for 15 minutes. Cells wererinsed for 30 minutes in three changes of 0.1 M phos-phate buffer, followed by treatment with 1% OsO4 in 0.1M phosphate-buffer for 1 hour. After being washed withdistilled water three times, the cells were dehydrated andembedded with epoxy resin. Ultrathin sections of 60 nmwere prepared. Electron photomicrographs were taken of

the ultrastructures of HepG2 cells under a transmissionelectron microscope.

Nile Red Staining. Nile red staining was used tospecifically stain the intracellular fat. The HepG2 cellswere treated with 1 mM of FFAs together with GA (0 to30 �M) for 24 hours. Cells were collected and incubatedwith Nile red (100 ng/mL) in PBS for 5 minutes. Afterbeing washed with PBS, the cells were resuspended in PBSand the fluorescence intensity was measured by flow cy-tometry at an excitation wavelength of 488 nm. Un-stained cells were used to adjust instrument settings.18

Apoptosis Analysis. HepG2 cells were treated with 1mM FFAs together with various concentrations of GA or20 �M of CA-074 methyl ester (Me) (a cathepsin B–spe-cific inhibitor) for 24 hours. Cells were collected andwashed with cold PBS, then resuspended in 400 �L ofreaction solution (10 mM of 4-[2-hydroxyethyl]-1-piper-azine ethanesulfonic acid, 140 mM of NaCl, 2.5 mM ofCaCl2, pH 7.4) and incubated with Annexin V-fluores-cein isothiocyanate (FITC) and propidium iodide (PI)staining solution according to the manufacturer’s instruc-tions. Cells stained with Annexin V-FITC and PI werefurther analyzed by two-color flow cytometry to quantifythe apoptotic cells. Annexin V and PI emissions weredetected in the FL1 and FL3 channels of a Cytinics FC500 flow cytometer. At least 10,000 cells were analyzed ineach sample.

Lysosomal Stability Analysis. Lysosomal stabilitywas assessed by using the acridine orange (AO)-uptakemethod.19 AO is a metachromatic fluorophore, whichshows red fluorescence at high (lysosomal) concentrationsand green fluorescence at low (nuclear and cytosolic) con-centrations. Rupture of lysosomes was monitored as anincrease in the number of cells with decreased AO uptake,as indicated by low red fluorescence. Cells were collectedafter treatment with 1 mM FFAs together with variousconcentrations of GA for 24 hours and were stained withAO (5 �g/mL) for 30 minutes at 37°C in the dark. Theintensity of red fluorescence was measured by flow cytom-etry, using the FL3 channel. Cells with decreased red flu-orescence were gated and their percentages wereindicated.

Measurement of �-Galactosidase Activity. To de-termine the effect of GA on lysosomal stability in liver, thefree form and membrane-bound form of �-galactosidaseactivities were measured. Increase of free �-galactosidaseactivity indicates the lysosomal rupture. Liver tissues werehomogenized in 0.3 M sucrose at 4°C and centrifuged at35,000g for 20 minutes. The supernatant with free en-zyme was collected. The pellets containing bound enzymewere suspended in 0.3 M sucrose containing 0.1% TritonX-100, and were incubated at 4°C for 24 hours. The

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supernatant containing bound enzyme was collected bycentrifugation at 35,000g for 20 minutes.20 The �-galac-tosidase activity was determined based on the degradationof 4-methylumbelliferyl-�-D-galactopyranoside using aTecan Safire2 Microplate Reader (excitation wave: 360nm, emission wave: 450 nm). The enzyme activity wasexpressed as nmol/mg protein.

Western Blot Analysis. The total cell lysates wereprepared and processed as described.16 The expressionlevels of cathepsin B, cytochrome c, p-JNK, and totalJNK were detected with specific antibodies. Both �-actinand �-tubulin were used as loading controls. The densi-ties of immunoblot bands were analyzed with Image Jcomputer software (National Institutes of Health).

Mitochondrial Transmembrane Potential. Themitochondrial transmembrane potential (��) was mea-sured by using the fluorescent probe 5,5�,6,6�-tetra-chloro-1,1�,3,3�-tetraethylbenzimidazolylcarbocyanineiodide (JC-1), which is able to selectively enter the mito-chondria and exists in a monomeric form emitting at 527nm with excitation at 490 nm. When JC-1 forms aggre-gates, the emission is shifted to 590 nm. When the mito-chondrial membrane is depolarized, the aggregatesdecrease and monomers increase. HepG2 cells weretreated with 1 mM of FFAs together with various concen-trations of GA for 24 hours. Cells were collected andstained with JC-1 (5 �g/mL) at 37°C in the dark for 15minutes. The fluorescence of FL-1 for JC-1 monomersand FL-2 for JC-1 aggregates was measured by flow cy-tometry.21

Measurement of Cathepsin B Activity. Cathepsin B(2 �g/mL) isolated from human liver (from Sigma Cat#C8571) was incubated with 200 �M of cathepsin B–spe-cific substrate Z-Arg-Arg-7-amido-4-methylcourmarinhydrochloride together with various concentrations ofGA or CA-074 in 60 �L of assay buffer (100 mM 2-(N-morpholino)ethanesulfonic acid, pH 5.5, 20 mM dithio-threitol) at 37°C for 30 minutes. The level offluorescence, generated through the hydrolysis of Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride by ca-thepsin B only, was measured on an excitation wavelengthof 355 nm and an emission wavelength of 460 nm.22 Tomeasure the cathepsin B activity in HepG2 cells and ratliver tissue, the whole-cell lysates and liver homogenateswere centrifuged at 15,000g for 10 minutes at 4°C. Thesupernatant containing cathepsin B was collected. Thecathepsin B activity was assayed as described above.

Cathepsin B Immunofluorescence. Animals wereperfused intracardially initially with 0.9% saline and thenwith 4.0% paraformaldehyde (pH 7.4). After the livercapsule was carefully dissected away, the segments of tis-sue were postfixed in 4% paraformaldehyde for 2 hours

and then in 30% sucrose overnight.22 Optimal cuttingtemperature (OCT)-embedded segments were sectioned(3 �m) and mounted onto slides at �30°C. The sectionswere blocked in blocking buffer (5% goat serum, 5%glycerol, 0.004% sodium azide) at 37°C for 30 minutes,then incubated for 2 hours with a rabbit polyclonal anti-cathepsin B antibody (1:200). After the sections werewashed with PBS three times, FITC-conjugated goat an-ti-rabbit antibody was added and sections were incubatedat 37°C for 45 minutes. Images were collected with afluorescence microscope.

Measurement of Intracellular ROS. IntracellularROS was measured using the 2,7-dichlorofluorescein di-acetate (DCFH-DA) method.23 DCFH-DA diffusesthrough the cell membrane and is enzymatically hydro-lyzed by intracellular esterases to DCFH, which can berapidly oxidized to highly fluorescent DCF in the pres-ence of ROS. After cells were incubated with 1 mM FFAstogether with GA for 24 hours, DCFH-DA was added toa final concentration of 5 mM and cells were incubated at37°C in darkness for 30 minutes. DCF fluorescence in-tensity was detected with an emission wavelength of 530nm and an excitation wavelength of 485 nm, using flowcytometry.

Statistical Analysis. All results were expressed asmean � standard error of the mean. One-way analysis ofvariance and Student t test were used to analyze the dif-ferences between different treatments. Statistics were per-formed using GraphPad Pro. P � 0.05 was consideredstatistically significant.

Results

Effect of GA on FFA-Induced Lipid Accumulationin HepG2 Cells. It is generally believed that fatty liverresults from an imbalance between the hepatic uptake ofFFAs, TG synthesis, and excretion. Recently, Gomez-Lechon et al.24 validated an in vitro model of steatosisusing HepG2 cells. HepG2 cells loaded with an FFA mix-ture containing oleate/PA (2:1 ratio) mimics benignchronic steatosis. FFA-overloaded HepG2 cells (1 mMFFAs) reached similar levels of maximal intracellular lipidaccumulation as found in human liver with steatosis.Therefore, we used 1 mM FFAs (oleate/PA, 2:1) in thepresent study.

First, we examined whether GA was able to preventFFA-induced lipid accumulation in HepG2 cells usingNile red staining. GA significantly reduced FFA-inducedlipid accumulation (Fig. 2). CA-074 Me exhibited astronger effect.

Inhibition of FFA-Induced Apoptosis by GA inHepG2 Cells. We initially confirmed FFA-induced apo-

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ptosis in HepG2 cells. We treated the cells with 1 mMFFAs for 4, 8, 12, and 24 hours, and detected apoptoticcells by Annexin V-FITC/PI staining. As expected, FFAinduced apoptosis, in a time-dependent fashion (data notshown). Then, we examined whether GA was able to in-hibit FFA-induced apoptosis in HepG2 cells. The simul-taneous treatment of cells with FFAs and GA for 24 hoursdose-dependently inhibited FFA-induced apoptosis (Fig.3A). CA-074 Me (20 �M), also partially attenuated FFA-induced apoptosis. We further used electron transmissionmicroscopy to confirm the characteristic of cell apoptosis,such as disruption of nuclear integrity and lysosomalstructure, chromatin margination and condensation, andlipid accumulation induced by 1 mM FFAs (Fig. 3B-b),and the protective effect of GA. GA significantly inhibitedFFA-induced lipid accumulation and apoptosis (Fig. 3B-c,d). In rat primary hepatocytes, GA showed the similarprotection against PA and stearate (SA)-induced apopto-sis (Supplementary Fig. S1)

Effect of HFD on Lipid Accumulation and Cathep-sin B Expression in Rat Liver. HFD-induced ratNAFLD models have been found to be useful models forhuman NAFLD.25 The hepatic histology of NAFLD canvary from isolated hepatic steatosis alone without histo-logical evidence of hepatocellular damage to steatohepa-titis with associated inflammation and hepatocytedamage. We fed the rats with HFD for 0, 4, 6, and 8weeks. The histological analysis demonstrated a time-de-

Fig. 2. Inhibition of FFA-induced lipid accumulation by GA in HepG2cells. Human HepG2 cells were treated with 1 mM FFA together withvarious concentrations of GA (0 to 30 �M) or CA-074 Me (20 �M) for24 hours. Intracellular lipids were stained with Nile red and measuredwith flow cytometry as described in Materials and Methods. The blackcurve represents normal control without FFA treatment. The red curverepresents the cells treated only with FFA, while the lime, purple, yellow,and blue curves represent the cells treated with FFA together with 20 �Mof CA-074 Me, or 3, 10, and 30 �M of GA, respectively. Flow cytometrydiagram is representative of three independent experiments.

Fig. 3. Inhibition of FFA-induced apoptosis by GA in HepG2 cells. (A)Human HepG2 cells were treated with 1 mM FFAs together with variousconcentrations of GA (0 to 30 �M) or CA-074 Me (20 �M) for 24 hours,and stained with Annexin V-FITC and PI. The apoptotic and necrotic cellswere detected by flow cytometry as described in Materials and Methods.The normal control, early apoptotic, late apoptotic, and necrotic cellswere present in the lower left, lower right, upper right, and upper leftquadrant, respectively. The percentage of cells in each quadrant isindicated. The flow cytometry diagram is representative of three inde-pendent experiments: (a) untreated cells; (b) cells treated with 1 mMFFAs; (c) cells treated with 1 mM FFAs and 3 �M GA; (d) cells treatedwith 1 mM FFAs and 10 �M GA; (e) cells treated with 1 mM FFAs and30 �M GA; (f) cells were treated with 1 mM of FFAs and 20 �M CA-074Me. (B) Representative transmission electron micrograph of ultrathinsection of HepG2 cells treated with FFAs and GA. (a) normal HepG2 cell;(b) cells treated with 1 mM FFAs for 24 hours; (c) cells treated with 1 mMof FFAs and 3 �M GA; and (d) cells treated with 1 mM of FFAs and 10�M GA. N, nucleus; M, mitochondria; L, lipid droplet.

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pendent induction of hepatic steatosis in these rats (Fig.4A). After feeding with HFD for 8 weeks, the character-istics of NASH developed (Fig. 4A-d). It has been dem-onstrated that cathepsin B is an important mediator ofFFA-induced hepatic injury.5,6,10,11 To further confirm ifHFD-induced lipotoxicity was correlated to the enzymeactivity of cathepsin B in cytosol, we measured cytosoliccathepsin B enzyme activities in rat livers. Cathepsin B

enzyme activities were significantly increased by HFDfeeding, which was time-dependent (Fig. 4B). Analysis ofthe correlation of cathepsin B activity and the histopatho-logical scores demonstrated that cathepsin B activity cor-related positively with the extent of hepatic injury (P �0.01) (data not shown). To determine if the increase ofcytosolic cathepsin B activities was due to release of ca-thepsin B from lysosomes, we detected the protein levelsof cathepsin B in cytosol by western blot analysis. Thecatalytically active fragments (p30 and p27) of cathepsinB in the cytosol were significantly increased after HFDfeeding (Fig. 4C).

Prevention of Lipid Accumulation and Lipotoxicityby GA in HFD-Induced Rat NAFLD Models. Toexamine whether GA has a protective effect on HFD-induced lipotoxicity in vivo, we gavaged rats with GA at alow dose (25 mg/kg) or high dose (50 mg/kg) for 8 weeks.GA had no significant effect on HFD-induced increase ofserum TG, total cholesterol, and low density lipoprotein-cholesterol levels, but inhibited HFD-induced decrease ofhigh density lipoprotein-cholesterol at high dose (Fig.5A). GA inhibited HFD-induced increase of serum ala-nine aminotransferase and aspartate aminotransferase lev-els (Fig. 5B). Histological analysis indicated that HFD fedrats displayed significant steatosis, acinar and portal in-flammation, infiltration of macrophages and lympho-cytes, and lipid droplet accumulation (Fig. 5C-b). GAtreatment significantly improved the hepatic histology.HFD-induced hepatic injury was decreased (Fig. 5C-c,d).

Effect of GA on Lysosomal Integrity in In Vitro andIn Vivo NAFLD Models. To identify the potentialcellular mechanisms of the protective effect of GA onFFA/HFD-induced hepatic injury, we first examined theeffect of GA on lysosomal stability using in vitro FFA-induced HepG2 models. By using the AO-uptakemethod, we were able to determine that FFAs treatmentsignificantly induced lysosomal rupture as indicated by anincreased number of “pale cells” (33.34%) (Fig. 6A-b).Simultaneous treatment with GA dose-dependently in-hibited FFA-induced lysosomal rupture (Fig. 6A-c-e). GAexhibited better a protective effect than CA-074 Me onFFA-induced lysosomal rupture (Fig. 6A-f).

To further ascertain the protective effect of GA, weexamined the effect of GA on lysosomal integrity using invivo rat NAFLD models by measuring free and bound�-galactosidase activities in rat liver. Free �-galactosidaseactivities were significantly increased, but the bound�-galactosidase activities were decreased after 8 weeks ofHFD feeding in rat livers (Fig. 6B). Treatment with GA(50 mg/kg) significantly inhibited HFD-induced increaseof free �-galactosidase activities and decrease of bound

Fig. 4. Effect of HFD on induction of steatosis and cathepsin Bexpression in rats. Rats were fed with HFD for 0, 4, 6, or 8 weeks. (A)Photomicrographs of hematoxylin and eosin stained liver sections rep-resenting each of the four time points, respectively. (a) normal dietcontrol group; (b) fed with HFD for 2 weeks; (c) fed with HFD for 4 weeks;and (d) fed with HFD for 8 weeks. (B) Effect of HFD on cathepsin Benzyme activity in rat liver. The enzyme activity of cathepsin B in rat livercytosol was measured as described in Materials and Methods. Statisticalsignificance relative to control group: *P � 0.05, **P � 0.01, ***P �0.001. (C) Effect of HFD on cathepsin B expression. Representativeimmunoblots against cathepsin B and �-tubulin from the rat liverhomogenates of each time point. �-tubulin was used as a proteinloading control.

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�-galactosidase activities, indicating that GA had a pro-tective effect on lysosomal stability.

Effect of GA on Mitochondrial Stability in In Vitroand In Vivo NAFLD Models. To determine if themitochondrial pathway was also involved in the GA-me-diated protective effect on lipotoxicity, we examined theeffect of GA on mitochondrial stability by measuringmembrane potential and cytosolic cytochrome c contentsin HepG2 NAFLD models. Treatment with 1 mM FFAsfor 24 hours significantly induced mitochondrial mem-

Fig. 5. Protective effects of GA on HFD-induced liver lipotoxicity inrats. Rats were fed with HFD and GA for 8 weeks as described inMaterials and Methods. (A) Effect of GA on serum lipid levels of the ratstreated with HFD. Each column represents the mean � standard error ofthe mean (SEM), n � 6. Statistical significance relative to vehiclecontrol: ##P � 0.01, ###P � 0.001; Statistical significance relative toHFD group: *P � 0.05. (B) Effect of GA on liver enzymes. Each columnrepresents the mean � SEM, n � 6. Statistical significance relative tovehicle control: ###P � 0.001; Statistical significance relative to HFDgroup: **P � 0.01, ***P � 0.001. (C) Photomicrographs of hematox-ylin and eosin stained liver sections representing each of the four groupsrespectively. (a) normal diet control group; (b) HFD group; (c) HFD � GA25 mg/kg group; (d) HFD�GA 50 mg/kg group. TG, triglycerides; TC,total cholesterol; HDL-C, high density lipoprotein-cholesterol; LDL-C, lowdensity lipoprotein-cholesterol; ALT, alanine aminotransferase; AST, as-partate aminotransferase.

Fig. 6. Effect of GA on the lysosomal membrane stability. (A) Effect ofGA on lysosomal membrane stability in HepG2 cells. Human HepG2 cellswere incubated with 1 mM FFAs in the absence or presence of GA orCA-074 Me for 12 hours and then stained with AO. The fluorescenceintensity was measured by flow cytometry as described in Materials andMethods. (a) normal HepG2 cells; (b) cells treated with 1 mM FFAs; (c-e)cells treated with 1 mM FFAs in the presence of 3, 10, and 30 �M of GA,respectively; (f) Cells treated with 1 mM FFAs and 20 �M of CA-074 Me.(B) Effect of GA on �-galactosidase activity in rat livers. The cytosolfraction and membrane fraction of liver tissue were separated and the�-galactosidase activity was measured as described in Materials andMethods. The enzyme activity in the cytosol fraction represents theenzymes released from lysosomes (free), the enzyme activity in themembrane fraction represents the enzymes retained in lysosomes(bound). Each column represents the mean � standard error of themean (SEM), n � 6. Statistical significance relative to vehicle control:#P � 0.05, ##P � 0.01; Statistical significance relative to FFA group:*P � 0.05.

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brane depolarization as indicated by decrease of JC-1 ag-gregates and increase of JC-1 monomers (Fig. 7A-b).Treatment with GA or CA-074 Me for 24 hours, theFFA-induced mitochondrial membrane depolarizationwas dose-dependently inhibited (Fig. 7A-c-f).

Release of mitochondrial cytochrome c into cytosol isanother indicator of perturbation of mitochondrial mem-brane stability. We further detected cytochrome c proteinlevels in cytosol using western blot analysis. FFAs signifi-cantly increased cytosolic cytochrome c level, which wasdose-dependently inhibited by GA in HepG2 cells (Fig.7B). We further confirmed the effect of GA on PA-in-

duced and SA-induced cytochrome c release in rat pri-mary hepatocytes. GA markedly inhibited PA and SA-induced increase of cytosolic cytochrome c (SupplementaryFig. S2). Similarly, in in vivo rat NAFLD models, treat-ment with GA inhibited HFD-induced cytochrome c re-lease from mitochondria (Fig. 7C).

Effects of GA on Cathepsin B Activity in In Vitroand In Vivo NAFLD Models. It has been shown thatcathepsin B plays a critical role in FFA-induced lipotox-icity.5,6,10,11 To further explore the underlying mecha-nisms by which GA exerts its protective effect onlipotoxicity, we examined the effect of GA on cathepsin Bactivity and protein expression using both in vitro and invivo NAFLD models. In an in vitro assay system, both GAand CA-074 dose-dependently inhibited cathepsin B en-zyme activity (Fig. 8A). The median inhibitory concen-tration (IC50) for GA was 12.5 �M; the IC50 for CA-074was 36.3 nM.

In HepG2 cells, FFAs not only increased cathepsin Benzyme activity, but also increased protein levels in cy-tosol (Fig. 8B and C). Increase of both active fragments ofcathepsin B (p30 and p27) in the cytosol after treatmentwith 1 mM FFAs for 24 hours provided further evidencethat FFAs induced cathepsin B release from lysosome tocytosol. Treatment with GA (10 and 30 �M) or CA-074Me (20 �M) significantly inhibited FFA-induced cathep-sin B expression and enzyme activity. GA also inhibitedPA-induced and SA-induced cathepsin B expression in ratprimary hepatocytes (Supplementary Fig. S3). In con-trast, both GA and CA-074 Me had no effect on cathepsinB activity in untreated HepG2 cells (data not shown).

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™Fig. 7. Effect of GA on mitochondrial membrane stability. (A) Human

HepG2 cells were treated with 1 mM FFAs in the absence or presence ofGA or CA-074 Me for 12 hours, and then stained with JC-1. Themitochondrial membrane potential was measured by flow cytometry asdescribed in Materials and Methods. (a) Normal HepG2 cells; (b) cellstreated with 1 mM FFAs; (c-e) cells treated with 1 mM FFAs in thepresence of 3, 10, or 30 M of GA respectively; (f) cells treated with 1 mMFFAs in the presence 20 mM of CA-074 Me. Flow cytometry diagram isrepresentative of three independent experiments. (B) Effect of GA onFFA-induced cytochrome c release in HepG2 cells. Cells were treated with1 mM FFAs in the absence or presence of various concentrations of GA(0, 3, 10, and 30 �M) or 20 �M of CA-074 Me for 24 hours. The proteinlevels of cytosolic cytochrome c were detected by western blot analysisas described in Materials and Methods. �-Tubulin was used as aprotein-loading control. Immunoblots are representative of three inde-pendent experiments. (C) Effect of GA on HFD-induced cytochrome crelease in rat livers. Rats were treated with HFD or HFD � GA (25 mg/kgor 50 mg/kg) for 8 weeks. At the end of the experiment, the liver tissuewas collected and homogenized in lysis buffer. The protein levels ofcytosolic cytochrome c were detected by western blot analysis as de-scribed in Materials and Methods. �-Tubulin was used as a protein-loading control. Immunoblots are representative of four differentexperiments.

1912 WU ET AL. HEPATOLOGY, June 2008

Similar to the observation in in vitro HepG2 models,treatment with GA also dose-dependently inhibitedHFD-induced increase of cathepsin B expression and en-zyme activities in rat NAFLD models. Treatment with 50mg/kg GA not only almost attenuated HFD-induced ca-thepsin B expression, but also markedly inhibited the en-zyme activity (Fig. 9).

The Effect of GA on Intercellular ROS. It has beenshown that progression of hepatic steatosis into steato-hepatitis can be promoted by oxidative stress.26 Intracel-

Fig. 8. Inhibition of cathepsin B activity and expression by GA inHepG2 cells. (A) Inhibition of cathepsin B activity by GA in in vitrocell-free assay. Cathepsin B isolated from human liver was incubatedwith cathepsin B specific substrate in the presence of various concen-trations of GA or CA-074. The enzyme activity was determined asdescribed in Materials and Methods. IC50 for GA is 12.5 �M; IC50 forCA-074 is 36.2 nM. (B) Inhibition of FFA-induced cathepsin B activity byGA in HepG2 cells. Cells were treated with 1 mM FFAs in the presenceof various concentrations of GA (0, 3, 10, and 30 �M) or 20 �M ofCA-074 Me for 24 hours; the cathepsin B activity in the cytosolic fractionwas measured as described in Materials and Methods. Data representthe average of three independent experiments. Statistical significancerelative to vehicle control: #P � 0.05; Statistical significance relative toFFA group: *P � 0.05. (C) Effect of GA on cathepsin B expression inHepG2 cells. Cells were treated as described above. The protein levels ofcathepsin B in the cytosol were detected by western blot analysis usingspecific antibody to cathepsin B as described in Materials and Methods.�-Tubulin was used as protein loading control. Immunoblots are repre-sentative of three different experiments.

Fig. 9. Inhibition of HFD-induced cathepsin B expression in rat liver. Ratswere fed with HFD and GA for 8 weeks as described in Materials andMethods. (A) Effect of GA on HFD-induced expression of cathepsin B in liver.Representative immunoblots against cathepsin B and �-tubulin from the ratliver homogenate of each group. �-tubulin was used as a protein loadingcontrol. (B) Immunofluorescent staining of cathepsin B in liver tissue. (a)Control group; (b) HFD group; (c) HFD�GA 25 mg/kg; (d) HFD�GA 50mg/kg; (e) negative control for immunofluorescent staining. Expression ofcathepsin B was detected using specific antibody against cathepsin B asdescribed in Materials and Methods. (C) Effect of GA on HFD-inducedenzyme activity of cathepsin B in liver. The cathepsin B activity of liverhomogenate was measured as described in Materials and Methods. Statis-tical significance relative to vehicle control: ###P � 0.001; Statisticalsignificance relative to HFD group: *P � 0.05, ***P � 0.001.

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lular ROS plays an important role in lipid peroxidation,inflammatory cytokine induction, and mitochondrialdysfunction.1 It also has been reported that GA has anti-inflammatory and antioxidant activities13,27,28 and FFAsinduce oxidative stress in liver. To examine whether GAwas able to reduce FFA-induced oxidative stress, we mea-sured the ROS in HepG2 cells. FFA-induced increase ofROS was significantly inhibited by GA and the effect wasdose-dependent (Fig. 10); however, CA-074 Me had nosignificant effect.

DiscussionFFAs are the key players in the pathogenesis of

NAFLD. The elevated FFAs levels are closely correlatedto the disease severity of NAFLD.4 Although it has beenfound that insulin resistance is a major risk factor forNAFLD, and a recent proof-of-concept study indicatedthat diet plus pioglitazone led to metabolic and histolog-ical improvement in NASH patients, more studies areneeded to assess the long-term beneficial effect of insulin-sensitizing agents on the prevention of disease progressionof NAFLD.29 Other significant factors leading to progres-sive liver injury remain to be identified and developmentof new therapeutic interventions is especially urgent.

An increasing amount of attention has been paid to theuse of complementary and alternative medicine as a partof the treatment for liver diseases and the complications

associated with current therapies. Licorice root has beenused in Chinese medicine for thousands of years to reduceliver injury associated with a number of clinical disordersincluding chronic hepatitis C infection.13,30 It also hasbeen reported that GA is a potent inhibitor of bile acid-induced cytotoxicity in hepatocytes.28 Although severalhypotheses have been proposed, such as inhibition ofoxidative stress, anti-inflammatory activities, and im-munomodulation, the precise cellular and molecularmechanisms accounting for its clinical benefits have notbeen identified. Whether GA has any beneficial effects onFFA-induced hepatic toxicity remains to be characterized.

In the present study, we provided the first evidence forthe protective effect of GA on FFAs/HFD-induced lipo-toxicity both in in vitro HepG2 and in in vivo NAFLDmodels. GA not only reduced FFA/HFD-induced lipidaccumulation, but also significantly inhibited FFA/HFD-induced apoptosis.

The lysosomal compartment is responsible for the re-cycling of organelles and long-lived proteins. During thelast decade, numerous studies have found that lysosomesare very vulnerable to various stress stimuli and release oflysosomal constituents into the cytosol may be an early orinitiating event in apoptosis induced by oxidative stress,oxidized lipids and p53.7,19 It is well accepted that lyso-some-associated apoptosis is mediated by specific pro-teases; that is, cathepsin. Although there are about a dozenidentified cathepsins in mammalian lysosomes, cathepsinB and D are the most prominent and stable under physi-ological conditions and are essential in hepatocyte apo-ptosis induced by bile-acids, TNF-�, and oxidativestress.26 It has been reported that inactivation of cathepsinB attenuates hepatic injuries induced by cholestasis,TNF-�, and cold ischemia–warm reperfusion.6,10,11 Re-distribution of cathepsin B from lysosomes to the cytosolwas also observed in liver biopsy specimens of NAFLDpatients.5 Consistent with a previous report,5 we alsofound that FFAs significantly increased cytosolic cathep-sin B protein levels and enzyme activities, which wereinhibited by GA and the specific cathepsin inhibitor, CA-074 Me, in HepG2 cells. In HFD-fed rat NAFLD mod-els, GA also significantly inhibited HFD-inducedcathepsin B release and enzyme activities. Furthermore,GA not only prevented the FFA-induced release of ca-thepsin B into cytosol, but also directly inhibited cathep-sin B enzyme activity with an IC50 of 12.5 �M.

Previous studies have demonstrated that FFAs pro-mote hepatic lipotoxicity by stimulating TNF-� expres-sion via a lysosomal pathway, activating mitogen-activated protein kinase cascades, especially JNK, andinducing oxidative stress.2,5 In the current study, wefound that GA inhibited FFA-induced expression of

Fig. 10. Inhibition of ROS by GA in HepG2 cells. Human HepG2 cellswere treated with vehicle control (dimethylsulfoxide [DMSO]) or 1 mMFFAs together with GA (3 or 10 �M) or CA-074 Me (20 �M) for 24 hours.Cells were incubated with DCFH-DA (5 mM) for 30 minutes at roomtemperature in the dark. The fluorescence intensity of DCF was measuredby flow cytometry. (A) Normal control, cells were treated with DMSO. (B)Positive control: cells were treated with H2O2 before incubation withDCFH-DA. (C) Cells were treated with 1 mM FFAs. (D,E) Cells were treatedwith 1 mM FFAs and 3 or 10 �M of GA. (F) cells were treated with 1 mMFFAs and 20 �M of CA-074 Me.

1914 WU ET AL. HEPATOLOGY, June 2008

proinflammatory cytokines (TNF-� and interleukin-6) inmacrophages (Supplementary Fig. S4). In addition, GA sig-nificantly inhibited FFA-induced JNK-2 activation both inHepG2 cells and primary rat hepatocytes (SupplementaryFig. S5). GA also inhibited PA-induced and SA-inducedJNK activation in primary rat hepatocytes (SupplementaryFig. S6). These results suggest that GA exhibited its protec-tive effect mainly by stabilizing lysosomal integrity and in-hibiting inflammatory response and cathepsin B activity.

Recent studies indicate that released lysosomal en-zymes promote mitochondrial ROS production and cy-tochrome c release. In the present study, we also observedthat FFAs significantly induced cytochrome c release andenhanced mitochondrial ROS production, which werealso inhibited by GA both in in vitro and in in vivoNAFLD models. However, the exact mechanisms bywhich FFAs induce lysosomal/mitochondrial dysfunctionremain to be identified. Our preliminary studies suggestthat activation of the endoplasmic reticulum stress andinnate immune response may be involved in FFA-in-duced lipotoxicity. Whether GA is able to modulate en-doplasmic reticulum stress and innate immunity remainsto be identified and is the focus of our ongoing project.

In summary, stabilization of lysosomal membrane, inhi-bition of cathepsin B expression and enzyme activity, andreduction of mitochondrial cytochrome c release representthe major cellular mechanisms that might account for GA’sprotective effects on FFA-induced hepatic lipotoxicity.

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