Phyllanthus urinaria ameliorates the severity of nutritional steatohepatitis both in vitro and in vivo

Authors

  • Bo Shen,

    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
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  • Jun Yu,

    Corresponding author
    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
    • Department of Medicine and Therapeutics, The Prince of Wales Hospital, Shatin, NT, Hong Kong
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    • fax: (852)-26321194

  • Shiyan Wang,

    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
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  • Eagle S.H. Chu,

    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
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  • V.W.S. Wong,

    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
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  • Xin Zhou,

    1. Department of Pharmacology, The Chinese University of Hong Kong, Hong Kong, China
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  • Ge Lin,

    1. Department of Pharmacology, The Chinese University of Hong Kong, Hong Kong, China
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  • Joseph J.Y. Sung,

    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
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  • Henry L.Y. Chan

    Corresponding author
    1. Institute of Digestive Disease, Department of Medicine and Therapeutics, Hong Kong, China
    2. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China
    • Department of Medicine and Therapeutics, The Prince of Wales Hospital, Shatin, NT, Hong Kong
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    • fax: (852) 26373852


  • Potential conflict of interest: Nothing to report.

Abstract

Hepatic oxidative stress plays a critical role in metabolic forms of steatohepatitis. Phyllanthus urinaria, an herbal medicine, has been reported to have potential antioxidant properties. We tested the effects of P. urinaria on nutritional steatohepatitis both in vitro and in vivo. Immortalized normal hepatocytes (AML-12) or primary hepatocytes were exposed to control, the methionine-and-choline-deficient (MCD) culture medium, in the presence or absence of P. urinaria for 24 hours. Hepatocyte triglyceride, release of alanine aminotransferase, lipoperoxides, and reactive oxygen species production were determined. Age-matched C57BL/6 and db/db mice were fed control or MCD diet for 10 days with or without P. urinaria. Hepatic steatosis, necroinflammation, triglycerides, and lipid peroxide levels were determined. Hepatic expression of inflammatory factors and lipid regulatory mediators were assayed. P. urinaria reduced steatosis and alanine aminotransferase (ALT) levels in culture of hepatocytes in a dose-dependent manner. Phyllanthus prevented MCD-induced hepatic fat accumulation and steatohepatitis in mice. This effect was associated with repressed levels of hepatic lipid peroxides, reduced expression of cytochrome P450-2E1, pro-inflammatory tumor necrosis factor alpha, interleukin-6, dampened activation of inflammatory c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB), increased expression of lipolytic cytochrome P450 (Cyp4a10), and suppressed transcriptional activity of lipogenic CCAAT/enhancer binding protein β (C/EBPβ). Hepatic acyl co-enzyme A oxidase that regulated hepatic beta-oxidation of fatty acid and other lipid regulators were not affected by P. urinaria. In conclusion, P. urinaria effectively alleviated the steatohepatitis induced by the MCD, probably through dampening oxidative stress, ameliorating inflammation, and decreasing lipid accumulation. (HEPATOLOGY 2008.)

The spectrum of nonalcoholic fatty liver disease extends from hepatic steatosis through steatohepatitis to liver fibrosis, the latter frequently progressing to cirrhosis.1 Nonalcoholic steatohepatitis (NASH) is a form of metabolic liver disease with steatosis, inflammation, hepatocyte injury, or hepatic fibrosis, not resulting from alcohol.1 Because the prevalence of nonalcoholic fatty liver disease/NASH is dramatically increasing in Western countries and recently in Asia,2, 3 it has become a worldwide public health issue in an “era of overeating.” However, the pathogenesis of this condition is not well understood, and specific treatment options are limited. It is generally believed that excessive accumulation of hepatic fat and oxidative stress play a critical role in pathogenesis of NASH.1, 4 Therefore, inhibition of fatty acid cytotoxicity and liver inflammatory change is an important goal in the treatment of NASH.

The traditional medicinal plant, Phyllanthus species, is common in tropical and subtropical regions of both hemispheres. The plant contains tannins and flavonoids, both active antioxidants.5Phyllanthus species have been widely used in Asian countries for the treatment of liver disorders.6, 7 Many studies have shown that Phyllanthus species protect the liver from damages by antagonizing oxidative stress,8, 9 preventing lipid peroxidation,10 and suppressing inflammation.11, 12 Moreover, Phyllanthus species have been proven to be effective in protecting against alcohol-induced liver injuries.13, 14 Whether Phyllanthus species would benefit NASH has not been assessed. We evaluated the effect and the underlying mechanism of Phyllanthus on the prevention of steatohepatitis both in vitro and in vivo.

Abbreviations

ACO, acyl co-enzyme A oxidase; ALT, alanine aminotransferase; AML-12, immortalized normal hepatocytes; C/EBPβ, CCAAT/enhancer binding protein β; CYP, cytochrome P450; DCF, oxidized fluorescent derivative 2′,7′-dichlorodihydrofluorescin; DMEM, Dulbecco's minimum essential medium; IL, interleukin; LCAD, long-chain acyl-CoA dehydrogenase; LXR, liver X receptors; MCD, methionine and choline deficient; mRNA, messenger RNA; NASH, non-alcoholic steatohepatitis; NF-κB, nuclear factor kappa B; P-JNK, phosphorylated C-jun N-terminal kinase; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; SCD, stearoyl coenzyme A desaturase; SD, standard deviation; TBARS, thiobarbituric acid reactive substances; TG, triglyceride; TNF-α, tumor necrosis factor alpha.

Materials and Methods

Phyllanthus urinaria koreanis.

Phyllanthus urinaria koreanis was obtained from Chen Hwang Pharmacy, 209-1 Songsan-dong, Mapo-gu, Seoul, Korea. Three major ingredients in the P. urinaria capsule include corilagin, flavonoids, and polysaccharides. Each capsule contains 480 mg Korean P. urinaria extracts.

Hepatocyte Isolation and Cell Culture.

Hepatocytes were isolated from male Wistar rats (approximately 250 g) using the collagenase perfusion method.15 Briefly, the liver was perfused progressively with perfusate solution (pH 7.2), Dulbecco's minimum essential medium (DMEM)/Ham's F12 (1:1, Invitrogen, Carlsbad, CA) containing 0.05% (wt/vol) collagenase NB 4 (Serva, Heidelberg, Germany), 4.2 mmol/L NaHCO3, and 1% penicillin-streptomycin. The minced liver was disrupted with blunt forceps and filtered through 2 layers of cheesecloth. After washing in DMEM/F12 medium and 1% penicillin-streptomycin (P/S), hepatocytes were collected by 50g centrifugation for 5 minutes.

Immortalized mouse normal hepatocytes (AML-12) or primary rat hepatocytes were grown in control medium, which is 1:1 mixture of DMEM and F12 (Invitrogen) with 10% fetal bovine serum (Invitrogen), 40 ng/mL dexamethasone (Sigma, St Louis, MO), and 1% P/S (Invitrogen). Equal amounts of cells (2 × 105) were seeded in 100-mm dishes. When the cultures reached 70%–80% confluence, serum-containing medium was replaced with serum-free medium for 24 hours' incubation. Then the quiescent cells were exposed to control medium, DMEM/F12 medium deficient in methionine and choline (MCD) or MCD medium containing different concentrations of P. urinaria (0.25, 0.5, or 1 mg/mL) for an additional 24 hours.16

Animals and Treatment.

C57BL/6 mice and age-matched db/db mice (8 weeks old) were housed in a 22°C controlled room under a 12-hour light–dark cycle, with free access to water. They were allowed to adapt to their food and environment for 1 week before starting the experiment. The C57BL/6 mice were divided into 5 groups (5 mice per group) and fed with control diet (ICN, Aurora, OH), MCD diet (ICN) or MCD diet supplemented with Phyllanthus (Chen Hwang Phamacy, Korea) at 500, 1000, or 2000 ppm, respectively. The dosage of P. urinaria was chosen based on the daily human dose, which was 50 mg/kg/day. Based on the results obtained from the C57BL/6 mice, a separate experiment was performed on db/db mice, with 3 groups feeding with control diet, MCD diet, and MCD diet supplemented with P. urinaria at 1000 ppm. After 10 days experiments, mice were sacrificed without fast. Blood was collected by cardiac puncture, and livers were weighed and fixed in 10% formalin for histological analysis or snap frozen in lipid nitrogen followed by storage at −80°C freezer until required. All the protocols and procedures were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong.

Histological Examination.

Hematoxylin-eosin–stained paraffin-embedded liver tissues isolated from mice (4 μm thick) were graded for hepatic steatosis and necroinflammation as described previously17: hepatic steatosis (percentage of liver cells containing fat) was graded as 0: 0%, 1: 1% to 25%, 2: 26% to 50%, 3: 51% to 75%, 4: >75%. Necroinflammation was graded as 0: no inflammatory foci, 1: mild, 2: moderate, 3: severe. The investigator who scored the histological slides was blinded to the treatment assignment.

Biochemical Assays.

Serum or culture medium alanine aminotransferase (ALT) levels were determined using spectrophotometric assay kits (Sigma). Total hepatic triglycerides (TG) were estimated using Wako E-test triglyceride Kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instruction.

Assays for Oxidative Stress.

Lipoperoxides were measured by thiobarbituric acid reactive substances (TBARS) with 1,1,3,3-tetramethoxypropane as a standard (Sigma)17 and by oxidized fluorescent derivative 2′,7′-dichlorodihydrofluorescin (DCF) as reported.18 The 5- and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate used as a DCF probe was purchased from Invitrogen.

Quantitation of Hepatic Messenger RNA Expression Levels.

Total RNA was extracted from frozen liver using TRIzol reagent (Invitrogen). Five microgram total RNA for each sample was reversed transcribed into complementary DNA with 15T/18T primer and 100 U MMLV reverse transcriptase (Promega, San Luis Obispo, CA). Real-time polymerase chain reaction was assayed using SYBR Green Master Mix (Applied Biosystems, Foster, CA). The reactions were performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Each Ct value was normalized to β-actin. Transcript levels of inflammatory factors including interleukin (IL)-6, IL-1β, tumor necrosis factor alpha (TNF-α), transforming growth factor beta, intercellular adhesion molecule-1, and lipid regulation factors acyl-coenzyme A oxidase (ACO), proliferator-activated receptor gamma (PPARγ), PPARα, cytochrome P450 2E1 (CYP2E1), CYP4A10, CYP4A14, long-chain acyl-coenzyme A dehydrogenase (LCAD), stearoyl coenzyme A desaturase-1 (SCD1), liver X receptors-alpha (LXRα), LXRβ, fatty acid synthase, were analyzed with the specific primers listed in Table 1.

Table 1. Primer Sequences Used for Amplification of mRNA by Real-Time PCR
mRNAAccession No.Forward PrimerReverse Primer
SCD1NM0091275′-GTACCGCTGGCACATCAACTT-3′5′-GGCTAAGACAGTAGCCTTAG-3′
LXRαNM0138395′-TCAGCATCTTCTCTGCAGACCGG-3′5′-TCATTAGCATCCGTGGGAACA-3′
LXRβNM0094735′-AAGCTGGTGAGCCTGCGC-3′5′-CGGCAGCTTCTTGTCCTG-3′
ACONM0157295′-GGAAGACTTCCAATCATGCGATAG-3′5′-GACAACAAAGGCATGTAACCCG-3′
PPARγNM0111465′-CAGGCTTGCTGAACGTGAAG-3′5′-GGAGCACCTTGGCGAACA-3′
LCADU214895′-CAGAGAAACATGGCGGCA-3′5′-AGCCAGCGCGTGTGCAATT-3′
FASNM0079875′-GCAAACCAGACTTCTACTGCG-3′5′-TTTGTATTGCTGGTTGCTGTG-3′
PPARαNM0111445′-TGGGGATGAAGAGGGCTGAG-3′5′-GGGGACTGCCGTTGTCTGT-3′
CYP2E1BC0426935′-AGTGTTCACACTGCACCTGG-3′5′-CCTGGAACACAGGAATGTCC-3′
CYP4A14NM0078225′-GCCAGAATGGAGGATAGGAACA-3′5′-ATGAATGTGTCCACCTCTGCAC-3′
CYP4A10NM010015′-ACACTGCTCCGCTTCGAACT-3′5′-CAAGTCGGGCTAAGGGCA-3′
IL-6NM0311685′-ACAACCACGGCCTTCCCTACTT-3′5′-GTGTAATTAAGCCTCCGACT-3′
TNF-αNM0136935′-CGTGCTCCTCACCCACAC-3′5′-GGGTTCATACCAGGGTTTGA-3′
IL-1βNM0083615′-TCAGGCAGGCAGTATCACTCA TT-3′5′-GGAAGGTCCACGGGAAAGA-3′
TGF-1βNM0115775′-CAACTTCTGTCTGGGACCCT-3′5′-TAGTAGACGATGGGCAGTGG-3′
ICAM-1NM0104935′-CCTGCCTCTGAAGCTCGGAT-3′5′-ACAGGAACTTTCCCGCCACC-3′
β-actinNM0311445′-TACTGCCCTGGCTCCTAGCA-3′5′-TGGACAGTGAGGCCAGGATAG-3′

Western Blot Analysis of Hepatic Proteins.

Liver tissue was homogenized in Tris-HCl (pH 7.4) buffer containing a protease inhibitor cocktail (Roche, Indiapolis, IN). Total protein was extracted and concentration was measured by the Bradford method (DC protein assay, Bio-Rad Laboratories, Hercules, CA). Twenty-five micrograms protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto equilibrated polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, UK) by electroblotting. Membranes were blocked using 5% skim milk for 1 hour, then incubated with specific antibodies against CYP2E1 (dilution 1: 5000, Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated C-jun N-terminal kinase (P-JNK) (1:1000) (Cell Signaling Technology, Danvers, MA), or glyceraldehyde 3-phosphate dehydrogenase (1:5000) (Abcam, Hong Kong, China) overnight at 4°C. After incubation with secondary antibody, proteins were detected by enhanced chemiluminescence (Amersham Corp.).

Nuclear protein was isolated from 100 mg fresh liver tissue. Fifty micrograms nuclear protein was electrophoresed through a 12% polyacrylamide gel, and immunoblots were incubated with primary antibodies against CCAAT/enhancer binding protein β (C/EBPβ) (1:1000, Cell Signaling Technology). Bands were detected by enhanced chemiluminescence as described previously.

Immunohistochemistry for Nuclear Factor κB Subunit p65.

Paraffin-embedded liver sections were deparaffined, rehydrated, and pretreated with 0.5% hydrogen peroxidase in phosphate-buffered saline buffer for 20 minutes. After blocking with 10% nonimmunized goat serum, sections were incubated with antibody to nuclear factor kappa B (NF-κB) subunit p65 (1:50, Santa Cruz Biotechnology) for 2 hours at room temperature. Then biotin-conjugated secondary antibody, avidin-biotin complex, and horseradish peroxidase were applied for 30 minutes at room temperature (Dako A/S, Glostrup, Denmark). Positive signals were visualized by diaminobenzidine and counterstained with hematoxylin. The number of p65 nuclear positive cells was counted per 1000 hepatocytes in 5 fields (100× magnification).

Determination of Methionine and Choline in P. urinaria Capsule.

The methionine and choline in the Phyllanthus capsule were measured using a combined high-performance liquid chromatography–tandem mass spectrometry system that consisted of a Perkin-Elmer series 200 liquid chromatograph connected to a QTRAP LC/MS/MS system (Applied Biosystem, MDS, SCIEX). The analysis was performed on a reversed-phase C18 column (Agilent Zorbax, 4.6 × 250 mm, 5 μm, Agilent, Santa Clara, CA) coupled with a C18 guard column and were detected using multiple reaction monitoring mode. The optimized parameters are shown in Table 2. High-pressure liquid chromatography-grade L-methionine and choline were purchased from Sigma Chemical Co. Methanol stock solutions containing methionine or choline were prepared with gradient elution for the construction of calibration curves. Control diet or P. urinaria was weighted and dissolved with water to 50 mg/mL. The mixture was centrifuged at 2,300g for 10 minutes, and the resultant solution was filtered, and the filtrate (30 μL) was subjected to high-performance liquid chromatography–tandem mass spectrometry analysis. The lower limit of quantification of 1.33 μg/g for methionine and 0.67 μg/g for choline was adequate for the analysis. High-performance liquid chromatography–tandem mass spectrometry assay showed no detectable methionine, and a very small amount of choline in P. urinaria (0.7 μg/mg); the latter was undetectable when it was mixed in the MCD diet at dosage of 500, 1000, or 2000 ppm (Table 2).

Table 2. Summary of Performing Parameter for HPLC-MS/MS
ParameterMethionineCholine
  1. Abbreviations: CEP, collision energy potential; CXP, collision cell exit potential; DP, declustering potential; EP, entrance potential; ESI, electrospray ionization; MRM, multiple reaction monitoring.

Mobile phaseWater/methanol (9:1)NH4OAc (50 mM)/methanol (9:1)
Flow rate0.5 mL/minute0.5 mL/minute
ESIPositivepositive
Nebulizing gas50 psi50 psi
Auxiliary gas50 psi50 psi
Curtain gas50 psi30 psi
DP16 V46 V
EP9.5 V11 V
CEP12 V12 V
CXP4V12 V
MRM (m/z) (precursor → product)→ 150/61→ 104/58

Statistical Analysis.

Data are presented as the mean ± standard deviation (SD). Comparisons between the different treatment groups were analyzed via the Student t test and 1-way analysis of variance. A 2-sided P value of less than 0.05 was considered statistically significant.

Results

Effect of P. urinaria on Hepatocyte Steatosis, Medium ALT, and Oxidative Injury.

We first evaluated the effects of P. urinaria on hepatocyte steatosis. Incubation of cultured primary hepatocytes or AML-12 hepatocytes with MCD medium resulted in significant increases in total cell TG content at 24 hours compared with the control medium (Fig. 1A and Fig. 2A). However, hepatocytes incubated in MCD medium in the presence of P. urinaria (0.25, 0.5, 1 mg/mL) produced a dose-dependent decrease of TG content (Fig. 1A). In parallel to the effect on TG content, this compound caused a dose-dependent suppression of reactive oxygen species (ROS) in both primary and immortalized hepatocytes detected by using DCF and TBARS assays, respectively (Fig. 1B and Fig. 2B), indicating a decrease in oxidative injury. ALT release was determined by measuring its activity in the conditioned medium. As shown in Fig. 1C, ALT release was significant higher in the hepatocytes cultured in the MCD medium, and was massively reduced in the MCD medium in the presence of 0.5 mg/mL and 1 mg/mL P. urinaria, respectively (Fig. 1C).

Figure 1.

The effects of P. urinaria (PU) on triglyceride accumulation, release of ALT, and lipoperoxide levels of AML-12 hepatocytes. AML-12 cells (1 × 106 cells) were exposed to control or MCD medium in the presence of P. urinaria (0, 0.25, 0.5, 1 mg/mL) for 24 hours. (A) The hepatocyte triglycerides, (B) lipoperoxides, and (C) ALT level in culture medium were measured as described in Materials and Methods. *P < 0.05, **P < 0.01, AML-12 cultured in MCD medium compared with control medium. #P < 0.05, ##P < 0.01, AML-12 in PU-treated MCD-medium compared with the MCD medium. Data are mean ± SD (n = 6).

Figure 2.

The effect of P. urinaria (PU) on triglyceride and ROS in primary rat hepatocytes. Primary hepatocytes were cultured in control or MCD medium in the presence of P. urinaria (0, 0.5, 1 mg/mL) for 24 hours. (A) The level of triglycerides was determined by TBARS assay, and (B) ROS production was quantified by the percentage increase of DCF fluorescence in hepatocytes. *P < 0.05, primary hepatocytes cultured in MCD medium compared with control medium. #P < 0.05, hepatocytes in PU-treated MCD-medium compared with the MCD medium. Data are mean ± SD (n = 6).

Effect of P. urinaria on Steatohepatitis in C57BL/6 Mice Induced by the MCD Diet.

In light of the observed antisteatosis and antioxidative injury effects of P. urinaria on the hepatocytes in vitro, we tested whether P. urinaria treatment could ameliorate steatohepatitis induced by MCD diet in vivo. C57BL/6 mice fed the MCD diet had similar body weight loss to those reported previously,19, 20 and supplement of P. urinaria did not rectify the weight loss. Despite the weight changes, all animals remained physically active throughout the experimental period. As shown by hematoxylin-eosin staining, mice fed the control diet had normal liver histology (Fig. 3A); however, mice fed with the MCD diet developed steatohepatitis with hepatocyte ballooning changes, scattered lobular inflammatory cells infiltration, and inflammatory foci (Fig. 3B). The MCD diet supplemented with 500 ppm P. urinaria failed to improve the liver histology (Fig. 3C), whereas MCD diet supplemented with 1000 ppm and 2000 ppm P. urinaria clearly reduced the severity of hepatic steatosis and inflammatory infiltration (Fig. 3D,E). Histological grading of liver sections confirmed that P. urinaria significantly ameliorated hepatic steatosis and necroinflammation (Table 3).

Figure 3.

Effect of P. urinaria on MCD diet–induced steatohepatitis in C57BL/6 mice. Hematoxylin-eosin–stained liver sections from mice fed: (A) the control diet—appearances are normal. (B) MCD diet. Liver shows foci of necroinflammation and macrovesicular fat droplets. (C) In mice fed the MCD diet, treatment with P. urinaria (500 ppm), the severity of steatohepatitis was not significantly improved. (D) In mice fed the MCD diet, treatment with P. urinaria (1000 ppm) and (E) with P. urinaria (2000 ppm) largely ameliorated steatohepatitis. Experiment lasted for up to 10 days. Slides are representative of 5 separate experiments (original magnification, ×100). The group data for steatosis and inflammatory scores are presented in Table 3.

Table 3. Effect of P. urinaria on Scores for Hepatic Steatosis and Necroinflammation
DietControlMCDMCD+PU 500 ppmMCD+PU 1000 ppmMCD+PU 2000 ppm
  • Abbreviation: PU, Phyllanthus urinaria. The severity of hepatic steatosis and necroinflammation were scored as described in Materials and Methods. Duration of this experiment is 10 days. Values of hepatic steatosis and necroinflammation are mean ± SD (n = 5/group).

  • *

    P < 0.0001 control versus MCD diet;

  • #

    P < 0.05;

  • ##

    P < 0.01 MCD versus MCD+PU-treated mice.

Steatosis0.0 ± 0.002.3 ± 0.45*2.3 ± 0.271.2 ± 0.45##1.0 ± 0.71##
Necroinflammation0.0 ± 0.001.8 ± 0.57*1.7 ± 0.450.7 ± 0.45##0.8 ± 0.57#

Effect of P. urinaria on Hepatic TGs and Lipid Peroxidation.

As shown in Fig. 4, intake of the MCD diet resulted in a prominent increase in TG contents (Fig. 4A) and TBARS (Fig. 4B) levels compared with that of the control diet. In contrast, the MCD diet supplemented with P. urinaria showed a dose-dependent inhibition on the accumulation of liver triacylglycerol fractions and lipoperoxides. Significant reduction both on the levels of TG and TBARS in mice treated with 2000 ppm P. urinaria was observed compared with the mice fed with MCD diet only (Fig. 4).

Figure 4.

Effects of the MCD diet and treatment with P. urinaria (PU) on hepatic triglyceride and lipoperoxide content in C57BL/6. (A) Hepatic triglyceride, and (B) lipoperoxide content were assessed in mice fed the control diet, MCD diet, or MCD diet treated with P. urinaria (500, 1000, and 2000 ppm) for 10 days. Data are mean ± SD, with 5 animals in each group. *P < 0.05, MCD compared with control diet–fed mice. #P < 0.05, P. urinaria–treated MCD-fed compared with the mice fed the MCD diet.

Effect of P. urinaria on the Expression of Proinflammatory Factors and Inflammatory Pathways.

To evaluate the mechanisms of the effect of P. urinaria on steatohepatitis, we investigated expression levels of proinflammatory factors including TNF-α, IL-6, IL-1β, transforming growth factor beta1, intracellular adhesion molecule-1, and P-JNK. Mice fed with MCD diet had a marked elevation of hepatic messenger RNA (mRNA) expression of TNF-α, IL-6, and IL-1β and protein expression of P-JNK as compared with mice fed with control diet. Conversely, treatment with P. urinaria at 1000 ppm or 2000 ppm in mice fed with MCD diet significantly lowered the mRNA expression of TNF-α and IL-6 (Fig. 5A) and reduced protein expression of P-JNK (Fig. 6F). Hepatic nuclear protein expression of p65, a key component of NF-κB, was significantly increased after 10 days of MCD dietary feeding, and this induction was blunted by P. urinaria as determined by immunohistochemistry assay (Fig. 6A-E).

Figure 5.

Effects of the MCD diet and treatment with P. urinaria on hepatic mRNA expression. (A) Expression of hepatic inflammatory mediators, and (B, C) fatty acid regulation genes were measured in mice fed the control diet, MCD diet, or MCD diet with P. urinaria 1000 or 2000 ppm for 10 days. Hepatic mRNA was assessed by real-time reverse transcription polymerase chain reaction, normalized to glyceraldehyde 3-phosphate dehydrogenase, and is expressed as fold-induction. Data are mean ± SD, with 5 animals in each group. *P < 0.05, MCD compared with control diet–fed mice. #P < 0.05, P. urinaria–treated MCD-fed compared with the mice fed the MCD diet.

Figure 6.

Effects of P. urinaria on hepatic CYP2e1, P-JNK, C/EBPβ protein expression and NF-κB activation in C57BL/6 mice. Immunohistochemistry for NF-κB subunit p65 in sections from mice fed with: (A) the control diet, little nuclear accumulation of p65 is present; (B) MCD diet, frequent p65 nuclear immunoreactivity was observed (arrows); (C) MCD diet supplement with 1000 ppm PU; and (D) MCD with 2000 ppm PU; the positive nuclear p65 was reduced. (E) A decrease in the number of nuclear p65-positive cells is evident in PU-treated mice. Representative slides obtained from 5 animals per group. (Original magnification ×400). *P < 0.05, MCD compared with control diet–fed mice. #P < 0.05, P. urinaria–treated MCD-fed mice compared with the mice fed the MCD diet. Data are mean ± SD. (F) Protein expression of CYP2e1 and P-JNK, and (G) nuclear protein expression of C/EBPβ were determined by western blotting. Glyceraldehyde 3-phosphate dehydrogenase was used as the internal control.

Effect of P. urinaria on the Expression of Genes Related to Fatty Acid Regulation.

To seek an explanation for the hepatic triglyceride-lowering effects of P. urinaria, we assessed the hepatic expression levels of the lipogenic genes PPARγ, fatty acid synthase, LXRα, LXRβ, SCD1, and C/EBPβ, and lipolytic genes involved in the β-oxidation of fatty acid, such as ACO, LCAD, PPARα and PPARα downstream target molecules Cyp4a10 and Cyp4a14 (Fig. 5B-D). Compared with mice fed with MCD diet alone, administration of P. urinaria with the MCD diet greatly reduced expression of C/EBPβ, which was highly induced by MCD diet (Fig. 6G) but had no effect on other lipogenic genes (Fig. 5C, D). Conversely, P. urinaria significantly induced mRNA levels for the lipolytic gene Cyp4A10 (4-fold) (Fig. 5B). Expression of ACO and LCAD involved in lipid beta-oxidation were repressed significantly by MCD diet. However, P. urinaria supplementation did not restore their expression (Fig. 5B, C).

Effect of P. urinaria on the Expression of CYP2e1.

The mRNA and protein expressions of CYP2e1, a major mediator of lipid peroxidation, were induced by MCD diet. Administration of P. urinaria at doses of 1000 ppm and 2000 ppm prevented the induction of CYP2e1 (Figs. 5C, 6F).

Effect of P. urinaria on Steatohepatitis in db/db Mice Induced by the MCD Diet.

To confirm the effects observed with P. urinaria in C57BL/6 mice, an additional experiment was performed on genetic db/db mice. In this genetic NASH model, P. urinaria attenuated MCD-induced steatohepatitis as indicated by improved histology (Fig. 7) and reduced TBARS levels (P. urinaria treated versus MCD; 9.81 ± 3.23 versus 5.16 ± 2.49, P < 0.05). Thus, the effect of P. urinaria on MCD diet–induced steatohepatitis in db/db mice was consistent with the results obtained from C57BL/6.

Figure 7.

Effect of P. urinaria on MCD diet–induced steatohepatitis in db/db mice. Hematoxylin-eosin–stained liver sections from db/db mice fed: (A) the control diet—appearances are normal. (B) MCD diet. Liver shows foci of necroinflammation and macrovesicular fat droplets. (C) In mice fed the MCD diet, treatment with P. urinaria (1000 ppm) largely ameliorated steatohepatitis. Experiment lasted for 10 days. Slides are representative of 5 separate experiments (original magnification, ×200).

Discussion

In the current study, we demonstrated that incubating cultured AML-12 hepatocytes with MCD medium increased cellular steatosis, release of ALT, and cellular TBARS. Treatment with P. urinaria led to a dose-dependent decrease in steatosis and ALT and a concomitant suppression of cellular TBARS. Because of immortalization of AML-12 by being stably transfected with transforming growth factor alpha, we also determined the effects of P. urinaria on cultured primary rat hepatocytes with MCD medium. Administration of P. urinaria appreciably protected cells from steatosis and ROS specifically probed by DCF. Having observed substantial suppression of steatohepatitis by P. urinaria treatment in vitro, we conducted experiments designed to test the potential for P. urinaria to exert preventive effects against steatohepatitis in vivo. C57BL/6 mice fed with MCD diet for 10 days developed steatohepatitis. The histology exhibited steatosis, cellular inflammatory infiltrate, and hepatocellular necrosis. Administering high-dose P. urinaria (1000 ppm or 2000 ppm) in MCD-fed mice resulted in attenuation of the steatohepatitis, as evident by diminished histological evidence of steatosis and inflammation. In addition, mice receiving P. urinaria had lower hepatic triglyceride content than in MCD diet-alone fed mice. P. urinaria was associated with a significant reduction of intrahepatic oxidative stress, and its antioxidant effect appears to play an important role in the attenuation of steatohepatitis. Moreover, in the genetic NASH model, db/db mice fed MCD diet with P. urinaria (1000 ppm) had improved liver histology with reduced cellular TBARS, compared with those fed MCD diet alone. The mechanisms for prevention of steatohepatitis by P. urinaria were investigated thereafter.

CYP2e1 is a major microsomal source of hydrogen peroxide and nicotinamide adenine dinucleotide phosphate, reduced form–dependent lipid peroxidation contributing to oxdative stress damage.20, 21 Up-regulation of CYP2e1 was reported both in human NASH and in experimental steatohepatitis, including in mice fed MCD.22–24 Hepatocytes from pyrazole-treated rats to induce CYP2e1 showed greater sensitivity to the toxicities of ethanol and polyunsaturated fatty acid.25 In the current work, we showed that administration of P. urinaria led to a dose-dependent reduction of CYP2e1 expression from approximately 2-fold to 5-fold compared with controls, thus alleviating lipid peroxidation. The effect of P. urinaria on preventing steatohepatitis and reducing lipid peroxidation is thought to be at least in part by down-regulating CYP2e1 expression. The regulation of CYP2e1 operates at both translational and post-translational levels.26 The promoter of CYP2e1 contained many transcription factor binding sites, including activator protein-1 (AP-1), hepatocyte nuclear factor, and C/EBP.27 One possible pathway of down-regulation of CYP2e1 by P. urinaria was through reducing nuclear accumulation of C/EBPβ. MCD diet–induced steatohepatitis is associated with elevation of both C/EBPβ and CYP2e1, whereas levels of CYP2e1 protein were lower in C/EBPβ−/− mice on MCD diet than in wild-type MCD diet–fed mice.28 C/EBPβ binding mediated the induction of CYP2e1 by lipopolysaccharide in astrocytes.29 Thus, C/EBPβ played an important role in control of CYP2e1 in MCD model and P. urinaria down-regulated CYP2e1 probably by effectively lowering C/EBPβ nuclear accumulation.

TNF-α and IL-6 are key inflammatory factors involved in the development of human NASH30, 31 and experimental steatohepatitis.17, 32 These pro-inflammatory regulators are mediated, at least in part, through oxidative stress.33 The level of TNF-α parallels the severity of human NASH.30, 31 As a key mediator, TNF-α plays a critical role in the evolution of steatohepatitis.32 Moreover, TNF-α aggravates oxidative stress and is involved in the pathogenesis of mitochondrial dysfunction, a predominant source of oxidative stress in NASH.35, 36 Treating cells with TNF-α dramatically increased ROS, decreased the expression of adenosine triphosphatase and cytochrome c oxidase,37 and impaired the electron flow of the mitochondrial respiratory chain.38, 39 IL-6 is another important mediator of inflammatory liver diseases, including fatty liver of obesity and cirrhosis.40–42 Long-term exposure of IL-6 sensitized the liver to injury and impaired liver regeneration because of activation of the IL-6/signal transducer and activator of transcription protein-3 pathway.43, 44 In this study, P. urinaria administration significantly suppressed oxidative stress and blunted TNF-α and IL-6 gene expression. The anti-inflammatory effects of P. urinaria may be partly related to inhibition of hepatic lipoperoxide and reductions in the mRNA expression levels of these regulators. Particularly, we also found that 2 major mediators in inflammatory response pathway, NF-κB and JNK, were highly active in MCD diet–fed mice but were substantially suppressed by P. urinaria administration. Both NF-κB and JNK were potently activated by a variety of stress signals such as TNF-α and ROS, and mediated the interaction among obesity, inflammation, and insulin resistance.45–47 It was well established that the JNK/c-Jun pathway mediated hepatocyte lipoapoptosis and liver injury from TNF-α, free fatty acids, and oxidative stress.48, 49 Therefore, NF-κB and JNK were functional as the cause and the consequence of increased inflammation and became self-reinforcing cycle factors.32, 47 In this regard, one of the potential mechanisms of P. urinaria on hepatic inflammation was that antioxidantive P. urinaria ameliorated cellular ROS production and blunted the origin of this vicious cycle, thereby suppressing NF-κB and JNK pathways. This hypothesis was supported by the evidence that JNK150 or NF-κB blockade51 protected mice from MCD diet–induced steatohepatitis with reduced hepatic triglyceride, inflammation, and liver injury.

An excessive accumulation of triglycerides in the hepatocytes of the liver is mostly attributable to enhanced uptake and synthesis of fatty acids and to inhibition of fatty acid oxidation. Administration of P. urinaria clearly lowered the nuclear accumulation of C/EBPβ, a newly established hepatic lipogenic regulator. A recent study showed that C/EBPβ−/− mice were protected against the development of obesity and the hepatic fat accumulation induced by a high-fat diet through increased energy expenditure and regulating many key genes in lipid metabolism.52 Moreover, genetic deletion of C/EBPβ dramatically reduced hepatic triglyceride accumulation and dampened steatohepatitis in C/EBPβ−/− mice fed with MCD28 and in the cross strain of C/EBPβ−/− and db/db mice.53 Thus, P. urinaria may have an effect on the control of fat input pathway through down-regulation of C/EBPβ. In addition, recent studies found that the classical inflammatory mediator JNK also played a lipogenic role. JNK activity was abnormally elevated in an obesity45, 54 and MCD diet model,50 whereas JNK1 deficiency resulted in reduced adiposity and plasma lipid in a high-fat diet–induced model of obesity45, 54 and lowered hepatic triglyceride accumulation in an MCD diet model.50 Therefore, P. urinaria–mediated inhibition of JNK activation may also contribute to the reduction of the lipid accumulation.

Conversely, hepatic fatty acid oxidation occurs via mitochondrial and peroxisomal β-oxidation and CYP4A-catalyzed ω-oxidation.55, 56 ACO and LCAD are the key enzymes of these 3 fatty acid oxidation systems in liver and are regulated by the lipolytic transcription factor PPARα.55, 56 In our study, supplementation of the MCD diet with P. urinaria increased the expression of Cyp4a10, a PPARα downstream molecule. It has been shown that PPARα stimulation is effective against steatohepatitis through enhancing the CYP4A expression in MCD-fed mice.19, 57 Moreover, Cyp4a10 was involved in the degradation of proinflammatory lipid mediators such as prostaglandins, leukotrienes, and end products of lipid peroxidation.57 Collectively, the dramatically enhanced expression of lipolytic CYP4A10 and suppression of lipogenic C/EBPβ and JNK by P. urinaria contributed to protection against the evolution of steatohepatitis.

In summary, P. urinaria clearly attenuated the liver injury of steatohepatitis in cultured hepatocytes in vitro and in MCD diet–fed mice in vivo. The mechanisms by which P. urinaria ameliorated steatohepatitis could be attributed to its antioxidant properties via suppression of CYP2e1, anti-inflammatory effects by suppressing inflammatory JNK and NF-κB pathways and by down-regulating critical inflammatory mediators TNF-α and IL-6, and induction of fatty acid oxidation through up-regulation of CYP4a10 and suppression of lipogenic transcription factor C/EBPβ (Fig. 8). Our findings support a potential beneficial role of P. urinaria for the prevention and treatment of NASH in humans, and future clinical studies are warranted for validation.

Figure 8.

Model for the mechanisms of protective effects of P. urinaria on MCD diet–induced steatohepatitis. MCD diet–induced hepatic TG overload triggers steatohepatitis via activating inflammatory JNK and NF-κB directly and by initiating CYP2e1-mediated oxidative stress, resulting in increased inflammatory recruitment. Phyllanthus relieves TG overload by promoting CYP4a10-catalyzed lipid o-oxidation and suppressing lipogenic regulator C/EBPβ. Conversely, P. urinaria can lower oxidative stress directly and via blocking CYP2e1-mediated lipid peroxidation, thereby dampening the inflammatory cycle along with reduced activation of JNK and NF-κB pathways and expression of TNF-α and IL-6.

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