Hepatoprotective mechanisms of Yan-gan-wan

Hepatology Research 32 (2005) 202–212

Hepatoprotective mechanisms of Yan-gan-wan Melissa D. Yang a , Qing-gao Deng a , Shuang Chen a , Shigang Xiong a , Dennis Koop b , Hidekazu Tsukamoto a,∗ a

Research Center for Alcoholic Liver and Pancreatic Diseases and Department of Pathology, Keck School of Medicine of the University of Southern California, VA Greater Los Angeles Healthcare System, 1333 San Pablo Street, MMR-412, Los Angeles, CA 90089-9141, USA b Department of Physiology and Pharmacology, Oregon Health and Science University, USA Received 11 March 2005; received in revised form 23 May 2005; accepted 8 June 2005

Abstract Background and aims: A herbal prescription, Yan-gan-wan (YGW), has been known to offer hepatoprotective effects in Asian countries for years. This study investigated its mechanisms of action. Methods: The effects of YGW on CCl4 induced liver damage were tested in mice and cultured hepatocytes. Microarray analysis screened genes affected by YGW. YGWs effects on the expression of cytochrome P450 (CYP) 2E1 and other isozymes were determined. YGWs effects on TNF␣ expression and NF-␬B activation in Kupffer cells (KC), and TNF␣ promoter activity in RAW264.7 cells, were also assessed. Results: Administration of YGW reduced the plasma ALT, centrilobular necrosis, neutrophilic infiltration, and TNF␣ mRNA in the livers of mice acutely given CCl4 . The in vivo herb treatment reduced ALT release and necrosis of isolated hepatocytes directly exposed to CCl4 . Microarray analysis demonstrated marked reductions in CYP4A10 and 4A14 by YGW but no changes in other CYP isozymes as confirmed by immunoblot analysis. The herb treatment suppressed LPS-stimulated TNF␣ release in vivo and by cultured KC. Direct addition of the aqueous herb extract suppressed NF-␬B activation by KC and TNF␣ promoter activity in RAW cells under LPS stimulation. This activity to suppress TNF␣ expression was largely separated into gel filtration fractions with the molecular size of 102–107 Da. YGW also attenuated liver fibrosis induced by chronic treatment of CCl4 or porcine serum. Conclusions: The protective effects of YGW on CCl4 hepatotoxicity are due in part to inhibition of KC NF-␬B activation and TNF␣ expression by small water soluble molecules, and may also be related to suppressed hepatic expression of CYP4A10 and 4A14 that are considered as alternative prooxidant cytochromes. © 2005 Elsevier B.V. All rights reserved. Keywords: Carbon tetrachloride; Liver fibrosis; Kupffer cells; TNF␣; CYP4A10; CYP4A14

1. Introduction Herbal medicine has been used in China for 1000 of years and has recently attracted the interest of modern scientific communities as alternative medicine. However, the efficacy of the herbal medicine is claimed largely based on clinical experiences and its active constituents and fundamental Abbreviations: YGW, Yan-gan-wan; CYP, cytochrome P450; LPS, lipopolysaccharide; TNF␣, tumor necrosis factor ␣; RT-PCR, reverse transcription-polymerase chain reaction; RNA, ribonucleic acid; cDNA, complimentary deoxyribonucleic acid; CCl4 , carbon tetrachloride; ALT, alanine aminotransferase; PPAR␥, peroxisome prolierator-activated receptor ␥ ∗ Corresponding author. Tel.: +1 323 442 5107; fax: +1 323 442 3126. E-mail address: [email protected] (H. Tsukamoto). 1386-6346/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hepres.2005.06.004

mechanisms have just begun to be investigated. Yan-gan-wan (YGW), an ancient herbal remedy [1] is a mixture of the extracts from eight medicinal herbs and its known ingredients include phenols (ferulic acid, coumaric acid, angelicin, paeonols), polyphenols (tannin, gallic acid), and flavonoids (quercetin, kaempferol). It is known to provide therapeutic and preventive efficacies for liver diseases in Asian nations for many years, and to date no adverse side effects have been reported. More recently, several case studies have been reported to attribute its therapeutic effects on acute hepatitis and chronic active hepatitis to its herbal components such as Chuan-Xiong (Ligusticum chuanxiong Hort), Dong-Quai (Angelica sinensis Diels) and Bai-Shan (Paeonia lactiflora Pall). These effects included improved ALT levels, jaundice,

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

recovery rate and liver fibrosis [2 for review]. However, the cellular and molecular mechanisms by which YGW exerts its hepatoprotective effects are largely unknown. The present study was aimed at understanding whether and how YGW prevents acute hepatotoxicity induced by CCl4 and liver fibrosis induced by repeated injection of CCl4 or porcine serum. Our results demonstrate that YGW indeed ameliorates the extent of hepatic necrosis and fibrosis induced by CCl4 . They further suggest that the hepatoprotective mechanisms of YGW against acute or chronic CCl4 injury are not due to alterations in the levels and activities of the enzymes involved in the metabolism of the drug. The mechanisms appear to reside in both parenchymal and Kupffer cells: the former possibly related to selective inhibition of the expression of CYP4A isoforms and the latter involving suppressed TNF␣ expression.

2. Materials and methods 2.1. In vivo acute CCl4 toxicity Animal protocol was approved by IACUC at the University of Southern California according to the NIH guidelines. Male C57BL/6 mice (Charles River Co., Wilmington, MA) were pre-treated with YGW or starch (placebo) via gastric gavage (300 mg/kg, Sheng-Pu Pharmaceuticals, Taipei) and a single dose of CCl4 (20 ␮l/kg as 0.2% in mineral oil, Sigma–Aldrich, St. Louis, MO) was injected intraperitoneally. Mice were sacrificed 16 h thereafter and blood was collected for determination of plasma ALT using a kit (Sigma, St. Louis, MO). Livers were fixed in 3% paraformaldehyde for hematoxylin and eosin staining and immunostaining of myeloperoxidase. 2.2. Microarray gene expression analysis Total RNA was extracted from the livers of mice pretreated with YGW or starch (300 mg/kg/day) [3]. Microarray analysis was performed on the RNA samples using GeneChip® expression analysis by Affymetrix. Changes of gene expression above two-fold were considered as significant. 2.3. TNFα RT-PCR assay Liver RNA (2 ␮g) was reverse transcribed into cDNA and the synthesized cDNA was amplified by 30 cycles of PCR using specific primers as described [4,5]. ␤-actin was used as a house keeping gene for which 25 cycles of amplification were performed. 2.4. In-vitro CCl4 toxicity in hepatocytes Hepatocytes were isolated by the Cell Culture Core of the USC Research Center for Liver Diseases by liver perfusion with collagenase [6] from mice pretreated with YGW

203

or starch (300 mg/kg/day for 2 weeks). CCl4 (66%, v/v in ethanol) was added to hepatocyte cultures (20 ␮l/ml) [7]. The media were collected for the determination of ALT at 10, 20, and 30 min. In a separate experiment, cultured hepatocytes (0.5 × 106 /27 cm2 ) were treated with CCl4 (1 ␮l/ml) for 1 h, nuclei were stained with Hoechst 33258 (8 ␮g/ml, Molecular Probes, Eugene, OR) [8] followed by staining of the necrotic nuclei with Sytox green (1 ␮M, Molecular Probes, Eugene, OR) [9]. 2.5. CYP isoform and glutathione assays Liver microsomes were prepared from the YGW or starch treated mice by differential centrifugation. Proteins (10 ␮g) were electrophoresed on 9% SDS-polyacrylamide gels and transferred to nitrocellulose. Blots were incubated with rabbit anti-rat CYP2E1 (from Dr. B.J. Song, NIAAA), rabbit anti-rat CYP3A, rabbit anti-CYP2B1 (from Dr. J. Halpert, University of Texas Medical Branch at Galveston, TX), rabbit anti pig P450 reductase (from Dr. B.S. Masters, University of Texas Health Science Center, San Antonio, TX) or sheep anti-rat CYP4A (from Dr. G. Gibson, University of Surrey School of Biological Science). Blots were incubated with goat anti-rabbit HRP or rabbit anti-goat HRP secondary antibodies (ICN Biomedicals, Costa Mesa, CA) and analyzed by enhanced chemiluminescence. The testosterone hydroxylation was used to determine the catalytic activity of CYP3A. Testosterone and metabolites were separated and quantified by a Waters HPLC system. The production of 6hydroxychlorzoxazone was used to determine the catalytic activity of CYP2E1 by HPLC as described by Barmada et al. [10]. CYP2B and CYP1A associated catalytic activities were determined by the dealkylation of pentoxyresorufin and ethoxyresorufin as described by Lubet et al. [11,12] and adapted for microtiter plates as described by Kennedy and Jones [13]. Total glutathione (GSH) content was determined in YGW or starch treated mouse livers without or with CCl4 treatment by recycling assay as previously described [14]. 2.6. Lipopolysaccharide (LPS)-induced TNFα release in vivo and in vitro Male Wistar rats were gavaged with YGW or starch (300 mg/kg/day) for 2 weeks and LPS was administered (50 ␮g/kg) (Sigma–Aldrich, St. Louis, MO) via a central venous catheter under general anesthesia. Blood was collected at 0, 0.5, 1, 2, 3 h for TNF␣ determination by an ELISA kit (R&D System, Minneapolis, MN). Kupffer cells were isolated from normal, YGW or starch treated rats by the Non-Parenchymal Liver Cell Core of the Research Center for Alcoholic Liver and Pancreatic Diseases as published [15]. The cells were treated with LPS (100 or 500 ng/ml) for 4 h for determination of TNF␣ release. The YGW or starch (placebo) extract was prepared by suspending and mixing in the serum-free media (35 mg/ml), centrifuging at 1500 × g for 15 min, and filter-sterilizing the supernatant. The extract

204

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

was added to Kupffer cell cultures for 16 h prior to stimulation with LPS (500 ng/ml).

System (Promega, Madison, WI). For electrophoretic mobility shift assay for NF-␬B binding, a probe encompassing a ␬B site in the murine TNF␣ promoter was used [16].

2.7. Effects of YGW on TNFα promoter and NF-κB binding

2.8. Fractionation of YGW extract and bioassay

RAW254.7 cells were transfected with a TNF␣ promoteror NF-␬B promoter-firefly luciferase construct using Targefect F-2 reagent (Targeting System, San Diego, CA) as described [16]. Renilla phRL-TK vector was used as an internal control for the transfection efficiency. The cells were incubated with the YGW or starch extract at 25% (v/v) for 16 h and stimulated with LPS (500 ng/ml) for 4 h. Cell lysate was collected and assayed by Dual-Luciferase Reporter Assay

The water YGW extract (350 mg/ml) was prepared and applied to Super Prep Grade gel in a XK 16/70 column (Amersham Pharmacia Biotech, Piscataway, NJ) using PBS as a mobile phase solvent. Elution fractions were collected, and glycine, triglycine, hexaglycine, substance P, gastrin, aprotinin were used as molecular standards (Sigma Chemical, St. Louis, MO). We pooled the fractions into 8 samples, each with 20 fractions to estimate the distribution of the biological

Fig. 1. YGW ameliorates acute CCl4 hepatotoxicity in vivo. (A) Plasma ALT levels and hepatic neutrophil accumulation in acutely CCl4 -treated mice were reduced by YGW (300 mg/kg) compared to the placebo (starch). Neutrophils were counted in eight random fields of liver sections after immunostaining. Each value represents mean ± S.E. per treatment group (n = 6 pairs). * p = 0.03 and ** p < 0.02 as compared to the starch + CCl4 group. (B) Histologic changes in the liver after acute CCl4 treatment in mice. Liver of CCl4 -treated, placebo mice (starch + CCl4 ) shows extensive centrilobular necrosis surrounded by several rows of ballooned hepatocytes. YGW clearly reduced the extent of necrosis. (H & E stain, 160×).

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

205

activity and then each fraction within a selected pooled sample was further tested for its ability to inhibit LPS-stimulated TNF␣ release by cultured Kupffer cells. 2.9. Liver fibrosis induced by repetitive injection of CCl4 or porcine serum Male C57BL/6 mice were orally administered YGW or starch (300 mg/kg/day) and injected subcutaneously with CCl4 (2 ␮l/g body weight, 1:1 v/v in mineral oil) or mineral oil twice a week and 0.05% (w/v) phenobarbital in H2 O provided as solely drinking water for 8 weeks [17,18]. Livers were fixed in for reticulin staining and morphometric analysis using the Nikon microscope with its Imaging System. For porcine serum (PS) induced liver fibrosis, male Wistar rats were given YGW or starch, and were injected intraperitoneally with 1 ml PS (Gibco BRL Life Technologies, Grand Island, NY) or saline twice a week for 8 weeks [19]. 2.10. Statistical analysis Data were expressed as mean ± S.E. The significant differences between the groups were assessed by standard or paired t-test. Differences were considered significant if p < 0.05.

Fig. 2. YGW suppresses TNF␣ mRNA expression in acute CCl4 hepatotoxicity. Liver RNA was extracted and RT-PCR analysis for TNF␣ and ␤-actin was performed. Note robust expression of TNF␣ mRNA in CCl4 -treated mice without (lanes 3 and 4) or with (lanes 5 and 6) the starch (placebo) treatment while it was undetectable in normal untreated mice (lanes 1 and 2). YGW treatment (lanes 7 and 8) suppressed TNF␣ mRNA levels as compared to the placebo group. ␤-actin served as an internal standard.

ner at 10, 20, and 30 min (Fig. 3A). The cells isolated from the YGW-treated mice showed significantly attenuated release of ALT at all time points. In a separate experiment, the cells were exposed to CCl4 and double stained with Hoechst 33258 and Sytox green. The former stained all hepatocyte nuclei regardless of whether the cell were alive or dead while the latter only

3. Results 3.1. YGW ameliorates acute CCl4 hepatotoxicity Carbon tetrachloride (CCl4 ) induces hepatotoxicity via the formation of a trichloromethyl (• CCl3 ) free radical catalyzed by microsomal CYP2E1 [7,20]. Using the mouse model of acute CCl4 hepatotoxicity [21,22], the present study tested whether and how YGW provides hepatoprotective effects. As reported [23], at 16 h following a single administration of CCl4 , the plasma ALT levels were markedly elevated (2171 ± 303 U/L, Fig. 1A) and severe centrilobular necrosis and neutrophilic infiltration ensued (Fig. 1A and B). Administration of YGW reduced the plasma ALT levels by 50% and the number of infiltrating neutrophils by 40% (Fig. 1A). The extent of liver necrosis was reduced in the YGW-treated mice (Fig. 1B). TNF␣ plays a pivotal role in CCl4 -induced liver damage as administration of soluble receptor for this cytokine [24] or the deficiency of TNF␣ type 1 receptor [25], ameliorates the hepatotoxicity. For this reason, the mRNA levels for TNF␣ were analyzed by RT-PCR. A robust induction of hepatic expression of TNF␣ mRNA was noted in CCl4 -treated mice without or with the placebo (starch) treatment (Fig. 2, lanes 3, 4 and 5, 6). The herb treatment suppressed this TNF␣ induction of (lanes 7 and 8). To further examine the protective effect of YGW against CCl4 -induced hepatotoxicity, hepatocytes were isolated from YGW or starch-treated mice and tested in culture for direct toxicity induced by CCl4 . Hepatocytes isolated from the starch-treated mice released ALT in a time-dependent man-

Fig. 3. In vivo YGW treatment ameliorates CCl4 toxicity in cultured hepatocytes. (A) CCl4 (20 ␮l/ml medium) was added to cultured hepatocytes isolated from mice treated with YGW or starch. Note significant reductions in the CCl4 -induced release of ALT by hepatocytes from YGW-treated mice. (n = 3, * p < 0.004). (B) YGW decreased the extent of hepatocyte necrosis caused by CCl4 treatment in culture. The percentage of necrotic hepatocytes was enumerated by Hoechst 33258 and Sytox green double staining. Data represent mean ± S.E. per group, (n = 7, ** p < 0.001).

206

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

stained necrotic nuclei. Approximately 45% (44.9 ± 2.35%) of hepatocytes from the starch-treated mice were necrotic after the CCl4 treatment while necrosis was significantly reduced to 29.7 ± 2.6% in hepatocytes from the YGW-treated mice (Fig. 3B). These direct in vitro evidence for the YGWs protective effects, suggested that the mechanism underlying the protection resides within hepatocytes. 3.2. YGW does not affect CYP2E1 or GSH but inhibits CYP4A To detect underlying protective changes within YGW-treated liver, we performed a microarray analysis (GeneChip® ,

Affymetrix) on two sets of pooled liver RNA samples from mice treated with YGW or starch (a placebo). Interestingly, there were only a few genes that are either upregulated or down-regulated more than two-fold. The analysis also showed no changes in antioxidant enzymes such as superoxide dismutase, glutathione (GSH)-S-transferase, and GSH peroxidase. However, a 9.5-fold reduction was noted for CYP4A10 and 4A14 in the YGW livers. Since CCl4 toxicity requires the generation of a toxic metabolite by CYP2E1, we also performed immunoblot analysis and metabolic assay for CYP2E1 along with CYP2A, 3A and reductase. Neither protein levels nor their activities, were altered by the herb treatment (Fig. 4A). These findings sug-

Fig. 4. YGW has no effects on CYP 2E1, 2A, 3A and reductase. (A) Livers from mice treated with the YGW or starch (300 mg/kg/day), were analyzed for the protein content of CYP isoforms and reductase by Western blot analyses and their activities by enzymatic assays using the specific substrates. Note no changes in CYP isoform protein levels or their activities between the two groups, suggesting that YGW does not affect the metabolism of CCl4 . (B) This Western blot analysis confirms marked reduction in the levels of CYP4A proteins in the livers of YGW-treated mice.

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

gest that the YGW’s protective effect on CCl4 induced liver injury is not mediated by suppressed bioconversion of the toxin. We also performed immunoblot analysis to validate the CYP4A microarray results using antibodies that recognize the CYP4A family members. The antibodies detected two bands, the upper band corresponding to CYP4A12 and the lower band for both CYP4A10 and 14 isoforms [26]. This immunoblot analysis demonstrated a striking reduction in the intensities of both bands of CYP4A isoenzymes (Fig. 4B). We also assessed the total liver content of GSH in YGW or starch treated mice to determine the herb treatment affected the level of this major antioxidant. The GSH content was not different between the two groups (YGWtreated: 6.06 ± 0.38 ␮mol/g liver versus starch-treated: 5.80 ± 1.27 ␮mol/g liver).

207

3.3. YGW inhibits Kupffer cell TNFα release We were intrigued by our observation that the YGW treatment reduced TNF␣ mRNA levels in the livers of CCl4 challenged mice (Fig. 2). This result may indicate that the herb suppresses TNF␣ expression as a protective mechanism against CCl4 hepatotoxicity. Alternatively, TNF␣ expression might have been suppressed as a consequence of reduced hepatic necrosis and inflammation mediated by different mechanisms. To test the former possibility, the rats were pre-treated with YGW or placebo (starch) and administered LPS intravenously for determination of the plasma TNF␣ levels. In the placebo group, the TNF␣ level was elevated to the peak value at 1.5 h and declined thereafter (Fig. 5A). In the YGW-treated rats, the peak level was significantly reduced and occurred at the earlier time point. These results suggested that YGW

Fig. 5. YGW inhibits LPS-stimulated Kupffer cell TNF␣ expression. (A) YGW treatment reduces plasma TNF␣ levels induced by LPS in vivo. LPS (50 ␮g/kg) was injected through a central venous catheter to YGW or starch pretreated rats, and plasma TNF␣ levels were assayed at 0, 0.5, 1, 1.5, 2 h time points. Each point represents the mean derived from three separate experiments. * p < 0.05. (B) YGW treatment in vivo decreases LPS-stimulated TNF␣ release by isolated and cultured Kupffer cells. Kupffer cells were isolated from YGW or starch treated rats and stimulated with LPS (100 and 500 ng/ml) for 4 h YGW significantly reduced TNF␣ release by 80% and 50% under stimulation with 100 and 500 ng/ml LPS, respectively (n = 8, * p < 0.02, ** p < 0.01). (C) Direct treatment of cultured Kupffer cells with the YGW extract, inhibits LPS-stimulated TNF␣ release. Kupffer cells were isolated from normal rats and cultured for 3 days. The YGW or starch extract was added at the different concentrations followed by stimulation with 500 ng/ml LPS (n = 5, control n = 3 * p < 0.05, ** p < 0.01).

208

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

pretreatment inhibited LPS-stimulated release of TNF␣ by macrophages for which Kupffer cells represented the largest population. Another possibility was increased clearance of TNF␣ in the YGW-treated rats. To test the former, we determined TNF␣ release by Kupffer cells isolated from the YGW or placebo (starch) treated rats. The cells were cultured after isolation and treated with LPS (100 and 500 ng/ml) for determination of TNF␣ release. TNF␣ protein released by Kupffer cells from the YGW-treated rats was significantly reduced by 80% and 50% upon stimulation with 100 and 500 ng/ml LPS,

respectively (Fig. 5B). To further test whether the YGW has direct inhibitory effects on TNF␣ release by Kupffer cells, the aqueous extract of the herb was added to Kupffer cell cultures established from un-treated, normal rats. The increasing percentage of the extract added to the culture media, resulted in a dose-dependent reduction in LPS-induced TNF␣ release (Fig. 5C). These results suggest that the observed suppression of TNF␣ release by in vivo treatment with YGW, may be attributable to this direct inhibitory action of the extracted ingredient(s) on Kupffer cells.

Fig. 6. YGW extract inhibits TNF␣ and NF-␬B promoter activities. (A) The YGW extract but not phenolic or flavonoid compounds inhibits LPS-induced TNF␣ promoter activity. RAW264.7 cells were transiently transfected with a TNF␣ promoter-luciferase construct, treated with the YGW or starch extract (25% v/v), quercetin (Q, 200 ␮M), feulic acid (F, 200 ␮M), kaempferol (K, 10 ␮M), or DMSO (a vehicle for Q, F, and K), and stimulated with LPS (500 ng/ml). * p < 0.05 B) The YGW extract inhibits LPS-stimulated NF-␬B promoter. RAW264.7 cells were transfected with a NF-␬B-luciferase construct, treated with the YGW or starch extract, and stimulated with LPS (500 ng/ml). * p < 0.05 as compared to no LPS stimulation; ** p < 0.05 compared to the starch extract treatment. (C) The YGW extract suppresses LPS-stimulated NF-␬B activation in Kupffer cells. Cultured, normal rat Kupffer cells were treated with the YGW or starch extract (25% v/v) over night followed by stimulation with LPS (500 ng/ml) for the indicated durations. Nuclear extracts were prepared and used for electrophoretic mobility shift assay using a probe specific for a ␬B site in the murine TNF␣ promoter.

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

209

3.4. YGW inhibits TNFα promoter activity via suppressed NF-κB activation We next examined whether YGW inhibits TNF␣ promoter activity in RAW264.7 cells, the murine macrophage cell line. The cells were transiently transfected with a TNF␣ promoterreporter gene construct and treated with the YGW or starch extract in the absence or presence of LPS (500 ng/ml). The LPS treatment increased the promoter activity 3.5-fold and the addition of the starch extract somehow increased it. However, the addition of the YGW extract at 25% v/v almost completely abrogated LPS-induced promoter activity (Fig. 6A). We also tested whether phenolic or flavonoid compounds contained in YGW (quercetin, ferulic acid, and kaempferol), inhibit the promoter. The former two were tested at 200 ␮M final concentration while the latter at 10 ␮M, the highest concentration that could be applied without crystallization. None of these compounds showed an inhibitory effect on the promoter. The 1.4 kb TNF promoter we used, has four known ␬B sites, two of which are known to serve as the critical enhancer elements [16]. To test whether NF-␬B promoter is specifically inhibited by the extract, we performed transient transfection using a NF-␬B-luciferase construct. As shown in Fig. 6B, LPS-stimulated NF-␬B promoter activity was inhibited 70% by the YGW extract while the starch extract had no effects. Next, we examined whether the YGW-mediated inhibition of NF-␬B promoter, is due to suppressed binding of NF-␬B to its cognate DNA binding site. As shown in Fig. 6C, YGW indeed inhibited LPS-induced NF-␬B binding to DNA, suggesting activation of this transcription factor was inhibited by the YGW extract. 3.5. Size fractionation of TNFα inhibitory activity We next pursued size characterization of the TNF␣ inhibitory activity of the YGW extract using gel filtration chromatography. The fractions were pooled into eight samples first to test the distribution of the inhibitory activity toward LPS-stimulated release of TNF␣ by cultured Kupffer cells. As shown in Fig. 7A, the pooled fraction #4 had the most notable inhibitory effect. We then tested individual fractions from 103 up to 120 within this pooled fraction. TNF␣ release is inhibited by the fractions between 107 and 111, and the fractions 110 and 111 showed the highest activity (Fig. 7B). Based upon calculation using a plot of the fractions against the logarithm of the known molecular masses of the standards, we estimate the molecular size of the fraction 111 and 110 to be 102–107 Da. 3.6. YGW prevents liver fibrosis Next, we tested whether YGW prevents liver fibrosis induced by repeated CCl4 injection. Morphometric analysis of reticulin staining was performed for comparison. After 8 weeks of repeated CCl4 injection, severe bridging fibrosis and incomplete micronodular cirrhosis were induced in

Fig. 7. Size fractionation of anti-TNF␣ activity. The YGW extract was applied to gel filtration chromatography and pooled fractions (A) or individual fractions in the pooled fraction #4 (B) were tested for its ability to suppress LPS-stimulated TNF␣ release by cultured Kupffer cells. Note the pooled fraction #4 containing fractions 101–120, had the most pronounced inhibitory activity (A). Testing of each fraction within the pooled fraction #4 showed the highest inhibitory activity toward LPS-induced TNF␣ release in the fractions 110 and 111 (B).

starch-treated mice (Fig. 8A). YGW-treated mice, on the other hand, showed only centrilobular and mild bridging fibrosis and the morphometric value was significantly reduced as compared to the placebo group (Fig. 8A: 1.43 ± 0.17 versus 2.42 ± 0.14, p < 0.001). These results demonstrate that YGW not only ameliorates CCl4 induced acute liver injury but also reduces the extent of liver fibrosis caused by chronic damage due to this toxin. The latter effect may be attributable to potential antioxidant effects of YGW. To address this question, the total hepatic content of GSH was measured after chronic CCl4 administration. However, no difference in this parameter was attained (data now shown). Nevertheless, the anti-fibrotic effect of YGW may merely reflect the consequence of reduced hepatocellular necrosis, rather than a specific inhibitory effect of YGW on liver fibrogenesis. To assess this question, we tested YGW in another liver fibrosis model induced by repetitive injection of porcine serum (PS). This model does not cause parenchymal damage or neutrophilic inflammation but develops liver fibrosis based on immune response incited by PS [19]. YGW administration also suppressed the development of liver fibrosis in this model as shown in a representative set of

210

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

Fig. 8. YGW reduces liver fibrosis induced by hepatotoxic and immunologic mechanisms. (A) YGW ameliorates CCl4 -induced liver fibrosis. Chronic CCl4 administration to mice for 8 weeks induced severe bridging fibrosis and incomplete cirrhosis in starch-treated mice (right panel, reticulin stain, 40×) while the fibrotic response was clearly attenuated in YGW-treated animals (left panel). Morphometric fibrosis score was significantly reduced in YGW-treated animals, ** p < 0.001. (B) YGW ameliorates porcine serum (PS)-induced liver fibrosis. PS administration to rats for 8 weeks produced incomplete or complete cirrhosis in starch-treated rats (right panel, reticulin stain, 40×) while only mild bridging fibrosis was induced in YGW-treated animals (left panel). Morphometric data confirmed significant reduction in liver fibrosis the YGW group, * p < 0.03.

reticulin staining and a summary of morphometric analysis (Fig. 8B).

4. Discussions Using CCl4 hepatotoxicity models in vivo and in culture, the present study demonstrates that YGW offers hepatic protection against this toxin through changes within hepatocytes. We demonstrate that these changes do not involve the expression or activity of CYP2E1, the principal enzyme that catalyzes the conversion of CCl4 to its toxic metabolite. However, YGW causes marked reductions in CYP4A proteins. Like CYP2E1, CYP4A enzymes are fatty acid hydroxylases and participate in the metabolism of fatty acids [27]. CYP4A genes are usually co-regulated with other genes that encode proteins involved in fatty acid ␤-oxidation and transport. However, the YGW’s effect on CYP4A isoenzymes is very selective and no effects are seen on other CYP enzymes. Although the actual contribution of CYP4A isozymes to CCl4 hepatotoxicity has not be defined to date, these enzymes are known to serve as alternative sources of oxidant stress in other models. For instance, in ob/ob obese mice deficient in leptin, the expression of CYP4A10 and 4A14 are increased [28]. The CYP2E1 deficiency neither prevents the development of nonalcoholic steatohepatitis caused by methionine- and cholinedeficient (MCD) diet, nor abates the increasing microsomal

NADPH-dependent lipid peroxidation in ob/ob mice, pointing to the role of non-CYP2E1 peroxidases. In these CYP2E1 knockout mice, CYP4A10 and 4A14 are upregulated and are considered as alternative sources of oxidative stress in the liver [29]. In light of this notion, the YGW’s ability to suppress CYP4A expression may serve as a potential mechanism underlying the observed protection against liver damage induced by CCl4 . YGW contains phenolic compounds and flavonoids that are known to serve as antioxidants. These antioxidants might have protected the liver against CCl4 induced hepatotoxicity. However, we observed the protective effect on hepatocytes in vitro with the use of the aqueous extract of YGW that should not contain these hydrophobic antioxidant components. In addition, the total live content of GSH was not affected by YGW in na¨ıve or CCl4 treated mice. Thus, although the antioxidant action of YGW cannot be completely ruled out by our present study, this mechanism may not be a sole and major reason for the observed hepatoprotection. Our findings suggest that the observed inhibition of TNF␣ expression by YGW in the acute CCl4 hepatotoxicity mode is the primary but not secondary effect, serving as another potential protective mechanism against this type of hepatotoxicity that is known to be mediated at least in part by TNF␣ [24,25]. Reduced neutrophilic infiltration in this model also supports this notion since TNF␣ induction is one of the earliest events for hepatic inflammation via induction

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

of chemokines [23,30] YGW’s anti-fibrotic effect seen in chronic CCl4 model is likely based on the demonstrated protection against acute CCl4 hepatotoxicity but may also involve its direct anti-TNF action since this cytokine is required for the progression of liver fibrosis induced by CCl4 [31] and dimethylnitrosamine [32]. Our results demonstrate YGW-mediated inhibition of TNF␣ expression is due likely to suppressed NF-␬B activation and TNF␣ promoter activity. This mechanism may also underlie YGW’s efficacy on liver fibrosis induced by porcine serum. This model induces fibrogenesis via immune response but not hepatocellular necrosis or neutrophilic inflammation. The observed prevention in the model may reflect YGWs ability to inhibit the expression of TNF␣ or other immune mediators that are positively regulated by NF-␬B. Indeed, TNF␣ stimulates cell proliferation of hepatic stellate cells (HSC), the key effector cell type in liver fibrogenesis [33] and downregualtes the activity of PPAR␥ [34], the transcription factor recently shown to facilitate HSC quiescence [35,36]. In addition, direct suppressive effects of YGW on HSC cannot be ruled out. To this end, our ongoing work demonstrates that the treatment of cultured HSC with the YGW aqueous extract suppresses HSC activation (unpublished observation). It is noteworthy that Gingo biloba extracts and its flavonoid component, quercetin, have been shown to inhibit expression of LPS-stimulated TNF␣ in the RAW264.7 [37]. However, our results demonstrate that quercetin, ferulic acid, and kaempferol, do not suppress TNF␣ promoter activity. Our chromatographic analysis reveals the major anti-TNF activity is confined to the molecular mass of 102–123 Da (between fractions 107 and 111) with the highest activity corresponding to 102–107 Da. Among small molecules known to inhibit LPS-induced TNF␣ expression, glycine and taurine achieve this effect in alveolar macrophages and Kupffer cells via attenuation of intracellular calcium response [38–40]. However, their molecular sizes (75 and 125 Da, respectively) do not fall into the range of molecular size shown by us to contain the highest anti-TNF activity (102–107 Da). Molecular characterization and identification of this anti-TNF activity is currently under investigation. In summary, our results demonstrate that YGW ameliorates acute CCl4 hepatotoxicity and liver fibrosis induced by hepatotoxic and immunological mechanisms. Its direct heptoprotective effects are not due to changes in toxin metabolism but at least in part to inhibition of Kupffer cell TNF␣ expression. Additionally, we demonstrated YGWs ability to selectively inhibit CYP4A10 and 4A14 expression in the liver. We propose that this unique effect may offer protection against oxidative damage since they are now considered as pivotal prooxidant cytochromes.

Acknowledgements This work was supported by NIH grants R37AA06603 (HT), R37AA08608 (DK), P50AA11999 (USC-UCLA

211

Research Center for Alcoholic Liver and Pancreatic Diseases), R24AA12885 (Non-Parenchymal Liver Cell Core), and Medical Research Service of Department of Veterans Affairs. References [1] Xie G. Chinese Medical Dictionary. Taipei: Shang Wu Press; 1984. [2] Hu-Zhan Z, Ze-Hong D, Jing S. Modern study of traditional Chinese medicine. Beijing: Xue Yuan Press; 1999. [3] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 1987;162(1):156–9. [4] Iimuro Y, Gallucci RM, Luster MI, Kono H, Thurman RG. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology 1997;26(6):1530–7. [5] Motomura K, Ohata M, Satre M, Tsukamoto H. Destabilization of TNF-alpha mRNA by retinoic acid in hepatic macrophages: implications for alcoholic liver disease. Am J Physiol Endocrinol Metab 2001;281(3):420–E429. [6] Zahlten RN, Stratman FW. The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique. Arch Biochem Biophys 1974;163(2):600–8. [7] Berger ML, Bhatt H, Combes B, Estabrook RW. CCl4 -induced toxicity in isolated hepatocytes: the importance of direct solvent injury. Hepatology 1986;6(1):36–45. [8] Simm A, Bertsch G, Frank H, Zimmermann U, Hoppe J. Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis. J Cell Sci 1997;110(Pt 7):819–28. [9] Roth BL, Poot M, Yue ST, Millard PJ. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 1997;63(6):2421–31. [10] Barmada S, Kienle E, Koop DR. Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life. Biochem Biophys Res Commun 1995;206(2):601–7. [11] Lubet RA, Mayer RT, Cameron JW, Nims RW, Burke MD, Wolff T, et al. Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat. Arch Biochem Biophys 1985;238(1):43–8. [12] Beebe LE, Fornwald LW, Alworth WL, Dragnev KH, Lubet RA. Effect of dietary Aroclor 1254 exposure on lung and kidney cytochromes P450 in female rats: evidence for P4501A2 expression in kidney. Chem Biol Interact 1995;97(3):215–27. [13] Kennedy SW, Jones SP. Simultaneous measurement of cytochrome P4501A catalytic activity and total protein concentration with a fluorescence plate reader. Anal Biochem 1994;222(1):217– 23. [14] Allen KG, Arthur JR. Inhibition by 5-sulphosalicylic acid of the glutathione reductase recycling assay for glutathione analysis. Clin Chim Acta 1987;162(2):237–9. [15] Kamimura S, Tsukamoto H. Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease. Hepatology 1995;22(4 Pt 1):1304–9. [16] Veal N, Hsieh CL, Xiong S, Mato JM, Lu S, Tsukamoto H. Inhibition of lipopolysaccharide-stimulated TNF-alpha promoter activity by S-adenosylmethionine and 5 -methylthioadenosine. Am J Physiol Gastrointest Liver Physiol 2004;287(2):G352–62. [17] Kovalovich K, DeAngelis RA, Li W, Furth EE, Ciliberto G, Taub R. Increased toxin-induced liver injury and fibrosis in interleukin-6deficient mice. Hepatology 2000;31(1):149–59. [18] Rechnagel RO, Glende Jr EA. Carbon tetrachloride hepatotoxicity: an example of lethal cleavage. CRC Crit Rev Toxicol 1973;2(3):263– 97.

212

M.D. Yang et al. / Hepatology Research 32 (2005) 202–212

[19] Bhunchet E, Eishi Y, Wake K. Contribution of immune response to the hepatic fibrosis induced by porcine serum. Hepatology 1996;23(4):811–7. [20] Sugihara A, Tsujimura T, Fujita Y, Nakata Y, Terada N. Evaluation of role of mast cells in the development of liver fibrosis using mast cell-deficient rats and mice. J Hepatol 1999;30(5):859–67. [21] Noguchi T, Fong KL, Lai EK, Alexander SS, King MM, Olson L, et al. Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical. Biochem Pharmacol 1982;31(5):615–24. [22] Recknagel RO, Glende Jr EA, Dolak JA, Waller RL. Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 1989;43(1):139–54. [23] Louis H, Van Laethem JL, Wu W, Quertinmont E, Degraef C, Van den BK, et al. Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice. Hepatology 1998;28(6):1607–15. [24] Czaja MJ, Xu J, Alt E. Prevention of carbon tetrachloride-induced rat liver injury by soluble tumor necrosis factor receptor. Gastroenterology 1995;108(6):1849–54. [25] Morio LA, Chiu H, Sprowles KA, Zhou P, Heck DE, Gordon MK, et al. Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice. Toxicol Appl Pharmacol 2001;172(1):44–51. [26] Heng YM, Kuo CS, Jones PS, Savory R, Schulz RM, Tomlinson SR, et al. A novel murine P-450 gene, Cyp4a14, is part of a cluster of Cyp4a and Cyp4b, but not of CYP4F, genes in mouse and humans. Biochem J 1997;325(Pt 3):741–9. [27] Ronis MJ, Lindros KO, Sundberg I. The CYP2E1 family. In: Ioannides C, editor. Cytochrome P-450 metabolic and toxicological aspects. Boca Raton: CRC Press; 1996. p. 211–39. [28] Enriquez A, Leclercq I, Farrell GC, Robertson G. Altered expression of hepatic CYP2E1 and CYP4A in obese, diabetic ob/ob mice, and fa/fa Zucker rats. Biochem Biophys Res Commun 1999;255(2):300–6. [29] Leclercq IA, Farrell GC, Field J, Bell DR, Gonzalez FJ, Robertson GR, CYP2E1. CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis. J Clin Invest 2000 Apr;105(8):1067–75. [30] Driscoll KE, Hassenbein DG, Howard BW, Isfort RJ, Cody D, Tindal MH, et al. Cloning, expression, and functional characterization of rat

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

MIP-2: a neutrophil chemoattractant and epithelial cell mitogen. J Leukoc Biol 1995;58(3):359–64. Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, et al. The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 2001;177(2):112–20. Kitamura K, Nakamoto Y, Akiyama M, Fujii C, Kondo T, Kobayashi K, et al. Pathogenic roles of tumor necrosis factor receptor p55mediated signals in dimethylnitrosamine-induced murine liver fibrosis. Lab Invest 2002;82(5):571–83. Matsuoka M, Pham NT, Tsukamoto H. Differential effects of interleukin-1 alpha, tumor necrosis factor alpha, and transforming growth factor beta 1 on cell proliferation and collagen formation by cultured fat-storing cells. Liver 1989;9(2):71–8. Sung CK, She H, Xiong S, Tsukamoto H. Tumor necrosis factoralpha inhibits peroxisome proliferator-activated receptor gamma activity at a posttranslational level in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2004;286(5):G722–9. Hazra S, Xiong S, Wang J, Rippe RA, Krishna V, Chatterjee K, et al. Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells. J Biol Chem 2004;279(12):11392–401. She H, Xiong S, Hazra S, Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellate cells. J Biol Chem 2005;280(6):4959–67. Wadsworth TL, McDonald TL, Koop DR. Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced signaling pathways involved in the release of tumor necrosis factoralpha. Biochem Pharmacol 2001;62(7):963–74. Wheeler M, Stachlewitz RF, Yamashina S, Ikejima K, Morrow AL, Thurman RG. Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J 2000;14(3):476–84. Stachlewitz RF, Seabra V, Bradford B, Bradham CA, Rusyn I, Germolec D, et al. Glycine and uridine prevent d-galactosamine hepatotoxicity in the rat: role of Kupffer cells. Hepatology 1999;29(3):737–45. Seabra V, Stachlewitz RF, Thurman RG. Taurine blunts LPS-induced increases in intracellular calcium and TNF-alpha production by Kupffer cells. J Leukoc Biol 1998;64(5):615–21.