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Geniposidic acid protects against d -galactosamine and lipopolysaccharide-induced hepatic failure in mice
Journal of Ethnopharmacology 146 (2013) 271–277
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Geniposidic acid protects against D-galactosamine and lipopolysaccharideinduced hepatic failure in mice So-Jin Kim a,1, Kang-Min Kim a,1, Juhyun Park a, Jong-Hwan Kwak a, Yeong Shik Kim b, Sun-Mee Lee a,n a b
School of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Republic of Korea College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
abstract
Article history: Received 24 June 2012 Received in revised form 23 October 2012 Accepted 14 December 2012 Available online 5 January 2013
Ethnopharmacological relevance: Geniposidic acid (GA) is an iridoid glucoside isolated from Gardeniae jasminoides Ellis (Rubiaceae) that has long been used to treat inflammation, jaundice and hepatic disorders. Aims of the study: This study examined the cytoprotective properties of GA against D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Materials and methods: Mice were given an intraperitoneal injection of GA (12.5, 25, 50 mg/kg) 1 h before receiving GalN (800 mg/kg)/LPS (40 mg/kg). Liver and blood samples were collected 1 and 8 h after GalN/LPS injection. Results: The survival rate of the GA group was significantly higher than the control. GalN/LPS increased serum aminotransferase activity, serum tumor necrosis factor-a level and hepatic lipid peroxidation and decreased hepatic glutathione content. These changes were attenuated by GA. GA augmented increases in serum interleukin-6 level, heme oxygenase-1 and NF-E2-related factor 2 protein expression. Mice treated with GA decreased cleaved caspase-8 and caspase-3 protein expression and showed significantly fewer apoptotic cells. GA increased Bcl-xL protein expression and decreased Bax protein expression. Moreover, GA treatment enhanced phosphorylation of signal transducer and activator of transcription 3. Conclusion: Our findings suggest that geniposidic acid alleviates GalN/LPS-induced liver injury by enhancing antioxidative defense system and reducing apoptotic signaling pathways. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Apoptosis Bcl-xL Geniposidic acid Heme oxygenase-1 Hepatic failure STAT3
1. Introduction Fulminant hepatic failure is a life-threatening clinical syndrome that results from severe impairment of liver function. It is associated with high mortality if not treated by liver transplantation. A low dose of lipopolysaccharide (LPS) in combination with D-galactosamine (GalN) induces an experimental liver injury that is similar to the acute hepatic failure that is observed clinically (Nakama et al., 2001). Tumor necrosis factor (TNF)-a, a key cytokine, induces massive hepatocyte injury in mice with GalN/ LPS-induced fulminant hepatic failure (Josephs et al., 2000). TNF-a causes production of reactive oxygen species (ROS) and activates apoptotic signaling pathways by binding to the TNF receptor on the hepatocyte surface. Hemeoxygenase (HO)-1 is an endogenous, cytoprotective enzyme that is upregulated under oxidative stress conditions. HO-1 exerts powerful anti-
n
Corresponding author. Tel.: þ82 31 290 7712; fax: þ82 31 292 8800. E-mail address: [email protected] (S.-M. Lee). 1 These authors contributed equally to this work.
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.12.042
inflammatory, antioxidant and anti-apoptotic effects. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that is crucial in a variety of biological functions including cell growth, suppression of apoptosis and cell motility (Akira, 1999). STAT3 protects against TNF-a-mediated hepatic apoptosis and induces HO-1 gene expression (Xing et al., 2011). The fruit of the gardenia plant (Gardenia jasminoides Ellis) is a traditional Chinese medicine that has long been used for the treatment of febrile diseases, jaundice, acute conjunctivitis and pyogenic infections, as well as sprains and painful swellings from blood stasis (Chang, 1987). Crude extracts of Gardenia jasminoides have biological and pharmacological activities such as antiinflammatory and antioxidative effects (Koo et al., 2004). Furthermore, glycoprotein isolated from Gardenia jasminoides has strong scavenging activity against oxygen free radicals and inhibits apoptosis of NIH/3T3 cells (Lee et al., 2006). Geniposidic acid ((1S,4aS,7aS)-1-(b-D-glucopyranosyloxy)-7(hydroxymethyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylicacid) is one of the major iridoid glucoside compounds of gardenia fruit. We screened a number of hepatoprotective
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components from Gardenia jasminides Ellis in GalN-treated primary cultures of rat hepatocytes, and GA was the most active component of the iridoid glucosides (unpublished results). GA efficiently protects PC-12 cells against amyloid-b peptide cytotoxicity (Zhou et al., 2009), and inhibits secondary inflammation and decreases serum TNF-a and interleukin (IL)-1b levels in adjuvant-induced arthritis rats (Jin et al., 2009). Since geniposide, one of the components of Gardenia jasminoides, which has similar structure with geniposidic acid, did not exhibit genotoxicity (Ozaki et al., 2002), it might provide novel therapeutic intervention. This study investigated the hepatoprotective effect and specific molecular mechanism of geniposidic acid, particularly on the oxidative stress and apoptotic pathways.
2. Material and methods 2.1. General experimental procedures
were injected intraperitoneally with GalN (800 mg/kg; Sigma Chemical Co., St Louis, MO, USA) and LPS (40 mg/kg Escherichia coli O111:B4; Sigma Chemical Co.) dissolved in phosphatebuffered saline, except for the normal control. The six treatment groups were: (a) vehicle-treated control, (b) vehicle-treated GalN/ LPS, (c–e) GA (12.5, 25, and 50 mg/kg)-treated GalN/LPS, and (f) silymarin (100 mg/kg)-treated GalN/LPS.The survival analysis of mice was monitored for 24 h after GalN/LPS injection. Mice were anesthetized with ketamine/xylazine and then sacrificed by decapitation at 1 and 8 h after GalN/LPS injection, and blood and liver samples were collected. 2.5. Survival and serum aminotransferase activity The survival of the animals was recorded for 24 h after GalN/ LPS injection. In the different set of survival experiment, the blood sample was collected and then ALT activity was determined by standard spectrophotometric procedures (Huang et al., 2006) using Chemi-Lab ALT assay kits (IVDLab Co., Uiwang, Korea) at 8 h after GalN/LPS injection.
NMR experiments were performed on a Varian Unity INOVA 500 spectrometer with the usual pulse sequences. ESI-MS spectrum was obtained on Agilent 1100 LC/MSD trap classic. Column chromatography was carried out on Silica gel 60 (230–400 mesh; Merck, Darmstadt, Germany), LiChroprep RP-18 (40–63 mm; Merck). TLC was performed on pre-coated Silica gel 60 F254 plates (20 20 cm2, 0.25 mm; Merck) and RP-18F254s plates (20 20 cm2, 0.25 mm; Merck).
Circulating levels of TNF-a and IL-6 were quantified as previously described (He et al., 2000 and Akiba et al., 2000, respectively) at 1 h after GalN/LPS injection using commercial mouse ELISA kits (BD Bioscience, San Diego, CA, USA), conducted according to the manufacturer’s instructions.
2.2. Plant material
2.7. Histological analysis and detection of apoptotic cells
Dried fruit of Gardenia jasminoides, Gardeniae Fructus, was purchased in April 2008 from a traditional medicine market, Kyung-dong yak-ryong-si, in Seoul, Korea. A voucher specimen (skku-08-18) is deposited in the School of Pharmacy, Sungkyunkwan University.
Liver specimens for the histopathological analysis were obtained 8 h after GalN/LPS injection. Samples were fixed in 10% neutral-buffered formalin and embedded in paraffin, sliced into 5 mm sections, and stained with hematoxylin–eosin for blind histological assessment. The degree of portal inflammation, hepatocellular necrosis, and inflammatory cell infiltration was evaluated (Frei et al., 1984). Histological changes were evaluated in randomly chosen histological fields at 200 magnification. Apoptotic cells were detected by the TUNEL method using an in situ apoptosis detection kit (TaKaRa Co., Shizo, Japan). The technique described by Tornusciolo et al. (1995) was used for the specific labeling of fragmented DNA. Sections were evaluated in nonconsecutive, randomly chosen histological fields at 200.
2.3. Extraction and isolation Dried fruits of Gardenia jasminoides (1.8 kg) were extracted with MeOH (7.7 L 2) at room temperature for 24 h. The MeOH extract was evaporated to dryness under reduced pressure, and the residue (538 g) was suspended in H2O (1.8 L). The resulting solution was consecutively partitioned with CH2Cl2, EtOAc, n-BuOH to give CH2CH2 (99 g), EtOAc (16 g), n-BuOH (88 g), and H2O (323 g) fractions. The n-BuOH fraction was fractionated on a silica gel column using a stepwise elution with EtOAc–MeOH– H2O (100:5:3, 100:10:7, 100:13:10, and 100:20:16). Fractions were combined based on their TLC pattern to yield fractions F1–F8. Fraction F6 was rechromatographed over a silica gel column with CH2Cl2–MeOH–H2O (40:10:1) and RP–C18 column with 5% MeOH–H2O as eluent to obtain geniposidic acid (GA, 482 mg). GA isolated from Gardeniae Fructus was identified by comparing its spectral data with literature values (Wang et al., 1999). 2.4. Animal treatment Male ICR mice weighing 25–30 g (DaehanBiolink Co., Eumseong, Korea) were made to fast for 18 h before experiments but given tap water ad libitum. All animal protocols were approved by the Animal Care Committee of Sungkyunkwan University and were performed in accordance with National Institutes of Health guidelines. In GA-treated group, mice were administered geniposidic acid (dissolved in saline) at 12.5, 25, and 50 mg/kg intraperitoneally 1 h before the GalN/LPS injection, while other groups received an equivalent volume of saline as the control. All animals
2.6. Serum TNF-a and IL-6 levels
2.8. Hepatic lipid peroxidation and glutathione content Liver homogenates were analyzed for malondialdehyde (MDA) by measuring the level of thiobarbituric acid-reactive substances spectrophotometrically at 535 nm using 1,1,3,3-tetraethoxypropane (Sigma Chemical Co.) as the standard (Buege and Aust, 1978). Total glutathione was determined by yeast– glutathione reductase, 5,50 -dithiobis (2-nitrobenzoic acid), and NADPH, at 412 nm in the liver homogenates after precipitation with 1% picric acid. Oxidized glutathione (GSSG) levels were determined by the same method in the presence of 2-vinylpyridine, and reduced glutathione (GSH) was the difference between total glutathione and GSSG (Brehe and Burch, 1976). 2.9. Preparation of protein extracts Fresh liver tissue was isolated and homogenized in PRO-PREP (iNtRON Biotechnology Co., Ltd., Seongnam, Korea) for whole protein samples and in NE-PER (Pierce Biotechnology, Inc., Rockford, IL, USA) for nuclear and cytosolic protein samples, according to the manufacturer’s instructions. The technique described by
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Table 1 Effect of geniposidic acid (GA)a on serum ALT activity in mice after GalN/LPS injection (means7 SEM, n¼ 10). Group
ALT (U/L)
Control GalN/LPS GA (12.5 mg/kg) þ GalN/LPS GA (25 mg/kg) þ GalN/LPS GA (50 mg/kg) þGalN/LPS
367 4 7237 7 825** 2766 7 318**,þþ 1566 7 205þþ 928 7 121þþ
a GA (12.5, 25, 50 mg/kg) was administered intraperitoneally 1 h before GalN/LPS injection. nn Significantly different (p o 0.01) from the control group. þþ Significantly different (p o0.01) from the GalN/LPS group.
Fig. 1. Effect of geniposidic acid on lethality induced by GalN/LPS. All groups consisted of 10 mice. Mice received intraperitoneal injection of vehicle, GA (12.5, 25 and 50 mg/kg) or silymarin (100 mg/kg) 1 h before GalN (800 mg/kg)/LPS (40 mg/kg) treatment.
Smith et al. (1985) was used for the protein assay. Protein concentration was determined by BCA Protein Assay kit (Pierce Biotechnology, Inc.) 2.10. Western blot immunoassay For western blot analysis freshly isolated liver tissue was homogenized in a lysis buffer. Liver proteins were separated on sodium dodecyl sulfate–polyacrylamide gels to determine levels of caspase-8, caspase-3, Bcl-xL, Bax, HO-1, Nrf2, and STAT3, and transferred to polyvinylidene fluoride membranes Transferred membranes were blocked for 1 h with 5% skim milk or 5% BSA in TBS/T at room temperature. Blots were probed overnight with antibodies against mouse caspase-8 (Cell Signaling Technology, Inc., Irvine, CA, USA), caspase-3 (Cell Signaling Technology, Inc.), Bcl-xL(Cell Signaling Technology, Inc.), Bax (Cell Signaling Technology, Inc.), HO-1 (StressGen Bioreagents Corp., Ann Arbor, MI, USA), Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or STAT3 (Abcam, Cambridge, UK). The next day, blots were probed with appropriate secondary antibodies and detected with an ECL detection system (iNtRON Biotechnology Co., Ltd.). Visualized bands were evaluated densitometrically with ImageQuant TL software (Amersham Biosciences/GE Healthcare, Piscataway, NJ, USA). The signals were normalized to that of b-actin (Sigma chemical Co.) for whole proteins or lamin B1 (Abcam) for nuclear protein.
LPS injection, increased the survival rate in a dose-dependent manner. With 50 mg/kg of GA, the survival rate was 80% at 19 h (Fig. 1). GA at 50 mg/kg was selected as the optimal effective dose for evaluating the molecular mechanisms of GA against GalN/LPSinduced hepatic failure. In the control group, the level of serum ALT, a marker of liver injury, was 3674 U/L. Serum ALT showed a marked increase to 80187899 U/L at 8 h after GalN/LPS injection, indicating severe hepatocellular damage. This increase was attenuated by GA 12.5, 25 and 50 mg/kg (Table 1). Histological observations of the liver samples strongly supported the release of ALT by damaged hepatocytes as well as the protective effect of GA. The histological features of the control group showed normal liver lobular architecture and cell structure. However, livers at 8 h after GalN/LPS treatment showed extensive areas of inflammation, broad cellular necrosis, and increased inflammatory cell infiltration. These pathological changes were ameliorated in GA-pretreated mice (Fig. 2).
3.2. Serum TNF-a and IL-6 levels As shown in Table 2, the serum TNF-a level in control animals were low. In control animals, serum TNF-a level was 68 78 pg/ mL. At 1 h after GalN/LPS injection, serum TNF-a level increased significantly to 15.4 times that observed in control animals. GA attenuated the increase in serum TNF-a level. The serum IL6 level in control group mice was low (3272 pg/mL). One hour after GalN/LPS injection, the serum IL-6 level was significantly increased to 283 times that observed in control group mice. This increased level was enhanced by pretreatment of GA.
2.11. Statistical analysis All results are presented as means7standard error of the mean (SEM). The overall significance of results was analyzed by one-way ANOVA. Differences between compared groups were considered statistically significant at p o0.05 with the appropriate Bonferroni correction for multiple comparisons. Survival data were analyzed by Kalplan–Meier curves and the log-rank test.
3. Results 3.1. Survival, serum alanine aminotransferase activity (ALT) and histological analysis Animals in a GalN/LPS-treated group began to die at 7 h after GalN/LPS injection, and the survival rate was 10% at 24 h. The survival rate of silymarin-treated group was 70% at 24 h. However, pretreatment with GA (12.5, 25, 50 mg/kg), 1 h before GalN/
3.3. Apoptotic cell detection and caspase-8, -3, Bcl-xLand Bax protein expression As shown in Fig. 3, at 8 h after GalN/LPS injection, the levels of caspase-8 and caspase-3 protein expression in the cytosolic fraction of the liver were increased to 1.9 and 22 times higher, respectively, than the control group. GA markedly suppressed the elevation of caspase-8 and caspase-3 protein expression. A more definitive characterization of the suppressive effect of GA on hepatocyte apoptosis was seen by a terminal deoxynucleotidyl transferase-mediated dUTP nick-labeling (TUNEL) assay. After 8 h, few TUNEL-positive hepatocytes were observed in livers from GAtreated mice (Fig. 4). At 1 h after GalN/LPS injection, the level of Bcl-xL increased 1.5-fold and the level of Baxincreased 1.6-fold in the liver cytosolic fraction compared to the control. GA augmented the increase in Bcl-xL protein expression and attenuated the increase in Bax protein expression (Fig. 5).
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Fig. 2. Effect of geniposidic acid on histological changes of mice liver after GalN/LPS injection (original magnification 200): (A) control, normal architecture of the liver; (B) GalN/LPS, extensive areas of inflammation, necrosis, and cell infiltration around the overall area; and (C) GA (50 mg/kg) þ GalN/LPS, minimal inflammation and inflammatory cell infiltration. Table 2 Effect of geniposidic acid (GA)a on serum TNF-a and IL-6 levels in mice afterGalN/ LPS injection (means 7SEM, n¼10). Group
TNF-a (pg/mL)
IL-6 (pg/mL)
Control GalN/LPS GA þ GalN/LPS
68 7 8 10407 197nn 504 7 62n, þ
32 72 8985 7678nn 1415971742nn,þþ
a
GA (50 mg/kg) was administered intraperitoneally 1 h before GalN/LPS injection. Significantly different (po0.05) from the control group. Significantly different (po0.01) from the control group. þ Significantly different (po0.05) from the GalN/LPS group. þþ Significantly different (po0.01) from the GalN/LPS group. n
nn
3.4. Hepatic lipid peroxidation and glutathione levels GalN/LPS increased the level of hepatic MDA, an end-product of lipid peroxidation, to 1.8 times higher than the normal control group; this was attenuated by GA. In contrast, reduced GSH decreased to 35% of control values at 8 h after the GalN/LPS injection; this was attenuated by GA treatment (Table 3). 3.5. HO-1 and NF-E2-related factor 2 (Nrf2) protein expression Nrf2 is a transcription factor that plays a critical role in the primary cellular defense against oxidative stress. As shown in Fig. 6, at 1 h after GalN/LPS injection, the GalN/LPS group had 1.3-fold higher hepatic HO-1 levels and 2.1-fold higher nuclear Nrf2 levels than the control. These increases in HO-1 and Nrf2 protein levels were significantly augmented by GA. 3.6. STAT3 protein expression The phosphorylation of Tyr705, which is essential for the activation of STAT3 was detected and quantified by western blot. At 1 h after GalN/LPS injection, the levels of both STAT3 and pSTAT3 protein were significantly higher than control group. Notably, the p-STAT3/STAT3 relative level was significantly increased 39-fold at 1 h after GalN/LPS injection; this increase was augmented by GA treatment (Fig. 7).
4. Discussion In this study, the protective effect of GA was examined using a model of GalN/LPS-induced hepatotoxicity. Fulminant hepatic failure is defined by the sudden onset of hepatic encephalopathy and often occurs in association with coagulopathy, jaundice and multisystem organ failure. The mortality of patients with advanced fulminant hepatic failure is high and the availability of liver transplantation has provided a means to save these patients (Sass and Shakil, 2005). GalN/LPS-induced liver failure
Fig. 3. Effect of geniposidic acid (50 mg/kg) on caspase-8 and caspase-3 protein expression after GalN/LPS injection. Values are presented as the means7 SEM of 10 mice per group. n,nnSignificantly different (p o 0.05, 0.01) from the control group. ]]Significantly different (p o0.01) from the GalN/LPS group.
is used widely as an experimental liver injury model for elucidating the mechanism of clinical liver dysfunction and for testing the efficacy of hepatoprotective agents (Nakama et al., 2001). The mice in the GalN/LPS-treated group began to die at 7 h, and the survival rate reached 10% by 24 h. Eight hours after the GalN/LPS injection, hepatic injury was occurred as a result of GalN/LPS injection. This was indicated by the significant increase in the serum ALT levels. These results show that GA protects mice from GalN/LPS-induced hepatic injury, as evidenced by the significant decrease in the mortality and serum ALT levels in treatment with GA 50 mg/kg. This result was also supported by results of histopathological examination that extensive inflammation, necrosis, and cell infiltration were improved dramatically in the GA-treated group. These results suggest that GA may have potential of clinical applications for treating liver injury. It is well known that TNF-a, a mediator of the hepatic acutephage response to inflammation, is rapidly produced by macrophages in response to tissue damage (Brouckaert and Fiers, 1996), and activates caspase-8-dependent apoptotic signals by binding to the surface of hepatocytes (Hishinuma et al., 1990). And then caspase-8 triggers the activation of caspase-3 through multiple apoptotic signaling pathways. In addition to necrosis, apoptosis is important in GalN/LPS-induced liver injury, and regulation of unwanted liver apoptosis is suggested as a therapeutic method for fulminant hepatic failure (Takamura et al., 2007). Our study
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Fig. 4. Detection of apoptotic hepatocytes in the liver of mice after GalN/LPS injection (original magnification 200). Values are presented as the means 7 SEM of 10 mice per group: (A) control, (B) GalN/LPS, and (C) GA (50 mg/kg) þGalN/LPS. nnSignificantly different (po 0.01) from the control group. ]Significantly different (p o 0.05) from the GalN/LPS group. Table 3 Effect of geniposidic acid (GA)a on lipid peroxidation and reduced glutathione levels in mice after GalN/LPS injection (means7 SEM, n ¼10). group
MDA (nmol/mg protein)
GSH (mmol/g liver)
Control GalN/LPS GA þGalN/LPS
0.47 70.04 0.83 70.10nn 0.56 70.06 þ
15.47 0.6 5.47 1.3nn 13.37 0.6þþ
a GA (50 mg/kg) was administered intraperitoneally 1 h before GalN/LPS injection. nn Significantly different (p o 0.01) from the control group. þ Significantly different (p o0.05) from the GalN/LPS group. þþ Significantly different (p o 0.01) from the GalN/LPS group.
Fig. 5. Effect of geniposidic acid (50 mg/kg) on Bcl-xL and Bax protein expression after GalN/LPS injection. Values are presented as the means7 SEM of 10 mice per group. nnSignificantly different (p o 0.01) from the control group. ],]]Significantly different (po 0.05, 0.01) from the GalN/LPS group.
confirmed the dramatic increase in the serum TNF-a level at 1 h after administration of GalN/LPS. These alterations were attenuated by the GA treatment. At 8 h after GalN/LPS injection, caspase-8 and caspase-3 protein expressions were markedly increased than control group and these elevations were significantly suppressed by GA treatment. Furthermore, TUNEL staining assay was examined to confirm the suppressive effect of GA on apoptosis. Bcl-2, Bcl-xL, and Bax are members of the Bcl-2 family
that play key roles in apoptosis regulation. Overexpression of Bcl-2 and Bcl-xL enhances cell survival by suppressing apoptosis in a number of cells subject to a wide range of apoptosis-inducing stimuli, but overexpression of Bax accelerates cell death (Hsu et al., 1997). A family of Bcl-2-related proteins regulates cell death and shares highly conserved BH1 and BH2 domains. The BH1 and BH2 domains of Bcl-2 are required for heterodimerization with Bax and for repressing apoptosis (Sedlak et al., 1995). Previous studies found that in a GalN/LPS-induced acute hepatic failure model, the Bcl-2-related protein Bcl-xL is a stronger protector against apoptosis than Bcl-2 (de la Coste et al., 1999). At 1 h after GalN/LPS injection, increased Bax and Bcl-xL protein expression by GalN/LPS were attenuated and augmented, respectively with treatment of GA. These results showed that GA suppressed the apoptotic signaling via modulation of TNF-a release and Bax and Bcl-xL protein expressions. The current biochemical and pathological studies suggest that ROS are important mediators in the pathophysiology of inflammatory liver
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Fig. 6. Effect of geniposidic acid (50 mg/kg) on HO-1 and nuclear Nrf2 protein expression after GalN/LPS injection. Values are presented as the means 7SEM of 10 mice per group. n,nnSignificantly different (p o 0.05, 0.01) from the control group.],]]Significantly different (p o0.05, 0.01) from the GalN/LPS group.
Fig. 7. Effect of geniposidic acid (50 mg/kg) on STAT3 phosphorylation after GalN/ LPS injection. Values are presented as the means7 SEM of 10 mice per group. nn Significantly different (po 0.01) from the control group. ]Significantly different (p o0.05) from the GalN/LPS group.
damage (Webb and Twedt, 2008). Release of ROS from activated Kupffer cells causes GalN/LPS-induced liver damage in rats (Shiratori et al., 1988). ROS attack biological membranes, and can lead to the oxidative destruction of polyunsaturated fatty acids through lipid peroxidation. HO is a rate-limiting enzyme that catalyzes the breakdown of heme into antioxidant and anti-inflammatory agents such as biliverdin, carbon monoxide, and iron. Previous studies have demonstrated that expression of HO-1 is rapidly upregulated in the liver after administration of toxins such as carbon tetrachloride (Nakahira et al., 2003), endotoxin, and GalN/LPS (Wan et al., 2008). This represents an adaptive response to the oxidative damage. Induction of HO-1 by a chemical inducer such as hemin is cytoprotective against GalN/LPS-induced acute hepatic injury in rats (Wen et al.,
2007). An antioxidant responsive element (ARE) is found in genes encoding a variety of proteins. It is upregulated by various stimuli such as oxidative stress. This study confirmed a significant increase in the HO-1 protein expression after GalN/LPS injection. These alterations were augmented by the GA treatment suggesting that GA induces HO-1 protein expression and/or suppresses the degradation of this protein. HO-1 protein expression is regulated by various transcription associated proteins. Among them, Nrf2 is an important transcription factor that regulates the expression of many antioxidant enzymes that provide additional cytoprotection (Yun et al., 2010). Nrf2 is reported to regulate ARE-driven genes such as glutathione S-transferase, quinone reductase and HO-1. These results were corelation with GSH and MDA level assay in this study. In GA-treatment group, reduced GSH and increased MDA levels by GalN/LPS injection were markedly attenuated. This suggests that GalN/LPS injection develops the oxidative stress circumstances being one of causes of hepatic injury, and GA may play an important role in reducing these oxidative damage by down-regulation of oxidative factors and upregulation of defense system against oxidative stress. On the basis of GSH and MDA levels, GalN/LPS injection induced oxidative stress which is causative of hepatic injury. Increased protein expression such as HO-1 and Nrf2 by GA treatment is construed as protective effect of GA by induction of oxidative defense system. Previous study demonstrated that various cytokines have the ability to protect cells from the byproducts of inflammation (Hideshima et al., 2001). IL-6 is typical inflammatory cytokine that leak from T cells and macrophages (especially Kupffer cells in liver) into bloodstream by stimulation such as LPS and plays a critical role as major regulator of the acute phase response in the liver. After GalN/LPS treatment, LPS directly actvates Kupffer cells to produce cytokines such as IL-6. In previous study, it has also been shown that IL-6 is essential for liver regeneration after partial hepatectomy and confers resistance to liver injury by hepatic toxins, and ischemic injury (Streetz et al., 2000). In a GalN/LPS-induced hepatitis model, IL-6 blockade aggravates liver injury and lethality (Barton and Jackson, 1993). STAT plays an important role in cell survival as transcription factor (Aggarwal et al., 2006). STAT proteins are phosphorylated by the Janus kinase family in response to a large number of cytokines, growth factors, and hormones, and they form dimers that translocate to the cell nucleus. STAT3, a STAT protein, is crucial in biological functions such as cell survival and carcinogenesis through regulation of proliferation and apoptosis. The importance of STAT3 in the inflammatory response was also supported by experiments in the STAT3-deficient mice (Takeda et al., 1999). Yamada et al. (2008) have reported that protection against GalN/LPS-induced injury is mediated by STAT3. STAT3 mediates the induction of gene expressions such as Bcl-xL and HO-1 by IL-6, one of the major STAT3 ligands (Deramaudt et al., 1999). IL-6 binds to its soluble receptor signals via gp130 and activates STAT3 through phosphorylation. STAT3 activation is impaired in IL-6-deficient mice (Alonzi et al., 1998) suggesting that STAT3 may mediate many of the effects of IL-6. This result was supported with that the anti-apoptotic proteins such as Bcl-2 and Bcl-xL protein expression were elevated in IL-6-treated livers (Kovalovich et al., 2001). In summary, our results showed that GA pretreatment may promote the induction of IL-6 protein expression and the activation of STAT3 transcription factor.
5. Conclusion In conclusion, geniposidic acid protects hepatocytes against GalN/LPS-induced injury through suppressing apoptotic signaling pathways and enhancing oxidative defense system. Thus, we
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propose that it might be useful as a potential therapeutic medication for attenuating fulminant hepatic failure.
Acknowledgment This work was supported by a Grant from the Korea Food and Drug Administration (Studies on the Identification of Efficacy of Biologically Active Components from Oriental Herbal Medicines).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2012.12.042.
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Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Geniposidic acid protects against D-galactosamine and lipopolysaccharideinduced hepatic failure in mice So-Jin Kim a,1, Kang-Min Kim a,1, Juhyun Park a, Jong-Hwan Kwak a, Yeong Shik Kim b, Sun-Mee Lee a,n a b
School of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Republic of Korea College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
abstract
Article history: Received 24 June 2012 Received in revised form 23 October 2012 Accepted 14 December 2012 Available online 5 January 2013
Ethnopharmacological relevance: Geniposidic acid (GA) is an iridoid glucoside isolated from Gardeniae jasminoides Ellis (Rubiaceae) that has long been used to treat inflammation, jaundice and hepatic disorders. Aims of the study: This study examined the cytoprotective properties of GA against D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Materials and methods: Mice were given an intraperitoneal injection of GA (12.5, 25, 50 mg/kg) 1 h before receiving GalN (800 mg/kg)/LPS (40 mg/kg). Liver and blood samples were collected 1 and 8 h after GalN/LPS injection. Results: The survival rate of the GA group was significantly higher than the control. GalN/LPS increased serum aminotransferase activity, serum tumor necrosis factor-a level and hepatic lipid peroxidation and decreased hepatic glutathione content. These changes were attenuated by GA. GA augmented increases in serum interleukin-6 level, heme oxygenase-1 and NF-E2-related factor 2 protein expression. Mice treated with GA decreased cleaved caspase-8 and caspase-3 protein expression and showed significantly fewer apoptotic cells. GA increased Bcl-xL protein expression and decreased Bax protein expression. Moreover, GA treatment enhanced phosphorylation of signal transducer and activator of transcription 3. Conclusion: Our findings suggest that geniposidic acid alleviates GalN/LPS-induced liver injury by enhancing antioxidative defense system and reducing apoptotic signaling pathways. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Apoptosis Bcl-xL Geniposidic acid Heme oxygenase-1 Hepatic failure STAT3
1. Introduction Fulminant hepatic failure is a life-threatening clinical syndrome that results from severe impairment of liver function. It is associated with high mortality if not treated by liver transplantation. A low dose of lipopolysaccharide (LPS) in combination with D-galactosamine (GalN) induces an experimental liver injury that is similar to the acute hepatic failure that is observed clinically (Nakama et al., 2001). Tumor necrosis factor (TNF)-a, a key cytokine, induces massive hepatocyte injury in mice with GalN/ LPS-induced fulminant hepatic failure (Josephs et al., 2000). TNF-a causes production of reactive oxygen species (ROS) and activates apoptotic signaling pathways by binding to the TNF receptor on the hepatocyte surface. Hemeoxygenase (HO)-1 is an endogenous, cytoprotective enzyme that is upregulated under oxidative stress conditions. HO-1 exerts powerful anti-
n
Corresponding author. Tel.: þ82 31 290 7712; fax: þ82 31 292 8800. E-mail address: [email protected] (S.-M. Lee). 1 These authors contributed equally to this work.
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.12.042
inflammatory, antioxidant and anti-apoptotic effects. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that is crucial in a variety of biological functions including cell growth, suppression of apoptosis and cell motility (Akira, 1999). STAT3 protects against TNF-a-mediated hepatic apoptosis and induces HO-1 gene expression (Xing et al., 2011). The fruit of the gardenia plant (Gardenia jasminoides Ellis) is a traditional Chinese medicine that has long been used for the treatment of febrile diseases, jaundice, acute conjunctivitis and pyogenic infections, as well as sprains and painful swellings from blood stasis (Chang, 1987). Crude extracts of Gardenia jasminoides have biological and pharmacological activities such as antiinflammatory and antioxidative effects (Koo et al., 2004). Furthermore, glycoprotein isolated from Gardenia jasminoides has strong scavenging activity against oxygen free radicals and inhibits apoptosis of NIH/3T3 cells (Lee et al., 2006). Geniposidic acid ((1S,4aS,7aS)-1-(b-D-glucopyranosyloxy)-7(hydroxymethyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylicacid) is one of the major iridoid glucoside compounds of gardenia fruit. We screened a number of hepatoprotective
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components from Gardenia jasminides Ellis in GalN-treated primary cultures of rat hepatocytes, and GA was the most active component of the iridoid glucosides (unpublished results). GA efficiently protects PC-12 cells against amyloid-b peptide cytotoxicity (Zhou et al., 2009), and inhibits secondary inflammation and decreases serum TNF-a and interleukin (IL)-1b levels in adjuvant-induced arthritis rats (Jin et al., 2009). Since geniposide, one of the components of Gardenia jasminoides, which has similar structure with geniposidic acid, did not exhibit genotoxicity (Ozaki et al., 2002), it might provide novel therapeutic intervention. This study investigated the hepatoprotective effect and specific molecular mechanism of geniposidic acid, particularly on the oxidative stress and apoptotic pathways.
2. Material and methods 2.1. General experimental procedures
were injected intraperitoneally with GalN (800 mg/kg; Sigma Chemical Co., St Louis, MO, USA) and LPS (40 mg/kg Escherichia coli O111:B4; Sigma Chemical Co.) dissolved in phosphatebuffered saline, except for the normal control. The six treatment groups were: (a) vehicle-treated control, (b) vehicle-treated GalN/ LPS, (c–e) GA (12.5, 25, and 50 mg/kg)-treated GalN/LPS, and (f) silymarin (100 mg/kg)-treated GalN/LPS.The survival analysis of mice was monitored for 24 h after GalN/LPS injection. Mice were anesthetized with ketamine/xylazine and then sacrificed by decapitation at 1 and 8 h after GalN/LPS injection, and blood and liver samples were collected. 2.5. Survival and serum aminotransferase activity The survival of the animals was recorded for 24 h after GalN/ LPS injection. In the different set of survival experiment, the blood sample was collected and then ALT activity was determined by standard spectrophotometric procedures (Huang et al., 2006) using Chemi-Lab ALT assay kits (IVDLab Co., Uiwang, Korea) at 8 h after GalN/LPS injection.
NMR experiments were performed on a Varian Unity INOVA 500 spectrometer with the usual pulse sequences. ESI-MS spectrum was obtained on Agilent 1100 LC/MSD trap classic. Column chromatography was carried out on Silica gel 60 (230–400 mesh; Merck, Darmstadt, Germany), LiChroprep RP-18 (40–63 mm; Merck). TLC was performed on pre-coated Silica gel 60 F254 plates (20 20 cm2, 0.25 mm; Merck) and RP-18F254s plates (20 20 cm2, 0.25 mm; Merck).
Circulating levels of TNF-a and IL-6 were quantified as previously described (He et al., 2000 and Akiba et al., 2000, respectively) at 1 h after GalN/LPS injection using commercial mouse ELISA kits (BD Bioscience, San Diego, CA, USA), conducted according to the manufacturer’s instructions.
2.2. Plant material
2.7. Histological analysis and detection of apoptotic cells
Dried fruit of Gardenia jasminoides, Gardeniae Fructus, was purchased in April 2008 from a traditional medicine market, Kyung-dong yak-ryong-si, in Seoul, Korea. A voucher specimen (skku-08-18) is deposited in the School of Pharmacy, Sungkyunkwan University.
Liver specimens for the histopathological analysis were obtained 8 h after GalN/LPS injection. Samples were fixed in 10% neutral-buffered formalin and embedded in paraffin, sliced into 5 mm sections, and stained with hematoxylin–eosin for blind histological assessment. The degree of portal inflammation, hepatocellular necrosis, and inflammatory cell infiltration was evaluated (Frei et al., 1984). Histological changes were evaluated in randomly chosen histological fields at 200 magnification. Apoptotic cells were detected by the TUNEL method using an in situ apoptosis detection kit (TaKaRa Co., Shizo, Japan). The technique described by Tornusciolo et al. (1995) was used for the specific labeling of fragmented DNA. Sections were evaluated in nonconsecutive, randomly chosen histological fields at 200.
2.3. Extraction and isolation Dried fruits of Gardenia jasminoides (1.8 kg) were extracted with MeOH (7.7 L 2) at room temperature for 24 h. The MeOH extract was evaporated to dryness under reduced pressure, and the residue (538 g) was suspended in H2O (1.8 L). The resulting solution was consecutively partitioned with CH2Cl2, EtOAc, n-BuOH to give CH2CH2 (99 g), EtOAc (16 g), n-BuOH (88 g), and H2O (323 g) fractions. The n-BuOH fraction was fractionated on a silica gel column using a stepwise elution with EtOAc–MeOH– H2O (100:5:3, 100:10:7, 100:13:10, and 100:20:16). Fractions were combined based on their TLC pattern to yield fractions F1–F8. Fraction F6 was rechromatographed over a silica gel column with CH2Cl2–MeOH–H2O (40:10:1) and RP–C18 column with 5% MeOH–H2O as eluent to obtain geniposidic acid (GA, 482 mg). GA isolated from Gardeniae Fructus was identified by comparing its spectral data with literature values (Wang et al., 1999). 2.4. Animal treatment Male ICR mice weighing 25–30 g (DaehanBiolink Co., Eumseong, Korea) were made to fast for 18 h before experiments but given tap water ad libitum. All animal protocols were approved by the Animal Care Committee of Sungkyunkwan University and were performed in accordance with National Institutes of Health guidelines. In GA-treated group, mice were administered geniposidic acid (dissolved in saline) at 12.5, 25, and 50 mg/kg intraperitoneally 1 h before the GalN/LPS injection, while other groups received an equivalent volume of saline as the control. All animals
2.6. Serum TNF-a and IL-6 levels
2.8. Hepatic lipid peroxidation and glutathione content Liver homogenates were analyzed for malondialdehyde (MDA) by measuring the level of thiobarbituric acid-reactive substances spectrophotometrically at 535 nm using 1,1,3,3-tetraethoxypropane (Sigma Chemical Co.) as the standard (Buege and Aust, 1978). Total glutathione was determined by yeast– glutathione reductase, 5,50 -dithiobis (2-nitrobenzoic acid), and NADPH, at 412 nm in the liver homogenates after precipitation with 1% picric acid. Oxidized glutathione (GSSG) levels were determined by the same method in the presence of 2-vinylpyridine, and reduced glutathione (GSH) was the difference between total glutathione and GSSG (Brehe and Burch, 1976). 2.9. Preparation of protein extracts Fresh liver tissue was isolated and homogenized in PRO-PREP (iNtRON Biotechnology Co., Ltd., Seongnam, Korea) for whole protein samples and in NE-PER (Pierce Biotechnology, Inc., Rockford, IL, USA) for nuclear and cytosolic protein samples, according to the manufacturer’s instructions. The technique described by
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Table 1 Effect of geniposidic acid (GA)a on serum ALT activity in mice after GalN/LPS injection (means7 SEM, n¼ 10). Group
ALT (U/L)
Control GalN/LPS GA (12.5 mg/kg) þ GalN/LPS GA (25 mg/kg) þ GalN/LPS GA (50 mg/kg) þGalN/LPS
367 4 7237 7 825** 2766 7 318**,þþ 1566 7 205þþ 928 7 121þþ
a GA (12.5, 25, 50 mg/kg) was administered intraperitoneally 1 h before GalN/LPS injection. nn Significantly different (p o 0.01) from the control group. þþ Significantly different (p o0.01) from the GalN/LPS group.
Fig. 1. Effect of geniposidic acid on lethality induced by GalN/LPS. All groups consisted of 10 mice. Mice received intraperitoneal injection of vehicle, GA (12.5, 25 and 50 mg/kg) or silymarin (100 mg/kg) 1 h before GalN (800 mg/kg)/LPS (40 mg/kg) treatment.
Smith et al. (1985) was used for the protein assay. Protein concentration was determined by BCA Protein Assay kit (Pierce Biotechnology, Inc.) 2.10. Western blot immunoassay For western blot analysis freshly isolated liver tissue was homogenized in a lysis buffer. Liver proteins were separated on sodium dodecyl sulfate–polyacrylamide gels to determine levels of caspase-8, caspase-3, Bcl-xL, Bax, HO-1, Nrf2, and STAT3, and transferred to polyvinylidene fluoride membranes Transferred membranes were blocked for 1 h with 5% skim milk or 5% BSA in TBS/T at room temperature. Blots were probed overnight with antibodies against mouse caspase-8 (Cell Signaling Technology, Inc., Irvine, CA, USA), caspase-3 (Cell Signaling Technology, Inc.), Bcl-xL(Cell Signaling Technology, Inc.), Bax (Cell Signaling Technology, Inc.), HO-1 (StressGen Bioreagents Corp., Ann Arbor, MI, USA), Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or STAT3 (Abcam, Cambridge, UK). The next day, blots were probed with appropriate secondary antibodies and detected with an ECL detection system (iNtRON Biotechnology Co., Ltd.). Visualized bands were evaluated densitometrically with ImageQuant TL software (Amersham Biosciences/GE Healthcare, Piscataway, NJ, USA). The signals were normalized to that of b-actin (Sigma chemical Co.) for whole proteins or lamin B1 (Abcam) for nuclear protein.
LPS injection, increased the survival rate in a dose-dependent manner. With 50 mg/kg of GA, the survival rate was 80% at 19 h (Fig. 1). GA at 50 mg/kg was selected as the optimal effective dose for evaluating the molecular mechanisms of GA against GalN/LPSinduced hepatic failure. In the control group, the level of serum ALT, a marker of liver injury, was 3674 U/L. Serum ALT showed a marked increase to 80187899 U/L at 8 h after GalN/LPS injection, indicating severe hepatocellular damage. This increase was attenuated by GA 12.5, 25 and 50 mg/kg (Table 1). Histological observations of the liver samples strongly supported the release of ALT by damaged hepatocytes as well as the protective effect of GA. The histological features of the control group showed normal liver lobular architecture and cell structure. However, livers at 8 h after GalN/LPS treatment showed extensive areas of inflammation, broad cellular necrosis, and increased inflammatory cell infiltration. These pathological changes were ameliorated in GA-pretreated mice (Fig. 2).
3.2. Serum TNF-a and IL-6 levels As shown in Table 2, the serum TNF-a level in control animals were low. In control animals, serum TNF-a level was 68 78 pg/ mL. At 1 h after GalN/LPS injection, serum TNF-a level increased significantly to 15.4 times that observed in control animals. GA attenuated the increase in serum TNF-a level. The serum IL6 level in control group mice was low (3272 pg/mL). One hour after GalN/LPS injection, the serum IL-6 level was significantly increased to 283 times that observed in control group mice. This increased level was enhanced by pretreatment of GA.
2.11. Statistical analysis All results are presented as means7standard error of the mean (SEM). The overall significance of results was analyzed by one-way ANOVA. Differences between compared groups were considered statistically significant at p o0.05 with the appropriate Bonferroni correction for multiple comparisons. Survival data were analyzed by Kalplan–Meier curves and the log-rank test.
3. Results 3.1. Survival, serum alanine aminotransferase activity (ALT) and histological analysis Animals in a GalN/LPS-treated group began to die at 7 h after GalN/LPS injection, and the survival rate was 10% at 24 h. The survival rate of silymarin-treated group was 70% at 24 h. However, pretreatment with GA (12.5, 25, 50 mg/kg), 1 h before GalN/
3.3. Apoptotic cell detection and caspase-8, -3, Bcl-xLand Bax protein expression As shown in Fig. 3, at 8 h after GalN/LPS injection, the levels of caspase-8 and caspase-3 protein expression in the cytosolic fraction of the liver were increased to 1.9 and 22 times higher, respectively, than the control group. GA markedly suppressed the elevation of caspase-8 and caspase-3 protein expression. A more definitive characterization of the suppressive effect of GA on hepatocyte apoptosis was seen by a terminal deoxynucleotidyl transferase-mediated dUTP nick-labeling (TUNEL) assay. After 8 h, few TUNEL-positive hepatocytes were observed in livers from GAtreated mice (Fig. 4). At 1 h after GalN/LPS injection, the level of Bcl-xL increased 1.5-fold and the level of Baxincreased 1.6-fold in the liver cytosolic fraction compared to the control. GA augmented the increase in Bcl-xL protein expression and attenuated the increase in Bax protein expression (Fig. 5).
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Fig. 2. Effect of geniposidic acid on histological changes of mice liver after GalN/LPS injection (original magnification 200): (A) control, normal architecture of the liver; (B) GalN/LPS, extensive areas of inflammation, necrosis, and cell infiltration around the overall area; and (C) GA (50 mg/kg) þ GalN/LPS, minimal inflammation and inflammatory cell infiltration. Table 2 Effect of geniposidic acid (GA)a on serum TNF-a and IL-6 levels in mice afterGalN/ LPS injection (means 7SEM, n¼10). Group
TNF-a (pg/mL)
IL-6 (pg/mL)
Control GalN/LPS GA þ GalN/LPS
68 7 8 10407 197nn 504 7 62n, þ
32 72 8985 7678nn 1415971742nn,þþ
a
GA (50 mg/kg) was administered intraperitoneally 1 h before GalN/LPS injection. Significantly different (po0.05) from the control group. Significantly different (po0.01) from the control group. þ Significantly different (po0.05) from the GalN/LPS group. þþ Significantly different (po0.01) from the GalN/LPS group. n
nn
3.4. Hepatic lipid peroxidation and glutathione levels GalN/LPS increased the level of hepatic MDA, an end-product of lipid peroxidation, to 1.8 times higher than the normal control group; this was attenuated by GA. In contrast, reduced GSH decreased to 35% of control values at 8 h after the GalN/LPS injection; this was attenuated by GA treatment (Table 3). 3.5. HO-1 and NF-E2-related factor 2 (Nrf2) protein expression Nrf2 is a transcription factor that plays a critical role in the primary cellular defense against oxidative stress. As shown in Fig. 6, at 1 h after GalN/LPS injection, the GalN/LPS group had 1.3-fold higher hepatic HO-1 levels and 2.1-fold higher nuclear Nrf2 levels than the control. These increases in HO-1 and Nrf2 protein levels were significantly augmented by GA. 3.6. STAT3 protein expression The phosphorylation of Tyr705, which is essential for the activation of STAT3 was detected and quantified by western blot. At 1 h after GalN/LPS injection, the levels of both STAT3 and pSTAT3 protein were significantly higher than control group. Notably, the p-STAT3/STAT3 relative level was significantly increased 39-fold at 1 h after GalN/LPS injection; this increase was augmented by GA treatment (Fig. 7).
4. Discussion In this study, the protective effect of GA was examined using a model of GalN/LPS-induced hepatotoxicity. Fulminant hepatic failure is defined by the sudden onset of hepatic encephalopathy and often occurs in association with coagulopathy, jaundice and multisystem organ failure. The mortality of patients with advanced fulminant hepatic failure is high and the availability of liver transplantation has provided a means to save these patients (Sass and Shakil, 2005). GalN/LPS-induced liver failure
Fig. 3. Effect of geniposidic acid (50 mg/kg) on caspase-8 and caspase-3 protein expression after GalN/LPS injection. Values are presented as the means7 SEM of 10 mice per group. n,nnSignificantly different (p o 0.05, 0.01) from the control group. ]]Significantly different (p o0.01) from the GalN/LPS group.
is used widely as an experimental liver injury model for elucidating the mechanism of clinical liver dysfunction and for testing the efficacy of hepatoprotective agents (Nakama et al., 2001). The mice in the GalN/LPS-treated group began to die at 7 h, and the survival rate reached 10% by 24 h. Eight hours after the GalN/LPS injection, hepatic injury was occurred as a result of GalN/LPS injection. This was indicated by the significant increase in the serum ALT levels. These results show that GA protects mice from GalN/LPS-induced hepatic injury, as evidenced by the significant decrease in the mortality and serum ALT levels in treatment with GA 50 mg/kg. This result was also supported by results of histopathological examination that extensive inflammation, necrosis, and cell infiltration were improved dramatically in the GA-treated group. These results suggest that GA may have potential of clinical applications for treating liver injury. It is well known that TNF-a, a mediator of the hepatic acutephage response to inflammation, is rapidly produced by macrophages in response to tissue damage (Brouckaert and Fiers, 1996), and activates caspase-8-dependent apoptotic signals by binding to the surface of hepatocytes (Hishinuma et al., 1990). And then caspase-8 triggers the activation of caspase-3 through multiple apoptotic signaling pathways. In addition to necrosis, apoptosis is important in GalN/LPS-induced liver injury, and regulation of unwanted liver apoptosis is suggested as a therapeutic method for fulminant hepatic failure (Takamura et al., 2007). Our study
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Fig. 4. Detection of apoptotic hepatocytes in the liver of mice after GalN/LPS injection (original magnification 200). Values are presented as the means 7 SEM of 10 mice per group: (A) control, (B) GalN/LPS, and (C) GA (50 mg/kg) þGalN/LPS. nnSignificantly different (po 0.01) from the control group. ]Significantly different (p o 0.05) from the GalN/LPS group. Table 3 Effect of geniposidic acid (GA)a on lipid peroxidation and reduced glutathione levels in mice after GalN/LPS injection (means7 SEM, n ¼10). group
MDA (nmol/mg protein)
GSH (mmol/g liver)
Control GalN/LPS GA þGalN/LPS
0.47 70.04 0.83 70.10nn 0.56 70.06 þ
15.47 0.6 5.47 1.3nn 13.37 0.6þþ
a GA (50 mg/kg) was administered intraperitoneally 1 h before GalN/LPS injection. nn Significantly different (p o 0.01) from the control group. þ Significantly different (p o0.05) from the GalN/LPS group. þþ Significantly different (p o 0.01) from the GalN/LPS group.
Fig. 5. Effect of geniposidic acid (50 mg/kg) on Bcl-xL and Bax protein expression after GalN/LPS injection. Values are presented as the means7 SEM of 10 mice per group. nnSignificantly different (p o 0.01) from the control group. ],]]Significantly different (po 0.05, 0.01) from the GalN/LPS group.
confirmed the dramatic increase in the serum TNF-a level at 1 h after administration of GalN/LPS. These alterations were attenuated by the GA treatment. At 8 h after GalN/LPS injection, caspase-8 and caspase-3 protein expressions were markedly increased than control group and these elevations were significantly suppressed by GA treatment. Furthermore, TUNEL staining assay was examined to confirm the suppressive effect of GA on apoptosis. Bcl-2, Bcl-xL, and Bax are members of the Bcl-2 family
that play key roles in apoptosis regulation. Overexpression of Bcl-2 and Bcl-xL enhances cell survival by suppressing apoptosis in a number of cells subject to a wide range of apoptosis-inducing stimuli, but overexpression of Bax accelerates cell death (Hsu et al., 1997). A family of Bcl-2-related proteins regulates cell death and shares highly conserved BH1 and BH2 domains. The BH1 and BH2 domains of Bcl-2 are required for heterodimerization with Bax and for repressing apoptosis (Sedlak et al., 1995). Previous studies found that in a GalN/LPS-induced acute hepatic failure model, the Bcl-2-related protein Bcl-xL is a stronger protector against apoptosis than Bcl-2 (de la Coste et al., 1999). At 1 h after GalN/LPS injection, increased Bax and Bcl-xL protein expression by GalN/LPS were attenuated and augmented, respectively with treatment of GA. These results showed that GA suppressed the apoptotic signaling via modulation of TNF-a release and Bax and Bcl-xL protein expressions. The current biochemical and pathological studies suggest that ROS are important mediators in the pathophysiology of inflammatory liver
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Fig. 6. Effect of geniposidic acid (50 mg/kg) on HO-1 and nuclear Nrf2 protein expression after GalN/LPS injection. Values are presented as the means 7SEM of 10 mice per group. n,nnSignificantly different (p o 0.05, 0.01) from the control group.],]]Significantly different (p o0.05, 0.01) from the GalN/LPS group.
Fig. 7. Effect of geniposidic acid (50 mg/kg) on STAT3 phosphorylation after GalN/ LPS injection. Values are presented as the means7 SEM of 10 mice per group. nn Significantly different (po 0.01) from the control group. ]Significantly different (p o0.05) from the GalN/LPS group.
damage (Webb and Twedt, 2008). Release of ROS from activated Kupffer cells causes GalN/LPS-induced liver damage in rats (Shiratori et al., 1988). ROS attack biological membranes, and can lead to the oxidative destruction of polyunsaturated fatty acids through lipid peroxidation. HO is a rate-limiting enzyme that catalyzes the breakdown of heme into antioxidant and anti-inflammatory agents such as biliverdin, carbon monoxide, and iron. Previous studies have demonstrated that expression of HO-1 is rapidly upregulated in the liver after administration of toxins such as carbon tetrachloride (Nakahira et al., 2003), endotoxin, and GalN/LPS (Wan et al., 2008). This represents an adaptive response to the oxidative damage. Induction of HO-1 by a chemical inducer such as hemin is cytoprotective against GalN/LPS-induced acute hepatic injury in rats (Wen et al.,
2007). An antioxidant responsive element (ARE) is found in genes encoding a variety of proteins. It is upregulated by various stimuli such as oxidative stress. This study confirmed a significant increase in the HO-1 protein expression after GalN/LPS injection. These alterations were augmented by the GA treatment suggesting that GA induces HO-1 protein expression and/or suppresses the degradation of this protein. HO-1 protein expression is regulated by various transcription associated proteins. Among them, Nrf2 is an important transcription factor that regulates the expression of many antioxidant enzymes that provide additional cytoprotection (Yun et al., 2010). Nrf2 is reported to regulate ARE-driven genes such as glutathione S-transferase, quinone reductase and HO-1. These results were corelation with GSH and MDA level assay in this study. In GA-treatment group, reduced GSH and increased MDA levels by GalN/LPS injection were markedly attenuated. This suggests that GalN/LPS injection develops the oxidative stress circumstances being one of causes of hepatic injury, and GA may play an important role in reducing these oxidative damage by down-regulation of oxidative factors and upregulation of defense system against oxidative stress. On the basis of GSH and MDA levels, GalN/LPS injection induced oxidative stress which is causative of hepatic injury. Increased protein expression such as HO-1 and Nrf2 by GA treatment is construed as protective effect of GA by induction of oxidative defense system. Previous study demonstrated that various cytokines have the ability to protect cells from the byproducts of inflammation (Hideshima et al., 2001). IL-6 is typical inflammatory cytokine that leak from T cells and macrophages (especially Kupffer cells in liver) into bloodstream by stimulation such as LPS and plays a critical role as major regulator of the acute phase response in the liver. After GalN/LPS treatment, LPS directly actvates Kupffer cells to produce cytokines such as IL-6. In previous study, it has also been shown that IL-6 is essential for liver regeneration after partial hepatectomy and confers resistance to liver injury by hepatic toxins, and ischemic injury (Streetz et al., 2000). In a GalN/LPS-induced hepatitis model, IL-6 blockade aggravates liver injury and lethality (Barton and Jackson, 1993). STAT plays an important role in cell survival as transcription factor (Aggarwal et al., 2006). STAT proteins are phosphorylated by the Janus kinase family in response to a large number of cytokines, growth factors, and hormones, and they form dimers that translocate to the cell nucleus. STAT3, a STAT protein, is crucial in biological functions such as cell survival and carcinogenesis through regulation of proliferation and apoptosis. The importance of STAT3 in the inflammatory response was also supported by experiments in the STAT3-deficient mice (Takeda et al., 1999). Yamada et al. (2008) have reported that protection against GalN/LPS-induced injury is mediated by STAT3. STAT3 mediates the induction of gene expressions such as Bcl-xL and HO-1 by IL-6, one of the major STAT3 ligands (Deramaudt et al., 1999). IL-6 binds to its soluble receptor signals via gp130 and activates STAT3 through phosphorylation. STAT3 activation is impaired in IL-6-deficient mice (Alonzi et al., 1998) suggesting that STAT3 may mediate many of the effects of IL-6. This result was supported with that the anti-apoptotic proteins such as Bcl-2 and Bcl-xL protein expression were elevated in IL-6-treated livers (Kovalovich et al., 2001). In summary, our results showed that GA pretreatment may promote the induction of IL-6 protein expression and the activation of STAT3 transcription factor.
5. Conclusion In conclusion, geniposidic acid protects hepatocytes against GalN/LPS-induced injury through suppressing apoptotic signaling pathways and enhancing oxidative defense system. Thus, we
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propose that it might be useful as a potential therapeutic medication for attenuating fulminant hepatic failure.
Acknowledgment This work was supported by a Grant from the Korea Food and Drug Administration (Studies on the Identification of Efficacy of Biologically Active Components from Oriental Herbal Medicines).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2012.12.042.
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