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Protection of a polysaccharide from Salvia miltiorrhiza, a Chinese medicinal herb, against immunological liver injury in mice
International Journal of Biological Macromolecules 43 (2008) 170–175
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Protection of a polysaccharide from Salvia miltiorrhiza, a Chinese medicinal herb, against immunological liver injury in mice Yu-Hong Song a , Qiang Liu b , Zhi-Ping Lv b,∗ , Yu-Yao Chen b , Ying-Chun Zhou a , Xue-Gang Sun b a b
Department of Traditional Chinese Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 21 March 2008 Received in revised form 23 April 2008 Accepted 25 April 2008 Available online 13 May 2008 Keywords: Salvia miltiorrhiza Polysaccharides Immunological liver injury
a b s t r a c t This study was designed to evaluate the hepatoprotective effects of S. miltiorrhiza polysaccharides (SMPS) in immunological liver injury induced by Bacille–Calmette–Guerin (BCG) and lipopolysaccharide (LPS) in mice. SMPS effectively improved the liver index, spleen index and thymus index, reduced the serum levels of alanine aminotransferase, aspartate aminotransferase and nitric oxide, and restored liver homogenate contents of tumor necrosis factor-␣ and interleukin-1. The histopathological analysis suggested that SMPS reduced the degree of liver injury. The results suggest that SMPS play a protective role against immunological liver injury, which may have important implications for our understanding on the immunoregulatory mechanisms of polysaccharides. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hepatic injury, with a high disease incidence, is a kind of a disease that is difficult to cure. The carrying rate of hepatitis B is about 10% in China and it is also a prevalent ailment worldwide. Severe liver injury leads to liver fibrosis, end stage cirrhosis and even liver cancer. This is a major public health problem, owing to lifethreatening complications of portal hypertension, liver failure and increased incidence of hepatocellular carcinoma. Viruses, toxins, alcohol, chemicals and other various adverse stimuli may trigger liver injury. Pathological courses of hepatic injury not only include direct effect of virus, but the immunologic disorders also play a key role. Botanical polysaccharides from a wide array of different species of flora, including higher plants, mushrooms, lichens and algae, are a class of macromolecules that can markedly enhance and activate immune responses, leading to immunomodulation, antitumor activity, wound-healing and other therapeutic effects [1]. For example, polysaccharides from a variety of traditional medicinal herbs have been shown to be immunomodulating both in vivo and in vitro [2–6]. Polysaccharides purified from certain mushrooms and lichens have anti-tumor activity via macrophage activation
∗ Corresponding author. Tel.: +86 20 61648241; fax: +86 20 87277771. E-mail address: lzping@fimmu.com (Z.-P. Lv). 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.04.012
[7–9]. Polysaccharides isolated from the algae exhibit anti-tumor and anti-metastatic activity [10,11]. S. miltiorrhiza belongs to the plant family oregano and is collected in spring and autumn. It was first indexed in the Shen Nong’s Materia Medica (206 b.c. to 8 a.d.) as a promoting blood flow–removing blood stasis and tonic herb of the nontoxic superior class, and has been used in traditional Chinese medicine (TCM) for more than 2000 years to prevent and treat various human diseases such as hepatitis, coronary artery disease, apoplexy, tumor growth and immunological disorders. According to ‘Fuzheng Guben’ and ‘Huoxue Huayu’, the major TCM therapeutic principles, S. miltiorrhiza is capable of strengthening body resistance and improving constitutive homeostasis. Various components of S. miltiorrhiza, including salviol, cryptotanshinone, tanshinol, protocatechualdehyde and salvianolic acid, were reported to exhibit diverse activity. Even though SMPS are major effective component of S. miltiorrhiza, the exact pharmacological effects of SMPS are still far from clear. Therefore, this study was designed to evaluate the therapeutic effects of SMPS on immunological liver injury in mice and we adopted the injection of Bacille–Calmette–Guerin (BCG) followed by lipopolysaccharide (LPS) as the experimental model. Since it was reported by Ferluga [12], it has been used as a classical experimental model for immunological liver injury. Many studies using this animal model have involved various factors, such as tumor necrosis factor-␣ (TNF-␣) [13–15], interleukin-1 (IL-1) [16,17] and nitric oxide (NO) [18] in the pathogenesis of the immunological hepatic failure, in which T cells play an important role [14,19,20]. So
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this paper explores the effects of SMPS on those factors in this model. 2. Materials and methods 2.1. Extraction of polysaccharides from S. miltiorrhiza Dried roots of S. miltiorrhiza were purchased in a local market. They were from Shandong province, a well-known production area for S. miltiorrhiza in China. S. miltiorrhiza roots were dried at 75 ◦ C and ground to fine powder. The ground powder samples were refluxed to remove lipids with acetoacetate: methanol solvent (1:1, v/v). After filtering, the residues were air-dried, and then refluxed again with 75% ethanol at 75 ◦ C to remove oligosaccharides. The residues were extracted three times in boiled water and filtered. The combined filtrates were concentrated by a rotvapor at 65 ◦ C, and then precipitated using 95% ethanol. After filtering and centrifuging, the precipitate was collected, vacuum-dried and represented a 3–3.5% yield of raw material dry weight. The obtained SMPS were stored in a refrigerator till further use. 2.2. Animals Male Kunming strain mice (18–22 g), 6–8 weeks old, were purchased from the Laboratory Animal Center of Southern Medical University. They were allowed food and water ad libitum. The mice were housed in plastic cages and maintained under standard conditions (12 h light/12h dark cycle; 25 ± 3 ◦ C; 35–60% relative humidity). 2.3. Reagents LPS was purchased from Sigma (St. Louis, MO, USA). BCG was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Commercial kits used for determining NO, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were obtained from the Jiancheng Institute of Biotechnology (Nanjing, China). Commercial kits used for determining TNF-␣ and IL-1 were obtained from R&D Systems (Minneapolis, MN, USA). Other chemicals used in these experiments of analytical grade were from commercial sources. 2.4. Experimental model and drug treatment Fifty Kunming strain mice were randomly divided into five groups (n = 10 in each group). A small dosage of LPS was injected into BCG-pretreated mice via the tail vein in order to induce immunological liver injury as previously reported [21]. Briefly, 0.2 ml of the BCG culture (approximately 5 × 107 viable units per mouse) was injected via the tail vein into mice, with the exception of the control group, who received saline alone. In the treatment groups, low-dose (90 mg/kg) of SMPS (SMPS-L), middle-dose (180 mg/kg) of SMPS (SMPS-M), and high-dose (360 mg/kg) of SMPS (SMPS-H) were given daily via gavage for a period of 12 days. The control group was given an equal volume of saline. Twelve days later, the mice were given 7.5 g of LPS in 0.2 ml saline (control received saline alone) via lateral tail vein injection. Ten hours after the injection of LPS, the mice were weighed and the blood was collected from mice orbit. Mice livers, spleens and thymus were immediately removed and weighed. Mice livers were partly fixed in neutralized formalin before being stained and analyzed for pathological changes. The remanent livers were stored at −70 ◦ C until required.
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2.5. Preparation of liver homogenates The frozen liver specimen were thawed at room temperature and washed with sterile isolating medium (0.25 mol/l sucrose, 1 mmol/l EDTA, 10 mmol/l Tris with pH buffered to 7.4, using HCl. Prior to use, the medium was sterilized at 120 ◦ C for 30 min). The liver was then weighed and homogenized with sterile PBS medium. The volumes of homogenates were adjusted to 10% (w/v) and centrifuged at 4000 rpm for 15 min. The clear supernatant was centrifuged again for 15 min at 4000 rpm. The sediment was discarded after each step. All steps were carried out at 4 ◦ C [22]. 2.6. Measurement of liver index, spleen index and thymus index, and serum levels of ALT and AST Liver index, spleen index and thymus index were calculated respectively as liver weight, spleen weight and thymus weight divided by 10 g body weight. Serum activity of ALT and AST were determined using commercial kits. Every step was operated according to the procedure. 2.7. Measurement of serum levels of NO and liver homogenate contents of TNF-˛ and IL-1ˇ Serum activity of NO was also determined using commercial kits. The liver homogenate contents of TNF-␣ and IL-1 were measured using commercially available ELISA kit. All samples and provided standards were analyzed in duplicate. A standard curve was constructed using standards provided in the kits, and the cytokine concentrations were determined from the standard curves using linear regression analysis. The lower limit of detection for each was as follows: 15.6 pg/ml for TNF-a and 7.8 pg/ml for IL1. 2.8. Histological examination The liver specimens were fixed with 10% neutral formalin and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed according to standard procedure. For histological evaluation of the liver injury, areas of necrotic lesions were microscopically evaluated using an MCID image analyzer. The degree of liver damage was categorized into four groups: Grade −, no necrosis; Grade +, necrotic area >0% and <2.5%; Grade ++, necrotic area >2.5% and <5%; and Grade +++, necrotic area >5% [21]. The slides were scored independently by three pathologists who had no prior knowledge of their source. Each sample was observed at 200 magnifications. The degree of liver damage was expressed as the mean of 10 different fields of view on each slide. 2.9. Statistical analysis All experiments described here have been repeated at three times. Quantitative data were expressed as mean ± S.D. and assessed by the one-way analysis of variance (ANOVA). If the variances between groups were homogenous (Levene’s test), groups were subjected to the multiple comparisons least significant differences (LSD) test. In case of no homogeneity variances, differences were evaluated by Kruskal–Wallis and the groups were subjected to the multiple comparison Dunnett’s T3 test. The Mann–Whitney rank-sum test was used for the degree of histopathological liver injury. The statistical significance between groups was indicated using superscript signs in the figures and tables. Significance was defined as P < 0.05.
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Table 1 Effects of SMPS on liver index, spleen index and thymus index in immunological liver injury mice Group
N
Liver indexa
Normal Model SMPS-H SMPS-M SMPS-L
10 10 10 10 10
442.67 709.70 605.60 653.91 673.10
± ± ± ± ±
40.94 52.82# 38.06** 60.44* 60.11
Spleen indexa 45.13 89.85 78.48 80.18 84.53
± ± ± ± ±
8.58 9.53# 5.87** 7.10* 8.85
3.3. Effects of SMPS on the contents of NO in serum and the contents of TNF-˛ and IL-1ˇ in liver homogenate of immunological liver injury in mice
Thymus indexa 29.97 17.69 24.34 22.20 20.39
± ± ± ± ±
7.71 5.68# 4.41* 5.83 8.19
# P < 0.01, compared with normal group. *P < 0.05, compared with model group. **P < 0.01, compared with model group. a Results were presented as mean ± S.D.
3. Results 3.1. Effects of SMPS on liver index, spleen index and thymus index in immunological liver injury mice Immunological liver injury was induced in the mice via lateral tail vein injection of LPS into BCG-pretreated mice. Table 1 showed that the elevated liver index and spleen index were markedly reduced by SMPS-H (P < 0.01) and SMPS-M (P < 0.05) treatment, and the reduction of thymus index was also significantly elevated by SMPS-H treatment (P < 0.05).
The effects of SMPS on BCG/LPS-induced production of NO, TNF␣ and IL-1 from serum and liver homogenate of immunological liver injury in mice were reported in Fig. 2D and E. Results showed that the level of NO was significantly decreased (P < 0.01) by SMPS treatment only at the high dose. TNF-␣ production was significantly inhibited by SMPS-H (P < 0.01) and SMPS-M (P < 0.05). Meanwhile IL-1 excretion was markedly decreased by SMPS treatment at three doses (P < 0.01). 3.4. Effects of SMPS on the pathological grading changes in immunological liver injury mice Significant histological differences were found in the experimental groups compared to the control group (Fig. 3). Liver histopathologic examination showed no histological abnormalities in normal mice (Fig. 3F). There were severe liver swelling, necrosis and monocyte infiltration after injecting LPS into the BCG-primed mice (Fig. 3G). The area, extent of necrosis and the infiltration of inflammatory cells were remarkably ameliorated by SMPS-H treatment (P < 0.05) (Fig. 3H, Table 2). 4. Discussion
3.2. Effects of SMPS on the serum activity of ALT and AST in immunological liver injury mice Immunological liver injury induced by BCG/LPS provoked a significant increment of ALT and AST contents (P < 0.01) in model group as compared to normal group (Fig. 1A and B). It indicated that we had successfully duplicated the immunological liver injury model in mice. Results showed that treatment with SMPS significantly restored ALT activity (P < 0.01) and reduced AST content (P < 0.01, P < 0.05).
The SMPS were tested for their capacities to lessen hepatic injury and to modulate immunological activity induced by LPS in mice primed with BCG. It is recognized that immune factors, such as autoimmune stimuli, virus or parasite infection, are the predominant reasons of hepatic damage especially under hepatitis [23]. To treat liver diseases, a hepatoprotective therapy is usually applied together with the anti-viral and the symptomatic therapies. Acute liver injuries induced chemically with CCl4 , d-galactosamine or thioacetamide, as well as the chronic models caused by the multi-
Fig. 1. Treatment with SMPS reduced immunological liver injury in mice. Serum levels of ALT (A) and AST (B) were analyzed as a measure of hepatocellular injury. Data represented mean ± S.D.; n = 10 mice per group. #P < 0.01, compared with normal group. *P < 0.05, compared with model group. **P < 0.01, compared with model group.
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Fig. 2. Effects of SMPS on BCG/LPS-induced production of NO, TNF-a and IL-1 from serum and liver homogenate. NO (C), TNF-a (D) and IL-1 (E) levels were implicated in immunological and inflammatory processes. Data represented mean ± S.D.; n = 10 mice per group. #P < 0.01, compared with normal group. *P < 0.05, compared with model group. **P < 0.01, compared with model group.
ple administrations of these chemicals, have been widely used for screening the hepatoprotective agents. However, these injuries are obviously caused and maintained by a foreign chemical but not by a host defense action seen in human hepatitis and thus are unsuitable for the evaluation of hepatic immunomodulating agents. Therefore, in this study, a BCG/LPS-induced immunological liver injury model was used to investigate the hepatoprotective effects of SMPS in mice. This liver injury model is similar to the pathophysiological course of human, so it is a suitable model to screen medicine of hepatic protection. Characteristics of impaired pathological histology are liver necrosis and a large number of monocyte infiltration [24]. The mechanism is that BCG activates T lymphocyte at first, and then especially activates Kupffer’s cells and macrophage, at last these assemble in the liver in a large amount. Macrophage is further activated after injecting LPS and releases a large number of cytotoxic factors including TNF-␣, IL-1, free radical, leukotriene and NO. NO as a free radical, reacts with O2− , and then turns into other free radicals which provoke the lipid peroxidation and cause the hepatocellular damage [24,13]. Results of our experimental studies have indicated a moderate protective effect of SMPS on serum transaminase levels and liver index in experimentally induced immunological liver injury in mice. Moreover, the degrees of histological changes of liver injury were also remarkably ameliorated after SMPS treatment indicating their positively hepatoprotective effects. Table 2 Effects of SMPS on the pathological grading changes in immunological liver injury mice Groupa
−b
+b
++b
+++b
Normal Model SMPS-H SMPS-M SMPS-L
10 0 0 0 0
0 0 3 2 1
0 4 5 4 4
0 6# 2* 4 5
#
P < 0.01, compared with normal group. *P < 0.05, compared with model group. a Each group consisted of 10 mice and figures represented number of mice per grade. b The signs −, +, ++, ++ + denoted the degree of severity of liver damage.
The actions of NO include vasodilation, inhibition of platelet such as aggregation and adhesion, cytotoxicity, cell-to-cell communication in the central nervous system and benefit to improving necrosis of the liver cells. Immunological liver injury is an important link of pathophysiological course in sepsis and systemic inflammatory response syndrome (SIRS). It causes a massive release of NO and inducible nitric oxide synthase (iNOS) expression in this course [25]. Immunological and inflammatory processes involve the production of NO induced by iNOS. Excess NO production from iNOS-mediated hepatotoxicity can be indirect via the activation of cytokines [18]. The generation of NO activated by Kupffer cells leads to production of TNF-␣, which is essential for the onset of liver injury [26]. The experimental result displayed the hepatic NO level of immunological liver injury in mice was markedly higher than that of normal mice. The elevated hepatic NO level was significantly down-regulated by the treatment with SMPS, which suggested that the hepatoprotective effect of SMPS may be related to the inhibition of NO release. Activated immune system has also been found to play an important role in the development of liver diseases [27]. In this regard, reports of abnormal behavior of various cytokines, in particular, TNF-a, released from inflammatory cells is considered to be essential for understanding in detail the mechanism of liver injury [28,29]. TNF-␣, which is thought to be a common early effector molecule in liver injury, is a pleiotropic pro-inflammatory cytokine produced chiefly by activated macrophages, and in smaller amounts by several other types of cells. It has been demonstrated that the course of viral hepatitis [30], alcoholic hepatitis [31], ischemia/reperfusion liver injury [32] and fulminant hepatic failure [28] implicate the activation of TNF-␣. A lot of pro-inflammatory mediators including NO, IL-1, IL-6, IL-8 and SIL-2R are stimulated by TNF-␣ which has direct cytotoxic effects [33–36]. Stimulation of these pro-inflammatory mediators is important for inflammation and consequent liver damage. IL-1 is an important cytokine, which is implicated in inflammation and other pathological processes such as rheumatoid arthritis. IL-1 is also an essential target as it leads to complex cascades along with other mediators such as TNF-␣ and NO leading severely inflammatory disease [37]. Even though IL-1 itself has little direct
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Fig. 3. Effects of SMPS administrations were displayed on histological image of liver injury induced by BCG/LPS. There was no histological abnormality in normal mice (F). The liver tissue of immunological liver injury in model mice showed serious degeneration of hepatocytes with marked swelling, necrosis and monocytes infiltration (G). The tissue primed with BCG was treated with SMPS-H followed by LPS challenge, the degree of tissue damage was significantly reduced. There was only swelling in liver tissue, necrosis and monocytes infiltration were hardly observed (H). The SMPS-M-administered group showed liver tissue swelling and focal necrosis (I). Swelling and lamellar necroses as well as monocytes infiltration were observed in SMPS-L group (J). The tissues were surgically excised and subjected to histological study by staining with HE. All the magnification was 200×.
damage on liver, its elevation could stimulate a great quantity of immunological and inflammatory cells to synthesize cytokines such as TNF-␣, IFN-␥, IL-6 and IL-8, which subsequently mediate the inflammatory and immunological injury [38]. Apart from these effects, IL-1 could additionally act on hepatocytes to increase the generation of NO and provoke the expression of iNOS mRNA, consequently resulting in further liver injury [39]. In our studies, the levels of liver homogenate contents of TNF-␣ and IL-1 were elevated, spleen index was significantly elevated and thymus index was markedly reduced in model group. It was also found that SMPS treatment significantly reduced the elevated levels of TNF-␣, IL1 and spleen index and enhanced the diminished thymus index. These present results not only showed the immunomodulatory function of SMPS, but also suggested an explanation for its mechanisms of hepatic protection in immunological liver injury induced by BCG/LPS. At present, specific drugs for the treatment of liver diseases are not available. A number of agents have been attempted in clinics to treat patients with liver injury. These include non-specific anti-inflammatory drugs, immunosuppressants such as glucocor-
ticoids and cyclophosphamide (either alone or in combination), levamisol and plant medicines [40]. However, such combination of drugs has proven limitations like poor solubility in water, administered intravenously and side effects. Therefore, the discovery and identification of new safe drugs, without severe side effects and conveniently to be administered, have become an important goal in the biomedical researches. In summary, these results suggest that SMPS protect against the immunological liver injury with high-dose and in the dose range of 90–360 mg/kg in a dose-dependent manner. The potential mechanisms of activity of SMPS might be related with its ability to reduce the generation of NO, TNF-␣ and IL-1 and to act as immunoregulator. Further study on the role of SMPS in immunomodulatory function is in progress in our laboratory. Acknowledgements We are grateful to Dr. Hui Li (Nanfang Hospital, Southern Medical University, Guangzhou, China) for excellent technical assistance and Professor Xiao Juan Li (School of Pharmacy, Southern Med-
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Protection of a polysaccharide from Salvia miltiorrhiza, a Chinese medicinal herb, against immunological liver injury in mice Yu-Hong Song a , Qiang Liu b , Zhi-Ping Lv b,∗ , Yu-Yao Chen b , Ying-Chun Zhou a , Xue-Gang Sun b a b
Department of Traditional Chinese Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 21 March 2008 Received in revised form 23 April 2008 Accepted 25 April 2008 Available online 13 May 2008 Keywords: Salvia miltiorrhiza Polysaccharides Immunological liver injury
a b s t r a c t This study was designed to evaluate the hepatoprotective effects of S. miltiorrhiza polysaccharides (SMPS) in immunological liver injury induced by Bacille–Calmette–Guerin (BCG) and lipopolysaccharide (LPS) in mice. SMPS effectively improved the liver index, spleen index and thymus index, reduced the serum levels of alanine aminotransferase, aspartate aminotransferase and nitric oxide, and restored liver homogenate contents of tumor necrosis factor-␣ and interleukin-1. The histopathological analysis suggested that SMPS reduced the degree of liver injury. The results suggest that SMPS play a protective role against immunological liver injury, which may have important implications for our understanding on the immunoregulatory mechanisms of polysaccharides. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hepatic injury, with a high disease incidence, is a kind of a disease that is difficult to cure. The carrying rate of hepatitis B is about 10% in China and it is also a prevalent ailment worldwide. Severe liver injury leads to liver fibrosis, end stage cirrhosis and even liver cancer. This is a major public health problem, owing to lifethreatening complications of portal hypertension, liver failure and increased incidence of hepatocellular carcinoma. Viruses, toxins, alcohol, chemicals and other various adverse stimuli may trigger liver injury. Pathological courses of hepatic injury not only include direct effect of virus, but the immunologic disorders also play a key role. Botanical polysaccharides from a wide array of different species of flora, including higher plants, mushrooms, lichens and algae, are a class of macromolecules that can markedly enhance and activate immune responses, leading to immunomodulation, antitumor activity, wound-healing and other therapeutic effects [1]. For example, polysaccharides from a variety of traditional medicinal herbs have been shown to be immunomodulating both in vivo and in vitro [2–6]. Polysaccharides purified from certain mushrooms and lichens have anti-tumor activity via macrophage activation
∗ Corresponding author. Tel.: +86 20 61648241; fax: +86 20 87277771. E-mail address: lzping@fimmu.com (Z.-P. Lv). 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.04.012
[7–9]. Polysaccharides isolated from the algae exhibit anti-tumor and anti-metastatic activity [10,11]. S. miltiorrhiza belongs to the plant family oregano and is collected in spring and autumn. It was first indexed in the Shen Nong’s Materia Medica (206 b.c. to 8 a.d.) as a promoting blood flow–removing blood stasis and tonic herb of the nontoxic superior class, and has been used in traditional Chinese medicine (TCM) for more than 2000 years to prevent and treat various human diseases such as hepatitis, coronary artery disease, apoplexy, tumor growth and immunological disorders. According to ‘Fuzheng Guben’ and ‘Huoxue Huayu’, the major TCM therapeutic principles, S. miltiorrhiza is capable of strengthening body resistance and improving constitutive homeostasis. Various components of S. miltiorrhiza, including salviol, cryptotanshinone, tanshinol, protocatechualdehyde and salvianolic acid, were reported to exhibit diverse activity. Even though SMPS are major effective component of S. miltiorrhiza, the exact pharmacological effects of SMPS are still far from clear. Therefore, this study was designed to evaluate the therapeutic effects of SMPS on immunological liver injury in mice and we adopted the injection of Bacille–Calmette–Guerin (BCG) followed by lipopolysaccharide (LPS) as the experimental model. Since it was reported by Ferluga [12], it has been used as a classical experimental model for immunological liver injury. Many studies using this animal model have involved various factors, such as tumor necrosis factor-␣ (TNF-␣) [13–15], interleukin-1 (IL-1) [16,17] and nitric oxide (NO) [18] in the pathogenesis of the immunological hepatic failure, in which T cells play an important role [14,19,20]. So
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this paper explores the effects of SMPS on those factors in this model. 2. Materials and methods 2.1. Extraction of polysaccharides from S. miltiorrhiza Dried roots of S. miltiorrhiza were purchased in a local market. They were from Shandong province, a well-known production area for S. miltiorrhiza in China. S. miltiorrhiza roots were dried at 75 ◦ C and ground to fine powder. The ground powder samples were refluxed to remove lipids with acetoacetate: methanol solvent (1:1, v/v). After filtering, the residues were air-dried, and then refluxed again with 75% ethanol at 75 ◦ C to remove oligosaccharides. The residues were extracted three times in boiled water and filtered. The combined filtrates were concentrated by a rotvapor at 65 ◦ C, and then precipitated using 95% ethanol. After filtering and centrifuging, the precipitate was collected, vacuum-dried and represented a 3–3.5% yield of raw material dry weight. The obtained SMPS were stored in a refrigerator till further use. 2.2. Animals Male Kunming strain mice (18–22 g), 6–8 weeks old, were purchased from the Laboratory Animal Center of Southern Medical University. They were allowed food and water ad libitum. The mice were housed in plastic cages and maintained under standard conditions (12 h light/12h dark cycle; 25 ± 3 ◦ C; 35–60% relative humidity). 2.3. Reagents LPS was purchased from Sigma (St. Louis, MO, USA). BCG was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Commercial kits used for determining NO, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were obtained from the Jiancheng Institute of Biotechnology (Nanjing, China). Commercial kits used for determining TNF-␣ and IL-1 were obtained from R&D Systems (Minneapolis, MN, USA). Other chemicals used in these experiments of analytical grade were from commercial sources. 2.4. Experimental model and drug treatment Fifty Kunming strain mice were randomly divided into five groups (n = 10 in each group). A small dosage of LPS was injected into BCG-pretreated mice via the tail vein in order to induce immunological liver injury as previously reported [21]. Briefly, 0.2 ml of the BCG culture (approximately 5 × 107 viable units per mouse) was injected via the tail vein into mice, with the exception of the control group, who received saline alone. In the treatment groups, low-dose (90 mg/kg) of SMPS (SMPS-L), middle-dose (180 mg/kg) of SMPS (SMPS-M), and high-dose (360 mg/kg) of SMPS (SMPS-H) were given daily via gavage for a period of 12 days. The control group was given an equal volume of saline. Twelve days later, the mice were given 7.5 g of LPS in 0.2 ml saline (control received saline alone) via lateral tail vein injection. Ten hours after the injection of LPS, the mice were weighed and the blood was collected from mice orbit. Mice livers, spleens and thymus were immediately removed and weighed. Mice livers were partly fixed in neutralized formalin before being stained and analyzed for pathological changes. The remanent livers were stored at −70 ◦ C until required.
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2.5. Preparation of liver homogenates The frozen liver specimen were thawed at room temperature and washed with sterile isolating medium (0.25 mol/l sucrose, 1 mmol/l EDTA, 10 mmol/l Tris with pH buffered to 7.4, using HCl. Prior to use, the medium was sterilized at 120 ◦ C for 30 min). The liver was then weighed and homogenized with sterile PBS medium. The volumes of homogenates were adjusted to 10% (w/v) and centrifuged at 4000 rpm for 15 min. The clear supernatant was centrifuged again for 15 min at 4000 rpm. The sediment was discarded after each step. All steps were carried out at 4 ◦ C [22]. 2.6. Measurement of liver index, spleen index and thymus index, and serum levels of ALT and AST Liver index, spleen index and thymus index were calculated respectively as liver weight, spleen weight and thymus weight divided by 10 g body weight. Serum activity of ALT and AST were determined using commercial kits. Every step was operated according to the procedure. 2.7. Measurement of serum levels of NO and liver homogenate contents of TNF-˛ and IL-1ˇ Serum activity of NO was also determined using commercial kits. The liver homogenate contents of TNF-␣ and IL-1 were measured using commercially available ELISA kit. All samples and provided standards were analyzed in duplicate. A standard curve was constructed using standards provided in the kits, and the cytokine concentrations were determined from the standard curves using linear regression analysis. The lower limit of detection for each was as follows: 15.6 pg/ml for TNF-a and 7.8 pg/ml for IL1. 2.8. Histological examination The liver specimens were fixed with 10% neutral formalin and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed according to standard procedure. For histological evaluation of the liver injury, areas of necrotic lesions were microscopically evaluated using an MCID image analyzer. The degree of liver damage was categorized into four groups: Grade −, no necrosis; Grade +, necrotic area >0% and <2.5%; Grade ++, necrotic area >2.5% and <5%; and Grade +++, necrotic area >5% [21]. The slides were scored independently by three pathologists who had no prior knowledge of their source. Each sample was observed at 200 magnifications. The degree of liver damage was expressed as the mean of 10 different fields of view on each slide. 2.9. Statistical analysis All experiments described here have been repeated at three times. Quantitative data were expressed as mean ± S.D. and assessed by the one-way analysis of variance (ANOVA). If the variances between groups were homogenous (Levene’s test), groups were subjected to the multiple comparisons least significant differences (LSD) test. In case of no homogeneity variances, differences were evaluated by Kruskal–Wallis and the groups were subjected to the multiple comparison Dunnett’s T3 test. The Mann–Whitney rank-sum test was used for the degree of histopathological liver injury. The statistical significance between groups was indicated using superscript signs in the figures and tables. Significance was defined as P < 0.05.
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Table 1 Effects of SMPS on liver index, spleen index and thymus index in immunological liver injury mice Group
N
Liver indexa
Normal Model SMPS-H SMPS-M SMPS-L
10 10 10 10 10
442.67 709.70 605.60 653.91 673.10
± ± ± ± ±
40.94 52.82# 38.06** 60.44* 60.11
Spleen indexa 45.13 89.85 78.48 80.18 84.53
± ± ± ± ±
8.58 9.53# 5.87** 7.10* 8.85
3.3. Effects of SMPS on the contents of NO in serum and the contents of TNF-˛ and IL-1ˇ in liver homogenate of immunological liver injury in mice
Thymus indexa 29.97 17.69 24.34 22.20 20.39
± ± ± ± ±
7.71 5.68# 4.41* 5.83 8.19
# P < 0.01, compared with normal group. *P < 0.05, compared with model group. **P < 0.01, compared with model group. a Results were presented as mean ± S.D.
3. Results 3.1. Effects of SMPS on liver index, spleen index and thymus index in immunological liver injury mice Immunological liver injury was induced in the mice via lateral tail vein injection of LPS into BCG-pretreated mice. Table 1 showed that the elevated liver index and spleen index were markedly reduced by SMPS-H (P < 0.01) and SMPS-M (P < 0.05) treatment, and the reduction of thymus index was also significantly elevated by SMPS-H treatment (P < 0.05).
The effects of SMPS on BCG/LPS-induced production of NO, TNF␣ and IL-1 from serum and liver homogenate of immunological liver injury in mice were reported in Fig. 2D and E. Results showed that the level of NO was significantly decreased (P < 0.01) by SMPS treatment only at the high dose. TNF-␣ production was significantly inhibited by SMPS-H (P < 0.01) and SMPS-M (P < 0.05). Meanwhile IL-1 excretion was markedly decreased by SMPS treatment at three doses (P < 0.01). 3.4. Effects of SMPS on the pathological grading changes in immunological liver injury mice Significant histological differences were found in the experimental groups compared to the control group (Fig. 3). Liver histopathologic examination showed no histological abnormalities in normal mice (Fig. 3F). There were severe liver swelling, necrosis and monocyte infiltration after injecting LPS into the BCG-primed mice (Fig. 3G). The area, extent of necrosis and the infiltration of inflammatory cells were remarkably ameliorated by SMPS-H treatment (P < 0.05) (Fig. 3H, Table 2). 4. Discussion
3.2. Effects of SMPS on the serum activity of ALT and AST in immunological liver injury mice Immunological liver injury induced by BCG/LPS provoked a significant increment of ALT and AST contents (P < 0.01) in model group as compared to normal group (Fig. 1A and B). It indicated that we had successfully duplicated the immunological liver injury model in mice. Results showed that treatment with SMPS significantly restored ALT activity (P < 0.01) and reduced AST content (P < 0.01, P < 0.05).
The SMPS were tested for their capacities to lessen hepatic injury and to modulate immunological activity induced by LPS in mice primed with BCG. It is recognized that immune factors, such as autoimmune stimuli, virus or parasite infection, are the predominant reasons of hepatic damage especially under hepatitis [23]. To treat liver diseases, a hepatoprotective therapy is usually applied together with the anti-viral and the symptomatic therapies. Acute liver injuries induced chemically with CCl4 , d-galactosamine or thioacetamide, as well as the chronic models caused by the multi-
Fig. 1. Treatment with SMPS reduced immunological liver injury in mice. Serum levels of ALT (A) and AST (B) were analyzed as a measure of hepatocellular injury. Data represented mean ± S.D.; n = 10 mice per group. #P < 0.01, compared with normal group. *P < 0.05, compared with model group. **P < 0.01, compared with model group.
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Fig. 2. Effects of SMPS on BCG/LPS-induced production of NO, TNF-a and IL-1 from serum and liver homogenate. NO (C), TNF-a (D) and IL-1 (E) levels were implicated in immunological and inflammatory processes. Data represented mean ± S.D.; n = 10 mice per group. #P < 0.01, compared with normal group. *P < 0.05, compared with model group. **P < 0.01, compared with model group.
ple administrations of these chemicals, have been widely used for screening the hepatoprotective agents. However, these injuries are obviously caused and maintained by a foreign chemical but not by a host defense action seen in human hepatitis and thus are unsuitable for the evaluation of hepatic immunomodulating agents. Therefore, in this study, a BCG/LPS-induced immunological liver injury model was used to investigate the hepatoprotective effects of SMPS in mice. This liver injury model is similar to the pathophysiological course of human, so it is a suitable model to screen medicine of hepatic protection. Characteristics of impaired pathological histology are liver necrosis and a large number of monocyte infiltration [24]. The mechanism is that BCG activates T lymphocyte at first, and then especially activates Kupffer’s cells and macrophage, at last these assemble in the liver in a large amount. Macrophage is further activated after injecting LPS and releases a large number of cytotoxic factors including TNF-␣, IL-1, free radical, leukotriene and NO. NO as a free radical, reacts with O2− , and then turns into other free radicals which provoke the lipid peroxidation and cause the hepatocellular damage [24,13]. Results of our experimental studies have indicated a moderate protective effect of SMPS on serum transaminase levels and liver index in experimentally induced immunological liver injury in mice. Moreover, the degrees of histological changes of liver injury were also remarkably ameliorated after SMPS treatment indicating their positively hepatoprotective effects. Table 2 Effects of SMPS on the pathological grading changes in immunological liver injury mice Groupa
−b
+b
++b
+++b
Normal Model SMPS-H SMPS-M SMPS-L
10 0 0 0 0
0 0 3 2 1
0 4 5 4 4
0 6# 2* 4 5
#
P < 0.01, compared with normal group. *P < 0.05, compared with model group. a Each group consisted of 10 mice and figures represented number of mice per grade. b The signs −, +, ++, ++ + denoted the degree of severity of liver damage.
The actions of NO include vasodilation, inhibition of platelet such as aggregation and adhesion, cytotoxicity, cell-to-cell communication in the central nervous system and benefit to improving necrosis of the liver cells. Immunological liver injury is an important link of pathophysiological course in sepsis and systemic inflammatory response syndrome (SIRS). It causes a massive release of NO and inducible nitric oxide synthase (iNOS) expression in this course [25]. Immunological and inflammatory processes involve the production of NO induced by iNOS. Excess NO production from iNOS-mediated hepatotoxicity can be indirect via the activation of cytokines [18]. The generation of NO activated by Kupffer cells leads to production of TNF-␣, which is essential for the onset of liver injury [26]. The experimental result displayed the hepatic NO level of immunological liver injury in mice was markedly higher than that of normal mice. The elevated hepatic NO level was significantly down-regulated by the treatment with SMPS, which suggested that the hepatoprotective effect of SMPS may be related to the inhibition of NO release. Activated immune system has also been found to play an important role in the development of liver diseases [27]. In this regard, reports of abnormal behavior of various cytokines, in particular, TNF-a, released from inflammatory cells is considered to be essential for understanding in detail the mechanism of liver injury [28,29]. TNF-␣, which is thought to be a common early effector molecule in liver injury, is a pleiotropic pro-inflammatory cytokine produced chiefly by activated macrophages, and in smaller amounts by several other types of cells. It has been demonstrated that the course of viral hepatitis [30], alcoholic hepatitis [31], ischemia/reperfusion liver injury [32] and fulminant hepatic failure [28] implicate the activation of TNF-␣. A lot of pro-inflammatory mediators including NO, IL-1, IL-6, IL-8 and SIL-2R are stimulated by TNF-␣ which has direct cytotoxic effects [33–36]. Stimulation of these pro-inflammatory mediators is important for inflammation and consequent liver damage. IL-1 is an important cytokine, which is implicated in inflammation and other pathological processes such as rheumatoid arthritis. IL-1 is also an essential target as it leads to complex cascades along with other mediators such as TNF-␣ and NO leading severely inflammatory disease [37]. Even though IL-1 itself has little direct
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Fig. 3. Effects of SMPS administrations were displayed on histological image of liver injury induced by BCG/LPS. There was no histological abnormality in normal mice (F). The liver tissue of immunological liver injury in model mice showed serious degeneration of hepatocytes with marked swelling, necrosis and monocytes infiltration (G). The tissue primed with BCG was treated with SMPS-H followed by LPS challenge, the degree of tissue damage was significantly reduced. There was only swelling in liver tissue, necrosis and monocytes infiltration were hardly observed (H). The SMPS-M-administered group showed liver tissue swelling and focal necrosis (I). Swelling and lamellar necroses as well as monocytes infiltration were observed in SMPS-L group (J). The tissues were surgically excised and subjected to histological study by staining with HE. All the magnification was 200×.
damage on liver, its elevation could stimulate a great quantity of immunological and inflammatory cells to synthesize cytokines such as TNF-␣, IFN-␥, IL-6 and IL-8, which subsequently mediate the inflammatory and immunological injury [38]. Apart from these effects, IL-1 could additionally act on hepatocytes to increase the generation of NO and provoke the expression of iNOS mRNA, consequently resulting in further liver injury [39]. In our studies, the levels of liver homogenate contents of TNF-␣ and IL-1 were elevated, spleen index was significantly elevated and thymus index was markedly reduced in model group. It was also found that SMPS treatment significantly reduced the elevated levels of TNF-␣, IL1 and spleen index and enhanced the diminished thymus index. These present results not only showed the immunomodulatory function of SMPS, but also suggested an explanation for its mechanisms of hepatic protection in immunological liver injury induced by BCG/LPS. At present, specific drugs for the treatment of liver diseases are not available. A number of agents have been attempted in clinics to treat patients with liver injury. These include non-specific anti-inflammatory drugs, immunosuppressants such as glucocor-
ticoids and cyclophosphamide (either alone or in combination), levamisol and plant medicines [40]. However, such combination of drugs has proven limitations like poor solubility in water, administered intravenously and side effects. Therefore, the discovery and identification of new safe drugs, without severe side effects and conveniently to be administered, have become an important goal in the biomedical researches. In summary, these results suggest that SMPS protect against the immunological liver injury with high-dose and in the dose range of 90–360 mg/kg in a dose-dependent manner. The potential mechanisms of activity of SMPS might be related with its ability to reduce the generation of NO, TNF-␣ and IL-1 and to act as immunoregulator. Further study on the role of SMPS in immunomodulatory function is in progress in our laboratory. Acknowledgements We are grateful to Dr. Hui Li (Nanfang Hospital, Southern Medical University, Guangzhou, China) for excellent technical assistance and Professor Xiao Juan Li (School of Pharmacy, Southern Med-
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