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Effect of silibinin and vitamin E on restoration of cellular immune response after partial hepatectomy
Journal of Ethnopharmacology 77 (2001) 227– 232 www.elsevier.com/locate/jethpharm
Effect of silibinin and vitamin E on restoration of cellular immune response after partial hepatectomy ´ . Horva´th a,*, R. Gonza´lez-Cabello b, Anna Bla´zovics a, Monika E Marco van der Looij c, Ire´n Barta a, Gyo¨rgyi Mu¨zes a, Pe´ter Gergely a, Ja´nos Fehe´r a a
Second Department of Medicine, Semmelweis Uni6ersity, Szentkira´lyi str. 46, H-1088 Budapest, Hungary b National Institute of Dermatology and Venerology, Budapest, Hungary c Department of Molecular Biology, National Institute of Oncology, Budapest, Hungary Received 5 March 2001; received in revised form 12 June 2001; accepted 26 June 2001
Abstract Our aim was to study the antioxidant and immunomodulatory effect of silibinin and vitamin E on the early postoperative course in rats that had undergone a partial hepatectomy (PHX). Male Wistar rats that were treated with silibinin (50 mg/b.w.kg i.p.) and/or vitamin E (500 mg/b.w.kg p.o.) were randomised to undergo 70% PHX. At 72 h after operation, Concanavalin A (Con-A) induced lymphocyte proliferation, and lipopolysaccharide (LPS) induced interleukin-1 (IL-1) mitogenicity and tumour necrosis factor-alpha (TNF-a) cytotoxicity were measured in the spleen. In addition, total free radical scavenger capacity of the liver was analysed. In PHX animals, Con-A induced lymphocyte proliferation was significantly decreased, and both LPS induced IL-1 and TNF-a activity were significantly increased as compared to Sham treated animals. Treatment with silibinin and vitamin E synergistically restored both lymphocyte proliferation (P B 0.01) and cytokine activity (PB 0.001) in PHX animals. In addition, silibinin and vitamin E synergistically (P B0.001) restored total hepatic free radical scavenger capacity as well as serum levels of AST and gGT, that were all markedly decreased in PHX animals. Our results suggest that preoperative treatment with silibinin and/or vitamin E modulates the cellular immunoresponse and restores impaired liver function following PHX, presumably through their antioxidant capacity. This may explain their beneficial effects on the postoperative course of liver repair. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; Silibinin; Partial hepatectomy; Free radical; Immune response
1. Introduction During liver regeneration quiescent hepatocytes undergo one or two rounds of replication and than return to a non-proliferative state (Fausto et al., 1996). It is proposed that the primary response after partial hepatectomy (PHX) involves priming of hepatocytes via the activation of transcription factors such as NF-kB. This process is triggered by many different stimuli including cytokines TNF-a and IL-1, and reactive oxidants (Liou and Baltimore, 1993; Anderson et al., 1994; Fausto et al., 1996). Major hepatectomy (70%) is accompanied by impaired liver function as a result of oxidative damage (Anderson et al., 1994) and a change in the host * Corresponding author. Tel.: +36-1-224-8600/1319; fax: +36-1224-8708. E-mail address: [email protected] (M.E´. Horva´th).
immune system (Hirano et al., 1993; Sato et al., 1999). Host injury due to liver resection promotes the release of liver tissue antigens, thereby altering both intrahepatic and peripheral leukocyte subsets and their sensibilisation level for cytokine production (Sato et al., 1999). Moreover, partial resection of the hepatic phagocytic system, namely the Kupffer cells, results in a reduced clearance of circulating potentially pathogenic particles (Vo and Chi, 1988), due to an impaired production of pro-inflammatory cytokines, of which IL-1 and TNF-a are the most proximal mediators (Luster et al., 1994). It is conceivable that the postoperative course of liver regeneration is determined by a balance between all pro-inflammatory mediators released after PHX including those with potential hepatotrophic properties, and their counter regulatory responses. The hepatoprotective effects of silibinin and vitamin E on hepatic lipid oxidative damage following PHX are
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well known and are partially attributed to their ability to scavenge oxygen free radicals (Fehe´ r et al., 1996; Dehmlow et al., 1996a). In addition, both silibinin and vitamin E appear to have immunoprotective and immunostimulatory functions (Bendich, 1990, 1993; Fehe´ r et al., 1996; Dehmlow et al., 1996b). Moreover, treatment with silibinin, promoted regeneration and by that the mitotic activity of hepatocytes in the regenerating liver tissue significantly increased (Sonnenbichler and Zetl, 1984). In the present study we investigated, whether pre-operative treatment with silibinin and/or vitamin E exerted a modulatory effect on the early cellular immune response during liver regeneration after PHX and whether this effect was mediated through their antioxidant properties.
2. Materials and methods
2.1. Partial hepatectomy Adult male albino Wistar rats weighing 150– 250 g were used in all experiments. Animals were fed standard laboratory food (Charles River, Hungary). 70% partial hepatectomy (lob. sin. lat., lob. sin. med., lob. dext. med.) was accomplished under ether anaesthesia as described previously (Higgins and Anderson, 1931). Rats were treated with silibinin and/or vitamin E, 5 days before operation and on each day after operation until termination of the experiment. Silibinin treatment consisted of intraperitoneal injection of 50 mg/b.w.kg. Vitamin E was supplemented in pellets containing 500 mg/b.w.kg. Rats were sacrificed by cervical dislocation on day 3 after operation.
2.2. Chemiluminometria Free radical formation was measured in rat liver homogenate by chemiluminometry using a CLD-1 Medicor-Medilab luminometer (Hungary; Bla´ zovics et al., 1992). Briefly, 200 ml reagent solution (0.7 mM luminol, 3.8 mM hemin) was added to 1 ml 25 w/v% tissue homogenate. The intensity of luminescence light, emitted on interaction with free radicals, was expressed in mV.
2.3. Serum parameters To assess the extent of liver damage, serum levels of aspartate-aminotransferase (AST) and gamma-glutamyl-transpeptidase (gGT) were measured using a Hitachi 717 Analyser (Fehe´ r et al., 1996).
2.4. Lymphocyte proliferation assay Lymphocytes were obtained by rat spleen perfusion and suspended in RPMI 1640 tissue culture medium (Gibco, USA) supplemented with 10% heat inactivated fetal calf serum (Phylaxia, Hungary), 25 mM HEPES buffer (Serva, GFR), 2 nM L-glutamine (Gibco, USA), 100 IU/ml penicillin, 100 ug/ml gentamicin and 7.5 mg/ml amphotericin B. 4 ×105 spleen cells in 200 ml medium were placed in each flat-bottomed microplate wells (Greiner, GFR) using four parallel samples. ConA (Pharmacia, Sweden) was added to the cells in doses of 0, 1, 5 and 10 ml/ml. Plates were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 72 h. Cultures were pulsed with 0.4 mCi [3H]-thymidine (UVVR, Czechoslovakia) 24 h before termination. Cells were harvested onto filter paper discs by an automated sample harvester (Skatron, Norway). Isotope determinations were made in a liquid scintillation counter (Nuclear Chicago Isocap 300, USA). Results were expressed in counts per minute (cpm) using the arithmetic mean of four replicate values.
2.5. Triggering IL-1 and TNF-h release Monocytes were isolated from a splenocyte suspension fraction and incubated (1.5× 106 cells/ml) in the presence or absence of 20 mg/ml lipopolysaccharide (LPS from E. coli 0111:B4, Sigma, USA). After 24 h, cell-free test supernatant was harvested by centrifugation and sterilised using 0.22 mm Millipore membrane filter (Mu¨ zes et al., 1989a).
2.6. IL-1 assay IL-1 activity was assayed in test supernatant as previously described (Mu¨ zes et al., 1989a). Briefly, thymocytes from 5 to 7 week-old C3H/HeJ mice were prepared as single cell suspensions and adjusted to a density of 1.5× 106 cells/well. Cells were cultured for 48 h at 37 °C in 96 well flat-bottomed microtitre plates in the presence of 1 mg/ml Con-A and test supernatant. After 48 h, the cultures were pulsed with [3H]thymidine. Thymidine incorporation was determined by a liquid scintillation counter. Results were expressed in cpm using the arithmetic mean of quadruplicate cultures.
2.7. TNF-h assay TNF-a cytotoxicity was measured by the ability of test supernatant to cause detachment from a monolayer of [3H]-thymidine labelled HEp-2 target cells (Mu¨ zes et al., 1989b). Briefly, HEp-2 target cells were cultured in Eagle’s MEM containing 10% heat-inactivated foetal calf serum, 25 mmol/l HEPES, 2 mmol/l L-glutamine
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Fig. 1. Effect of silibinin and vitamin E treatment on the natural H2O2/ OH scavenger capacity of the liver measured by chemoluminescence at 72 h after PHX. Values are expressed in mean 9SEM (N= 10). Treatment versus PHX group: *P B0.01; ** PB 0.001.
and antibiotics. After discarding detached dead cells with the supernatant medium HEp-2 cells were resuspended with 0.5 ml of 0.1% trypsin (Sigma, USA) in TC 199 medium, then washed twice. Resuspended HEp-2 target cells were placed in 96-well flat bottom microtitre plates (2.5×103 cells/well), then labelled with 0.4 mCi/well of [3H]-thymidine. After 24 h test supernatant was added to each well. The medium containing detached Hep-2 cells were removed from each well and the remaining adherent cells were frozen at − 20 °C. After thawing, the content of wells was sucked off onto filter mats with a cell harvester. Results from six replicate values were expressed as mean cpm. Cytotoxic values are expressed in mean% label measured in detached cells as percentage of total label (attached and detached cells).
Plasma AST and gGT levels were significantly (PB 0.001) increased in PHX operated as compared to Sham operated animals (Fig. 2A and 2B). In both silibinin and vitamin E treated PHX animals, AST and
2.8. Statistical analysis All results are expressed as mean9SEM and statistical significance was assessed by the Student’s t-test. Confidence limits were added at P B0.05. 3. Results Total free radical level in rat liver tissue, as measured by chemiluminometry, increased approximately twofold in PHX operated as compared to Sham operated animals (Fig. 1). In PHX animals treated with silibinin or vitamin E free radical level was significantly (P B0.001) reduced as compared to control PHX animals. There was a strong synergistic effect of combined silibinin/vitamin E treatment as free radical level decreased to less than half Sham level (P B 0.001).
Fig. 2. Effect of silibinin and vitamin E treatment on serum gGT (A) and AST (B) levels measured at 72 h after PHX. Values are expressed in mean 9SEM (N =10). Treatment versus PHX group: *PB 0.01; **PB0.001; NS =not significant.
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Fig. 3. The effect of silibinin and vitamin E treatment on Con-A (5 mg/ml) induced blast transformation of spleen cells measured at 72 h after PHX. Mitogenic effect was assessed by [3H]-thymidine incorporation and expressed as mean cpm 9 SEM (N= 10). Treatment versus PHX group: *P B0.01; **PB0.001.
gGT levels were lower as compared to untreated PHX animals, whereas combined silibinin/vitamin E treatment synergistically restored PHX plasma levels of both liver enzymes to approximately Sham level (P B 0.001). Concanavalin A (0, 1, 5 and 10 mg/ml) induced immune reactivity of rat splenic lymphocytes was analysed in a pilot experiment at time of operation and at 72 h and 3 weeks after operation. Lymphocyte proliferation activity was significantly reduced at 72 h after PHX at all Con-A concentrations and restored to control PHX level at 3 weeks after operation (data not shown). The most effective Con-A concentration at 72 h, was 5 mg/ml (PB 0.001). Therefore, 72 h time interval and 5 mg/ml Con-A concentration were used in subsequent experiments. In both silibinin and vitamin E treated PHX animals Con-A (5 mg/ml) induced spleen lymphocyte proliferation was significantly (P B 0.001) elevated as compared to untreated PHX animals (Fig. 3). There was also a significant (P B0.01) effect of combined silibinin/vitamin E treatment. Changes in splenocyte IL-1 activity and TNF-a cytotoxicity, as measured in supernatants of LPS stimulated splenocyte cultures, was assessed in pilot experiments at 0 h, 72 h and 3 weeks after PHX. PHX significantly increased LPS stimulated IL-1 (P B0.01) and TNF-a (PB 0.05) activity of spenocyte cultures as compared to Sham operated animals, with highest levels at 72 h after operation (data not shown). At this timepoint, vitamin E significantly (PB 0.001) decreased LPS stimulated splenocyte IL-1 mitogen activity as compared to untreated PHX animals (Fig. 4). Silibinin treatment had no significant effect. Therefore, reduction of IL-1 mitogen activity to Sham level in animals receiving combined silibinin/vitamin E treatment (P B0.01) was due to vitamin E alone. In contrast, TNF-a cytotoxicity was
Fig. 4. Effect of silibinin and vitamin E on IL-1 mitogen activity of LPS (20 mg/ml) treated splenocyte cultures prepared from rats at 72 h after PHX (N =10). IL-1 activity was assessed by the ability of culture supernatants to induce [3H]-thymidine incorporation of cultured C3H/HeJ mice target thymocytes. Values are expressed as mean cpm 9SEM (N =10). Treatment versus PHX group: **PB 0.01; ***PB0.001; NS = not significant.
significantly (PB 0.05) decreased by silibinin as compared to untreated PHX animals (Fig. 5), whereas vitamin E treatment had no significant effect. However, combined silibinin/vitamin E treatment was most effective, reducing TNF-a cytotoxicity to below Sham level (P B0.001). 4. Discussion Partial hepatectomy (PHX) in rats has been shown to induce not only proliferation of hepatocytes, but also of lymphoid tissues (Lai et al., 1996). Although it is not clear what mechanism terminates this suddenly initiated process, the restoration of post-operative impairment of immunocompetence is thought to play a role. We have tested this hypothesis and found evidence for the in-
Fig. 5. Effect of silibinin and vitamin E on TNF-a cytotoxicity of LPS (20 mg/ml) treated splenocyte cultures prepared from rats at 72 h after PHX (N =10). TNF-a cytotoxicity was measured by the ability of culture supernatants to induce detachment of cultured [3H]thymidine labeled HEp-2 target cells. Values are expressed in mean%9 SEM (N =10) label measured in detached cells as percentage of total label (attached and detached Hep-2 cells). Treatment versus PHX group: *PB 0.05; **PB0.01; NS =not significant.
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volvement of the cellular peripheral immune system in the early post-operative course of liver regeneration as measured at 72 h after PHX, presumably through interaction with reactive oxygen intermediates (ROIs). As a consequence of massive loss and damage of liver tissue, we could demonstrate a marked decrease of total hepatic natural free radical scavenger capacity. In addition, high concentrations of AST and gGT were measured in the systemic circulation as a result of acute destruction of hepatocytes. Treatment by silibinin and vitamin E restored the levels of these indicators of liver damage, confirming that ROIs are involved. We demonstrated that PHX caused a change in the systemic cellular immune response as assessed in the spleen. First, mitogen induced splenic lymphocyte proliferation markedly decreased following PHX. Both silibinin and vitamin E, and most importantly their combination, were very effective in restoring the lymphocyte proliferation rate, presumably through their antioxidant capacity. It is known that ROIs are associated with damage of cells of the immune system (Deshago et al., 1981; Erickson et al., 1983; Grunder et al., 1986). Antioxidants, by inactivating ROIs, protect membrane lipids, receptors and other cellular components of the immune system from oxidation (Bendich, 1990; Fehe´ r et al., 1996; Bla´ zovics et al., 1992). However, Oonishi et al. (1995) found that macrophages play an important role in the proliferation of splenic lympocytes following in vitro incubation with vitamin E, suggesting that vitamin E may also act as a direct immunomodulator. Second, endotoxin induced splenocyte IL-1 and TNF-a production significantly increased following PHX. IL-1 production could be restored by silibinin whereas TNF-a production could be restored by vitamin E. Especially the combined treatment was very effective. It is difficult to assess whether these changes in immunoreactivity are a direct consequence of, or an early response to PHX. Several cytokines shown to be present in normal spleen increased in the early phase after PHX (Hirano et al., 1993; Sato et al., 1999) and extrahepatic cytokines IL-1 and TNF-a were previously shown to modulate liver repair following hepatotoxic damage or regeneration after PHX (Akerman et al., 1992). The notion that both cytokines exert pleiotropic roles in the modulation of liver regeneration is further supported by a number of other reports (Akerman et al., 1992; Beyer and Theologides, 1993; Straatsburg et al., 1996; Boulton et al., 1997; Kimura et al., 1997). Our results suggest that alterations in the systemic cellular immune responses are related to liver injury after PHX due to a decrease in the natural hepatic ROI scavenger defence. Whether these are two independent phenomena or one follows the other remains to be investigated.
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5. Conclusion We conclude that changes in splenocyte responsiveness to mitogen stimulation are a prominent feature of the post-operative impairment of immunocompetence and that these changes are related to free radical formation during PHX. The beneficial synergistic antioxidant and immunomodulatory effects of silibin and vitamin E may have future therapeutic consequences in the pretreatment of liver resection, presumably by decreasing the dysfunction period of remnant regenerating liver.
Acknowledgements This work was supported by the Ministery of Welfare ETT 02-517 and by the Ph.D. program of Semmelweis University, Budapest.
References Akerman, P., Cote, P., Yang, S.Q., McLain, C., Nelson, S., Bagby, G.J., et al., 1992. Antibodies to tumour necrosis factor alpha inhibit liver regeneration after partial hepatectomy. American Journal of Physiology. 263, G579 – G585. Anderson, M.T., Staal, F.J., Gitler, C., Herzenberg, L.A., Herzenberg, L.A., 1994. Separation of oxidant-initiated and redox-regulated steps in the NF-kB signal transduction pathway. Proceedings of the National Academy of Sciences USA 91, 11527 – 11531. Bendich, A., 1990. Antioxidant micronutrients and immune responses. Annals of the New York Academy of Science. 587, 168 – 180. Bendich, A., 1993. Physiological role of antioxidants in the immune system. Journal of Dairy Sciences. 76, 2789 – 2794. Beyer, H.S., Theologides, A., 1993. Tumour necrosis factor alpha is a direct hepatocyte mitogen in rat. Biochemical and Molecular Biology International. 29, 1 – 4. Bla´ zovics, A., Fehe´ r, E., Fehe´ r, J., 1992. Role of free radical reactions in experimental hyperlipidemia in the pathomechanism of fatty liver. In: Csomo´ s, G., Fehe´ r, J. (Eds.), Free Radicals and Liver. Springer, Berlin, pp. 98 –126. Boulton, R., Woodman, A., Calnan, D., Selden, C., Tam, F., Hodgson, H., 1997. Non-parenchymal cells from regenerating rat liver generate interleukin-1alpha and -1beta: a mechanism of negative regulation of hepatocyte proliferation. Hepatology 26, 49 –58. Dehmlow, C., Murawski, N., de Groot, H., 1996a. Scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism by silibinin in human cell. Life Sciences 58, 1591 – 1600. Dehmlow, C., Erhard, J., de Groot, H., 1996b. Inhibition of Kupffer cell function as an explanation for the hepatoprotective properties of silibinin. Hepatology 23, 749 – 754. Deshago, R.D., Ewel, C., London, O.S., Metzger, Z., Hoffeld, J.T., Oppenheim, J.J., 1981. Evidence for the involvement of monocyte-derived toxic oxygen metabolites in the lymphocyte dysfunction of Hodgkin‘s disease. Clinical and Experimental Immunology 46, 313 –317. Erickson, K.L., Adams, D.A., McNeill, C.J., 1983. Dietary lipid modulation of immune responsiveness. Lipids 18, 468 – 474.
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Fausto, N., Laird, A.D., Webber, M., 1996. Role of growth factors and cytokines in hepatic regeneration. FASEB 10, 413 –427. Fehe´ r, J., Bla´ zovics, A., Gonza´ lez-Cabello, R., Horva´ th, M.E., Gergely, P., 1996. Effect of silibinin and vitamin E on lipid peroxidation and cellular immunreactivity in experimentaly induced fatty liver of rats. Medical Science Monitor 2, 397 – 403. Grunder, S., Volk, H., Falck, P., Von Baehr, R., 1986. The influence of phagocytic stimuli on the expression of HLA-DR antigens; role of reactive oxygen intermediates. European Journal of Immunology 6, 212 – 217. Higgins, G.M., Anderson, R.M., 1931. Experimental pathology of the liver. 1. Restoration of the liver of the white rat following surgical removal. Archives of Pathology 12, 1986 –2002. Hirano, F., Sakamoto, T., Mabuchi, A., Norose, Y., Yokomuro, K., 1993. Modification of T-lymphocytes subsets in peripheral blood, the liver, and the spleen during liver regeneration after partial hepatectomy of mice. Regulatory Immunology 5, 293 – 298. Kimura, T., Sakaida, I., Terai, S., Matsumura, Y., Uchida, K., Okita, K., 1997. Inhibition of tumour necrosis factor-alpha production retards liver regeneration after partial hepatectomy in rats. Biochemical and Biophysical Research Communication 231, 557 – 560. Lai, H.S., Chen, W.J., Chen, K.M., 1996. Changes in the Tlymphocyte subpopulations and serum lymphokine concentrations after partial hepatectomy in rats. Nutrition 12, 700 – 705. Liou, H.-C., Baltimore, D., 1993. Regulation of the NF-kB/Rel transcription factor and IkB inhibitor system. Current Opinion in Cell Biology 5, 477 – 487. Luster, M.I., Germolec, D.R., Yoshida, T., Kayama, F., Thompson, M., 1994. Endotoxin-induced cytokine gene expression and excre-
tion in the liver. Hepatology 19, 480 – 488. Mu¨ zes, Gy., Dea´ k, Gy., La´ ng, I., Gonza´ lez-Cabello, R., Gergely, P., Fehe´ r, J., 1989a. Depressed monocyte production of interleukin-1 and tumour necrosis factor alpha in patients with alcoholic liver cirrhosis. Liver 9, 302 – 306. Mu¨ zes, Gy., Vien, C.V., Gonza´ lez-Cabello, R., Gergely, P., Fehe´ r, J., 1989b. A simple assay for tumour necrosis factor using Hep-2 target cells. Journal of Clinical Laboratory Immunology 30, 41 – 44. Oonishi, K., Moriguchi, S., Kishino, Y., 1995. The role of macrophages in increased mitogen response of rat splenic lymphocytes following in vitro incubation with vitamin E. Journal of Nutritional Science and Vitaminology, Tokyo 41, 445 –453. Sato, Y., Farges, O., Buffello, D., Bismuth, H., 1999. Intra- and extrahepatic leukocytes and cytokine mRNA expression during liver regeneration after partial hepatectomy in rats. Digestive Disease and Science 44, 806 – 816. Sonnenbichler, J., Zetl, I., 1984. Mechanism of action of silibinin. Effect of silibinin on the synthesis of ribosomal RNA, mRNA and tRNA in rat livers in vivo. Hoppe-Seyler’s Zeitschrift fu¨ r Physiologische Chemie 365, 555 – 566. Straatsburg, I.H., Boermeester, M.A., Houdijk, A.P., Frederiks, W.M., Wesdorp, R.I., Van Leeuwen, P.A., et al., 1996. Endotoxin and cytokine-mediated effects on liver cell proliferation and lipid metabolism after partial hepatectomy: a study with recombinant N-terminal bactericidal/permeability-increasing protein and interleukin-1 receptor antagonist. Journal of Pathology 179, 100 –105. Vo, N.M., Chi, D.S., 1988. Effect of hepatectomy on the reticuloendothelial system of septic rats. Journal of Trauma 28, 852 –854.
Effect of silibinin and vitamin E on restoration of cellular immune response after partial hepatectomy ´ . Horva´th a,*, R. Gonza´lez-Cabello b, Anna Bla´zovics a, Monika E Marco van der Looij c, Ire´n Barta a, Gyo¨rgyi Mu¨zes a, Pe´ter Gergely a, Ja´nos Fehe´r a a
Second Department of Medicine, Semmelweis Uni6ersity, Szentkira´lyi str. 46, H-1088 Budapest, Hungary b National Institute of Dermatology and Venerology, Budapest, Hungary c Department of Molecular Biology, National Institute of Oncology, Budapest, Hungary Received 5 March 2001; received in revised form 12 June 2001; accepted 26 June 2001
Abstract Our aim was to study the antioxidant and immunomodulatory effect of silibinin and vitamin E on the early postoperative course in rats that had undergone a partial hepatectomy (PHX). Male Wistar rats that were treated with silibinin (50 mg/b.w.kg i.p.) and/or vitamin E (500 mg/b.w.kg p.o.) were randomised to undergo 70% PHX. At 72 h after operation, Concanavalin A (Con-A) induced lymphocyte proliferation, and lipopolysaccharide (LPS) induced interleukin-1 (IL-1) mitogenicity and tumour necrosis factor-alpha (TNF-a) cytotoxicity were measured in the spleen. In addition, total free radical scavenger capacity of the liver was analysed. In PHX animals, Con-A induced lymphocyte proliferation was significantly decreased, and both LPS induced IL-1 and TNF-a activity were significantly increased as compared to Sham treated animals. Treatment with silibinin and vitamin E synergistically restored both lymphocyte proliferation (P B 0.01) and cytokine activity (PB 0.001) in PHX animals. In addition, silibinin and vitamin E synergistically (P B0.001) restored total hepatic free radical scavenger capacity as well as serum levels of AST and gGT, that were all markedly decreased in PHX animals. Our results suggest that preoperative treatment with silibinin and/or vitamin E modulates the cellular immunoresponse and restores impaired liver function following PHX, presumably through their antioxidant capacity. This may explain their beneficial effects on the postoperative course of liver repair. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; Silibinin; Partial hepatectomy; Free radical; Immune response
1. Introduction During liver regeneration quiescent hepatocytes undergo one or two rounds of replication and than return to a non-proliferative state (Fausto et al., 1996). It is proposed that the primary response after partial hepatectomy (PHX) involves priming of hepatocytes via the activation of transcription factors such as NF-kB. This process is triggered by many different stimuli including cytokines TNF-a and IL-1, and reactive oxidants (Liou and Baltimore, 1993; Anderson et al., 1994; Fausto et al., 1996). Major hepatectomy (70%) is accompanied by impaired liver function as a result of oxidative damage (Anderson et al., 1994) and a change in the host * Corresponding author. Tel.: +36-1-224-8600/1319; fax: +36-1224-8708. E-mail address: [email protected] (M.E´. Horva´th).
immune system (Hirano et al., 1993; Sato et al., 1999). Host injury due to liver resection promotes the release of liver tissue antigens, thereby altering both intrahepatic and peripheral leukocyte subsets and their sensibilisation level for cytokine production (Sato et al., 1999). Moreover, partial resection of the hepatic phagocytic system, namely the Kupffer cells, results in a reduced clearance of circulating potentially pathogenic particles (Vo and Chi, 1988), due to an impaired production of pro-inflammatory cytokines, of which IL-1 and TNF-a are the most proximal mediators (Luster et al., 1994). It is conceivable that the postoperative course of liver regeneration is determined by a balance between all pro-inflammatory mediators released after PHX including those with potential hepatotrophic properties, and their counter regulatory responses. The hepatoprotective effects of silibinin and vitamin E on hepatic lipid oxidative damage following PHX are
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 2 9 8 - 7
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well known and are partially attributed to their ability to scavenge oxygen free radicals (Fehe´ r et al., 1996; Dehmlow et al., 1996a). In addition, both silibinin and vitamin E appear to have immunoprotective and immunostimulatory functions (Bendich, 1990, 1993; Fehe´ r et al., 1996; Dehmlow et al., 1996b). Moreover, treatment with silibinin, promoted regeneration and by that the mitotic activity of hepatocytes in the regenerating liver tissue significantly increased (Sonnenbichler and Zetl, 1984). In the present study we investigated, whether pre-operative treatment with silibinin and/or vitamin E exerted a modulatory effect on the early cellular immune response during liver regeneration after PHX and whether this effect was mediated through their antioxidant properties.
2. Materials and methods
2.1. Partial hepatectomy Adult male albino Wistar rats weighing 150– 250 g were used in all experiments. Animals were fed standard laboratory food (Charles River, Hungary). 70% partial hepatectomy (lob. sin. lat., lob. sin. med., lob. dext. med.) was accomplished under ether anaesthesia as described previously (Higgins and Anderson, 1931). Rats were treated with silibinin and/or vitamin E, 5 days before operation and on each day after operation until termination of the experiment. Silibinin treatment consisted of intraperitoneal injection of 50 mg/b.w.kg. Vitamin E was supplemented in pellets containing 500 mg/b.w.kg. Rats were sacrificed by cervical dislocation on day 3 after operation.
2.2. Chemiluminometria Free radical formation was measured in rat liver homogenate by chemiluminometry using a CLD-1 Medicor-Medilab luminometer (Hungary; Bla´ zovics et al., 1992). Briefly, 200 ml reagent solution (0.7 mM luminol, 3.8 mM hemin) was added to 1 ml 25 w/v% tissue homogenate. The intensity of luminescence light, emitted on interaction with free radicals, was expressed in mV.
2.3. Serum parameters To assess the extent of liver damage, serum levels of aspartate-aminotransferase (AST) and gamma-glutamyl-transpeptidase (gGT) were measured using a Hitachi 717 Analyser (Fehe´ r et al., 1996).
2.4. Lymphocyte proliferation assay Lymphocytes were obtained by rat spleen perfusion and suspended in RPMI 1640 tissue culture medium (Gibco, USA) supplemented with 10% heat inactivated fetal calf serum (Phylaxia, Hungary), 25 mM HEPES buffer (Serva, GFR), 2 nM L-glutamine (Gibco, USA), 100 IU/ml penicillin, 100 ug/ml gentamicin and 7.5 mg/ml amphotericin B. 4 ×105 spleen cells in 200 ml medium were placed in each flat-bottomed microplate wells (Greiner, GFR) using four parallel samples. ConA (Pharmacia, Sweden) was added to the cells in doses of 0, 1, 5 and 10 ml/ml. Plates were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 72 h. Cultures were pulsed with 0.4 mCi [3H]-thymidine (UVVR, Czechoslovakia) 24 h before termination. Cells were harvested onto filter paper discs by an automated sample harvester (Skatron, Norway). Isotope determinations were made in a liquid scintillation counter (Nuclear Chicago Isocap 300, USA). Results were expressed in counts per minute (cpm) using the arithmetic mean of four replicate values.
2.5. Triggering IL-1 and TNF-h release Monocytes were isolated from a splenocyte suspension fraction and incubated (1.5× 106 cells/ml) in the presence or absence of 20 mg/ml lipopolysaccharide (LPS from E. coli 0111:B4, Sigma, USA). After 24 h, cell-free test supernatant was harvested by centrifugation and sterilised using 0.22 mm Millipore membrane filter (Mu¨ zes et al., 1989a).
2.6. IL-1 assay IL-1 activity was assayed in test supernatant as previously described (Mu¨ zes et al., 1989a). Briefly, thymocytes from 5 to 7 week-old C3H/HeJ mice were prepared as single cell suspensions and adjusted to a density of 1.5× 106 cells/well. Cells were cultured for 48 h at 37 °C in 96 well flat-bottomed microtitre plates in the presence of 1 mg/ml Con-A and test supernatant. After 48 h, the cultures were pulsed with [3H]thymidine. Thymidine incorporation was determined by a liquid scintillation counter. Results were expressed in cpm using the arithmetic mean of quadruplicate cultures.
2.7. TNF-h assay TNF-a cytotoxicity was measured by the ability of test supernatant to cause detachment from a monolayer of [3H]-thymidine labelled HEp-2 target cells (Mu¨ zes et al., 1989b). Briefly, HEp-2 target cells were cultured in Eagle’s MEM containing 10% heat-inactivated foetal calf serum, 25 mmol/l HEPES, 2 mmol/l L-glutamine
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Fig. 1. Effect of silibinin and vitamin E treatment on the natural H2O2/ OH scavenger capacity of the liver measured by chemoluminescence at 72 h after PHX. Values are expressed in mean 9SEM (N= 10). Treatment versus PHX group: *P B0.01; ** PB 0.001.
and antibiotics. After discarding detached dead cells with the supernatant medium HEp-2 cells were resuspended with 0.5 ml of 0.1% trypsin (Sigma, USA) in TC 199 medium, then washed twice. Resuspended HEp-2 target cells were placed in 96-well flat bottom microtitre plates (2.5×103 cells/well), then labelled with 0.4 mCi/well of [3H]-thymidine. After 24 h test supernatant was added to each well. The medium containing detached Hep-2 cells were removed from each well and the remaining adherent cells were frozen at − 20 °C. After thawing, the content of wells was sucked off onto filter mats with a cell harvester. Results from six replicate values were expressed as mean cpm. Cytotoxic values are expressed in mean% label measured in detached cells as percentage of total label (attached and detached cells).
Plasma AST and gGT levels were significantly (PB 0.001) increased in PHX operated as compared to Sham operated animals (Fig. 2A and 2B). In both silibinin and vitamin E treated PHX animals, AST and
2.8. Statistical analysis All results are expressed as mean9SEM and statistical significance was assessed by the Student’s t-test. Confidence limits were added at P B0.05. 3. Results Total free radical level in rat liver tissue, as measured by chemiluminometry, increased approximately twofold in PHX operated as compared to Sham operated animals (Fig. 1). In PHX animals treated with silibinin or vitamin E free radical level was significantly (P B0.001) reduced as compared to control PHX animals. There was a strong synergistic effect of combined silibinin/vitamin E treatment as free radical level decreased to less than half Sham level (P B 0.001).
Fig. 2. Effect of silibinin and vitamin E treatment on serum gGT (A) and AST (B) levels measured at 72 h after PHX. Values are expressed in mean 9SEM (N =10). Treatment versus PHX group: *PB 0.01; **PB0.001; NS =not significant.
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Fig. 3. The effect of silibinin and vitamin E treatment on Con-A (5 mg/ml) induced blast transformation of spleen cells measured at 72 h after PHX. Mitogenic effect was assessed by [3H]-thymidine incorporation and expressed as mean cpm 9 SEM (N= 10). Treatment versus PHX group: *P B0.01; **PB0.001.
gGT levels were lower as compared to untreated PHX animals, whereas combined silibinin/vitamin E treatment synergistically restored PHX plasma levels of both liver enzymes to approximately Sham level (P B 0.001). Concanavalin A (0, 1, 5 and 10 mg/ml) induced immune reactivity of rat splenic lymphocytes was analysed in a pilot experiment at time of operation and at 72 h and 3 weeks after operation. Lymphocyte proliferation activity was significantly reduced at 72 h after PHX at all Con-A concentrations and restored to control PHX level at 3 weeks after operation (data not shown). The most effective Con-A concentration at 72 h, was 5 mg/ml (PB 0.001). Therefore, 72 h time interval and 5 mg/ml Con-A concentration were used in subsequent experiments. In both silibinin and vitamin E treated PHX animals Con-A (5 mg/ml) induced spleen lymphocyte proliferation was significantly (P B 0.001) elevated as compared to untreated PHX animals (Fig. 3). There was also a significant (P B0.01) effect of combined silibinin/vitamin E treatment. Changes in splenocyte IL-1 activity and TNF-a cytotoxicity, as measured in supernatants of LPS stimulated splenocyte cultures, was assessed in pilot experiments at 0 h, 72 h and 3 weeks after PHX. PHX significantly increased LPS stimulated IL-1 (P B0.01) and TNF-a (PB 0.05) activity of spenocyte cultures as compared to Sham operated animals, with highest levels at 72 h after operation (data not shown). At this timepoint, vitamin E significantly (PB 0.001) decreased LPS stimulated splenocyte IL-1 mitogen activity as compared to untreated PHX animals (Fig. 4). Silibinin treatment had no significant effect. Therefore, reduction of IL-1 mitogen activity to Sham level in animals receiving combined silibinin/vitamin E treatment (P B0.01) was due to vitamin E alone. In contrast, TNF-a cytotoxicity was
Fig. 4. Effect of silibinin and vitamin E on IL-1 mitogen activity of LPS (20 mg/ml) treated splenocyte cultures prepared from rats at 72 h after PHX (N =10). IL-1 activity was assessed by the ability of culture supernatants to induce [3H]-thymidine incorporation of cultured C3H/HeJ mice target thymocytes. Values are expressed as mean cpm 9SEM (N =10). Treatment versus PHX group: **PB 0.01; ***PB0.001; NS = not significant.
significantly (PB 0.05) decreased by silibinin as compared to untreated PHX animals (Fig. 5), whereas vitamin E treatment had no significant effect. However, combined silibinin/vitamin E treatment was most effective, reducing TNF-a cytotoxicity to below Sham level (P B0.001). 4. Discussion Partial hepatectomy (PHX) in rats has been shown to induce not only proliferation of hepatocytes, but also of lymphoid tissues (Lai et al., 1996). Although it is not clear what mechanism terminates this suddenly initiated process, the restoration of post-operative impairment of immunocompetence is thought to play a role. We have tested this hypothesis and found evidence for the in-
Fig. 5. Effect of silibinin and vitamin E on TNF-a cytotoxicity of LPS (20 mg/ml) treated splenocyte cultures prepared from rats at 72 h after PHX (N =10). TNF-a cytotoxicity was measured by the ability of culture supernatants to induce detachment of cultured [3H]thymidine labeled HEp-2 target cells. Values are expressed in mean%9 SEM (N =10) label measured in detached cells as percentage of total label (attached and detached Hep-2 cells). Treatment versus PHX group: *PB 0.05; **PB0.01; NS =not significant.
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volvement of the cellular peripheral immune system in the early post-operative course of liver regeneration as measured at 72 h after PHX, presumably through interaction with reactive oxygen intermediates (ROIs). As a consequence of massive loss and damage of liver tissue, we could demonstrate a marked decrease of total hepatic natural free radical scavenger capacity. In addition, high concentrations of AST and gGT were measured in the systemic circulation as a result of acute destruction of hepatocytes. Treatment by silibinin and vitamin E restored the levels of these indicators of liver damage, confirming that ROIs are involved. We demonstrated that PHX caused a change in the systemic cellular immune response as assessed in the spleen. First, mitogen induced splenic lymphocyte proliferation markedly decreased following PHX. Both silibinin and vitamin E, and most importantly their combination, were very effective in restoring the lymphocyte proliferation rate, presumably through their antioxidant capacity. It is known that ROIs are associated with damage of cells of the immune system (Deshago et al., 1981; Erickson et al., 1983; Grunder et al., 1986). Antioxidants, by inactivating ROIs, protect membrane lipids, receptors and other cellular components of the immune system from oxidation (Bendich, 1990; Fehe´ r et al., 1996; Bla´ zovics et al., 1992). However, Oonishi et al. (1995) found that macrophages play an important role in the proliferation of splenic lympocytes following in vitro incubation with vitamin E, suggesting that vitamin E may also act as a direct immunomodulator. Second, endotoxin induced splenocyte IL-1 and TNF-a production significantly increased following PHX. IL-1 production could be restored by silibinin whereas TNF-a production could be restored by vitamin E. Especially the combined treatment was very effective. It is difficult to assess whether these changes in immunoreactivity are a direct consequence of, or an early response to PHX. Several cytokines shown to be present in normal spleen increased in the early phase after PHX (Hirano et al., 1993; Sato et al., 1999) and extrahepatic cytokines IL-1 and TNF-a were previously shown to modulate liver repair following hepatotoxic damage or regeneration after PHX (Akerman et al., 1992). The notion that both cytokines exert pleiotropic roles in the modulation of liver regeneration is further supported by a number of other reports (Akerman et al., 1992; Beyer and Theologides, 1993; Straatsburg et al., 1996; Boulton et al., 1997; Kimura et al., 1997). Our results suggest that alterations in the systemic cellular immune responses are related to liver injury after PHX due to a decrease in the natural hepatic ROI scavenger defence. Whether these are two independent phenomena or one follows the other remains to be investigated.
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5. Conclusion We conclude that changes in splenocyte responsiveness to mitogen stimulation are a prominent feature of the post-operative impairment of immunocompetence and that these changes are related to free radical formation during PHX. The beneficial synergistic antioxidant and immunomodulatory effects of silibin and vitamin E may have future therapeutic consequences in the pretreatment of liver resection, presumably by decreasing the dysfunction period of remnant regenerating liver.
Acknowledgements This work was supported by the Ministery of Welfare ETT 02-517 and by the Ph.D. program of Semmelweis University, Budapest.
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