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The American Journal of Chinese Medicine, Vol. 42, No. 5, 1199–1213 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X1450075X
Antagonistic Interaction Between Cordyceps sinensis and Exercise on Protection in Fulminant Hepatic Failure Yu-Jung Cheng,* Woei-Cherng Shyu,‡ Yi-Hsien Teng,* Yu-Hsuan Lan† and Shin-Da Lee*,§ *Department
of Physical Therapy and Graduate Institute of Rehabilitation Science †
School of Pharmacy, China Medical University Taichung, Taiwan, ROC
‡
Center for Neuropsychiatry and Graduate Institute of Immunology China Medical University and Hospital, Taichung, Taiwan §Department
of Healthcare Administration Asia University, Taichung, Taiwan Published 17 September 2014
Abstract: Herb supplements are widely used by Asian athletes; however, there are no studies evaluated the co-effects of exercise and herb supplements on hepatic failure. In this study, DGalN/LPS-induced fulminant hepatic failure was used to examine whether there are synergistic or antagonistic effects of exercise and Cordyceps sinensis (CS). Mice were randomly divided into eight groups: control, swimming exercise for four weeks, D-GalN/LPS challenge, swimming exercise plus D-GalN/LPS, 20 mg/kg or 40 mg/kg CS pretreated for four weeks plus D-GalN/LPS, and swimming exercise combined with 20 mg/kg or 40 mg/kg CS pretreatment plus D-GalN/LPS. Either exercise or 40 mg/kg CS pretreatment alone significantly decreased D-GalN/LPS-induced TNF-, AST, NO, apoptotic-related proteins, and hepatocyte apoptosis. Exercise or 40 mg/kg CS alone increased the IL-10 and D-GalN/LPSsuppressed Superoxide Dismutase (SOD) level. However, no protective or worse effect was observed in the mice treated with exercise preconditioning combined 40 mg/kg CS compared to those receive exercise alone or CS alone. TNF-, AST, NO level, caspase-3 activity, and hepatocytes apoptosis were not significantly different in the exercise combined with 40 mg/ kg CS compared to mice challenged with D-GalN/LPS. The IL-10 level was significantly decreased after D-GalN/LPS stimulation in the mice received exercise combined with 40 mg/ kg CS, indicating the combination strongly reduced the anti-inflammatory effect. In summary, preconditioning exercise or CS pretreatment alone can protect mice from septic liver damage, but in contrast, the combination of exercise and CS does not produce any benefit. Correspondence to: Dr. Shin-Da Lee, Department of Physical Therapy and Graduate Institute of Rehabilitation Science, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402, ROC. Taichung, Tel: (þ886) 4-2205-3366 (ext. 7300), Fax: (þ886) 4-2206-5051, E-mail: [email protected]
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The antagonistic interactions between exercise and CS imply taking CS is not recommended for people who undertake regular exercise. Keywords: Apoptosis; Cordyceps sinensis; Inflammation; Lipopolysaccharide; Liver; Physical Activity.
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Introduction Herbal supplements have been widely used for centuries, particularly in Asia, and increasing numbers of Asian athletes are using herbal supplements to improve their performance (Chen et al., 2012). Cordyceps sinensis (CS) has been reported to promote exercise endurance capacity and performance (Chen et al., 2010; Kumar et al., 2011). In addition its beneficial effects on sports, CS have been used to treat inflammatory and autoimmune diseases (Won and Park, 2005; Kim et al., 2006). The anti-inflammatory effect of CS might result from the inhibition of TNF- (Cheng et al., 2014). Among 50 compounds found in CS, more than 10 compounds have anti-inflammatory effects (Yang et al., 2011). In general, regular exercise can enhance immunity and anti-oxidant function (Mackinnon, 1994; Ho et al., 2013), but acute and exhaustive exercise can reduce immune function, increase infection risk and increase immune cell apoptosis (Kakanis et al., 2010; Pereira et al., 2012). Chen et al. (2007) suggested that exercise training attenuates septic responses and protects organs from damage in sepsis. Exercise training is beneficial and counteracts cardiovascular abnormalities and pulmonary edema in septic animals (Mehanna et al., 2007). The mechanisms of exercise-associated immune changes are multifactorial and include multiple neuroendocrinological factors. Age, metabolism and nutrition contribute to exercise-associated changes in immune function (Pedersen et al., 1999). Because CS or exercise preconditioning can attenuate septic responses and protect organs from damage, the present study was designed to investigate whether exercise preconditioning combined with CS has a synergistic effect in attenuating hepatic injury in sepsis. Materials and Methods Chemicals LPS (derived from E. coli, serotype 055:B5) and D-GalN were obtained from Sigma Chemical (St. Louis, MO, USA). Water extracts of CS were purchased from TCM Biotech. International Corp., Taiwan. Treatments and Animals Male C57BL/6 mice weighing 20–25 g were obtained from the National Laboratory Animal Center (NLAC) and housed in the laboratory animal center at China Medical University. Animals were housed individually in a room with a 12-h light/dark cycle and
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central air-conditioning (25 C, 70% humidity), fed with standard food ad libitum, and cared for according to the appropriate nationally approved guidelines. Thirty-two mice were randomized into eight groups. For the CS treatment, mice were treated with saline or an extract of CS (20 mg/kg or 40 mg/kg) by oral gavage, five days per week, for a total of four weeks. For exercise preconditioning, 4 h after CS treatment, swimming exercise was performed. The exercise protocol consists of swimming exercise for 30 min/day, five days/ week for four weeks in 26–28 C water. For the preparation of mice with GalN/LPS-induced fulminant hepatic failure, mice were administered an intraperitoneal injection of GalN (800 mg/g body weight; Sigma-Aldrich, St. Louis, MO, USA). Three or six hours later, serum or liver tissues were collected for further analysis. Histology Liver tissue obtained from mice treated with GalN/LPS for 6 h was fixed in 3.7% buffered formalin and embedded in paraffin wax. Five-m sections were stained with hematoxylin and eosin Y (H&E stain). Photomicrographs were obtained using Zeiss Axiophot microscopes. A third pathologist examined the H&E-stained slides to verify the diagnosis. The degeneration/necrosis and hemorrhage status were scored. This scoring system provided a representation of the grade of degeneration/necrosis and hemorrhage from 1 to 5. Scores of 1 represent histological normality (non-degenerate), and scores of 5 represent severe degeneration. Blood Collection and Biochemistry Study The blood of mice was collected from the inferior vena cava under chloral hydrate anesthesia, drawn into serum separation tubes, allowed to clot for 10 min at room temperature, and then centrifuged (1000 g, 10 min, 4 C). Serum samples were stored at 70 C. To determine the serum concentrations of aspartate aminotransferase (AST), sera were spotted onto slides (Fuji Dri-Chem; Fujifilm, Kanagawa, Japan) and evaluated by a slide analyzer (Fuji Dri-Chem 3500S; Fujifilm). Enzyme-Linked Immunosorbent Assay (ELISAs) TNF- and IL-10 were measured using antibody enzyme-linked immunosorbent assays (ELISAs). The experiment was repeated with blood collected at 3 and 6 h for cytokine analysis by intracardiac puncture and immediately centrifuged for 10 min at 5000 g. The liver tissue was collected for analysis 6 h after GalN/LPS treatment. Liver tissue was homogenized and centrifuged for 20 min at 5000 g. The supernatant was stored at 80 C for the determination of IL-10 and TNF- concentration using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The optical density was measured at a wavelength of 450 nm.
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Measurement of Serum Nitrite Concentration
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The nitrite concentrations in sera were measured after the Griess reaction by incubating 20L samples with 20 l of Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) and 160 L of deionized water at room temperature for 20 min. The absorbance was measured at 550 nm. Nitrite concentration was calculated via comparison with a standard solution of known sodium nitrite concentration. Superoxide Dismutase (SOD) Assay The enzyme activity of SOD was measured using a kit from Cayman Chemical (Ann Arbor, MI, USA). The method utilizes tetrazolium salt to quantify superoxide radicals generated by xanthine oxidase and hypoxanthine. The standard curve was generated using a quality-controlled SOD standard, and optical density was measured at 450 nm. Western Blot Analysis Mice liver tissue was harvested 6 h after GalN/LPS treatment. The tissue was homogenized and solubilized with ice-cold buffer containing 25 mM Hepes (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM DTT, 100 g/ml PMSF, and 2 g/ml leupeptin. Approximately 40 g of protein was separated by electrophoresis in a 10% sodium-dodecyl-sulfate polyacrylamide gel. After electrophoresis, the protein was electro-transferred onto polyvinylidene fluoride membranes (Life Science, Boston, MA). Membranes were probed with antibodies specific for phosphor-ERK, ERK, phospho-p38, p38, cleaved caspase-3, cleaved caspase-6 or PARP (New England Biolabs). After probing with a horseradish peroxidase-conjugated secondary antibody, protein signals were visualized using enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL) and exposed on Kodak X-ML film. Terminal Deoxynucleotide Transferase-Mediated dUTP Nick End Labeling (TUNEL) Apoptosis in the liver was detected with an ApopTag Detection Kit (Millipore, Billerica, MA, USA). Paraffin wax slides of liver were deparaffinized and soaked to quench the endogenous peroxidase in a solution containing 3% H2O2. The slides were then rinsed with PBS once more. To add the residues of the digoxigenin-nucleotide to the 3 0 -OH end of the DNA catalytically, terminal deoxynucleotidyl transferase (TdT) with reaction buffer was used, and the specimens were mounted for 1 h. The reaction was terminated by applying a stop solution for 30 min at 37 C, and the specimens were washed with PBS. Before rinsing with PBS, the anti-digoxigenin-peroxidase solution was applied for 15 min to the sections to join the chromogenic substrates to the complex between the DNA and the digoxigenin-nucleotide. To detect positive staining, specimens were soaked in DAB and counterstained with hematoxylin.
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Statistical Analysis The data are presented as mean SEM. Statistical evaluation was performed with one-way ANOVA, two-way ANOVA and Tukey’s post hoc test. A value of p < 0:05 was considered to be statistically significant.
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Results Effects of CS and Exercise Preconditioning on Liver Damage in LPS/D-GalN-Treated Mice Light microscopy revealed no infiltration or sequestration of neutrophils in the liver of the control group, whereas more overt sequestration of neutrophils and coagulative necrosis tissues were observed in mice treated with LPS/D-GalN. The neutrophil infiltration and liver necrosis were significantly reduced by exercise preconditioning or 40 mg of CS. However, exercise preconditioning combined with 40 mg of CS significantly increased neutrophil infiltrations and liver necrosis compared to 40 mg of CS alone (Figs. 1A–1C). Similar results were found for AST levels. AST expression in sera was measured 6 h after the LPS/D-GalN challenge to examine the effect of CS and exercise preconditioning (Fig. 1D). The AST levels in LPS/D-GalN-treated groups were significantly increased compared with the control group and significantly decreased with 40 mg of CS pretreatment. Exercise preconditioning also strongly decreased AST levels compared with the LPS group; however, exercise preconditioning combined with 40 mg of CS significantly increased AST concentration after LPS/D-GalN stimulation compared with mice that received 40 mg of CS alone or exercise preconditioning alone. Effects of CS and Exercise Preconditioning on Serum and Tissue TNF- Concentration in LPS/D-GalN-treated Mice TNF- is the main mediator leading to hepatocyte apoptosis. We next examined the level of TNF- in serum and liver (Fig. 2A). The serum TNF- concentration 3 h after a LPS/DGalN challenge was significantly increased, and an increased dose of CS significantly decreased serum TNF- in the non-exercise group (all p < 0:05). Exercise preconditioning significantly reduced the LPS/D-GalN-induced TNF- concentration. In contrast, the combination of exercise and 40 mg of CS increased the TNF- concentration compared with exercise preconditioning alone or 40 mg of CS alone. Two-way ANOVA revealed that there were negative interactions between CS dose and exercise training (p < 0:05). The tissue TNF- level was determined 6 h after the LPS/D-GalN challenge. Similar results were found in tissue. Exercise and CS alone can reduce TNF-, but the combined treatment had antagonistic effects (Fig. 2B).
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(D) Figure 1. Effects of CS pretreatment and exercise preconditioning on liver damage in LPS/D-GalN-treated mice. Mice were orally pretreated with 20 mg or 40 mg of CS combined with, or without, swimming exercise (30 min/ day) for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/D-GalN. Mice were sacrificed after 6 h, and histological examination of liver tissue was performed with H&E staining (A). The scores for degeneration/necrosis (B) and hemorrhage (C) were also measured. Serum AST (D) was evaluated 6 h after LPS/D-GalN treatment. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
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Figure 2. Effects of CS pretreatment and exercise preconditioning on serum and tissue TNF- and IL-10 concentration in LPS/D-GalN-treated mice. Either 20 or 40 mg of CS was orally administered in combination with, or without, swimming exercise (30 min/day) to mice daily for four weeks. After injection with LPS/D-GalN, mice were sacrificed after 6 h, and serum TNF- (A), liver tissue TNF- (B), serum IL-10 (C), and liver tissue IL-10 (D) were evaluated by ELISA. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
Effects of CS and Exercise Preconditioning on Serum and Tissue IL-10 Concentration in LPS/D-GalN-Treated Mice IL-10, which is an anti-inflammatory cytokine, was further examined. After a 6-h challenge of LPS/D-GalN, the IL-10 level was determined both in serum and liver tissue by ELISA (Figs. 2C and 2D). Serum and tissue IL-10 concentrations were significantly increased after the LPS/D-GalN challenge, and 40 mg of CS significantly increased serum and tissue IL-10. Exercise preconditioning also significantly increased serum IL-10 after the LPS/D-GalN challenge. However, exercise preconditioning combined with 40 mg of CS decreased serum LPS/D-GalN-induced IL-10 concentration, compared with 40 mg of CS alone.
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(C) Figure 3. Effects of CS pretreatment and exercise preconditioning on NO production and SOD levels in LPS/DGalN-treated mice. The mice were orally pretreated with 20 mg or 40 mg of CS daily in combination with, or without, swimming exercise (30 min/day) for four weeks. After injection with LPS/D-GalN, the mice were sacrificed after 6 h, and their serum nitrite (A), liver tissue nitrite (B), and liver tissue SOD (C) were evaluated. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
Effects of CS and Exercise Preconditioning on Serum and Tissue NO Production in LPS/D-GalN-Treated Mice To examine the effect of CS and exercise preconditioning on NO production, nitrite concentration in serum and liver tissue was measured using the Griess Reaction (Figs. 3A and 3B) after 3 h. Serum and tissue nitrite concentrations 3 h after the LPS/D-GalN challenge were significantly increased, and an increased dose of CS significantly decreased serum and tissue nitrite in the non-exercise group. The administration of 40 mg of CS strongly decreased LPS/D-GalN-induced nitrite elevation. Exercise preconditioning also decreased serum and tissue nitrite concentration, but exercise preconditioning combined with 40 mg of CS increased nitrite concentration after LPS/D-GalN stimulation compared to 40 mg of CS without exercise. Similar to previous results, exercise preconditioning combined with 40 mg of CS does not display any benefit compared with exercise or CS alone.
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Effects of CS and Exercise Preconditioning on SOD Levels in LPS/D-GalN-Treated Mice SOD is responsible for the quenching of superoxide radicals. We next examined the effect of CS and exercise preconditioning on SOD production (Fig. 3C). The SOD concentration after the LPS/D-GalN challenge was significantly decreased, and when mice were treated with CS, they displayed significantly increased SOD levels in a dose-dependent manner. Exercise preconditioning also significantly increased SOD concentrations after the LPS/DGalN challenge, but combined exercise with 40 mg of CS did not have beneficial effects.
Effects of CS and Exercise Preconditioning on Caspase-3/6 Activity in LPS/D-GalN-Treated Mice We further measured whether CS and exercise preconditioning might alter the activity of the apoptotic pathways. The cleaved caspase-3, cleaved caspase-6 and cleaved-PARP were detected by western blotting to determine caspase pathway activation. Liver tissue was harvested, and the homogenate was analyzed after 6 h of LPS/D-GalN infusion. As shown in Fig. 4, cleaved caspase-3, caspase-6, and PARP were markedly increased after the LPS/D-GalN challenge. Either exercise preconditioning alone or 40 mg of CS pretreatment alone significantly decreased LPS/D-GalN-induced caspase-3, caspase-6, and PARP levels. The combination of exercise preconditioning and 40 mg of CS attenuated the anti-apoptotic effect of exercise preconditioning alone or 40 mg of CS pretreatment alone. Effects of CS and Exercise Preconditioning on LPS/D-GalN-Induced Hepatic Apoptosis To examine the effect of CS and exercise preconditioning on LPS/D-GalN-induced hepatic apoptosis, liver tissues were processed for TUNEL staining. Administration of LPS/DGalN induced apoptosis of hepatocytes (Fig. 5A). TUNEL staining indicated significant hepatic apoptosis 6 h after LPS/D-GalN-treatment (control, 0:23 0:016; LPS, 6:7 0:5; p < 0:05; Fig. 5B). Pretreatment with CS dose-dependently attenuated the percentage of LPS/D-GalN-induced TUNEL-positive staining (LPS, 6:7 0:5; 20 mg of CS þ LPS, 4:9 0:48; 40 mg of CSþLPS, 0:9 0:24; p < 0:05). In addition, exercise preconditioning reduced LPS/D-GalN-stimulated apoptosis (LPS, 6:7 0:5; Ex, 4:8 0:4; p < 0:05). However, the combination of exercise preconditioning and CS did not prevent apoptosis on LPS/D-GalN-treated mice compared with exercise alone or CS alone (40 mg of CS þ LPS, 0:9 0:24; Ex þ 40 mg of CS þ LPS, 7:6 0:3; p < 0:05). Effects of CS and Exercise Preconditioning on p38 MAPK Protein Phosphorylation in LPS/D-GalN-Treated Mice The effect of CS and exercise preconditioning on the activation of the p38 pathway was measured by western blotting (Fig. 6). Compared with the control and LPS/D-GalN
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(D) Figure 4. Effects of CS pretreatment and exercise preconditioning on caspase-3/6 activity in LPS/D-GalN-treated mice. Either 20 or 40 mg of CS was orally administered in combination with, or without, swimming exercise (30 min/day) to mice daily for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/D-GalN. Mice were sacrificed after 6 h and tissue cleaved caspase-3, caspase-6, and PARP were evaluated through western blotting (A). The quantification of related cleaved caspase-3 (B), cleaved caspase-6 (C) and cleaved PARP are shown. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
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(B) Figure 5. Effects of CS pretreatment and exercise preconditioning on LPS/D-GalN-induced hepatic apoptosis. Mice were orally pretreated with 20 mg or 40 mg of CS daily in combination with, or without, swimming exercise (30 min/day) for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/DGalN. Mice were sacrificed after 6 h, and cell apoptosis was evaluated by TUNEL staining (A). The quantification of TUNEL þ cell number is shown (B). Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were statistically significant ( p < 0:05).
challenged groups, 40 mg of CS significantly decreased LPS/D-GalN-induced p38 protein phosphorylation. Exercise preconditioning alone did not change the level of phosphor-p38; however, the combination of exercise and CS induced a 2-fold increase in p38 phosphorylation, compared with CS or exercise preconditioning alone. Discussion The influence of physical exercise on pharmacokinetics has been well investigated in many aspects (van Baak, 1990; Ylitalo, 1991; Khazaeinia et al., 2000). Some studies indicate physical exercise impairs the absorption or bioactivity of drugs (Arends et al., 1986; Carter et al., 1992; Stromberg et al., 1992; Chien et al., 2012). Although many studies indicate that herbal supplements have an ergogenic effect on physical performance (Chen et al., 2012), there is no evidence to indicate that physical exercise might affect the bioactivity of herbal supplements. Consistent with the findings for some drugs, four weeks of swimming
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(B) Figure 6. Effects of CS pretreatment and exercise preconditioning on p38 MAPK protein phosphorylation in LPS/ D-GalN-treated mice. Mice were orally pretreated with 20 mg or 40 mg of CS daily in combination with, or without, swimming exercise (30 min/day) for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/D-GalN. Mice were sacrificed after 6 h, and tissue phosphor-p38 MAPK and p38 MAPK were evaluated by western blotting (A). The quantification of the related phosphor-p38 MAPK is shown (B). Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using oneway ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
training reduced the protective effects of CS on D-GalN/LPS-induced hepatic failure, as indicated by AST level and morphology changes. An increased number of apoptotic cells were also detected in the livers of the B6 mice pretreated with the combination of exercise and 40 mg of CS. Notably, the pro-inflammatory mediators, TNF- and NO were increased, and the anti-inflammatory cytokine IL-10 was decreased for both herb treatment alone and the exercise þ CS combination. This finding suggests that the imbalance of proinflammatory and anti-inflammatory effects plays a key role in the antagonistic effects of exercise on CS. Hepatocyte apoptosis was the main consequence of D-GalN/LPS-induced hepatic failure (Morikawa et al., 1996). Thus, prevention of hepatic apoptosis is important in the protection of mice from liver injury. We have demonstrated that either exercise preconditioning or CS can protect mice from D-GalN/LPS-induced fulminant hepatic failure through reducing hepatic apoptosis. The combination of exercise preconditioning and CS did not act synergistically and instead offset their protective effects. It is worth addressing the observation that exercise or a high dose of CS alone directly reduced hepatocyte apoptosis or reduced TNF- production in the serum and liver, but a combination of exercise and CS resulted in their mutual inhibitory effects on TNF-, leading to severe
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hepatocyte apoptosis. Previous studies have suggested that exercise might reduce LPSinduced TNF- production in rats (Chen et al., 2007) and that CS inhibits LPS-stimulated bronchoalveolar lavage fluids from releasing TNF- (Kuo et al., 2001). In the same manner, serum and tissue TNF- were profoundly reduced in the mice that received exercise or 40 mg of CS pretreatment in our animal model. TNF-, after LPS stimulation in vivo, has been identified as the main mediator leading to hepatic apoptosis (Leist et al., 1995). We also found that exercise þ CS resulted in a lost ability to inhibit TNF- expression, which resulted in liver damage, including AST and histology findings. In addition, TNF--induced hepatocyte apoptosis depends on the p38–MAPK signal pathway (Pastorino et al., 2003). Consistent with these results, the up-regulation of the p38 pathway was also found in the mice receiving exercise þ CS after LPS/D-GalN stimulation. Although the changes of NO level were very similar to TNF-, the role of NO on LPSinduced hepatocyte apoptosis is still controversial (Liu et al., 2002; Farghali et al., 2003). Thus, we speculate the changes of TNF- affected by exercise or CS are the key mediators of hepatocyte apoptosis. The controversial results of exercise combined with CS treatment indicate that physical exercise is a new host factor with an impact on drug pharmacokinetics. Physical exercise might affect the absorption, distribution, metabolism, and excretion of a drug. The gastrointestinal pH and gastric emptying are factors of exercise that may affect drug absorption of those drugs delivered orally (Costill and Saltin, 1974; Nielsen et al., 1995). However, swimming exercise was performed for 4 h after CS treatment in our study. The effects of exercise on the GI can generally be ruled out. The other possibility is that physical exercise might change the distribution of CS in the liver. A reduction of liver blood flow amounting to 60% has been reported (Rowell, 1974) and induces a diminished elimination of high-clearance drugs (Dossing, 1985). According to pharmacokinetic studies, CS will be quickly cleared from the blood to liver after i.v. injection (Tsai et al., 2010). Thus, the effects of exercise on the blood flow to the liver might lead to changes in CS efficacy and toxicity. In summary, pretreatment with a high dose of CS and exercise preconditioning alone both reduced sepsis-induced liver damage, but the combination had no beneficial effects, which might result from the high expression of TNF-, NO, and caspase activation. These results imply that taking CS is not recommended for athletes or people who undertake regular exercise. Acknowledgments This study was supported by National Science Council of Taiwan grants NSC 99-2314-B039-004-MY3, and CMU98-N2-12 and CMU99-N2-04-1 from the China Medical University, Taiwan. We would like to thank Dr. Timothy Williams for proofreading the manuscript. References Arends, B.G., R.O. Bohm, J.E. van Kemenade, K.H. Rahn and M.A. van Baak. Influence of physical exercise on the pharmacokinetics of propranolol. Eur. J. Clin. Pharmacol. 31: 375–377, 1986.
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Carter, J.H., J.G. Nutt and W.R. Woodward. The effect of exercise on levodopa absorption. Neurology 42: 2042–2045, 1992. Chen, C.K., A.S. Muhamad and F.K. Ooi. Herbs in exercise and sports. J. Physiol. Anthropol. 31: 4, 2012. Chen, H.I., S.Y. Hsieh, F.L. Yang, Y.H. Hsu and C.C. Lin. Exercise training attenuates septic responses in conscious rats. Med. Sci. Sports Exerc. 39: 435–442, 2007. Chen, S., Z. Li, R. Krochmal, M. Abrazado, W. Kim and C.B. Cooper. Effect of Cs-4 (Cordyceps sinensis) on exercise performance in healthy older subjects: A double-blind, placebo-controlled trial. J. Altern. Complement. Med. 16: 585–590, 2010. Cheng, Y.J., S.M. Cheng, Y.H. Teng, W.C. Shyu, H.L. Chen and S.D. Lee. Cordyceps sinensis prevents apoptosis in mouse liver with D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure. Am. J. Chin. Med. 42: in press, 2014. Chien, K.Y., C.C. Huang, K.F. Hsu, C.H. Kuo and M.C. Hsu. Swim training reduces metformin levels in fructose-induced insulin resistant rats. J. Pharm. Pharm. Sci. 15: 85–93, 2012. Costill, D.L. and B. Saltin. Factors limiting gastric emptying during rest and exercise. J. Appl. Physiol. 37: 679–683, 1974. Dossing, M. Effect of acute and chronic exercise on hepatic drug metabolism. Clin. Pharmacokinet. 10: 426–431, 1985. Farghali, H., N. Canova, T. Kucera, J. Martinek and K. Masek. Nitric oxide synthase inhibitors modulate lipopolysaccharide-induced hepatocyte injury: Dissociation between in vivo and in vitro effects. Int. Immunopharmacol. 3: 1627–1638, 2003. Ho, R.T., C.W. Wang, S.M. Ng, A.H. Ho, E.T. Ziea, V.T. Wong and C.L. Chan. The effect of t’ai chi exercise on immunity and infections: A systematic review of controlled trials. J. Altern. Complement. Med. 19: 389–396, 2013. Kakanis, M.W., J. Peake, E.W. Brenu, M. Simmonds, B. Gray, S.L. Hooper and S.M. MarshallGradisnik. The open window of susceptibility to infection after acute exercise in healthy young male elite athletes. Exerc. Immunol. Rev. 16: 119–137, 2010. Khazaeinia, T., A.A. Ramsey and Y.K. Tam. The effects of exercise on the pharmacokinetics of drugs. J. Pharm. Pharm. Sci. 3: 292–302, 2000. Kim, H.G., B. Shrestha, S.Y. Lim, D.H. Yoon, W.C. Chang, D.J. Shin, S.K. Han, S.M. Park, J.H. Park, H.I. Park, J.M. Sung, Y. Jang, N. Chung, K.C. Hwang and T.W. Kim. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur. J. Pharmacol. 545: 192–199, 2006. Kumar, R., P.S. Negi, B. Singh, G. Ilavazhagan, K. Bhargava and N.K. Sethy. Cordyceps sinensis promotes exercise endurance capacity of rats by activating skeletal muscle metabolic regulators. J. Ethnopharmacol. 136: 260–266, 2011. Kuo, Y.C., W.J. Tsai, J.Y. Wang, S.C. Chang, C.Y. Lin and M.S. Shiao. Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis. Life Sci. 68: 1067–1082, 2001. Leist, M., F. Gantner, I. Bohlinger, G. Tiegs, P.G. Germann and A. Wendel. Tumor necrosis factorinduced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am. J. Pathol. 146: 1220–1234, 1995. Liu, T.H., E.K. Robinson, K.S. Helmer, S.D. West, A.A. Castaneda, L. Chang and D.W. Mercer. Does upregulation of inducible nitric oxide synthase play a role in hepatic injury? Shock 18: 549–554, 2002. Mackinnon, L.T. Current challenges and future expectations in exercise immunology: Back to the future. Med. Sci. Sports Exerc. 26: 191–194, 1994.
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Mehanna, A., D.C. Vitorino, C. Panis, E.E. Blanco, P. Pinge-Filho and M.C. Martins-Pinge. Cardiovascular and pulmonary effects of NOS inhibition in endotoxemic conscious rats subjected to swimming training. Life Sci. 81: 1301–1308, 2007. Morikawa, A., T. Sugiyama, Y. Kato, N. Koide, G.Z. Jiang, K. Takahashi, Y. Tamada and T. Yokochi. Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model. Infect. Immun. 64: 734–738, 1996. Nielsen, H.B., L.B. Svendsen, T.H. Jensen and N.H. Secher. Exercise-induced gastric mucosal acidosis. Med. Sci. Sports Exerc. 27: 1003–1006, 1995. Pastorino, J.G., N. Shulga and J.B. Hoek. TNF-alpha-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8. Am. J. Physiol. 285: G503–G516, 2003. Pedersen, B.K., H. Bruunsgaard, M. Jensen, A.D. Toft, H. Hansen and K. Ostrowski. Exercise and the immune system — influence of nutrition and ageing. J. Sci. Med. Sport 2: 234–252, 1999. Pereira, G.B., J. Prestes, R.A. Tibana, G.E. Shiguemoto, J. Navalta and S.E. Perez. Acute resistance training affects cell surface markers for apoptosis and migration in CD4(þ) and CD8(þ) lymphocytes. Cell Immunol. 279: 134–139, 2012. Rowell, L.B. Human cardiovascular adjustments to exercise and thermal stress. Physiol. Rev. 54: 75– 159, 1974. Stromberg, C., J. Vanakoski, K.T. Olkkola, A. Lindqvist, T. Seppala and L.A. Laitinen. Exercise alters the pharmacokinetics of midazolam. Clin. Pharmacol. Ther. 51: 527–532, 1992. Tsai, Y.J., L.C. Lin and T.H. Tsai. Pharmacokinetics of adenosine and cordycepin, a bioactive constituent of Cordyceps sinensis in rat. J. Agric. Food Chem. 58: 4638–4643, 2010. van Baak, M.A. Influence of exercise on the pharmacokinetics of drugs. Clin. Pharmacokinet. 19: 32–43, 1990. Won, S.Y. and E.H. Park. Anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J. Ethnopharmacol. 96: 555–561, 2005. Yang, M.L., P.C. Kuo, T.L. Hwang and T.S. Wu. Anti-inflammatory principles from Cordyceps sinensis. J. Nat. Prod. 74: 1996–2000, 2011. Ylitalo, P. Effect of exercise on pharmacokinetics. Ann. Med. 23: 289–294, 1991.
The American Journal of Chinese Medicine, Vol. 42, No. 5, 1199–1213 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X1450075X
Antagonistic Interaction Between Cordyceps sinensis and Exercise on Protection in Fulminant Hepatic Failure Yu-Jung Cheng,* Woei-Cherng Shyu,‡ Yi-Hsien Teng,* Yu-Hsuan Lan† and Shin-Da Lee*,§ *Department
of Physical Therapy and Graduate Institute of Rehabilitation Science †
School of Pharmacy, China Medical University Taichung, Taiwan, ROC
‡
Center for Neuropsychiatry and Graduate Institute of Immunology China Medical University and Hospital, Taichung, Taiwan §Department
of Healthcare Administration Asia University, Taichung, Taiwan Published 17 September 2014
Abstract: Herb supplements are widely used by Asian athletes; however, there are no studies evaluated the co-effects of exercise and herb supplements on hepatic failure. In this study, DGalN/LPS-induced fulminant hepatic failure was used to examine whether there are synergistic or antagonistic effects of exercise and Cordyceps sinensis (CS). Mice were randomly divided into eight groups: control, swimming exercise for four weeks, D-GalN/LPS challenge, swimming exercise plus D-GalN/LPS, 20 mg/kg or 40 mg/kg CS pretreated for four weeks plus D-GalN/LPS, and swimming exercise combined with 20 mg/kg or 40 mg/kg CS pretreatment plus D-GalN/LPS. Either exercise or 40 mg/kg CS pretreatment alone significantly decreased D-GalN/LPS-induced TNF-, AST, NO, apoptotic-related proteins, and hepatocyte apoptosis. Exercise or 40 mg/kg CS alone increased the IL-10 and D-GalN/LPSsuppressed Superoxide Dismutase (SOD) level. However, no protective or worse effect was observed in the mice treated with exercise preconditioning combined 40 mg/kg CS compared to those receive exercise alone or CS alone. TNF-, AST, NO level, caspase-3 activity, and hepatocytes apoptosis were not significantly different in the exercise combined with 40 mg/ kg CS compared to mice challenged with D-GalN/LPS. The IL-10 level was significantly decreased after D-GalN/LPS stimulation in the mice received exercise combined with 40 mg/ kg CS, indicating the combination strongly reduced the anti-inflammatory effect. In summary, preconditioning exercise or CS pretreatment alone can protect mice from septic liver damage, but in contrast, the combination of exercise and CS does not produce any benefit. Correspondence to: Dr. Shin-Da Lee, Department of Physical Therapy and Graduate Institute of Rehabilitation Science, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402, ROC. Taichung, Tel: (þ886) 4-2205-3366 (ext. 7300), Fax: (þ886) 4-2206-5051, E-mail: [email protected]
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The antagonistic interactions between exercise and CS imply taking CS is not recommended for people who undertake regular exercise. Keywords: Apoptosis; Cordyceps sinensis; Inflammation; Lipopolysaccharide; Liver; Physical Activity.
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Introduction Herbal supplements have been widely used for centuries, particularly in Asia, and increasing numbers of Asian athletes are using herbal supplements to improve their performance (Chen et al., 2012). Cordyceps sinensis (CS) has been reported to promote exercise endurance capacity and performance (Chen et al., 2010; Kumar et al., 2011). In addition its beneficial effects on sports, CS have been used to treat inflammatory and autoimmune diseases (Won and Park, 2005; Kim et al., 2006). The anti-inflammatory effect of CS might result from the inhibition of TNF- (Cheng et al., 2014). Among 50 compounds found in CS, more than 10 compounds have anti-inflammatory effects (Yang et al., 2011). In general, regular exercise can enhance immunity and anti-oxidant function (Mackinnon, 1994; Ho et al., 2013), but acute and exhaustive exercise can reduce immune function, increase infection risk and increase immune cell apoptosis (Kakanis et al., 2010; Pereira et al., 2012). Chen et al. (2007) suggested that exercise training attenuates septic responses and protects organs from damage in sepsis. Exercise training is beneficial and counteracts cardiovascular abnormalities and pulmonary edema in septic animals (Mehanna et al., 2007). The mechanisms of exercise-associated immune changes are multifactorial and include multiple neuroendocrinological factors. Age, metabolism and nutrition contribute to exercise-associated changes in immune function (Pedersen et al., 1999). Because CS or exercise preconditioning can attenuate septic responses and protect organs from damage, the present study was designed to investigate whether exercise preconditioning combined with CS has a synergistic effect in attenuating hepatic injury in sepsis. Materials and Methods Chemicals LPS (derived from E. coli, serotype 055:B5) and D-GalN were obtained from Sigma Chemical (St. Louis, MO, USA). Water extracts of CS were purchased from TCM Biotech. International Corp., Taiwan. Treatments and Animals Male C57BL/6 mice weighing 20–25 g were obtained from the National Laboratory Animal Center (NLAC) and housed in the laboratory animal center at China Medical University. Animals were housed individually in a room with a 12-h light/dark cycle and
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central air-conditioning (25 C, 70% humidity), fed with standard food ad libitum, and cared for according to the appropriate nationally approved guidelines. Thirty-two mice were randomized into eight groups. For the CS treatment, mice were treated with saline or an extract of CS (20 mg/kg or 40 mg/kg) by oral gavage, five days per week, for a total of four weeks. For exercise preconditioning, 4 h after CS treatment, swimming exercise was performed. The exercise protocol consists of swimming exercise for 30 min/day, five days/ week for four weeks in 26–28 C water. For the preparation of mice with GalN/LPS-induced fulminant hepatic failure, mice were administered an intraperitoneal injection of GalN (800 mg/g body weight; Sigma-Aldrich, St. Louis, MO, USA). Three or six hours later, serum or liver tissues were collected for further analysis. Histology Liver tissue obtained from mice treated with GalN/LPS for 6 h was fixed in 3.7% buffered formalin and embedded in paraffin wax. Five-m sections were stained with hematoxylin and eosin Y (H&E stain). Photomicrographs were obtained using Zeiss Axiophot microscopes. A third pathologist examined the H&E-stained slides to verify the diagnosis. The degeneration/necrosis and hemorrhage status were scored. This scoring system provided a representation of the grade of degeneration/necrosis and hemorrhage from 1 to 5. Scores of 1 represent histological normality (non-degenerate), and scores of 5 represent severe degeneration. Blood Collection and Biochemistry Study The blood of mice was collected from the inferior vena cava under chloral hydrate anesthesia, drawn into serum separation tubes, allowed to clot for 10 min at room temperature, and then centrifuged (1000 g, 10 min, 4 C). Serum samples were stored at 70 C. To determine the serum concentrations of aspartate aminotransferase (AST), sera were spotted onto slides (Fuji Dri-Chem; Fujifilm, Kanagawa, Japan) and evaluated by a slide analyzer (Fuji Dri-Chem 3500S; Fujifilm). Enzyme-Linked Immunosorbent Assay (ELISAs) TNF- and IL-10 were measured using antibody enzyme-linked immunosorbent assays (ELISAs). The experiment was repeated with blood collected at 3 and 6 h for cytokine analysis by intracardiac puncture and immediately centrifuged for 10 min at 5000 g. The liver tissue was collected for analysis 6 h after GalN/LPS treatment. Liver tissue was homogenized and centrifuged for 20 min at 5000 g. The supernatant was stored at 80 C for the determination of IL-10 and TNF- concentration using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The optical density was measured at a wavelength of 450 nm.
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Measurement of Serum Nitrite Concentration
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The nitrite concentrations in sera were measured after the Griess reaction by incubating 20L samples with 20 l of Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) and 160 L of deionized water at room temperature for 20 min. The absorbance was measured at 550 nm. Nitrite concentration was calculated via comparison with a standard solution of known sodium nitrite concentration. Superoxide Dismutase (SOD) Assay The enzyme activity of SOD was measured using a kit from Cayman Chemical (Ann Arbor, MI, USA). The method utilizes tetrazolium salt to quantify superoxide radicals generated by xanthine oxidase and hypoxanthine. The standard curve was generated using a quality-controlled SOD standard, and optical density was measured at 450 nm. Western Blot Analysis Mice liver tissue was harvested 6 h after GalN/LPS treatment. The tissue was homogenized and solubilized with ice-cold buffer containing 25 mM Hepes (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM DTT, 100 g/ml PMSF, and 2 g/ml leupeptin. Approximately 40 g of protein was separated by electrophoresis in a 10% sodium-dodecyl-sulfate polyacrylamide gel. After electrophoresis, the protein was electro-transferred onto polyvinylidene fluoride membranes (Life Science, Boston, MA). Membranes were probed with antibodies specific for phosphor-ERK, ERK, phospho-p38, p38, cleaved caspase-3, cleaved caspase-6 or PARP (New England Biolabs). After probing with a horseradish peroxidase-conjugated secondary antibody, protein signals were visualized using enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL) and exposed on Kodak X-ML film. Terminal Deoxynucleotide Transferase-Mediated dUTP Nick End Labeling (TUNEL) Apoptosis in the liver was detected with an ApopTag Detection Kit (Millipore, Billerica, MA, USA). Paraffin wax slides of liver were deparaffinized and soaked to quench the endogenous peroxidase in a solution containing 3% H2O2. The slides were then rinsed with PBS once more. To add the residues of the digoxigenin-nucleotide to the 3 0 -OH end of the DNA catalytically, terminal deoxynucleotidyl transferase (TdT) with reaction buffer was used, and the specimens were mounted for 1 h. The reaction was terminated by applying a stop solution for 30 min at 37 C, and the specimens were washed with PBS. Before rinsing with PBS, the anti-digoxigenin-peroxidase solution was applied for 15 min to the sections to join the chromogenic substrates to the complex between the DNA and the digoxigenin-nucleotide. To detect positive staining, specimens were soaked in DAB and counterstained with hematoxylin.
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Statistical Analysis The data are presented as mean SEM. Statistical evaluation was performed with one-way ANOVA, two-way ANOVA and Tukey’s post hoc test. A value of p < 0:05 was considered to be statistically significant.
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Results Effects of CS and Exercise Preconditioning on Liver Damage in LPS/D-GalN-Treated Mice Light microscopy revealed no infiltration or sequestration of neutrophils in the liver of the control group, whereas more overt sequestration of neutrophils and coagulative necrosis tissues were observed in mice treated with LPS/D-GalN. The neutrophil infiltration and liver necrosis were significantly reduced by exercise preconditioning or 40 mg of CS. However, exercise preconditioning combined with 40 mg of CS significantly increased neutrophil infiltrations and liver necrosis compared to 40 mg of CS alone (Figs. 1A–1C). Similar results were found for AST levels. AST expression in sera was measured 6 h after the LPS/D-GalN challenge to examine the effect of CS and exercise preconditioning (Fig. 1D). The AST levels in LPS/D-GalN-treated groups were significantly increased compared with the control group and significantly decreased with 40 mg of CS pretreatment. Exercise preconditioning also strongly decreased AST levels compared with the LPS group; however, exercise preconditioning combined with 40 mg of CS significantly increased AST concentration after LPS/D-GalN stimulation compared with mice that received 40 mg of CS alone or exercise preconditioning alone. Effects of CS and Exercise Preconditioning on Serum and Tissue TNF- Concentration in LPS/D-GalN-treated Mice TNF- is the main mediator leading to hepatocyte apoptosis. We next examined the level of TNF- in serum and liver (Fig. 2A). The serum TNF- concentration 3 h after a LPS/DGalN challenge was significantly increased, and an increased dose of CS significantly decreased serum TNF- in the non-exercise group (all p < 0:05). Exercise preconditioning significantly reduced the LPS/D-GalN-induced TNF- concentration. In contrast, the combination of exercise and 40 mg of CS increased the TNF- concentration compared with exercise preconditioning alone or 40 mg of CS alone. Two-way ANOVA revealed that there were negative interactions between CS dose and exercise training (p < 0:05). The tissue TNF- level was determined 6 h after the LPS/D-GalN challenge. Similar results were found in tissue. Exercise and CS alone can reduce TNF-, but the combined treatment had antagonistic effects (Fig. 2B).
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(D) Figure 1. Effects of CS pretreatment and exercise preconditioning on liver damage in LPS/D-GalN-treated mice. Mice were orally pretreated with 20 mg or 40 mg of CS combined with, or without, swimming exercise (30 min/ day) for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/D-GalN. Mice were sacrificed after 6 h, and histological examination of liver tissue was performed with H&E staining (A). The scores for degeneration/necrosis (B) and hemorrhage (C) were also measured. Serum AST (D) was evaluated 6 h after LPS/D-GalN treatment. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
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Figure 2. Effects of CS pretreatment and exercise preconditioning on serum and tissue TNF- and IL-10 concentration in LPS/D-GalN-treated mice. Either 20 or 40 mg of CS was orally administered in combination with, or without, swimming exercise (30 min/day) to mice daily for four weeks. After injection with LPS/D-GalN, mice were sacrificed after 6 h, and serum TNF- (A), liver tissue TNF- (B), serum IL-10 (C), and liver tissue IL-10 (D) were evaluated by ELISA. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
Effects of CS and Exercise Preconditioning on Serum and Tissue IL-10 Concentration in LPS/D-GalN-Treated Mice IL-10, which is an anti-inflammatory cytokine, was further examined. After a 6-h challenge of LPS/D-GalN, the IL-10 level was determined both in serum and liver tissue by ELISA (Figs. 2C and 2D). Serum and tissue IL-10 concentrations were significantly increased after the LPS/D-GalN challenge, and 40 mg of CS significantly increased serum and tissue IL-10. Exercise preconditioning also significantly increased serum IL-10 after the LPS/D-GalN challenge. However, exercise preconditioning combined with 40 mg of CS decreased serum LPS/D-GalN-induced IL-10 concentration, compared with 40 mg of CS alone.
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(C) Figure 3. Effects of CS pretreatment and exercise preconditioning on NO production and SOD levels in LPS/DGalN-treated mice. The mice were orally pretreated with 20 mg or 40 mg of CS daily in combination with, or without, swimming exercise (30 min/day) for four weeks. After injection with LPS/D-GalN, the mice were sacrificed after 6 h, and their serum nitrite (A), liver tissue nitrite (B), and liver tissue SOD (C) were evaluated. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
Effects of CS and Exercise Preconditioning on Serum and Tissue NO Production in LPS/D-GalN-Treated Mice To examine the effect of CS and exercise preconditioning on NO production, nitrite concentration in serum and liver tissue was measured using the Griess Reaction (Figs. 3A and 3B) after 3 h. Serum and tissue nitrite concentrations 3 h after the LPS/D-GalN challenge were significantly increased, and an increased dose of CS significantly decreased serum and tissue nitrite in the non-exercise group. The administration of 40 mg of CS strongly decreased LPS/D-GalN-induced nitrite elevation. Exercise preconditioning also decreased serum and tissue nitrite concentration, but exercise preconditioning combined with 40 mg of CS increased nitrite concentration after LPS/D-GalN stimulation compared to 40 mg of CS without exercise. Similar to previous results, exercise preconditioning combined with 40 mg of CS does not display any benefit compared with exercise or CS alone.
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Effects of CS and Exercise Preconditioning on SOD Levels in LPS/D-GalN-Treated Mice SOD is responsible for the quenching of superoxide radicals. We next examined the effect of CS and exercise preconditioning on SOD production (Fig. 3C). The SOD concentration after the LPS/D-GalN challenge was significantly decreased, and when mice were treated with CS, they displayed significantly increased SOD levels in a dose-dependent manner. Exercise preconditioning also significantly increased SOD concentrations after the LPS/DGalN challenge, but combined exercise with 40 mg of CS did not have beneficial effects.
Effects of CS and Exercise Preconditioning on Caspase-3/6 Activity in LPS/D-GalN-Treated Mice We further measured whether CS and exercise preconditioning might alter the activity of the apoptotic pathways. The cleaved caspase-3, cleaved caspase-6 and cleaved-PARP were detected by western blotting to determine caspase pathway activation. Liver tissue was harvested, and the homogenate was analyzed after 6 h of LPS/D-GalN infusion. As shown in Fig. 4, cleaved caspase-3, caspase-6, and PARP were markedly increased after the LPS/D-GalN challenge. Either exercise preconditioning alone or 40 mg of CS pretreatment alone significantly decreased LPS/D-GalN-induced caspase-3, caspase-6, and PARP levels. The combination of exercise preconditioning and 40 mg of CS attenuated the anti-apoptotic effect of exercise preconditioning alone or 40 mg of CS pretreatment alone. Effects of CS and Exercise Preconditioning on LPS/D-GalN-Induced Hepatic Apoptosis To examine the effect of CS and exercise preconditioning on LPS/D-GalN-induced hepatic apoptosis, liver tissues were processed for TUNEL staining. Administration of LPS/DGalN induced apoptosis of hepatocytes (Fig. 5A). TUNEL staining indicated significant hepatic apoptosis 6 h after LPS/D-GalN-treatment (control, 0:23 0:016; LPS, 6:7 0:5; p < 0:05; Fig. 5B). Pretreatment with CS dose-dependently attenuated the percentage of LPS/D-GalN-induced TUNEL-positive staining (LPS, 6:7 0:5; 20 mg of CS þ LPS, 4:9 0:48; 40 mg of CSþLPS, 0:9 0:24; p < 0:05). In addition, exercise preconditioning reduced LPS/D-GalN-stimulated apoptosis (LPS, 6:7 0:5; Ex, 4:8 0:4; p < 0:05). However, the combination of exercise preconditioning and CS did not prevent apoptosis on LPS/D-GalN-treated mice compared with exercise alone or CS alone (40 mg of CS þ LPS, 0:9 0:24; Ex þ 40 mg of CS þ LPS, 7:6 0:3; p < 0:05). Effects of CS and Exercise Preconditioning on p38 MAPK Protein Phosphorylation in LPS/D-GalN-Treated Mice The effect of CS and exercise preconditioning on the activation of the p38 pathway was measured by western blotting (Fig. 6). Compared with the control and LPS/D-GalN
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(D) Figure 4. Effects of CS pretreatment and exercise preconditioning on caspase-3/6 activity in LPS/D-GalN-treated mice. Either 20 or 40 mg of CS was orally administered in combination with, or without, swimming exercise (30 min/day) to mice daily for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/D-GalN. Mice were sacrificed after 6 h and tissue cleaved caspase-3, caspase-6, and PARP were evaluated through western blotting (A). The quantification of related cleaved caspase-3 (B), cleaved caspase-6 (C) and cleaved PARP are shown. Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
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(B) Figure 5. Effects of CS pretreatment and exercise preconditioning on LPS/D-GalN-induced hepatic apoptosis. Mice were orally pretreated with 20 mg or 40 mg of CS daily in combination with, or without, swimming exercise (30 min/day) for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/DGalN. Mice were sacrificed after 6 h, and cell apoptosis was evaluated by TUNEL staining (A). The quantification of TUNEL þ cell number is shown (B). Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using one-way ANOVA. The differences between treatments with different letters were statistically significant ( p < 0:05).
challenged groups, 40 mg of CS significantly decreased LPS/D-GalN-induced p38 protein phosphorylation. Exercise preconditioning alone did not change the level of phosphor-p38; however, the combination of exercise and CS induced a 2-fold increase in p38 phosphorylation, compared with CS or exercise preconditioning alone. Discussion The influence of physical exercise on pharmacokinetics has been well investigated in many aspects (van Baak, 1990; Ylitalo, 1991; Khazaeinia et al., 2000). Some studies indicate physical exercise impairs the absorption or bioactivity of drugs (Arends et al., 1986; Carter et al., 1992; Stromberg et al., 1992; Chien et al., 2012). Although many studies indicate that herbal supplements have an ergogenic effect on physical performance (Chen et al., 2012), there is no evidence to indicate that physical exercise might affect the bioactivity of herbal supplements. Consistent with the findings for some drugs, four weeks of swimming
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(B) Figure 6. Effects of CS pretreatment and exercise preconditioning on p38 MAPK protein phosphorylation in LPS/ D-GalN-treated mice. Mice were orally pretreated with 20 mg or 40 mg of CS daily in combination with, or without, swimming exercise (30 min/day) for four weeks. To induce fulminant hepatic failure, mice were intraperitoneally injected with LPS/D-GalN. Mice were sacrificed after 6 h, and tissue phosphor-p38 MAPK and p38 MAPK were evaluated by western blotting (A). The quantification of the related phosphor-p38 MAPK is shown (B). Data are expressed as mean SE (n ¼ 4). Significant differences of measured traits were analyzed using oneway ANOVA. The differences between treatments with different letters were significant ( p < 0:05).
training reduced the protective effects of CS on D-GalN/LPS-induced hepatic failure, as indicated by AST level and morphology changes. An increased number of apoptotic cells were also detected in the livers of the B6 mice pretreated with the combination of exercise and 40 mg of CS. Notably, the pro-inflammatory mediators, TNF- and NO were increased, and the anti-inflammatory cytokine IL-10 was decreased for both herb treatment alone and the exercise þ CS combination. This finding suggests that the imbalance of proinflammatory and anti-inflammatory effects plays a key role in the antagonistic effects of exercise on CS. Hepatocyte apoptosis was the main consequence of D-GalN/LPS-induced hepatic failure (Morikawa et al., 1996). Thus, prevention of hepatic apoptosis is important in the protection of mice from liver injury. We have demonstrated that either exercise preconditioning or CS can protect mice from D-GalN/LPS-induced fulminant hepatic failure through reducing hepatic apoptosis. The combination of exercise preconditioning and CS did not act synergistically and instead offset their protective effects. It is worth addressing the observation that exercise or a high dose of CS alone directly reduced hepatocyte apoptosis or reduced TNF- production in the serum and liver, but a combination of exercise and CS resulted in their mutual inhibitory effects on TNF-, leading to severe
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hepatocyte apoptosis. Previous studies have suggested that exercise might reduce LPSinduced TNF- production in rats (Chen et al., 2007) and that CS inhibits LPS-stimulated bronchoalveolar lavage fluids from releasing TNF- (Kuo et al., 2001). In the same manner, serum and tissue TNF- were profoundly reduced in the mice that received exercise or 40 mg of CS pretreatment in our animal model. TNF-, after LPS stimulation in vivo, has been identified as the main mediator leading to hepatic apoptosis (Leist et al., 1995). We also found that exercise þ CS resulted in a lost ability to inhibit TNF- expression, which resulted in liver damage, including AST and histology findings. In addition, TNF--induced hepatocyte apoptosis depends on the p38–MAPK signal pathway (Pastorino et al., 2003). Consistent with these results, the up-regulation of the p38 pathway was also found in the mice receiving exercise þ CS after LPS/D-GalN stimulation. Although the changes of NO level were very similar to TNF-, the role of NO on LPSinduced hepatocyte apoptosis is still controversial (Liu et al., 2002; Farghali et al., 2003). Thus, we speculate the changes of TNF- affected by exercise or CS are the key mediators of hepatocyte apoptosis. The controversial results of exercise combined with CS treatment indicate that physical exercise is a new host factor with an impact on drug pharmacokinetics. Physical exercise might affect the absorption, distribution, metabolism, and excretion of a drug. The gastrointestinal pH and gastric emptying are factors of exercise that may affect drug absorption of those drugs delivered orally (Costill and Saltin, 1974; Nielsen et al., 1995). However, swimming exercise was performed for 4 h after CS treatment in our study. The effects of exercise on the GI can generally be ruled out. The other possibility is that physical exercise might change the distribution of CS in the liver. A reduction of liver blood flow amounting to 60% has been reported (Rowell, 1974) and induces a diminished elimination of high-clearance drugs (Dossing, 1985). According to pharmacokinetic studies, CS will be quickly cleared from the blood to liver after i.v. injection (Tsai et al., 2010). Thus, the effects of exercise on the blood flow to the liver might lead to changes in CS efficacy and toxicity. In summary, pretreatment with a high dose of CS and exercise preconditioning alone both reduced sepsis-induced liver damage, but the combination had no beneficial effects, which might result from the high expression of TNF-, NO, and caspase activation. These results imply that taking CS is not recommended for athletes or people who undertake regular exercise. Acknowledgments This study was supported by National Science Council of Taiwan grants NSC 99-2314-B039-004-MY3, and CMU98-N2-12 and CMU99-N2-04-1 from the China Medical University, Taiwan. We would like to thank Dr. Timothy Williams for proofreading the manuscript. References Arends, B.G., R.O. Bohm, J.E. van Kemenade, K.H. Rahn and M.A. van Baak. Influence of physical exercise on the pharmacokinetics of propranolol. Eur. J. Clin. Pharmacol. 31: 375–377, 1986.
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Y.-J. CHENG et al.
Carter, J.H., J.G. Nutt and W.R. Woodward. The effect of exercise on levodopa absorption. Neurology 42: 2042–2045, 1992. Chen, C.K., A.S. Muhamad and F.K. Ooi. Herbs in exercise and sports. J. Physiol. Anthropol. 31: 4, 2012. Chen, H.I., S.Y. Hsieh, F.L. Yang, Y.H. Hsu and C.C. Lin. Exercise training attenuates septic responses in conscious rats. Med. Sci. Sports Exerc. 39: 435–442, 2007. Chen, S., Z. Li, R. Krochmal, M. Abrazado, W. Kim and C.B. Cooper. Effect of Cs-4 (Cordyceps sinensis) on exercise performance in healthy older subjects: A double-blind, placebo-controlled trial. J. Altern. Complement. Med. 16: 585–590, 2010. Cheng, Y.J., S.M. Cheng, Y.H. Teng, W.C. Shyu, H.L. Chen and S.D. Lee. Cordyceps sinensis prevents apoptosis in mouse liver with D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure. Am. J. Chin. Med. 42: in press, 2014. Chien, K.Y., C.C. Huang, K.F. Hsu, C.H. Kuo and M.C. Hsu. Swim training reduces metformin levels in fructose-induced insulin resistant rats. J. Pharm. Pharm. Sci. 15: 85–93, 2012. Costill, D.L. and B. Saltin. Factors limiting gastric emptying during rest and exercise. J. Appl. Physiol. 37: 679–683, 1974. Dossing, M. Effect of acute and chronic exercise on hepatic drug metabolism. Clin. Pharmacokinet. 10: 426–431, 1985. Farghali, H., N. Canova, T. Kucera, J. Martinek and K. Masek. Nitric oxide synthase inhibitors modulate lipopolysaccharide-induced hepatocyte injury: Dissociation between in vivo and in vitro effects. Int. Immunopharmacol. 3: 1627–1638, 2003. Ho, R.T., C.W. Wang, S.M. Ng, A.H. Ho, E.T. Ziea, V.T. Wong and C.L. Chan. The effect of t’ai chi exercise on immunity and infections: A systematic review of controlled trials. J. Altern. Complement. Med. 19: 389–396, 2013. Kakanis, M.W., J. Peake, E.W. Brenu, M. Simmonds, B. Gray, S.L. Hooper and S.M. MarshallGradisnik. The open window of susceptibility to infection after acute exercise in healthy young male elite athletes. Exerc. Immunol. Rev. 16: 119–137, 2010. Khazaeinia, T., A.A. Ramsey and Y.K. Tam. The effects of exercise on the pharmacokinetics of drugs. J. Pharm. Pharm. Sci. 3: 292–302, 2000. Kim, H.G., B. Shrestha, S.Y. Lim, D.H. Yoon, W.C. Chang, D.J. Shin, S.K. Han, S.M. Park, J.H. Park, H.I. Park, J.M. Sung, Y. Jang, N. Chung, K.C. Hwang and T.W. Kim. Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur. J. Pharmacol. 545: 192–199, 2006. Kumar, R., P.S. Negi, B. Singh, G. Ilavazhagan, K. Bhargava and N.K. Sethy. Cordyceps sinensis promotes exercise endurance capacity of rats by activating skeletal muscle metabolic regulators. J. Ethnopharmacol. 136: 260–266, 2011. Kuo, Y.C., W.J. Tsai, J.Y. Wang, S.C. Chang, C.Y. Lin and M.S. Shiao. Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis. Life Sci. 68: 1067–1082, 2001. Leist, M., F. Gantner, I. Bohlinger, G. Tiegs, P.G. Germann and A. Wendel. Tumor necrosis factorinduced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am. J. Pathol. 146: 1220–1234, 1995. Liu, T.H., E.K. Robinson, K.S. Helmer, S.D. West, A.A. Castaneda, L. Chang and D.W. Mercer. Does upregulation of inducible nitric oxide synthase play a role in hepatic injury? Shock 18: 549–554, 2002. Mackinnon, L.T. Current challenges and future expectations in exercise immunology: Back to the future. Med. Sci. Sports Exerc. 26: 191–194, 1994.
Am. J. Chin. Med. 2014.42:1199-1213. Downloaded from www.worldscientific.com by UNIVERSITY OF AUCKLAND LIBRARY - SERIALS UNIT on 10/15/14. For personal use only.
INTERACTION OF CS AND EXERCISE IN LIVER FAILURE
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Mehanna, A., D.C. Vitorino, C. Panis, E.E. Blanco, P. Pinge-Filho and M.C. Martins-Pinge. Cardiovascular and pulmonary effects of NOS inhibition in endotoxemic conscious rats subjected to swimming training. Life Sci. 81: 1301–1308, 2007. Morikawa, A., T. Sugiyama, Y. Kato, N. Koide, G.Z. Jiang, K. Takahashi, Y. Tamada and T. Yokochi. Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model. Infect. Immun. 64: 734–738, 1996. Nielsen, H.B., L.B. Svendsen, T.H. Jensen and N.H. Secher. Exercise-induced gastric mucosal acidosis. Med. Sci. Sports Exerc. 27: 1003–1006, 1995. Pastorino, J.G., N. Shulga and J.B. Hoek. TNF-alpha-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8. Am. J. Physiol. 285: G503–G516, 2003. Pedersen, B.K., H. Bruunsgaard, M. Jensen, A.D. Toft, H. Hansen and K. Ostrowski. Exercise and the immune system — influence of nutrition and ageing. J. Sci. Med. Sport 2: 234–252, 1999. Pereira, G.B., J. Prestes, R.A. Tibana, G.E. Shiguemoto, J. Navalta and S.E. Perez. Acute resistance training affects cell surface markers for apoptosis and migration in CD4(þ) and CD8(þ) lymphocytes. Cell Immunol. 279: 134–139, 2012. Rowell, L.B. Human cardiovascular adjustments to exercise and thermal stress. Physiol. Rev. 54: 75– 159, 1974. Stromberg, C., J. Vanakoski, K.T. Olkkola, A. Lindqvist, T. Seppala and L.A. Laitinen. Exercise alters the pharmacokinetics of midazolam. Clin. Pharmacol. Ther. 51: 527–532, 1992. Tsai, Y.J., L.C. Lin and T.H. Tsai. Pharmacokinetics of adenosine and cordycepin, a bioactive constituent of Cordyceps sinensis in rat. J. Agric. Food Chem. 58: 4638–4643, 2010. van Baak, M.A. Influence of exercise on the pharmacokinetics of drugs. Clin. Pharmacokinet. 19: 32–43, 1990. Won, S.Y. and E.H. Park. Anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J. Ethnopharmacol. 96: 555–561, 2005. Yang, M.L., P.C. Kuo, T.L. Hwang and T.S. Wu. Anti-inflammatory principles from Cordyceps sinensis. J. Nat. Prod. 74: 1996–2000, 2011. Ylitalo, P. Effect of exercise on pharmacokinetics. Ann. Med. 23: 289–294, 1991.
