Inhibitive effect on apoptosis in splenic lymphocytes of mice pretreated with Lingzhi (Ganoderma Lucidum) spores

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1 Online Submissions: J Tradit Chin Med 2014 April 15; 34(2): info@journaltcm.com ISSN JTCM. All rights reserved. EXPERIMENTAL STUDY TOPIC Inhibitive effect on apoptosis in splenic lymphocytes of mice pretreated with Lingzhi (Ganoderma Lucidum) spores Quanxi Wang, Yifan Huang, Baocheng Wu, Jingliang Mei, Honglei Zhang, Baomin Qi aa Quanxi Wang, Yifan Huang, Baocheng Wu, Jingliang Mei, Baomin Qi, College of Animal Science, Fujian Agriculture and Forestry University, Fujian , China Honglei Zhang, Pharmacological Laboratory, Fujian Institute for Drug Control, Fujian , China Supported by the Natural Science Foundation of Fujian Province (No. 2013J01069; No. 2012J01067 ) Correspondence to: Prof. Yifan Huang, College of Animal Science, Fujian Agriculture and Forestry University, Fujian , China. zjhyfang@163.com; Prof. Baomin Qi, College of Animal Science, Fujian Agriculture and Forestry University, Fujian , China. qbm64@163.com Telephone: Accepted: July 1, 2013 Abstract OBJECTIVE: To investigate how the pretreatment of mice with Ganoderma spores affected the apoptosis of their splenic lymphocytes induced by dexamethasone after 19 days treatment. METHODS: Sixty Kunming mice were randomly divided into six groups: blank control groupdrenched with normal saline; a drug control group drenched with 150 mg/ml Ganoderma spores; a model group treated with saline; a low dose group with 50 mg/ml Ganoderma spores; a moderate dose group with 100 mg/ml Ganoderma spores; and a high dose group with 150 mg/ml Ganoderma spores. The effect of Ganoderma spores on apoptosis in spleen lymphocytes was analyzed. All groups were treated for 19 days. On day 20, the model group and the 3 treatment groups were intraperitoneally injected dexamethasone to induce apoptosis. Splenic index and apoptosis indes were employed to measure cell apoptosis. RESULTS: The results showed that Ganoderma spores reduced the splenic index to different degrees in each group and the best effect was seen in the high dose group (P<0.05). Terminal dexynucleotidyl transferase (TdT)-mediated 2'-Deoxyuridine 5'-Triphosphate nick end labeling staining revealed that the apoptotic index in all groups administered Ganoderma spores differed significantly from the model group, and a dose-response was observed. Flow cytometric analysis indicated that spleen lymphocyte apoptosis in the model group was extensive. Each dose of Ganoderma spores inhibited dexamethasone-induced apoptosis in spleen lymphocytes, and a dose-response was observed as well. The highest dose of Ganoderma spores decreased Malondialdehyde content in serum induced by dexamethasone (P<0.05). CONCLUSION: The findings imply that the pretreatment of the mice with Ganoderma spores could reduce the apoptosis rate induced by dexamethasone in their splenic lymphocytes JTCM. All rights reserved. Key words: Ganoderma; Spleen; Lymphocytes; Apoptosis; Dexamethasone INTRODUCTION The dynamic balance of immune cell proliferation and death enables the body to maintain stable physiological activities. The abnormal proliferation can lead to tumor formation, and excessive apoptosis may lead to im- JTCM www. journaltcm. com 173 April 15, 2013 Volume 34 Issue 2

2 munosuppression. Lymphocyte apoptosis has been reported to lead to immune suppression in animal diseases. 1 Lingzhi (Ganoderma Lucidum) and its extracts play a role in the regulation of lymphocyte apoptosis by inducing apoptosis in tumor cells and inhibiting apoptosis in normal immune cells, and different doses of Lingzhi (Ganoderma Lucidum) extract were able to induce apoptosis in lymphoma cells. The germ cells of mature Lingzhi (Ganoderma Lucidum), known as Lingzhi (Ganoderma Lucidum) spores, also possess immunomodulatory, anti-tumor and antioxidant properties. 2-4 In this study, the effects of Lingzhi (Ganoderma Lucidum) spores on dexamethasone-induced apoptosis in mice spleen lymphocytes were studied in situ with terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and flow cytometry to demonstrate the immunoregulatory activity of Lingzhi (Ganoderma Lucidum) spores. MATERIALS AND METHODS Drugs and reagents The powder extracted from Lingzhi (Ganoderma Lucidum) spore was purchased from Fujian Xianzhilou Biotechnology Co., Ltd., (Fuzhou, China). Dexamethasone Sodium Phosphate Injection was purchased from Fuzhou Neptunus Pharmaceutical Co., Ltd. (Fuzhou, China). Malondialdehyde (MDA) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjin, China). TUNEL kit (Product No , alkaline phosphatase-labeled) and Proteinase K was purchased from Fuzhou Maixin Biotechnology Development Co., Ltd. (Fuzhou, China). Animals Sixty healthy Kunming mice (30 femal and 30 male ) of clean grade, one-month old, weighing (20.0±0.5) g were provided by Fujian Medical University Laboratory Animal Center (Certificate of quality No. SCXK (min) ). The study was approved by the experimental animal ethics committee of Fujian Agriculture and Forestry University. Totally 60 mice were randomly divided into 6 groups by random number table: blank control group (10), drug control group (10), model group (10), 3 treatment groups (10 each). The mice in the blank control group were drenched with normal saline, 0.2 ml/d, for 19 consecutive days. The drug control mice were drenched with a concentration of 150 mg/ml Lingzhi (Ganoderma Lucidum) spore powder aqueous solution, 0.2 ml/d, for 19 consecutive days. The mice in the model group were fed with saline, 0.2 ml/d, for 19 consecutive days. Mice in the treatment groups were fed 150 mg/ml (high dose), 100 mg/ ml (moderate dose) or 50 mg/ml (low dose) Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days. On the 20th day, the model group and 3 treatment groups were intraperitoneally injected with 40 mg/kg dexamethasone, the blank control group and the drug control group with normal saline. After 15 h, the mice in each group were weighed, sacrificed, and the spleens were harvested and weighed. Half of each spleen was fixed with 4% paraformaldehyde, and the other half was used to prepare a cell suspension. Determination of the splenic index Splenic index was calculated according to the following formula: Splenic index=spleen wet weight (mg)/body weight (g). Determination of the apoptotic index in spleen Spleens were fixed in 4% paraformaldehyde fixing solution for 4 h at 4. The tissues were dehydrated, made transparent and embedded according to routine histological methods. Samples were cut into 5 μm semi-serial cross-sections. For each tissue sample, one of every 10 sections was stained immunohistochemically for apoptotic cells. Positive cell nuclei were stained dark-blue, while the negative cell nuclei were stained red. The average apoptotic index was calculated from five randomly chosen fields in each slice under a microscope. Apoptotic index AI (%) =(positive cell number/ total number of cells) 100%. Determination of serum MDA At the end of experiement, the mice were slaughtered and blood samples were collected in heparinized tubes and centrifuged at 2000 g for 20 min at 4. Analysis of apoptosis by flow cytometry The cell suspension was filtered through a 400 mesh filter, centrifuged, precipitated, and stored at 4 in 70% cold ethanol. Then 1 ml of PI dye was added and the cells were incubated at 4 in the dark for 30 min prior to flow cytometric analysis. Statistical analysis All data were analyzed by one-way analysis of variance using SPSS 10.0 (vers 10, SPSS Institute, Chicago, IL, USA) and multiple comparisons were performed using the LSD test. P<0.05 was considered to be significant. RESULTS Splenic index The apoptosis rate was significantly different (P<0.01) between the blank control group and the model group, which suggests that dexamethasone-induced apoptosis was successful. The splenic index of high dose group was significantly higher than that of the model group (P<0.05), but not higher than that of the blank control group (P>0.05). The indexes of moderate and low dose groups were insignificantly better than that of the model group (P>0.05, Table 1). JTCM www. journaltcm. com 174

3 Wang QX et al. / Experimental Study the 3 groups treated with the extracted powder in terms of apoptotic rate. Apoptotic index in spleen by TUNEL assay Compared with the blank control group, apoptotic rate was significantly increased (P<0.01) in the model group (Figure 1). The rate in high dose group was not significantly different from that in the blank control group (P>0.05, Table 1). A dose-response was seen in The apoptosis in spleen by flow cytometry As shown in Figure 2, the results further confirmed that the Apoptotic rate in high dose group was signifi- Table 1 Inhibitive effect on apoptosis in splenic lymphocytes of mice pretreated with Lingzhi (Ganoderma Lucidum) spores Dose (mg/ml) Splenic index (mg/g) Apoptotic rate (TUNEL) (%) Apoptotic rate (flow cytometry) (%) MDA (nmol/ml) Model group ± ± ± ±6.78 Blank control group ± ± ± ±4.39 Drug control group ± ± ± ±5.69 High dose group ± ± ± ±2.80 Moderate dose group ± ± ± ±4.84 Low dose group ± ± ± ±3.68 Group Notes: model group were fed with saline, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg dexamethasone; blank control group were drenched with normal saline, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; drug control group were drenched with a concentration of 150 mg/ml Lingzhi (Ganoderma Lucidum) spore powder aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; high dose group were fed with 150 mg/ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; moderate dose group were fed with 100 mg/ ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; low dose group were fed with 50 mg/ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline. MDA: malondialdehyde; TUNEL: terminal-deoxynucleoitidyl transferase mediated nick end labeling. A B C D E F Figure 1 Apoptosis detection by TUNEL in spleen ( 200) A: control group; B: model group; C: drug control group; D: high-dose group; E: moderate dose group; F: low dose group. Model group were fed with saline, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg dexamethasone; blank control group were drenched with normal saline, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; drug control group were drenched with a concentration of 150 mg/ml Lingzhi (Ganoderma Lucidum) spore powder aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; high dose group were fed with 150 mg/ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; moderate dose group were fed with 100 mg/ ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; low dose group were fed with 50 mg/ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline. TUNEL: terminal-deoxynucleoitidyl transferase mediated nick end labeling. JTCM www. journaltcm. com 175 April 15, 2014 Volume 34 Issue 2

4 Count A 1.84% B 10.10% % C Count % D % FL3 FL3 FL3 Figure 2 Apoptosis detection by flow cytometry in spleen A: control group; B: model group; C: drug control group; D: high-dose group; E: moderate dose group; F: low dose group. Model group were fed with saline, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg dexamethasone; blank control group were drenched with normal saline, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; drug control group were drenched with a concentration of 150 mg/ml Lingzhi (Ganoderma Lucidum) spore powder aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; high dose group were fed with 150 mg/ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; moderate dose group were fed with 100 mg/ ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline; low dose group were fed with 50 mg/ml Lingzhi (Ganoderma Lucidum) spores in aqueous solution, 0.2 ml/d, for 19 consecutive days, on the 20th day, injected with 40 mg/kg normal saline. E % F cantly lower than those in moderate-dose, low-dose groups, and the model group (P<0.05, Table 1). Serum MDA Apoptosis induced by free radicals leads to the formation of MDA. To further investigate how Lingzhi (Ganoderma Lucidum) spore powder suppresses apoptosis induced by dexamethasone in spleen, we examined the serum malondialdehyde levels. The results showed that the MDA level in high dose group was lower than those in the model, low-dose, and moderate-dose groups (Table 1). DISCUSSION Glucocorticoid is a classical immunosuppressant that can induce apoptosis in lymphocytes. In 1980, Wyllie demonstrated that glucocorticoid induced thymocyte apoptosis. 5 In this study, we established a lymphocyte-apoptosis model by injection of 40 mg/kg dexamethasone for 15 h. Lingzhi (Ganoderma Lucidum) spores are the reproductive cells of Lingzhi (Ganoderma Lucidum); the spores contain higher amounts of polysaccharides, peptides, enzymes, selenium (Se) elements and other ingredients than Lingzhi (Ganoderma Lucidum). 6 Studies have shown that Lingzhi (Ganoderma Lucidum) spores can significantly improve the splenic index of glucocorticoid immunosuppressed mice. 7 In this study, pretreatment of immunosuppressed mice with Lingzhi (Ganoderma Lucidum) spores significantly produced better splenic indexes. These results suggest that Lingzhi (Ganoderma Lucidum) spores had a positive role in facilitating the suppressed immune function to recover or reducing the suppression to some extent. Water-soluble extracts are the popular forms of Traditional Chinese Medicine. Polysaccharides are the main components of the water-soluble extract of Lingzhi (Ganoderma Lucidum). 8 In recent years, modern pharmacological research on Lingzhi (Ganoderma Lucidum) polysaccharides has clearly demonstrated their immuno-modulatory and anti-tumor activities. 9,10 The immunomodulatory effects of Lingzhi (Ganoderma Lucidum) polysaccharides include the promotion of humoral immunity, cellular immunity and the mononuclear phagocyte system. Many reports have demonstrated that Lingzhi (Ganoderma Lucidum) polysaccharides can in- JTCM www. journaltcm. com 176

5 duce cytokine production by the differentiation of lymphocytes, 11 the maturation of cultured murine bone marrow-derived dendritic cells and the immune response initiated by dendritic cells, 12 the proliferation of immunoglobulin production by murine splenic B cells 13 and the activation of natural killer cells, which finally results in the activation of the immune system. This study suggested, Lingzhi (Ganoderma Lucidum) spores could reduced the rate of lymphocyte apoptosis induced by dexamethasone, but the high dose showed better effect. A previous study demonstrated that various free radicals mediate apoptosis in various ways in vivo. 14 Reactive oxygen radicals can affect signal transduction by damaging DNA, as well as participating in the regulation of gene expression pathways mediated by apoptosis. 15 MDA is the final product of lipid peroxidation. The accumulation of free radicals in the body can accelerate lipid peroxidation and produce large amounts of MDA, which causes damage to cells and tissues. Determination of the MDA content is often used to reflect the degree of lipid peroxidation, 16 and therefore the degree of accumulation of free radicals. This study implied that Lingzhi (Ganoderma Lucidum) spore powder could inhibit lipid peroxidation caused by dexamethasone. This suggested that Lingzhi (Ganoderma Lucidum) spores may perform an antioxidant role by scavenging free radicals as reported by Li et al. 17 REFERENCES 1 Qi BM, Chen XY, Wu BC, Yao JS, Zhang HL. The observation of the apoptosis induced by muscovy duck reovirus. Xu Mu Shou Yi Xue Bao 2010; 41(4): Zhou Y, Qu ZQ, Zeng YS, et al. Neuroprotective effect of preadministration with Ganoderma lucidum spore on rat hippocampus. Exp Toxicol Pathol 2012; 64(7): Gao P, Hirano Tomoya, Chen ZQ, Yasuhara Tadashi, Nakata Yoshihiro, Sugimoto Akiko. Isolation and identification of C-19 fatty acids with anti-tumor activity from the spores of Ganoderma lucidum (reishi mushroom). Fitoterapia 2012; 83(3): Zhu XY, Chen X, Xie J, Wang P, Su WK. Mechanochemical-assisted extraction and antioxidant activity of polysaccharides from Ganoderma lucidum spores. Int J Food Sci Tech 2012; 47(5): Wyllie AH. Glucocorficoid-induced thymocyte apoptesis is associated with endogenous endonuclease activation. Nature 1980; 284(5756): Boh B, Berovic M, Zhang J, Li ZB. Ganoderma lucidum and its pharmaceutically active compounds. Biotechnol Annu Rev 2007; 13(1): Wei R, Li Z, Zhong ZQ. Regulation of Ganoderma lucidum spores on glucocorticoid in the immunosuppressive mice model. Zhong Guo Mian Yi Xue Za Zhi 1980; 23 (11): Ma C, Guan SH, Yang M, Liu X, Guo DA. Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum. Phytomedicine 2008; 15(4): Lin ZB. Cellular and molecular mechanisms of immuno-modulation by Ganoderma lucidum. J Pharmacol Sci 2005; 99(2): Paterson RR. Ganoderma-a therapeutic fungalbiofactory. Phytochemistry 2006; 67(18): Hsu HY, Hua KF, Lin CC, Lin CH, Hsu J, Wong CH. Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways. J Immunol 2004; 173(10): Cao LZ, Lin ZB. Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides. Immunol Lett 2002; 83(3): Lin KI, Kao YY, Kuo HK, et al. Reishi polysaccharides induce immunoglobulin production through the TLR4/ TLR2-mediated induction of transcription factor Blimp-1. J Biol Chem 2006; 281(38): Wu R, Lian XZ, Fu YL, Xia C, Kang SL. Relation of free radical and apoptosis in duckling with selenium poisoning. Zhong Guo Shou Yi Ke Xue 2007; 27(5): He XM, Mo XM, Gu Q, et al. Effect of diazoxide on oxygen free radicals and cell apoptosis in brain tissue after deep hypothermia cerebral ischemia reperfusion injury in young rats. Zhong Guo Wai Ke Xue 2010; 48(2): Yao J, Luo GQ, Zhang YF, Cui DW, Yang LR. Determination and significance of the level of super oxide dismutase and lipid per oxidation and tumor necrosis factor-α in serum of patients with nasopharyngeal carcinoma. Zhong Guo Mian Yi Xue Za Zhi 2003; 9(3): Li Y, Liu XY, Lou X. The effect of Ganoderma Lucidum on exhausted mice's resistance to oxidation and injury. Jilin Ti Yu Xue Yuan Xue Bao 2008; 24(15): JTCM www. journaltcm. com 177