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Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapy-related fatigue in mice
Accepted Manuscript Title: Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapy-related fatigue in mice Author: Ming-zi Ouyang Li-zhu Lin Wen-jiao Lv Qian Zuo Zhuo Lv Jie-shan Guan Shu-tang Wang Ling-ling Sun Han-rui Chen Zhi-wei Xiao PII: DOI: Reference:
S0141-8130(16)30401-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.04.084 BIOMAC 6053
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
1-2-2016 19-4-2016 29-4-2016
Please cite this article as: Ming-zi Ouyang, Li-zhu Lin, Wen-jiao Lv, Qian Zuo, Zhuo Lv, Jie-shan Guan, Shu-tang Wang, Ling-ling Sun, Han-rui Chen, Zhi-wei Xiao, Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapyrelated fatigue in mice, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.04.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapy-related fatigue in mice
Ming-zi Ouyanga,b, Li-zhu Lina,b*[email protected], Wen-jiao Lva,b, Qian Zuoa,b, Zhuo Lva,b, Jie-shan Guana, Shu-tang Wanga, Ling-ling Suna,Han-rui Chena, Zhi-wei Xiaoa
a
Department of the oncology centre, First Affiliated Hospital, Guangzhou University of Traditional
Chinese Medicine, Guangzhou, Guangdong 510407, China b
First Clinic Medical School, Guangzhou University of Traditional Chinese Medicine，Guangzhou,
Guangdong 510405, China
*
Corresponding authors: Department of the oncology centre, First Affiliated Hospital, Guangzhou
University of Traditional Chinese Medicine, 16 Airport Road, Baiyun District, Guangzhou, Guangdong 510407, China. Tel.: +86 20 36596356.
Abstract The weight-loaded swimming capability, tumor growth, survival time and biochemical markers of Ganoderma lucidum polysaccharides (GLPs) in a chemotherapy-related fatigue mouse model were tested in the present study. The results showed that the middle-dose GLPs(GLP-M) and the high-dose GLPs(GLP-H) could increase the exhausting swimming time, which was observed to decrease in the cisplatin control group(PCG) and the tumor control group(TCG).The GLP-M and the GLP-H had reduced serum levels of tumor necrosis factor-αand interleukin-6, which were up-regulated by cisplatin. Cisplatin and the presence of tumor significantly enhanced the malondialdehyde(MDA) content and inhibited the activity of superoxide dismutase(SOD) in the muscle. Administration of GLPs at a high dose decreased the levels of MDA and up-regulated the SOD activity. The high-dose GLPs + cisplatin group presented a decreased tendency of tumor volume and a lower tumor weight compared with PCG. Moreover, the mice in the GLP-M and GLP-H groups had longer survival times compared with the mice in the TCG and PCG.The levels of creatinine and serum blood urea nitrogen, which are up-regulated by cisplatin, were significantly reduced by GLP-M and GLP-H. Therefore, these results suggest that GLPs might improve chemotherapy-related fatigue via regulation of inflammatory responses, oxidative stress and reduction of nephrotoxicity.
Keyword Ganoderma lucidum polysaccharides; Chemotherapy-Related Fatigue; Mouse model
1. Introduction Lung cancer is the leading cause of cancer-related death in China, with the mortality increasing by 464.84% over the past three decades[1]. Approximately 80% of lung cancer cases are non-small cell lung cancer (NSCLC) and are typically in an inoperable advanced stage when diagnosed. Therefore, systemic chemotherapy continues to be the mainstay of treatment for NSCLC patients. Cisplatin is the most common and active anti-cancer agent used in the combination treatment regimen for NSCLC[2]. However, 80-96% of patients are estimated to have experienced chemotherapy-related fatigue(CRF)[3, 4].Fatigue is a key reason for patients to discontinue treatment[5],and it has a profound negative impact on the quality of life of the patients after completion of treatment[6]. Methylphenidate is the only pharmacologic treatment for fatigue that is recommended by the National Comprehensive Cancer Network guidelines; however, as a central nervous system stimulant, the most commonly observed adverse effects of methylphenidate, such as appetite loss, dry mouth, anxiety/nervousness, nausea and insomnia, will reduce the quality of life of the fatigue patients[7, 8]. In this regard, more attention should be given to CRF. Additionally, new strategies are needed for CRF treatment that does not have side effects. Ganoderma lucidum (LEYSS, ex FR., G. lucidum), a genus of polypore mushrooms, is a traditional Chinese medicinal herb that is widely used in China, Japan, Korea and other Asian countries. Recorded in the Chinese medical classic Shennong’s Herba, it is considered to be a top grade herb in China for promoting longevity and improvingvigor without appreciable adverse effects [9].G. lucidum contains species of bioactive components such as polysaccharidess, triterpenoids and alkaloids[10]. Modern research has indicated that polysaccharides are the main chemical components related to the bioactivities and pharmacological properties of G. lucidum [11-13]. Over the past two decades, numerous studies have demonstrated that G. lucidum polysaccharides(GLPs) is an effective medication to improve the quality of life and nutritional status of individuals[12, 14], and some clinical studies of G.
lucidum have shown beneficial effects on CRF and the quality of life among breast cancer patients[15].Furthermore, GLPs has been shown to exhibit significant anti-tumor effects by its immunomodulatory function both in vitro and in vivo[16, 17]. Based on these findings, it is possible that GLPs may represent an alternative therapy to CRF. However, there have been no reports of the effect of GLPs in a cisplatin-induced CRF animal model. In the present study, we have focused on the weight-loaded swimming capability, the tumor growth, the survival time and the biochemical marker levels of GLPs in an A549 cell-inoculated lung cancer model with cisplatin treatment to evaluate if GLPs has beneficial effects on CRF.
2. Materials and Methods 2.1. Reagents Ganoderma lucidum was purchased from Guangdong Tianchen Decoction Pieces of Traditional Chinese Medicine Co., Ltd. (Guangdong, China). Cisplatin was purchased from Haosen Pharmaceutical Co., Ltd. (Jiangsu, China). Reagent kits for the determination of TNF-α, IL-6 and IL-1β were purchased from Qiyun Biotechnology Co. (Guangdong, China). Assay kits for the determination of serum blood urea nitrogen(BUN),
creatinine(CRE), superoxide dismutase (SOD) activity and
malondialdehyde (MDA) were acquired from Zuoke Biotechnology Co. (Guangdong, China). 2.2. Cell culture The A549 cells were acquired from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI 1640 containing 10% (v/v) heat-inactivated fetal calf serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) at 37°C in a humidified atmosphere with 5% CO2. 2.3. Animals Male BALB/c-nu mice (five-six weeks old, weighing 16-20 g) were purchased from
Medical Laboratory Animal Center of Guangdong province (Approval No. SCXK (Yue) 2013-0092, Foshan, China). Mice were acclimatized for one week prior to the experiment and housed at room temperature (23°C ± 1°C and under a 12h-light-and-12h-dark cycle. Food and water were available ad libitum. Mice were treated in compliance with the current laws of the Guiding Principles for the Care and Use of Laboratory Animals approved by the Animal Research Ethics Committee of Guangzhou University of Chinese Medicine. 2.4. Preparation of GLPs As in previously studies[18, 19],GLPs was extracted in boiling water for 8 to 12 h from the G. lucidum, then the whole extract water was fractionated by ethanol precipitation, dialysis and protein depletion using the Sevag method. The crude polysaccharides obtained from G. lucidum were determined by gel permeation chromatography and HPLC for the purity and molecular weight distribution [20]. The total yield of GLPs from starting crude materials was 1.04% (w/w). As a peptide-bound polysaccharides, the GLPs consisted of approximately 94.2% polysaccharides and 5.8% peptides. GLPs had a molecular weight of 372,190, consisting of D-mannose, D-fructose, D-rhamnose, D-xylose, D-galactose, D-glucose and uronic acid. GLPs was freeze-dried as a hazel-colored, water-soluble powder and stored at 4°C until further use. 2.5. Chemotherapy-Related Fatigue (CRF) animal model and drug treatment Mice underwent a weight-loaded swimming test before the experiment, and the mice that swam for the longest and shortest period were excluded. A549 cells (5×106 cells/0.2 ml/mouse) cultured in RPMI 1640 and matrigel were subcutaneously injected in the right flanks of the mice. All mice were inoculated, and controls were injected with phosphate buffered saline. Tumors formed within seven days, and the treatment started when the tumor size reached 80-100 mm3.The mice were then randomly divided into nine groups: the normal control group(NCG), the tumor control group (TCG), the low-dose GLPs group (GLPs, p.o., once daily, 50 mg/kg) (GLP-LG), the middle-dose GLPs group (GLPs, p.o., once daily, 100 mg/kg) (GLP-MG), the high-dose GLPs group (GLPs, p.o., once daily, 200 mg/kg) (GLP-HG), the cisplatin
control group(cisplatin, i.p., every two days, 10 mg/kg and normal saline, p.o., once daily, the same dose as GLP) (PCG), the low-dose GLPs + cisplatin group (GLPs, p.o., once daily, 50 mg/kg and cisplatin, i.p., every two days, 10 mg/kg) (GLP-L+PG), the middle-dose GLPs + cisplatin group (GLPs, p.o., once daily, 100 mg/kg and cisplatin, i.p., every two days, 10 mg/kg) (GLP-M+PG)and the high-dose GLPs + cisplatin group (GLPs, p.o., once daily, 200 mg/kg and cisplatin, i.p., every two days, 10 mg/kg) (GLP-L+PG). The TCG and the NCG received normal saline in volumes equivalent to those used for injection and intragastric administration of the drugs. The duration of treatment was 21 days. In each group, 20 mice were randomly divided into two subgroups. 2.6. Weight-loaded swimming test All of the mice in each group underwent a weight-loaded swimming test[21] every seven days as follows: each mouse was loaded with a tin wire (7 % of body weight) on the tail root and placed individually in a swimming pool (25 cm in diameter, 30 cm high), in which the mice could only support themselves by touching the bottom with their feet (at 25°C±1°C). The mice were determined to be exhausted when they failed to rise to the surface of water to breathe within a 10 s period, and the time spent by the mouse floating in the water with struggling and making necessary movements until exhausting its strength was negatively correlated with the level of fatigue. At the end of the session, the mice were removed from the water, dried with a paper towel and placed back in their original cages. Water in the container was drained after each session. 2.7. Determination of blood biomarkers After 21 days, the mice in subgroup 1 were randomly removed from each group for analyses of blood biochemical parameters. Mice were fasted for 8 h and were then anesthetized using pentobarbital sodium. The blood samples of the mice were collected by removal of the left eyeball and stored in tubes without anticoagulant. Serum was prepared by centrifugation at 1,000×g at 4°C for 15 min. The serum was tested to determine the level of nephrotoxicity (BUN and CRE) and the expression of inflammatory cytokines (TNF-α, IL-6 and IL-1β). The biomarkers were tested
following the recommended procedures provided in the kits. 2.8. Determination of muscle SOD activity and MDA After the blood was collected, the gastrocnemius muscle of the mice was immediately dissected and stored in liquid nitrogen for the following analysis. The levels of SOD activities and MDA were measured with a kit following the manufacturer’s protocol. 2.9. Observation of tumor volume, tumor weight and survival time Tumors sizes of each group were measured every three days using calipers, and the tumor volume was calculated using the following formula: tumor volume (mm3) = L×W2×0.50, where L is the longest diameter and W is the perpendicular short diameter. After the blood was collected, tumors were excised from the mice and weighed, and the mice in subgroup 2(the remaining 10 mice of each group) were observed for survival analysis. 2.10. Statistical analysis Statistical analyses were conducted using SPSS 17.0 software. The results are expressed as the mean ± standard error (SE) in the tables and indicated by vertical bars in the figures. Differences between the groups were determined by ANOVA and Student’s t-test. For the survival time of animals, Kaplan-Meier curves were established for each group, and the survival times were compared by a log-rank test. Probability value P less than 0.05 was considered significant.
3. Results and discussion 3.1. Effects on the Weight-Loaded Swimming Test In the present study, BALB/c-mice were inoculated with A549 cells and were treated with cisplatin in a tumor-bearing state to mimic cancer and chemotherapy-related fatigue scenarios observed in human patients. The weight-loaded swimming time was used for the evaluation of fatigue, and the model was used to evaluate the effect of GLPs on the fatigue caused by tumor presence and chemotherapy. As shown in Figure 1(a, b, c), compared with the NCG, the weight-loaded swimming time was significantly reduced in response to administration of cisplatin starting on the 7th day
after treatment and persisted for as long as 21 days, consistent with prior studies[22].However, the swimming time of the TCG gradually decreased from Day 14,but was longer than observed in the PCG (Figure 1(a), P<0.05-0.01). Moreover, compared with the PCG, the swimming time of the GLP-M+PG and GLP-H+PG significantly increased from Day 14 (Figure 1(c), P< 0.05-0.01), meanwhile, the swimming time of the GLP-MG and GLP-HG had a increased trend from Day 7 and showed significantly enhancement until Day 21 compared with the TCG (Figure 1(b), P< 0.01).These results suggest that both tumor and cisplatin could reduce the weight-loaded swimming time of mice, demonstrating that this animal model is well established for the evaluation of drug efficacy on fatigue. It was also observed that compared with the fatigue induced by the presence of a tumor, the fatigue induced by cisplatin occurred earlier and was more severe, and the effect of GLPs on improving the fatigue induced by cisplatin was more obvious and earlier, which confirming the anti-chemotherapy-related fatigue property of GLPs. 3.2. Effects on serum TGF-α, IL-6 and IL-1β It has been speculated that the increase in production of inflammatory cytokines, which is induced by common cancer treatments, especially chemotherapy, may be correlated with CRF[23-27]. In these cytokines, the TNF-α, IL-1β and IL-6 play important roles in the development of fatigue[23, 28-31]. Previous reports have demonstrated that GLPs could modulate immune function according to suppressing the secretion of the inflammatory cytokines TNF-α, IL-6 and inflammatory mediator nitric oxide [32, 33]. Similarly, as shown in Figure 2(a, b, c), the results of the present study demonstrated that cisplatin induced an significant increase in the levels of TNF-α, IL-6 and IL-1β than the NCG and TCG(P<0.01). GLPs reduced the serum levels of TNF-α and IL-6 (P<0.05-0.01), but did not decrease the level of IL-1β, which is up-regulated by cisplatin. The levels of TNF-α and IL-6 in TCG were also higher than in the NCG (P<0.05), but there was no significant increase in the level of IL-1β.Compaired with TCG, GLP-LG, GLP-MG and GLP-HG had the trend to down-regulate the levels of TNF-α and IL-6, but there had no statistical significance. These observations suggested that the increase in production of TNF-α, IL-6 and
IL-1β, which was induced by cisplatin, might be correlated with CRF , the anti-fatigue effect of GLPs might be correlated with the modulation of levels of TNF-α and IL-6. As in a prior study, these cytokines further stimulate the production of several important inflammatory mediators, such as reactive oxygen species (ROS) [34], which could cause oxidative damage to cellular components. 3.3. Effects on muscle SOD activity and MDA In recent years, many studies have shown that ROS may be responsible for, or contribute to, muscle weakness and development of fatigue[35-38] by increasing lipid peroxidation, which can ultimately produce MDA and intoxicate cells and cell membranes. The natural cellular antioxidant enzyme SOD is accepted as one of the most important physiological antioxidants that mediates free radicals and prevents subsequent lipid peroxidation by increasing the rate at which they are removed [37]. There has been increasing evidence that some polysaccharides isolated from plants, herbs and fungi have beneficial activities as antioxidants by scavenging free radicals[39-41].As shown in a prior study, GLPs exhibited hydrogen oxide radical scavenging function with SOD-like activity[32]. In the present study, as shown in Figure 3(a, b), both of the PCG and TCG significantly enhanced the MDA content and inhibited the activity of SOD in muscle (P<0.05-0.01), and the regulate effect of the PCG was more obvious than the TCG (P<0.05-0.01) Administration of GLP-M and GLP-H decreased the levels of MDA and up-regulated the SOD activity caused by cisplatin (P<0.05-0.01), however, only the administration of GLP-H decreased the levels of MDA and up-regulated the SOD activity caused by tumor (P<0.05). These results suggested that the changes of MDA levels and SOD activity in the muscle which induced by cisplatin treatment and tumor presence might have direct contact with the weight-loaded swimming ability of mice. GLPs might operate by SOD-like activity directly, or it may interact with endogenous antioxidants to inhibit lipid peroxidationin muscle tissues to defend against fatigue. 3.4. Effects on serum BUN and CRE Cisplatin is toxic and might cause kidney damage, as indicated by high CRE and BUN levels [42], which are also closely related to fatigue [43]. In the present study,
CRE and BUN levels were measured to evaluate kidney damage. As shown in Figure 4(a, b), the groups treated with cisplatin exhibited higher levels of CRE and BUN compared with the NCG and the other tumor bearing mice, and the cisplatin-induced levels of CRE and BUN were significantly reduced by treatment with GLP-M and GLP-H (P<0.05-0.01). These observations suggested that GLPs might improve fatigue by reducing nephrotoxicity which was induced by cisplatin. 3.5. Effects on tumor weight and volume The antitumor activity of GLPs in the implant mouse model of A549 cells in combination with cisplatin treatment was measured. As shown in Figure 5 and Table 1,until the end of the experiment, the tumor volume and weight in the cisplatin control group and the combination groups(PCG, GLP-L+PG, GLP-M+PG and GLP-H+PG) were significantly lower than that of the TCG(P<0.01).The GLP-H+PG also presented a decreased tendency of tumor volume beginning on Day 15 and exhibited a lighter tumor weight at the end in comparison with PCG (P<0.05).The tumor inhibition rates of PCG, GLP-L+PG, GLP-M+PG and GLP-H+PG were 60.3%, 62.4%, 65.5% and 69.9%, respectively(compared with TCG, P<0.05-0.01).These results suggested that the GLPs might have the synergistic effects on antitumor activity with cisplatin. 3.6. Effects on the survival time of mice As shown in Figure 6,compared with mice in the TCG and PCG, the survival time in the GLP-MG, GLP-HG, GLP-M+PG and GLP-H+PG were significantly prolonged (P<0.05-0.01). These observations showed that GLPs might be an effective medication to improve the quality of life as show in the prior studies[12, 15].
4. Conclusion In summary, these findings suggest that GLPs may improve chemotherapy-related fatigue via the regulation of inflammatory responses, oxidative stress and reduction of nephrotoxicity, the results also presented the synergistic effects of GLPs on antitumor activity with cisplatin and the role of improving quality of life. However, this experiment focused only on body fatigue, and further research is needed to assess the
effect of GLPs on central fatigue.
Abbreviations GLPs: Ganoderma lucidum polysaccharides; NCG: the normal control group; TCG: the tumor control group; GLP-LG: the low-dose GLP group; GLP-MG: the middle-dose GLP group; GLP-HG: the high-dose GLP group; PCG: the cisplatin control group; GLP-L+PG: the low-dose GLP + cisplatin group; GLP-M+PG :the middle-dose GLP + cisplatin group; GLP-L+PG: the high-dose GLP + cisplatin group; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; IL-1β: interleukin-1β; SOD: superoxide dismutase; MDA: malondialdehyde; BUN: serum blood urea nitrogen; CRE: creatinine; ROS: reactive oxygen species.
Competing interests The authors declare that there are no conflicts of interest.
Author contributions Ming-zi Ouyang designed the study, conducted the experiments and wrote the manuscript. Wen-jiao Lv, Qian Zuo, Zhuo Lv, Jie-shan Guan, Shu-tang Wang, Han-rui Chen, Zhi-wei Xiao, performed the experiments and analyzed the data. Li-zhu Lin provided the initial idea. All authors read and approved the final manuscript.
Acknowledgments This work was supported by the Natural Science Foundation of China (Grant NO.81403340), the Natural Science Foundation of China (Grant NO. 81273963) and the Natural Science Foundation of Guangdong Province, China(Grant NO. 2014A030310063).
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Figure1a. Effects of A549 cell inoculation and cisplatin treatment on the Weight-Loaded Swimming Test. Each value represents the mean±SE. Compared with the TCG, *P<0.05, **P<0.01.Compared with the PCG, # P<0.05，## P<0.01.
Figure1b. Effects of GLPs on the Weight-Loaded Swimming Test of the A549 cell inoculated mice. Each value represents the mean±SE. Compared with the TCG, *P<0.05，**P<0.01.
Figure1c. Effects of
GLPs on the Weight-Loaded Swimming Test of the A549 cell inoculated and
cisplatin treated mice .Each value represents the mean±SE. Compared with the PCG, #P<0.05， ##
P<0.01.
Figure2a. Effects of GLPs on the TGF-α of the A549 cell inoculated and cisplatin treated mice . Each value represents the mean±SE. Compared with the NCG,
P<0.05， P<0.01.Compared with
△
△△
the TCG, * P<0.05，** P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure2b. Effects of GLPs on the IL-6 of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure2c. Effects of GLPs on the IL-1β of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, * P<0.05，** P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure3a. Effects of GLPs on the SOD of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure3b. Effects of GLPs on the MDA of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, * P<0.05，** P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure4a. Effects of GLPs on the BUN of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure4b. Effects of GLPs on the CRE of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure5. Effects of GLPs on the tumor volume of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01
Figure6. Effects of GLPs on the survival time of mice.Compared with the TCG, *P<0.05， **P<0.01.Compared with the PCG, #P<0.05，##P<0.01
Table 1. The weight of tumor in each groups ( x ± s ，n=10)
Tumor weight（ （g） ）
Groups
N
TCG
10
1.730±0.519##
--
GLP-LG
10
1.406±0.478##
18.7
GLP-MG
10
1.404±0.338##
18.8
GLP-HG
10
1.371±0.180##
20.7
PCG
10
0.687±0.159**
60.3
GLP-L+PG
10
0.650±0.247**
62.4
GLP-M+PG
10
0.596±0.143**#
65.5
GLP-H+PG
10
0.519±0.158**#
69.9
Compared with TCG,
*
Tumor inhibition rate(%)
P<0.05，** P<0.01, compared with PCG, #P<0.05，##P<0.01.
S0141-8130(16)30401-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.04.084 BIOMAC 6053
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
1-2-2016 19-4-2016 29-4-2016
Please cite this article as: Ming-zi Ouyang, Li-zhu Lin, Wen-jiao Lv, Qian Zuo, Zhuo Lv, Jie-shan Guan, Shu-tang Wang, Ling-ling Sun, Han-rui Chen, Zhi-wei Xiao, Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapyrelated fatigue in mice, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.04.084 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of the polysaccharides extracted from Ganoderma lucidum on chemotherapy-related fatigue in mice
Ming-zi Ouyanga,b, Li-zhu Lina,b*[email protected], Wen-jiao Lva,b, Qian Zuoa,b, Zhuo Lva,b, Jie-shan Guana, Shu-tang Wanga, Ling-ling Suna,Han-rui Chena, Zhi-wei Xiaoa
a
Department of the oncology centre, First Affiliated Hospital, Guangzhou University of Traditional
Chinese Medicine, Guangzhou, Guangdong 510407, China b
First Clinic Medical School, Guangzhou University of Traditional Chinese Medicine，Guangzhou,
Guangdong 510405, China
*
Corresponding authors: Department of the oncology centre, First Affiliated Hospital, Guangzhou
University of Traditional Chinese Medicine, 16 Airport Road, Baiyun District, Guangzhou, Guangdong 510407, China. Tel.: +86 20 36596356.
Abstract The weight-loaded swimming capability, tumor growth, survival time and biochemical markers of Ganoderma lucidum polysaccharides (GLPs) in a chemotherapy-related fatigue mouse model were tested in the present study. The results showed that the middle-dose GLPs(GLP-M) and the high-dose GLPs(GLP-H) could increase the exhausting swimming time, which was observed to decrease in the cisplatin control group(PCG) and the tumor control group(TCG).The GLP-M and the GLP-H had reduced serum levels of tumor necrosis factor-αand interleukin-6, which were up-regulated by cisplatin. Cisplatin and the presence of tumor significantly enhanced the malondialdehyde(MDA) content and inhibited the activity of superoxide dismutase(SOD) in the muscle. Administration of GLPs at a high dose decreased the levels of MDA and up-regulated the SOD activity. The high-dose GLPs + cisplatin group presented a decreased tendency of tumor volume and a lower tumor weight compared with PCG. Moreover, the mice in the GLP-M and GLP-H groups had longer survival times compared with the mice in the TCG and PCG.The levels of creatinine and serum blood urea nitrogen, which are up-regulated by cisplatin, were significantly reduced by GLP-M and GLP-H. Therefore, these results suggest that GLPs might improve chemotherapy-related fatigue via regulation of inflammatory responses, oxidative stress and reduction of nephrotoxicity.
Keyword Ganoderma lucidum polysaccharides; Chemotherapy-Related Fatigue; Mouse model
1. Introduction Lung cancer is the leading cause of cancer-related death in China, with the mortality increasing by 464.84% over the past three decades[1]. Approximately 80% of lung cancer cases are non-small cell lung cancer (NSCLC) and are typically in an inoperable advanced stage when diagnosed. Therefore, systemic chemotherapy continues to be the mainstay of treatment for NSCLC patients. Cisplatin is the most common and active anti-cancer agent used in the combination treatment regimen for NSCLC[2]. However, 80-96% of patients are estimated to have experienced chemotherapy-related fatigue(CRF)[3, 4].Fatigue is a key reason for patients to discontinue treatment[5],and it has a profound negative impact on the quality of life of the patients after completion of treatment[6]. Methylphenidate is the only pharmacologic treatment for fatigue that is recommended by the National Comprehensive Cancer Network guidelines; however, as a central nervous system stimulant, the most commonly observed adverse effects of methylphenidate, such as appetite loss, dry mouth, anxiety/nervousness, nausea and insomnia, will reduce the quality of life of the fatigue patients[7, 8]. In this regard, more attention should be given to CRF. Additionally, new strategies are needed for CRF treatment that does not have side effects. Ganoderma lucidum (LEYSS, ex FR., G. lucidum), a genus of polypore mushrooms, is a traditional Chinese medicinal herb that is widely used in China, Japan, Korea and other Asian countries. Recorded in the Chinese medical classic Shennong’s Herba, it is considered to be a top grade herb in China for promoting longevity and improvingvigor without appreciable adverse effects [9].G. lucidum contains species of bioactive components such as polysaccharidess, triterpenoids and alkaloids[10]. Modern research has indicated that polysaccharides are the main chemical components related to the bioactivities and pharmacological properties of G. lucidum [11-13]. Over the past two decades, numerous studies have demonstrated that G. lucidum polysaccharides(GLPs) is an effective medication to improve the quality of life and nutritional status of individuals[12, 14], and some clinical studies of G.
lucidum have shown beneficial effects on CRF and the quality of life among breast cancer patients[15].Furthermore, GLPs has been shown to exhibit significant anti-tumor effects by its immunomodulatory function both in vitro and in vivo[16, 17]. Based on these findings, it is possible that GLPs may represent an alternative therapy to CRF. However, there have been no reports of the effect of GLPs in a cisplatin-induced CRF animal model. In the present study, we have focused on the weight-loaded swimming capability, the tumor growth, the survival time and the biochemical marker levels of GLPs in an A549 cell-inoculated lung cancer model with cisplatin treatment to evaluate if GLPs has beneficial effects on CRF.
2. Materials and Methods 2.1. Reagents Ganoderma lucidum was purchased from Guangdong Tianchen Decoction Pieces of Traditional Chinese Medicine Co., Ltd. (Guangdong, China). Cisplatin was purchased from Haosen Pharmaceutical Co., Ltd. (Jiangsu, China). Reagent kits for the determination of TNF-α, IL-6 and IL-1β were purchased from Qiyun Biotechnology Co. (Guangdong, China). Assay kits for the determination of serum blood urea nitrogen(BUN),
creatinine(CRE), superoxide dismutase (SOD) activity and
malondialdehyde (MDA) were acquired from Zuoke Biotechnology Co. (Guangdong, China). 2.2. Cell culture The A549 cells were acquired from Shanghai Institute of Biochemistry and Cell Biology of Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI 1640 containing 10% (v/v) heat-inactivated fetal calf serum and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) at 37°C in a humidified atmosphere with 5% CO2. 2.3. Animals Male BALB/c-nu mice (five-six weeks old, weighing 16-20 g) were purchased from
Medical Laboratory Animal Center of Guangdong province (Approval No. SCXK (Yue) 2013-0092, Foshan, China). Mice were acclimatized for one week prior to the experiment and housed at room temperature (23°C ± 1°C and under a 12h-light-and-12h-dark cycle. Food and water were available ad libitum. Mice were treated in compliance with the current laws of the Guiding Principles for the Care and Use of Laboratory Animals approved by the Animal Research Ethics Committee of Guangzhou University of Chinese Medicine. 2.4. Preparation of GLPs As in previously studies[18, 19],GLPs was extracted in boiling water for 8 to 12 h from the G. lucidum, then the whole extract water was fractionated by ethanol precipitation, dialysis and protein depletion using the Sevag method. The crude polysaccharides obtained from G. lucidum were determined by gel permeation chromatography and HPLC for the purity and molecular weight distribution [20]. The total yield of GLPs from starting crude materials was 1.04% (w/w). As a peptide-bound polysaccharides, the GLPs consisted of approximately 94.2% polysaccharides and 5.8% peptides. GLPs had a molecular weight of 372,190, consisting of D-mannose, D-fructose, D-rhamnose, D-xylose, D-galactose, D-glucose and uronic acid. GLPs was freeze-dried as a hazel-colored, water-soluble powder and stored at 4°C until further use. 2.5. Chemotherapy-Related Fatigue (CRF) animal model and drug treatment Mice underwent a weight-loaded swimming test before the experiment, and the mice that swam for the longest and shortest period were excluded. A549 cells (5×106 cells/0.2 ml/mouse) cultured in RPMI 1640 and matrigel were subcutaneously injected in the right flanks of the mice. All mice were inoculated, and controls were injected with phosphate buffered saline. Tumors formed within seven days, and the treatment started when the tumor size reached 80-100 mm3.The mice were then randomly divided into nine groups: the normal control group(NCG), the tumor control group (TCG), the low-dose GLPs group (GLPs, p.o., once daily, 50 mg/kg) (GLP-LG), the middle-dose GLPs group (GLPs, p.o., once daily, 100 mg/kg) (GLP-MG), the high-dose GLPs group (GLPs, p.o., once daily, 200 mg/kg) (GLP-HG), the cisplatin
control group(cisplatin, i.p., every two days, 10 mg/kg and normal saline, p.o., once daily, the same dose as GLP) (PCG), the low-dose GLPs + cisplatin group (GLPs, p.o., once daily, 50 mg/kg and cisplatin, i.p., every two days, 10 mg/kg) (GLP-L+PG), the middle-dose GLPs + cisplatin group (GLPs, p.o., once daily, 100 mg/kg and cisplatin, i.p., every two days, 10 mg/kg) (GLP-M+PG)and the high-dose GLPs + cisplatin group (GLPs, p.o., once daily, 200 mg/kg and cisplatin, i.p., every two days, 10 mg/kg) (GLP-L+PG). The TCG and the NCG received normal saline in volumes equivalent to those used for injection and intragastric administration of the drugs. The duration of treatment was 21 days. In each group, 20 mice were randomly divided into two subgroups. 2.6. Weight-loaded swimming test All of the mice in each group underwent a weight-loaded swimming test[21] every seven days as follows: each mouse was loaded with a tin wire (7 % of body weight) on the tail root and placed individually in a swimming pool (25 cm in diameter, 30 cm high), in which the mice could only support themselves by touching the bottom with their feet (at 25°C±1°C). The mice were determined to be exhausted when they failed to rise to the surface of water to breathe within a 10 s period, and the time spent by the mouse floating in the water with struggling and making necessary movements until exhausting its strength was negatively correlated with the level of fatigue. At the end of the session, the mice were removed from the water, dried with a paper towel and placed back in their original cages. Water in the container was drained after each session. 2.7. Determination of blood biomarkers After 21 days, the mice in subgroup 1 were randomly removed from each group for analyses of blood biochemical parameters. Mice were fasted for 8 h and were then anesthetized using pentobarbital sodium. The blood samples of the mice were collected by removal of the left eyeball and stored in tubes without anticoagulant. Serum was prepared by centrifugation at 1,000×g at 4°C for 15 min. The serum was tested to determine the level of nephrotoxicity (BUN and CRE) and the expression of inflammatory cytokines (TNF-α, IL-6 and IL-1β). The biomarkers were tested
following the recommended procedures provided in the kits. 2.8. Determination of muscle SOD activity and MDA After the blood was collected, the gastrocnemius muscle of the mice was immediately dissected and stored in liquid nitrogen for the following analysis. The levels of SOD activities and MDA were measured with a kit following the manufacturer’s protocol. 2.9. Observation of tumor volume, tumor weight and survival time Tumors sizes of each group were measured every three days using calipers, and the tumor volume was calculated using the following formula: tumor volume (mm3) = L×W2×0.50, where L is the longest diameter and W is the perpendicular short diameter. After the blood was collected, tumors were excised from the mice and weighed, and the mice in subgroup 2(the remaining 10 mice of each group) were observed for survival analysis. 2.10. Statistical analysis Statistical analyses were conducted using SPSS 17.0 software. The results are expressed as the mean ± standard error (SE) in the tables and indicated by vertical bars in the figures. Differences between the groups were determined by ANOVA and Student’s t-test. For the survival time of animals, Kaplan-Meier curves were established for each group, and the survival times were compared by a log-rank test. Probability value P less than 0.05 was considered significant.
3. Results and discussion 3.1. Effects on the Weight-Loaded Swimming Test In the present study, BALB/c-mice were inoculated with A549 cells and were treated with cisplatin in a tumor-bearing state to mimic cancer and chemotherapy-related fatigue scenarios observed in human patients. The weight-loaded swimming time was used for the evaluation of fatigue, and the model was used to evaluate the effect of GLPs on the fatigue caused by tumor presence and chemotherapy. As shown in Figure 1(a, b, c), compared with the NCG, the weight-loaded swimming time was significantly reduced in response to administration of cisplatin starting on the 7th day
after treatment and persisted for as long as 21 days, consistent with prior studies[22].However, the swimming time of the TCG gradually decreased from Day 14,but was longer than observed in the PCG (Figure 1(a), P<0.05-0.01). Moreover, compared with the PCG, the swimming time of the GLP-M+PG and GLP-H+PG significantly increased from Day 14 (Figure 1(c), P< 0.05-0.01), meanwhile, the swimming time of the GLP-MG and GLP-HG had a increased trend from Day 7 and showed significantly enhancement until Day 21 compared with the TCG (Figure 1(b), P< 0.01).These results suggest that both tumor and cisplatin could reduce the weight-loaded swimming time of mice, demonstrating that this animal model is well established for the evaluation of drug efficacy on fatigue. It was also observed that compared with the fatigue induced by the presence of a tumor, the fatigue induced by cisplatin occurred earlier and was more severe, and the effect of GLPs on improving the fatigue induced by cisplatin was more obvious and earlier, which confirming the anti-chemotherapy-related fatigue property of GLPs. 3.2. Effects on serum TGF-α, IL-6 and IL-1β It has been speculated that the increase in production of inflammatory cytokines, which is induced by common cancer treatments, especially chemotherapy, may be correlated with CRF[23-27]. In these cytokines, the TNF-α, IL-1β and IL-6 play important roles in the development of fatigue[23, 28-31]. Previous reports have demonstrated that GLPs could modulate immune function according to suppressing the secretion of the inflammatory cytokines TNF-α, IL-6 and inflammatory mediator nitric oxide [32, 33]. Similarly, as shown in Figure 2(a, b, c), the results of the present study demonstrated that cisplatin induced an significant increase in the levels of TNF-α, IL-6 and IL-1β than the NCG and TCG(P<0.01). GLPs reduced the serum levels of TNF-α and IL-6 (P<0.05-0.01), but did not decrease the level of IL-1β, which is up-regulated by cisplatin. The levels of TNF-α and IL-6 in TCG were also higher than in the NCG (P<0.05), but there was no significant increase in the level of IL-1β.Compaired with TCG, GLP-LG, GLP-MG and GLP-HG had the trend to down-regulate the levels of TNF-α and IL-6, but there had no statistical significance. These observations suggested that the increase in production of TNF-α, IL-6 and
IL-1β, which was induced by cisplatin, might be correlated with CRF , the anti-fatigue effect of GLPs might be correlated with the modulation of levels of TNF-α and IL-6. As in a prior study, these cytokines further stimulate the production of several important inflammatory mediators, such as reactive oxygen species (ROS) [34], which could cause oxidative damage to cellular components. 3.3. Effects on muscle SOD activity and MDA In recent years, many studies have shown that ROS may be responsible for, or contribute to, muscle weakness and development of fatigue[35-38] by increasing lipid peroxidation, which can ultimately produce MDA and intoxicate cells and cell membranes. The natural cellular antioxidant enzyme SOD is accepted as one of the most important physiological antioxidants that mediates free radicals and prevents subsequent lipid peroxidation by increasing the rate at which they are removed [37]. There has been increasing evidence that some polysaccharides isolated from plants, herbs and fungi have beneficial activities as antioxidants by scavenging free radicals[39-41].As shown in a prior study, GLPs exhibited hydrogen oxide radical scavenging function with SOD-like activity[32]. In the present study, as shown in Figure 3(a, b), both of the PCG and TCG significantly enhanced the MDA content and inhibited the activity of SOD in muscle (P<0.05-0.01), and the regulate effect of the PCG was more obvious than the TCG (P<0.05-0.01) Administration of GLP-M and GLP-H decreased the levels of MDA and up-regulated the SOD activity caused by cisplatin (P<0.05-0.01), however, only the administration of GLP-H decreased the levels of MDA and up-regulated the SOD activity caused by tumor (P<0.05). These results suggested that the changes of MDA levels and SOD activity in the muscle which induced by cisplatin treatment and tumor presence might have direct contact with the weight-loaded swimming ability of mice. GLPs might operate by SOD-like activity directly, or it may interact with endogenous antioxidants to inhibit lipid peroxidationin muscle tissues to defend against fatigue. 3.4. Effects on serum BUN and CRE Cisplatin is toxic and might cause kidney damage, as indicated by high CRE and BUN levels [42], which are also closely related to fatigue [43]. In the present study,
CRE and BUN levels were measured to evaluate kidney damage. As shown in Figure 4(a, b), the groups treated with cisplatin exhibited higher levels of CRE and BUN compared with the NCG and the other tumor bearing mice, and the cisplatin-induced levels of CRE and BUN were significantly reduced by treatment with GLP-M and GLP-H (P<0.05-0.01). These observations suggested that GLPs might improve fatigue by reducing nephrotoxicity which was induced by cisplatin. 3.5. Effects on tumor weight and volume The antitumor activity of GLPs in the implant mouse model of A549 cells in combination with cisplatin treatment was measured. As shown in Figure 5 and Table 1,until the end of the experiment, the tumor volume and weight in the cisplatin control group and the combination groups(PCG, GLP-L+PG, GLP-M+PG and GLP-H+PG) were significantly lower than that of the TCG(P<0.01).The GLP-H+PG also presented a decreased tendency of tumor volume beginning on Day 15 and exhibited a lighter tumor weight at the end in comparison with PCG (P<0.05).The tumor inhibition rates of PCG, GLP-L+PG, GLP-M+PG and GLP-H+PG were 60.3%, 62.4%, 65.5% and 69.9%, respectively(compared with TCG, P<0.05-0.01).These results suggested that the GLPs might have the synergistic effects on antitumor activity with cisplatin. 3.6. Effects on the survival time of mice As shown in Figure 6,compared with mice in the TCG and PCG, the survival time in the GLP-MG, GLP-HG, GLP-M+PG and GLP-H+PG were significantly prolonged (P<0.05-0.01). These observations showed that GLPs might be an effective medication to improve the quality of life as show in the prior studies[12, 15].
4. Conclusion In summary, these findings suggest that GLPs may improve chemotherapy-related fatigue via the regulation of inflammatory responses, oxidative stress and reduction of nephrotoxicity, the results also presented the synergistic effects of GLPs on antitumor activity with cisplatin and the role of improving quality of life. However, this experiment focused only on body fatigue, and further research is needed to assess the
effect of GLPs on central fatigue.
Abbreviations GLPs: Ganoderma lucidum polysaccharides; NCG: the normal control group; TCG: the tumor control group; GLP-LG: the low-dose GLP group; GLP-MG: the middle-dose GLP group; GLP-HG: the high-dose GLP group; PCG: the cisplatin control group; GLP-L+PG: the low-dose GLP + cisplatin group; GLP-M+PG :the middle-dose GLP + cisplatin group; GLP-L+PG: the high-dose GLP + cisplatin group; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; IL-1β: interleukin-1β; SOD: superoxide dismutase; MDA: malondialdehyde; BUN: serum blood urea nitrogen; CRE: creatinine; ROS: reactive oxygen species.
Competing interests The authors declare that there are no conflicts of interest.
Author contributions Ming-zi Ouyang designed the study, conducted the experiments and wrote the manuscript. Wen-jiao Lv, Qian Zuo, Zhuo Lv, Jie-shan Guan, Shu-tang Wang, Han-rui Chen, Zhi-wei Xiao, performed the experiments and analyzed the data. Li-zhu Lin provided the initial idea. All authors read and approved the final manuscript.
Acknowledgments This work was supported by the Natural Science Foundation of China (Grant NO.81403340), the Natural Science Foundation of China (Grant NO. 81273963) and the Natural Science Foundation of Guangdong Province, China(Grant NO. 2014A030310063).
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Figure1a. Effects of A549 cell inoculation and cisplatin treatment on the Weight-Loaded Swimming Test. Each value represents the mean±SE. Compared with the TCG, *P<0.05, **P<0.01.Compared with the PCG, # P<0.05，## P<0.01.
Figure1b. Effects of GLPs on the Weight-Loaded Swimming Test of the A549 cell inoculated mice. Each value represents the mean±SE. Compared with the TCG, *P<0.05，**P<0.01.
Figure1c. Effects of
GLPs on the Weight-Loaded Swimming Test of the A549 cell inoculated and
cisplatin treated mice .Each value represents the mean±SE. Compared with the PCG, #P<0.05， ##
P<0.01.
Figure2a. Effects of GLPs on the TGF-α of the A549 cell inoculated and cisplatin treated mice . Each value represents the mean±SE. Compared with the NCG,
P<0.05， P<0.01.Compared with
△
△△
the TCG, * P<0.05，** P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure2b. Effects of GLPs on the IL-6 of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure2c. Effects of GLPs on the IL-1β of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, * P<0.05，** P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure3a. Effects of GLPs on the SOD of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure3b. Effects of GLPs on the MDA of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, * P<0.05，** P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure4a. Effects of GLPs on the BUN of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure4b. Effects of GLPs on the CRE of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the NCG,
△
P<0.05，
P<0.01.Compared with the
△△
TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01.
Figure5. Effects of GLPs on the tumor volume of the A549 cell inoculated and cisplatin treated mice .Each value represents the mean±SE. Compared with the TCG, *P<0.05，**P<0.01.Compared with the PCG, #P<0.05，##P<0.01
Figure6. Effects of GLPs on the survival time of mice.Compared with the TCG, *P<0.05， **P<0.01.Compared with the PCG, #P<0.05，##P<0.01
Table 1. The weight of tumor in each groups ( x ± s ，n=10)
Tumor weight（ （g） ）
Groups
N
TCG
10
1.730±0.519##
--
GLP-LG
10
1.406±0.478##
18.7
GLP-MG
10
1.404±0.338##
18.8
GLP-HG
10
1.371±0.180##
20.7
PCG
10
0.687±0.159**
60.3
GLP-L+PG
10
0.650±0.247**
62.4
GLP-M+PG
10
0.596±0.143**#
65.5
GLP-H+PG
10
0.519±0.158**#
69.9
Compared with TCG,
*
Tumor inhibition rate(%)
P<0.05，** P<0.01, compared with PCG, #P<0.05，##P<0.01.