Whole body radioprotective activity of an acetone–water extract from the seedpod of Nelumbo nucifera Gaertn. seedpod

Food and Chemical Toxicology 48 (2010) 3374–3384

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Whole body radioprotective activity of an acetone–water extract from the seedpod of Nelumbo nucifera Gaertn. seedpod Y. Duan a,⇑, H. Zhang a, B. Xie b, Y. Yan c, J. Li a, F. Xu a, Y. Qin a a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China Department of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China c School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, China b

a r t i c l e

i n f o

Article history: Received 6 January 2010 Accepted 5 September 2010

Keywords: Irradiation injury Lotus seedpod Procyanidins Radioprotective activity

a b s t r a c t Procyanidins extracted with acetone–water from lotus (Nelumbo nucifera Gaertn.) seedpod (LSPCs) were evaluated for in vivo radioprotective activity against whole body gamma irradiation in Swiss albino mice. Pretreated with LSPCs 200 mg/kg by intragastric (i.g.) for 15 days was found to be the most effective dose in preventing radiation sickness, reducing radiation-induced mortality, increasing mean survival time and elevating radiation median lethal dose (LD50) from 8.9 to 10.5 Gy, indicating a dose modifying factor (DMF) of 1.18. Further, administered LSPCs at a dose of 200 mg/kg could effectively maintain spleen index close to normal, stimulate endogenous spleen colony forming units, promote the levels of red blood cells (RBC), white blood cells (WBC), platelets and hemoglobin in peripheral blood, and prevent spleen and skin damage in irradiated mice, reduce the level of radiation-induced micronucleated polychromatic erythrocytes in bone marrow, maintain the polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) ratio (P/N ratio) and significantly decrease bone marrow chromosomal damage. Alternatively, pretreated with LSPCs (200 mg/kg) significantly decreased the lipid peroxidation (LPO) level, and elevated the activities of endogenous antioxidant enzymes in liver after irradiation. Thus LSPCs possess a strong whole body radioprotective activity, and it may be used as a radioprotector. Published by Elsevier Ltd.

1. Introduction Ionizing radiation is an electromagnetic wave or particle capable of producing ions in its passage through matter, causing immediate chemical alterations in biological tissues. These alterations disrupt metabolic pathways, which can lead, after days or weeks, to cell damage and, potentially, cell dysfunction and death (Hosseinimehr, 2007). Radiation attenuates the endogenous antioxidant enzymes, which are considered as the first line defense mechanism in the maintenance of redox balance and normal biochemical processes (Sun et al., 1998). The exposure of mammals to ionizing radiation, such as gamma-radiation, can cause the

Abbreviations: CAT, catalase; CFU, colony forming units; DMF, dose modifying factor; GSH-Px, glutathione peroxidase; HE, hematoxylin and eosin; i.g., intragastric; LD50, median lethal dose; LPO, lipid peroxidation; LSPCs, procyanidins from lotus seedpod; MPCE, micronucleated polychromatic erythrocytes; NCE, normochromatic erythrocytes; NS, normal saline; P/N, polychromatic erythrocytes/ normochromatic erythrocytes; PCE, polychromatic erythrocytes; RBC, red blood cells; ROS, reactive oxygen species; SOD, superoxide dismutase; WBC, white blood cell. ⇑ Corresponding author. Tel./fax: +86 511 88780201. E-mail address: [email protected] (Y. Duan). 0278-6915/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.fct.2010.09.008

development of a complex, dose-dependent series of potentially fatal physiologic and morphologic changes, known as hematopoietic syndrome (Lahouel et al., 1987). Radiation-induced destruction of lymphoid and hematopoietic systems are the primary cause of septicemia and death. Enhanced susceptibility to infections with opportunistic microbes occurs in parallel with progressive radiation-induced atrophy of lymph nodes and the spleen (Orsolic´ et al., 2007). Recently, the synthetic agents WR2721 (amifostine), OK-432 (picibanil) and ethiofos have been investigated for their efficacy in protecting against radiation-induced tissue damage (Hosseinimehr, 2007). However, these agents have the potential to cause serious side effects including decreased cellular function, nausea, hypotension and death (Bogo et al., 1985; Satoh et al., 1982). Alternatively, natural plant extracts that can protect cells and tissues against ionizing radiation, without obvious side effects, would be a considerable adjunct to successful radiotherapy (Hosseinimehr et al., 2003, 2009; Jagetia and Baliga, 2004; Park et al., 2008). Such as procyanidins extracted from grape seed exhibit a radio-protective effects against chromosomal damage induced in vivo by X-rays (Castillo et al., 2000). Nelumbo nucifera Gaertn., commonly known as lotus, is a perennial aquatic plant grown and consumed throughout Asia. Almost

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all parts of the plant are eaten as vegetable and are used for various medicinal purposes in oriental medicine. Recently several bioactive compounds such as alkaloid (Kashiwada et al., 2005; Lu et al., 2008; Sugimoto et al., 2010; Zheng et al., 2010), triterpenoid (Chaudhuri and Singh, 2009), flavonoids (Jung et al., 2003; Lin et al., 2009a,b; Deng et al., 2009; Guo et al., 2010), and polyphenols (Park et al., 2009) have been extracted from rhizomes, seeds, flowers and leaves of this plant, which can account for the pharmacological effects of different parts of N. nucifera Gaertn. (Mukherjee et al., 2009), including antidepressant (Sugimoto et al., 2010), antifertility activity of seed of N. nucifera (Mazumder et al., 1992), anti-HIV (Kashiwada et al., 2005), anti-obesity (Ono et al., 2006; Ohkoshi et al., 2007; Wu et al., 2010), antioxidant activity (Wu et al., 2003; Rai et al., 2006; Jung et al., 2003; Lin et al., 2009a,b; Sohn et al., 2003), hypoglycemic activity (Mukherjee et al., 1997b; Mani et al., 2010), anti-inflammatory (Liu et al., 2004; Mukherjee et al., 1997a), anti-diarrheal (Talukder and Nessa, 1998), sedative effects (Sugimoto et al., 2008), antipyretic potential (Sinha et al., 2000), immunomodulatory activity (Mukherjee et al., 2010), anti-atherosclerosis (Ho et al., 2010), hepatoprotective effects (Sohn et al., 2003; Lin et al., 2009b), and enhancing learning and memory (Yang et al., 2008). Lotus seedpod is usually discarded, except when occasionally used as a traditional medicine with hemostatic function and eliminating bruise. It has been reported that lotus seedpod is an another important natural source of oligomers and polymers of catechin and epicatechin, which are also denominated procyanidins (Ling and Xie, 2002). Procyanidins from lotus seedpod (LSPCs) were first isolated and characterized by our laboratory, which were constituted by a variable number of flavan-3-ols units linked together through C4–C8 (or C6) interflavanoid bonds, and the oligomeric procyanidins are considered to be the main active constituents of LSPCs (Ling et al., 2005). Previous research indicated that LSPCs contain monomers, dimers, and tetramers of proanthocyanidins, in which the amounts of dimmers are greatest, and catechin and epicatechin are the base units (Ling et al., 2005). As a part of our research, we have found LSPCs possess a wide range of biological effects including scavenging free radicals (such as hydroxyl radical, hydrogen peroxide, hydroperoxyl radical, and superoxide anion radical), inhibiting the formation of lipid peroxidation (LPO) in soybean oil system, or from erythrocyte, liver mitochondria, and liver homogenates of rat in vitro, increasing the activities of SOD, GST and decreasing the level of LPO in liver and plasma of CCl4 toxic mice (Ling and Xie, 2002; Duan and Xie, 2003; Ling et al., 2005), keeping red blood cell membrane from lipid peroxidation and promoting the regeneration of natural vitamin E (Duan et al., 2005), protecting against experimental myocardial injury (Zhang et al., 2004) and ethanol-induced liver damage (Li et al., 2005), suppressing the growth and inducing apoptosis in the cancer cells in vitro (Duan et al., 2010), and improving learning and memory abilities (Gong et al., 2008; Xu et al., 2009). It has been confirmed that radiation-induced normal tissue damage is manifested as a result of the increased production of reactive oxygen species, such as hydrogen peroxide, hydroxyl radical, hydroperoxyl radical and superoxide anion radical, due to the radiolysis of water (Pandey et al., 2006). Considering the excellent antioxidant capacity and radical scavenging activities, LSPCs may be a promising radioprotective compound. However, the effect of LSPCs on irradiated animal survival and hemopoietic system has not been established. Therefore, we investigated the effect of LSPCs administration to Swiss albino mice before whole body gamma irradiation on the radioprotective activities of LSPCs through histopathology, micronuclei assay, chromosomal analysis, and the measurement of lipid peroxidation and antioxidant enzymes levels in this study.

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2. Materials and methods 2.1. Preparation of LSPCs Lotus seedpod was collected from Honghu Lantian Lake (Hubei, China). This variety of N. nucifera Gaertn. was named Number 2 Wuhan plant and authenticated by the Department of Botany, Wuhan Plant Institute of the Chinese Academy of Science. LSPCs is a kind of procyanidins compounds extracted from lotus seedpod. Commonly, these phenolic compounds are extracted with aqueous-ethanol. However, our previous research work indicated that aqueous-ethanol was defective in extracting oligomeric procyanidins in lotus seedpod, although the total phenolic compounds content was higher than the other solvents, such as aqueous-methanol, ethyl acetate and acetone–water. Conversely, acetone–water possessed obvious advantage in extracting oligomeric procyanidins. Oligomeric procyanidins are the main activated substance with special physiological functions, and it has been reported acetone–water can be used to extract oligomeric procyanidins (Palazzo de Mello et al., 1999). Therefore, we select acetone–water as the extraction solvent to extract LSPCs from lotus seedpod. LSPCs was extracted, purified, and characterized by the method described previously (Ling et al., 2005). Briefly, the lotus seedpod was extracted three times with acetone/water (V/V, 7:3), then the acetone–water extract was purified by Sephadex LH-20 column chromatography, with a purity of >98%, and the main molecular weight distribution of LSPCs was confirmed to be in the range 291.1–1155.3, the LSPCs polymerization was 64 and contained monomers, dimers and tetramers of procyanidins in which the amounts of dimers were greatest and catechin and epicatechin were the base units, which were consistent with Ling et al. (2005). For all experiments, final concentrations (50, 100 and 200 mg/kg) of the tested compound were prepared by diluting the stock with normal saline. The residue of acetone in the extract (LSPCs) was determined by headspace capillary gas chromatography (HP5890 type, Hewlett-Packard, US). The chromatographic column was HP-Wax, bonding polyethylene glycol 30 m  0.32 mm  0.50 lm. A temperature program starting at 50 °C for 3 min followed by a 20 °C/min ramp to 120 °C for 4 min, and the temperature of detector and injector was 200 °C. The carrier gas was nitrogen, and the detector was flame ionization detector (FID). Under these conditions, the minimum detection limit of acetone was 0.002%, and the acetone residue in LSPCs was not detected (tested three times). According to the regulations of International Conference on Harmonization (ICH), the residue of acetone in the pharmaceuticals for human use is restricted of 60.5%. Consequently, the security maybe posed from acetone has been excluded. 2.2. Animals All experiments were carried out on random bred male Swiss albino mice, aged 6–8 weeks and weighing 25 ± 2 g, from the Laboratory Animal Research Center of Hubei Province. The mouse colony was maintained under conditions of controlled temperature (23 ± 2 °C) and humidity (50 ± 5%), and a 12 h light/dark cycle. The animals were housed in sanitized polypropylene cages containing autoclaved paddy husk as bedding. They had free access to standard mouse food and water. Animals were treated humanely in compliance with the guidelines of the National Institutes of Health, and the protocol conformed to the Institutional Animal Ethical Committee. 2.3. Irradiations Mice were placed in well ventilated Perspex boxes of dimensions 23.5 cm  23.5 cm  3.5 cm, partitioned into 3 cm  3 cm  11 cm cells for individual animals. They were exposed to whole body irradiation from a 60Co Gammatron teletherapy unit (Theratron-780, Hubei Academy of Agricultural Sciences) at a dose rate of 1.14 Gy/min and a source to surface distance of 100 cm. The irradiation facility was provided by Hubei Academy of Agricultural Sciences Atomic Energy Graduate School. 2.4. Treatment of mice with LSPCs and survival studies To determine whether LSPCs conferred an advantage after lethal whole body irradiation, the effect of LSPCs on the survival of mice was investigated. LSPCs dissolved in NS were used for administration (i.g.) daily at doses of 50, 100 and 200 mg/kg to animals for 15 consecutive days before irradiation. The number of surviving mice was recorded daily up to 30 days post-irradiation, and the data were expressed as percentage survival. Radiation doses between 8 and 10 Gy were used to study the effect of LSPCs on survival, and a dose of 4 Gy was used to evaluate the effect of LSPCs on bone marrow, lipid peroxidation and antioxidant enzymes (Coleman et al., 2003). All of the mice in the experiments were randomly divided into six groups as follow: Group I (control) and II (radiation alone) orally received normal saline but no LSPCs; Group III was only given with LSPCs 200 mg/kg (LSPCs200); Group IV was radiation plus LSPCs 50 mg/kg (radiation + LSPCs50); Group V was radiation plus LSPCs 100 mg/kg (radiation + LSPCs100); Group VI was radiation plus LSPCs 200 mg/kg (radiation + LSPCs200). Each group consisted of 12 mice.

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2.5. Hematological study

2.9. Estimation of lipid peroxidation and antioxidant enzymes

Experimental grouping and treatment used for periphery blood analysis consulted in Section 2.4. Mice were pretreated with NS or LSPCs (50, 100 and 200 mg/kg, i.g.) for 15 consecutive days before irradiation (8 Gy). In addition, 0.1 ml of blood was collected from the tip of mouse tail and transferred to vials containing 0.5 M ethylene diamine tetraacetic acid at before whole body irradiation and 1, 3, 5, 7, 14, 21 and 30 days after irradiation. The total numbers of RBC, WBC, platelets and the hemoglobin level were determined using standard procedures. To determine the spleen index and spleen colony trials, all of mice were sacrificed by cervical dislocation on the 10th day after irradiation (8 Gy). The spleen was removed and the spleen index was calculated (spleen weight/body weight  100). Spleen colonies were counted manually in the same spleen (Uma Devi and Prasanna, 1995). The experiment was repeated three times with a minimum of three animals in each group.

According to Section 2.4, animals were divided into six experimental groups and administered NS or LSPCs with different concentrations (50, 100 and 200 mg/ kg, i.g.) before whole body irradiation (4 Gy). At 24 h post-irradiation, all animals were sacrificed by cervical dislocation. The liver was dissected out after transcardial perfusion with ice-cold saline. And then the liver was weighed and a 10% homogenate was prepared with ice-cold potassium chloride (150 mM) using a homogenizer (Yamato LSG LH-21, Japan). The homogenate and blood were used for the estimation of LPO (Konings and Driver, 1979), superoxide dismutase (SOD) (Misra and Fridovich, 1972), catalase (CAT) (Aebi, 1984) and glutathione peroxidase (GSH-Px) (Moran et al., 1979). The amount of antioxidant enzyme was expressed as units/ mg of protein or lmol/mg of protein (Parihar et al., 2007).

2.6. Histopathological study In the histological studies, portions of spleen and skin tissue were fixed with 10% phosphate-buffered neutral formalin, dehydrated in graded (50–100%) alcohol and embedded in paraffin. Thin sections (4–5 lm) were cut and stained with hematoxylin and eosin (HE) stain. They were analyzed by a light microscope and images were captured with a digital microscope camera (Leica DC300F).

2.7. Micronuclei assay of mouse bone marrow Micronuclei assay was carried out on six groups according to Section 2.4. Animals were treated with LSPCs (50, 100 and 200 mg/kg, i.g.) or NS for 15 consecutive days before whole body irradiation at a dose of 4 Gy. At 24 h post-irradiation animals were sacrificed. The femurs of each animal were dissected out, the adherent tissues were cleaned, and the bone marrow was flushed out into saline. The cell suspension was centrifuged and the pellet was resuspended in a few drops of fetal calf serum. Cell smears were made on pre-cleaned, pre-coded, air-dried slides and fixed in absolute methanol. The slides containing the cells were stained with 4% Giemsa and observed under a light microscope [Olympus IX70, Japan] using a 40 objective. Giemsa staining method employed in the micronuclei assay of mouse bone marrow conforms to the requirements of relative National Standard (GB 15193.51994) of China. The numbers of PCE, NCE with micronuclei (micronucleated polychromatic erythrocytes [MPCE] and normochromatic erythrocytes [MNCE]) were recorded. The ratio of PCE to NCE (P/N) was also calculated. May Granwald/Giemsa (MGG) staining is commonly used to mount and stain tissues, which combines the advantages of Giemsa staining and May Granwald staining. Somewhat differently, cytoplasm can be stained well by May Granwald staining, while the cell nucleus is much easier to be stained by Giemsa staining. Further, Giemsa staining is widely used for its simplicity, speediness and cheapness. In the experiment, the object of observation was the micronuclei of mouse bone marrow. At the beginning, we compared the results of different staining methods. Then we found the slides stained with Giemsa were clear enough, and there was no significant difference (P > 0.05) between May Granwald and Giemsa staining method. Also, there have been some instances of Giemsa staining application (Prasad et al., 2005; Ivanova et al., 2006). Thus, we select Giemsa staining as stained method for its special applications.

2.8. Chromosomal analysis of mouse bone marrow Administering of various doses of LSPCs and the six experimental groups were in accordance with Section 2.4. Cell chromosomes with multiple lesions were observed (Mantena et al., 2008; Ganasoundari et al., 1998), mice were treated with LSPCs (50, 100 and 200 mg/kg, i.g.) or normal saline for 15 days consecutively before whole body irradiation at a dose of 4 Gy. Twenty-two hours after irradiation, each mouse was injected with 0.3 ml of 0.025% colchicine and killed humanely 2 h later by cervical dislocation. The femurs were dissected out and cleaned free of adhering tissue. Bone marrow from the femurs was used for chromosome preparation. The metaphase plates were prepared using the routine air-drying method (Uma Devi et al., 1998). Briefly, the bone marrow from femurs was aspirated and washed in saline, treated hypotonically (0.559% potassium chloride), fixed in methanol/acetic acid (V/V, 3:1), dried and stained with 4% Giemsa. Chromosomal aberrations were scored under a light microscope. A total of 500 metaphase plates were scored per animal. Different types of aberration such as chromatid breaks, chromosome breaks, fragments, rings and dicentrics, as well as cells showing polyploidy and severe damage (cells with 10 or more aberrations of any type) were scored. The aberrations were identified using the criteria given by Savage (1976). When breaks involved both chromatids, they were termed ‘‘chromosome type” aberrations, while ‘‘chromatid type” aberrations involved only one chromatid. If the deleted portion had no apparent relation to a specific chromosome, it was called a fragment (Bender et al., 1988).

2.10. Statistical analysis Data were reported as means ± S.D. The results were analyzed using the Student’s t-test. Statistical analysis was carried out using the Statistical Package for Social Science (SPSS 11.5).

3. Results 3.1. Time course analysis of the administration for LSPCs In order to determine the appropriate time of administration for LSPCs, a time course study on it was carried out according to Sections 2.4 and 2.9. The results indicated that pretreatment with LSPCs for consecutive 15 days could decrease the level of LPO, and increase the activities of antioxidant enzymes including SOD, CAT, and GSH-Px to a stable level at 24 h post-irradiation (4 Gy). As shown in Fig. 1, there were apparently three distinguishable phases of each 5-day span in the duration of administration for LSPCs within the 30-day observation period. The duration of administration for LSPCs 1-day was low, i.e., the activities of the antioxidant enzymes drastically to decrease in the first period. In the second period, pretreatment with LSPCs for consecutive 5–15 days, the duration of administration was prolonged, and the levels of the antioxidant enzymes were increased gradually, and then a plateau of them were reached in the third period (15–30 days). Therefore, consecutive 15 days administration for LSPCs was used to investigate the whole body radioprotective activity of LSPCs in the present study. 3.2. Survival studies Animals in the radiation-alone group exhibited signs of radiation sickness within 2–4 days after exposure to different radiation doses (8–10 Gy). The severity of the symptoms increased and advanced with the increase in radiation doses. The exposure of mice to higher radiation doses resulted in the early appearance of symptoms. The main symptoms included a reduction in food and water intake, epilation, weight loss, emaciation, lethargy, diarrhea and ruffled hair. A few animals also exhibited facial edema between 1 and 2 weeks after exposure to radiation at dose of 10 Gy. Some of the animals exhibited paralysis and difficulty in locomotion during the second week after exposure to higher doses of radiation. The results of the survival studies were expressed as percent survival after exposure to various doses of radiation. No mortality was seen at the dose of 8 Gy alone, while an increase in the radiation doses resulted in a steep rise in mortality. The 30-day mortality rose from 30% at 8.5 Gy to 80% at 9.5 Gy (Fig. 2A). All animals exposed to 10 Gy died within 2 weeks after irradiation. Probit analysis of survival data showed a linear dose-dependent decrease in the radiation-alone group (Fig. 2B). Pretreatment with LSPCs at doses of 50, 100 and 200 mg/kg for 15 consecutive days before irradiation decreased the 10 Gy radiation-induced mortality compared with radiation-alone group. The severity of radiation sickness reduced in the LSPCs treated groups, with fewer animals showing facial edema and diarrhea. Obviously,

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Fig. 1. Effects of time on antioxidant enzymes and lipid peroxidation (LPO) in livers of mice administrated with LSPCs before irradiation. Panel A: SOD; Panel B: CAT; Panel C: GSH-Px; Panel D: LPO.

LSPCs was effective in protecting animals from hemopoietic death and to a lesser extent against gastrointestinal death. Pretreatment with LSPCs significantly increased the survival levels of animals (Fig. 2B). They were 100% at 8.5 Gy, 80% at 9 Gy, 70% at 9.5 Gy and 60% at 10 Gy for LSPCs treated groups, as compared with 70%, 40%, 20% and 0%, respectively, in the corresponding radiation-alone groups (Fig. 2A). In the 200 mg/kg pretreatment group, LSPCs elevated the radiation LD50 from 8.9 to 10.5 Gy, indicating a DMF of 1.18 (Fig. 2C). 3.3. Effects of LSPCs on the peripheral blood in mice In the present study, alterations in RBC, WBC, platelets count and the hemoglobin level were found to show a parallel pattern in all groups. These parameters were markedly suppressed in the irradiated (8 Gy) animals and had not returned to normal levels after radiation day 30. Prior administering LSPCs at a dose of 50 mg/kg enhanced the recovery in those parameters, which had returned to normal values by day 30 post-irradiation. The minimum of white blood cells was recorded on day 3 post-irradiation, the RBC, platelets counts, and hemoglobin were at minimum levels on day 7 post-irradiation, with a progressive increase beyond this time point until the last autopsy interval on day 30. However, a return to normal values was not observed in the radiation-alone group (Fig. 3). An increasing pattern was also recorded beyond day 7 in radiation plus LSPCs-treated mice at doses of 100 and 200 mg/kg, and these four parameters had returned to normal lev-

els by day 21 post-irradiation (Fig. 3), which suggested there is a dose–response relationship between the levels of hemoglobin, red blood cells, white blood cells, platelets and the concentration of LSPCs in general. 3.4. Hemopoietic study The results of hemopoietic study were presented in Table 1. As shown in Table 1, a significant reduction (P < 0.01) in the spleen index (0.23) and spleen weight was observed in radiation-alone group. Pretreatment with LSPCs at a dose of 200 mg/ kg maintained the spleen index close to normal value (0.42) in irradiated mice. To confirm the radio-protective effect of LSPCs on hematopoietic stem cells damaged by ionizing radiation, an endogenous CFU assay was performed. The number of endogenous CFUs is considered to be an indicator of hematopoiesis, which is a critical survival factor (Uma Devi et al., 1999). In the radiation plus LSPCs groups at doses of 100 and 200 mg/kg, the numbers of endogenous CFUs were significantly increased (P < 0.05 and P < 0.01, respectively) compared with radiationalone group (Table 1), which indicated LSPCs possess the ability to restore hematopoietic ability by stimulating the formation of endogenous CFUs. Thus, the present data on spleen index and spleen colonies demonstrated that LSPCs has a significant protective effect on hemopoietic system. No significant changes in spleen weight, spleen index and CFU were observed in the group of LSPCs200 alone.

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3.5. Histopathological study The morphological observation results for mouse spleen are presented in Fig. 4A–D. Splenic corpuscles with lymphocytes inside were stained with HE. They had a uniform distribution and clear boundaries, and appeared uniformly blue in normal mice (Fig. 4A). Splenic corpuscles in mice from the radiation-alone group were deflated and their number decreased (Fig. 4B). Compared with radiation-alone group, the splenic corpuscle in mice treated with 100 and 200 mg/kg LSPCs before irradiation, increased and distributed uniformly (Fig. 4C and D). As shown in Fig. 4E, the epithelial lamina was thin and composed of stratified squamous epithelium cells, in which keratinocytes were few or no, while the dermis stratum was thick and the hair follicles, sebaceous glands, sweat glands were integrity in control mice. An increase in squamous cells of mice skin in the radiation-alone group resulted in a thickening of the epithelial lamina and dermis stratum. Dense staining and large-scale edema were observed and the numbers of hair follicles, sebaceous glands and sweat glands reduced in the dermis stratum (Fig. 4F). Compared with control, the epithelial cells shrank, skin wrinkled and the dermis between hair follicles was still slightly swollen in radiation + LSPCs100 group (Fig. 4G), while there was no obvious abnormalities in radiation + LSPCs200 group (Fig. 4H). 3.6. Bone marrow micronuclei assay As shown in Table 1, significant increases in the frequency of MPCE and MNCE were observed obviously in mice exposed to

whole body gamma irradiation (4.0 Gy) (Fig. 5). A drastic fall in the P/N ratio was also observed in the radiation-alone group compared with control. However, there was no difference (P > 0.05) between control and the LSPCs200 group in terms of the formation of MPCE and MNCE. Pretreatment with 50, 100 and 200 mg/kg LSPCs for 15 consecutive days prior to irradiation reduced the frequency of MPCE and MNCE formation and prevented the fall in the P/N ratio (Table 1).

3.7. Bone marrow chromosomal damage assay A significant increase in the frequency of aberrant metaphases and different types of aberrations were observed in the mice treated with radiation alone. The number of aberrant metaphases/500 cells in the radiation-alone group reached 279.3, which was 71.3 times higher than that of control group (Table 2). In the mice pretreated with LSPCs a decrease in the number of chromosomal aberrations induced by radiation (4 Gy) was observed. At a dose of 4 Gy, the aberrations were mainly simple aberrations including breaks, fragments and chromosomal exchanges such as rings and dicentrics. Further, cells with multiple aberrations and polyploids increased significantly (P < 0.01) compared with normal control values (Table 2). Pretreatment with LSPCs at doses of 100 and 200 mg/kg resulted in a significant decrease (P < 0.01) in the percentage of aberrant metaphases as well as a significant reduction in the different aberrations involved breaks, fragments, rings and dicentrics, polyploids and severe damage.

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Fig. 3. Effects of LSPCs on white blood cells (A), red blood cells (B), platelets (C) and hemoglobin (D) in the peripheral blood of mice. Different doses of LSPCs administered to mice for 15 consecutive days before whole body irradiation at a dose of 8 Gy. The peripheral blood study was carried on 1, 3, 5, 7, 14, 21 and 30 days post-radiation. Values are expressed as means ± S.D. of 12 mice.

Table 1 Effect of LSPCs on spleen index, colony forming units (CFU) and micronuclei formation in mice exposed to whole body irradiation.* Groups

Spleen weightA (mg)

Spleen indexA

CFUA

MPCE/1000B (cells ± S.D.)

MNCE/1000B (cells ± S.D.)

P/N ratioB

Control LSPCs200 Radiation alone Radiation + LSPCs50 Radiation + LSPCs100 Radiation + LSPCs200

137 ± 5.64 135 ± 4.81 74 ± 3.26c 80 ± 7.21c 109 ± 4.85d 131 ± 3.69e

0.42 ± 0.05 0.39 ± 0.09 0.23 ± 0.03c 0.26 ± 0.07c 0.35 ± 0.09d 0.41 ± 0.02e

0 0 5.2 ± 0.4c 6.8 ± 0.3c 9.3 ± 0.8d 13.4 ± 0.5e

4.71 ± 0.34 3.85 ± 0.76 74.23 ± 4.59c 65.43 ± 3.21c 46.58 ± 4.65d 32.24 ± 3.47e

0.14 ± 0.02 0.16 ± 0.01 5.23 ± 0.03c 3.26 ± 0.07c 2.35 ± 0.09d 1.41 ± 0.02e

1.31 ± 0.05 1.26 ± 0.02 0.54 ± 0.04c 0.68 ± 0.03c 0.93 ± 0.08d 1.14 ± 0.05e

A

Whole body irradiation at a dose of 8 Gy. Animals were sacrificed at 24 h after whole body irradiation at a dose of 4 Gy. Femurs were dissected out, bone marrow was flushed and slides were prepared. The number of polychromatic erythrocytes and normochromatic erythrocytes with micronuclei (MPCE and MNCE) was recorded. * Values are expressed as means ± S.D., numbers of the same period with different letters are significantly different (dP < 0.05; eP < 0.01). B

3.8. Effects of LSPCs on lipid peroxidation and antioxidant enzymes in vivo Radiation treatment significantly (P < 0.01) increased the LPO level in the livers of mice exposed to a dose of 4.0 Gy (Table 3). Conversely, the LPO levels significantly decreased in the radiation plus LSPCs (100 and 200 mg/kg) groups (P < 0.05 and P < 0.01) compared with the radiation-alone group. No significant difference (P > 0.05) was found between LSPCs200 group and control with regard to lipid peroxidation levels. A significant (P < 0.01) reduction in the level of antioxidant enzymes involving SOD, CAT and GSHPx was observed in the radiation-alone group at 24 h post-irradiation (Table 3). On the contrary, pretreatment with 100 and 200 mg/ kg of LSPCs significantly (P < 0.05 and P < 0.01) increased those antioxidant enzymes level at 24 h post-irradiation. No significant

difference (P > 0.05) was found in the level of superoxide dismutase, catalase and glutathione peroxidase in LSPCs200 group compared with control (Table 3). At a lower dose (50 mg/kg), the recovery of enzyme activities ranged from 7.1% for GSH-Px to 12.6% for SOD, while with higher dose (200 mg/kg) of LSPCs, the recovery of enzyme activities ranged from 51.5% for SOD to 60.2% for CAT. This situation is quite similar to our previous work (Duan et al., 2010), in which we found that there was no significant difference (P > 0.05) in the level of SOD, CAT and GSH-Px in the mouse treated only with LSPCs (120 mg/kg bwt, i.g.) compared with control during the course of 15 days. But to the B16 melanoma-bearing (BMB) mice treated with LSPCs (90 and 120 mg/kg bwt, i.g.), a significant increase of SOD, CAT and GSH-Px activities was observed (P < 0.05 and P < 0.01, respectively) compared with the BMB group

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Fig. 4. Morphological observation of spleen (Panels A–D, 4  3.3) and skin (Panel E–H, 10  3.3) using a light microscope. Mice were sacrificed by cervical dislocation on the 10th day after whole body irradiation at a dose of 8 Gy. Portions of spleen and skin tissues were fixed, embedded, cut into sections and stained with hematoxylin and eosin for observation using a light microscope. Control (A and E); radiation alone (B and F); radiation + LSPCs100 (C and G); radiation + LSPCs200 (D and H).

without LSPCs. This phenomenon indicated that treated with LSPCs, either before or after injury, may protect mice against injury by maintaining the enzymes levels. 4. Discussion Exposure to ionizing radiation induces the production of reactive oxygen species (ROS), which include superoxide, hydroxyl radicals, singlet oxygen and hydrogen peroxide (Ewing and Jones, 1987). These free radicals react with critical cellular components, such as DNA, RNA, proteins, and membranes resulting in cell dysfunction and death (Cerutti, 1985). When DNA is damaged, it is followed by altered cell division, cell death, depletion of stem cell pools, organ system dysfunction and, if the radiation

dose is sufficiently high, the organism will die. The elimination of the free radical species from the cell environment can inhibit the side effects induced by irradiation (Hosseinimehr et al., 2007). In terms of radiation sickness and mortality, the results after lethal whole body irradiation followed a similar pattern to that of earlier studies (Uma Devi and Prasanna, 1995). The higher the radiation dose (8–10 Gy) is, the more of the mortality is. The primary cause of mortality during the early phases of radiation-induced haematopoietic syndrome is sepsis, resulting from opportunistic infection, due to low numbers of neutrophils and increased translocation of bacteria across the gastrointestinal mucosa. This is complicated by thrombocytopenia and concomitant haemorrhage and defects in the adaptive immune system resulting

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Fig. 5. Photographs of PCE and NCE with micronuclei (1000). (A) and (B) PCE (1) and NCE (2); (C) and (D) intracellular MPCE (3); (E) and (F) extracellular MPCE (3).

Table 2 Effect of LSPCs on the induction of chromosomal aberrations in the bone marrow of mice exposed to whole body irradiation at a dose of 4 Gy.* Groups

Number of aberrant cells/500 metaphases

Chromatid breaks

Chromosome breaks

Fragments

Rings + dicentrics

Polyploidy

SDC

Control LSPC200 Radiation alone Radiation + LSPC50 Radiation + LSPC100 Radiation + LSPC200

3.92 ± 0.46 3.57 ± 0.81 279.3 ± 4.26a 240.5 ± 3.21a 196.3 ± 3.16b 139.5 ± 2.23c

0 0 35.3 ± 2.2a 29.2 ± 1.7a 23.4 ± 2.5b 18.5 ± 1.2c

0 0 11.2 ± 0.8a 9.5 ± 0.7a 6.7 ± 0.4b 3.6 ± 0.2c

5.2 ± 1.1 6.4 ± 0.8 454.2 ± 4.9a 392.4 ± 3.5a 335.8 ± 5.6b 261.2 ± 4.7c

0 0 22.3 ± 1.1a 18.2 ± 0.7a 13.5 ± 0.9b 10.1 ± 0.6c

0 0 9.5 ± 0.4a 8.6 ± 0.7a 6.9 ± 0.8b 4.3 ± 0.3c

0 0 37.3 ± 1.6a 30.4 ± 0.8a 23.1 ± 1.3b 17.5 ± 0.9c

Values are expressed as means ± S.D. of six mice, numbers of the same period with different letters are significantly different (bP < 0.05; cP < 0.01) compared to radiationalone group.

*

from apoptosis of lymphocytes and deficient lymphopoiesis (Whitnall et al., 2000). The DMF of 1.18 obtained with LSPCs for a range of lethal doses (8–10 Gy) was lower than a DMF of 1.6– 1.8 obtained for different thiols (Weiss and Landauer, 2000). In

general, DMF with values lower than 1.4 using a 30-day survival endpoint have been reported for some naturally occurring radioprotectors such as Ocimum phenolic, flavonoids, polysaccharides and cytokines (Weiss and Landauer, 2009).

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Table 3 Effects of LSPCs on lipid peroxidation (LPO) and antioxidant enzymes in mice liver.* Group

SOD (units/mg)

CAT (lmol/mg)

GSH-Px (lmol/mg)

LPO (nmol/mg)

Control LSPCs200 Radiation alone Radiation + LSPCs50 Radiation + LSPCs100 Radiation + LSPCs200

79.43 ± 7.58 81.15 ± 9.61 45.62 ± 6.25a 51.35 ± 9.38a 63.24 ± 8.13b 77.81 ± 7.29c

249.12 ± 16.21 253.36 ± 19.02 156.42 ± 11.51a 173.16 ± 13.84a 201.32 ± 18.85b 250.65 ± 17.56c

167.76 ± 14.23 171.23 ± 10.31 106.70 ± 11.05a 114.26 ± 16.61a 138.83 ± 13.18b 163.44 ± 12.22c

2.58 ± 0.15 2.52 ± 0.14 3.94 ± 0.19a 3.27 ± 0.12a 2.93 ± 0.10b 2.64 ± 0.11b

The unit used for SOD was 50% inhibition of auto oxidation of pyrogallol per min per mg protein; for CAT, micromoles of H2O2 liberated per min per mg protein; and for GSHPx, micromoles of GSH oxidized per min per mg protein. Values in a column with different superscripts were significantly different (bP < 0.05; cP < 0.01 compared to radiationalone group). * Values are expressed as means ± S.D. (n = 12) for each group.

Hematopoietic stem cells are highly sensitive to ionizing radiation, as well as chemotherapeutic drugs administered to cancer patients (Park et al., 2008). In fact, myelosuppression and hematopoietic dysfunction are the most common clinical complications of these treatments (Chen et al., 2007). Therefore, an important adjunct to their use is to promote the recovery of hematopoiesis (Neta et al., 1986). In our experiments, irradiated mice treated with LSPCs recovered from whole body irradiation, as established by an increase in splenic CFU numbers relative to irradiated mice not receiving LSPCs. The enhancement of endogenous CFUs in mice received LSPCs before undergoing irradiation indicated that LSPCs can protect and/or stimulate the proliferation of hematopoietic stem cells. Furthermore, LSPCs at doses of 100 and 200 mg/kg kept the spleen index close to normal and initiated endogenous spleen colony forming units, which indicated that LSPCs has a hematopoietic protection effect. This important mechanism of LSPCs-mediated survival described here indicates that LSPCs may possess a strong potential for clinical application. Haematopoietic recovery depends on the percentage of residual haematopoietic stem cells. The higher the radiation dose is, the weaker the efficacy of haematopoietic growth factors is. The results of the investigation revealed that, after exposure to 10 Gy, the erythrocyte count exhibited a fall that could be attributed to the inhibition of new cells entering into the blood, loss through hemorrhage and/or radiation-induced injury (Sasaki and Matsubara, 1977). Further, the hemoglobin level was found to be declined significantly after radiation exposure following a similar pattern to that of red blood cells in general, which did not approach normal levels until the 30th day after radiation. The minimum value for hemoglobin level was found on the 7th day after irradiation in all radiation groups (10 Gy). The decrease in hemoglobin content was attributed to a decline in the number of red blood cells. Similar findings were proposed earlier by Daga et al., who found noticeable depletion in hemoglobin concentration in Swiss albino mice exposed to 3.6 Gy gamma irradiation (Daga et al., 1995). In LSPCspretreated irradiated groups, hemoglobin levels were higher than that of in radiation-alone group, which suggested a significant protection of red blood cells by LSPCs. Similarly, the levels of white blood cells and platelets declined significantly after radiation exposure, and their minimum values appeared on the 3rd and 7th day, respectively. While in the LSPCs-pretreated groups, white blood cells and platelet values were higher than that of in radiation-alone group. LSPCs-pretreated groups exhibited a significant protection and/or stimulation of hematopoietic stem cell proliferation, which was consistent with LSPCs increasing splenic endogenous CFUs. Bone marrow and organs like intestines, skin and spleen are particularly sensitive to ionizing radiation due to the relatively high numbers of proliferative cells undergoing deoxyribonucleic acid synthesis (Hosseinimehr, 2007). As the body’s first line of defense and the largest organ, skin is involved in a wide range of physiological functions. It has been confirmed inflammation plays

a key role in radiation-induced skin damage. This damage, when sufficiently severe, is expressed as follows: skin flushing, edema, itching, blisters, erosions, exudate and ulceration (Srinivasan et al., 2007). In the present investigation, serious skin damage such as epidermal thickening, large areas of dermal edema, and a reduction in the numbers of hair follicles, sweat glands and sebaceous glands occurred after whole body irradiation at a dose of 10 Gy. However, the skin damage of mice pretreated with LSPCs was found to be alleviated in relation to the radiation-alone group. Consequently, LSPCs can be considered as a potential radioprotector against skin injury caused by radiation. Most of radiation-induced damage to biomolecules in aqueous media, such as those prevailing in living systems, is caused by the formation of free radicals resulting from the radiolysis of water (Orsolic´ et al., 2007). Reactive oxygen species mediated cascading chain reactions and redox imbalances have been well documented in radiation toxicity studies. Radiation toxicity and repair mechanisms depend on the status of endogenous antioxidant enzymes (Sun et al., 1998). LSPCs can reduce radiation toxicity in Swiss albino mice by elevating endogenous antioxidant enzymes levels and reducing lipid peroxidation formation. The results of cytogenetic studies showed that pretreatment with LSPCs (100 and 200 mg/kg) was highly effective in reducing simple chromosomal aberrations, and protecting against singlestrand breaks. A significant decrease in the complex aberrations like rings, dicentrics, and severely damaged cells after radiation (4 Gy) also suggested that LSPCs may confer a good protection against double-strand breaks and decreased the proportion of cells with multiple deoxyribonucleic acid lesions. Enhancement of micronuclei frequency in the bone marrow of irradiated mice has been previously reported (Uma Devi et al., 1999). However, in the present study, pretreated with LSPCs (100 and 200 mg/kg) significantly reduced the radiation-induced micronuclei frequency in both PCEs and NCEs, which supported their radioprotective activity. The P/N ratio is an indicator of cell proliferation rate, which is an expression of early radiation effects on the cell cycle and suppression of erythropoiesis decreasing at 24 h post-irradiation (Chaubey et al., 1993). In the investigation, LSPCs pretreatment prevented the fall in the P/N ratio induced by radiation, which demonstrated its radioprotective activity. It is very important to develop radioprotective agents for protecting patients from the side effects of radiotherapy, as well as the public from unwanted irradiation. At the present, there are two strategies to find an approved radioprotective agent: immunomodulators and natural herbal medicine. One approach is to find a safe chemical or biological compound capable of binding cytokine receptors and hence, stimulating the release of two or more cytokines and indirectly regenerating haematopoietic stem cells. Another strategy is to find natural products with actions as free radical scavengers and capable of inducing bone marrow recovery (Hosseinimehr, 2007). In the present study, we demonstrated that

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LSPCs can protect mouse against whole body gamma irradiation, and LSPCs may be a promising adjunct treatment for patients exposed to radiation, as well as to a hazardous radiation environment. However, our investigation was limited to research the radioprotective activity of LSPCs, and the results only demonstrated the security of the extract (LSPCs) in part. Consequently, definitely more independent corroboration is necessary, such as the appropriate method of drug administration, and the safety and toxicology assessment of LSPCs, including acute toxicology experiment, subacute toxicology experiment, genetic toxicology and cancer risk assessment, etc.

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