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Effect of lentinan on membrane-bound protein expression in splenic lymphocytes under chronic low-dose radiation
International Immunopharmacology 22 (2014) 505–514
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Effect of lentinan on membrane-bound protein expression in splenic lymphocytes under chronic low-dose radiation Ying-Hua Liu a,1, Shou-Dong Ma a,1, Qing-Jie Fu a, Li-Yan Zhao a, Yi Li b, Hai-Qing Wang b, Ming-Chun Li a,⁎ a b
Department of Pharmacy, No. 401 Hospital of Chinese People's Liberation Army, Qingdao 266071, China Radiation Oncology Department, No. 401 Hospital of Chinese People's Liberation Army, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 12 March 2014 Received in revised form 2 July 2014 Accepted 21 July 2014 Available online 4 August 2014 Keywords: Lentinan Lymphocytes Radioprotection
a b s t r a c t We investigated the protective effects of lentinan against damages to chronic and low-dose radiation (CL-radiation) by using mouse models. The mice were randomized divided into four groups: normal control mice (Ctr), mice exposed to radiation (Rad), irradiated mice treated with low-dose lentinan (0.1 mg/(kg.d), RL), and irradiated mice treated with high-dose lentinan (0.5 mg/(kg.d), RH). All the mice were injected intraperitoneally once a day at a dose of 0.5 mL (Ctr and Rad with normal sodium while RL and RH with lentinan). The success of radiation models was confirmed by HE stain and cell morphology by a transmission electron microscope (TEM). On the basis of radiation models, we investigated the expression of proteins on the membrane of splenic cells through MALDI-TOF-MS/MS. The results demonstrated that both RT-radiation and lentinan affected the expression of membrane proteins, but lentinan protected the splenic cells and tissue from the injuries caused by CLradiation. Therefore, we speculated that CL-radiation mainly damages the genetic materials and membranebound proteins, while lentinan protects membrane-bound proteins by regulating signal transduction and the appearance of the cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction With the development of technology and the wide use of electronic appliance, a growing number of people are exposed to radiation, so radiation hazard has become a serious problem and a major health threat worldwide [1,2]. It has been increasingly realized that radiation can cause injuries to multiple organs, especially in those who work in nuclear medicine, nuclear industry etc., resulting in immunodeficiency, tumors, aging and hematologic diseases [3–5]. Radiation damage is mainly caused by excessive production of free radicals that are produced from endosomatic water. As a result, free radicals are often used as an indicator to detect radiation-induced damage [6–8]. At present, the majority of radiation protection agents are sulfur-containing drugs, hormones, cytokines and natural drugs [9]. Among them, natural drugs, especially polysaccharides, have been extensively studied and paid a significant attention because of their edibility, few side effects, and bioavailability. Lentinan, a [1–3]-beta-D-glucan extracted from lentinus edodes (shiitake), has been well recognized as an immunomodulator [10–12]. It has been reported that it can regulate immunity, resist oxidization and mutations, and reduce blood lipid [13–16]. However, although the ⁎ Corresponding author. E-mail address: [email protected] (M.-C. Li). 1 These two authors contributed equally to this work and should be considered as cofirst authors.
http://dx.doi.org/10.1016/j.intimp.2014.07.027 1567-5769/© 2014 Elsevier B.V. All rights reserved.
immunological regulation of lentinan has been well established, its radioprotective effect remains elusive. The purpose of our study was to investigate the protective capacity of lentinan against CL-radiation and the molecular mechanisms involved. 2. Materials and methods 2.1. Animals Kunming mice (male; 5 weeks; 20–24 g) were obtained from the Experimental Animal and Animal Studies Center of Qingdao, China, and kept under the pathogen free condition. All our experiments were performed in accordance with the regulation laid down by Chinese Association for Laboratory Animal Science. 2.2. Reagents Lentinan (1 mg/2 mL, 110103–3) was purchased from Jinling Pharmaceutical Co. Ltd. (Nanjing, China). It was further diluted to 0.004 mg/mL, and 0.02 mg/mL with saline before the injections. ProteoExtract Transmembrane Protein Extraction Kit was purchased from EMD Chemicals Inc. (Darmstadt, Germany). iTRAQ kit was purchased from Applied Biosystems (California, US). BSA standards kit, Commassie blue standing kit and the others were purchased from Sigma-Aldrich Co. LLC (Shanghai, China).
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2.3. Animal models The mice were randomly divided into four groups (n = 20 each): normal control mice (Ctr), mice exposed to radiation (Rad), irradiated mice treated with low-dose (0.1 mg/(kg.d), RL) and high-dose (0.5 mg/(kg.d), RH) lentinan. The mice in the Ctr and Rad groups were injected with 0.5 mL normal sodium, and those in the RL and RH groups were injected with 0.5 mL lentinan in different concentrations as mentioned above. All the mice, expect those in the Ctr group, were exposed for 15 min to systematical radiation of 0.4 Gy from medical linear accelerator with an absorbed dose rate of 3 Gy/min. The source to skin distance was 150 cm, and exposure field was 20 cm × 20 cm. The radiation was given from Monday to Friday for 40 days. All the mice were euthanized by cervical vertebra dislocation after the last radiation and sterilized in 75% ethanol for 30 s. Their spleens were then removed. 2.4. Pathological examination of the animal model After the removal of the spleens, ten spleens of each group were sent to the Pathology Department of our hospital for histological examination. HE staining was used to observe the changes in tissue structures. TEM was used to observe the damages to the splenic lymphocytes [17]. 2.5. Expression of membrane proteins in lymphocytes The inspection of the animal model showed that lentinan was more protective against irradiation in the RH group than in the RL group. As a result, expressions of membrane proteins in lymphocytes were analyzed only in the Ctr, Rad and RH groups in the following experiments. 2.5.1. Protein extraction The ten spleens in each group were milled with the 200 mesh stainless steel cell sieve in the culture medium, followed by centrifugation at 1500 r/min for 15 min. The supernatant was discarded. RBC lysis buffer was added into the centrifuge tube for 2–3 times. After removing the supernatant, we got lymphocytes of different populations. Cells were
incorporated within the same group, and the concentrations were regulated into 5 × 106/mL [18]. Transmembrane protein extraction kit was used to get transmembrane protein. The extraction was preserved at −20 °C. 2.5.2. Sample preparation The protein concentrations were determined with Bradford Regent. Six times the volumes of acetone (−20 °C) were added to the sample tubes (4 °C), which were turned over three times. The sample tubes were incubated at − 20 °C for at least 30 min until the appearance of precipitation. The acetone was decanted and dissolution buffer added for concentration of 2 μg/μL in each sample. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) [19] was done to detect the differences in the expressions of protein samples. Hoefer miniVE was used to prepare 70 × 100 × 1 mm3 polyacrylamide gels with 13% separating gels (pH 8.8) and 4% stacking gels (pH 6.8). Loading buffer (10 μL), the sample (10 μL) and 2μL βmercaptoethanol were mixed and heated at 95 °C for 3 min for denaturation. 5× Running Buffer (1 L) was prepared with 15 g Tris base, 72 g glycine, and 5 g SDS. The gels were run at 10 mA for 30 min, and then changed to 30 mA for 1 h. Coomassie blue staining [20]. 2.5.3. iTRAQ labeling and digestion [21] 53.325 μg sample of each group was made according to the above steps. After lyophilization, 20 μL TTLB was added to dissolve the protein samples. Then, 1 μL 2% SDS was added into the kit and vortex to mix. 2 μL 50 mM TECP was added to each sample tube, vortex to mix, and the tubes were incubated at 60 °C for 1 h. 1 μL 200 mM MMTS was added to each tube, vortex to mix, and the tubes were incubated at room temperature for 10 min. 20 μg trypsin was reconstituted with 40 μL ultrapure water, vortex to mix. The trypsin solution was added to each sample tube (50:1 proteins: enzyme), vortex to mix. The tubes were incubated at 37 °C for 12 h then spanned in order to bring the sample digest to the bottom of the tube. allowed each vial of iTRAQ reagent required to room temperature, then spanned to bring the reagent to the bottom of the tubes. Then,
Fig. 1. The picture of SCX-HPLC resolution.
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70 μL of ethanol was added to each reagent vial, vortex to mix. The reagent 114 was transferred to the protein samples of Ctr, reagent 115 to Rad, and the reagent 116 to LNT. Each tube was vortexed to mix. The tubes were incubated at room temperature for 1 h, and each tube was spanned at room temperature every 15 min. All peptides were preserved at 20 °C. 2.5.4. 2D-LC separation [22] 2.5.4.1. Strong cationic exchange high performance liquid chromatography (SCX-HPLC). The mobile phase A (5 mM KH2PO4/20% HCN, pH = 3.0) and mobile phase B (5 mM KH2PO4/350 mM KCl/20% HCN, pH = 3.0) were prepared. The protein samples were diluted with mobile phase A. Conditioning of SCX (Thermo Biobasic SCX, 4.6 mm × 250 mm × 5 μm): the flow rate was set to 700 μL/min, the wavelength to 214 nm, and the pressure to 15 MPa. The mobile phase A was run at 0–35 min, 50% mobile A and mobile phase B at 35–45 min, 100% mobile phase B at 45–50 min, 0–100% mobile phase B at 50–51 min, and 100% mobile phase A at 51 min. The samples were loaded and injected, then peptides were fractionated and eluted as shown in Fig. 1. The fractions were collected and pooled into 10 tubes according to SCX elution chromatogram. 2.5.4.2. Reverse phase high-performance liquid chromatography (RPHPLC). Mobile A (5% KH2PO4/95% HCN/0.1% KCl) and mobile B (95% KH2PO4/5% HCN/0.1% KCl) were prepared. All fractions were lyophilized to remove SCX Solvent A, and then diluted with 80–100 μL RP Solvent A for each fractions and centrifuged for 15 min at 10,000 rpm. Conditioning of RP-HPLC (MICHROM Bioresources/MAGIC C18AQ 0.2 × 150 mm 3 μ 200 A): The cartridge was wetted with 10 μL 100% HCN for 10 times, wetted with 10 μL 50% ACN for 10 times, and equilibrated with 10 μL
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0.1% TFA for 10 times. The flow rate was set to 0.2 μL/min, the wavelength to 214 nm and 280 nm, and the pressure to 15 MPa; 90% phase A and 10% phase B were run at 0–90 min, 50% phase A and 50% phase B at 90–120 min, 0–100% phase B at 120–140 min, 100% phase B at 140–145 min, and 90% phase A and 10% phase B at 145 min. MS/MS: The fresh MALDI matrix solution (5 mg CHCA dissolved with 50% HCN/0.1% TFA) was prepared. The spotting was initiated at 35 min when the first peptide peaks were eluted. The column effluent was mixed directly with MALDI matrix solution. The fractions were automatically deposited every 30 s onto the MALDI target plate using a Probot micro-fraction collector. MS range was 700–4000 Da, total shot/spectrum was 1000, and fixed laser intensity was 3400; MS/MS range was 10–1360 Da, total shot/spectrum was 2000, and fixed laser intensity was 380. 2.5.5. ABI 4800 MALDI MS/MS analysis and database searching MALDI plates were analysed on ABI 4800 MS/MS. The LC-MALDI template was selected, and Mascot software was used for peptide identification. The parameters were set for protein identification as follows: fixed modifications were (N_term)_iTRAQ, Lysine (k) _iTRAQ and MMTS (c); variable modifications were oxidation (M); precursor tolerance was 0.2 Da; maximum peptide rank was 2. The definition of value for differentially expressed proteins was 116/114 (116/115) N 1.5 times or 116/114 (116/115) b 0.5 times, and P b 0.05. Then peak areas of different groups were automatically extracted. The quantitative information was processed according to correction factor. 2.5.6. Western-blot analysis Protein concentrations were determined using the BCA protein assay kit (CWBIO, China), and samples were separated on SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane
Fig. 2. HE slices after radiation and lentinan treatment (×100). 2-1 (Ctr group): the splenic tissues retained normal. 2-2 (Rad group): severe bleeding and remote hemorrhage existed in the interstitium. 2-3 (RL group): megakaryocytes increased and hemorrhage was present. 2-4 (RH group): the tissues were basically normal, with a slight decrease of megakaryocytes.
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(MILLIPORE, US). The membranes were reacted with antibodies against phosphorylated or nonphosphorylated Igh-6 Ig mu chain C region secreted form, Ryr2 Cardiac Ca2 + release channel or Ncam2 Isoform Short of Neural cell adhesion molecule 2(Cell Signaling Technology, US). Then, specific antigen/antibody complexes were made with horseradish peroxidase-conjugated secondary antibodies (Rabbit IgG, Cell Signaling Technology, US) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, US). The images were digitized from the membrane and the band intensity of each protein was quantified with NIH image software. 3. Results 3.1. Pathological changes of the tissues Pathological changes have been observed following the exposure to CL-radiation. Histology showed scattered mature megakaryocytes in the splenic tissues, but no erythrocyte stasis in splenic sinus in the Ctr group. Compared with the Ctr group, severe blood loss in the
interstitium and obvious congestion were observed in the Rad group; no obvious injury or mild congestion was displayed in the RL group. 3.2. Changes in cell structure Injuries were also observed in the ultrastructure of the spleen. Compared with the Ctr group, minute projections on the membrane disappeared and the cells were swelled seriously and even disrupted, and pyknosis was emerged in the nuclei and cytoplasm in the Rad group. The ultrastructure was essentially normal, the projections were complete, and mild swelling was occasionally found in the RH group. All the pathological changes are shown in Fig.2. Ctr group: As shown in Fig. 3-1, there were no injuries in the cell membrane and the nuclear membrane. The projections were found on the cell membrane. Within the cells, there were large round or ellipse nuclei in the center, containing mostly euchromatin. Abundant cytoplasm, many free-ribosomes, and smaller cell organs were observed. Clear and intact cristae were preserved in mitochondria as indicated by the arrow in Fig. 3-1-1.
Fig. 3. Electron microscopic sections after radiation and lentinan treatment. 3-1 (Ctr group): the nucleus was big and in the center. The double-cellular membrane and nuclear membrane were visible. The cristae were evident in the chondriosome. 3-1-1 (Ctr group): mitochondria were clear and intact cristae, as shown at the arrow. 3-2 (Rad group): in the center were megameric chromosomes. Vacuoles existed in the cytoplasm, as shown at the arrow. 3-3 (Rad group): in the center were megameric chromosomes. The cells were mainly heterogeneous. The cell organ was abnormal. 3-4 (Rad group): in the center were megameric chromosomes. The membrane was broken, and the kytoplasm outflew, as shown at the arrow. 3-5 (RL group): cellular structure was essentially normal, and the nucleus was big and in the center. The projections were present. 3-5-1 (RL group): the mitochondria were essentially normal, as shown in the circle, 3-6 (RH group): the cellular structure and kytoplasm were normal.
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Fig. 3 (continued).
Rad group: The structures of cells were injured (as shown in Figs. 3-2, 3-3, and 3-4). Their morphology was irregular. The cells became swollen and even broken, the projections disappeared, cytoplasm outflew, and karyopyknosis was observed. Under an electron microscope, there were a lot of cell debris, most of which were wrapped with broken membrane, and vacuoles were present in cytoplasm of the debris. Some debris was suspected as apoptotic bodies, but no obvious sign has been acquired. RL group: As shown in Figs. 3-5 and 3-5-1, the cell structures remained relatively complete, membranes were smooth, and fewer projections were invisible. Some cells were still swollen, and the structure of mitochondria was essentially normal as circled in Fig. 3-5-1. Heterochromatin existed in the nuclei. RH group: The cell structures of the RH were almost the same as those of the Ctr group, as shown in Fig. 3-6. More projections were observed on the membrane, with no vacuoles in the cytoplasm and large cytoblast in the cell center.
3.3.2. Identification results A total of 506 proteins were identified in our experiment, of which 169 different proteins were detected. All the proteins are shown in Table 1.
3.3. Bioinformatical analysis for proteins
3.3.4. Protein expression detected by western-blot The result of western-blot shows that the expression level of proteins such as Sos1—Son of sevenless homolog 1 and Igh-6—Ig mu chain C region secreted form and in agreement with the result of mass spectrometric analysis as shown in Fig. 6.
3.3.1. Electrophoregram After the purification of the sample proteins, SDS-PAGE was done to detect any differences in different groups. The result is shown in Fig. 4.
3.3.3. Biological function analysis We distinguished all proteins by their functions with GOfact (http:// 61.50.138.118/GOFACT) and identified 83 proteins with intimate biological functions. Most of these proteins have binding and catalytic functions, including 73 binding proteins and 32 catalytic proteins. Of the 73 binding proteins, 35 were protein binding, including receptor binding and cytoskeletal binding proteins. The 32 catalytic proteins comprised transferase and hydrolase. The others were 3 transporter proteins, 4 transcription regulator proteins, 4 molecular transducer proteins, and 3 enzyme regulator proteins. All the proteins are shown in Fig. 5.
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A
B
Ctr
C
Rad
Lnt
A B C Fig. 6. The expression levels of 3 proteins differently expressed among Ctr, Rad and Lnt groups were detected by western-blot. A: Igh-6 Ig mu chain C region secreted form. B: Ryr2 Cardiac Ca2+ release channel. C: Ncam2 Isoform Short of Neural cell adhesion molecule 2.
Fig. 4. Electrophoregram of SDS. A: Membrane proteins from splenic lymphocyte in the LNT group. B: Membrane proteins from splenic lymphocyte in the RAD group. C: Membrane proteins from splenic lymphocyte in the Ctr group.
4. Discussion Ionizing radiation causes damage to various organs and systems, especially to the immune system, which is linked to a higher proliferative activity of the immune cells [23]. Radiation can change morphology and functions of the cells of immune organs. As the largest peripheral immune organ, the spleen contains about 25% of the lymphoid tissues, playing an important role in immune regulation. It is not only an important place for the mature lymphocytes to settle, to proliferate and to differentiate, but also a place to secret interferon, complement, cytokines and other important immunologic active materials [24–26]. Therefore, we chose the spleen as the experimental target and investigated the injuries of long-term low dose radiation to the spleen and the protective effect of lentinan. The damage by ionizing radiation is mainly due to the free radicals generated by radiation, because of oxidative damage to the membranes [27]. Proteins, lipids, and carbohydrates in the biomembrane can be affected by free radicals, leading to the damage of molecular structure, especially to the oxidative scission of membrane lipid unsaturated bonds [28]. One of the necessary conditions of the living cells is the cell membrane fluidity, which mainly depends on interconversion of cis and trans conformation of the unsaturated bonds. It is the unsaturation that makes the bonds easily attacked by free radicals, losing electrons and resulting in oxidation reaction, which ultimately change the rigidity of cell membrane and permeability, increase their brittleness, and
transcription regulator activity
decrease the fluidity [29]. Moreover, there are a variety of protein substances on the cell membrane, which play an important role in the transmission of cell information and transfer of matters [30]. Considering the membrane sensitivity of splenic cells to radiation, we explored the damage by long-term low-dose radiation and the protective effects of lentinan in the spleen lymphocytes. There are no standard LD-radiation models currently. Based on the previous literature, the model of “0.4 Gy × 40 d” was considered as the optimal condition, which pledges the surviving rate of the mice and causes injuries for tissues [31]. The model was used in our experiment to observe the protective effects of lentinan on the mice. We found that oozing blood and monocytes existed extensively in the splenic tissues, indicating radiation-induced injuries in the interstitial microcirculation. We also found that the injuries were present in RL and RH groups. Furthermore, lymphocytes were seriously injured after CL-radiation as compared with the Ctr group. Most cells were broken with periplast efflux. However, only mild injuries were observed in the RL and RH groups, and the cell morphology maintained normal. These results suggest that lentinan can protect lymphocytes against CL-radiation. We investigated the expression of membrane-binding proteins on the basis of the successful generation of radiation-induced injury model. MALDI-TOF/TOF was used to analyze the proteins both qualitatively and quantitatively. A total of 506 proteins were identified, of which 169 underwent changes in their expression levels. 83 proteins with previous known biological function were detected with GOfact database. Most of them have receptor and cytoskeleton binding (n = 73) and catalytic (n = 32) properties. In addition, we found 3 transporter proteins, 4 transcription regulator proteins, 4 molecular transducer molecular transducer activity
enzyme regulator activity catalytic activity
transporter activity
binding
Fig. 5. Function classification of the proteins according to GOfact.
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Table 1 The differentially regulated proteins in Rad and RH groups. Protein name Up-regu in both groups (28) Spnb2 MKIAA4049in Gsto2 Glutathione S-transferase omega-2 Cltc Clathrin, heavy polypeptide Gm6636 40S ribosomal protein S19-like 2810422J05Rik Uncharacterized protein Gm10119 Uncharacterized protein - Protocadherin gamma (fragment) Arhgap11a Rho GTPase activating protein 11A Rpl7a 60S ribosomal protein L7a Zfp532 Isoform 1 of zinc finger protein 532 - 20 kDa protein Rbbp6 Isoform 1 of E3 ubiquitin-protein ligase RBBP6 Ndufs2 Uncharacterized protein - 58 kDa protein Plxna4 Plexin A4, isoform CRA_b Rpl18 60S ribosomal protein L18 G6pd2 Glucose-6-phosphate 1-dehydrogenase 2 2410089E03Rik Uncharacterized protein Aldh2 Aldehyde dehydrogenase, mitochondrial Ctsg Cathepsin G Canx Calnexin Gm9602 hypothetical protein LOC673676 2210023G05Rik carboxylesterase 2-like Smek1 Serine/threonine-protein phosphatase 4 regulatory subunit 3A isoform 2 Pon3 Serum paraoxonase/lactonase 3 Clip1 Protein 1200016B10Rik Isoform 2 of Transcriptional protein SWT1 Hsp90b1 Putative uncharacterized protein Down-regulated in both groups (44) Gm6115 High mobility group protein B1-like Dennd4a DENN/MADD domain containing 4A Fem1b Protein fem-1 homolog B Adamts6 Uncharacterized protein Dek Uncharacterized protein Nono Putative uncharacterized protein Xirp2 Isoform 2 of Xin actin-binding repeat-containing protein 2 Liph Isoform 1 of lipase member H Hspe1 10 kDa heat shock protein, mitochondrial Zfp119 Zinc finger protein 119 LOC100504093 Zinc finger protein 845-like Arhgef37 Isoform 2 of Rho guanine nucleotide exchange factor 37 Hnrnpa0 Heterogeneous nuclear ribonucleoprotein A0 B230315N10Rik hypothetical protein LOC237411 Cep290 Uncharacterized protein Evpl Envoplakin Gprin1 G protein-regulated inducer of neurite outgrowth 1 Erh 14 kDa protein Zfp773 Zinc finger protein LOC76373 Lrrc49 Uncharacterized protein Lrrk1 58 kDa protein Samhd1 SAM domain and HD domain-containing protein 1 isoform 1 Galnt3 Polypeptide N-acetylgalactosaminyltransferase 3 Tacc3 Transforming acidic coiled-coil containing protein 3b 9630025I21Rik Regulator of sex-limitation candidate 16 Phf3 PHD finger protein3 2210408I21Rik Isoform 1 of uncharacterized protein KIAA0825 homolog Zfp78 Zinc finger protein 78 isoform 1 Gatad2b Isoform 2 of transcriptional repressor p66-beta Gm7052 Uncharacterized protein H2afx Histone H2A.x Hnrnpa1 Isoform long of heterogeneous nuclear ribonucleoprotein A1 Gm4916 Low quality protein: hypothetical protein LOC237030 Myo7a Myosin-VIIa - 13 kDa protein Zfp185 Zinc finger protein 185 isoform b Hmgb2 High mobility group protein B2 Mocs3 Adenylyltransferase and sulfurtransferase MOCS3 Hbb-b2 Hemoglobin subunit beta-2 Acin1 Apoptotic chromatin condensation inducer 1 Ankrd26 Uncharacterized protei Herc1 hect domain and RCC1-like domain 1 Zfp54 zinc finger protein 54 Zfp709 zinc finger protein 709
Accession no.
iTRAQ ratio Rad/Ctr
iTRAQ ratio RH/Ctr
IPI00831369 IPI00459308 IPI00648173 IPI00985865 IPI00895479 IPI00473521 IPI00463081 IPI00756581 IPI00330363 IPI00411014 IPI00988982 IPI00551482 IPI00830766 IPI00985776 IPI00876097 IPI00555113 IPI00228867 IPI00660087 IPI00111218 IPI00118696 IPI00119618 IPI00848976 IPI00320204 IPI00930798 IPI00121114 IPI00856350 IPI00453902 IPI00652998
1.579 1.593 1.638 1.645 1.706 1.718 1.751 1.77 1.846 1.932 2.067 2.117 2.412 2.426 2.447 2.532 2.812 2.851 2.868 3.142 3.196 3.215 3.273 3.406 4.48 4.699 4.809 5.092
1.551 2.105 2.147 2.944 1.637 10.614 6.347 3.863 2.158 1.799 1.959 1.526 2.259 1.532 1.615 1.861 2.138 2.112 2.029 2.493 2.823 2.499 2.553 3.406 3.755 2.32 1.987 3.377
IPI00761360 IPI00938460 IPI00469092 IPI00660859 IPI00856853 IPI00987483 IPI00845587 IPI00229926 IPI00263863 IPI00120383 IPI00985765 IPI00894812 IPI00109813 IPI00848582 IPI00798578 IPI00410954 IPI00138232 IPI00988949 IPI00111956 IPI00858029 IPI00856434 IPI00875198 IPI00652440 IPI00473397 IPI00461552 IPI00377615 IPI00672647 IPI00480556 IPI00453989 IPI00850144 IPI00230264 IPI00817004 IPI00884497 IPI00125593 IPI00990539 IPI00776202 IPI00462291 IPI00277554 IPI00316491 IPI00918977 IPI00875424 IPI00676574 IPI00132925 IPI00121318
0.466 0.133 0.185 0.188 0.191 0.214 0.223 0.231 0.267 0.305 0.336 0.337 0.339 0.36 0.361 0.361 0.361 0.367 0.37 0.391 0.397 0.402 0.405 0.406 0.411 0.415 0.419 0.423 0.427 0.436 0.437 0.439 0.442 0.45 0.452 0.453 0.455 0.456 0.461 0.471 0.472 0.492 0.495 0.496
0.304 0.427 0.238 0.213 0.175 0.161 0.456 0.382 0.118 0.309 0.439 0.454 0.305 0.343 0.41 0.421 0.462 0.367 0.344 0.436 0.374 0.24 0.25 0.423 0.288 0.333 0.392 0.249 0.32 0.419 0.212 0.352 0.45 0.472 0.377 0.237 0.328 0.381 0.375 0.347 0.468 0.391 0.448 0.478 (continued on next page)
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Table 1 (continued) Protein name Only down-regulated in Rad group (18) Igsf10 Immunoglobulin superfamily member 10 - Uncharacterized protein Ezr Ezrin Mmp12 Putative uncharacterized protein Pwwp2a Isoform 1 of PWWP domain-containing protein 2A Auh Protein Smchd1 Structural maintenance of chromosomes flexible hinge domain-containing protein 1 Ryr2 Cardiac Ca2+ release channel Mthfd1l Monofunctional C1-tetrahydrofolate synthase, mitochondrial Spag17-ps Sperm-associated antigen 17 Polr1a DNA-directed RNA polymerase I subunit RPA1 Loxhd1 Uncharacterized protein LOC100502716 hypothetical protein LOC100502716 Rp1 Uncharacterized protein Ttn Isoform 3 of Titin Gm7426; H3f3c Histone H3.3C Dst Uncharacterized protein Gm8008 Zinc finger protein 660-like Only up-regulated in Rad group (25) Plekhh1 Isoform 1 of Pleckstrin homology domain-containing family H member Myh8 Myosin-8 Ltf Lactotransferrin Srrm2 Isoform 1 of serine/arginine repetitive matrix protein 2 Myh2 Myosin, heavy polypeptide 2, skeletal muscle, adult Mybpc1 Uncharacterized protein Styxl1 Serine/threonine/tyrosine-interacting-like protein 1 Zfp422 Zinc finger protein 22 LOC100046684; D10Jhu81e ES1 protein homolog, mitochondrial Atf6 Activating transcription factor 6 Neb Uncharacterized protein Bcap31 B-cell receptor-associated protein 31, isoform CRA_c Scp2 Isoform SCPx of Non-specific lipid-transfer protein Dnahc7c;Dnahc7b Uncharacterized protein Srrm2 Serine/arginine repetitive matrix protein 2 Fmo4 Dimethylaniline monooxygenase [N-oxide-forming] 4 Lrrc45 Leucine-rich repeat-containing protein 45 Ccpg1 Isoform 3 of cell cycle progression protein 1 Gripap1 Gripap1 protein Smg7 Isoform 2 of protein SMG7 Hells Isoform 2 of lymphocyte-specific helicase Kif12 Kinesin-like protein KIF12 Calr Calreticulin 4932429P05Rik Novel DUF625 domain containing protein Nlrp4a NACHT, LRR and PYD domains-containing protein 4A Only down-regulated in LNT group (29) Samd11 Sterile alpha motif domain-containing protein 11 Hnrnpab Heterogeneous nuclear ribonucleoprotein A/B isoform 1 Hnrnpd Isoform 1 of heterogeneous nuclear ribonucleoprotein D0 Hmgb3 High mobility group protein B3 H2afv 14 kDa protein Hist3h2a Histone H2A type 3 Suclg2 Uncharacterize protein Hist3h2bb-ps Histone H2B type 3-B Erbb3 Receptor tyrosine-protein kinase erbB-3 Zfp418 Zinc finger protein 418 Ccdc110 Coiled-coil domain-containing protein 110 Clic6 Chloride intracellular channel protein 6 Slk Isoform 2 of STE20-like serine/threonine-protein kinase Ubr5 E3 Ubiquitin-protein ligase UBR5 isoform 2 Trip12 Thyroid hormone receptor interactor 12 Fam83b Protein Ttc28 Tetratricopeptide repeat protein 28 Myh7 Myosin-7 Entpd5 Ectonucleoside triphosphate diphosphohydrolase 5 isoform a - 36 kDa protein Chd2 Chromodomain helicase DNA binding protein 2 Numa1 Nuclear mitotic apparatus protein 1 Ncam2 Isoform short of neural cell adhesion molecule 2 Ep300 Ep300 protein Myh13 Myosin, heavy polypeptide 13, skeletal muscle Zfp329 Zinc finger protein 329 Hmgn1;LOC100044391 Non-histone chromosomal protein HMG-14 Rbm42 Isoform 1 of RNA-binding protein 42 Rhof Rho-related GTP-binding protein RhoF Only up-regulated in LNT group (23)
Accession no.
iTRAQ ratio Rad/Ctr
iTRAQ ratio RH/Ctr
IPI00405810 IPI00985852 IPI00330862 IPI00403307 IPI00876148 IPI00880589 IPI00137433 IPI00338309 IPI00875833 IPI00421240 IPI00313726 IPI00785327 IPI00986541 IPI00874717 IPI00753639 IPI00108200 IPI00973613 IPI00986309
0.202 0.266 0.282 0.284 0.297 0.305 0.315 0.35 0.354 0.377 0.408 0.412 0.43 0.43 0.44 0.457 0.472 0.495
0.518 0.522 0.502 0.857 0.777 0.863 1.13 0.535 0.625 0.721 0.665 0.647 0.574 0.891 0.641 0.565 0.509 0.698
IPI00460337 IPI00265380 IPI00323235 IPI00785240 IPI00649292 IPI00895129 IPI00118956 IPI00124708 IPI00133284 IPI00278602 IPI00720238 IPI00828225 IPI00134131 IPI00283755 IPI01008483 IPI00128288 IPI00955107 IPI00226182 IPI00775775 IPI00678743 IPI00808497 IPI00135586 IPI00123639 IPI00355933 IPI00225222
1.549 1.564 1.6 1.619 1.62 1.624 1.629 1.651 1.657 1.676 1.696 1.723 1.738 1.751 1.797 1.813 1.826 1.865 1.872 1.899 1.931 1.943 1.995 2.749 3.738
0.896 1.362 1.244 1.304 0.92 1.331 1.151 1.002 0.985 1.091 1.201 1.23 1.244 1.353 1.343 1.044 1.058 1.135 1.416 1.52 1.284 1.21 1.504 1.291 0.605
IPI00153184 IPI00277066 IPI00330958 IPI00228879 IPI00555055 IPI00221463 IPI00665996 IPI00229539 IPI00468814 IPI00323205 IPI00129608 IPI00221825 IPI00749669 IPI00876390 IPI00623570 IPI00352394 IPI00944724 IPI00130653 IPI00262024 IPI00989722 IPI00845761 IPI00263048 IPI00322617 IPI00461822 IPI00468665 IPI00990416 IPI00338745 IPI00853826 IPI00227191
0.493 0.523 0.548 0.549 0.551 0.553 0.56 0.56 0.56 0.572 0.589 0.59 0.602 0.618 0.623 0.629 0.64 0.678 0.748 0.755 0.77 0.786 0.79 0.79 0.838 0.942 0.988 1.041 1.237
0.421 0.429 0.276 0.443 0.308 0.356 0.3 0.363 0.378 0.488 0.465 0.423 0.493 0.445 0.481 0.48 0.43 0.343 0.337 0.448 0.45 0.4 0.39 0.479 0.424 0.379 0.476 0.364 0.433
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Table 1 (continued) Protein name
Accession no.
iTRAQ ratio Rad/Ctr
Gm10260 40S ribosomal protein S18-like Dnahc3 Isoform 2 of dynein heavy chain 3, axonemal Suz12 Polycomb protein Suz12 Ush2a Isoform 3 of usherin Wdr76 Isoform 3 of WD repeat-containing protein 76 Tek Tek protein Sacs Isoform 1 of sacsin Parp14 Poly [ADP-ribose] polymerase 14 Slc12a9 Solute carrier family 12 member 9 - Protein Igh-6 Ig mu chain C region secreted form Sos1 Son of sevenless homolog 1 Camp Cathelin-related antimicrobial peptide Fndc3a Protein Recql5 ATP-dependent DNA helicase Q5 Anxa1 Uncharacterized protein Cacna1d Isoform 1 of voltage-dependent L-type calcium channel subunit alph Lcp1 Uncharacterized protein Fmnl2 Isoform 3 of Formin-like protein 2 Chi3l3 Chitinase-3-like protein 3 Tfrc Transferrin receptor protein 1 Lcp1 Plastin-2 Tapbpl Tapasin-related protein precursor Up-regulated in Rad group and down-regulated in LNT group Trpc4 Isoform beta of short transient receptor potential channel 4 Down-regulated in Rad group and up-regulated in LNT group Rps3 Uncharacterized protein
IPI00620156 IPI00880351 IPI00396676 IPI00742431 IPI00674950 IPI00403931 IPI00816925 IPI00776127 IPI01008484 IPI00986896 IPI00468055 IPI00311611 IPI00875797 IPI00970067 IPI00129287 IPI00652811 IPI00855172 IPI00856578 IPI00345373 IPI00157508 IPI00124700 IPI00118892 IPI01007807
0.639 0.695 0.7 0.714 0.947 0.961 0.989 0.999 1.039 1.064 1.193 1.227 1.252 1.255 1.265 1.294 1.31 1.323 1.395 1.41 1.42 1.453 1.689
2.141 1.525 2.036 1.712 2.149 1.9 2.11 1.563 2.472 2.034 4.152 1.811 1.872 1.782 1.969 2.189 2.5 1.583 1.594 1.788 1.774 2.431 3.514
IPI00229745
1.59
0.342
IPI00856866
0.372
2.448
proteins, and 3 enzyme regulator proteins, which were differentially expressed. Biological functional analysis of these proteins shows that, in the Ctr group, the up-regulated expression was mainly found in repairing proteins (mainly ubiquitin and HSP family proteins) and cytoskeleton proteins (mainly were calmodulin), while the down-regulated expression was mainly found in nucleolemma binding proteins (zinc finger protein, DNA and RNA binding proteins, histone proteins). CL-radiation mainly causes injuries to genetic materials and proteins of the lymphocytes. Lentinan can repair the damages by regulating cystoskeleton and signal transduction, but not by repairing the genetic materials. Based on the above mentioned results, we speculate that CL-radiation could trigger related repairing functions, such as change of cell morphology and signal regulation, in order to escape from the injuries to biomacromolecules. But because of the weak repairing effects on genic materials, CL-radiation induces the breakage or death of the cells. The administration of lentinan resulted in up-regulation of the proteins related to the cell repairing machinery (mainly ubiquitin and of HSP family), cell morphology, signal transduction, oxidizing reaction, and stress reaction, and down-regulation of the proteins related to nucleolemma-binding (mainly zinc finger protein). Our results suggest that the protective effects of lentinan against CL-radiation are attributed mainly by repairing the damaged proteins, regulating signal transduction through membrane, instead of protecting genetic materials. Acknowledgments We appreciate the National Natural Science Foundation of China (81102410) for their financial support. We also thank Yong WANG and Qian SUN for their help in the experiments. Moreover, we greatly appreciate the help from the Department of Pathology of No. 401 Hospital of Chinese People's Liberation Army. References [1] Santovito A, Cervella P, Delpero M. Increased frequency of chromosomal aberrations and sister chromatid exchanges in peripheral lymphocytes of radiology technicians chronically exposed to low levels of ionizing radiations. Environ Toxicol Pharmacol 2013;37:396–403.
iTRAQ ratio RH/Ctr
[2] Osipenkova-vichtomova TK. Forensic medical implications of histomorphological changes in the bone and cartilage tissues under effect of radiation. Sud Med Ekspert 2013;56:16–21. [3] Giugni U. Scrap metal and ionizing radiation hazard: prevention and protection. G Ital Med Lav Ergon 2012;34:584–7. [4] Fliedner TM, Graessle DH, Meineke V, Feinendegen LE. Hemopoietic response to low dose-rates of ionizing radiation shows stem cell tolerance and adaptation. Dose Response 2012;10:644–63. [5] Sakly A, Ayed Y, Chaari N, Akrout M, Bacha H, Cheikh HB. Assessment of chromosomal aberrations and micronuclei in peripheral lymphocytes from Tunisian hospital workers exposed to ionizing radiation. Genet Test Mol Biomarkers 2013;17: 650–5. [6] Arakali AV, Paul CR, French JB, Box HC. Comparison of free radical damage in dinucleoside monophosphate via autoxidation processes and by ionizing radiation. Radiat Res 1987;112:183–6. [7] Smith JT, Willey NJ, Hancock JT. Low dose ionizing radiation produces too few reactive oxygen species to directly affect antioxidant concentrations in cells. Biol Lett 2012;8:594–7. [8] Chuai Y. Molecular hydrogen and radiation protection. Free Radic Res 2012;46:1061–7. [9] Bernard ME, Kim H, Berhane H, Epperly MW, Franicola D, Zhang X, et al. GSnitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines. Radiat Res 2011;176:603–12. [10] Hao T, Wang YM, Li JJ, Du ZY, Duan CM, Wang CY, et al. Effect of lentinan against immunosuppression of lymphocytes cultured in simulated microgravity environment. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2012;20:182–6. [11] Patchen ML, MacVittie TJ, Brook I. Glucan-induced hemopoietic and immune stimulation: therapeutic effects in sublethally and lethally irradiated mice. Methods Find Exp Clin Pharmacol 1986 Mar;8(3):151–5. [12] Schwartz GN, Patchen ML, Neta R. Radioprotection of mice with interleukin-1: relationship to the number of erythroid and granulocyte-macrophage colony-forming cells. Radiat Res 1990 Feb;121(2):220–6. [13] Ina K. The use of lentinan for treating gastric cancer. Anticancer Agents Med Chem 2013;13:681–8. [14] Liu M, Hu J, Ma T, Wang S, Ding H. Application of a disposable screen-printed electrode to depression diagnosis for laboratory rats based on blood serotonin detection. Anal Sci 2011;27:839–43. [15] Kurita K, Matsumura Y, Takahara H, Hatta K, Shimojoh M. Synthesis and macrophage activation of lentinan-mimic branched amino polysaccharides: curdlans having NAcetyl-d-glucosamine branches. Biomacromolecules 2011;12:2267–74. [16] Shi L, Fu Y. Isolation, purification, and immunomodulatory activity in vitro of three polysaccharides from roots of Cudrania tricuspidata. Acta Biochim Biophys Sin (Shanghai) 2011;43:418–24. [17] Liu Ying-hua. Inhibitory effect of lentinan on visceral organs in subchronic radiationinjured mice. Pharm J Chin PLA 2012;28:200–4. [18] Qing-jie Fu FU. The effect of lentinan on the gene expression of major ion channels in T lymphocytes isolated from mice exposed to chronic radiation. Nucl Technol Radiat Prot 2013;28:204–11. [19] Shi J, Feng H, Lee J, Ning Chen W. Comparative proteomics profile of lipidcumulating oleaginous yeast: an iTRAQ-coupled 2-D LC-MS/MS analysis. PLoS One 2013;8:e85532.
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[20] Zhuang Lu. Study on enrichment and quantitation strategy for core fucosylated glycoprotein in large scale[D]. Beijing: Beijing Institute of Technology; 2009. [21] Bjarnadóttir SG, Hollung K, Høy M, Bendixen E, Codrea MC, Veiseth-Kent E. Changes in protein abundance between tender and tough meat from bovine longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis. J Anim Sci 2012;90: 2035–43. [22] Martyniuk CJ, Popesku JT, Chown B, Denslow ND, Trudeau VL. Quantitative proteomics in teleost fish: insights and challenges for neuroendocrine and neurotoxicology research. Gen Comp Endocrinol 2012;176:314–20. [23] Noda M, Uema M. Current topics on inactivation of norovirus. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku 2011;129:37–54. [24] Luo-Owen X, Pecaut MJ, Rizvi A, Gridley DS. Low-dose total-body γ irradiation modulates immune response to acute proton radiation. Radiat Res 2012;177:251–64. [25] Gridley DS, Freeman TL, Makinde AY, Wroe AJ, Luo-Owen X, Tian J, et al. Comparison of proton and electron radiation effects on biological responses in liver, spleen and blood. Int J Radiat Biol 2011;87:1173–81.
[26] Gridley DS, Pecaut MJ, Green LM, Sanchez MC, Kadhim MA. Strain-related differences and radiation quality effects on mouse leukocytes: gamma-rays and protons (with and without aluminum shielding). In Vivo 2011;25:871–80. [27] Lu W, Xue YM, Zhu B, Lian X, Liu N. The relationship between oxidative injury induced by low glucose and mitochondrial membrane potential in HUVEC-12 cells. Zhonghua Nei Ke Za Zhi 2011;50:873–6. [28] Kidd P. Astaxanthin, cell membrane nutrient with diverse clinical benefits and antiaging potential. Altern Med Rev 2011;16:355–64. [29] Greig FH, Kennedy S, Spickett CM. Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation. Free Radic Biol Med 2012;52:266–80. [30] Azalea-Romero M, González-Mendoza M, Cáceres-Pérez AA, Lara-Padilla E, CáceresCortés JR. Low expression of stem cell antigen-1 on mouse haematopoietic precursors is associated with erythroid differentiation. Cell Immunol 2012;279:187–95. [31] Patchen ML, MacVittie TJ. Dose-dependent responses of murine pluripotent stem cells and myeloid and erythroid progenitor cells following administration of the immunomodulating agent glucan. Immunopharmacology 1983 Apr;5(4):303–13.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Effect of lentinan on membrane-bound protein expression in splenic lymphocytes under chronic low-dose radiation Ying-Hua Liu a,1, Shou-Dong Ma a,1, Qing-Jie Fu a, Li-Yan Zhao a, Yi Li b, Hai-Qing Wang b, Ming-Chun Li a,⁎ a b
Department of Pharmacy, No. 401 Hospital of Chinese People's Liberation Army, Qingdao 266071, China Radiation Oncology Department, No. 401 Hospital of Chinese People's Liberation Army, Qingdao 266071, China
a r t i c l e
i n f o
Article history: Received 12 March 2014 Received in revised form 2 July 2014 Accepted 21 July 2014 Available online 4 August 2014 Keywords: Lentinan Lymphocytes Radioprotection
a b s t r a c t We investigated the protective effects of lentinan against damages to chronic and low-dose radiation (CL-radiation) by using mouse models. The mice were randomized divided into four groups: normal control mice (Ctr), mice exposed to radiation (Rad), irradiated mice treated with low-dose lentinan (0.1 mg/(kg.d), RL), and irradiated mice treated with high-dose lentinan (0.5 mg/(kg.d), RH). All the mice were injected intraperitoneally once a day at a dose of 0.5 mL (Ctr and Rad with normal sodium while RL and RH with lentinan). The success of radiation models was confirmed by HE stain and cell morphology by a transmission electron microscope (TEM). On the basis of radiation models, we investigated the expression of proteins on the membrane of splenic cells through MALDI-TOF-MS/MS. The results demonstrated that both RT-radiation and lentinan affected the expression of membrane proteins, but lentinan protected the splenic cells and tissue from the injuries caused by CLradiation. Therefore, we speculated that CL-radiation mainly damages the genetic materials and membranebound proteins, while lentinan protects membrane-bound proteins by regulating signal transduction and the appearance of the cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction With the development of technology and the wide use of electronic appliance, a growing number of people are exposed to radiation, so radiation hazard has become a serious problem and a major health threat worldwide [1,2]. It has been increasingly realized that radiation can cause injuries to multiple organs, especially in those who work in nuclear medicine, nuclear industry etc., resulting in immunodeficiency, tumors, aging and hematologic diseases [3–5]. Radiation damage is mainly caused by excessive production of free radicals that are produced from endosomatic water. As a result, free radicals are often used as an indicator to detect radiation-induced damage [6–8]. At present, the majority of radiation protection agents are sulfur-containing drugs, hormones, cytokines and natural drugs [9]. Among them, natural drugs, especially polysaccharides, have been extensively studied and paid a significant attention because of their edibility, few side effects, and bioavailability. Lentinan, a [1–3]-beta-D-glucan extracted from lentinus edodes (shiitake), has been well recognized as an immunomodulator [10–12]. It has been reported that it can regulate immunity, resist oxidization and mutations, and reduce blood lipid [13–16]. However, although the ⁎ Corresponding author. E-mail address: [email protected] (M.-C. Li). 1 These two authors contributed equally to this work and should be considered as cofirst authors.
http://dx.doi.org/10.1016/j.intimp.2014.07.027 1567-5769/© 2014 Elsevier B.V. All rights reserved.
immunological regulation of lentinan has been well established, its radioprotective effect remains elusive. The purpose of our study was to investigate the protective capacity of lentinan against CL-radiation and the molecular mechanisms involved. 2. Materials and methods 2.1. Animals Kunming mice (male; 5 weeks; 20–24 g) were obtained from the Experimental Animal and Animal Studies Center of Qingdao, China, and kept under the pathogen free condition. All our experiments were performed in accordance with the regulation laid down by Chinese Association for Laboratory Animal Science. 2.2. Reagents Lentinan (1 mg/2 mL, 110103–3) was purchased from Jinling Pharmaceutical Co. Ltd. (Nanjing, China). It was further diluted to 0.004 mg/mL, and 0.02 mg/mL with saline before the injections. ProteoExtract Transmembrane Protein Extraction Kit was purchased from EMD Chemicals Inc. (Darmstadt, Germany). iTRAQ kit was purchased from Applied Biosystems (California, US). BSA standards kit, Commassie blue standing kit and the others were purchased from Sigma-Aldrich Co. LLC (Shanghai, China).
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2.3. Animal models The mice were randomly divided into four groups (n = 20 each): normal control mice (Ctr), mice exposed to radiation (Rad), irradiated mice treated with low-dose (0.1 mg/(kg.d), RL) and high-dose (0.5 mg/(kg.d), RH) lentinan. The mice in the Ctr and Rad groups were injected with 0.5 mL normal sodium, and those in the RL and RH groups were injected with 0.5 mL lentinan in different concentrations as mentioned above. All the mice, expect those in the Ctr group, were exposed for 15 min to systematical radiation of 0.4 Gy from medical linear accelerator with an absorbed dose rate of 3 Gy/min. The source to skin distance was 150 cm, and exposure field was 20 cm × 20 cm. The radiation was given from Monday to Friday for 40 days. All the mice were euthanized by cervical vertebra dislocation after the last radiation and sterilized in 75% ethanol for 30 s. Their spleens were then removed. 2.4. Pathological examination of the animal model After the removal of the spleens, ten spleens of each group were sent to the Pathology Department of our hospital for histological examination. HE staining was used to observe the changes in tissue structures. TEM was used to observe the damages to the splenic lymphocytes [17]. 2.5. Expression of membrane proteins in lymphocytes The inspection of the animal model showed that lentinan was more protective against irradiation in the RH group than in the RL group. As a result, expressions of membrane proteins in lymphocytes were analyzed only in the Ctr, Rad and RH groups in the following experiments. 2.5.1. Protein extraction The ten spleens in each group were milled with the 200 mesh stainless steel cell sieve in the culture medium, followed by centrifugation at 1500 r/min for 15 min. The supernatant was discarded. RBC lysis buffer was added into the centrifuge tube for 2–3 times. After removing the supernatant, we got lymphocytes of different populations. Cells were
incorporated within the same group, and the concentrations were regulated into 5 × 106/mL [18]. Transmembrane protein extraction kit was used to get transmembrane protein. The extraction was preserved at −20 °C. 2.5.2. Sample preparation The protein concentrations were determined with Bradford Regent. Six times the volumes of acetone (−20 °C) were added to the sample tubes (4 °C), which were turned over three times. The sample tubes were incubated at − 20 °C for at least 30 min until the appearance of precipitation. The acetone was decanted and dissolution buffer added for concentration of 2 μg/μL in each sample. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) [19] was done to detect the differences in the expressions of protein samples. Hoefer miniVE was used to prepare 70 × 100 × 1 mm3 polyacrylamide gels with 13% separating gels (pH 8.8) and 4% stacking gels (pH 6.8). Loading buffer (10 μL), the sample (10 μL) and 2μL βmercaptoethanol were mixed and heated at 95 °C for 3 min for denaturation. 5× Running Buffer (1 L) was prepared with 15 g Tris base, 72 g glycine, and 5 g SDS. The gels were run at 10 mA for 30 min, and then changed to 30 mA for 1 h. Coomassie blue staining [20]. 2.5.3. iTRAQ labeling and digestion [21] 53.325 μg sample of each group was made according to the above steps. After lyophilization, 20 μL TTLB was added to dissolve the protein samples. Then, 1 μL 2% SDS was added into the kit and vortex to mix. 2 μL 50 mM TECP was added to each sample tube, vortex to mix, and the tubes were incubated at 60 °C for 1 h. 1 μL 200 mM MMTS was added to each tube, vortex to mix, and the tubes were incubated at room temperature for 10 min. 20 μg trypsin was reconstituted with 40 μL ultrapure water, vortex to mix. The trypsin solution was added to each sample tube (50:1 proteins: enzyme), vortex to mix. The tubes were incubated at 37 °C for 12 h then spanned in order to bring the sample digest to the bottom of the tube. allowed each vial of iTRAQ reagent required to room temperature, then spanned to bring the reagent to the bottom of the tubes. Then,
Fig. 1. The picture of SCX-HPLC resolution.
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70 μL of ethanol was added to each reagent vial, vortex to mix. The reagent 114 was transferred to the protein samples of Ctr, reagent 115 to Rad, and the reagent 116 to LNT. Each tube was vortexed to mix. The tubes were incubated at room temperature for 1 h, and each tube was spanned at room temperature every 15 min. All peptides were preserved at 20 °C. 2.5.4. 2D-LC separation [22] 2.5.4.1. Strong cationic exchange high performance liquid chromatography (SCX-HPLC). The mobile phase A (5 mM KH2PO4/20% HCN, pH = 3.0) and mobile phase B (5 mM KH2PO4/350 mM KCl/20% HCN, pH = 3.0) were prepared. The protein samples were diluted with mobile phase A. Conditioning of SCX (Thermo Biobasic SCX, 4.6 mm × 250 mm × 5 μm): the flow rate was set to 700 μL/min, the wavelength to 214 nm, and the pressure to 15 MPa. The mobile phase A was run at 0–35 min, 50% mobile A and mobile phase B at 35–45 min, 100% mobile phase B at 45–50 min, 0–100% mobile phase B at 50–51 min, and 100% mobile phase A at 51 min. The samples were loaded and injected, then peptides were fractionated and eluted as shown in Fig. 1. The fractions were collected and pooled into 10 tubes according to SCX elution chromatogram. 2.5.4.2. Reverse phase high-performance liquid chromatography (RPHPLC). Mobile A (5% KH2PO4/95% HCN/0.1% KCl) and mobile B (95% KH2PO4/5% HCN/0.1% KCl) were prepared. All fractions were lyophilized to remove SCX Solvent A, and then diluted with 80–100 μL RP Solvent A for each fractions and centrifuged for 15 min at 10,000 rpm. Conditioning of RP-HPLC (MICHROM Bioresources/MAGIC C18AQ 0.2 × 150 mm 3 μ 200 A): The cartridge was wetted with 10 μL 100% HCN for 10 times, wetted with 10 μL 50% ACN for 10 times, and equilibrated with 10 μL
507
0.1% TFA for 10 times. The flow rate was set to 0.2 μL/min, the wavelength to 214 nm and 280 nm, and the pressure to 15 MPa; 90% phase A and 10% phase B were run at 0–90 min, 50% phase A and 50% phase B at 90–120 min, 0–100% phase B at 120–140 min, 100% phase B at 140–145 min, and 90% phase A and 10% phase B at 145 min. MS/MS: The fresh MALDI matrix solution (5 mg CHCA dissolved with 50% HCN/0.1% TFA) was prepared. The spotting was initiated at 35 min when the first peptide peaks were eluted. The column effluent was mixed directly with MALDI matrix solution. The fractions were automatically deposited every 30 s onto the MALDI target plate using a Probot micro-fraction collector. MS range was 700–4000 Da, total shot/spectrum was 1000, and fixed laser intensity was 3400; MS/MS range was 10–1360 Da, total shot/spectrum was 2000, and fixed laser intensity was 380. 2.5.5. ABI 4800 MALDI MS/MS analysis and database searching MALDI plates were analysed on ABI 4800 MS/MS. The LC-MALDI template was selected, and Mascot software was used for peptide identification. The parameters were set for protein identification as follows: fixed modifications were (N_term)_iTRAQ, Lysine (k) _iTRAQ and MMTS (c); variable modifications were oxidation (M); precursor tolerance was 0.2 Da; maximum peptide rank was 2. The definition of value for differentially expressed proteins was 116/114 (116/115) N 1.5 times or 116/114 (116/115) b 0.5 times, and P b 0.05. Then peak areas of different groups were automatically extracted. The quantitative information was processed according to correction factor. 2.5.6. Western-blot analysis Protein concentrations were determined using the BCA protein assay kit (CWBIO, China), and samples were separated on SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane
Fig. 2. HE slices after radiation and lentinan treatment (×100). 2-1 (Ctr group): the splenic tissues retained normal. 2-2 (Rad group): severe bleeding and remote hemorrhage existed in the interstitium. 2-3 (RL group): megakaryocytes increased and hemorrhage was present. 2-4 (RH group): the tissues were basically normal, with a slight decrease of megakaryocytes.
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(MILLIPORE, US). The membranes were reacted with antibodies against phosphorylated or nonphosphorylated Igh-6 Ig mu chain C region secreted form, Ryr2 Cardiac Ca2 + release channel or Ncam2 Isoform Short of Neural cell adhesion molecule 2(Cell Signaling Technology, US). Then, specific antigen/antibody complexes were made with horseradish peroxidase-conjugated secondary antibodies (Rabbit IgG, Cell Signaling Technology, US) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, US). The images were digitized from the membrane and the band intensity of each protein was quantified with NIH image software. 3. Results 3.1. Pathological changes of the tissues Pathological changes have been observed following the exposure to CL-radiation. Histology showed scattered mature megakaryocytes in the splenic tissues, but no erythrocyte stasis in splenic sinus in the Ctr group. Compared with the Ctr group, severe blood loss in the
interstitium and obvious congestion were observed in the Rad group; no obvious injury or mild congestion was displayed in the RL group. 3.2. Changes in cell structure Injuries were also observed in the ultrastructure of the spleen. Compared with the Ctr group, minute projections on the membrane disappeared and the cells were swelled seriously and even disrupted, and pyknosis was emerged in the nuclei and cytoplasm in the Rad group. The ultrastructure was essentially normal, the projections were complete, and mild swelling was occasionally found in the RH group. All the pathological changes are shown in Fig.2. Ctr group: As shown in Fig. 3-1, there were no injuries in the cell membrane and the nuclear membrane. The projections were found on the cell membrane. Within the cells, there were large round or ellipse nuclei in the center, containing mostly euchromatin. Abundant cytoplasm, many free-ribosomes, and smaller cell organs were observed. Clear and intact cristae were preserved in mitochondria as indicated by the arrow in Fig. 3-1-1.
Fig. 3. Electron microscopic sections after radiation and lentinan treatment. 3-1 (Ctr group): the nucleus was big and in the center. The double-cellular membrane and nuclear membrane were visible. The cristae were evident in the chondriosome. 3-1-1 (Ctr group): mitochondria were clear and intact cristae, as shown at the arrow. 3-2 (Rad group): in the center were megameric chromosomes. Vacuoles existed in the cytoplasm, as shown at the arrow. 3-3 (Rad group): in the center were megameric chromosomes. The cells were mainly heterogeneous. The cell organ was abnormal. 3-4 (Rad group): in the center were megameric chromosomes. The membrane was broken, and the kytoplasm outflew, as shown at the arrow. 3-5 (RL group): cellular structure was essentially normal, and the nucleus was big and in the center. The projections were present. 3-5-1 (RL group): the mitochondria were essentially normal, as shown in the circle, 3-6 (RH group): the cellular structure and kytoplasm were normal.
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Fig. 3 (continued).
Rad group: The structures of cells were injured (as shown in Figs. 3-2, 3-3, and 3-4). Their morphology was irregular. The cells became swollen and even broken, the projections disappeared, cytoplasm outflew, and karyopyknosis was observed. Under an electron microscope, there were a lot of cell debris, most of which were wrapped with broken membrane, and vacuoles were present in cytoplasm of the debris. Some debris was suspected as apoptotic bodies, but no obvious sign has been acquired. RL group: As shown in Figs. 3-5 and 3-5-1, the cell structures remained relatively complete, membranes were smooth, and fewer projections were invisible. Some cells were still swollen, and the structure of mitochondria was essentially normal as circled in Fig. 3-5-1. Heterochromatin existed in the nuclei. RH group: The cell structures of the RH were almost the same as those of the Ctr group, as shown in Fig. 3-6. More projections were observed on the membrane, with no vacuoles in the cytoplasm and large cytoblast in the cell center.
3.3.2. Identification results A total of 506 proteins were identified in our experiment, of which 169 different proteins were detected. All the proteins are shown in Table 1.
3.3. Bioinformatical analysis for proteins
3.3.4. Protein expression detected by western-blot The result of western-blot shows that the expression level of proteins such as Sos1—Son of sevenless homolog 1 and Igh-6—Ig mu chain C region secreted form and in agreement with the result of mass spectrometric analysis as shown in Fig. 6.
3.3.1. Electrophoregram After the purification of the sample proteins, SDS-PAGE was done to detect any differences in different groups. The result is shown in Fig. 4.
3.3.3. Biological function analysis We distinguished all proteins by their functions with GOfact (http:// 61.50.138.118/GOFACT) and identified 83 proteins with intimate biological functions. Most of these proteins have binding and catalytic functions, including 73 binding proteins and 32 catalytic proteins. Of the 73 binding proteins, 35 were protein binding, including receptor binding and cytoskeletal binding proteins. The 32 catalytic proteins comprised transferase and hydrolase. The others were 3 transporter proteins, 4 transcription regulator proteins, 4 molecular transducer proteins, and 3 enzyme regulator proteins. All the proteins are shown in Fig. 5.
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A
B
Ctr
C
Rad
Lnt
A B C Fig. 6. The expression levels of 3 proteins differently expressed among Ctr, Rad and Lnt groups were detected by western-blot. A: Igh-6 Ig mu chain C region secreted form. B: Ryr2 Cardiac Ca2+ release channel. C: Ncam2 Isoform Short of Neural cell adhesion molecule 2.
Fig. 4. Electrophoregram of SDS. A: Membrane proteins from splenic lymphocyte in the LNT group. B: Membrane proteins from splenic lymphocyte in the RAD group. C: Membrane proteins from splenic lymphocyte in the Ctr group.
4. Discussion Ionizing radiation causes damage to various organs and systems, especially to the immune system, which is linked to a higher proliferative activity of the immune cells [23]. Radiation can change morphology and functions of the cells of immune organs. As the largest peripheral immune organ, the spleen contains about 25% of the lymphoid tissues, playing an important role in immune regulation. It is not only an important place for the mature lymphocytes to settle, to proliferate and to differentiate, but also a place to secret interferon, complement, cytokines and other important immunologic active materials [24–26]. Therefore, we chose the spleen as the experimental target and investigated the injuries of long-term low dose radiation to the spleen and the protective effect of lentinan. The damage by ionizing radiation is mainly due to the free radicals generated by radiation, because of oxidative damage to the membranes [27]. Proteins, lipids, and carbohydrates in the biomembrane can be affected by free radicals, leading to the damage of molecular structure, especially to the oxidative scission of membrane lipid unsaturated bonds [28]. One of the necessary conditions of the living cells is the cell membrane fluidity, which mainly depends on interconversion of cis and trans conformation of the unsaturated bonds. It is the unsaturation that makes the bonds easily attacked by free radicals, losing electrons and resulting in oxidation reaction, which ultimately change the rigidity of cell membrane and permeability, increase their brittleness, and
transcription regulator activity
decrease the fluidity [29]. Moreover, there are a variety of protein substances on the cell membrane, which play an important role in the transmission of cell information and transfer of matters [30]. Considering the membrane sensitivity of splenic cells to radiation, we explored the damage by long-term low-dose radiation and the protective effects of lentinan in the spleen lymphocytes. There are no standard LD-radiation models currently. Based on the previous literature, the model of “0.4 Gy × 40 d” was considered as the optimal condition, which pledges the surviving rate of the mice and causes injuries for tissues [31]. The model was used in our experiment to observe the protective effects of lentinan on the mice. We found that oozing blood and monocytes existed extensively in the splenic tissues, indicating radiation-induced injuries in the interstitial microcirculation. We also found that the injuries were present in RL and RH groups. Furthermore, lymphocytes were seriously injured after CL-radiation as compared with the Ctr group. Most cells were broken with periplast efflux. However, only mild injuries were observed in the RL and RH groups, and the cell morphology maintained normal. These results suggest that lentinan can protect lymphocytes against CL-radiation. We investigated the expression of membrane-binding proteins on the basis of the successful generation of radiation-induced injury model. MALDI-TOF/TOF was used to analyze the proteins both qualitatively and quantitatively. A total of 506 proteins were identified, of which 169 underwent changes in their expression levels. 83 proteins with previous known biological function were detected with GOfact database. Most of them have receptor and cytoskeleton binding (n = 73) and catalytic (n = 32) properties. In addition, we found 3 transporter proteins, 4 transcription regulator proteins, 4 molecular transducer molecular transducer activity
enzyme regulator activity catalytic activity
transporter activity
binding
Fig. 5. Function classification of the proteins according to GOfact.
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Table 1 The differentially regulated proteins in Rad and RH groups. Protein name Up-regu in both groups (28) Spnb2 MKIAA4049in Gsto2 Glutathione S-transferase omega-2 Cltc Clathrin, heavy polypeptide Gm6636 40S ribosomal protein S19-like 2810422J05Rik Uncharacterized protein Gm10119 Uncharacterized protein - Protocadherin gamma (fragment) Arhgap11a Rho GTPase activating protein 11A Rpl7a 60S ribosomal protein L7a Zfp532 Isoform 1 of zinc finger protein 532 - 20 kDa protein Rbbp6 Isoform 1 of E3 ubiquitin-protein ligase RBBP6 Ndufs2 Uncharacterized protein - 58 kDa protein Plxna4 Plexin A4, isoform CRA_b Rpl18 60S ribosomal protein L18 G6pd2 Glucose-6-phosphate 1-dehydrogenase 2 2410089E03Rik Uncharacterized protein Aldh2 Aldehyde dehydrogenase, mitochondrial Ctsg Cathepsin G Canx Calnexin Gm9602 hypothetical protein LOC673676 2210023G05Rik carboxylesterase 2-like Smek1 Serine/threonine-protein phosphatase 4 regulatory subunit 3A isoform 2 Pon3 Serum paraoxonase/lactonase 3 Clip1 Protein 1200016B10Rik Isoform 2 of Transcriptional protein SWT1 Hsp90b1 Putative uncharacterized protein Down-regulated in both groups (44) Gm6115 High mobility group protein B1-like Dennd4a DENN/MADD domain containing 4A Fem1b Protein fem-1 homolog B Adamts6 Uncharacterized protein Dek Uncharacterized protein Nono Putative uncharacterized protein Xirp2 Isoform 2 of Xin actin-binding repeat-containing protein 2 Liph Isoform 1 of lipase member H Hspe1 10 kDa heat shock protein, mitochondrial Zfp119 Zinc finger protein 119 LOC100504093 Zinc finger protein 845-like Arhgef37 Isoform 2 of Rho guanine nucleotide exchange factor 37 Hnrnpa0 Heterogeneous nuclear ribonucleoprotein A0 B230315N10Rik hypothetical protein LOC237411 Cep290 Uncharacterized protein Evpl Envoplakin Gprin1 G protein-regulated inducer of neurite outgrowth 1 Erh 14 kDa protein Zfp773 Zinc finger protein LOC76373 Lrrc49 Uncharacterized protein Lrrk1 58 kDa protein Samhd1 SAM domain and HD domain-containing protein 1 isoform 1 Galnt3 Polypeptide N-acetylgalactosaminyltransferase 3 Tacc3 Transforming acidic coiled-coil containing protein 3b 9630025I21Rik Regulator of sex-limitation candidate 16 Phf3 PHD finger protein3 2210408I21Rik Isoform 1 of uncharacterized protein KIAA0825 homolog Zfp78 Zinc finger protein 78 isoform 1 Gatad2b Isoform 2 of transcriptional repressor p66-beta Gm7052 Uncharacterized protein H2afx Histone H2A.x Hnrnpa1 Isoform long of heterogeneous nuclear ribonucleoprotein A1 Gm4916 Low quality protein: hypothetical protein LOC237030 Myo7a Myosin-VIIa - 13 kDa protein Zfp185 Zinc finger protein 185 isoform b Hmgb2 High mobility group protein B2 Mocs3 Adenylyltransferase and sulfurtransferase MOCS3 Hbb-b2 Hemoglobin subunit beta-2 Acin1 Apoptotic chromatin condensation inducer 1 Ankrd26 Uncharacterized protei Herc1 hect domain and RCC1-like domain 1 Zfp54 zinc finger protein 54 Zfp709 zinc finger protein 709
Accession no.
iTRAQ ratio Rad/Ctr
iTRAQ ratio RH/Ctr
IPI00831369 IPI00459308 IPI00648173 IPI00985865 IPI00895479 IPI00473521 IPI00463081 IPI00756581 IPI00330363 IPI00411014 IPI00988982 IPI00551482 IPI00830766 IPI00985776 IPI00876097 IPI00555113 IPI00228867 IPI00660087 IPI00111218 IPI00118696 IPI00119618 IPI00848976 IPI00320204 IPI00930798 IPI00121114 IPI00856350 IPI00453902 IPI00652998
1.579 1.593 1.638 1.645 1.706 1.718 1.751 1.77 1.846 1.932 2.067 2.117 2.412 2.426 2.447 2.532 2.812 2.851 2.868 3.142 3.196 3.215 3.273 3.406 4.48 4.699 4.809 5.092
1.551 2.105 2.147 2.944 1.637 10.614 6.347 3.863 2.158 1.799 1.959 1.526 2.259 1.532 1.615 1.861 2.138 2.112 2.029 2.493 2.823 2.499 2.553 3.406 3.755 2.32 1.987 3.377
IPI00761360 IPI00938460 IPI00469092 IPI00660859 IPI00856853 IPI00987483 IPI00845587 IPI00229926 IPI00263863 IPI00120383 IPI00985765 IPI00894812 IPI00109813 IPI00848582 IPI00798578 IPI00410954 IPI00138232 IPI00988949 IPI00111956 IPI00858029 IPI00856434 IPI00875198 IPI00652440 IPI00473397 IPI00461552 IPI00377615 IPI00672647 IPI00480556 IPI00453989 IPI00850144 IPI00230264 IPI00817004 IPI00884497 IPI00125593 IPI00990539 IPI00776202 IPI00462291 IPI00277554 IPI00316491 IPI00918977 IPI00875424 IPI00676574 IPI00132925 IPI00121318
0.466 0.133 0.185 0.188 0.191 0.214 0.223 0.231 0.267 0.305 0.336 0.337 0.339 0.36 0.361 0.361 0.361 0.367 0.37 0.391 0.397 0.402 0.405 0.406 0.411 0.415 0.419 0.423 0.427 0.436 0.437 0.439 0.442 0.45 0.452 0.453 0.455 0.456 0.461 0.471 0.472 0.492 0.495 0.496
0.304 0.427 0.238 0.213 0.175 0.161 0.456 0.382 0.118 0.309 0.439 0.454 0.305 0.343 0.41 0.421 0.462 0.367 0.344 0.436 0.374 0.24 0.25 0.423 0.288 0.333 0.392 0.249 0.32 0.419 0.212 0.352 0.45 0.472 0.377 0.237 0.328 0.381 0.375 0.347 0.468 0.391 0.448 0.478 (continued on next page)
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Table 1 (continued) Protein name Only down-regulated in Rad group (18) Igsf10 Immunoglobulin superfamily member 10 - Uncharacterized protein Ezr Ezrin Mmp12 Putative uncharacterized protein Pwwp2a Isoform 1 of PWWP domain-containing protein 2A Auh Protein Smchd1 Structural maintenance of chromosomes flexible hinge domain-containing protein 1 Ryr2 Cardiac Ca2+ release channel Mthfd1l Monofunctional C1-tetrahydrofolate synthase, mitochondrial Spag17-ps Sperm-associated antigen 17 Polr1a DNA-directed RNA polymerase I subunit RPA1 Loxhd1 Uncharacterized protein LOC100502716 hypothetical protein LOC100502716 Rp1 Uncharacterized protein Ttn Isoform 3 of Titin Gm7426; H3f3c Histone H3.3C Dst Uncharacterized protein Gm8008 Zinc finger protein 660-like Only up-regulated in Rad group (25) Plekhh1 Isoform 1 of Pleckstrin homology domain-containing family H member Myh8 Myosin-8 Ltf Lactotransferrin Srrm2 Isoform 1 of serine/arginine repetitive matrix protein 2 Myh2 Myosin, heavy polypeptide 2, skeletal muscle, adult Mybpc1 Uncharacterized protein Styxl1 Serine/threonine/tyrosine-interacting-like protein 1 Zfp422 Zinc finger protein 22 LOC100046684; D10Jhu81e ES1 protein homolog, mitochondrial Atf6 Activating transcription factor 6 Neb Uncharacterized protein Bcap31 B-cell receptor-associated protein 31, isoform CRA_c Scp2 Isoform SCPx of Non-specific lipid-transfer protein Dnahc7c;Dnahc7b Uncharacterized protein Srrm2 Serine/arginine repetitive matrix protein 2 Fmo4 Dimethylaniline monooxygenase [N-oxide-forming] 4 Lrrc45 Leucine-rich repeat-containing protein 45 Ccpg1 Isoform 3 of cell cycle progression protein 1 Gripap1 Gripap1 protein Smg7 Isoform 2 of protein SMG7 Hells Isoform 2 of lymphocyte-specific helicase Kif12 Kinesin-like protein KIF12 Calr Calreticulin 4932429P05Rik Novel DUF625 domain containing protein Nlrp4a NACHT, LRR and PYD domains-containing protein 4A Only down-regulated in LNT group (29) Samd11 Sterile alpha motif domain-containing protein 11 Hnrnpab Heterogeneous nuclear ribonucleoprotein A/B isoform 1 Hnrnpd Isoform 1 of heterogeneous nuclear ribonucleoprotein D0 Hmgb3 High mobility group protein B3 H2afv 14 kDa protein Hist3h2a Histone H2A type 3 Suclg2 Uncharacterize protein Hist3h2bb-ps Histone H2B type 3-B Erbb3 Receptor tyrosine-protein kinase erbB-3 Zfp418 Zinc finger protein 418 Ccdc110 Coiled-coil domain-containing protein 110 Clic6 Chloride intracellular channel protein 6 Slk Isoform 2 of STE20-like serine/threonine-protein kinase Ubr5 E3 Ubiquitin-protein ligase UBR5 isoform 2 Trip12 Thyroid hormone receptor interactor 12 Fam83b Protein Ttc28 Tetratricopeptide repeat protein 28 Myh7 Myosin-7 Entpd5 Ectonucleoside triphosphate diphosphohydrolase 5 isoform a - 36 kDa protein Chd2 Chromodomain helicase DNA binding protein 2 Numa1 Nuclear mitotic apparatus protein 1 Ncam2 Isoform short of neural cell adhesion molecule 2 Ep300 Ep300 protein Myh13 Myosin, heavy polypeptide 13, skeletal muscle Zfp329 Zinc finger protein 329 Hmgn1;LOC100044391 Non-histone chromosomal protein HMG-14 Rbm42 Isoform 1 of RNA-binding protein 42 Rhof Rho-related GTP-binding protein RhoF Only up-regulated in LNT group (23)
Accession no.
iTRAQ ratio Rad/Ctr
iTRAQ ratio RH/Ctr
IPI00405810 IPI00985852 IPI00330862 IPI00403307 IPI00876148 IPI00880589 IPI00137433 IPI00338309 IPI00875833 IPI00421240 IPI00313726 IPI00785327 IPI00986541 IPI00874717 IPI00753639 IPI00108200 IPI00973613 IPI00986309
0.202 0.266 0.282 0.284 0.297 0.305 0.315 0.35 0.354 0.377 0.408 0.412 0.43 0.43 0.44 0.457 0.472 0.495
0.518 0.522 0.502 0.857 0.777 0.863 1.13 0.535 0.625 0.721 0.665 0.647 0.574 0.891 0.641 0.565 0.509 0.698
IPI00460337 IPI00265380 IPI00323235 IPI00785240 IPI00649292 IPI00895129 IPI00118956 IPI00124708 IPI00133284 IPI00278602 IPI00720238 IPI00828225 IPI00134131 IPI00283755 IPI01008483 IPI00128288 IPI00955107 IPI00226182 IPI00775775 IPI00678743 IPI00808497 IPI00135586 IPI00123639 IPI00355933 IPI00225222
1.549 1.564 1.6 1.619 1.62 1.624 1.629 1.651 1.657 1.676 1.696 1.723 1.738 1.751 1.797 1.813 1.826 1.865 1.872 1.899 1.931 1.943 1.995 2.749 3.738
0.896 1.362 1.244 1.304 0.92 1.331 1.151 1.002 0.985 1.091 1.201 1.23 1.244 1.353 1.343 1.044 1.058 1.135 1.416 1.52 1.284 1.21 1.504 1.291 0.605
IPI00153184 IPI00277066 IPI00330958 IPI00228879 IPI00555055 IPI00221463 IPI00665996 IPI00229539 IPI00468814 IPI00323205 IPI00129608 IPI00221825 IPI00749669 IPI00876390 IPI00623570 IPI00352394 IPI00944724 IPI00130653 IPI00262024 IPI00989722 IPI00845761 IPI00263048 IPI00322617 IPI00461822 IPI00468665 IPI00990416 IPI00338745 IPI00853826 IPI00227191
0.493 0.523 0.548 0.549 0.551 0.553 0.56 0.56 0.56 0.572 0.589 0.59 0.602 0.618 0.623 0.629 0.64 0.678 0.748 0.755 0.77 0.786 0.79 0.79 0.838 0.942 0.988 1.041 1.237
0.421 0.429 0.276 0.443 0.308 0.356 0.3 0.363 0.378 0.488 0.465 0.423 0.493 0.445 0.481 0.48 0.43 0.343 0.337 0.448 0.45 0.4 0.39 0.479 0.424 0.379 0.476 0.364 0.433
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Table 1 (continued) Protein name
Accession no.
iTRAQ ratio Rad/Ctr
Gm10260 40S ribosomal protein S18-like Dnahc3 Isoform 2 of dynein heavy chain 3, axonemal Suz12 Polycomb protein Suz12 Ush2a Isoform 3 of usherin Wdr76 Isoform 3 of WD repeat-containing protein 76 Tek Tek protein Sacs Isoform 1 of sacsin Parp14 Poly [ADP-ribose] polymerase 14 Slc12a9 Solute carrier family 12 member 9 - Protein Igh-6 Ig mu chain C region secreted form Sos1 Son of sevenless homolog 1 Camp Cathelin-related antimicrobial peptide Fndc3a Protein Recql5 ATP-dependent DNA helicase Q5 Anxa1 Uncharacterized protein Cacna1d Isoform 1 of voltage-dependent L-type calcium channel subunit alph Lcp1 Uncharacterized protein Fmnl2 Isoform 3 of Formin-like protein 2 Chi3l3 Chitinase-3-like protein 3 Tfrc Transferrin receptor protein 1 Lcp1 Plastin-2 Tapbpl Tapasin-related protein precursor Up-regulated in Rad group and down-regulated in LNT group Trpc4 Isoform beta of short transient receptor potential channel 4 Down-regulated in Rad group and up-regulated in LNT group Rps3 Uncharacterized protein
IPI00620156 IPI00880351 IPI00396676 IPI00742431 IPI00674950 IPI00403931 IPI00816925 IPI00776127 IPI01008484 IPI00986896 IPI00468055 IPI00311611 IPI00875797 IPI00970067 IPI00129287 IPI00652811 IPI00855172 IPI00856578 IPI00345373 IPI00157508 IPI00124700 IPI00118892 IPI01007807
0.639 0.695 0.7 0.714 0.947 0.961 0.989 0.999 1.039 1.064 1.193 1.227 1.252 1.255 1.265 1.294 1.31 1.323 1.395 1.41 1.42 1.453 1.689
2.141 1.525 2.036 1.712 2.149 1.9 2.11 1.563 2.472 2.034 4.152 1.811 1.872 1.782 1.969 2.189 2.5 1.583 1.594 1.788 1.774 2.431 3.514
IPI00229745
1.59
0.342
IPI00856866
0.372
2.448
proteins, and 3 enzyme regulator proteins, which were differentially expressed. Biological functional analysis of these proteins shows that, in the Ctr group, the up-regulated expression was mainly found in repairing proteins (mainly ubiquitin and HSP family proteins) and cytoskeleton proteins (mainly were calmodulin), while the down-regulated expression was mainly found in nucleolemma binding proteins (zinc finger protein, DNA and RNA binding proteins, histone proteins). CL-radiation mainly causes injuries to genetic materials and proteins of the lymphocytes. Lentinan can repair the damages by regulating cystoskeleton and signal transduction, but not by repairing the genetic materials. Based on the above mentioned results, we speculate that CL-radiation could trigger related repairing functions, such as change of cell morphology and signal regulation, in order to escape from the injuries to biomacromolecules. But because of the weak repairing effects on genic materials, CL-radiation induces the breakage or death of the cells. The administration of lentinan resulted in up-regulation of the proteins related to the cell repairing machinery (mainly ubiquitin and of HSP family), cell morphology, signal transduction, oxidizing reaction, and stress reaction, and down-regulation of the proteins related to nucleolemma-binding (mainly zinc finger protein). Our results suggest that the protective effects of lentinan against CL-radiation are attributed mainly by repairing the damaged proteins, regulating signal transduction through membrane, instead of protecting genetic materials. Acknowledgments We appreciate the National Natural Science Foundation of China (81102410) for their financial support. We also thank Yong WANG and Qian SUN for their help in the experiments. Moreover, we greatly appreciate the help from the Department of Pathology of No. 401 Hospital of Chinese People's Liberation Army. References [1] Santovito A, Cervella P, Delpero M. Increased frequency of chromosomal aberrations and sister chromatid exchanges in peripheral lymphocytes of radiology technicians chronically exposed to low levels of ionizing radiations. Environ Toxicol Pharmacol 2013;37:396–403.
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