Immunity Promotion and Proteomic Identification in Mice upon Exposure to Manganese Superoxide Dismutase Expressed in Silkworm Larvae Wan-fu Yue,† Jian-mei Liu,† Jian-tong Sun,† Guang-li Li,† Xing-hua Li,† Xiao-feng Wu,† Hong-xiang Sun,‡ Ji-yong Zhou,‡ and Yun-gen Miao*,† Department of Special Economic Animals, and Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310029, P. R. China Received February 10, 2007
With manganese superoxide dismutase (SOD) expressed in silkworm larvae, Bomby mori L, we investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dosedependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine. Keywords: silkworm ( Bombyx mori L.) • manganese superoxide dismutase (SOD) • splenocyte proliferation • PFC assay • phagocytosis • proteomic identification • mouse
Introduction Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme, which catalyzes the conversion of the superoxide radicals into molecular O2 and H2O2 and thus forms a crucial part of the cellular antioxidant defense mechanism.1 The amount of SOD present in cellular and extracellular environment is crucial for the prevention of disease linked to oxidative stress. Mutations in SOD account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases.2 SOD also appears to be important in the prevention of other neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.3-5 Three types of SODs have been characterized according to their metal content: copper/zinc (Cu/Zn), manganese (Mn), and iron (Fe). SOD is widely distributed in both plants and animals. There are three forms of SOD: cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD, and extracellular SOD.6 In the fruit fly, Drosophila melanogaster, the overexpression of SOD was found to extend its lifespan.7 Since the discovery of SOD, numerous direct and indirect methods for the extraction and assay of SOD have been * To whom correspondence should be addressed. College of Animal Sciences, Zhejiang University, Hangzhou 310029, P. R. China. Tel: +86 571 86971659. E-mail:
[email protected]. † Department of Special Economic Animals. ‡ Institute of Preventive Veterinary Medicine. 10.1021/pr070073i CCC: $37.00
2007 American Chemical Society
developed. The SOD were extracted or purified widely from animal serums and livers such as pig, cattle, horse, chicken, and dog, etc., through biochemical technology. However, it is anxious about the risk practically because of bird influenza and bovine spongiform encephalopathy (BSE). SOD also was extracted from plants such as vegetables, fruits, and cereals. But the low content and efficiency of the extraction from plants became the bottleneck for utilization. We used the silkworm, Bombyx mori, larvae as a bio-reactor and expressed the Mn-SOD by the recombinant bacmid baculoviruses expression system.8 To study the function of SOD and potential practical development, we investigated the immunological function of SOD in the plasma of treated mice with SOD-including silkworm larvae powder, suggesting the silkworm larvae powder containing SOD play a positive role in immunity promotion in mice. Furthermore, we adopted a proteomics approach to identify the protein changes that occur in the plasma of treated mice with SOD-including silkworm larvae powder. The relation between the immunity promotion and protein changes in mice was discussed.
Experimental Procedures Preparation of Silkworm Powder Including Superoxide Dismutase (SOD). The fifth instar silkworm larvae expressed SOD 96 h post-infection with recombinant virus (rBacmid/ Journal of Proteome Research 2007, 6, 1875-1881
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research articles BmNPV/SOD) were collected8 and dried with a vacuum dryer (Brocher CHRIST Beta-16, Germany) under a low temperature of -56 °C. The dried larvae were homogenized to powder and stored at -20 °C up to use. Animal and Breeding Conditions. Seventy-five male ICR mice (Animal experiment center of Medical College, Zhejiang University, China) weighing 18-22 g were used. The animals were housed in individual stainless steel cages in an airconditioned room under a 12:12 h light:dark cycle. A commercial pellet diet and water were provided throughout the experiment. All procedures were conducted in accordance with the P. R. China legislation under No. 8910M047 on the use and care of laboratory animals and with the guidelines established by the Institute for Experimental Animals of Zhejiang University and were approved by the university committee for animal experiments. Animal Experiments. The mice were randomly divided into groups with 10 or 15 animals in each group; normal mice received distilled water (Control); treated groups, administrated through feeding with silkworm powder including SOD at the level of 100 mg/kg body weight/day (low-dosage group, GroupL), 200 mg/kg body weight/day (middle-dosage group, GroupM), and 400 mg/kg body weight/day (high-dosage group, Group-H), and a positive control were set as oral feeding of Huangqi fluid (Astragalus membranaceus (Fisch.) Bunge) at the level of 0.3 mL/day (Positive-C). Assay of Immunity Promotion in Mice upon Exposure to Manganese Superoxide Dismutase Expressed in Silkworm Larvae. In Vivo Splenocyte Proliferation Assay. Spleen of mice exposure to Mn-SOD expressed in silkworm larvae was collected under aseptic conditions, in Hank’s balanced salt solution (Sigma), was minced using a pair of scissors and passed through a fine steel mesh to obtain a homogeneous cell suspension, and the erythrocytes were lysed with NH4Cl (0.8% (w/v)). After centrifugation (1500× g at 4 °C for 10 min), the pelleted cells were washed three times with phosphate buffer saline (PBS) and resuspended in RPMI 1640 complete medium (supplemented with 12 mm HEPES (pH 7.1), 0.05 mm 2-sulfanylethanol, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% FCS). Cell numbers were counted with a hemocytometer by the trypan blue dye exclusion technique. Cell viability exceeded 95%. Splenocyte proliferation was assayed as described by Pan et al. (2005).9 Briefly, splenocytes were seeded into a 96-well flat-bottom microtiter plate at 1 × 106 cell/mL in 100 µL of complete medium, and then the Con A (final concentration 5 µg/mL), RPMI 1640 medium was added to give a final volume of 200 µL (tetraplicate wells). The plate was incubated at 37 °C in a humidified atmosphere with 5% CO2. After 44 h, 50 µL of MTT solution (5 µg/mL) was added to each well and incubated for a further 4 h. The plates were centrifuged (1400× g, 5 min), and the untransformed MTT was removed carefully by pipetting. Two-hundred microliters of acidic isopropanol solution (192 µL of isopropanol with 8 µL of 1N HCl) were added to each well, and the absorbance was evaluated in an ELISA reader at 570 nm with a 630 nm reference after 15 min. The stimulation index (SI) was calculated based on the following formula: SI ) the absorbance value for mitogen cultures divided by the absorbance value for nonstimulated cultures. Anti SRBC Plague-Forming Cells (PFC) Assay. To compare antibody production between mice upon exposure to Mn-SOD expressed in silkworm larvae, antibody responses to sheep red blood cells (SRBC) were measured. The SRBC was taken from 1876
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sheep and triple washed with saline, centrifugated at 400× g for 10 min, and adjusted to a final concentration of 2%. Each animal was given a single intravenous injection of 0.2 mL of 2% SRBC. On day 4 after the SRBC injection, spleens were removed and placed into individual Petri dishes containing sterile Hanks’ solution. Then the spleens were minced using a pair of scissors to obtain a single cell suspension. The single cell suspension was collected with a 23-gauge needle and a 3-mL syringe through a mesh into a sterile 15 mL tube. After centrifugation (1500× g at 4 °C for 10 min), the pelleted cells were washed two times with Hanks’ solution and resuspended with 2 mL of RPMI 1640 complete medium. Cell numbers were counted with a hemocytometer by the trypan blue dye exclusion technique (cell viability exceeded 95%) and adjusted to a final concentration of 5 × 106 cell/mL. Aliquots of spleen cells in RPMI1640 medium were added to glass test tubes that contained 10% SRBC (v/v, with saline). The contents of each test tube were mixed by vortexing and poured into two chambers. The chambers were incubated for 1 h in a 37 °C, 5% CO2 incubator, and the number of plagues was counted. The data were presented as the number of PFC/ spleen cells. In Vitro Macrophage Phagocytosis of Mouse to Chicken Erythrocyte. Each animal was given a single intravenous injection of 0.2 mL of 2% SRBC and sacrificed in day 4 after treatment. The 5 mL of Hanks’ solution containing 5% FCS was injected into the abdominal cavity and lightly pressed 30 times to release macrophages. The macrophages were collected into a sterile 2 mL tube for use. Chicken red blood cells (0.5 mL) (CRBC, 1% in Hanks’ solution) and 0.5 mL of mouse macrophages were added into a test tube and mixed by vortexing. The above mixture suspension sample (0.5 mL) was spread on slide glass and incubated for 20 min a 37 °C, 5% CO2 incubator. After incubation, the glass was washed with saline to remove unattached cells. Then the glass was treated with methyl alcohol for 5 min and dyed with 4% (v/v) Giemsa-PBS buffer solution. The number of macrophages that ingested chicken red blood cells was calculated under a microscope. The data were presented as phagocytic rate (%) (macrophages that ingested chicken red blood cells/total macrophages) and phagocytic index (ingested chicken red blood cells/total macrophages). Proteomic Identification. Blood plasma samples were obtained with anesthesia by resection of the terminal 1-2 mm of the mice’s tails; a total of 0.5 mL of blood was drawn into sodium EDTA-added tubes. Plasma was separated by centrifugation (3000× g, 10 min) and then stored at -30 °C until analysis. The protein content of plasma samples was determined using the Bradford method with protein assay dye reagent concentrate (Bio-Rad, Hercules, CA). The 2-D electrophoresis (2-DE), gel visualization, 2-D protein image acquisition and analysis, in-gel digestion, matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and protein database searching were conducted as described in our previous paper.10 Briefly, after 2-D electrophoresis and in-gel digestion, 1 µL of peptide sample mixed with an equal volume of 10 mg/mL (R)-cyano-4-hydroxycinnamic acid (Sigma) saturated with 50% acetonitrile in 0.1% trifluoroacetic acid was used for peptide mass fingerprinting (PMF) analysis utilized by MALDI-TOF-MS equipment. Protein
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Effect of SOD Table 1. In Vitro Splenocyte Proliferation Assay in Mouse upon Exposure to Manganese Superoxide Dismutase Expressed in Silkworm Larvae treatment
animal number
stimulation index (SI)a
P valueb
Group-L Group-M Group-H control
10 10 10 10
2.55 ( 0.55 2.99 ( 0.79 2.78 ( 0.49 2.33 ( 0.33
* *
a Values are shown as the mean ( SD of 10 animals. b *, P < 0.05 in comparison between treatments.
Figure 2. Phagocytosis photos of mouse macrophages to chicken erythrocyte administrated through feeding with silkworm powder including SOD. Macrophages (arrow) were presented. Table 2. Phagocytosis of Mouse Macrophages to Chicken Erythrocyte Administrated through Feeding with Silkworm Powder Including Superoxide Dismutase (SOD) treatment
animal number
phagocytic ratea (%)
Group-L Group-M Group-H Positive-C control
12 12 12 12 12
45.3 ( 9.9 52.0 ( 8.0 85.4 ( 15.6 41.4 ( 11.8 40.5 ( 8.4
P valueb
* ** **
phagocytic index
1.31 ( 0.29 1.26 ( 0.11 1.15 ( 0.10 1.30 ( 0.24 1.21 ( 0.17
a Values Are Shown as the Mean ( SD of 10 Animals. b *, P < 0.05; **, P < 0.01 in comparisons between treatments.
Figure 1. Body weight and anti-SRBC plague-forming cells (PFC) assay in mice upon exposure to manganese superoxide dismutase (SOD) expressed in silkworm larvae. Values are shown as the mean ( SD of 10 animals. *, P < 0.05; **, P < 0.01
identification was performed by the MASCOT search engine (http://www.matrixscience.com) against the NCBI protein database. Statistical Analysis. The data were expressed as mean ( S.D. and examined for their statistical significance of difference with Student’s t test, P < 0.05 being considered significant.
Results Immunity Promotion in Mice upon Exposure to Manganese Superoxide Dismutase Expressed in Silkworm Larvae. The in vivo effect of Mn-SOD expressed in silkworm larvae on ConA-stimulated splenocyte proliferation is shown in Table 1. The ConA-stimulated splenocyte proliferations of all three treated groups were higher than that of the control. Among them, the stimulation indexes of 200 mg SOD contained silkworm powder-treated mouse splenocyte (Group-M) and 400 mg SOD contained silkworm powder-treated mouse splenocyte (Group-H) were significantly enhanced respectively compared to the control. To compare antibody production between mice treated with silkworm larvae powder containing Mn-SOD and controlled
mice, antibody responses to SRBC were measured. Figure 1 showed the result of body weight and the PFC assay. The mean body weights in treated mice were significantly heavier than that of control (distilled water given orally) (Figure 2a). Meanwhile, the mean antibody responses in the PFC assay in mice upon exposure to Mn-SOD expressed in silkworm larvae were also significantly higher than those of controlled mice (Figure 2b). The resident macrophages of the liver and macrophages in the peritoneal fluid play a major role in regulating local inflammatory reactions by releasing pro- and anti-inflammatory molecules and by supporting the primary defense mechanism via phagocytosis. Table 2 presents the results of phagocytosis of mouse macrophages to chicken erythrocyte administrated through feeding with silkworm powder including SOD. After 30-day oral giving for mice with silkworm powder including SOD, all three treated groups were enhanced in the phagocytosis of mouse macrophages to chicken red blood cells (CRBC) than that of controlled mice. Especially the phagocytic rate (%) of mice macrophages to CRBC in Group-M (200 mg/kg body weight/ day) and Group-H (400 mg/kg body weight/day) were significantly higher than that of the control (P < 0.01). Figure 2 shows the photograph of phagocytosis of mouse macrophages to CRBC. In Group-M and Group-H, a clear phagocytic image was observed, which showed the mouse macrophages is engulfing, or has engulfed, the CRBC. As in Journal of Proteome Research • Vol. 6, No. 5, 2007 1877
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Figure 3. Two-dimensional (2-D) electrophoresis of mouse plasma protein. The 2-D PAGE image from plasma of controlled mice was used as a master gel and reference map. Protein spots identified by MALDI-TOF MS (arrow) are marked by their spot numbers.
the control or in positive control, there are few CRBC gobbled up by the mouse macrophages. 2-D Electrophoresis and Protein Identification. A proteomics approach was used to determine the differential expression of proteins in mouse administrated through feeding with silkworm powder including SOD. Three microliters of blood plasma of mouse from the controlled group or administrated through feeding with silkworm powder including SOD at the level of 100 mg/kg body weight/day (low dosage group, GroupL) was used for 2-D analysis, and the separated proteins were visualized by silver staining and analyzed using the ImageMaster 2D software. Figure 3 shows as many as 140 of the protein spots that were expressed in mouse. Among them, most of the proteins were concentrated in pI 5.0-9.0, their number was 95, which reached 67% of the total protein spots. As for the protein molecular sizes, 105 of the protein spots concentrated between 30 and 67 KD, which comes to 75% of the total spots.
Figure 4. PMF of spot 10 extracted from 2-DE gel. 1878
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Analysis of Peptide Mass Fingerprinting (PMF) Patterns and Homology Searching. The protein spots of interest were excised manually from the silver-stained gels with a clean scalpel blade. The spots’ gels were rehydrated in 10-20 µL of trypsin solution (20 ng/µL in 40 mM NH4HCO3 in 9% acetonitrile) and incubated at 37 °C overnight for digestion. The digested peptide mixture was analyzed by a Voyager-DE STR MALDI-TOF-MS using a delayed ion extraction and ion mirror reflector mass spectrometer. A total of 25 spots expressed in controlled and treated mouse were analyzed through MALDITOF-MS. Protein identification using peptide mass fingerprinting (PMF) was performed by the MASCOT search engine (http:// www.matrixscience.com) against the NCBI protein database. The results showed that six spots were related to the immunity of mice and four spots were considered as regulation proteins. Here we listed spot numbers 10 and 22, which were identified as immunoglobulin superfamily receptor translocation associated protein 2a and fibrinogen, respectively. Figure 4 and Table 3 present the result of spot 10 using PMF. The matched peptides for spot 10 are shown in bold font as follows: 1 MNQITAVVFL IASLILQAPL SVFEGDFVVL RCRAKAEVTL NTMYKNELFS 51 RPVLRASSSQ PTNGSPVTLT CETRLSLERS EVQLQFCFFR DGQTLGSGWS 101 SCPKFQISAM RSEDSWSFWD WNFTLIPITV DDACKRPCAP RAPWEVGSLT 151 PGKSFQQKGS KNYPSSLDNW MGLYSLFIKF SEP Similarly, spot 22 was identified as fibrinogen. Figure 5 and Table 4 show the peptide mass fingerprinting and its database searching results. The matched peptides for spot 22 are shown in bold font as follows: 1 MSWSLQPPSF LLCCLLLLFS PTGLAYVATR DNCCILDERF GSFCPTTCGI 51 ADFLSSYQTD VDNDLRTLED ILFRAENRTT EAKELIKAIQ VYYNPDQPPK 101 PGMIDSATQK SKKMVEEIVK YEALLLTHET SIRYLQEIYN SNNQKITNLK 151 QKVAQLEAQC QEPCKDSVQI HDTTGKDCQE IANKGAKESG LYFIRPLKAK 201 QQFLVYCEID GSGNGWTVLQ KRIDGSLDFK KNWIQYKEGF GHLSPTGTTE
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Figure 5. PMF of spot 22 extracted from 2-DE gel. Table 3. PMF Database Searching Results of Protein Spot 10 homolohous protein name
similar to immunoglobulin superfamily receptor translocation associated protein 2a
NCBI accession no.
calculated pI
nominal mass (Mr)
sequence GFcoverage
gi|114560265
8.23
20599
62%
start-end
observed
Mr (expt)
Mr (calc)
delta
sequence
1-33 36-45 91-104 112-135 142-153 162-183
3663.1312 1169.4805 1479.7443 2932.3184 1241.5303 2624.0118
3662.1239 1168.4733 1478.7370 2931.3111 1240.5230 2623.0045
3662.0091 1168.5798 1478.6460 2931.3061 1240.6451 2623.2304
0.1149 -0.1065 0.0911 0.0050 -0.1222 -0.2259
-.MNQITAVVFLIASLILQAPLSVFEGDFVVLRCR.A K.AEVTLNTMYK.N R.DGQTLGSGWSSCPK.F R.SEDSWSFWDWNFTLIPITVDDACK.R R.APWEVGSLTPGK.S K.NYPSSLDNWMGLYSLFIKFSEP.-
Table 4. PMF Database Searching Results of Protein Spot 22 homolohous protein name
fibrogen, gamma polypeptide
NCBI accession no.
calculated pI
nominal mass (Mr)
sequence coverage
gi|19527078
5.54
49360
45%
start-end
observed
Mr (expt)
Mr (calc)
delta
sequence
31-39 40-66 88-110 114-133 188-198 201-221 238-257 258-272 273-281 282-291 347-381 364-381
1194.4442 3086.2687 2561.1474 2374.0755 1322.6464 2441.9359 2206.9093 1742.8153 1129.4995 1162.4442 3884.6530 1985.7473
1193.4369 3085.2615 2560.1401 2373.0682 1321.6391 2440.9286 2205.9020 1741.8080 1128.4922 1161.4369 3883.6457 1984.7401
1193.4805 3085.3433 2560.2631 2373.2613 1321.7394 2441.1685 2206.0330 1741.9549 1128.6040 1161.5124 3883.6313 1984.8962
-0.0436 -0.0819 -0.1230 -0.1931 -0.1003 -0.2399 -0.1310 -0.1469 -0.1117 -0.0755 0.0144 -0.1561
R.DNCCILDER.F R.FGSFCPTTCGIADFLSSYQTDVDNDLR.T K.AIQVYYNPDQPPKPGMIDSATQK.S K.MVEEIVKYEALLLTHETSIR.Y K.ESGLYFIRPLK.A K.QQFLVYCEIDGSGNGWTVLQK.R K.EGFGHLSPTGTTEFWLGNEK.I K.IHLISMQSTIPYALR.I R.IQLKDWNGR.T R.TSTADYAMFR.V K.FEGNCAEQDGSGWWMNKCHAGHLNGVYHQGGTYSK.S K.CHAGHLNGVYHQGGTYSK.S
251 FWLGNEKIHL ISMQSTIPYA LRIQLKDWNG RTSTADYAMF RVGPESDKYR 301 LTYAYFIGGD AGDAFDGYDF GDDPSDKFFT SHNGMQFSTW DNDNDKFEGN 351 CAEQDGSGWW MNKCHAGHLN GVYHQGGTYS KSSTTNGFDD GIIWATWKSR 401 WYSMKETTMK IIPFNRLSIG EGQQHHMGGS KQAGDV
Six spots, which are considered as related with immunity, together with four regulation proteins are presented herein with protein names, observed masses, and matched peptide sequence coverage (Table 5). The expression profiles of the six immunity-related proteins with significant (P < 0.05) differential expression are visualized as Figure 6. All six matched proteins related immunity showed Journal of Proteome Research • Vol. 6, No. 5, 2007 1879
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Table 5. List of Proteins Expressed in Mouse Plasma Identified by MALDI-TOF MS Analysis groups
immunity-related proteins
regulation proteins
spot no.
protein name
accession no.
pI/Mr (Da)
sequence coverage (%)
4
MHC class I antigen
gi|32400585
5.76/18845
86
6 10 13 15 22 1
protein-tyrosine kinase immunoglobulin superfamily receptor Apoa4 protein XRCC6 binding protein fibrinogen, gamma polypeptide GTP cyclohydrolase I feedback regulatory protein trypsin inhibitor albumin vitamin D-binding protein
gi|57582 gi|114560265 gi|29477189 gi|27754038 gi|19527078 gi|16975333
6.82/56948 8.23/20599 5.55/42889 7.17/21696 5.54/49360 6.14/9535
50 62 48 45 45 71
gi|49259463 gi|55391508 gi|193446
8.70/23318 6.09/68714 5.26/53051
59 35 51
11 12 23
the increase of expression level in plasma of mouse administrated with silkworm powder including SOD at the level of 100 mg/kg body weight/day, compared to that of control.
Discussion SODs are metalloenzymes that catalyze the dismutation of the superoxide anion (O2-) to elemental molecular oxygen (O2) and hydrogen peroxide (H2O2) and, thus, form a crucial part of the cellular antioxidant defense mechanism.11 In fact, overexpression of SOD protects murine fibrosarcoma cells from apoptosis and promotes cell differentiation.12 SOD also inhibits adriamycin-induced apoptosis in murine peritoneal macrophages.13 Mn-SOD cDNAs have been cloned from diverse organisms.14-16 The domesticated silkworm, Bombyx mori, is an economically important animal that was used for silk production for more than 5000 years. With the development of biotechnology, B. mori has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES).17-19 In our last report, we used a practical BmNPV bacmid system to express the MnSOD enzyme protein in silkworm larvae by the recombinant bacmid baculoviruses. As silkworms can be conveniently reared
with mulberry leaves at much lower cost or with artificial diet throughout the year, large scale and successive production of SOD is possible.8 The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for its potential application in medicine. To study the function of SOD and potential practical development, we investigated here immunity promotion and proteomic identification in mice upon exposure to Mn-SOD expressed in silkworm larvae. These experiments were designed to investigate the effects of SOD expressed in silkworm on the immune system of mice both in vivo and in vitro. The present study focused on three immune parameters: in vivo splenocyte proliferation, anti SRBC PFC assay, and in vitro macrophage phagocytosis. These are of great value in understanding the pharmacological effects of SOD on the immune system. The spleen is an important immunological organ that contains mainly B-lymphocytes. Hence, the spleen can indirectly reflect humoral immunity. The higher the immunity index, the stronger the immune capability. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control (Table 1), which is in agreement with previous reports.20 To compare antibody production between mice treated with silkworm larvae powder containing Mn-SOD and controlled mice, antibody responses to SRBC were measured. The results of PFC assay also revealed that antibody production was higher in all three treated groups than in controlled mice.
Figure 6. Analysis of immunity-related proteins in mouse plasma with significant differential expression by treatment at the level of 100 mg/kg body weight/day. The expression profiles of the individual proteins were classified by cluster analysis. 1880
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Macrophages are mononuclear phagocytes that are found in various tissues through maturation and differentiation of immigrating blood monocytes. Macrophages, in general, are critical effectors of the body’s immune system. Macrophages play a critical role in cellular host defense against infection and tissue injury.21-22 In response to stimuli, macrophages undergo a series of inflammatory processes, including chemotaxis, phagocytosis, intracellular killing, and release of inflammatory cytokines.21,23 To evaluate the potential of SOD to activate macrophages, we treated mouse with varying concentrations of SOD-containing silkworm larvae powder and investigated the phagocytosis of mouse macrophages. Its treatment led to a dose-dependent increase of phagocytic activity; especially the phagocytic rate (%) of mice macrophages to CRBC in Group-M (200 mg/kg body weight/day) and Group-H (400 mg/kg body weight/day) were significantly higher than that of control (P < 0.01) (Table 2 and Figure 2). Therefore, the activation of
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phagocytosis by SOD may be a general phenomenon, and it could be used as an activator of macrophage function. Proteomics is a large-scale study of the gene expression at the protein level, which ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins. As the SOD-containing silkworm larvae powder enhanced the immunity response of treated mouse in in vivo splenocyte proliferation, anti-SRBC PFC assay, and in vitro macrophage phagocytosis, a proteome approach was conducted to investigate the relative protein profiles. Using 2-D and PMF, we identified six proteins that related to the immunity of mice. The data showed that all these six matched proteins related immunity showed the increase of expression level in plasma of mouse administrated with silkworm powder including SODs at the level of 100 mg/kg body weight/day, compared to that of control. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in an enhancement of immunity activities in mouse. The results also suggested that the SOD expressed in silkworm maybe have a potential application in medicine.
Acknowledgment. This work was supported by the HiTech Research and Development Program of China (No. 2006AA10A119), a key project of Zhejiang Government (No. 2005C22042), and National Natural Science Foundation of China (No. 30625030). References (1) Weisiger, R. A.; Fridovich, I. Superoxide dismutase: organelle specificity. J. Biol. Chem. 1973, 248, 3582-3592. (2) Louvel, E.; Hugon, J.; Doble, A. Therapeutic advances in amyotrophic lateral sclerosis. Trends Pharm. Sci. 1997, 18(6), 196203. (3) Maier, C. M.; Chan, P. H. Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 2002, 8(4), 323-334. (4) Liu, D. The roles of free radicals in amyotrophic lateral sclerosis. J. Mol. Neurosci. 1996, 7, 159-167. (5) Kushleika, J.; Checkoway, H.; Woods, J. S. Selegiline and lymphocyte superoxide dismutase activity in Parkinson’s disease. Ann. Neurol. 1996, 39(3), 378-381. (6) Sandstrom, J.; Nilsson, P.; Karlsson, K. 10 fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain. J. Biol. Chem. 1994, 269(29), 19163-19166. (7) Orr, W. C.; Sohal, R. S. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 1994, 263, 1128-1130.
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