Cloning and Functional Analysis of the Full Length cDNA Sequence of Eukaryotic Translation Initiation Factor 5 in Schistosoma japonicum

The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by the bioinformatics method. The overlapping sequences were assembled into one that includes the complete open reading frame （GenBank accession number： AY686501）. The full-length cDNA of SjeIF5 was cloned into a pET-28c^（＋） vector, which generated a prokaryotic expression plasmid, and a fusion protein of 18 kDa was induced in Escherichia coll. The recombinant expression of eIF5 protein of Schistosoma japonicum was purified. The immunoprotection test against schistosomiasis demonstrated that the recombinant protein worked to a certain extent, especially in the reduction of eggs in the liver of the host.

Osteopontin expression is associated with hepatopathologic changes in Schistosoma japonicum infected mice

AIM:To investigate osteopontin expression and its association with hepatopathologic changes in BALB/C mice infected with Schistosoma japonicum.METHODS:The schistosomal hepatopathologic mouse model was established by abdominal infection with schistosomal cercaria.Liver samples were obtained from mice sacrif iced at 6,8,10,14,and 18 wk after in-fection.Liver histopathological changes were observed with hematoxylin-eosin and Masson trichrome staining.The expression of osteopontin was determined with im-munohistochemistry,reverse transcription-polymerase chain reaction,and Western blotting.The expressionof α-smooth muscle actin(α-SMA)and transforming growth factor-β1(TGF-β1)were determined by im-munohistochemistry.Correlations of osteopontin ex-pression with other variables(α-SMA,TGF-β1,hepato-pathologic features including granuloma formation and degree of liver f ibrosis)were analyzed.RESULTS:Typical schistosomal hepatopathologic changes were induced in the animals.Dynamic changes in the expression of osteopontin were observed at week 6.The expression increased,peaked at week 10(P<0.01),and then gradually decreased.Positive correla-tions between osteopontin expression and α-SMA(r=0.720,P<0.01),TGF-β1(r=0.905,P <0.01),granu-loma formation(r=0.875,P<0.01),and degree of liver f ibrosis(r=0.858,P<0.01)were also observed.CONCLUSION:Osteopontin may play an important role in schistosomal hepatopathology and may promote granuloma formation and liver fi brosis through an un-explored mechanism.

The efficacy of NP11-4-derived immunotoxin scFv-artesunate in reducing hepatic fibrosis inducedby Schistosoma japonicum in mice

Schistosomiasis is one of the most prevalent parasitic diseases in China, and hepatic fibrosis caused by schistosome infection is the principal cause of death. The aim of this study was to evaluate the efficacy of NPll-4- derived immunotoxin scFv-artesunate on Schistosoma japonicum-induced hepatic fibrosis. A single-chain variable fragment （scFv） was generated from the murine anti-Schistosoma japonicum （S. japanicum） monoclonal antibody NP11-4. The scFv was expressed as a soluble protein and purified by Ni-affinity chromatography. After conjuga- tion with artesunate, the binding ability with soluble egg antigens （SEA） was determined by an enzyme-linked immunosorbent assay （ELISA）. The biological activity of purified scFv, scFv-artesunate （immunotoxin）, and artesunate was detected in vivo. Image-Pro Plus software was used to analyze the size of egg granuloma and the extent of liver fibrosis. The recombinant scFv expession vector was constructed and expressed successfully. After purification by a His-trap Ni-affinity column, the scFv yield was approximately 0.8 mg/L of culture medium. ELISA results showed that chemical conjugation did not affect the binding activity of the immunotoxin. Our animal experiments indicated that the immunotoxin could significantly reduce the size of egg granuloma in the liver and inhibit hepatic fibrosis. The immunotoxin could be used as a promising candidate in the targeted therapy of S. .japonicum-induced hepatic fibrosis.

Schistosoma japonicum attenuates dextran sodium sulfateinduced colitis in mice via reduction of endoplasmic reticulum stress

AIM To elucidate the impact of Schistosoma(S.) japonicum infection on inflammatory bowel disease by studying the effects of exposure to S. japonicum cercariae on dextran sodium sulfate(DSS)-induced colitis. METHODS Infection was percutaneously established with 20 ± 2 cercariae of S. japonicum, and colitis was induced by administration of 3% DSS at 4 wk post infection. Weight change, colon length, histological score(HS) and disease activity index(DAI) were evaluated. Inflammatory cytokines, such as IL-2, IL-10 and IFN-γ, were tested by a cytometric bead array and real-time quantitative polymerase chain reaction(RT-PCR). Protein and m RNA levels of IRE1α, IRE1β, GRP78, CHOP, P65, P-P65, P-IκBα and IκBα in colon tissues were examined by Western blot and RT-PCR, respectively. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling positive cells, cleaved-caspase 3 expression and Bcl2/Bax were investigated to assess the apoptosis in colon tissues.RESULTS Mice infected with S. japonicum cercariae were less susceptible to DSS. Mice infected with S. japonicum cercariae and treated with DSS showed decreased weight loss, longer colon, and lower HS and DAI compared with mice treated with DSS alone. A substantial decrease in Th1/Th2/Th17 response was observed after infection with S. japonicum. Endoplasmic reticulum(ER) stress and the nuclear factor-kappa B(NF-κB) pathway were reduced in mice infected with S. japonicum cercariae and treated with DSS, along with ameliorated celluar apoptosis, in contrast to mice treated with DSS alone. CONCLUSION Exposure to S. japonicum attenuated inflammatory response in a DSS-induced colitis model. In addition to the Th1/Th2/Th17 pathway and NF-κB pathway, ER stress was shown to be involved in mitigating inflammation and decreasing apoptosis. Thus, ER stress is a new aspect in elucidating the relationship between helminth infection and inflammatory bowel disease(IBD), which may offer new therapeutic methods for IBD.

Orthotopic liver transplantation from a donor with Schistosoma japonicum

To the Editor：Despite of the rapid increase of donation after cardiac death （DCD） in China, the shortage of organs continues to be a major problem. Every organ procured is so valuable that it should never be discarded easily, especially a liver that could save a patient＇s life in an emergency. This leads to the use of grafts from donors with unrecognized Here and unusual diseases, including schistosomiasis. we reported a case of orthotopic liver transplantation （OLT） from a donor with Schistosorna japonicurn to a patient with end-stage cirrhosis due to HBV infection.

Recombinant protein Schistosoma japonicum-derived molecule attenuates dextran sulfate sodium-induced colitis by inhibiting miRNA-217-5p to alleviate apoptosis

BACKGROUND Inflammatory bowel disease(IBD)affects millions of people worldwide and has emerged as a growing problem in industrialized nations.The lack of therapeutic targets has limited the treatment of IBD.Studies found that parasitic nematode infections can ameliorate clinical and experimental colitis.Our previous study found that rSj16,a 16-kDa secreted protein of Schistosoma japonicum produced by Escherichia coli,has protective effects on dextran sulfate sodium(DSS)-induced colitis in mice.Apoptosis is an important factor in the pathogenesis of colitis.However,it is not clear whether the effect of rSj16 on colitis is related to apoptosis.AIM To investigate whether the protective effects of rSj16 on colitis is related to apoptosis and its mechanism.METHODS In-vivo,colitis was induced by DSS.The severity of colitis was assessed.WB was used to detect the changes of apoptosis-related genes in colon tissues.Q-PCR was used to detect the changes of miRNA-217-5p and HNF1B.In-vitro,WB was used to detect the changes of apoptosis-related genes in intestinal epithelial cells.TUNNEL staining and flow cytometry were used to detect cell apoptosis.RESULTS rSj16 attenuates clinical activity in DSS-induced colitis mice.TUNNEL staining and WB results showed that apoptosis was increased in colon tissue after treatment with DSS,and the apoptosis of colon tissue was significantly reduced after treatment with rSj16.Compared with normal mice,the expression of miR-217-5p was increased in colon tissue of DSS-induced colitis mice.In addition,the miR-217-5p target gene hnf1b was decreased after administration of DSS.After treatment with rSj16,the expression of miR-217-5p was decreased and the expression of HNF1B was increased compared with the DSS-treated group.When Etoposide was used in combination with miR-217-5p mimic on MODE-K cells,the expression of cleaved-Caspase-3 and Bax was increased,and Bcl-2 was decreased compared with only Etoposide treatment,the expression of HNF1B was significantly reduced,suggesting that miR-217-5p acts as a pro-apoptotic in colon epithelial cells and down-regulates the target gene hnf1b.After rSj16 administration in MODE-K cells,miR-217-5p expression was significantly decreased,HNF1B expression was increased,and apoptosis was reduced.CONCLUSION The protective effects of rSj16 on colitis is related to apoptosis and miRNA-217-5p may be a further target for therapeutic intervention against IBD.

Schistosoma japonicum ova maintains epithelial barrier function during experimental colitis

AIM:To evaluate the impacts of Schistosoma japoni-cum(S.japonicum) ova on the tight junction barriers in a trinitrobenzenesulfonic acid(TNBS)-induced colitis model.METHODS:Balb/c mice were randomly divided into three groups:control group;TNBS + ovagroup and TNBS + ova + group.TNBS was used intracolonic to in-duce colitis and mice of the TNBS + ova + group were pre-exposed to S.japonicum ova as a prophylactic intervention.Colon inflammation was quantified using following variables:mouse mortality,weight loss,co-lon extent and microscopic inflammation score.Serum expression of tumor necrosis factor-αand interferon-γ were assessed to evaluate the systemic inflamma-tory response.NOD2 and its mRNA were also tested.Bacterial translocations were tested by culturing blood and several tissues.ZO-1 and occludin were chosen as the representations of tight junction proteins.Both the proteins and mRNA were assessed.RESULTS:Ova pre-treatment contributed to the reliefof colitis and decreased the mortality of the models.NOD2 expression was significantly downregulated when pretreated with the ova.The TNBS injection caused a significant downregulation of ZO-1 and oc-cludin mRNA together with their proteins in the colon;ova pre-exposure reversed these alterations.Treat-ment with S.japonicum ova in the colitis model caused lower intestinal bacterial translocation frequency.CONCLUSION:S.japonicum ova can maintain epithelial barrier function through increasing tight junction pro-teins,thus causing less exposure of NOD2 to the lumi-nal antigens which may activate a series of inflamma-tory factors and induce colitis.

Effect of artmether,hemin and Fe^(3+) on recombinant lactate dehydrogenase from Schistosoma japonicum

Objective:To explore antischitosome effects of artemether,hemin and Fe on S/LDH.Methods: Enzyme activity of rS/LDH was assayed in the standard reaction system by adding different concentration of reagents(0.00-0.10 mM artemether,0.00-0.02 mM hemin,0.00-0.50 mM Fe^(3+)). Same solvents of the each reagent were used as control.Results:There was no enzyme activity inhibition observed at 0.10 mM artemther:obivious inhibition for lactate oxidation reaction and pyruvate reduction reaction were detected at 0.002 mM and 0.004 mM of hemin,respectively: comparing with that of the control(P<0.05).The relative enzymatic activity inhibitions for pyruvate reduction reaction and lactate oxidation reaction at 0.02 mM hemin were 93.48%and 100.00%,respectively,comparing with that of the control(P<0.01):both pyruvate reduction and lactate oxidation reaction were inhibited completely at 0.50 mM Fe^(3+),comparing with that of the control(P<0.01).Conclusions:The results implied that SjLDH was not the direct molecular target of artemether.Hemin and Fe are inhibitors of SjLDH.

The Construction of Schistosoma Japonicum Vaccine BCG-Sj26GST and Its Identification

Summary: The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette Guerin (BCG), Mycobacterium ( M. smegmatis ) and Escherichia coli ( E. coli ) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST in E. coli as template. The Sj26GST cDNA was cloned into the downstream of human M. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together into E. coli Mycobacteria shuttle plasmid pBCG 2000 to construct the expression shuttle plasmid pBCG Sj26. The recombinant BCG and M. smegmatis mc 2 155, which were electroplated with pBCG Sj26, could express Sj26GST and the recombinant Schistosoma Japonicum vaccine BCG Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15 % and 10 % of total bacterial protein in BCG and M. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.

Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene （Sj26）, and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells, The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro, By using Western blot, the expression of 26 ku glutathione S-transferase （GST） was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24×10^8. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells, It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Effect of RNA interference on WD101 gene of Schistosoma japonicum

Objective: To study the effect of RNA interference(RNAi) on WD101 gene and its effect on the expression of WD101 mRNA and protein in Schistosoma japonicum.Methods: Doublestranded RNA(dsRNA) WD101 gene and control gene(lacZ) were generated by in vitrotranscription and transfected into mechanically transformed schistosomula.The total RNA and protein were isolated simultaneously using TRIzol reagent.The expression levels of mRNA and the protein were determined by quantitative real-time PCR(qPCR) and Western blotting, respectively.After injected dsRNA-electroporated schistosomula into BALB/c mouse six weeks, the male and female reproductive organs were observed and measured under the confocal laser scanning microscope.Results: After 1, 3 and 5 d of RNAi, WD101 mRNA level was decreased by 15%, 39%, and 58% in experiment group compared to that in control group; meanwhile, WD101 protein level was decreased by 11%, 28%, and 43% in experiment group compared to that in control group.There were significantly more sperms in testicular lobes in experiment group than that in control group, while there were no significant differences in terms of ovary and vitelline glands between two groups.Conclusions: The ds WD101-RNAi can effectively induce suppression of WD101 gene expression at both mRNA and protein levels.WD101 gene might be a reproduction-related gene in Schistosoma japonicum.

Ultraviolet-attenuated cercariae ofSchistosoma japonicum fail to effectively induce a Th1 response in spite of up-regulating expression of cytotoxicity-related genes in C57BL/6 mice

Objective： To better understand the reason that Schistosoma japonicurn （S. japonicum） ultraviolet （UV）- radiated cercariae could not induce high level of protection in C57BL/6 mice. Methods： Microarray technology was performed to investigate the gene transcription profile in skin draining lymph nodes （sdLNs） at 1 w after exposure to attenuated cercariae （AC） or normal cercariae （NC） of S. japonicum in C57BL/6 mice. The expressions of some representative genes were further confirmed by real-time PCR. Subsequently, the expressions of Th1/Th2 cytokine genes, cytotoxicity-related genes, as well as co-stimulator genes in spleens from AC-vaccinated and NC- infected mice were analyzed by real-time PCR at w 3 and 6 post-exposure. Results： The gene expressions of Th1 cytokines, including interferon-y （IFN-γ）, interleukin （IL）-12 and tumor necrosis factor-α （TNF-α） in the sdLNs were significantly lower in AC-vaccinated mice than in NC-infected mice. Furthermore, the gene expressions of Th1- and Th2- cytokines, including IFN-γ, IL-12, TNF-α, IL-4 and IL-10, in the spleens from AC-vaccinated mice showed little changes at w 3 and 6 post-vaccination. In addition, cytotoxicity-related molecules including granzyme A, granzyme B, granzyme K, perforin 1 and Fas L were up-regulated from the early stage of vaccination, and peaked at the 3rd w after vaccination with UV-AC. Conclusion： UV-AC of S. japonicum could not ef- fectively induce a Thl response in C57BL/6 mice, which may be an explanation for the low protection against parasite challenge, and the role played by up-regulated expression of cytotoxicity-related genes in mice needs to be further investigated.

Antigen presenting cells may be able to distinguish between normal and radiatedSchistosoma japonicum cercaria:anin vitro observation

Objective： To observe the discrepancies of responses induced by Schistosoma japonicum （S. japonicum） normal cercaria antigen （NCA） and ultraviolet （UV） -radiation-attenuated cercaria antigen （UVACA） in an in vitro system. Methods： S. japonicum cercariae were collected and UVACA and NCA were prepared. Mouse macro- phage model cells （RAW 264.7） were treated with medium, NCA （40 μg/mL） or UVACA （40 μg/mL） in the presence or absence of recombinant mouse interferon gamma （rmIFN-γ; 4 ng/mL） for 48 h. Cell surface staining and flow cytometry were used to assess the major histocompatibility complex （MHC） Ⅱ expression, and data were expressed as mean fluorescence intensities （MFI）. Interleukin （IL） -10, IL-6 and prostaglandin E2 （PGE2） in cell culture supernatant were evaluated by commercial enzyme-linked immunosorbent assays. Results： NCA significantly suppressed IFN-7-induced MHC Ⅱ expression on RAW 264.7 cells. In the presence of 1FN-7, NCA significantly promoted IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells. In the presence of IFN-γ, UVACA significantly promoted IL-10 but not IL-6 and PGE2 secretion from RAW 264.7 cells and showed no effect on IFN-γ-induced MHC Ⅱ expression. Compared with UVACA, NCA significantly suppressed IFN-γ-induced MHC Ⅱ expression and significantly promoted IL-6, PGE2 and IL-10 secretion from RAW 264.7 cells. Conclusion： RAW 264.7 cells respond differently to NCA and UVACA. NCA can significantly suppress IFN-γ-induced MHC Ⅱ expression and significantly promote IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells compared with UVACA.

Effects of Ferulic Sesquiterpene Compounds on Schistosoma japonicum at Different Developmental Stages in Infected Juvenile Rabbits

[Objectives]This study was conducted to explore the effects of ferulic sesquiterpene compounds on juvenile worms and adults of Schistosoma japonicum at different developmental stages in infected juvenile rabbits.[Methods]Juvenile rabbits were infected with S.japonicum quantitatively,and administered with 500 mg/kg ferulic sesquiterpene compounds at 2 h,1,2,6,11,13,15,24,36,and 41 d after infection by gavage,1 time/d,and a control group was administrated with normal saline.After 4 weeks of administration,the juvenile rabbits were dissected,and the worms were collected by portal vein perfusion for the calculation of worm reduction rate.[Results]The reduction rates of ferulic sesquiterpene compounds on 1,2,6,11,13,15,24,36,and 41-day-old juveniles or adults were 15.8%,17.0%,60.2%,50.2%,36.9%,31.3%,45.3%,57.0%,and 25.2%,respectively,and it was found that worms at the age of 6-36 d were most sensitive to the drug.[Conclusions]Ferulic sesquiterpene compounds had a killing effect on S.japonicum at different developmental stages in juvenile rabbits,and the killing effect on 6-36-day-old worms was more significant.

Expression and Analysis of Microtus fortis Against Schistosoma japonicum CD72

The total RNA was extracted from Microtus fortis liver tissue which before being infected and after being infected 10 d and 15 d by the Schistosoma japonicum cercariae. Using rattus norvegicus CD72 gene probes were used to hybridize analysis of CD72 difference expression in the Microtus fortis liver tissues which were infected with Schistosoma japonicum before and after being infected. At the same time, the cDNA sequence and encoded amino acid sequence of the rattus norvegicus CD72 gene and CD72 protein structural domains were analyzed by using bioinformatics. The results showed that the CD72 expression levels in the liver tissue of Microtus fortis after being infected was significantly higher than before being infected. The rattus norvegicus CD72 cDNA sequence of a total length is 1479 bp and encode 364 amino acid residues and rattus norvegicus CD72 protein containing a CD72 superfamily domain.

Screening and Primary Characterization of NewAntigen Genes of Schistosoma Japonicum

To find Schistosoma japonicum(S.j)new antigen gene thus provide more useful vaccine candidates, the cDNA library of S.j adult worm was screened with sera of rabbits immunized with the membrane antigens of Schistosoma japonicum hepato-portal schistosomula (SjHmAg). The positive clones were amplified by PCR and sequenced, then the sequences of clones were compared with all sequences in GenBank database using Blast process. The new clones were submitted to GenBank for accession numbers. Fifteen positive clones were obtained after three rounds of immunoscreening. The size of S.j cDNA fragments in positive clones ranged from 0.7?kb-3.0?kb after automatically excised with the helper phage. Sequence analysis revealed that partial sequence of clone M5 had significant homology with S.j mitochondria mRNA, the other positive clones were new S.j genes. M2 clone sequence (GenBank accession number AF502579) was 730?bp long it had a 117?bp open reading frame (ORF). The sequence of M15 (GenBank accession number AF502582) has no transmembrane region and encodes 92 amino acids, and its protein contains a ferredoxins iron-sulfur binding region signature and two VWFC signal regions. The size of M1、M8、M9、M12(GenBank accession numbers: AF502578, AF502580, AF500622, AF502581) ranges from 402?bp to 766?bp. It concluded that the sera from rabbit immunized with SjHmAg could recognize S.j specific antigens molecules, and these antigens may induce the protective immunity against S.j infection.

Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54% of egg reduction rate in mice, revealing a significant difference (P<0.001) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display peptide library can elicit partial protection against this pathogen.

Isolation and Characterization of 44.6 kDa Protein from Schistosoma japonicum Male Worm

Soluble male worm antigen of Schistosoma japonicum ( Sj ) was investigated for development of new vaccine candidate. SDS-PAGE and Western blotting were performed to compare the difference between soluble antigens from worms of different sex. Mice vaccination with the testing purified protein was followed by Sj cercariae challenge to detect the protective effect against Sj . Sixteen bands were seen for the soluble male worm antigen and 12 for the female worm. In addition, a distinct band of 44.6 kDa from the male worm antigen was observed, and its antigenicity was demonstrated by Western blotting. This 44.6 kDa protein could induce significant worm and egg reduction rate in mice (39.31%, 41.98%, P <0.001). In this study a 44.6 kDa protein was isolated and partially characterized. Its antigenicity, immunogenicity and the partial immune protection suggest its potential vaccine candidte against Sj .

Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis inSchistosoma japonicum-infected micevia transforming growth factor-β/Smad signaling

AIM: To investigate the antifibrotic effects of bone morphogenetic protein-7 (BMP-7) on Schistosoma japonicum (S. japonicum )-induced hepatic fibrosis in BALB/C mice. METHODS: Sixty BALB/C mice were randomly divided into three groups, including a control group (group A, n = 20), model group (group B, n = 20) and BMP-7 treated group (group C, n = 20). The mice in group B and group C were abdominally infected with S. japonicum cercariae to induce a schistosomal hepatic fibrosis model. The mice in group C were administered human recombinant BMP-7. Liver samples were extracted from mice sacrificed at 9 and 15 wk after modeling. Hepatic histopathological changes were assessed using Masson's staining. Transforming growth factor-beta 1 (TGF-β1), alpha-smooth muscle actin (α-SMA), phosphorylated Smad2/3 (pSmad2/3) and Smad7 protein levels and localization were measured by Western blotting and immunohistochemistry, respectively, and their mRNA expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). RESULTS: The schistosomal hepatic fibrosis mouse model was successfully established, as the livers of mice in group B and group C showed varying degrees of typical schistosomal hepatopathologic changes such as egg granuloma and collagen deposition. The degree of collagen deposition in group C was higher than that in group A (week 9: 22.95±6.66vs 2.02±0.76; week 15: 12.84±4.36 vs 1.74±0.80; P<0.05), but significantly lower than that in group B (week 9: 22.95±6.66 vs 34.43±6.96; week 15: 12.84±4.36 vs 18.90±5.07;P<0.05) at both time points. According to immunohistochemistry data, the expressions of α-SMA, TGF-β1 and pSmad2/3 protein in group C were higher than those in group A (α-SMA: week 9: 21.24±5.73 vs 0.33±0.20; week 15: 12.42±4.88 vs 0.34±0.27; TGF-β1: week 9: 37.00±13.74 vs 3.73±2.14; week 15: 16.71±9.80 vs 3.08±2.35; pSmad2/3: week 9: 12.92±4.81 vs 0.83±0.48; week 15: 7.87±4.09 vs 0.90±0.45; P<0.05), but significantly lower than those in group B (α-SMA: week 9: 21.24±5.73 vs 34.39±5.74; week 15: 12.42±4.88 vs 25.90±7.01; TGF-β1: week 9: 37.00±13.74 vs 55.66±14.88; week 15: 16.71±9.80 vs 37.10±12.51; pSmad2/3: week 9: 12.92±4.81 vs 19.41±6.87; week 15: 7.87±4.09vs 13.00±4.98;P<0.05) at both time points; the expression of Smad7 protein in group B was higher than that in group A and group C at week 9 (8.46±3.95 vs 1.00±0.40 and 8.46±3.95 vs 0.77±0.42; P<0.05), while there were no differences in Smad7 expression between the three groups at week 15 (1.09±0.38 vs 0.97±0.42 vs 0.89±0.39; P>0.05). Although minor discrepancies were observed, the results of RT-PCR and Western blotting were mainly consistentwith the immunohistochemical results. CONCLUSION: Exogenous BMP-7 significantly decreased the degree of hepatic fibrosis in both the acute and chronic stages of hepato-schistosomiasis, and the regulatory mechanism may involve the TGF-β/Smad signaling pathway.

Bioinformatics analysis of the structure and linear B-cell epitopes of aquaporin-3 from Schistosoma japonicum

Objective:To analyze the structure of aquaporins-3(AQP-3) from Schistosoma japonicum(SJAQP-3) using bioinformalical methods,and to provid of references for vaccine targets research.Methods:Protparam,BepiPred,TMHMM Server,MLRC,Geno3d,DNA star software packages were used to predict the physical and chemical properties,hydrophilicity plot, flexibility regions,antigenic index,surface probability plot,secondary structure,and tertiary structure of amino acid sequence of SJAQP-3.Results:SJAQP-3 had six transmembrane regions and two half-spanning helices that form a central channel.The half-spanning helices fold into the centre of the channel.Either of the half-spanning helix had a conserved motif of NPA common to all aquaporins.Predicted linear B-Cell epitopes were most likely at the N-terminal amino acid residues of Saa-7aa,59aa- 62aa,225aa-230aa,282aa -288aa,294aa -29Saa and 305aa -307aa area.59aa- 62aa,22Saa-230aa located outside the membrane,the others located inside the cell.Conclusions:SJAQP-3 is a integral membrane protein in Schistosoma japonicum tegument.There are six potential epitopes in SJ AQP-3.It might be a potential molecular target for the development of vaccines.