OBJECTIVES: To explore the therapeutic mechanism of compound Centella asiatica formula (CCA) for alleviating Schistosoma japonicum (Sj)-induced liver fibrosis in mice. METHODS: The active components and targets of CCA were identified using the TCMSP database with cross-analysis of Sj-related liver fibrosis targets. A "drug-component-target-pathway-disease" network was constructed using Cytoscape 3.9.1. Functional enrichment analysis (GO/KEGG) was performed using DAVID. Molecular docking study was carried out to validate interactions between the core targets and the key compounds. For experimental validation of the results, 36 mice were divided into control group, Sj-infected model group, and CCA-treated groups. In the latter two groups, liver fibrosis was induced via abdominal infection with Sj cercariae for 8 weeks, followed by 8 weeks of daily treatment with CCA decoction or saline. Hepatic pathology of the mice was assessedwith HE and Masson staining, and hepatic expressions of collagen-I and collagen-III were detected using immunohistochemistry; serum IL-6 and TNF-α levels were determined with ELISA. Hepatic expressions of TLR4 and MyD88 proteins were analyzed with Western blotting. RESULTS: We identified a total of 107 bioactive CCA components and 791 targets, including 37 intersection targets linked to Sj-induced fibrosis. The core targets included TNF, TP53, JUN, MMP9, and CXCL8, involving the IL-17 signaling, lipid metabolism, TLR4/MyD88 axis, and cancer pathways. Molecular docking study confirmed strong binding affinity between quercetin (a primary CCA component) and TNF/TP53/JUN/MMP9. In Sj-infected mouse models, CCA treatment significantly attenuated hepatic inflammatory cell infiltration, reduced collagen-I and collagen-III deposition, improved tissue architecture, reduced serum IL-6 and TNF-α levels, and downregulated TLR4 and MyD88 expressions in the liver. CONCLUSIONS CCA mitigates Sj-induced liver fibrosis by targeting TNF, TP53, JUN, and MMP9 to modulate the TLR4/MyD88 pathway, thereby suppressing pro-inflammatory cytokine release, inhibiting hepatic stellate cell activation, reducing collagen deposition, and preventing granuloma formation in the liver.
Animals ; Toll-Like Receptor 4/metabolism* ; Mice ; Myeloid Differentiation Factor 88/metabolism* ; Schistosoma japonicum ; Liver Cirrhosis/parasitology* ; Schistosomiasis japonica ; Signal Transduction ; Molecular Docking Simulation ; Inflammation ; Centella/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVETo examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits.

METHODSNew Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas.

RESULTSThe level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas.

CONCLUSIONSThe transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.

Animals ; Antibodies, Monoclonal ; Antigens, Helminth ; metabolism ; Fluorescent Antibody Technique ; Gene Expression Profiling ; Granuloma ; parasitology ; Helminth Proteins ; metabolism ; Liver ; parasitology ; RNA, Messenger ; Rabbits ; Schistosoma japonicum ; Schistosomiasis japonica

Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a(+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control
Animals ; Antibodies, Helminth ; blood ; Blotting, Western ; Cloning, Molecular ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Genetic Vectors ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Schistosomiasis japonica ; prevention & control ; Vaccines ; immunology

OBJECTIVETo study whether the infection of Schistosomiasis japanicum (S. japanicum) is related to enhanced proliferation and migration of cancer cells, and the molecular mechanism pertains to cancer cell metastasis in human host.

METHODSThe gene of S. japanicum glutathione transferase (sjGST) cloned from S. japanicum was expressed, purified and applied in a series of assays to explore the effect of sjGST on proliferation and migration of MDA-MB-435S, and the expression of MMP2 and MMP9. Immunofluorescence assay for the binding of sjGST to MDA-MB-435S was also carried out.

RESULTSResults showed that sjGST enhanced proliferation and migration in human breast cancer cell MDA-MB-435S signifycantly at 50-200 nM, but did not enhance them in human lung cancer cell A549. Immunofluorescence assay for the binding of sjGST to MDA-MB-435S and A549 showed that GST was readily bound to the breast cancer cells, but showed almost no binding to human lung cancer cells. The assays for gelatinase activity showed that both MMP2 and MMP9 activities were increased significantly in the presence of sjGST (50-200 nM) in MDA-MB-435S, but they were not significant in A549.

CONCLUSIONSOur current results show strongly that S. japanicum GST binds to MDA-MB-435S probably via its receptor, and enhances proliferation and migration of the cancer cells by up-regulatory expression of MMP2 and MMP9.

Breast Neoplasms ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glutathione Transferase ; genetics ; metabolism ; pharmacology ; Humans ; Matrix Metalloproteinase 2 ; analysis ; metabolism ; Matrix Metalloproteinase 9 ; analysis ; metabolism ; Protozoan Proteins ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Schistosomiasis japonica ; enzymology ; genetics

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.
Animals ; Antigens, Helminth ; immunology ; Cloning, Molecular ; Cyclophilins ; biosynthesis ; genetics ; immunology ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Schistosoma japonicum ; genetics ; immunology ; Schistosomiasis japonica ; prevention & control ; Vaccines, Synthetic ; biosynthesis ; immunology

OBJECTIVETo explore therapeutic effect of Haobieyangyinruanjianfang (HBYYRJ) on mouse liver fibrosis by schistosomiasis.

METHODSMice except for normal control were infected with Japanese schistosome cercarias, after 12 weeks, infected mice were divided into 7 groups: low HBYYRJ group, middle HBYYRJ group, high HBYYRJ group, Fufangbiejiaruangan tablet (FFBJRG) group, colchicine group, 3 months infection group and 6 months infection group. Hepatic fibrosis was found in 3 months infection group. Liver hydroxyproline (Hyp) was determined, matrix metalloproteinase-2 and 9 (MMP-2, MMP-9) were detected with gelatin zymography, serum hyaluronic acid (HA) and precollagen III (PC-III) were detected using RIA.

RESULTSHBYYRJ obviously reduced hepatic fibrosis (probability value less than 0.01). Collagen and HA in 3 months infection group and 6 months infection group were higher than that in normal group (probability value less than 0.01), collagen in high and middle HBYYRJ groups and HA in middle and low HBYYRJ groups were lower than that in 6 months infection group (P less than 0.01, probability value less than 0.05). The expression of MMP-9 and MMP-2 in 3 months infection group and 6 months infection group was higher than that in normal group (probability value less than 0.01), The expression of MMP-9 in three HBYYRJ groups and the expression of MMP-2 in high HBYYRJ group were lower than that in 6 months infection group (probability value less than 0.05).

CONCLUSIONHBYYRJ can reduce liver fibrosis caused by schistosomiasis.

Animals ; Collagen Type III ; blood ; Disease Models, Animal ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Female ; Hyaluronic Acid ; blood ; Hydroxyproline ; metabolism ; Liver ; drug effects ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; drug therapy ; etiology ; metabolism ; pathology ; Male ; Materia Medica ; isolation & purification ; pharmacology ; therapeutic use ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Schistosoma japonicum ; Schistosomiasis japonica ; complications ; Severity of Illness Index ; Sex Factors ; Treatment Outcome

BACKGROUNDType 2 cytokine interleukin (IL)-13 and its decoy receptor, IL-13 receptor (R) alpha2 appear to play a major role in tissue fibrosis of schistosomiasis and asthma. IL-13 is a key regulator of the extracellular matrix (ECM). It is known to signal to cells by binding to the IL-13Ralpha1, which then heterodimerizes with IL-4Ralpha. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 is known to down-regulate granulomatous inflammation and prolong host survival in Schistosoma mansoni (S. mansoni) infection, but little is known about the location and expression level of IL-13Ralpha2 in the context of S. japonicum infection.

METHODSWe established S. japonicum-infected mouse models. Kinetic serum levels of IL-13Ralpha2 were examined with ELISA. IL-13Ralpha2 mRNA and protein of liver tissues were determined by PCR and immunoblotting analysis, respectively. Detection of IL-13Ralpha2 expression and location in macrophages was performed by TaqMan PCR and fluorescent immunocytochemistry technique, respectively.

RESULTSA marked elevation of mRNA and protein expression of IL-13Ralpha2 was observed in mice during S. japonicum infection. An enhanced expression of IL-13Ralpha2 was further demonstrated in primary macrophages of murine schistosomiasis.

CONCLUSIONSIL-13Ralpha2 in macrophages may be a critical contributor to pathogenesis of schistosomiasis. The data highlight the potential importance of cell signaling and antifibrotic gene therapeutics in T helper 2 cell (Th2)-mediated diseases.

Animals ; Blotting, Western ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Interleukin-13 Receptor alpha2 Subunit ; metabolism ; Macrophages ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Schistosoma japonicum ; pathogenicity ; Schistosomiasis japonica ; immunology ; microbiology

This study examined endogenous cannabinoid (ECB)-anandamide (AEA) and its cannabinoid receptors (CBR) in mice liver with the development of schistosoma japonicum. Mice were infected with schistosoma by means of pasting the cercaria onto their abdomens. Liver fibrosis was pathologically confirmed nine weeks after the infection. High performance liquid chromatography (HPLC) was employed to determine the concentration of AEA in the plasma of mice. Immunofluorescence was used to detect the expression of CBR1 and CBR2 in liver tissue. Morphological examination showed typical pathological changes, with worm tubercles of schistosoma deposited in the liver tissue, fibrosis around the worm tubercles and infiltration or soakage of inflammatory cells. Also, CBR1 and CBR2 were present in hepatocytes and hepatic sinusoids of the two groups, but they were obviously enhanced in the schistosoma-infected mice. However, the average optical density of CBR1 in the negative control and fibrosis group was 13.28+/-7.32 and 30.55+/-7.78, and CBR2 were 28.13+/-6.42 and 52.29+/-4.24 (P<0.05). The levels of AEA in the fibrosis group were significantly increased as compared with those of the control group. The concentrations of AEA were (0.37+/-0.07) and (5.67+/-1.34) ng/mL (P<0.05). It is concluded that the expression of endocannabinoids AEA and its cannabinoid receptor CBR were significantly increased in schistosoma-infected mice. Endogenous endocannabinoids may be involved in the development of schistosoma-induced liver fibrosis.
Arachidonic Acids/*metabolism ; Endocannabinoids/*metabolism ; Liver Cirrhosis/etiology ; Liver Cirrhosis/*metabolism ; Liver Cirrhosis/parasitology ; Polyunsaturated Alkamides/*metabolism ; Random Allocation ; Receptor, Cannabinoid, CB1/*metabolism ; Receptor, Cannabinoid, CB2/*metabolism ; Schistosomiasis japonica/*complications ; Schistosomiasis japonica/metabolism

Phosphoglycerate mutase (PGAM) is a key enzyme in glycolytic pathways. With PCR technique based on an EST identified in our lab, a novel gene named SjPGAM (GenBank Accession No. EU374631) was cloned. Sequence analysis revealed that the ORF of SjPGAM gene contained 753 nucleotides, encoding 250 amino acids, and the molecular weight was about 28.26 kD. Real-time PCR analysis showed that the mRNA level of SjPGAM was much higher in the 14 days and 19 days schistosomula than other stages, suggesting that the gene was a schistosomula stage differential expression gene. The SjPGAM cDNA fragment was subcloned into an expression vector pET-28a (+) and transformed into Escherichia coli BL21 cells. In the presence of IPTG, the 31 kD fusion protein was expressed in included bodies. Western blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the mechanism of the PGAM in the glycolytic pathways of Schistosoma japonnicum.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Male ; Phosphoglycerate Mutase ; genetics ; immunology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Schistosoma japonicum ; enzymology ; genetics ; Schistosomiasis japonica ; immunology ; parasitology

To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5alpha. The amplified library contained more than 400 positive bacterial clone, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully, the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.
Animals ; Base Sequence ; Blotting, Northern ; DNA, Complementary ; genetics ; Gene Expression Regulation ; genetics ; Gene Library ; Hepatocytes ; metabolism ; Liver ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; genetics ; Schistosoma japonicum ; Schistosomiasis japonica ; genetics