Prp5-Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription.

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

Potential role of IL-37 signaling pathway in feedback regulation of autoimmune Hashimoto thyroiditis.

IL-37, the anti-inflammatory cytokine of the IL-1 family, plays several key roles in the regulation of autoimmune diseases. Yet, its role in Hashimoto's thyroiditis (HT) is not clear. In the present study, we found that, in tissues from HT patients, most of the follicular epithelial cells were positive for both IL-37 and single Ig IL-1-related receptor (SIGIRR) by immunohistochemical staining, while the infiltrating lymphocytes and other inflammatory cells hardly expressed any. Meanwhile, mRNA expression levels of IL-37 in peripheral blood mononuclear cells (PBMC) of HT patients were significantly higher than those in normal controls measured by quantitative real-time PCR. Finally, we studied the possible role of IL-37 in IFN-γ-stimulated rat FRTL-5 cells. The results showed that IL-1ß, TNF-α, and MCP-1 mRNA levels were significantly decreased, while the expression of IL-4 mRNA was dramatically up-regulated in IFN-γ-stimulated rat thyroid cell line FRTL-5 pre-treated with IL-37. The current study, for the first time, demonstrated that the IL-37 network is involved in Hashimoto's thyroiditis, and IL-37 signaling pathway may ameliorate the excessive autoimmune responses in this chronic lymphocytic thyroiditis.

An Efficient Schistosoma japonicum Bivalent Membrane Protein Antigen DNA Vaccine Against Schistosomiasis in Mice.

BACKGROUND Schistosomiasis is one of the most important infectious parasitic diseases in the world. The most important was to control schistosomiasis is through a combination of medical therapy and immunization. The membrane antigens Tsp2 and 29 from Schistosoma are promising anti-schistosomiasis vaccine candidates. MATERIAL AND METHODS In this study, the pcDNA3.1(+)-SjTsp2, pcDNA3.1(+)-Sj29, and pcDNA3.1 (+)-SjTsp2-29 eukaryotic expression vectors were successfully constructed as DNA vaccines, and the protective abilities of these vaccines were evaluated in mice. RESULTS The results showed that vaccination with SjTsp2, Sj29, and SjTsp2-29 reduced parasite burden and hepatic pathology compared to the control group, and the protective effect of the bivalent SjTsp2-29 DNA vaccine was better than that of the univalent SjTsp2 or Sj29 DNA vaccines. We also found high levels of IgG, IgG1, and IgG2a against SjTsp2, Sj29, and SjTsp2-29 DNA vaccines, with high expression of IFN-γ and no IL-4 in the mice. CONCLUSIONS The double-membrane antigen DNA vaccine SjTsp2-29 elicited protection against Schistosoma infection and might serve as a vaccine candidate.

Schistosoma japonicum: Tsunagi/Y14 protein plays a critical role in the development of the reproductive organs and eggs.

Tsunagi/Y14 is an evolutionarily conserved RNA-binding protein that is required for the maintenance of oogenesis and the masculinization of the germ-line in many animal models. We speculated that Tsunagi/Y14 might also regulate reproductive organ development in Schistosoma japonicum (S. japonicum, Sj). Sj Tsunagi/Y14 and control double-stranded RNAs were introduced into schistosomula by electroporation respectively. These transfected schistosomula were cultured in vitro for 1, 3 or 5 days. The mRNA and protein levels of the target gene in the cultured schistosomula were significantly suppressed compared with those of the control group. Furthermore, BALB/c mice were infected with the transfected schistosomula for 6 weeks and were sacrificed to harvest the adult worms. We found that the silencing of Sj Tsunagi/Y14 led to defects in reproductive organs development in both male and female worms. Moreover, it also affected the size, quantity and activity of the eggs in the mice liver. Our findings indicated that Tsunagi/Y14 plays a critical role in the development of reproductive organs and eggs in S. japonicum.

Worm morphology of Schistosoma japonicum using confocal laser scanning microscopy.

Male and female Schistosoma japonicum worms have dissimilar appearances in their final host. In this study, a morphometric and morphological assessment of whole worms derived from unisexual and mixed infections in mice was conducted using confocal laser scanning microscopy. Worms from mixed infections showed significant morphological changes between 15 and 25 days post-infection (PI). On the fifteenth day PI, 33% of males had formed the conspicuous gynecophoric canal, but only 8% of them had testicular lobes containing a few germinative cells; 13% of females had incipient ovaries with a few immature ovarian cells inside. On the twentieth day PI, the testicular lobes contained more germinative cells in all male worms, while female worms presented vitelline glands. On the twenty-fifth day PI, more germinative cells were observed in the male testicular lobes, and differentiated cells were present in the female ovaries. All worms had fully developed reproductive organs from 30 days PI onwards. Morphometric analysis showed significant differences between mixed and unisexual infections at 35 days PI. Ovaries of worms from unisexual infections contained cells in one stage of maturation and vitelline glands had undifferentiated cells. Our study of S. japonicum provides a detailed comparison of different morphological traits from worms of mixed and unisexual infections throughout development.

[Preliminary function study on Mago nashi gene of Schistosoma japonicum].

OBJECTIVE: To study the function of Mago nashi gene in reproductive system of Schistosoma japonicum. METHODS: dsRNA products of SjMago nashi gene and control gene (lacZ) were generated by in vitro transcription. SjMago nashi dsRNA and control (lacZ) dsRNA were electroporated into mechanically transformed schistosomula. Aliquots of parasites (1000) were harvested at day 1, 3, and 5 after electroporation, respectively. Total RNA and proteins were isolated simultaneously using TRIzol reagent. Levels of SjMago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis, respectively. About 1000 dsRNA-electroporated schistosomula were injected into each BALB/c female mouse. Six weeks later the worms were collected, fixed, stained, clarified, dehydrated and mounted. The male and female reproductive organs were observed and measured under the confocal laser scanning microscope. RESULTS: At day 1, 3 and 5 post-electroporation, 22%, 69%, and 80% reduction in Mago nashi mRNA levels were detected respectively in SjMago nashi dsRNA-electroporated schistosomula (experiment group) compared to parasites treated with control dsRNA (control group); and schistosomula of experiment group exhibited 12%, 39%, and 56% decreased in Mago nashi protein expression levels in comparison to the control group, respectively. In experiment group there were many spermatozoa in testicular lobes and no changes were observed in ovary and vitelline gland. Compared to control group, adult worms in experiment group were smaller in the body width, the width and length of testicles and ovaries (P < 0.05). CONCLUSION: Mago nashi dsRNA can specifically inhibit the expression of target gene and protein. SjMago nashi gene is a reproduction-related gene.

[Evaluation of recombinant 29,000 extra membranous protein for the immunodiagnosis of schistosomiasis japonica].

OBJECTIVE: To evaluate the recombinant membranous protein 29000 (rSj29) of Schistosoma japonicum in the immunodiagnosis of schistosomiasis by indirect ELISA. METHODS: 394 serum samples collected from two low-endemic villages in Anhui Province were tested by rSj29-ELISA and AWA-ELISA using adult worm antigen (AWA) of S. japonicum. Meanwhile all subjects were each tested by 9 examinations with 3 fecal samples by modified Kato-Katz method, and by indirect hemagglutination assay (IHA). RESULTS: It was found that the positive detection rates of stool examination, IHA, rSj29-ELISA and AWA-ELISA were 4.8% (19/394), 62.2% (245/394), 68.3% (269/394), and 89.9% (354/394), respectively. There was no statistical difference between rSj29-ELISA and IHA (chi2 = 3.2, P > 0.05) with the coincidence of 80.7% (318/394). The coincidence of IHA, rSj29-ELISA and AWA-ELISA with stool examination was 42.6% (168/394), 36.0% (142/394), and 15.0% (59/394), respectively. CONCLUSION: The indirect ELISA with rSj29 antigen shows similar effect as IHA for the diagnosis of schistosomiasis.

[Construction of DNA vaccine pcDNA3.1(+)/tetraspanin 2-A against Schistosoma japonicum and its immune-protective effect in mice].

Tetraspanin 2-A (SjTsp2-A) gene was amplified by PCR. pcDNA3.1(+)/SjTsp2-A recombinant plasmids were constructed and transformed into E. coli DH5alpha. Twenty four BALB/c mice were randomly divided into pcDNA3.1(+)/ -SjTsp2-A group (A), pcDNA3.1(+)/SjGST group (B) and pcDNA3.1(+) group (C). Each mouse was injected through musculus quadriceps femoris by three times (two weeks interval) respectively with 100 microg pcDNA3.1(+)/SjTsp2-A, pcDNA3.1 (+)/SjGST, or pcDNA3.1(+). At two weeks after the final inoculation, mice were each challenged by 40 +/- 2 cercariae of S. japonicum. Forty-five days after infection, all mice were sacrificed, the number of worms collected and eggs in liver tissue was counted. Anti-pcDNA3.1(+)/SjTsp2-A antibody was detected by ELISA and protein expression in quadriceps muscle by immunohistochemical staining. The worm reduction rate (44.4%) and egg reduction rate (28.4%) of group A was higher than those of group B and C (P < 0.05), but no significant difference between groups B (3.9%, 19.3%) and C. Higher antibody titer (1:25 600) was detected in sera of group A. Immunohistochemistry analysis showed an expression of specific antigens in quadriceps muscles of groups A and B. The DNA candidate vaccine induces partial protective immunity against S japonicum in BALB/c mice.

Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference.

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.

[Cloning, expression and identification of membrane protein Tetraspanin 2-A of Schistosoma japonicum].

OBJECTIVE: To clone and express a membrane protein (Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2). METHODS: A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. RESULTS: Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1:32,000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. CONCLUSION: SjTsp2 has been expressed and shows certain antigenicity.

[Morphological observation on the reproductive system of adult Schistosoma japonicum under confocal laser scanning microscopy].

BALB/c mice were infected with Schistosoma japonicum cercariae (40+/-2 per mouse) through abdominal skin. Mice were sacrificed after 35 days to acquire the adult worms which were then fixed, stained, clarified, dehydrated and mounted. The specimens were observed under the confocal laser scanning microscope. The overall morphology of the adult worms was displayed distinctly, especially the testicular lobes, seminal vesicle and genital pore of the male, reproductive system, and the ovary, vitelline glands, oviduct, vitelline duct, seminal receptacle, ootype, mehlis gland, uterus, genital pore and eggs of female reproductive system. The confocal laser scanning microscopy is an alternative method to research organs, tissues and cell structure of schistosome worm.

Development of monoclonal antibodies against Sj29 and its possible application for schistosomiasis diagnosis.

OBJECTIVE: Timely Schistosoma japonicum detection improves outcomes in schistosomiasis. Here, we established a double antibody sandwich ELISA to detect Schistosoma japonicum. METHODS: Sj29 polyclonal and monoclonal antibodies were developed and identified. A Sj29 double antibody sandwich ELISA was evaluated. RESULTS: Assay sensitivity for detecting Schistosoma japonicum circulating antigen Sj29 was 76.7% (23/30), 54.5% (18/33) and 50.0% (18/36) in patients with acute, chronic and advanced schistosomiasis. No false positives or cross-reactivity was observed in healthy controls or patients with clonorchiasis, paragonimiasis, or ancylostomiasis, respectively. By contrast, false positives (5.7%) and cross-reactivity (6.5%-10%) were detected using an AWA-ELISA. The circulating antigen positive rates decreased significantly faster than that of the antibody detection after 6 months treatment (22.2%, 4/18 and 88.9%, 16/18). Chi-Square Tests revealed that Sj29 sandwich ELISA had lower sensitivity than AWA indirect ELISA in the detection of S. japonicum infected patients (p<0.05). Although our assay detection specificity in patients infected with other parasites or healthy controls appeared higher, the difference between the assays was insignificant. However, our assay showed significantly better results in monitoring praziquantel therapeutic effects (p=0.001), with antigen-positive rates decreasing significantly faster than antibody detection rates after 6 months of treatment (22.2%, 4/18 versus 88.9%, 16/18). CONCLUSIONS: Sj29 double antibody sandwich ELISA was established. The specificity of this method for detecting healthy sera was 100%. Meanwhile, Sj29 sandwich ELISA may have a potential diagnostic capability to distinguish current from past infections and assess drug treatment responses.

Prevention of early renal injury by mycophenolate mofetil and its mechanism in experimental diabetes.

Previously it was shown that treatment with mycophenolate mofetil (MMF) attenuated renal inflammation and glomerular injury in a model of diabetes. However, the mechanism involved in the renoprotective effects of MMF in experimental diabetes has not been clearly delineated. Diabetes was induced by injection of streptozotocin (STZ) after uninephrectomy. Diabetic animals received no treatment or treatment with MMF (10 mg/kg once daily by gastric gavage) for 8 weeks, non-diabetic rats served as controls. Level of malondialdehyde (MDA) in renal tissue and urine as well as the activity of antioxidant enzymes (AOE) in renal tissue was determined. Renal injury was evaluated. Immunohistochemistry for ED-1 macrophages marker, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) was performed. Expression of transforming growth factor (TGF)-beta1 protein was determined by Western blotting analysis. Treatment with MMF had no effect on blood glucose level, but did prevent increased urinary albumin excretion and glomerular damage in diabetic rats. Oxidative stress was reduced by MMF treatment, as indicated by a reduction in MDA level in renal tissue and urine. Activity of AOE such as glutathione peroxidase (GSH-PX) was markedly elevated while superoxide dismutase (SOD) and catalase (CAT) were not changed by MMF treatment. In diabetic animals receiving no treatment, there were increases in ED-1-positive cells, ICAM-1 expression and MCP-1 expression in glomeruli and tubulointerstitium, which were effectively suppressed by MMF treatment. Western blotting analysis showed that the expression of TGF-beta1 protein was increased by 1.92-fold in renal tissue in diabetic rats, and MMF treatment significantly reduced the increased expression of TGF-beta1 protein by 45%. Our data suggest that MMF treatment ameliorates early renal injury via the inhibition of oxidative stress and overexpression of ICAM-1, MCP-1 and TGF-beta1 in renal tissue in diabetic rats.

Renoprotective effect of breviscapine through suppression of renal macrophage recruitment in streptozotocin-induced diabetic rats.

BACKGROUND/AIMS: Experimental and clinical evidence has consistently demonstrated that renal macrophage infiltration is one of the most important events for the progression of diabetic nephropathy. Breviscapine is a flavonoid extracted from the Chinese herb Erigeron breviscapus. Previously, it was shown that treatment with breviscapine attenuated renal injury in the diabetic rats. The purpose of this study is to investigate whether the renoprotective effect of breviscapine is through suppression of renal macrophage recruitment in diabetic rats. METHODS: Diabetes was induced bystreptozotocin injection, and breviscapine was administered orally at a dose of 20 mg/kg/day for 8 weeks. Control rats received vehicle or breviscapine with the same schedule. RESULTS: Breviscapine treatment markedly inhibited both an increase of albuminuria and glomeruli hypertrophy and tubulointerstitial injury without modifying mean arterial blood pressure and creatinine clearance. Levels of malondialdehyde and protein kinase C activities were markedly higher and antioxidant enzyme activities such as superoxide dismutase, catalase as well as glutathione peroxidase were significantly lower in the kidneys of diabetic rats than of the control group, breviscapine administration markedly remitted these changes. ED-1-positive cells and expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in glomeruli and tubulointerstitium were all markedly elevated but were significantly reduced by breviscapine. Western blot analysis noted that the expression of transforming growth factor beta1 protein was increased 1.8-fold in the kidney in diabetic rats, breviscapine treatment could reduce increased expression of TGF-beta1 protein by 47%. CONCLUSION: This study describes a novel mechanism by which breviscapine confers a renoprotective effect.

[Immunoscreening of schistosomulum cDNA library of Schistosoma japonicum and preliminary identification of membrane-associated protein abundant in tegument].

OBJECTIVE: To obtain genes encoding the novel molecules for diagnosis of schistosomiasis. METHODS: Juvenile S. japonicum cDNA library was immunoscreened to obtain positive clones. By DNA sequencing and sequence analysis, the target gene was amplified by PCR and subcloned into prokaryotic plasmid pET28a. The recombinant plasmid was transformed into E. coli BL21 followed by expression of the protein induced by IPTG. The protein was identified by Western blotting. RESULTS: 34 positive clones were obtained, 24 of which were chosen to be sequenced, 13 of which were Sj22600 gene. The protein were recognized with sera of infected rabbits and patients with acute and chronic schistosomiasis by Western blotting. CONCLUSION: The gene coding for Sj22600 membrane protein was screened with high frequency in the cDNA library. The E. coli BL21 transformed with the recombinant plasmid can express the fusion protein, which shows immunoactivity.

[Experimental study of canine bone marrow mesenchymal stem cells combined with calcium phosphate cement for repair of mandibular bone defects in Beagle dogs].

PURPOSE: To evaluate the effects of bone marrow mesenchymal stem cells (BMSCs) combined with calcium phosphate cement (CPC) scaffold for repair of mandibular defect in Beagle dogs. METHODS: BMSCs were isolated from Beagle dogs and cultured in DMEM plus 10% FBS. The induction effect was determined using alizarin red staining or alkaline phosphate staining at 14-day of culture. BMSCs were added to the CPC scaffold for animal experiments. In vivo, three critical size bone defects were surgically created in each side of the mandible. The bone defects were repaired with BMSCs-CPC (scaffolds with composite seeding cells), CPC (scaffold alone) or no materials (blank group). Two dogs were sacrificed at 4-week and 8-week after operation. Gross observation, X-ray imaging, histologic and histometric analyses were performed to evaluate the level of bone formation. RESULTS: Newly formed bones were detected within all defect sites after operation. The BMSCs-CPC group and CPC group showed increased bone formation compared with the blank group. The BMSCs-CPC group exhibited more bone formation and degradation of the material than the CPC group. The percentage of new bone in the BMSCs-CPC and CPC treated group were significantly higher than that in the control group (P<0.05), while the percentage of new bone in the BMSCs-CPC sites was higher than that in the CPC sites (P<0.01); the percentage of residual material in the BMSCs-CPC sites was lower than that in the CPC sites (P<0.01) 4 weeks and 8 weeks after operation. CONCLUSIONS: Using the theory of tissue engineering, BMSCs composite CPC compound is an effective method in promoting new bone regeneration, which has a positive influence on the bone space preservation.

Effects of total glucosides of paeony on immune regulatory toll-like receptors TLR2 and 4 in the kidney from diabetic rats.

TLRs are a family of receptors that play a critical role in the pathogenesis of diabetic nephropathy. TGP have been shown to have anti-inflammatory and immuno-regulatory activities. However, the relation between TGP and TLRs on diabetic nephropathy remains unknown. In this study, we examined effects of TGP on immune regulatory TLR2 and 4 in the kidney from streptozotocin-induced diabetic rats. TGP decreased the levels of 24h urinary albumin excretion rate significantly in diabetic rats. Western blot analysis showed that TGP significantly inhibited the expression of TLR2 and 4, MyD88, p-IRAK1, NF-κB p65, p-IRF3, TNF-α and IL-1ß. Quantitative real-time PCR analysis showed that the significantly increased levels of TLR2 and 4, and MyD88mRNA in the kidneys of diabetic rats were significantly suppressed by TGP treatment. Macrophages infiltration were also markedly increased in the kidneys of the diabetic rats, but were significantly inhibited by TGP in a dose-dependent manner. These results suggest that TGP has protective effects on several pharmacological targets in the progress of diabetic nephropathy by selectively blocking TLRs activation in vivo.

Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats.

Breviscapine is a flavonoid extracted from a Chinese herb Erigeron breviscapus, previously it was shown that treatment with breviscapine attenuated renal injury in the diabetic rats. The purpose of this study was to investigate whether breviscapine combined with enalapril (an ACE inhibitor) have superior renoprotective effects against diabetic nephropathy. Rats were randomly separated into five groups: control, diabetes, diabetes treated with enalapril, diabetes treated with breviscapine, or diabetes treated with combined enalapril with breviscapine. Twenty-four hours urinary AER and the levels of 3-NT in renal tissue and MDA in renal tissue and urine as well as activities and expression of PKC in renal tissue were determined, and renal tissue morphology were observed by light microscopy after 8 weeks. Expression of TGFß1 protein was performed by immunohistochemistry method. Increased AER and kidney pathologic injury were attenuated by treatment with either enalapril or breviscapine and further reduced by the combination of the two. Elevated 3-NT in renal tissue and MDA levels in renal tissue and urine were reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine. PKC activities and expression were higher in renal tissue in diabetic rats than those of the control group, which were reduced by both monotherapies, and further abrogated by combination therapy in both cases. Overexpression of TGFß1 protein observed in the glomeruli and tubulointerstitium of diabetic rats was attenuated by enalapril or breviscapine to a similar lever and further reduced by the combination of the two. The combination of enalapril and breviscapine confers superiority over monotherapies on renoprotection, which mechanism may be at least partly correlated with synergetic suppression on increased oxidative stress and PKC activities as well as overexpression of TGFß1 in renal tissue.

[Comparison of collagen fiber staining between Van-Gieson staining and Masson trichrome staining of hepatic specimens in mice with Schistosoma japonicum infection].

OBJECTIVE: To compare the effects of collagen fiber staining between Van-Gieson staining and Masson trichrome staining of hepatic specimens in mice with Schistosoma japonicum infection. METHODS: A model of hepatic granuloma and fibrosis was established by infecting mice with S. japonicum cercariae, then the hepatic specimens were taken and Van-Gieson staining and Masson trichrome staining were performed. Eventually, the area of granuloma and fibrosis were measured by imaging analysis software. RESULTS: When the time of staining was 3-7 min, there was no significant difference of the fibrosis areas between the two methods (P > 0.05); when the time of staining was more than 10 min, the staining area showed by Masson's staining was significantly larger than that showed by Van-Gieson staining, and the difference was statistically significant (P < 0.05). CONCLUSION: The operation procedures of Van-Gieson staining are simpler and easier to master than those of Masson trichrome staining, therefore Van-Gieson staining is a better method to display collagen.

FK506 ameliorates renal injury in early experimental diabetic rats induced by streptozotocin.

Calcineurin (CaN) plays an important role in glomerular hypertrophy and extracellular matrix accumulation in early diabetic nephropathy. Cyclosporine (CSA), a CaN inhibitor, has been shown to reduce renal injury in streptozotocin-induced diabetic rats. We examined whether FK506, which immunosuppressive action was 10-100 times of CSA, inhibits progression of diabetic nephropathy in experimental diabetic rats. Diabetes was induced with streptozotocin in rats, and FK506 (0.5 or 1.0mg/kg) was orally administered once a day for 4 weeks. Increased relative kidney weight was significantly reduced by FK506 treatment with 1.0mg/kg (p<0.05), and elevated 24 hour urinary albumin excretion rate was markedly attenuated by FK506 treatment with 0.5 and 1.0mg/kg (p<0.05, 0.01). Elevated glomerular volume was significantly attenuated by FK506 treatment with 0.5 and 1.0mg/kg (p<0.05), and increased indices for tubulointerstitial injury were only ameliorated by FK506 treatment with 1.0mg/kg (p<0.01). Western blot analysis noted that the expression of CaN protein was increased 2.4 fold in the kidney from diabetic rats, and FK506 treatment with 0.5 and 1.0mg/kg could reduce increased expression of CaN protein by 38.0% and 73.2%. The expression of 1α (IV) collagen, p65, p-p65, OPN, α-SMA and TGF-ß1 protein in kidney was significantly increased in diabetic rats and reduced by FK506 treatment (p<0.05, 0.01). Our results show that FK506 could ameliorate renal injury in early experimental diabetic rats, which mechanism may be at least partly correlated with suppression on increased CaN in renal tissue in diabetic rats.