[Experimental study on effect of rhizoma drynariae flavone on bone destruction of collagen inducted arthritis rat].

OBJECTIVE: Through establishing the rat model of CIA to evaluate the effect and mechanism of Rhizoma Drynariae Flavone on bone destruction of CIA rat. METHODS: Subcutaneous injection of bovine type II collagen was used to induce Wistar rats to fall ill, and then established the rat model of CIA. The rats whose inflammation scores reached to two points or above were randomly divided into four groups, and were treated accordingly. The effect of Rhizoma Drynariae Flavone on bone destruction was evaluated. RESULTS: At 12 weeks after treatment, bone trabecular area percentage and bone trabecular number in Rhizoma Drynariae Flavone group, Rhizoma Drynariae Flavone-1/2 Etanercept group, Etanercept group was obviously higher than that of sterilization water group (P < 0.05); and the trabecular resolving power of these groups was obviously less than that of sterilization water group (P < 0.05). CONCLUSION: Rhizoma Drynariae Flavone can obviously inhibit inflammation of joint bone destruction of CIA rats,the effect may be related with bone trabecular number reduction and trabecular resolving power increasing.

[Effect of Bushen Qianggu decoction on the proliferation of synovial fibroblasts and expression of PCNA and Bcl-2].

OBJECTIVE: To observe the effects of Bushen Qianggu decoction proliferation and PCNA and Bcl-2 expression. METHODS: Serum containing BQD was made and synovial fibroblasts were separated and cultured and passaged in vitro. Four groups were divided as 20% blank control group, serum containing 20% Tripterygium wilfordii multi-glycosides drug (TWMD), 20% of serum containing high and low of BQD, respectively. Serum containing drugs of different concentration were added into the synovial fibroblasts of the third generation, and then the synovial fibroblasts were cultured continued. The effects of different drugs on synovial fibroblasts and PCNA and Bcl-2 expression were observed. RESULTS: Compared with the control serum, BQD-containing serum promoted the apoptosis of synovial fibroblasts (P < 0.000 1); especially, high dose could inhibit proliferation. The expression of PCNA and Bcl-2 was significantly lower in BQD-containing serum (P < 0.000 1 vs control group). CONCLUSION: BQD can promote the apoptosis of synovial fibroblasts by improving of expression of PCNA and Bcl-2, which may be one of the mechanisms of BQD in preventing and treating osteoporosis of rheumatoid arthritis.

[Effects of Drynaria total flavonoids on apoptosis of osteoblasts mediated by tumor necrosis factor-α].

OBJECTIVE: To investigate the influence of Drynaria total flavonoids on proliferation and apoptosis of osteoblasts in tumor necrosis factor-α (TNF-α)- mediated medium, so as to explore the mechanism of Drynaria total flavonoids in preventing and treating osteoporosis of rheumatoid arthritis. METHODS: Twenty Wistar rats with average weight of (200±20) g were randomly divided into two groups: blank control group and Qianggu capsule (Drynaria total flavonoids) group. Rats in Qianggu capsule group were fed with 75 mg Qianggu capsule daily for continuous 3 d. One hour after the last feed, blood samples were collected. The in vitro experiment of four groups was designed: blank control serum group, Drynaria total flavonoids-containing serum group, blank control serum plus TNF-α group and Drynaria total flavonoids-containing serum plus TNF-α group. Methyl thiazolyl tetrazolium method was used to detect the proliferation of osteoblasts. Flow cytometry was used to detect the apoptosis of osteoblasts and real-time fluorescent quantitative polymerase chain reaction to detect the expressions of Bcl-2 and Bax mRNAs in osteoblasts. RESULTS: Compared with the control serum, Drynaria total flavonoids-containing serum promoted the proliferation and decreased the apoptosis of osteoblasts in TNF-α-mediated inflammatory environment (P<0.05), and increased the ratio of Bcl-2 mRNA to Bax mRNA. CONCLUSION: In TNF-α-mediated inflammatory environment, Drynaria total flavonoids can promote the proliferation and decrease the apoptosis of osteoblasts by improving the ratio of Bcl-2 mRNA to Bax mRNA, which may be one of the mechanisms of Drynaria total flavonoids in preventing and treating osteoporosis of rheumatoid arthritis.

Heme oxygenase-1 attenuates ovalbumin-induced airway inflammation by up-regulation of foxp3 T-regulatory cells, interleukin-10, and membrane-bound transforming growth factor- 1.

Cumulative evidence suggests the up-regulation of interleukin (IL)-10 and T-regulatory (Treg) cells is implicated in anti-inflammatory effect of heme oxygenase-1 (HO-1). Thus, we postulated that induction of HO-1 could augment IL-10 and transforming growth factor (TGF)-beta production and foxp3+CD4+CD25+ Treg cell function, thereby leading to attenuation of airway inflammation. In this study, CD4+CD25+ Treg cells isolated from mouse spleen were either transfected with a HO-1 expression vector (pcDNA3HO-1) or treated with a HO-1 inducer (hemin). Up-regulation of HO-1 enhanced foxp3 expression and IL-10 secretion in the Treg cells in vitro. Next, BALB/c, C57/B6.129, and IL-10-deficient B6.129P2-Il10tm1Cgn/J mice were challenged by ovalbumin to induce airway inflammation. Consistent with in vitro findings, hemin treatment resulted in induction of HO-1 and foxp3 and production of IL-10 and membrane-bound TGF-beta1 in vivo. This was further correlated with decrease of ovalbumin-specific immunoglobulin E level and eosinophil infiltration in bronchial alveolar lavage fluid from the asthmatic mice. Furthermore, hemin significantly enhanced the biological activity of CD4+CD25+ Treg cells. This protective effect was specifically blocked by Sn-protoporphyrin, a HO-1 enzymatic inhibitor. Finally, hemin failed to up-regulate the function of CD4+CD25+ Treg cells from IL-10-deficient mice. Our study indicates that HO-1 exerts its protective effect on asthma through a mechanism mediated by foxp3+CD4+CD25+ Treg cells, IL-10, and membrane-bound TGF-beta1.

[Effects of Astragalus on expression of renal angiopoietin receptor Tie-2 in diabetic rats].

OBJECTIVE: To study the expression of angiopoietin receptor Tie-2 in the renal tissue of diabetic rats and the effects of Astragalus. METHODS: SD rats were randomly divided into normal control group, diabetes group and Astragalus-treated group. The expression of receptor Tie-2 in the renal tissue was assessed by using real-time quantitative polymerase chain reaction and immunohistochemical method. RESULTS: Glomerule Tie-2 protein expression was significantly elevated in the diabetes group as compared with the normal control group (P<0.01). Glomerule Tie-2 protein expression in the Astragalus-treated group was decreased as compared with the diabetes group (P<0.01). CONCLUSION: Tie-2 may play an important role in the pathogenesis of the early stage diabetic renal injury. The reno-protection effect of Astragalus may be mediated by down-regulating the expression of Tie-2 in the kidney tissue of diabetic rats.

Role of soluble Fas ligand in autoimmune diseases.

AIM: To investigate the role of soluble Fas ligand in autoimmune diseases. METHODS: RT-PCR was performed to amplify sFasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. DNA fragments were cloned into PCR vector. After sequenced, sFasL gene fragments were inserted into pQE-31 vector and expressed in E. Coli M15 respectively. Proteins were purified through affinity chromatography column with ligand of 6XHis tag and identified by SDS-PAGE and Western blot. Mice were immunized with sFasL protein and specific anti-serum was harvested 6 wk after immunization. Monoclonal anti-human FasL antibody was made from the immunized mice. Serum level of sFasL in different patients was detected using anti-FasL antibodies from the immunized mice. RESULTS: The protein expressed was 24 ku by SDS-PAGE electrophrosis. The protein was specially bound to anti-human FasL antibody by Western blot analysis. The sFasL protein could induce Jurket cell apoptosis in vitro. The concentration of serum sFasL in patients with autoimmune diseases was higher than that in normal individuals. sFasL could reduce arthritis in collagen induced arthritis (CIA) mice model by subcutaneous injection. CONCLUSION: sFasL may be involved in either induction of apoptosis or autoimmune diseases. Furthermore, sFasL may have potential application in treatment of autoimmune diseases.

[Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris].

To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.

Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation.

To lay background for studying rejection mechanisms in xenotransplantation and developing the strategies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their accession numbers AY102467-AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A*0201) are better than those between pigs and mice (H-2Db/H-2Kb). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA P1 protein could respond quite well in vitro to the class I-positive swine chondrocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.

[The expression and application of human Fas ligand in E.coli].

AIM: To express recombinant human FasL molecule in E.coli. METHODS: RT-PCR was applied to amplify FasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR2.1 vector. After sequencing, the FasL gene was inserted into pQE-31 vector and expressed in E.coli M15. The FasL protein was purified through Ni-ATA affinity chromatography column and identified by SDS-PAGE and Western blot. The mice were immunized with the FasL protein and the specific anti-serum was harvested 6 weeks after immunization. The serum level of FasL from with different kinds of diseases patients were detected using the anti-FasL antibodies from the immunized mice. RESULTS: The expressed protein could be recognized by anti-human FasL antibody in Western-blot analysis with M(r)40 000. This protein could induce Jurket cells apoptosis. anti-FasL serum prepared from mouse could detect the serum FasL as sensitive as commercial ELISA kits. CONCLUSION: The human FasL protein is obtained. It lays the foundation for the further detecting the concentration of FasL and sFasL of patients.