Sea Snakes Ethanol Extract Of Collagen-induced Arthritis Rat Synovial Angiogenesis Regulatory Factor

ObjectiveThe present study was to explore the therapeutic mechanism of seasnake alcohol extracts on collagen induced arthritis (CIA) in rats from the aspect of antiangiogenesis in synovium.Materials and methods1 MaterialsThirty male DA rats were randomly divided into three groups: normal control group, collagen induced arthritis group(CIA) and Seasnake-treated group, 10 rats each group. CollagenⅡ(2mg/ml) was dissolved by 0．1mol/L acetic acid and then CFA was added. Compound was completely emulsified .All the model DA rats were injected perculaneously in the root of tail with 150μg collagenⅡ, which is the compound of 75μl 0．1mol/L acetic acid emulsified in an equal volume of complete Freund's adjuvant, to induce arthritis. Drugs were given Fifteen days after models establishment, Seasnake-treated group was fed with Seasnake alcohol extracts, 1．84g/kg·d, 1ml/100g and the rats in the model group and normal group were fed with physiological saline, 1ml/100g. Forty-five days after models establishment, the blood sample was obtained under anesthesia condition and then the rats were sacrificed by decapitation, knee joints were taken following removing skin and hair. Specimens containing bone were fixed for 36 hours in 4% paraformaldehyde and subsequently decalaified for at least seven weeks in 10%EDTA·Na₂．2 Methods2．1 Measurement of Arthritis IndexFifteen days after models establishment, rats were observed twice a week for the presence of distal joint swelling and erythema. Each limb was scored on a scale from 0 to 4 (0, absence of arthritis; 1, erythema and mild swelling of phalangeal joints; 2, moderate erythema and swelling of phalangeal and metatarsal joints; 3, severe swelling of the whole paw below ankle; 4, severe swelling of ankles and the whole paw). A maximum arthritic index (MAI) was obtained for each mouse by summing the greatest score recorded for each limb (0, no disease; 16, highest possible score).2．2 Observation of knee jointsKnee joints were fixed in 4% paraformaldehyde for 36 hours and 10%EDTA·Na for another 35 days which was used to remove calcium within joint. Samples taking, dehydration, paraffin embedding, mounting and HE staining were carried out according to analomical surface. The pathology of synovium, cartilage and bone can be observed under light microscope.2．3 Measurement of T cell subsets in the blood50μl anticoagulant blood and 10μl corresponding antibody was added into tube and then after being incubated for 30min at 4℃refrigerator, 1ml 0．84% NH₄Cl hemolysin was added. Samples were placed at at room temperature avoiding light, 5min later centrifugated 1,200prm for 3min and then supernatant was abandoned. Samples into which 1ml PBS were added were centrifugated 1,200prm for 3min, alongside supernatant was abandoned (rpeated). Then 400μl PBS was added for measurement.2．4 Measurement of TNF-α, IFN-γand anti-collagenⅡantibody derived from serum by ELISAMeasurement of TNF-α, IFN-γ: Coating: The plates were coated with coating antibody solution overnight at 4℃, wash once with Wash Buffer. Blocking: Add 250μl of Assay Buffer to each well and allow the binding reaction to take place for two hours at room temperature. Wash the plate twice. 50μl of Sample Diluent and 50μl of each Sample were added to the sample wells, alongside 50μl of diluted Biotin-Conjugate to all wells .And then Covered with a Plate Cover and incubated at room temperature for 2 two hours on a microplate shaker set at 200rpm. Wash the plate forth. 100μl of diluted Streptavidin-HRP was added to all wells for one hour at room temperature. Wash the plate third. 100μl of mixed TMB Substrate Solution was pipetted to all wells for about 20min at room temperature, avoiding direct exposure to intense light. The reaction was stopped with stop solution and read at 450nm in a spectrophotometer. Measurement of anti-collagenⅡantibody: Coating: The plates were coated with CⅡantigen overnight at 4℃, wash once with Wash Buffer. Blocking: Add 100μl of 1%BSA--PBS to each well and allow the binding reaction to take place for an hour at room temperature. Wash the plate twice. 100μl of Sample Dilution were added to the sample wells for two hours at room temperature, alongside 50μl of diluted Biotin goat anti-rat antibody to all wells. And then Covered with a Plate Cover and incubated at room temperature for an hour on a microplate shaker set at 200rpm. Wash the plate forth. 100μl of diluted Streptavidin-HRP was added to all wells for 30min at room temperature. Wash the plate third. 100μl of mixed TMB Substrate Solution was pipetted to all wells for about 20min at room temperature, avoiding direct exposure to intense light. The reaction was stopped with stop solution and read at 450nm in a spectrophotometer.2．5 Expression of VEGF, Flk-1, Flt-4, Ang-2, Tie-2, TNF-αand IFN-γwere assessed by Immunohistochemical methodsKnee joints were fixed for in 4% paraformaldehyde and subsequently decalaified in 10%EDTA·Na₂．Specimens were processed for Samples taking, dehydration, paraffin embedding and 4 or 5μm serial sections were cut for immunohistochemical staining using an immunoperoxidase technique with diaminobenzidine (DAB) as the chromogen.Immunohistochemical staining was performed, using the PV-6001 method. Briefly, 5μm paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded ethanols, followed by incubation with 3% H₂O₂ for 15minutes at room temperature (RT) to eliminate endogenous peroxidase activity. And then trypsin antigen retrieval to paraffin sections were performed. For detection of non-specific antigen expression, the sections were incubated with 10% normal goat serum for 20 minutes at RT and then incubated with rabbit monoclonal antibody, overnight at 4℃(PBS substitute rabbit monoclonal antibody as a negative control). Then the sections were incubated for 30 minutes at 37℃with a goat anti-rabbit immunoglobulin G (IgG) antibody-HRP polymer. The reaction products were visualized using diaminobenzidine. Six visions were randomly selected for each slice to calculate the percentage value of positive area as for every vision. The result is theaverage of percentage values.2．6 Statistical AnalysisResults are presented as Mean±SE. Differences between groups were analyzed usingq-test by one-way ANOVA.Results1 Arthritis Index: It was showed that from the twenty-forth day the arthritis indexes of CIA group were significantly higher than that of Seasnake-treated group which decreased gradually.2 Pathological changes: There were no pathological changes in the knees joints of normal control group. As for the CIA model group, erosion of cartilage and bone, proliferation of synoviocytes and capillary vessels, the hyperemia and edema in synovium and the formation of pannus, the inflammatory cells could be observed. In the Seasnake-treated group we found that there were slight pathological changes in synovial tissue and cartilage.3 T cell subsets in the blood: Forty-five days after models establishment, The levels of CD4⁺ and CD8⁺ lymphocyte of CIA group were significantly higher than that of normal control group. Compared with CIA group, The levels of CD4⁺ and CD8⁺ lymphocyte of Seasnake-treated group decreased, especially CD4⁺ lymphocyte. There was no statistically significant difference between Seasnake-treated group and normal control group.4 Amount of anti-collagenⅡantibody in serum: It was shown that the amount of anti-collagenⅡantibody of CIA group was higher than that of normal control group, whereas Seasnake could significantly lessen the higher level of anti-collagenⅡantibody in serum.5 Amount of TNF-αand IFN-γin serum: TNF-αlevel in serum of CIA group increased significantly, which indicated that TNF-αpromote the progress of the disease and played an important role in angiogenesis. In contrast with CIA group, TNF-αlevel in serum of Seasnake-treated group decreased significantly. Furthermore, IFN-γlevel in serum of CIA group was significantly higher than that of normal control group. It was the direct evidence that CIA just as RA was the disease that Th1 inflammatory response dominated. Findings shew that IFN-γlevel in serum of Seasnake-treated group was lower than that of CIA group. There were statistically significant differences.6 Expression of VEGF, Flk-1, Flt-4, Ang-2, Tie-2, TNF-αand IFN-γin synovium : Our results demonstrated that the expression of VEGF, Flk-1, Flt-4 , Ang-2 and Tie-2 in the rats of CIA group were significantly higher than that of normal control group . There was also a significant correlation between VEGF and Ang-2．Furthermore, TNF-α, IFN-γexpression of CIA group increased significantly compared with the normal control group. Our results also indicated that TNF-a expression within synovial tissues might play an important role in angiogenesis. A significant correlation between TNF-αand VEGF exists. The expression of VEGF, Flk-1, Flt-4 , Ang-2, Tie-2, TNF-αand IFN-γin the rats of Seasnake-treated group were less than that of CIA group. There were statistically significant differences.ConclusionThis study shows that effective treatment of RA with Seasnake is associated with anti-angiogenesis. Seasnake alcohol extracts can inhibit angiogenesis via the following mechanisms: Directly Seasnake alcohol extracts inhibit VEGF/VEGFR signaling and the angiopoietin-2/Tie-2 system. Indirectly Seasnake alcohol extracts regulate immune system to suppress inflammatory factors. Detailedly, Seasnake alcohol extracts can significantly reduce the amount of IFN-γand TNF-αeither in synovium or in blood .