The Immunopharmacological Study Of HGT-5A And The Evaluation Of Its Anti-rheumatoid Arthritis Effect

Rheumatoid arthritis (RA) is a chronic inflammatory systemic autoimmune disease. Growing evidence suggests that T lymphocytes abnormality proliferation and the imbalance of Th1 and Th2 may play a key role in the pathogenesis of RA. Among the pro-inflammatory cytokines, tumor necrosis factor- (TNF- ), interleukin 1β(IL-1β), and IL-17 are expressed at high levels in the rheumatoid joint and are central to the mechanisms of the inflammatory response.Therapy for RA rests on two principle approaches: symptomatic treatment with non-steroidal anti-inflammatory drugs (NSAIDs), treatment with disease-modifying anti-rheumatic drugs (DMARDs) and Glucocorticoids. Although these drugs can retard or halt disease progression or delay disease onset but have multiple effects, some of which are undesirable, and toxicity also over the long term. These facts illustrate the need for novel therapeutic approaches for preventing the inflammatory and autoimmune components of the disease, promoting restoration of immune tolerance, and for satisfactory prolonged treatment of RA disease.Periploca forrestii Schltr. (HGT), one of Asclepiadaceae family Periploca Linn plant is a traditional ethnic herbal medicine, which is widely used to treat rheumatoid arthritis by the Hmong in Southwest China. It is efficacious to alleviate turgescence, and treat injuries from falls, contusions and strains. In our previous studies, we found out a polysaccharose part HGT-5A with low toxicity and relative good immunocompetence from HGT. We isolated and purified the ingredients of HGT-5A, obtained neutral polysaccharose HP1 and acidic polysaccharose HP2. HP1 mainly composed of homogeneous polysaccharides HP1-1,HP1-3,HP1-4, HP2 mainly composed of homogeneous polysaccharides HP2-2,HP2-3,HP2-4. The aim of the current study is to investigate the immunological activity of the extraction of HGT-5A and its possible active components, and evaluate its treatment on rheumatoid arthritis.Assessment of immunocompetence of HGT-5A and its ingredients in vitro The in vitro immunological activity and cell viability were determined by 3[H]-thymidine uptake assay and MTT assay, respectively. HGT-5A enhanced the primary proliferation of splenocyte, significantly suppressed concanavalin A (Con A) and anti-CD3 antibody stimulated T cell proliferation, but had no effect on lipopolysaccharide (LPS) stimulated B cell proliferation and IgG secretion in vitro. And then we found that HGT-5A showed little effect on splenocyte survival. So we evaluate HGT-5A shows immunosuppressive activity on Con A induced T cell proliferation.Then we found the ingredients of HGT-5A,such as neutral polysaccharose HP1,HP1-1,HP1-3,HP1-4 and acidic polysaccharose HP2,HP2-2,HP2-3,HP2-4 exhibit similar immunosuppressive activity on Con A induced T cell proliferation. Follow above-mentioned results, we presumed that homogeneous polysaccharides HP1-1~HP1-4 and HP2-2~HP2-4 may contribute partly to the immune inhibition activity of HGT. And HGT-5A may be one of active parts of Periploca forrestii Schltr.Evaluation immunological function of HGT-5A in vivo To assess the immunosuppressive property in vivo, we employed the delayed type hypersensitivity (DTH) model , ovalbumin (OVA)-immunized mice model and collagen II -immunized mice.DTH is CD4+T cell-mediated cellular immune response. In this study we employed Prednisone and Polyglycosides of tripterygium wilfordii Hook (LGT) as control. We found that Prednisone (15 mg/kg) and LGT (15 mg/kg) significantly inhibited DTH; HGT-5A 50 and 100 mg/kg also inhibited DTH by decreasing ear swelling, and suppressing Con A stimulated lymphocyte proliferation. Compared with model group, percentages of CD3+, CD4+ and CD8+ lymphocytes in spleens increased, and the ratio of CD4+/CD8+ increased in HGT-5A group. These results suggest that HGT-5A shows immunosuppressive activity on cellular immune response and regulates the imbalance of immunological function.OVA-immunized mice model is Th2 dominate immune response. The serum level of IgG1 and IgG2a can reflect Th1 or Th2 cytokines, Since Th1 cytokines mediate the switching of the IgM to IgG2a isotype, Th2 cytokines mediate the switching of the IgM to IgG1 isotype. Compared with the high level of OVA-specific antibody IgG1 and IgG2a in model group, HGT-5A 100 mg/kg ig suppressed the secretion of OVA-specific antibody IgG1 and IgG2a. This indicated that HGT-5A has regulatory effect on the specific-antigen mediated Th1/Th2 balance.Collagen II immunized mice model is Th1 dominated and cell-mediated inflammatory immune responses. The pro-inflammatory cytokines, tumor necrosis factor- (TNF- ), interleukin 1β(IL-1β), IL-17 and C II -specific antibody are expressed at high levels in mice and are central to the mechanisms of the inflammatory response. HGT-5A significantly suppressed the secretion of IFN-γand IL-4, and down regulation the ratio of IFN-γ/IL-4. Since changes in the balance between IgG2a and IgG1 levels in vivo provide evidence of the biological effect of changes in the Th1 and Th2 cytokine milinu. HGT-5A elevated the level of IgG1 and had no effect on IgG2a at 50,250 mg/kg ig for 4 weeks. This manifested that the mice immunity station shifting from Th1 to Th2 and HGT-5A has regulatory effect on the Th1/Th2 balance. Compared with model group, HGT-5A conspicuously suppressed the secretion of TNF-?, IL-1? and IL-17 at 50, 250 mg/kg ig for 4 weeks. It is known that high titer of C II -specific antibody is one representation for CollagenⅡ-immunized mice, but HGT-5A has little effect on the level of C II -specific IgG. We presume that HGT-5A perhaps has no effect on humoral immunity.Follow above-mentioned results, we suppose HGT-5A has immunosuppressive activities on T cell mediated cellular immunity response in vivtro or in vivo, and has little effect on humoral immunity. However, HGT-5A regulates Th1/Th2 balance which is mediated by specific-antigen, and suppresses the secretion of TNF-?, IL-1? and IL-17. So we presumed that it is worth for us to study HGT-5A as anti-rheumatoid arthritis candidate drug.Effect of HGT-5A on Collagen-induced arthritis Collagen-induced arthritis (CIA) was first established by Trentham in 1977, and has proven to be a powerful model of human rheumatoid arthritis (RA). Because of the important similarities between CIA and rheumatoid arthritis, this experimental model of autoimmune arthritis has been the subject of extensive investigation for drug evaluation and pathogenesis research in the international laboratories. So we used CIA to evaluate the anti-arthritic effect of HGT-5A.Since about 27th day after the first immunity, the thickness of mice feet and arthritis score had been increasing. HGT-5A 250 mg/kg depressed thickness of feet , reduced arthritis scores and ameliorated arthritis symptom at ig prophase. Further study manifested that HGT-5A 250 mg/kg significantly suppressed CII stimulated specific-T lymphocyte proliferation.In conclusion, HGT-5A shows immunosuppressive activity on T cell proliferation, and neutral polysaccharose HP1 and its homogeneous polysaccharides HP1-1,HP1-3,HP1-4, acidic polysaccharose HP2 and its homogeneous polysaccharides HP2-2,HP2-3,HP2-4 are possible active components. HGT-5A has immunosuppressive activities on T cell mediated cellular immunity response in vivtro or in vivo, and has little effect on humoral immunity. However, HGT-5A regulates specific-antigens mediated Th1/Th2 balance, and suppresses the secretion of TNF-?, IL-1? and IL-17, and exhibits some beneficial effect on collagen-induced arthritis. It is worth to further study HGT-5A as anti-rheumatoid arthritis candidate drug.