The Therapeutic Effects Of Pentapeptide Compound CMS010.26 On Graft Rejection & Rheumatoid Arthritis And Its Mechanisms

Objective:To investigate the therapeutical effect of hexapeptide CMSO 10.26 on homograft rejection and rheumatoid arthritis. To approach its probable mechanisms of therapeutic effect by researching on p56^lck and PI3K signal transduction pathway of T cells.Methods:1. The effect of CMSO 10.26 on one-way MLR was assayed by MTT method;Tail-back skin and pinna aurium allogeneic transplantation model were established, calculate grafts' mean survival time (MST). The effect of CMSO 10.26 on allogeneic graft pathological damage was observed with HE stain, and on graft T lymphocyte infiltration was observed with immunohistochemistry method.2. Complete Freund's adjuvant (10mg/ml) was injected intradermally in the right hind footpad of Wistar rats to induce AA. Intraperitoneal injection with given drugs was commenced daily from the seventh day after immunization. Swelling of right and left ankle jionts in A A rats were measured every four days. The effect of CMS010.26 on the pathology damage of ankle joints was assayed by HE stain. ConA induced AA rats T lymphocytes proliferation was assayed by MTT method. T lymphocyte infiltrating to ankle joints and T lymphocyte activation was assayed by immunohistochemistry.3. The effect of CMSO 10.26 on CD3 McAb induced Jurkat T cell proliferation was assayed with MTT method. Protease activity of p56^lck in Jurkat T cells was assayed by immunoprecipitate technique and ELISA method. p56^lck protein expression in Jurkat T cells was assayed by western blot method. Protease activity of PI3K in Jurkat T cells was assayed by immunoprecipitate technique and ELISA method.Regulatory subunit P85a mRNA and catalytic subunit PI 10a mRNA were assayed by Real-time PCR. P85 and P85a protein expression in Jurkat T cells was assayed by western blot.4. Protease activity of p56lck in AA spleen cells was assayed by immunoprecipitate technique and ELISA method. p56lck protein expression in AA spleen cells was assayed by western blot. Protease activity of PI3K in AA spleen cells was assayed by immunoprecipitate technique and ELISA method. Regulatory subunit P85a mRNA and catalytic subunit PI 10a mRNA were assayed by Real-time PCR. P85 and P85a protein expression in AA spleen cells was assayed by western blot.Results:1. CMSO 10.26 (l-100ug/ml) can significantly inhibit T lymphocyte proliferation in one-way MLR. CMSO 10.26 (200ug/kg/d, lOOug/kg/d) can significantly prolong the MST of skin and cardiac grafts (PO.01). Prolongation rates of skin grafts are 41.4% and 40.5% respectively. Prolongation rates of cardiac grafts are 34.4% and 39.6% respectively. CMSO 10.26 can reduce pathological damage and T lymphocyte infiltrating to the grafts.2. CMSO 10.26 can relieve ankle mortise swelling at dose of 200ug/kg/d and 100ug/kg/d (PO.05). CMSO 10.26 can reduce pathological damage and T lymphocyte infiltrating to the joints, and inhibit T lymphocyte proliferation of AA rats.3. CMS010.26(0. l-100ug/ml) can significantly inhibit CD3 McAb induced Jurkat T cell proliferation (PO.05). Optimal concentration were lOug/ml and lug/ml, with the inhibitory rate 28.81% and 35.48% respectively. CMS010.26(10ug/ml, lug/ml) can significantly inhibit p56lck protease activity and protein expression in CD3 McAb induced Jurkat T cells (P<0.05). CMS010.26 (10ug/ml, lug/ml) cansignificantly inhibit PI3K protease activity, PI 10a and P85a mRNA, P85 and P85a protein expression in CD3 McAb induced Jurkat T cells (P<0.05). 4. CMSO 10.26 (200|ig/kg/d, lOOug/kg/d) can significantly inhibit p56lck protease activity and protein expression in T lymphocyte of AA rats (PO.05). CMSO 10.26 (200ug/kg/d, lOOug/kg/d) can significantly inhibit PI3K protease activity, PI 10a and P85a mRNA, P85 and P85a protein expression in T lymphocyte of AA rats (PO.05). CMS010.26 (200ug/kg/d, lOOug/kg/d) can significantly inhibit PI3K Protease activity, PI 10a and P85a mRNA, P85 and P85a protein expression in T lymphocyte of AA rats (PO.05). Conclusions:CMSO 10.26 can significantly prolong the MST of homografts, reduce pathological damage and T lymphocyte infiltrating to the graft. CMSO 10.26 could relieve the primary joints swelling, ameliorate pathological damage and diminish T lymphocyte infiltrating in AA rats. CMSO 10.26 probably suppress the abnormal activation of T lymphocyte by blocking its p56lck and PI3K signal transduction pathways, and so it can treat graft rejection and rheumatoid arthritis.