The Therapeutic Effects Of Tetrapeptides Compound CMST1 On Rheumatoid Arthritis And Its Therapeutic Mechanisms

Objective:To research the therapeutic effects of tetrapeptides compound CMSTl on rheumatoid arthritis and its possible mechanisms. Methods:1. The inhibiting effect of CMST1 in vivo and in vitro on ConA induced T lymphocytes proliferation was assayed by MTT method; The effect of CMSTl in vitro on MLR(mix lymphocytes reaction) was assayed by MTT method; DNFB can induce DTH (delayed type hypersensitivity), the effect of CMSTl in vivo was assayed by ear edema weights.2. Complete Freund's adjuvant(10mg/ml)was injected 0.1ml in right the hind footpad per Wistar was used to induce AA. From 10th days after injected Complete Freund's adjuvant ,they were given drugs, primary and secondary paw swelling of AA rats were measured, HE colorization to observe the effect of CMSTl on the pathology damage of ankle. ConA induced T lymphocytes proliferation were assayed by MTT method. TNF-α IL-1β secreted from LPS induced peritoneal macrophages of AA was assayed by ELISA.3. Establish human synovial cell cultivation system in vitro; The inhibiting effect of CMSTl on the proliferation of synovial cell was assayed by MTT method; The effect of CMSTl on the VEGF secreted from synovial cell was assayed by ELISA.4. Establish human umbilical endothelium cell (HUVEC)cultivation system in vitro for further research; The inhibiting effect of CMSTl on the proliferation of HUVEC was assayed by MTT method; The effect of CMSTl on the ICAM-1 and VCAM-1 expressed by HUVEC was assayed by cell-ELISA.Results:1. Tetrapeptides compound CMSTl (0.01-100ug/ml) in vitro can inhibit ConA induced T lymphocytes proliferation(P<0.05), inhibiting rate is 22.1-44.9%. CMSTl (20ug/kgAL 40ug/kg/d) in vivo(i.p.) can significantly inhibit ConA induced T lymphocytes proliferation(P<0.05); CMSTl(1-lOOug/ml) in vitro can significantly inhibit MLR of BALB/c and C57BL/6J(P<0.05),inhibiting rate is 18-56.4%. CMSTl(40ug/kg/d, 20ug/kg/d)(i.p.) in vivo can significantly inhibit ear edema using DNFB to induced DTH(P<0.05, PO.01).2. Tetrapeptides compound CMSTl (2-20^g/kg/d)(i.p.)was given continuously for 4 days can significantly diminish the primary hind paw swelling,given for 8 days can significantly diminish secondary hind paw swelling(P<0.05), Given continuing for 26 days, CMSTl (2-20ug/kg/d) can ameliorate ankle pathology damage.CMSTl (2-20ug/kg/d)can significantly inhibit the ConA (5ug/ml) induced T lymphocytes proliferation(P<0.05) and reduce the production of TNF-a ^ IL-ip secreted by LPS (lOug/ml) induced peritoneal macrophages in AA rats(P<0.05).3. Type II collagenase and trypsin digest synovial tissue of RA patients and successfully cultured synovial cell.CMSTl(l-100(ig/ml ) in vitro can significantly inhibit synovial cell proliferation,the highest inhibiting rate is 33.6%, CMSTl (l-100|j,g/ml ) in vitro can significantly inhibit VEGF secreted by basic and TNF-a induced synovial cell(P<0.05),but has no effect on VEGF induced by IL-ip(P>0.05).4. HUVEC was successfully cultured by Type I collagenase digestion.CMSTl (0.1-100|xg/ml) in vitro can significantly inhibit VEGF induced HUVEC proli-feration(,P<0.05,P<0.01). CMSTl(l-100[ig/ml) in vitro can significantly inhibit ICAM-1 induced by TNF-a(P<0.05, P<0.01), CMSTl(lOOug/ml) in vitro can significantly inhibit VCAM-1 induced by TNF-a.(P<0.01).