Studies On Pharmacodynamics And Mechanism Of Sinomenine Ameliorating Acute Experimental Autoimmune Encephalolmyelitis

BACKGROUNDMultiple sclerosis (MS) is an autoimmune disease of the centralnervous system (CNS), characterized by inflammatory demyelinating, whichis the major disabling disease of young individuals. Relapse, the mainclinical manifestation, is the major cause of progressive deficiencies. Butuntil now there is no ideal curative therapy for MS in the world.The alkaloid sinomenine (SIN) is extracted from the Chinese medicalplant Sinomenium acutum, which has been utilized to treat rheumatoidarthritis (RA).SIN has been shown to have effects on anti-inflammation andimmune suppression. Some drugs such as immunoglobulins or anantirheumatic drug (TAK-603) have protective effect against arthritis andMS which involve T cell mediated disorders and that beneficial effect isassociated with a decreased proliferation of T cells specific for theimmunizing antigen and reduction of tissue inflammation. So we have thehypothesis that SIN could be an ideal drug to ameliorate relapse andprogressive of MS.AIMSTo induce animal model of MS, experimental autoimmuneencephalomyelitis (EAE), clinical evaluation, histopathology, proliferationassays and cytokine analysis of splenocytes, expression of cytokine andchemokines in spinal cords were investigated. Chemokines include monocyte chemoattractant protein(MCP-1), macrophage inflammatoryprotein-1α(MIP-1α) and regulated upon activation normal T cell expressedand secreted(RANTES). To explore the effects of SIN on MS treatment, themechanism of these effects was discussed.Transcription factor T-bet plays a critical role in differentiation of Thlcells and IFN-γinduction. We have demonstrated that treatment of SIN isassociated with inhibition of proliferation of splenocytes and secretion ofIFN-γand TNF-α. So our further studies focus on T-bet to discuss themechanism.METHODS1. EAE induction: EAE was induced by myelin basic protein peptide68-82 (MBP_68-82) in Lewis rat. Mean clinical score, incidence, mean day ofonset, disease index, mean maximum severity were used to evaluate severityof EAE, as well as the weights of rats. Histological changes were detectedby HE and Luxol fast blue/periodic acid-Schiff (LFB/PAS) stain in spinalcords of EAE rats.2. Effects of SIN on EAE. Five groups: Control rats were immunizedonly with 0.2ï¼…DMSO; EAE rats were immunized only with peptideMBP_68-82; SIN200 group treated with SIN 200mg/kg.d was immunized withpeptide MBP_68-82; SIN100 group treated with SIN 100mg/kg.d wasimmunized with peptide MBP_68-82; SIN50 group treated with SIN 50mg/kg.dwas immunized with peptide MBP_68-82. The Lewis rats were treated withdifferent doses of SIN with intraperitoneal injection from day -1 to 3 for 5consecutive days after immunized. The therapeutic effect on disease activitywas observed during 21 days after immunization. Infiltration of mononuclear cells in spinal cord was detected by HE stain. Splenocyteswere isolated and used to test proliferation; cytokine levels of splenocytessupematant and spinal cords were measured by ELISA; expression of iNOSwas measured by western blot; epression of chemokines mRNA wasmeasured by RT-PCR; plasma concentration of SIN was evaluated by highperformance liquid phase(HPLC) in each group.3. Effects of SIN on T-bet expression in vitro. Groups: Control group,splenocytes were isolated from normal Lewis rats; Induced group,splenocytes were stimulated with anti-CD3 antibody and rmIL-12; SINtreated group, splenocytes were cultured with both stimulates and SIN. T-betexpressions of splenocytes in every group was measured by RT-PCR atdifferent time points; levels of IFN-γin splenocytes supernatant weremeasured by ELISA.RESULTS1. The incidence of EAE is 20/20, mean day of onset is (6.5±1.2);disease index is (1.9±0.4); mean maximum severity is (4.6±0.4); the ratio ofbody weight change is (70.4±5.0)ï¼…. EAE rats show mononuclear cellssurrounding vascellum by HE stain. Compared with Control group, thenumber of lymphocytes in spinal cords surrounding vascellum in EAE ratssignificantly increased [(43.4±9.0)/mm³ vs (7.1±5.5)/mm³, P＜0.01]2. Effects of SIN on clinical evaluation.(1) Compared with EAE group, treatment with daily dose of 200mg/kg.d SIN was effective in reducing incidence of EAE (15/20: 20/20,P＜0.05); 100mg/kg.d and 200mg/kg.d SIN treated respectively led tosignificant delay of disease progression; rats given 50 mg/kg.d, 100 mg/kg.d and 200mg/kg.d SIN showed significantly reduction of disease index;disease severity of rats with 100mg/kg.d and 200 mg/kg.d SIN wassignificantly reduced; treatment with 50mg/kg.d, 100mg/kg.d and 200mg/kg.d SIN was effective in reducing the loss of body weight.(2) Effect of SIN on inflammation in the spinal cordHE stain showed dose-dependent correlation. Compared with Controlgroup, inflammatory cells in surround vascellum of spinal cord with SINtreated groups showed significantly reduced [(12.5±7.0), (18.5±5.5) and(25.5±8.5) vs (43.4±9.0), P＜0.05].(3) Effects of SIN treatment on stimulated responses by specificantigen1) Proliferation of splenocytes stimulated with PHA or MBP_68-82 inEAE and SIN treated rats were obviously increased in comparison withunstimulated. Compared with EAE rats, proliferation of splenocytes in SINtreated rats (200mg/kg.d, 100 mg/kg.d and 50 mg/kg.d, respectively) weresignificantly inhibited(P＜0.05). Furthermore the inhibition wasdose-dependent. Besides, SIN also inhibited secretion of IFN-γand TNF-αin splenocytes supematant (P＜0.05).2) In comparison with PHA and MBP_68-82 stimulation in EAEsplenocytes of SIN treated rats, the inhibition of proliferation stimulatedwith MBP_68-82 was more obvious than that with PHA. The inhibition ofTNF-αsecretion stimulated with PHA in SIN200 and SIN100 group wasmore obvious than that stimulated with MBP_68-82.(4) Compared with EAE group, SIN (200 mg/kg.d, 100mg/kg.d and50mg/kg.d, respectively) treated rats inhibited secretion of IFN-γand TNF-αin spinal cord by dose-dependent (p＜0.05); Compared with EAE group, SIN treated rats inhibited expression of iNOS in spinal cord.(5) SIN treated rats inhibited expression of RANTES, MCP-1 andMIP-1αin spinal cord by partly dose-dependent (p＜0.05).(6) With the dose increase of SIN, the drug concentration in theplasma dectected with HPLC increases consecutively. The correlationcoefficient between SIN concentration and mean maximum severity is-0.6227 (P＜0.05); The correlation coefficient between SIN concentrationand ratio of weigh change is -0.7198 (P＜0.01)(7) There was T-bet expression in vitro at 12h, 24h and 48h afterinduction with the peak point at 12h reached. T-bet mRNA expression ofsplenocytes pretreated with SIN in vitro was significantly inhibited, so as thelevel of IFN-γ.CONCLUSIONS1. EAE induced with peptide MBP_68-82 in Lewis rats, whose incidence,mean day of onset, disease progression and severity were coincide with withmany other reports.2. We demonstrated that SIN could ameliorate EAE by inhibitingproliferation of antigen specific T cells, reducing inflammatory cytokinessecretion, inhibiting myelin attack by T cells and inhibiting expression ofcytokines and chemokines in spinal cord.3. SIN could reduce IFN-γsecretion by inhibiting T-bet expression.