Sinomenine Reduces INOS Expression In Experimental Autoimmune Encephalomyelitis Rats Via Inhibiting The T-bet /IFN-γ Pathway

Objective:This study was designed to explore the effect of Sinomenine on the treatment of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To understand the possible mechanism about the effect of Sinomenine on EAE with animal model in vivo including clinical evaluation, histopathology and expression of cytokine and in vitro using astrocytes and splenocytes culture, so as to approach the Sinomenine's application in clinic.Methods1. In vivo(1) Preparation for EAE modelFemales Lewis rats were immunized with MBP68-82 plus complete Freund's adjuvant (CFA) and Pertussis Toxin(PTX)to make up the model.(2)GroupsFive groups: Control rats,EAE rats,SIN50mg/kg/d treated EAE rats,SIN 100mg/kg/d treated EAE rats,SIN 200mg/kg/d treated EAE rats. The Lewis rats were treated with different doses of SIN with intraperitoneal injection from day -1 to 3 for 5 consecutive days after immunization.(3)Effects of SIN on EAE and the mechanisms of the effectsThe incident condition, clinical score and weight lose were used to evaluated severity of EAE. Histological changes were detected by HE and Luxol fast blue/periodic acid-Schiff (LFB/PAS) stain in spinal cords of each group rats. Splenocytes were isolated and used to test proliferation response with 3H-TdR and cytokine levels of splenocytes supernatant and spinal cord with ELISA. Using RT-PCR to test the expression of T-bet, IFN-γand iNOS mRNA level in spinal cords, T-bet and iNOS protein level were measured with Western Blot.2. In Vitro(1) Astrocyte cell culture and iNOS inductionAstrocytes were incubated with IFN-γ(10ng/mL) and TNF-α(10ng/mL) to induce expression of iNOS with or without SIN (1mM) or selective iNOS inhibitor, L-canavanine (1mM). SIN (1mM) or L-canavanine (1mM) was added 30 minutes ahead. These cells were cultured for 6 hours for RT-PCR and 12 hours for Western Blot.(2) Splenocyte culture and T-bet, IFN-γinductionNa?ve splenocytes were stimulated with plate-coated anti-CD3 antibody (2μg/ml) and recombinant rat IL-12 (10ng/ml). To determine the effects of SIN, the cells were pretreated with SIN (1mM) before being added to the plates for 30 minutes. After 48 hours, the culture supernatants were collected and assayed for IFN-γproduction by ELISA. Cells were analyzed for IFN-γand T-bet mRNA by RT-PCR at 24 hours and T-bet protein by western blot at 48 hours.(3) The supernatants of rat splenocytes stimulated with anti-CD3 antibody (2μg/ml) and rmIL-12 (10ng/ml) in the presence and absence of SIN (1mM) were collected and then used to induce iNOS production by primary astrocytes. After being cultured for either 6 hours or 12 hours, the astrocytes were collected for preparation of RNA and protein extracts. RT-PCR was performed with the cellular RNA extracts to determine the expression levels of iNOS mRNA. Western blot was performed with the cellular protein extracts to assay the level of iNOS protein.Results:1. In vivo:(1)The condition of the incident and clinical evaluation in each group ratsThe rats in EAE group begin to fall ill after 7 days, the fiftieth day to the peak. The clinic manifestations are: losing weight, limbs palsy, even on the brink of death. The rats in control group have no incidence of EAE. The peak time of SIN treated group delayed 1 day, on the sixth day after immunization. The condition is likely the EAE group, but the clinical situation is better than the EAE group and the clinical scores are significantly reduced compared with EAE group (p<0.05).(2) Histopathology change in each group ratsHE stain showed inflammatory cell infiltration is obviously in EAE rats, the number of mononuclear cells in spinal cords surrounding vascellum in EAE rats significantly increased. The spinal cords from SIN-treated rats showed reduced infiltration of inflammatory cells in the lumbar segments.(3)Proliferation responses by stimulation with MBP68-82 and secretion of IFN-γand TNF-αin the supernatants of splenocytes in each group ratsCompared with unstimulated splenocytes of EAE rats, proliferation responses by MBP68-82 were obviously increased. Compared with EAE rats, proliferation of splenocytes in SIN treated rats were significantly inhibited (P<0.05). Besides, SIN also inhibited secretion of IFN-γand TNF-αin splenocytes supernatant (P<0.05).(4) T-bet,IFN-γ,iNOS mRNA and protein levels in each group ratsMBP-induced EAE resulted in high expression levels of T-bet, IFN-γand iNOS compared with control rats. Compared with EAE rats, 50 mg/kg, 100 mg/kg and 200 mg/kg SIN inhibited the iNOS mRNA and protein level in spinal cord, but only 100 mg/kg and 200 mg/kg SIN blocked the T-bet and IFN-γmRNA and protein levels in spinal cord.2. In Vitro(1) iNOS induction in primary astrocytes cultureThe primary astrocytes stimulated with combined IFN-γand TNF-αhad a high expression of iNOS mRNA and protein levels compared unstimulated cells. However, the levels of iNOS in the primary astrocytes cultures were reduced by L-canavanine but not by SIN (1mM).(2) T-bet/ IFN-γinduction in primary splenocytes cultureThe splenocytes cultured with anti-CD3 antibody and rmIL-12 in the presence or absence of SIN (1mM). Na?ve splenocytes expressed T-bet and IFN-γat basal levels. However, stimulation with CD3 antibody under polarized conditions with rmIL-12 induced their expression significantly. Treatment with SIN (1mM) markedly inhibited the expression of both IFN-γand T-bet.(3) The ability of the splenocytes supernatants treated with anti-CD3 antibody and rmIL-12 in the presence of SIN to induce iNOS production by primary astrocytes is reducedThe supernatants from splenocytes stimulated with anti-CD3 antibody and rmIL-12 in the presence of SIN (1mM) induced significantly lower production of iNOS by primary astrocytes than stimulated only with anti-CD3 antibody and rmIL-12, without SIN (1mM).Conclusion1. EAE induced with peptide MBP68-82, complete Freund's adguvant and Pertussis Toxin in Lewis rats, whose incidence condition, mean day of onset, disease progression and severity were coincide with with many other reports.2. SIN could effectively ameliorate EAE by reducing clinical severity and inflammatory infiltration, proliferation of antigen specific T cells and inflammatory cytokines, which exserted an immunesuppression property.3. SIN could inhibit T-bet/IFN-γ/iNOS axis in spinal cord. The anti-iNOS effect of SIN is not associated with the directly suppression iNOS induction of astrocytes but correlated with its inducer IFN-γblocking, which resulted from the inhibition of the transcription factor T-bet.