The Immunologic Mechasnism Of Action Of EAE In Wistar Rats By Using Matrine And The Preventive Treatment

Objective:In order to investigate the preventive treatment and potential mechanism of matrine (MAT) in experiment autoimmune encephalomyelitis (EAE), we observed the effects of matrine on the rat model of EAE, which include clinical symptoms of EAE, the degree of histopathologic changes in spinal cord, the expression of ICAM-1、VCAM-1、CCL3、CCL5、Nrf2、HO-1、TLR4、MD-2、IL-4、IL-5、 IL-10、TGF-β1in rats blood and spinal cord.Methods:1Induction of animal Model:The EAE models were induced in female Wistar rats by being immuned with guinea pig spinal cord homogenate (GPSCH) extracts together with complete Freund adjuvant (CFA). The clinical symptoms of EAE rats、 body weights and the actions were observed every day after immunization.2Animal groups and administration:Fifty,Clean,female,Wistar rats were randomly divided into five equal group:EAE model group (M), low dose MAT treatment group (MAT-L), middle dose MAT treatment group (MAT-M), high dose MAT treatment group (MAT-H) and normal control group (N).M group and N group were intraperitoneally injected with6.7mL-kg-1normal saline injection for16uninterrupted days after immunization, at the same time MAT-L group were treated MAT at150mg-kg-1, MAT-M group received MAT at200mg-kg-1, and MAT-H group received MAT at250mg-kg-1by intraperitoneally injected for16uninterrupted days after immunization. 3Sample collections:the rats were killed at the17th day postimmunization.The blood was collected via posteriororbital venous plexus approach and then the serum was separated. The spinal cord were removed after heartperfusion and stored in4%paraformaldehyde solution.4Detection of indexes:The degree of histopathologic changes in spinal cord was assessed directly by hematoxylin-eosin (HE) and chromotrope-2R-brilliant green (C-2R-BG). ICAM-1、VCAM-1、Nrf2、HO-land TLR4in spinal cord were determined by immunohisto-chemical techniques(IHC) and Reverse transcription-polymerase chain reaction (RT-PCR) respectively. Enzyme linked immunosorbent assay(ELISA) method was used for determination of CCL3、CCL5、 MD-2、IL-4、IL-5、IL-10and TGF-β1in serum level.Results:1Incidence rate and the changes of clinical score, body weight:There was no ill rat in N group;the incidence rate of MAT-H、MAT-M and MAT-L groups were lower than M group (p<0.05); comparing to MAT-H、MAT-M group, the incidence rate of MAT-L group is higher (p<0.05). The body weight of N group was increased constantly in the whole experimental period, the mean weight change of M,MAT-H、MAT-M and MAT-L group were declined;the mean weight change of MAT-H、MAT-M and MAT-L groups were lower than M group (p<0.05); the mean weight change of MAT-H、MAT-M group and MAT-L group have significantly differences(P<0.05).Compared to the MAT-H、MAT-M and MAT-L group, the clinical scores of M group was significantly increased(p<0.05); the clinical scores of MAT-treated group have significantly differences(p<0.05).2The observation of histopathologic changes:There were no inflammatory infiltration and demyelination in N group rats,but the M, MAT-treated group rats all have inflammatory infiltration and demyelination; compared to M group, the inflammatory infiltration and demyelination in MAT-treated group were reduced(p <0.05); the inflammatory infiltration and demyelination in MAT-H、MAT-M group was reduced than MAT-L group(p<0.05).3The expression of ICAM-1and VCAM-1in spinal cord:Compared to N group,the expression of ICAM-1and VCAM-1in the M, MAT-treated group are increased(p<0.05); compared to the M group, the expression of ICAM-1and VCAM-1in MAT-treated group is declined(p<0.05), the expression of ICAM-1and VCAM-1in MAT-treated group have significantly differences(p<0.05).4The changes of CCL3、CCL5in serum:Compared to N group,the expression of CCL3、CCL5in the M, MAT-treated group are increased(p<0.05); compared to M group, the expression of CCL3、CCL5in MAT-treated group is declined(p<0.01); the expression of CCL3、 CCL5in MAT-treated group have significantly differences(p <0.05).5The expression of Nrf2and HO-1in spinal cord:Compared to N group,the expression of Nrf2and HO-1in the M, MAT-treated group are increased(p<0.05); compared to the M group, the expression of Nrf2and HO-1in MAT-treated group are increased (p<0.05), the expression of Nrf2and HO-1in MAT-treated group have significantly differences(p<0.05).6The changes of TLR4、MD-2in spinal cord and in serum:Compared to N group,the expression of TLR4、MD-2in the M, MAT-treated group are increased(p <0.05); compared to M group, the expression of TLR4、MD-2in MAT-treated group is declined(p<0.01); the expression of TLR4、MD-2in MAT-treated group have significantly differences(p<0.05).7The changes of IL-5、IL-10in serum:Compared to N group,the expression of IL-5、IL-10in the M, MAT-treated group are increased(p<0.05); compared to M group, the expression of IL-5、IL-10in MAT-treated group are increased (p<0.01); the expression of IL-5IL-10in MAT-treated group have significantly differences(p <0.05).8The changes of IL-4、TGF-β1in serum:Compared to M group,the expression of IL-4-. TGF-β1in the N, MAT-treated group are increased(p<0.05); compared to N group, the expression of IL-4、TGF-31in MAT-treated group are increased (p<0.01); the expression of IL-4、TGF-β1in MAT-treated group have significantly differences(p<0.05).Conclusion:MAT possesses obvious preventive treatment to EAE model in Wistar rats, which were induced by being immuned with spinal cord extracts to together with complete Freund adjuvant.the possible mechanism of action is related to inhibit the expression of ICAM-1、VCAM-1、CCL3、CCL5、TLR、MD-2in rats blood and spinal cord, up-regulating of Nrf2、HO-1、IL-4、TGF-β1、IL-5、IL-10expression,thus improve the clinical symptom of EAE rats.