Immunoregulatory Effect Of Buyang Huanwu Decoction On Mononuclear Macrophages In Mice With Experimental Autoimmune Encephalomyelitis

Objective:In this study,we used Buyang Huanwu decoction?BYHWD?to treat the female C57BL/6 mice with experimental autoimmune encephalomyelitis?EAE?.The EAE mice were induced by myelin oligodendrocyte glycoprotein peptides 35-55(MOG _35-55).The clinical symptoms,pathological changes and the influence of BYHWD on the macrophages of central nervous system?CNS?and peripheral immune system were observed in the control group and the treatment group.Our study provides the evidence that BYHWD has the role of immune regulation of mononuclear macrophages in EAE.Methods:The mice which immunized with MOG35-55 were randomly divided into EAE control group and BYHWD treatment group,each group had a total of 21 mice.On day 3post immunization?p.i?,the mice were orally administrated with BYHWD,while normal saline was given to the control mice.The experiment lasted for only 500?l/d?with the BYHWD raw material 1g?.The clinical score and body mass were recorded every other day.On day 17 p.i,9 mice in each group sacrificed and the spleen were isolated.Five mice were used to prepare the spleen mononuclear cell suspension.M1 and M2 mononuclear macrophage phenotypes were detected by flow cytometry?FCM?.The spleens from four mice were made into frozen sections and stained with immunohistochemistry to detect the expression of iNOS and Arginase-1.Frozen sections of the spinal cord were stained by HE staining and myelin staining to observe the infiltration of inflammatory cells,and demyelination ratio expression of the spinal cord.The protein expression of i NOS,TNF-?,arginase and IL-10 of the macrophages in the spinal cord was determined by immunofluorescence staining and Western blotting.The experimental results were analyzed by the software Graphpad Prism 6.Results:1.The EAE model of C57BL/6 mice induced by MOG_35-555-55 antigen emulsion was successfully established.Compared with the average onset time of EAE control group,the onset time of BYHWD group was significantly delayed,and clinical symptom score decreased significantly.At the same time,along with the severity of clinical symptom score,BYHWD treatment can significantly inhibit the weight loss of EAE mice?p<0.01?.2.BYHWD can relieve spinal cord demyelination in EAE?p<0.05?.3.HE staining and immunofluorescence histochemical staining showed that EAE had different degrees of inflammatory cell infiltration in spinal cord.BYHWD can reduce the infiltration of CD68⁺mononuclear macrophages and inflammatory cells in CNS?p<0.05?.4.BYHWD can significantly inhibit the expression of iNOS,TNF-?,CD16/32,while promoting the expression of Arg-1,IL-10,CD206?p<0.05?in spinal cord and spleen.Conclusion:1.BYHWD has a good therapeutic effect on EAE,can delay the onset of EAE,relieve the clinical symptoms of EAE?that is traditional Chinese medical science"atrophy disease"?mice such as listlessness,loss of appetite,weak limbs atrophy or even paralysis.2.BYHWD can reduce demyelination,inhibit inflammatory cell infiltration and promote remyelination of EAE mice.3.BYHWD promotes the transformation of M1 type macrophages into M2 type in the spinal cord and spleen.BYHWD has therapeutic potential in EAE possibly through inducing the inhibition of inflammatory responses and the transformation of M1 type macrophages into type M2.