A Study On The Mechanism Of Immunoregulation By Combination Of Catalpol And Rhein In Mice With Experimental Autoimmune Encephalomyelitis

ObjetiveThe mechanisms of immunoregulation by catalpol and rhein in experimental autoimmune encephalomyelitis(EAE) were observed in the study. HE, ELISA, q RT-PCR, Western Blot were carried out to test levels of cytokines and transcription factors expression. The study was to unveil mechanisms of catalpol and rhein on the therapeutic immoregulation in EAE mice. The study provides scientific basis for traditional Chinese medicine treatment for MS. MethodsExperiment 1: C57BL/6 mice that was SPF and female were randomly divided into five groups: normal group, model group, catalpol and rhein high-dose group, catalpol and rhein middle-dose group, catalpol and rhein low-dose group. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to establish EAE. The body weight and neurological score of mice were observed in this study, which is used to determine the optimal therapeutic dose of rhein.Experiment 2: we can know the treatment of catalpol and rhein low-dose is best from the first experiment. Then submitting the above method to establish EAE model again, we design four groups: normal group, model group, PA group and catalpol and rhein low-dose group. On day 6, 20 and 40, the brain and spinal cord were taken from mice. HE for observing the pathological changes, q RT-PCR and Western Blot for testing gene and protein expression of T-bet, GATA3, ROR-γt and Foxp3, ELISA for detecting IL-2, IL-4, IL-10 and IL-17 A in cerebral and spinal cord.ResultsExperiment 1: Through the analysis of clinical symptoms, weight and neurological score of mice, we draw a concequence that catalpol and rhein low-dose has an optimization.Experiment 2: First, Hematoxylin and eosin stain observed by light microscopy showed that EAE model group had inflammatory cells surrounding the small blood vessels and nerve nucleus pyknotic on the day 20. Meanwhile, lymphocytes gathered in blood vessels and formed the typical vascular. On day 40, Inflammatory infiltration is relieved. Secondly, an obvious improvement of the expression of IL-4, IL-10 was found in catalpol compatibility rhein low-dose group. On the contrast, the levels of IL-2, IL-17 A were reduced. Finally, by detecting gene and protein, catalpol compatibility rhein play a considerable role on increasing the expression of GATA3, Foxp3, which is better than PA. However, the influence for ROR-γt and Foxp3 was negative. ConclusionCatalpol and rhein low-dose can improve the injury of neural function and alleviate inflammatory reaction in EAE mice. Their compatibility may exerts positive effect on Th2 cell and Treg cell differentiation, while may preventing Th1 cell and Th17 cell from differentiating. In addition, the combination of catalpol and rhein low-dose can also relieve the degree of inflammation by regulating the secretion of many inflammatory factors.