Therapeutic Effects Of Icariin On EAE And Its Influence On The HPA Axis

Background: Multiple sclerosis (experimental allergic encephalomyelitis, MS) isa common demyelinating disease of the central nervous system white matter. Its symptomstake on the complex features which include recurrent seizures, progressive increase inmostly repeated episodes and high disability. High-dose methylprednisolone (the methylprednisolone MP) have significant effects on relieving symptoms, but there is no generallyaccepted drug treatment can prevent the recurrence of progress, and high-doseglucocorticoids (glucocorticoid, GC) can lead to bone osteoporosis, osteonecrosis, uppergastrointestinal bleeding and other serious adverse reactions. Domestic and foreign researchconfirmed that the MS is more prevalent in women, and recurrence progress is closelyrelated to estrogen levels, although supplementary estrogen is good for the control ofrelapse, and the GC has a positive regulatory role, the risk of the presence of tumorinitiation, lower immunity and thrombotic diseases still exists. Therefore, this study selectsestrogen and icariin (icariin ICA) which have the verified role of estrogen therapy bytaking estrogen as the starting point, and discusses the differences in drug efficacy, dose-dependency as well as the relationship between efficacy and hypothalamus-pituitary-adrenal (pitaitary the hypothalamic-adrenal HPA) axis with the help of expressions of EAEmodel nerve damage, pathology, cytokines, hormone levels and related receptor protein. Atlast, further explores the estrogen pathway-based Longspur hopane glycosides intervention in EAE mechanism.Objective:The experimental autoimmune encephalomyelitis (experimental allergicencephalomyelitis, EAE),which is based on the MS animal model,analyzes ICAtherapeutic effect on EAE and the relationship between them which is based on the EAEmice treatment by estrogen, estrogen receptor inhibitors and different doses of estrogen-likeeffect of icariin from different aspects of clinical score, the immune mechanisms cytokinesand histopathological changes perspectively.This paper examines the effect brought by different doses of ICA to the expressionlevel of HPA axis hormones by multidimensional analysis of peripheral blood, the targetorgan, central molecular level and protein levelsICA estrogen-like effects are researched by the detection of different doses of ICA roleof endogenous estrogen levels. Moreover, this paper also studies the ICA treatment on EAEby comparing ER antagonist group with the other group, and explores the role of thistreatment and the correlation of the activation of the HPA axis, which development of newdrugs and provides new ideas for Integrative Medicine MS.Methods:1.Myelin oligodendrocyte glycoprotein peptide (myelin oligodendrocyteglycoprotein, MOG35-55) was diluted into600ug/ml, mixed the diluted solution with anequal volume of complete freund’s adjuvant(final concentration of extinguishingMycobacterium tuberculosis5mg/ml) as an antigen, and then inoculated in C57BL/6micewith bilateral lateral abdominal wall and the spine on both sides subcutaneous (four points,each part of50ul), strengthened immunization at the7d after immunization (dose MOG/CFA), carried out the injection of500ng pertussis toxin at the0D after immunization andthe48h intraperitoneal and the model of mice induction EAE.2. The EAE attacked mice were randomly divided into eight groups,which relied onthe clinical nerve injury, at least six for one group. which acute group were divided intogroup A, B for the model control group, make it the natural course of development, C is theestrogen group diethylstilbestrol0.2mg/kg.d of gavage; the ICA big D for the onset of thepeak period put to death; dose group, gavage ICA300mg/kg.d; the E is ICA dose group, gavage be ICA150mg/kg.d; the F group is called ICA low-dose group, given50mg/kg.dorally; G is ICA ICI182,780groups, given ICA150mg/kg.d gavage plus ICI182,subcutaneous injection, M is blank control group, give a mouse model and capacitycarboxymethyl cellulose suspension gavage; N for the normal control group, of apacitycarboxymethyl cellulose suspension orally. Immunization began nine days after the onset of14days to reach the peak incidence administration, administered for5consecutive days,weighed daily at the day before immunization, and observed the mouse nervous systemsymptoms and rated according to the score of the packet, administered in the ninth day ofthe immunization. About14days, the acute group were sacrificed, each group weresacrificed at19days, about40days the model control group (group B) were sacrificed,blood,brain, spinal cord tissue samples were collected.3. Demyelinated the Mice spinal cord lumbar enlargement HE staining, myelin stainedwith Luxol Fast Blue（LFB）, and sliced the demyelination score;3’end of DNA transferase(TdT)-mediated dUTP nick end labeling (Terminaldeoxynucleotidyl teansferac-mediatedOridine3’-Tri-phosphatebiotinNick End Labeling, TUNEL) apoptosis was observed spinalcord tissue; the other half of the brain tissue extracted protein by Western blot (Western-blot,WB) detection of brain tissue glucocorticoidthe expression of the hormone receptor(glucocorticoid receptor, GR).4.Collect blood and measure the serum Interleukin-17A (interleukin-17A, IL-17A)content by enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay,ELISA). And the serum e corticosterone (corticosterone, CORT) content was measured byradioimmunoassay.5.Give a clinical symptom score to each group of drug treatment of mice and themodel group, weigh the control group mice and count inflammatory lesions apoptotic cell,cytokine content, the hormone content analysis to assess the efficacy of different doses ofICA with estrogen on EAE, as well as the influence of ER antagonists for the efficacy ofICA on EAE. And analyze the mice in each group efficacy and GR expression correlation.Results:1.Established EAE model mice showed symptoms of neurologicalimpairment, HE staining, myelin staining at the lumbar enlargement of the spinal cord, which are in accordance with the pathological and clinical characteristics of the typicalEAE model, there is relief–recurrence phenomenon in model control group (B group);2.The body weight after treatment was higher than before treatment in estrogen group(C group), ICA high-dose group (D group), the ICA middle dose group (E group), but itwas not statistically significant. There were significant differences in the low-dose group (Fgroup)(P <0.01); The body weight was lower than before treatment in the G group, but p>0.05. The Clinical symptoms score showed that group B, C, D, E, each group hasdeclined, the most remarkable decrease in the C group and D group amongthem.,meanwhile,the means seems no difference in group F.However,there were significantdifferences in means of score change in the C group and F group,(P <0.01). The score hasincreased in the t group M and group G.3.It was observed some phenomenon from the picture of spinal cord cell-apoptosis,the number of neuronal apoptosis order as follows: the model control group (B)> ICAlow-dose group (F group)> ICA high-dose group (D group)> the normal control group (Ngroup), however, owing to little sample, it has no statistics.4.The level of IL-17A, has increased in each model group compared with the normalgroup, it was remarkably increased in group A, B, G,P＜0.05; while Comparing with groupA,B, G, it was lower group C and group D, and it has reduced greatly in the group C, therewere significant differences between them(P＜0.05);It has deceased in the group C whilecomparing with group D, but there was no significantly difference between them. Howeverthe level of IL-17A was negatively correlated (r=-0.8581, P=0.0288) between thepretherapy and post-treatment5.The CORT level varies greatly between the C group and D group, which has thesame relief situation in the clinical symptoms. Compared with the normal control group, ithas increased in the C group and decreased in the D group, and comparing the C group andD group, P <0.0001.There was positively correlated between the level ofIL-17A and CORTin group D.(r=0.812, P=0.0496)6.In contrast with the normal control group, GR protein expression level in the braintissue has increased in the A, B,C,D,E and M group, B, C, D, M group increased significantly, P＜0.01respectively. In contrast with group M (blank control group), themeans of the rest groups have increased except group G. But there was statisticallysignificant difference only in the group C (P <0.05). Cpmpared with E,the group Ddecressde little,but there was no statistically significant.The above statistics are based onbilateral P <0.05, which was considered statistically significant.Conclusions:1.ICA can give a certain therapeutic effect in a certain range of dose on EAE mousemodel. ER antagonism agent can reduce this function of ICA.2.High-dose of ICA reduced the level of IL-17, indicates that large doses of ICA hasobvious anti-inflammatory effects.3.ICA can increase the expression of GR in the brain tissue, but the level of CORT arelow on EAE, which suggests that it may promote the function of the HPA axis,;prompt thatunite a certain dose of ICA and glucocorticoid may get a synergy in the clinicalapplication.4.The possible mechanism of the treatment of ICA is combined with ER, its quasiestrogen-like effect directly exert anti-inflammatory; Secondly, activating the downstreamHPA axis initiation factor, improving the HPA axis function, enhancing the body’santi-inflammatory and immunosuppressive function.