Study On The Effect Of Icariin In The Treatment Of EAE To CREB And Estrogen Related Tumor Inducing Factors And Its Mechanisms

BackgroundMultiple sclerosis(MS)is a kind of central nervous system(CNS)autoimmune disease white matter demyelination as the main pathological changes.Occur in young adults,more women than men,often involving the periventricular white matter,optic nerve,spinal cord,brain stem and cerebellum,clinically with visual impairment,limb weakness,paresthesia,ataxia,autonomic dysfunction and other peripheral nerve damage mainly.Due to the lack of specific therapeutic drugs,patients often suffer from varying degrees of disability and seriously affect the quality of life of patients.Because the etiology and pathogenesis of MS are not yet clear,there are no specific treatment methods,clinically,high-dose glucocorticoid shock therapy is still the mainstay.In recent years,the protective effect of estrogen on CNS inflammation has been widely concerned.Estrogen also has a protective effect on the pathogenesis of experimental autoimmune encephalomyelitis(EAE)in animal models of MS.However,estrogen can metabolize 4-hydroxyl estradiol(4-OHE2),and oxygen free radicals produced by 4-OHE2 during oxidative metabolism can bind to DNA,resulting in DNA damage,leading to the occurrence of cancer.In addition,estrogen can promote the expression of vascular endothelial growth factor(VEGF)through multiple signaling pathways,VEGF can promote angiogenesis,the growth of tumors requires a continuous supply of blood,which is highly expressed in tumors.Therefore,high-dose or long-term use of estrogen has the risk of inducing tumors,making its clinical application limited.The phytoestrogen(PE)is considered to be a good endogenous estrogen replacement with estrogenic effects and less adverse reactions,but whether phytoestrogen does not increase the incidence of cancer compared to estrogen the advantages of risk,determine the key of phytoestrogens to become endogenous estrogen replacement therapy drugs,and it is also the key to good application prospect of phytoestrogen.Previous studies confirmed that icariin(ICA),a major component of epimedium,which has the effect of estrogen,has a therapeutic effect on EAE.In this study,based on the previous studies,the termination of signaling pathways and the c AMP response element binding protein(CREB)as the starting point of the junction were taken as the starting point to investigate whether ICA regulates the core site of signaling pathway by CREB protein versus EAE,the model exerts a therapeutic effect,at the same time,from the perspective of estrogen-related tumor-induced estrogen metabolites 4-OHE2 and tumor-associated VEGF,it is possible to investigate the possible mechanism of ICA as compared with estrogen does not increase tumor incidence.ObjectiveIn the experiment,female C57BL/6 mice characterized by relapsing remission were used to construct an animal model of MS-EAE mouse model.After observing the pathogenesis of EAE mice,the effect of ICA treatment on the expression of CREB in the nerve tissue of EAE mice was verified,it was verified that ICA exerts its therapeutic effect on EAE by up-regulating the expression of core protein CREB protein in signal pathway.Analyze and compare the effects of ICA and estrogen therapy on the level of 4-OHE2 and VEGF,the estrogen metabolites in plasma,and the effect of VEGF protein expression in the uterus,and investigate whether phytoestrogen ICA may increase the risk associated with tumor pathogenesis,factors 4-OHE2 and VEGF levels in order to verify that phytoestrogen is superior to endogenous estrogens without increasing the safety of tumors.Methods Part one60 6-8 weeks old female C57BL/6 mice were selected at the SPF level,of which 51 were used to construct the EAE model and 9 were used as the normal control group.The myelin oligodendrocyte glycoprotein 35-55(MOG35-55)polypeptide was diluted with equal amount of complete freund's adjuvant(CFA)(inactivated tuberculosis the final concentration of bacillus was 5 mg/ml),the antigen emulsion was mixed well into a water-in-oil state and inoculated subcutaneously on both sides of the spine of anesthetized C57BL/6 female mice at a dose of 300 ?g per mouse MOG35-55 polypeptide(four subcutaneous injection sites,each site of 50 ?l),were boosted once more on the 7th day after the initial vaccination(equal doses of MOG35-55/CFA).500 ng of pertussis toxin was injected intraperitoneally at 0 and 48 h after immunization to induce EAE model mice.After the immunization,the mice were observed and recorded at the same time every day,and the mice were dynamically observed for neurological symptoms and their neurological impairment scores were recorded.At the peak of mouse onset,3 EAE mouse models with stable disease and 3 normal mice were randomly selected and subjected to anesthesia for perfusion of the heart,spinal cord lumbar enlargement was used for hematoxylin-eosin(HE)staining and luxol fast blue(LFB)myelin staining,the above stained sections were observed with spinal cord tissue using a fluorescence microscope,pathological changes determine whether the EAE model mice have established success.Part twoAccording to the principle of neurological impairment score,the EAE model mice were randomly assigned to 3 groups,respectively ICA group,estrogen group,EAE model group,and normal control group(blank control group),a total of 4 groups.ICA group and estrogen group were given ICA 300 mg/kg.d,diethylstilbestrol 0.2 mg/kg.d,model group and normal control group were given equal volume of 0.5% sodium carboxymethyl cellulose orally administered continuously for 5 days,the mice were anesthetized using mice with eye method were preserved in sterile EDTA blood in the blood after the mice were quickly taken down the uterus,spinal cord and brain white matter,into the labeled 1.5 ml sterile EP tube saved to-80?.The white matter,spinal cord and uterus were homogenized,proteins were extracted,and the expression of CREB protein in the white matter and spinal cord and the expression of VEGF protein in the uterus were detected by Western blot(WB);Mouse blood collection in 30 min at 4? high speed refrigerated centrifuge in 3000 r/min centrifugal 15 min,extraction supernatant after centrifugation to mark 1.5 ml sterile EP tube saved to-80?,enzyme-linked immunosorbent assay(ELISA)was used to detect 4-OHE2 and VEGF levels in plasma.Results Part oneThe mouse EAE model constructed by MOG35-55/CFA antigen emulsion,the onset of the disease in 13-14 days after immunization,3-5 days after the disease reached peak,the highest neurological score of 5 points,the initial onset of symptoms for atonic tail,and tail tension disappeared,and unilateral hind hind limbs weakness,paralysis,severe paralysis of limbs,and weight decreased mice in normal control group have no obvious abnormal.The HE staining of spinal cord in EAE model mice showed that a large number of lymphocytes infiltrated in the spinal cord,and the most peripheral vessels were "cuff like" inflammatory structural changes.LFB myelin staining biopsy showed spinal cord lumbar enlargement of spinal cord myelin leukoaraiosis and large patchy depigmentation,normal mice had no obvious pathological change.According to C57BL/6 mice showed neurological impairment symptoms,spinal cord lumbar enlargement HE staining and LFB myelin staining,are consistent with the typical EAE model of clinical and pathological features.Part two1.Changes in neurological function scores in mice: the neurological scores before and after treatment in the ICA group and the estrogen group were significantly improved(P<0.01),there was no significant difference between the ICA group and the estrogen group(P>0.05),the scores of neurological function after treatment in the ICA group and the estrogen group were significantly lower than those in the model control group(P<0.01).2.Changes in body weight in mice: body weights of mice before and after treatment in the ICA group and the estrogen group were significantly increased(P<0.01),there was no significant difference between the ICA group and the estrogen group(P>0.05),the body weight of the ICA group was higher than that of the model control group(P<0.05),and the body weight of the estrogen group was significantly higher than that of the model control group(P<0.01).3.Changes of CREB protein expression in mouse brain white matter: after EAE mice were treated,the expression of CREB protein in ICA group was significantly higher than that in model control group(P<0.01),and it was significantly higher than that in normal control group(P<0.01);the expression levels of CREB in the hormone group and the model control group were higher(P<0.05),and there was no significant difference compared with the normal control group.4.Changes of CREB protein expression in mouse spinal cord: after EAE mice were treated,the expression of CREB protein in spinal cord of ICA group was significantly higher than that in model control group(P<0.01),although the protein content of CREB in ICA group was higher than that in normal control group,the difference was not statistically significant(P>0.05);the expression of CREB protein in the estrogen group was higher than that in the model control group(P<0.05),and there was no significant difference compared with the normal control group(P>0.05).5.Changes of VEGF protein expression in mouse uterus: the expression of VEGF protein in uterus of EAE mice of ICA group was significantly lower than that of estrogen group(P<0.05),the VEGF protein content in ICA group was lower than that in model control group and normal control group,but the difference was no statistical significance(P>0.05);the expression of VEGF protein in the estrogen group was higher than that in the model control group and the normal control group,but the difference was not statistically significant(P>0.05).6.Changes in plasma 4-OHE2 levels: after treatment with EAE mice,plasma 4-OHE2 levels in the ICA group were significantly higher than those in the model control group(P<0.01);plasma 4-OHE2 levels in the EAE mice of the estrogen group were higher than those in the model control group.The normal control group was significantly elevated(P<0.01);compared with the estrogen group in the ICA group,the 4-OHE2 level in the ICA group was decreased,but the difference was not statistically significant(P>0.05).7.Changes in plasma VEGF levels: plasma VEGF levels in EAE mice of the ICA group were significantly lower than those in the estrogen group(P<0.01),and were not significantly different from the model control group and the normal control group(P>0.05);plasma VEGF in the EAE mice of the estrogen group the level was significantly higher than the model control group and the normal control group(P<0.01).Conclusions1.ICA has a therapeutic effect on EAE,which may exert its neuroprotective,immunoregulatory,and anti-inflammatory effects on EAE by up-regulating the expression of core signaling factor CREB protein,thereby improving neurological impairment in model rats.2.After ICA treatment of EAE mice,plasma 4-OHE2 was slightly lower than estrogen,and VEGF expression and plasma VEGF levels in the reproductive organs were not increased.Therefore,ICA is superior to endogenous estrogens without increasing the safety of tumors.