Effect Of Berberine On SPHK1/S1P/S1PR1,3 Pathway Of Astrocytes In EAE Mice

BackgroundMultiplesclerosis?MS?isaninflammatorydemyelinating autoimmune disease of the central nervous system?CNS?.It often affects young and middle-aged people.MS has a chronic course of disease with multiple characteristics at different time and space and often causing severe neurological deficits.Major pathological changes of MS include demyelination,gliosis,axonal injury and neuronal necrosis.At present the etiology of MS is unclear and the treatment is limited without cure.To elucidate the pathogenesis and ascertain various therapeutic methods is a subject of intense concern.Animal model most commonly used in the study of MS is the experimental autoimmune encephalomyelitis?EAE?.Astrocytes play a key role in the pathogenesis of MS and EAE which upon activation lead to amplification and maintenance of inflammation in the central nervous system?CNS?.The degree of astrocyte proliferation?gliosis?is closely related to neurological dysfunctions and clinical manifestations.Activated astrocytes release various cytokines leading to inflammation,and T cell activation.In addition,activated astrocytes can damage the blood-brain barrier?BBB?and facilitate the infiltration of toxic T lymphocytes into the CNS.Sphingosine 1-phosphate?S1P?widely exists in many human cells and is an important second signal molecule produced from sphingosine via sphingosine kinase?SPHK?enzyme.Binding of S1P to its various receptors on astrocytes are considered an important event in the pathogenesis of MS among which S1P binding with S1PR1 and S1PR3receptors are central to astrocyte activation and pathogenesis of MS and EAE.Berberine?BBR?is a natural isoquinoline alkaloid extracted from traditional Chinese medicine Coptis chinensis having a wide range of biological activities.Even though specific mechanism of BBR is unclear,it can alleviate neurological deficit symptoms in EAE mice as shown by previous studies.Elucidating the therapeutic mechanism of BBR on EAE model can provide a theoretical basis for its clinical application.ObjectiveBased on the research background above and the discovery of the effects of BBR on the SPHK/S1P/S1PR1 pathway in renal cells,our team proposed a hypothesis:BBR inhibits glial proliferation and reduces inflammation and demyelination of the CNS by directly acting on the SPHK/S1P/S1PR1,3 pathway on astrocytes.In the present study,firstly,the changes of SPHK/S1PR1,3 pathway in astrocytes of CNS were studied in vivo animal model?EAE mice?which were then administered different doses of BBR to observe the effect of the drug on this pathway.Secondly,different doses of BBR were given after lipopolysaccharide?LPS?activation of mouse astrocytes in vitro to verify and explore the mechanism of BBR action,MethodsExperimental animal groups were divided as?i?CFA group{animal modelsinduced by saline instead of MOG_35-555-55 polypeptide fragments?n=12?},?ii?EAE group{animal models induced by MOG_35-555-55 polypeptide fragments,?n=12?}and?iii?BBR intervention groups{animal model induced by MOG_35-555-55 peptide fragments and treated with different doses of BBR?50 mg,100 mg and 300 mg/kg/d dose of BBR,n=12 in each group?}.BBR was administered by gastric perfusion on the tenth day after immunization with MOG_35-555-55 peptide fragments.Mice were observed for the activity,appetite and weight while neurological function was assessed by Neurological deficits score daily.Thirty days after immunization,six mice in each group were randomly selected for cardiac perfusion fixation with paraformaldehyde.The infiltration of inflammatory cells in brain and spinal cord was observed by HE staining,the demyelination in brain and spinal cord by LFB staining while glial hyperplasia by fluorescence immunoassay.The expression of SPHK1,SPHK2,S1PR1 and S1PR3 in astrocytes of brain and spinal cord of mice were detected by double immunofluorescenceimmunoassayinvivo.Thedetectionand measurement of levels of S1P,IL-1?,IL-6 brain and spinal cord of mice were detected by ELISA.Cell experiment:Brain tissue samples were taken from the fetal mice within 48 hours of birth under aseptic condition for primary astrocyte culture.Experimental groups were divided into given six groups?i?astrocytes?ii?astrocyte plus LPS activated(astrocyte plus 100 ng of LPS?iii?LPS activated astrocytes with different doses of BBR intervention{incubated with different doses of BBR(1,3,10u1 after activation by LPS}and?iv?astrocytes without activation by LPS plus BBR?10u1?groups.The expression of SPHK1,SPHK2,S1PR1 and S1PR3 in the astrocytes of each group were detected by western blot and the levels of S1P,IL-1?,IL-6 were tested by ELISA.Results1.CFA mice did not show any clinical manifestations of disease while EAE group showed significant clinical features such as decreased appetite,decreased activity and weight loss.On the 30th day of intervention,the average body weight of EAE group was lower than that of CFA group?P<0.01?.BBR-treated groups with 100 mg/kg/d and 300mg/kg/d showed significant increase in bodyweight as compared to EAE group?P<0.01?.EAE group showed progressive deterioration after the onset of the disease as evident by increase in the neurological function score.The highest neurological function score and cumulative neurological function score in the 100 mg/kg/day and 300 mg/km?is it km or kg?/d BBR-treated group were significantly lower when compared to the EAE group.?P<0.01?;2.The infiltration of inflammatory cells in brain and spinal cord sections of mice:Inflammatory cells infiltration in brain and spinal cord in CFA group were few whereas it was significantly higher in the EAE mice.On the other hand,the inflammatory cells infiltration in the 100,300mg/kg/d BBR-treated groups were significantly lower than that of the EAE group.?P<0.01?.3.Demyelination as observed by LFB staining:No significant demyelinating changes were observed in brain and spinal cord in CFA group.EAE group showed apparent,demyelination,sparse spacing and vacuoles.The 50 mg/kg/d BBR intervention group also showed more demyelination,which was slightly lesser than that of EAE group and the difference was not significant?P>0.05?.On the other hand,100 mg/kg/d and 300 mg/kg/d BBR intervention group showed significantly less demyelination?P<0.01?.4.Gliosis in brain and spinal cord:The expression of GFAP?glial fibrillary acidic protein?in brain and spinal cord astrocytes was significantly higher in EAE group than that of CFA group?P<0.01?whereas the expression of GFAP was significantly lower in different doses of berberine treated group than that of EAE group?P<0.01?.There was a decreasing tendency of expression of GFAP in each dose intervention group?P<0.01?,which suggested that berberine inhibited glial proliferation in a dose-dependent manner.5.Expression of SPHK1 and SPHK2 in astrocytes of brain and spinal cord:The expression of SPHK1 in astrocytes of brain and spinal cord of mice in the EAE group was significantly increased when compared with CFA group?P<0.01?.The expression of SPHK1 in different doses of BBR treated group was significantly lower than that of EAE group?P<0.01?.However,the decrease in SPHK1 expression was evident only among 100mg/kg/d and 300 mg/kg/d BBR treated group?P<0.01?.There was no significant difference in the expression of SPHK2 on astrocytes in brain and spinal cord of each group?P>0.05?.6.Expression of S1PR1 and S1PR3 receptors on astrocytes in brain and spinal cord:The expression of S1PR1 and S1PR3 receptors on astrocytes in brain tissue and spinal cord of mice in EAE group was higher than that in CFA group?P<0.01?whereas the expression of S1PR1 and S1PR3 receptors among the mice treated with different dose of BBR was significantly lower than that in the EAE group?P<0.01?.There was significant difference in the expression of SPR1 and SPR3 in spinal cord among the intervention groups of BBR which shows that there is dose-dependent characteristics.7.Levels of S1P,IL-1?and IL-6 in brain and spinal cord as measured by ELISA assay:The level of S1P,IL-1?and IL-6 in brain and spinal cord in EAE group were significantly higher than those in CFA group?P<0.01?.The level of S1P in 100 mg/kg/d and 300 mg/kg/d BBR-treated groups were lower than those in EAE group?P<0.01?.Similarly,the level of IL-1?and IL-6 in brain and spinal cord of mice treated with different doses BBR were lower than those in EAE group?P<0.01?.8.Expression of SPHK1 and SPHK2 in each primary astrocytes groups:The expression of SPHK1 in activated primary astrocytes was significantly higher than that in non-activated astrocytes?P<0.01?.After the intervention with BBR,the expression of SPHK1 in activated astrocytes decreased in different dose groups?P<0.01?.The expression of SPHK1 in the cells treated with BBR at the dose of 10?l was lower than that in the 1?l group?P<0.01?.There was no significant difference in SPHK1expression between 3?l and 10?l groups of berberine?P>0.05?.No significant difference in the expression of SPHK2 was found between each groups?P>0.05?.9.The expression of S1PR1 and S1PR3 in primary astrocytes of each group:The expression of S1PR1 and S1PR3 in LPS-activated primary astrocytes was significantly higher when compared with non-activated astrocytes?P<0.01?.The expression of S1PR1 and S1PR3 in the cells of BBR intervention of 3,10?l dosage were lower than those in the 1 ul dose group?P<0.01?.The expression of S1PR1 and S1PR3 in non-activated astrocytes treated with BBR were lower than those in all groups after LPS activation with or without BBR intervention?P<0.01?.There was no significant difference in the expression of S1PR1 and S1PR3 between 1?l BBR-treated astrocytes and LPS activated astrocytes group?P>0.05?.10.S1P,IL-1?and IL-6 levels in primary astrocytes in each group tested by ELISA:The level of S1P,IL-1?and IL-6 of primary astrocytes were significantly increased than compared to those in activated group?P<0.01?.S1P,IL-1?and IL-6 levels of astrocytes treated with different doses of BBR were lower than those of non-intervention group?P<0.01?.The level of IL-1?decreased in a dose-dependent manner among the intervention groups.The level of S1P,IL-1?and IL-6 in the non-activated astrocytes treated with BBR were lower than those in the activated groups treated with or without BBR?P<0.01?.Conclusion1.Berberine can improve the clinical manifestations and decrease clinical neurological function score of EAE mice.2.Berberine inhibits the activation of astrocytes and reduces the degree of gliosis in brain and spinal cord of EAE mice,which correlates with clinical manifestations and neurological function score.3.Berberine inhibits the SPHK1/S1P/S1PR1,3 pathway of astrocytes in brain and spinal cord of EAE mice,which was also confirmed in vitro in our study,suggesting that berberine could inhibit astrocyte activation and glial proliferation.4.The effect of berberine on the SPHK1/S1P/S1PR1,3 pathway in astrocytes is expected to provide clinical thinking and therapeutic strategies for the study of MS.