The Study On The Role And Mechanism Of Ganoderic Acid A Via Activation Of FXR Receptor To Ameliorate Central Demyelination Disease

Background and ObjectiveDemyelination in central nervous system is a symptom for neurodegenerative disease with uncertain etiology and significant pathological features.There is no drug to cure this disease clinically.The development of drugs for the treatment of central demyelinating diseases by means of promoting remyelination will bring hope for the complete cure of these diseases.Previous studies showed that the farnesoid X receptor(FXR)play a protective role in autoimmune mediated inflammatory demyelination disease.In this study,we chose the FXR as a target and ganoderic acid A(GAA)as an activator of FXR,the possible effects and mechanisms of GAA to regulate neuroimmune and promote remyelination were studied systematically at the cellular and animal levels,providing a potential target and candidate compound for clinical treatment of central demyelination disease.MethodsThe study on LPS-induced neuroinflammatory model of BV2 microglia:The Lipopolysaccharide(LPS)was used to stimulate BV2 microglia cells to induce inflammatory response.The CCK-8 method was used to determine the effect of GAA on LPS induced proliferation of BV2 microglia.The cyto-immunofluorescence staining and Western blotting were used to evaluate the effect of GAA on LPS induced neuro-inflammatory model of BV2 microglia,including Iba1,i NOS,Arg-1,TNF-?,IL-1?,IL-6,BDNF and FXR.Z-guglesterone(GS),an antagonist of FXR receptor,was selected to block FXR.In LPS stimulated BV2 microglia cells,the effects of GAA on the expression of TNF-?and BDNF were evaluated after FXR receptor was blocked.The study on CPZ-induced central demyelination model mice and MOG35-55 induced EAE model mice.First,the effect of GAA on the central demyelinating disease model mice induced by cuprizone(CPZ)was evaluated.The rotarod test was used to evaluate the locomotor coordination of mice.LFB staining and MBP staining were used to evaluate changes of expression in myelin.Iba1,GFAP,Olig2 were used as makers for immunofluorescence staining to evaluate the expression of microglia,astrocytes and oligodendrocytes in callosum corpus.Western blotting analysis was applied to evaluate the expression of IL-6 and FXR in the mice.The potential mechanism of GAA was further investigated on demyelinating mice after using FXR receptor antagonist GS and FXR gene knockout mice to knock-down FXR receptor expression.Secondly,the effect of GAA on MOG_35-55 induced EAE model mice was evaluated.The rotarod test was used to evaluate the locomotor coordination of mice.The spleen morphology and HE staining were used to evaluate the autoimmune response and inflammatory cell infiltration.LFB staining and MBP staining were used to evaluate changes of expression in myelin.Iba1 and GFAP were used as makers for immunofluorescence staining to evaluate the expression of microglia and astrocytes in white matter of spinal cord.Results:The study on the neuro-inflammation model of BV2 microglial cells induced by LPS:(1)GAA significantly inhibited LPS-induced proliferation of BV2microglia cells,which is revealed by the CCK-8 method.(2)The immunofluorescence staining and western blotting analysis showed that GAA significantly inhibited the increase of expression of Iba1,i NOS,IL-6,IL-1?and TNF-?stimulated by LPS in BV2 microglia cells.In contrast,GAA prominently promoted the expression of FXR,Arg-1 and BDNF in LPS-stimulated BV2microglia cells.(3)The western blotting analysis showed that the regulatory effect of GAA on TNF-?and BDNF expression in LPS-stimulated BV2 microglial cells was significantly inhibited after FXR was blocked with GS.The study on mouse model of CPZ-induced demyelination:(1)After C57BL/6 mice were fed with standard rodent chow containing 0.2%CPZ for 5weeks,we successfully established the central demyelination mouse model.Pathological manifestations mainly included:body weight loss,motor dysfunction,myelin loss,the number of Iba1⁺,GFAP⁺and Olig2⁺positive cells were significantly increased in the callosum corpus;(2)The continuous treatment with GAA for 7 days significantly improved the decreased locomotor coordination of CPZ model mice by rotarod test.The LFB and MBP staining results showed that GAA significantly inhibited myelin loss of model mice.The immunofluorescence staining showed that GAA prominently inhibited the increase of Iba1⁺,GFAP⁺and Olig2⁺positive cells in the callosum corpus of model mice.The western blotting analysis showed that GAA significantly inhibited the upregulation of IL-6 expression and downregulation of FXR expression in CPZ-exposed mice.(3)After FXR receptor was blocked with GS or down-regulated in FXR deficiency mice,the beneficial effect of GAA on the central demyelination disease model mice induced by CPZ was significantly inhibited,which was evaluated by rotarod test,immunofluorescence staining,and western blotting etc.Research of the EAA effect on MOG35-55 induced EAE animal model:The clinical score observation and rotarod test showed that GAA significantly decreased the clinical score and improved the motor function loss of EAE model mice.The spleen morphology and HE staining showed that GAA significantly inhibited the splenomegaly and inflammatory cell infiltration in EAE model mice.The LFB and MBP staining results showed that GAA promoted the expression of myelin content in the spinal cord white matter of EAE model mice.The immunofluorescence staining showed that GAA inhibited the increase of Iba1⁺and GFAP⁺positive cells in the spinal cord white matter of EAE model mice.ConclusionThe present study showed that:GAA significantly improved the central demyelinating disease by activating FXR,inhibiting the occurrence of neuroinflammation,improving motor function and promoting remyelination,providing a novel potential candidate for the treatment of central demyelination-associated disease.