The Neuroprotective Effect And Mechanism Of Icariin On CPZ-induced Central Nervous System Demyelinating Mice

Central nervous system（CNS）demyelinating disease is a kind of autoimmune system disease mainly characterized by multifocal inflammatory demyelination of the CNS,which includes multiple sclerosis（MS）,neuromyelitis optica（NMO）,acute disseminated encephalomyelitis（ADEM）,etc.The most common demyelinating disease is MS,an inflammatory demyelinating and neurodegenerative disease of the CNS triggered by autoimmune mechanisms,in which the characteristic pathological change is the shedding of multiple myelin debris in the white matter of the CNS.At present,the etiology and pathogenesis of such demyelinating diseases,including MS,have not yet been fully clarified,and hence,they are difficult to cure,easy to relapse,and there are serious side effects of the drugs during the treatment.However,it is found that the onset of MS is related to the accumulation of myelin debris,and the oxidative stress in the brain caused by myelin debris is related to the neuroinflammatory microenvironment,which interferes with the regeneration and repair of myelin.Therefore,improving the local oxidative and inflammatory environment and removing myelin debris is essential for the treatment of MS.Microglia play an important role in improving the microenvironment of the brain and removing myelin debris,which is a key step to allow remyelination.The encephalopathy in Traditional Chinese medicine refers to a large group of diseases in which various pathogenic factors directly or indirectly act on the brain and spinal cord to cause dysfunction or abnormality.The clinical manifestations are complex and changeable.Studies have showed that patients with demyelinating diseases often have symptoms such as headache,dizziness,upset,forgetfulness,lack of breath,weak waist and knees,and depression.The onset of the diseases is related to many factors such as exogenous,internal injury,trauma,congenital,poisoning and so on.In terms of Chinese medicine,deficiency of qi and blood,deficiency of liver and kidney yin and deficiency of spleen and kidney are more common.In summary,the disease is closely related to the liver,spleen,and kidneys based on internal deficiency of qi and blood,therefore to yield visceral dysfunction,and internal injuries and external feelings,and excessive fatigue.Traditional Chinese medicine（TCM）plays a certain role in CNS demyelinating diseases such as MS by improving patients’s overall conditioning,regulating yin and yang,as well as having multiple targets and all-round advantages.Epimedium,the whole plant of the epimedium of the Berberis family,is a tonic medicine in the TCM.Icariin（ICA）is its main active ingredient,belongs to8-prenylflavonol glycosides in structure and is one of the hot TCM monomers studied at home and abroad,molecular formula of which is C₃₃H₄₀O₁₅and relative molecular weight is 676.67.In recent years,there has been increasing research on the mechanism of ICA on neuroprotection.It is showed that ICA inhibits oxidant stress and inflammatory responses,affects the release of monoamine and amino acid neurotransmitters,and increases neurotrophic effects.A good preventive effect on CNS diseases such as Alzheimer’s disease（AD）,depression and stroke has been showed.Therefore,in this study,firstly,we used DPPH,ABST and FRAP in vitro free radical scavenging methods to test the ability of ICA to scavenging free radicals.Secondly,we used lipopolysaccharide（LPS）in vitro to induce microglia/macrophages and cuprizone in vivo to induce demyelination to prove that ICA promotes the repair and regeneration of myelin by inhibiting the oxidant and inflammatory responeses in microglia/macrophages.Finally,we examined whecher ICA may promote microglia/macrophage phagocytosis of myelin debris and improve local oxidation and inflammation in both in vivo and in vitro experiments,and explore possible cellular and molecular mechanisms of action.Part â… .Cell-independent free radical scavenging capacity of ICAObjective:The free radical scavenging ability of ICA was detected with DPPH,ABST and FRAP in vitro.Methods:All samples were tested for scavenging free radicals with DPPH,ABTS and FRAP with slight modifications according to the reported method.In a 96-well plate,270μl of DPPH,ABTS and FRAP solutions were added.30μl of ICA solutions of different concentrations（1.56,3.12,6.25,12,25,50,100μg/m L）were incubated in the darkness,and absolute ethanol was used as blank control.After 30 minutes,the color was detected in a multifunctional microplate reader,and each sample was subjected to 3 independent repeated experiments.Results:The free radical scavenging activity of ICA in DPPH varied from 14.763%to 81.086%,and the IC₅₀was 17.98μg/ml.The free radical scavenging activity in FRAP was 15.477%to 75.783%,and the IC50 of FRAP was 20.7μg/ml.The free radical scavenging activity in ABTS was from 13.435%to 61.863%,and the IC50 is36.5μg/ml.Conclusion:ICA has a strong ability to remove DPPH,ABTS and FRAP,and is positively related to its concentration within a certain range,that is,as the concentration increases,the antioxidant capacity increases,which shows that ICA has the ability to scavenge free radicals,demonstrating the effect of antioxidants.Part 2 ICA promoted myelin regeneration by regulating the dynamic balance between antioxidation and inflammationObjective:In this experiment,we observed the therapeutic effect of ICA on the C57BL/6 mouse model of central demyelination induced by dicyclohexanone oxalyl dihydrazone（Cuprizone,CPZ）,and explored the possible mechanism of action.Methods:This study includes two parts of in vivo and in vitro experiments.（1）In vitro experiment.ICA of different concentrations（1.5625,3.125,6.25,12.5,25,50,100μg/m L）was added to cultured BV2 cells for 24h,and cell proliferation was detected by MTT.BV2,primary microglia and BMDMs were seeded on a 6-well plate at a density of 1×10⁶,and after adherence,LPS or LPS+ICA were added,and incubated at 37°C for 24 hours to establish a microglia/macrophage oxidation and inflammation model.The content of oxidative stress products NO,MDA and H₂O₂and antioxidant enzymes SOD,CAT and GSH-Px was detected by kit,and TNF-α,IL-1β,IL-10 and TGF-βwere detected by ELISA.（2）In vivo experiment.C57BL/6 mice were randomly divided into Normal group,CPZ group,and CPZ+ICA group.Control group was fed with normal feed for 6 week,CPZ group and CPZ+ICA group were fed with 0.2%CPZ feed for 6 weeks to induce demyelination.At the end of the week,CPZ mice were intraperitoneally injected with NS for two weeks,while the CPZ+ICA mice were intraperitoneally injected with ICA（50 mg/kg）for two weeks.After 6 weeks of modeling,the mice were tested for behavior;then the animals were collected and the slices were stained with black gold（Black GoldⅡ）and MBP immunopathological staining to detect the degree of demyelination.NO,MDA,H₂O₂,SOD,CAT,GSH-Px,TNF-α,IL-1β,IL-10 and TGF-βin brain proteins were detected by a kit and ELISA.The expressions of Iba-1,i NOS,Arg-1,TLR4,NF-κB,Nrf2,HO-1,PDGF-Rα,Ki67 and CNPase in brain tissue were detected by immunofluorescence staining.Results:1.Screening the optimal concentration of ICA in in vitro experiments.MTT results showed that there were statistical differences between the Control group and the 12.5μg/m L ICA group,the Control group and the 25μg/m L ICA group（p<0.05）,Therefore,we chose 15μg/m L for subsequent in vitro experiments.2.LPS interferes with BV2 cells,primary microglia and BMDMs cells to induce oxidative stress and neuroinflammation.It was found that:（1）For oxidative stress,the productions of NO and MDA of BV2 cells were significantly reduced in the LPS+ICA group（p<0.05 and p<0.01,respectively）,but the levels of antioxidant enzymes SOD,CAT,and GSH-Px were increased（p<0.05,p<0.01 and p<0.001,respectively）.The productions of NO and H₂O₂of primary microglia were significantly reduced in LPS+ICA group（all p<0.05）,but the levels of antioxidant enzymes SOD,CAT and GSH-Px increased（p<0.01,p<0.01,and p<0.001,respectively）.The productions of NO and H₂O₂of BMDMs were also significantly reduced in LPS+ICA group（p<0.05and p<0.01,respectively）,but the levels of antioxidant enzymes SOD and CAT were increased（both p<0.05）.（2）For neuroinflammation:ELISA showed that TNF-αfrom microglia/macrophages was increased significantly in the LPS group（p<0.01,p<0.01,p<0.001,respectively）,and the content of IL-1βalso increased to varying degrees（p<0.05,P<0.001,p<0.001,respectively）,while the LPS+ICA group decreased the content of TNF-α（p<0.05,P<0.05,p<0.01,respectively）and IL-1β（p<0.05,P<0.01,p<0.01,respectively）;the primary microglia and BMDMs of the LPS+ICA group had a significant increase in IL-10 content（both p<0.05）.3.ICA ameliorated the abnormal behavior and demyelination of CPZ-induced demyelinating mice.Forced swimming and elevated plus maze test showed that the total rest time to enter the closed arm and the time to reach the platform were significantly reduced in CPZ+ICA group（p<0.001,p<0.05,respectively）.Black gold II staining showed that compared with the CPZ group,the degree of demyelination of the corpus callosum was reduced in CPZ+ICA group（p<0.01）.Immunohistochemical MBP staining also showed that the intensity of myelin MBP staining was increased in CPZ+ICA group（p<0.01）.4.ICA inhibits the oxidative stress and neuroinflammatory response of CPZ demyelinating mice.Consistent with in vitro experiments,compared with CPZ-fed mice,ICA treatment effectively inhibited the levels of oxidation products NO,H₂O₂and MDA in the brain of demyelinated mice（p<0.05,p<0.01,p<0.05,respectively）,inhibited the inflammatory cytokine TNF-αand IL-1β（both p<0.01）,increased the content of SOD,CAT and GSH-Px antioxidant products（p<0.001,p<0.05,p<0.05,respectively）,and promoted the anti-inflammatory cytokines IL-10 and TGF-β（both p<0.05）.5.ICA inhibited the activation and enrichment of microglia.Immunofluorescence staining of Iba-1 showed that,compared with CPZ group,the number of Iba-1⁺microglia in the corpus callosum was effectively reduced in CPZ+ICA group（p<0.01）.6.ICA induced the polarization of the M2 phenotype of microglia in vivo.Immunofluorescence staining showed that compared with CPZ group,the expression of i NOS in the corpus callosum region was reduced（p<0.01）and the expression of Arg-1 was increased in CPZ+ICA group（p<0.001）.7.ICA activated Nrf2/HO-1 antioxidant pathways.Immunofluorescence results showed that,compared with CPZ group,the expressions of Nrf2 and HO-1 were significantly increased in CPZ+ICA group（p<0.001,p<0.01,respectively）.8.ICA inhibited the expressions of TLR4 and P-NF-k B/p65 in microglia.Immunofluorescence staining showed that,compared with CPZ group,Iba-1⁺microglia expressing TLR4 and P-NF-k B/p65 were significantly inhibited in CPZ+ICA group（both p<0.001）.9.ICA induced the regeneration and differentiation of OPCs.Compared with CPZ group,both PDGF-Rα⁺and Ki67⁺co-expression and CNPase⁺cells were significantly increased in CPZ+ICA group（both p<0.001）.Conclusion:Oxidative stress and neuroinflammatory responses are pathophysiological processes closely related to the demyelination.Our results show that ICA can reduce the oxidation and inflammation of microglia/macrophages,improve the brain microenvironment,therefore,being beneficial to the protection and regeneration of myelin sheath.Part 3 ICA promoted microglial phagocytosis of myelin debris and reduced inflammatory response and oxidative stressObjective:Based on the antioxidant and anti-inflammatory effects of ICA on microglia,it is assumed that ICA promotes the phagocytosis of myelin debris by microglia to induce myelin protection and regeneration.Therefore,in this study,we observed the potential of ICA to promote microglia/macrophages to engulf myelin debris and improve local oxidation and inflammation,and explore possible cellular and molecular mechanisms of action.Methods:This study includes two parts of in vivo and in vitro experiments.（1）In vitro experiment.After the adherence of cultured BV2 and BMDMs in a48-well plate at a density of 1.3×10⁵,CFSE-myelin debris and ICA were added,and incubated at 37°C for 24h in order to establish phagocytosis myelin debris model of microglia/macrophages.Detect whether ICA can promote the phagocytosis of myelin debris by microglia by fluorescence microscope The expressions of Iba-1,MBP,TREM2,i NOS,Arg-1,TLR4,and Nrf2 were detected tby immunofluorescence staining.The secretions of NO,ROS,RNS,TNF-α,IL-1β,and 6 were detected in the cell supernatants.（2）In vivo experiment.C57BL/6 mice were randomly divided into Normal group,CPZ+Myelin group,and CPZ+Myelin+ICA group.The mice in control group were fed with normal feed for 6 weeks,the mice in CPZ+Myelin group and CPZ+Myelin+ICA group were fed with 0.2%CPZ feed for 6 weeks to induce demyelination.At the end of the fourth week,CFSE-labeled myelin debris were injected into the unilateral corpus callosum area of the mice in CPZ+Myelin group and the CPZ+Myelin+ICA group through stereotaxic.NS was intraperitoneally injected in the mice of CPZ+Myelin group for two weeks,while ICA（50 mg/kg）was intraperitoneally injected into the mice of CPZ+Myelin+ICA group two weeks.After 6 weeks of modeling,the animals were sacrificed,and the expressions of Iba-1,MBP,TREM2,i NOS,Arg-1,TLR4,and Nrf2 in brain were detected by immunofluorescence staining and Western blot.Results:1.ICA promoted microglia phagocytosis of myelin debris.In vivo immunofluorescence staining revealed that there was a co-localization of Iba-1 and MBP in the corpus callosum of the CPZ+Myelin+ICA mice,and the co-localization part also wrapped CFSE-labeled myelin fragments.Then,in vitro experiments showed that there were obvious green particles in the cytoplasm of BV2 and BMDMs,which was more in Myelin+ICA group than that in Myelin group（p<0.05,p<0.01,respectively）.2.ICA promoted the phagocytosis of myelin debris by inducing M2 polarization of microglia.In vivo experiments with immunofluorescence staining and Western blot showed that,compared with CPZ+Myelin group,the expression of i NOS in the process of promoting microglia to engulf myelin debris was reduced in CPZ+Myelin+ICA group（p<0.001,p<0.01,respectively）,and the expression of Arg-1increased（both p<0.01）.These results were verified by in vitro experiments.It was found that compared with the Myelin group,the number of cells co-expressing Iba-1/i NOS/CFSE-Myelin debris in Myelin+ICA group was reduced（p<0.01,p<0.001,respectively）and the number of Iba-1/Arg-1/CFSE-Myelin debris co-expressed cell increased（both p<0.01）.3.ICA promoted microglia phagocytosis of myelin debris by upregulating TREM2receptors.The results of immunofluorescence staining from in vivo experiments showed that,compared with the CPZ+Myelin group,the expression of TREM2 was increasd（p<0.001,p<0.01,respectively）in CPZ+Myelin+ICA group.ICA was used to interfere with BV2/BMDMs cells in vitro.Compared with Myelin group,significantly higher expression of TREM2 receptor was found in Myelin+ICA group（both p<0.01）.4.ICA promoted microglia phagocytosis of myelin debris and alleviated inflammatory responses.In vivo experiments showed that the expression of TLR4 in the CPZ+Myelin+ICA group was reduced compared with the CPZ+Myelin group（both p<0.01）.Immunofluorescence staining of BV2/BMDMs showed the same results as in vivo.The secretions of inflammatory cytokines of cultured cell supernatants were detected and the results showed that the secretions of TNF-α（p<0.05,p<0.001,respectively）,IL-1β（p<0.01,p<0.05,respectively）and IL-6（all p<0.05）in the Myelin+ICA group were significantly reduced compared with the Myelin group.5.ICA promoted the phagocytosis of myelin debris by microglia and reduced oxidative stress.The expression of Nrf2 in the CPZ+Myelin+ICA group was reduced compared with the CPZ+Myelin group（p<0.001,p<0.01,respectively）.In cultured BV2/BMDMs cells,immunofluorescence showed that the expression of Nrf2 in the Myelin+ICA group was significantly increased（p<0.05,p<0.01,respectively）.In BMDM,the productions of NO,ROS and RNS in Myelin+ICA group were greatly reduced compared with the Myelin group（p<0.05,p<0.05,p<0.01,respectively）.Conclusion:ICA reduced neuroinflammation and oxidative stress,improved the microenvironment in the brain,therefore,promotes the phagocytosis of myelin debris by microglia/macrophages via the up-regulation of TREM2 receptors,down-regulation of TLR4/TNF-αand up-regulation of Nrf2.In this case,a favorable environment for the repair and regeneration of myelin is formed.Concluding RemarksEpimedium is a traditional Chinese herbal medicine for invigorating the kidney and strengthening yang,and its modern pharmacological effects are extensive,especially its role in CNS diseases is getting more and more attention.In this research,the methods of biology and immunology were applied to research,and meaningful discoveries were made.It is proved that ICA has the ability of scavenging free radicals and antioxidants,and is positively correlated with its concentration within a certain range;ICA can treat CPZ-induced demyelination by regulating the dynamic balance between Nrf2-mediated antioxidant and NF-κB-mediated neuroinflammation ICA can also up-regulate the TREM2 signaling pathway and down-regulate the TLR4/Nrf2 signaling pathway to promote the phagocytosis of myelin fragments by microglia/macrophages,creating a good environment for myelin repair and regeneration.These results provide meaningful clues for the in-depth study of Epimedium,especially the role of ICA in CNS demyelinating diseases.