| Background:Multiple sclerosis is a kind of autoimmune diseases in central nervous system,which is characterized by inflammatory demyelinating lesions,the etiology and pathogenesis are not fully clear,a variety of factors such as heredity,environment,virus infection are related to it,the recurrence rate and disabled rate is higher,optic neuritis as the most common first symptoms of MS,occurs in Asian populations,can lead to optic nerve axons and myelin damage,it is often presented as visual impairment such as blurred vision,ocular movement pain,retrobulbous pain and so on.It is one of the difficulties and hot spots in current neuroscience research and seriously reduces the quality of life of patients.Due to the optic nerve and the retina are senior nerve tissue and a part of the brain extends outward,it can cause the pathological changes of the cell tissue necrosis and degeneration easily,and optic nerve sheath outside membrane package,by three layers of meningeal(dura mater,arachnoid,pia mater)continue to CNS inflammatory reaction by organizing further clearance glia involving the optic nerve and retina,cause visual function damage,such as infiltration of macrophages,activation of microglia,and apoptosis of oligodendrocyte(OL)and retinal ganglion cell(RGC)are important factors in visual impairment in MS/experimental autoimmune encephalomyelitis(EAE)models.Studies have confirmed that the kidney and phlegm and activating blood method can improve the CNS inflammatory environment by regulating T lymphocyte subsets in the redistribution of lesion area,inhibiting inherent MG cell activation in CNS and promoting nerve repair,Bushen Yisui capsules based on the method of tonifying kidney,reducing phlegm and promoting blood circulation can control inflammatory reaction of EAE/MS,reduce EAE/MS myelin and axon damage.More and more studies have proved the effect of MS/EAE pathological changes on the structural and functional changes of the optic nerve and retina.As a functional part of the brain,the optic nerve and retina are easy to obtain by exposure,and have been widely used as a model to study nerve regeneration in the CNS system.Objective:By constructing EAE model,comparing the activation of optic nerve and retinal glial cells(M?,MG,Muller glial cells,astrocytes)between different EAE groups,myelin depigmentation and axon damage degree and apoptosis of OL and RGC cell,the mechanism of Bushen Yisui capsules in treating visual impairment of EAE by regulating the inflammatory stress response of CNS glial cells was studied systematically and deeply,Further elucidation of the cellular mechanisms of CNS associated with myelin regeneration failure and neurotoxicity,to provide experimental data and scientific basis for the treatment of kidney and phlegm and activating blood method and Bushen Yisui capsules to MS/EAEResearch methods:1.The specified pathogen free(SPF)grade C57BL/6 female mice were 80,6-8 weeks old,18-22g in body weight.They were randomly divided into 4 groups(n=20 per group):normal control group,model group,fingolimod group,and Bushen Yisui capsule group.EAE mice were immunized with myelin oligodendrocyte glycoprotein 35-55(MOG35-55)to induce retinal and optic nerve inflammatory response.2.Body weight and neurological function score of mice in each group were recorded daily,and neurological function score was performed on a 5-point scale.3.On the 35th day after immunization,the mice were anesthetized with 10%chloral hydrate(0.3ml/100g body weight).Ten mice in each group were infused with 4%paraformaldehyde.Luxol fast blue staining(LFB)was used to observe the changes of myelin sheath and axons in the tissue sections of mouse optic nerve.The expressions of CCI,cleaved caspase 3,GFAP,1BA1,and TMEM119 in optic nerve and Brn3A,cleaved caspase 3,vimentin,GFAP,IB A1,and TMEM119 in retina were detected by immunofluorescence.The reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expression of Brn3A,cleaved caspase 3,vimentin,GFAP and IBA1 genes in the retina of mice,and the expression of cleaved caspase 3,GFAP and IBA1 genes in the visual nerve of mice.Result:1.Influence of body weight of each group mice after modelingAfter modeling,the body weight of mice in the model group was lower than that in the normal group,and the difference was most obvious on the 17th day after modeling(P<0.01),and compared with model group,both the body weight of the fingolimod group and the Bushen Yisui capsule group were increased(P<0.01),compared with the fingolimod group,the difference of body weight was more significant in Bushen Yisui capsule group(P<0.01).2.Influence of neurological function scores in each group after modelingAfter 7 days of modeling,all the immunized mice began to show EAE symptoms,and the neurological function of the mice reached the peak on the 17th day,and then entered the stable phase,indicating the success of modeling.The scores of fingolimod group and Bushen Yisui capsule group were lower than those of model group(P<0.05,P<0,01),indicating that Bushen Yisui capsule can reduce the neurological function scores of EAE mice.3.Inflammatory cell infiltration in HE staining of mice in each groupThe results of HE staining showed that the infiltration of inflammatory cells in the optic nerve and retina of the model group was obvious,with cavitation like change.After the intervention of fingolimod and Bushen Yisui capsule,the infiltration trend of inflammatory cells was significantly reduced.4.The status of myelin sheath and axon in the optic nerve of mice staining with LFB and silverThe results of LFB and silver staining showed intact myelin sheath and axon in normal mice comparing EAE mice,axon entanglement,demyelination of myelin sheath and morphologic evacuation were observed in EAE mice,LFB gray scale was used to analyze the demyelination of optic nerve in mice,compared with normal group,the model group had more severe demyelination(P<0.001),compared with model group,EAE demyelination was significantly alleviated in Bushen Yisui capsule group(P<0.001),indicating that Bushen Yisui capsule can significantly relieve the demyelination and axonal degeneration of optic nerve.5.Immunofluorescence staining resultsCompared with the normal group,the number of oligodendrocytes in the optic nerve of EAE mice was significantly reduced(P<0.001)and the proportion of CC1+ cleaved caspase 3+to CC1+ cells was significantly increased(P<0.001),suggesting increased oligodendrocytes apoptosis in immunized mice.Compared with the model group,the number of oligodendrocytes in optic nerve increased significantly after treatment with Bushen Yisui capsule and fingolimod,and the proportion of CC1+ cleaved caspase 3+ to CC1+ cells was decreased(P<0.001,P<0.001),suggesting that drug intervention can reduce the apoptosis of EAE optic nerve oligodendrocytes.Compared with the normal group,the ratio of Brn3A+ cleaved caspase 3+ to Brn3A+cells in RGL of EAE mice was significantly increased(P<0.001),suggesting an increased rate of RGC cell apoptosis in immunized mice.Compared with model group,the RGC apoptosis rate of mice in Bushen Yisui capsule group and fingolimod group was significantly decreased(P<0.001,P<0.001),suggesting that Bushen Yisui capsule could reduce the apoptosis of retinal RGC cells.Compared with the normal group,Vimentin+ cells in the model group had longer protrusions extending from the outer plexiform layer(OPL)to the deeper outer nuclear layer(ONL)and retinal ganglion layer(RGL).Compared with the EAE group,the expression of Vimentin protein in the whole depth of the retina was significantly reduced in the drug group.The gray value results showed that the expression of Vimentin protein in Bushen Yisui capsule group was significantly lower than that in model group(P<0.001),and the expression of Vimentin protein in Bushen Yisui capsule group was lower than that in fingolimod group(P<0.05).Compared with normal group,Fluorescence staining showed that the astrocytes in the model group were hypertrophic,glial hyperplasia was obvious,and the gray value of GFAP in the retinal RGL layer and OPL layer and the optic nerve were increased(P<0.001,P<0.001,P<0.001),compared with model group,GFAP grayscale value of Bushen Yisui capsule group decreased obviously(P<0.05,P<0.001,P<0.001),suggesting that Bushen Yisui capsule can inhibit the proliferation of astrocytes in EAE mice.Compared with the normal group,the total number of microglia and macrophages in the retina of EAE mice was significantly increased(P<0.001,P<0.01),and the total number of microglia and macrophages in the optic nerve was significantly increased(P<0.001,P<0.001).Compared with the EAE group,the number of microglia and macrophages decreased significantly in the drug group,but there was no significant difference between the Bushen Yisui capsule group and the fingolimod group,indicating that both of them could alleviate the overactivation of microglia in the optic nerve and retina of EAE mice.6.Real-Time PCR resultsCompared with the normal group,mRNA expression of cleaved caspase 3,GFAP,IBA1,Brn3A,and Vimentin in the retina of model group was significantly increased(P<0.05,P<0.05,P<0.001,P<0.05,P<0.05),the mRNA expressions of cleaved caspase 3,GFAP,IBA1 and Vimentin in Bushen Yisui capsule group were significantly lower than those in model group(P<0.05,P<0.001,P<0.001,P<0.01),the difference was statistically significant.Compared with the normal group,the mRNA expressions of cleaved caspase 3,GFAP,and IBA1 in the model group were significantly increased(P<0.001,P<0.01,P<0.001),the mRNA expressions of cleaved caspase 3,GFAP and IBA1 in Bushen Yisui capsule group were significantly lower than those in model group(P<0.001,P<0.05,P<0.001).The mRNA expression of optic nerve and retina-related glial cells in fingolimod group was also significantly lower than that in model group,but there was no statistical difference between fingolimod group and Bushen Yisui capsule group,indicating that both of the two intervention drugs could play an important role in the inflammatory infiltration of optic nerve and retina.Conclusion:1.Bushen Yisui capsule can reduce the EDSS neurological function score in EAE model,alleviate the weight loss caused by immunity in model mice,and increase the body weight of mice;2.Bushen Yisui capsule can alleviate the inflammatory infiltration of the optic nerve and retina of EAE mice,reduce the expression of RGL layer and OPL layer of retina,and the expression of astrocytes,microglial and macrophages of optic nerve,as well as the expression of Muller glia cells of retina,reduce the apoptosis of optic nerve OL and retinal RGC cells,further alleviate the loss of optic nerve myelin sheath and axon,and plays a role in protecting the visual function of EAE;3.Compared with fingolimod,Bushen Yisui capsule has advantages in reducing the expression of retinal Muller glial cells,but other experimental results showed no statistical difference.The results indicating that Bushen Yisui capsule has important significance in maintaining retinal homeostasis and remodeling in EAE mice. |
PDF Full Text Request