| Background:Multiple sclerosis(MS)is an autoimmune disease characterized by demyelination of the central nervous system(CNS).The pathological hallmarks include inflammatory cell infiltration,myelin sheath destruction,axonal damage and glial cell proliferation.In recent years,the main clinical use of immunosuppressive therapy for MS has achieved short-term efficacy,but the long-term treatment results are still poor.The main reason is that most immunosuppressants inhibit the immune system's attack on the CNS,but they can not effectively promote the damaged nerve repair,which makes MS progressively worse.It has been demonstrated that the expression of Kvl.3 channel is significantly increased in MS,and Kvl.3 potassium channel has become a potential target for the treatment of MS.Kv1.3 voltage-gated potassium channel is a member of Kv1.x subfamily,which is expressed both in immune cells and nerve cells.Kv1.3 channels play a vital role in regulating the physiological functions of immune cells and nerve cells.After the repeated activation by autoantigens,T-cells crosses the blood-brain barrier and causes CNS inflammatory responses,which is one of the most important causes of MS.During this process,the expression of Kvl.3 channel increased significantly.Although studies have confirmed that Kv1.3 channel blockers can alleviate the symptoms of MS rat models,the research on Kvl.3 channel blockers is mostly focused on the suppression of T cell-mediated inflammatory responses,and there are few studies on its involvement in the protection of the CNS and the regulation of other immune cell functions.Microglia as an innate immune cell in the CNS are involved in the development and progression of MS.In MS,activated microglial cells along with a significant increase in Kv1.3 channel expression.However,the relationship and its mechanism between the inflammatory activation of microglia and the expression of Kvl.3 channels remain unclear.In the previous work,we screened out a highly potent and specific Kv1.3 channel blocker ImKTx88.In this study,we first investigate the neuroprotective effect of ImKTx88 on MS animal model,experimental autoimmune encephalomyelitis(EAE).Further discussion about the effect and molecular mechanism that ImKTx88 inhibits microglia inflammatory activation to reduce nerve damage will provide the theoretical and experimental basis for the treatment of MS.Objective:To study the neuroprotective effect of the Kv1.3 blocker ImKTx88 in EAE rats,and reveal the potential mechanism how ImKTx88 can suppress the inflammatory activation of microglia and thereby reduce nerve cells damage.Methods:(1)Sprague-Dawley rats were immunized twice with cerebrospinal homologous homogenate on the Oth and 7th days to establish EAE rat models.The rats were randomly divided into control group,EAE group and ImKTx88-treatment group.On the 12th day,rats were given subcutaneous injection of ImKTx88 daily at a dose of 100 ?g/kg(treatment group)or the same volume of PBS(control group and EAE group).Double-blind method was used to observe and record the behavioral scores of rats in each group daily.The rats were sacrificed to obtain spinal cord tissue on the 23rd day.(2)Paraffin sections were made from the lumbosacral spinal cord tissue block.Immunohistochemistry staining was performed to observe oligodendrocyte lineage cell loss,axonal damage and inflammatory cell infiltration in the white matter areas.Immunohistochemistry for oligodendrocyte in the early differentiation stage(NG2),oligodendrocyte in the late differentiation stage(CC-1)and mature oligodendrocytes(MBP)were used to observe the loss of myelin cells.NF200 and APP staining were used to observe axonal injury.CDS,GFAP,MPO and CD68/Iba-1 staining were used to observe T lymphocyte,astrocyte,neutrophil and microglia infiltration,respectively.Double immunofluorescence labeling of CD68 and Kv1.3 was used to detect the Kvl.3 channel expression of activated microglia in the spinal cord of each group.(3)Paraffin sections from the lumbosacral spinal cord tissue of each group were subjected to LFB staining to observe demyelination.Myelination and axonal structure in the white matter of spinal cord was analyzed by transmission electron microscopy for ultrastructural analysis.(4)ELISA was used to detect the levels of pro-inflammatory cytokines(IL-2,IFN-y,TNF-a and IL-6)in rat spinal cord homogenate of each group.BV2 microglia were treated by LPS and with or without different concentrations of ImKTx88,ELISA was used to detect secretion levels of proinflammatory cytokines(TNF-a and IL-6)in the supernatant of BV2 microglia culture.(5)Primary oligodendrocytes and neurons were cultured with supernatants from the BV2 microglia culture with LPS and with or without different concentration of ImKTx88 treatment for 24 h.CCK8 was used to detect oligodendrocytes and neurons survival,and TUNEL was used to detect apoptosis of oligodendrocytes and neurons.(6)The expression of Kv1.3 channel in BV2 microglia of control group,LPS group and LPS+ImKTx88-treatment group were detected by qPCR,Western blot and immunofluorescence staining respectively.Western blot was used to further detect the protein expression of key molecules MyD88?TRAF6 and NF-?B p65 which were mainly involved in signaling transduction pathway.Results:(1)Rats from control group appear no neurological signs during the experimental course.EAE rats begin to show limbs paralysis symptom from the 12th day after the first immunization and progressively worsened.ImKTx88 treatment significantly alleviates the symptoms in EAE rats,and lowers their behavioral scores.(2)The immunohistochemistry results for oligodendrocytes in different stages of differentiation revealed that the number of NG2+,CC1+ and MBP+ oligodendrocytes in EAE group was decreased compared to control group.However,the number of above positive cells in ImKTx88-treatment group was significantly increased compared to EAE group.(3)The results of immunohistochemical staining of axons and ultrastructure analyzed by electron microscope showed that the NF200 expression in EAE group was significantly lower than that in control group,and the(3-APP expression was significantly increased in the lesion areas.The NF200 expression in ImKTx88 treatment group was significantly increased,and the ?-APP expression in the lesion areas was decreased.Electron microscopy observation of the spinal cord ultrastructure revealed that in EAE group,axonal density and myelin sheath thickness were significantly decreased,and G-ratio value was increased compared to control group.However,ImKTx88 significantly increased axonal density,myelin sheath thickness and decreased G-ratio value.The results suggest that ImKTx88 reduces the loss and destruction of myelin cells and axons.(4)The number of infiltrated CD3,GFAP,MPO and CD68/Iba-1 positive cells in EAE group was significantly increased compared to control group,while the number of inflammatory cells above in ImKTx8 8-treatment group was significantly lower than that in EAE group.The ELISA results showed that the concentration of IL-6,TNF-a,IL-2 and IFN-'y of the spinal cord in EAE group were significantly higher than that in control group.However,the concentration of indicated cytokines in ImKTx88-treatment group were significantly reduced compared to EAE group.The results suggest that ImKTx88 can alleviate CNS inflammatory responses of in EAE rats.(5)Double immunofluorescence labeling showed that the Kvl.3 channel expression in activated microglia in the spinal cord of EAE group was significantly increased.However,the expression of Kvl.3 channel in microglia was significantly decreased in ImKTx88-treatment group.At the level of cytology,the expression of Kvl.3 in LPS-activated BV2 microglia was significantly increased.After ImKTx88 treatment,the expression of Kvl.3 channels in LPS-activated BV2 microglia was significantly decreased.In addition,the results of ELISA test for the levels of proinflammatory cytokines showed that the levels of IL-6 and TNF-a in BV2 microglia culture supernatant were significantly increased after LPS treatment.However,IL-6 and TNF-a levels were significantly reduced after ImKTx88 treatment.qPCR results showed that ImKTx88 inhibited the transcription of IL-6 and TNF-? in LPS-activated BV2 microglia.In the meanwhile,the results of CCK8 and TUNEL staining showed that ImKTx88 inhibited oligodendrocytes and neurons apoptosis caused by inflammatory supernatant damage.(6)The results of the expression of signaling pathway protein in BV2 microglia by western blot showed that the expression of MyD88,TRAF6 and p-p65 protein in LPS group were significantly increased compared to control group,However,the proteins above were decreased in LPS+ImKTx88 group compared to LPS group.The results suggest that ImKTx88 may suppress microglial activation through inhibiting MyD88/TRAF6/NF-?B signaling pathway.Conclusion:This study demonstrates that the Kvl.3 channel blocker ImKTx88 can alleviate the symptoms of EAE rats,suppress inflammatory response,reduce the loss of myelin sheath and the damage of axons.Study has further shown that ImKTx88 suppresses the activation of microglia by blocking Kv1.3 channel,and reduces neuronal cell injury caused by the inflammatory activation of microglia,its mechanism may be down-regulation of MyD88/TRAF6/NF-KB signaling pathway in microglia.The above studies suggest that the Kv1.3 channel blocker ImKTx88 has potential application prospects for the clinical treatment of MS. |
PDF Full Text Request