Protective Effects And Mechanisms Of ImKTx88 On The Blood-brain Barrier Of Experimental Autoimmune Encephalomyelitis Rats

Background:Multiple sclerosis（MS）is a common autoimmune inflammatory disease of central nervous system（CNS）.Disruption of blood-brain barrier（BBB）and subsequent infiltration of autoreactive T lymphocytes are major characteristics of multiple sclerosis（MS）and its animal model experimental autoimmune encephalomyelitis（EAE）.It has been reported that large amounts of activated Th17 cells are the main inducer of BBB disruption and neuronal degeneration,while Kvl.3 channel plays a key role in Th17 activation.Kvl.3 channel blocker is reported to have a potential therapeutic effect on MS patients and EAE rats.However,most of the studies focused on the mechanisms of immunosuppressive effects,while very few on the BBB protection.We have obtained a Kvl.3 peptide blocker-ImKTx88 in our previous work,which exhibited high competence and selectivity and may have a potential therapeutic effect on MS and its rat EAE models.In this study,we will further explore its role in the protective effects and mechanism of BBB injury which induced in EAE rats,and provide theoretic and experimental basis on the prevention and treatment of MS.Objective:In this study,we used peptide ImKTx88,a high selective Kvl.3 channel blocker,to explore its role in protective effects and mechanism of BBB in a rat EAE model.Methods:（1）6-8 weeks old female Sprague-Dawley rats were randomly chosed and divided into Control group,EAE group,Prevention group and Treatment group.The rats were subcutaneously injected at the footpad with autoimmune antigens to induce EAE model.In the prevention group,the rats were daily subcutaneous injected with ImKTx88（100 μg/kg in 1 ml PBS）from day 0 to day 23.In the treatment group,the rats was administered with ImKTx88（100 μg/kg in 1 ml PBS）from day 12 to day 23.In the Control group and EAE group,the rats received PBS only.The behavioral changes were observed and recorded daily.On day 23 after EAE induction,rats were sacrificed and the brains were removed to observe histopathological changes of cerebellum by hematoxylin-eosin（HE）and Luxol fast blue（LFB）staining.（2）The evaluation of blood-brain barrier permeability in rats.Evans blue dye was slowly injected into the tail vein of the rats in each group and was allowed to circulate for 1 h,brains and the lumbar enlargement of spinal cords were immersed and homogenized in formamide to quantify the leakage of Evans Blue dye for BBB permeability evaluation.（3）The mRNA and protein level of tight junctional proteins（Occludin,ZO-1,Claudin-5）in cerebellar tissues were detected by RT-qPCR and western blot（WB）respectively,and the changes in morphology and distribution of cerebellar tissues in each group were detected by immunofluorescence（IF）;the expression and distribution of ICAM-1 and VCAM-1 in the cerebellar tissues were detected by immunohistochemistry（IHC）.（4）The mRNA and protein level of angiopoietin-1（Ang-1）and its specific receptor Tie-2 in cerebellar tissues in each group were detected by RT-qPCR and western blot（WB）respectively.（5）The mRNA and proteins level of IL-17 in cerebellar tissues were investigated by RT-qPCR and enzyme-linked immune sorbent assay（ELISA）respectively.While,the inhibitory effect of Kvl.3 blocker ImKTx88 on Thl7 cell activation was detected by the in-vitro T cell activation assay and IL-17 level of the supernatants from peripheral activated T cells was investigated by ELISA.Results:（1）The result of clinical score showed that both the prevention and treatment of ImKTx88 can effectively reduce clinical scores in EAE rats.The HE and LFB stainings experiments indicated that the EAE rats exhibited extensive perivascular cuffing fulled with inflammatory cells and lots of demyelination around the white matter of cerebellum compared to control group.ImKTx88 can effectively decrease the extent of the inflammatory cells infiltration and demyelination after EAE induction.（2）The result of Evans Blue dye injection indicated that compared to the control group,Evans Blue dye leakage of brain and spinal cord in EAE rats was increased and ImKTx88 can reduce the leakage of Evans Blue dye in CNS.（3）RT-qPCR and WB indicated that compared to control group,both the mRNA and protein levels of tight junctional complex（Occludin,ZO-1 and Claudin-5）in cerebellar white matter were decreased significantly in EAE group,and IF showed that the distribution of Claudin-5 was changed;ImKTx88 can greatly increase the expression level of Occludin,ZO-1 and Claudin-5,and restore the distributed Claudin-5.IHC indicated that the expression and distribution of cellar adhesion molecules ICAM-1 and VCAM-1 were increased greatly in cerebellar white matter of EAE rats,and ImKTx88 can greatly decrease the expression and distribution of ICAM-1 and VCAM-1.（4）RT-qPCR and WB indicated that compared to control group,the mRNA and proteins level of Ang-1/Tie-2 in the cerebellar white matter of EAE rats were decreased and ImKTx88 can promote the expression of Ang₁/Tie-2 axis in EAE rats.（5）The experiments in vivo indicated that ImKTx88 can decrease the mRNA and proteins level of IL-17 in cerebellum and the T cell activation assay further confirmed that ImKTx88 can decrease the expression of IL-17 in vitro.Conclusions:ImKTx88 can effectively ameliorate EAE disease,and exert protective effects against the BBB destruction:On one hand,ImKTx88 can protect BBB integrity by restoring the loss and distribution of tight junctional proteins;on the other hand,it can also inhibit the expression of cellular adhesion molecular and decrease the activation of endothelial cells,therefore reducing the adhesion and migration of inflammatory cells.We futher found that these protective effects on BBB were probably achieved by blocking the Kvl.3 channel,suppressing Th17 activation and decreasing IL-17 production,which increased the expression of Ang-1/Tie-2 axis.All of these indicated that this newly Kvl.3 selective peptide may provide an important value on novel drug research and development in MS.