| Background:Kv1.3 channel blockers have been widely used on therapeutic research of many human diseases.However,comparing to small molecular drugs,peptide Kv1.3 channel blockers with higher selectivity lack blood brain barrier(BBB)penetrating ability.BBB,as a complicated natural barrier,is mainly responsible for maintaining the stability of the central nervous system(CNS).On the one hand,the barrier protects the body.On the other hand,the barrier limits the possibility of many drugs to play therapeutic roles in the CNS.Therefore,it is emergent to find a transport vector that can help functional moleculars cross the BBB.It’s reported that cell penetrating peptides(CPPs)can carry a variety of macromolecules into cells or tissues,including peptides.Meanwhile,dNP2,as a novel CPP,is reported to have higher delivery efficiency than classical CPPs.In our previous work,we screened a highly selective Kv1.3 channel blocker,peptide ImKTx88,or ImK for short.It can effectively ameliorate the symptoms of experimental autoimmune encephalomyelitis(EAE),a classic rat model of multiple sclerosis(MS),by inhibiting T cell activation.However,as the disease is ameliorated and the damage of BBB is repaired,peptide ImK can not continue to enter the CNS and play an anti-inflammatory role.This study will construct and express the Kv1.3 channel blocker,peptide dNP2-ImK with BBB penetrating potential and anti-inflammatory effects by using dNP2,which provides a theoretical and experimental basis for further application of Kv1.3 channel blockers in the CNS disease and CPPs in drug delivery system.Objective:To construct a Kv1.3 channel blocker dNP2-ImK with BBB penetrating potential.To compare the difference between dNP2-ImK and ImK in the aspect of cell penetrating abilities,BBB penetrating potential and anti-inflammatory effects.Method:(1)The pEGX-6p-1 plasmid containing gene sequence of ImK was used as template.The gene sequence of dNP2-ImK was obtained by using PCR primers.The prokaryotic expression vector pEGX-6p-1-dNP2-ImK was constructed through double enzymes digestion reaction and coupled reaction.After that,positive clones were identified and sequenced.(2)After the plasmid with the correct amino acid sequences was transformed into Escherichia coli Rosetta(DE3)cells,purified dNP2-ImK was obtained by isopropyl β-D-Thiogalactoside(IPTG)induction,ultrasonic wave breaking,GST affinity chromatography,ultrafiltration,enzyme digestion reaction,purification by high performance liquid chromatography(HPLC)and freeze-drying.A small amount of peptide sample was used to reflect the process of protein expression by Tricine-SDS-PAGE.Freeze-dried peptide was used to accurately measure the molecular weight by Mass Spectrum.(3)293T cells were cultured in vitro and mKv1.3 plasmid was transfected into the cells.The effect of dNP2-ImK on Kv1.3 current was detected by whole-cell patch clamp technique,and its function on Kv1.3 channel was evaluated.Rat peripheral blood monouclear cells(PBMCs)were isolated in vitro.With the intervention of dNP2-ImK(100pM,1nM,10nM,100nM)in advance,PBMCs were stimulated by Concanavalin A(ConA).Enzyme-linked immunosorbent assay(ELISA)was used to detect the effects of dNP2-ImK on the secretion of inflammatory cytokines IL-2,IFN-γ and IL-17A.Under the same conditions,10nM ImK and 10nM dNP2-ImK were used to intervene respectively.Real-time quantitative polymerase chain reaction(RT-qPCR)and ELISA were used to detect the effects of ImK and dNP2-ImK on the genes and proteins expression of IL-2,IFN-γ and IL-17A.Then the anti-inflammatory effects of ImK and dNP2-ImK were evaluated.(4)Human brain microvascular endothelial cells(HBMECs)were cultured in vitro.The experimental groups were divided into Ctrl group,Dye group,Dye-ImK group and Dye-dNP2-ImK group.HBMECs were treated with PBS containing 0.5%DMSO,μM Dye,1μM Dye-ImK and 1μM Dye-dNP2-ImK for 4 hours individually.After going through fixation by paraformaldehyde and dyeing with Hoechst 33258,the fluorescence densities of cells were detected by confocal microscopy.Then the cell penetrating abilities of ImK and dNP2-ImK were evaluated.Meanwhile,HBMECs were laid on the upper layer of Transwell’s ventricle with 0.4 micron aperture.And PBS was added to the lower layer.PBS containing 0.5%DMSO,1μM Dye,1μM Dye-ImK and 1μM Dye-dNP2-ImK were added to the upper layer individually after cell adherence.The fluorescence density of liquid on the lower layer was measured in 24 hours.Then the BBB penetrating potential of ImK and dNP2-ImK was evaluated.(5)HBMECs,293T cells,primary rat astrocytes and PBMCs were cultured in vitro.The experimental groups were divided into Ctrl group and treatment group.Cells in each group were treated with PBS or different concentrations of dNP2-ImK(10nM,100nM,1μM)for 24 hours.After that,cell supernatants were collected for lactate dehydrogenase(LDH)test.Meanwhile,cell counting kit-8(CCK8)was used to detect cell viability.The effects of dNP2-ImK on cell viability and cell membrane integrity were evaluated.Results:(1)Sequencing results of positive clones indicated that the amino acid sequences were consistent with what we expected after translating.Expression vector pEGX-6p-1-dNP2-ImK was constructed successfully.(2)The results of HPLC indicated that the peptide we need and fusion protein were separated,and the peak time was 14 min and 28 min respectively.The results of Tricine-SDS-PAGE showed the whole process of protein expression.The result of Mass spectrum indicated that mass-to-charge ratios was 7068.4 m/Z,which represented the molecular mass of the purified peptide was 7067.4 Da.It’ s consistent with what we expected.(3)The results of whole-cell patch clamp indicated that dNP2-ImK could significantly reduce Kv1.3 currents in 293T cells,and the currents would recover after washout,which showed that dNP2-ImK retained the inhibitory effect of ImK on Kv1.3 channel.The results of ELISA indicated that in rat PBMCs,dNP2-ImK could significantly inhibit the secretion of ConA-induced inflammatory cytokines IL-2,IFN-γ and IL-17A in a concentration-dependent manner.RT-qPCR and ELISA results indicated that both ImK and dNP2-ImK could significantly inhibit the gene and protein expression of IL-2,IFN-γ and IL-17A induced by ConA in rat PBMCs.And their anti-inflammatory abilities were similar.(4)The results of confocal microscopy indicated that the intracellular fluorescence intensity of Dye-dNP2-ImK group was significantly higher than that of Dye-ImK group.Statistics showed that the intracellular fluorescence density of Dye-dNP2-ImK group was significantly higher than that of Dye-ImK group,which showed that comparing to ImK,dNP2-ImK represented stronger cell penetrating ability.The results of transwell indicated that fluorescence intensity of liquid on the lower layer in Dye-dNP2-ImK group was significantly higher than that in Dye-ImK group,which meant dNP2-ImK had better potential to cross BBB than ImK.(5)The results of CCK8 and LDH release assay indicated that dNP2-ImK treatment(1OnM,100nM,1μM)would not affect the cell viability and cell membrane integrity in 293T cells,PBMCs,rat primary astrocytes and HBMECs.Conclusions:(1)dNP2-ImK can significantly decrease Kv1.3 currents in 293T cells and effectively inhibit the genes and proteins expression of ConA-induced inflammatory cytokines IL-2,IFN-γ and IL-17A in rat PBMCs.In addition,there’s no difference in the aspect of inhibitory effects of inflammatory cytokines between dNP2-ImK and ImK.And dNP2-ImK treatment did not affect cell viability.(2)Without affecting cell membrane integrity,dNP2-ImK represented better cell penetrating ability and BBB penetrating potential than ImK,which established the foundation for our following-up study about the therapeutic effects of dNP2-ImK in EAE animal models,and suggested that it could be a potential strategy for drug design to combine CPPs with functional drugs which lack cell penetrating abilities. |
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