The Neurological Mechanism Of Chronic Itch Induced By Polyamine In Psoriasis

Objectives:Firstly,we establish a mouse model of chronic itch induced by imiquimod cream to mimic psoriasis itch and examine the stability and pathology of the model.Secondly,we detect the spontaneous itch andtouch-evoked itchin psoriasis mice and investigate the content of spermine and expression of polyamine synthetase in the psoriasis model.Thirdly,we explore the molecular mechanisms of polyamine-induced itch by technique in pharmacology,molecular biology and live cell imaging in mice and cells.Methods:(1)ICR mice were randomly divided into two groups.One group was depilated and treated with imiquimod,the other group was solely depilated as a control.Psoriasis-related inflammatory factors,skin keratinocyte proliferation and mast cell migration were detected by q-PCR,HE staining and toluidine blue staining,respectively.The scratching behaviors were observed in mice at a fixed time point;and the mice were treated with touch or not to detect the scratching behaviors of mice after imitation of imiquimod.The expression of polyamine and genes for their synthesis and metabolism were also checked.(2)RTX(20 μg/kg,70 μg/kg,100 μg/kg),compound 48/80(25 μg/200 g,60μg/200 g,125 μg/200 g,200 μg/200 g)and DFMO(0.5%)were administered before imiquimod treatment.Then,we compared itch and pathological features between the model group and the treatment group.(3)According to the results of pharmacological study,the mRNA expression of TRPA1,TRPV1and Cav3.2 were detected by real-time PCR.(4)To check the appropriate doses of polyamine,ICR mice were randomly divided to receive spermine(100-1200 μg),spermidine(50-300 μg)and putrescine(50-500 μg)treatment in the nape of the neck by intradermal injection,6-8 mice per group.ICR mice were randomly injected with spermine(500 μg),spermidine(200 μg)or putrescine(200 μg)in the nape of the neck,respectively.ICR mice were treated by spermine(500μg),spermidine(200 μg)and putrescine(200 μg)in the cheeks of mice,respectively.Moreover,pre-treatment with morphine(10 mg/kg)and naloxone(10 mg/kg)to determine if polyamine can induce itch.(5)To explore the molecular mechanism of polyamine-induced itch,mice were treated with RTX,compound 48/80,chlorpheniramine(1 mg/kg),TRPV1 blocker(CPZ),TRPA1 blocker(HC030031)and glutamate receptor antagonist,respectively.(6)The TRPA1-or TRPVI-plasmids were transfected into HEK-293 cell line.Then,we use living cell workstation and microplate reader to detect the level of intracellular calcium when polyamines were added.(7)After injection of spermine(500 μg),spermidine(200 μg)or putrescine(200μg),the spinal cord and DRG were taken within 5 minutes.And the expression of p-ERK was measured by western blotting.To determine the assocaiton between p-ERK and polyamine-induced itch,we also checked the itch behavior and expression of p-ERK by pretreatment of U0126(1 nmol)and MEK inhibitor.Results:(1)After treatment of imiquimod,mice showed psoriasis pathological features:markedly increased expression of inflammatory factors,significant proliferation of skin keratinocytes and obviousspontaneous itch and touch-evoked itch behaviors.(2)The results of q-PCR showed that genes related to the polyamine synthesis and metabolism were significantly changed in psoriasis mice,and the immunofluorescence results showed that the content of spermine and spermidine was significantly increased.Pre-treatment of DFMO,compound 48/80 and RTX could significantly reduce psoriasis-induced itch.(3)Three polyamines could induce itch behaviorsin the cheek model.In the neck model,the numbers of scratching were in a dose-dependent manner.(4)RTX,compound 48/80 and TRPA1 and TRPV1 antagonists could significantly inhibit polyamine-induced itch,whereas chlorpheniramine and glutamate receptor antagonists did not alter polyamine-induced itch.(5)Results from living cell workstations and microplate reader showed that polyamines significantly increased the calcium concentration in HEK-293 cells transfected with TRPA1 and TRPV1,(6)Polyamine could induce the expression of p-ERK in spinal cord,while U0126 could significantly inhibit the expression of p-ERK and polyamine-induced itch.Conclusion:(1)Psoriasis mice are accompanied withspontaneous itch and touch-evoked itch.(2)Polyamines are involved in psoriasis-induced itch.(3)Polyamines induce itch bymediating calcium influx through activating TRPA1 and TRPV1 channels and up-regulation of p-ERK expression.