The Molecular Mechanism Underlying 5-HT Receptor Subtype 5-HT1F Mediated Acute And Chronic Itch In Mice

Objective:Itch is an unpleasant sensation that can induce scratching behavior.Acute itch as an early warning signal protects the body from external harm,while chronic itch causes great suffering to the patient.The mechanism of pruritus is complex and diverse.5-HT is an important mediator involved in itch regulation through the corresponding 5-HT receptor.There are 15 5-HT receptors.It has been reported that 5-HT1F receptor agonists could induce itch,but the mechanism of itch is not clear.The present study aims to clarify the link between 5-HT1F receptor and itch.So a series of behavioral,pharmacological and molecular biological methods were used to explore the molecular and cellular mechanisms of 5-HT1F receptor-mediated acute and chronic itch,which provides a new target for clinical treatment of chronic itch.Methods:1.ICR adult mice were randomly divided into groups of 6-8 mice.Then the mice were injected intradermally 5-HT1F receptor agonists LY334370 and LY334864(30 μg,50 μg,100 μg,200 μg)in the neck to detect scratching behaviors.The mice were then grouped into groups of 5-7 each,and the 5-HT1F receptor agonist(100 μg,200 μg)was injected into the cheek to detect the scratches or wipes.2.The mice were injected with Morphine(10 mg/kg),Naloxone(10 mg/kg),RTX,Compound 48/80,Chloropheniramine(1 mg/kg),TRPV4 antagonist(HC067047),TRPV1 antagonist(Capsazepine)and TRPA1 antagonist(HC030031)to detect whether opioid receptors,C fibers,mast cells,histamine receptors and TRP family are involved in the pruritus induced by 5-HT1F receptor activation.3.The wild-type and knockout mice(TRPV1-/-,TRPA1-/-and TRPV4-/-)were used to inject with 5-HT1F receptor agonists LY334370 into the neck to detect the change of itch,in order to determine the relationship between 5-HT1F induced pruritus and the TRP family4.ICR mice were established the chronic itch model.Q-PCR determined the mRNA levels of 5-HT receptor subtype and TRP family in the DRG of the two chronic itch model.Western blotting determined the protein levels of 5-HT receptor subtype and TRP family in the DRG of the two chronic itch model5.The psoriasis chronic itch model of mice was used to inject 5-HT1F receptor agonist LY334370 and TRPV4 antagonist HC067047 on the 3rd,5th,and 7th day to detect the change of chronic itch6.The FISH(Fluorescence in situ hybridization)technique detected the co-localization of 5-HT1F and TRPV4,and the distribution of 5-HT1F in the spinal cord7.ICR mice were injected with 5-HT1F receptor agonist 100 μg LY334370 in the neck.The mouse DRG and spinal cord were taken at 10min and 30min after inject.The phosphorylation of ERK was detected by western blotting.At the same time,the pharmacological method was used to inject the MEK inhibitor U0126(lnmol)to detect the number of scratches in ’mice.8.HEK293A cells were transfected with 5-HT1F plasmid,and then calcium imaging technique was used to detect whether administration of the 5-HT1F agonist LY334370 would mediate an increase in intracellular calcium concentrationResults:1.In the neck model,intradermal inject the two agonists of 5-HT IF receptor LY334370/LY334864 could induce acute itch in a dose dependent manner;in the cheek model,intradermal injections the agonism of 5-HT IF receptor could induce scratches,not wipes.2.The opioid receptor,mast cell and C-fibers are involved in activating 5-HT1F receptor inducing itch,activation of 5-HTI F receptor evokes histamine-independent itch in mice3.The TRPV4 antagonist HC067047 and TRPV4-/-mice could reduce scratching behaviors by activating 5-HT1F receptor,but TRPV1 and TRPA1 not.4.The results of Q-PCR showed that the expression of 5-HT1F mRNA and TRPV4 mRNA were up-regulated in the DRG of chronic itch model(PSO and AEW),the results of western blotting showed that he expression of 5-HT1F protein and TRPV4 protein were increased in the DRG of chronic itch model(PSO and AEW)5.Intradermal injection the agonist of 5-HT1F receptor LY334370 could induce chronic itch in the PSO,intradermal injections the TRPV4 antagonist HC067047 could reduce chronic itch in the PSO.6.Fluorescence in situ hybridization results showed that 5-HT1F receptor and TRPV4 were co-expressed in DRG.5-HT1F was mainly distributed in the CGRP+neurons in the DRG and dorsal horn of spinal cord.7.Western blotting results showed that LY334370 significantly induced the expression of p-ERK in DRG and spinal cord,indicating that p-ERK may be involved in itch by activating 5-HT1F receptor.8.Calcium imaging results showed that HEK293A cells transfected with 5-HT1F plasmid and TRPV4 plasmid,administration of LY334370 caused an increase in intracellular calcium.Conclusions:1.5-HT1F receptor is involved in acute itch and chronic itch.2.5-HT1F induces phosphorylation of ERK by activating the TRPV4 ion channel,which in turn induces pruritus.