The Effects And Mechanism Of P2X₃ Receptor In Acute And Chronic Pruritus In Mice

Background:Itch,also called pruritus,is an annoying and impatient sensation that provokes a desire to scratch.Innocuous and noxious touch impacts our daily life,activities,and communication with other people.At the same time,the treatment to the itch is not satisfactory.Itch can be divided into acute and chronic itch according to the duration.In recent years,a variety of itch models have been established with the gradual deepening of research on itch.Itch sensation originates from the peripheral afferent primary sensory neurons of the skin,and the cell body is located in the dorsal root ganglion?DRG?and trigeminal ganglion,which is then transmitted to the sensory center of the brain via the spinal cord.External noxious stimuli can cause the increase of adenosine triphosphate?ATP?released by neurons.As a neurotransmitter ATP acts on purine 2 receptor?P2?on neurons.P2X₃ receptor is an important sensor for nociceptive stimuli.Previous studies have shown that P2X₃ receptor is involved in neuropathic pain,acute pain,inflammatory pain and cancer pain,and refers to inflammation and immune responses.It has been reported that the expression level of P2X₃ mRNA is up-regulated in the AEW model.These findings suggest that P2X₃receptor is not only involved in pain response,but may play a role in pruritus neuropathy.However,how the P2X₃ receptor participates in the itching process and its mechanism is still unclear.Objective:The purpose of this study was to investigate the pathophysiological effect and possible mechanism of P2X₃ receptor in pruritus model by combining the whole and molecular levels,thus providing a new theoretical basis for the prevention and treatment of acute and chronic pruritus.Methods:1.Observe the pathological changes of P2X₃ receptor in dry skin model.A dry skin model of mice was established.Mice were randomly divided into six groups:control group?Ctrl?,AEW group?acetone-ether-water,AEW?,AEW mice treated with P2X₃ shRNA group?AEW+P2X₃ shRNA?,AEW mice treated with scramble shRNA negative control group?AEW+NC shRNA?,AEW mice treated with antagonist?AEW+A317491?and AEW mice treated with phosphate buffer solvent?AEW+PBS?.?1?Calculate the number of scratches in mice to observe changes in itching behavior.?2?Detect the expression of P2X₃ in DRG by Real-time PCR,western bloting?WB?and immunohistochemistry?IHC?.?3?Observe the pathologic changes of the skin in the modeling site by hematoxylin-eosin?HE?staining.?4?Observe the changes of protein expression levels of IL-1?and TNF-?in stellate ganglion by Real-time PCR and Western blotting.?5?The phosphorylation of ERK1/2in DRG was detected by Western blotting.?6?The co-expression of gastrin-releasing peptide receptor?GRPR?and P2X₃ receptor in DRG was observed by immunofluorescence double labeling technique.2.Detect the signal pathway of P2X₃ receptors in pruritus.An acute pruritus model of mice was established.Mice were divided into control group?Ctrl?,P2X₃shRNA group?P2X₃ shRNA?,control mice treated with scramble shRNA negative control group?NC shRNA?.The mice in each group were respectively injected with compound 48/80,which promoted histamine release,and chloroquine,a histamine-independent pruritus agent,to observe the changes of pruritus behavior in each group.3.Detect the role of P2X₃ receptor-specific agonists??,?-methylene ATP,?,?-meATP?and specific antagonists?A317491?in pruritus.The cheek model was established,then the changes of pruritus behavior in each group were observed by setting different concentration gradients of?,?-meATP and A317491.Results:1.Pathological changes of P2X₃ receptor in dry skin model.?1?The results of animal behavior showed that the number of scratches of AEW group was significantly higher than those in Ctrl group?p<0.01?.The number of scratches was significantly reduced after treated with P2X₃ shRNA or A317491?p<0.01?.There was no significant difference among AEW+NC shRNA group,AEW+PBS group and AEW group?p>0.05?.?2?The results of Real-time PCR,WB and IHC showed that the expression of P2X₃ mRNA and protein in DRG of AEW group were significantly higher than those in Ctrl group?p<0.01?.The expression levels of P2X₃ mRNA and protein in DRG of AEW+P2X₃ shRNA group and AEW+A317491 group were significantly lower than those in AEW group?p<0.01?,but no significant difference were found among the AEW+NC shRNA,AEW+PBS group and AEW group?p>0.05?.?3?HE staining showed that the epidermis of AEW group was significantly thicker than that in Ctrl group.After treated with P2X₃ shRNA or A317491,the thickness of the epidermis was alleviated.?4?The expression levels of IL-1?and TNF-?protein in DRG were detected by Real-time PCR and Western blotting.Compared with the Ctrl group,the expression levels of IL-1?and TNF-?protein in the AEW group were significantly increased?p<0.01?.After P2X₃ shRNA or A317491 treatment,the expression levels of IL-1?and TNF-?protein were significantly lower than those in the AEW group?p<0.01?,there was no significant difference among the AEW+NC shRNA,AEW+PBS group and AEW group?p>0.05?.?5?Compared with the Ctrl group,the phosphorylation of ERK1/2 protein in the AEW group increased?p<0.01?.After P2X₃ shRNA or A317491 treatment,the phosphorylation of ERK1/2 decreased?p<0.01?.?6?Double-labeled immunofluorescence results showed that P2X₃ receptor in dorsal horn of spinal cord was co-expressed with GRPR in neuron.The fluorescence intensity of AEW,AEW+NC shRNA group and AEW+PBS group was higher than that in Ctrl group?p<0.01?,and the fluorescence intensity of AEW+P2X₃ shRNA group and AEW+A317491 group was lower than that in AEW group?p<0.01?.2.P2X₃ receptor is involved in histamine-independent itch.?1?After injection compound 48/80 in the nape of the neck,there was no significant difference in the number of scratches per 5 minutes or in 30 minutes among Ctrl group,P2X₃ shRNA group and NC shRNA group?p>0.05?.?2?After injection chloroquine,the number of scratches of P2X₃ shRNA group at 5,10,15 and 20 minutes?a time period every 5minutes?were decreased compared with those in Ctrl group or NC shRNA group?p<0.05,P<0.05,P<0.01,P<0.01?.The number of scratches in P2X₃ shRNA group decreased significantly compared with Ctrl or NC shRNA group in total 30 minutes?P<0.01?.3.P2X₃ receptor specific agonist or antagonist can activate or inhibit the P2X₃receptor respectively and participate in the pruritic process.Different concentrations of?,?-meATP affected the frequency of scratch,and the number of scratches in concentration of 0.5?mol/L was significantly increased compared with 0?the total number of the first 5 minutes??p<0.05?.A317491 inhibited the pruritus induced by chloroquine.The number of scratches was significantly decreased when concentration of A317491 is 100 nmol/L compared with 0?p<0.05??total 0-30 minutes?.Conclusion:P2X₃ receptor is a key signaling molecule for acute and chronic pruritus.The P2X₃ receptor is involved in histamine-independent pruritus.Agonist or antagonist can activate or inhibit the P2X₃ receptor respectively and participate in the pruritic process.All these indicating that the P2X₃ receptor may be a potential target for pruritus treatment.