Study On Hypothalamic Central Regulatory Mechanism Of Itch Induced By Bombesin In Mice

Objective: To study the pharmacological characteristics of Bombesin(BN)-induced itch and explore the central regulation mechanism in mice.Methods:(1)Record the number of itch-related behaviors induced by KM mice after intracerebroventricular(i.c.v.)administration of BN(0.04μg,0.4μg,4μg),including grooming,scratching,licking;(2)Compared the number of scratching in mice after i.c.v.administration of BN(0.04μg,0.4μg,4μg)with those by tail intravenous injection(i.v.)of BN(0.4μg,4μg,400μg);(3)Compared the effect of subcutaneous(s.c.)injection of 5μg/kg or 50 μg/kg anti-itching drug κ-opioid receptor(KOR)agonist nalfurafine(Nal)on scratching induced by histamine,chloroquine and BN;(4)Constructed the NBD-BN,in which the BN were coupled with NBD(a fluorescent label,7-nitrobenz-2-oxa-1,3-diazole),Then traced the dynamic distribution of NBD-BN(i.c.v.)with fluorescence signal at 5,10,20,30 min in the brain to determine the target regions of the BN distribution;(5)Immunohistochemistry and western blotting were used for analyzing the expression of Neuromedin B receptor(NMBR)and gastrin-releasing peptide receptors(GRPR)in hypothalamus;(6)Immunofluorescence and western blotting were used to detect c-fos protein expression in the brain after i.c.v.administration of BN;(7)Compare the number of scratching induced by hypothalamic in situ injection(i.s.i.)of BN and i.c.v.administration of BN;(8)Signal pathways associated with biological effects of BN were screened by RNA-seq and verified using western blotting;(9)Compare the number of scratching induced by BN after administration of NMBR antagonist PD168368(0.15-1.5μg),GRPR antagonist RC3095(0.01-1μg),and the PKA antagonist Rp-cAMPS(0.02-0.2μg).Results:(1)Scratching,one of the itch-related behavior,was main behavior in KM mice after administration of BN,the number of scratching induced by 0.04μg BN accounted for73.21% of its total number of itch-related behaviors during the 30 min observation period,0.4μg BN accounted for 66.07%,4μg BN accounted for 66.07%;(2)Mice showed intense scratching behavior after i.c.v.administration of BN(0.04μg,0.4μg,4μg)in a dose-dependent manner.Compared to the vehicle group,during the 30 min observation period,the number of scratching was significantly increased after i.c.v.administration of BN 0.04μg(p <0.05),0.4μg(p <0.01),4μg(p<0.001).No observable scratching behavior was observed in the mice after i.v.administration of BN(0.4μg,4μg,400μg).Compared to the vehicle group,no significant difference in the number of scratching;(3)After mice were pretreated with 5μg / kg nalfurafine,Compared to the vehicle pretreatment group,a significant decrease in the number of scratching induced by histamine(p <0.001)and chloroquine(p <0.001),no significant difference in the number of scratching induced by BN.After mice were pretreated with 50μg / kg nalfurafine,Compared to the vehicle pretreatment group,a significant decrease in the number of scratching induced by histamine(p <0.001)and chloroquine(p <0.001),no significant difference in the number of scratching induced by BN;(4)Fluorescent tracing results showed that the NBD-BN distribute in the hypothalamus observed at 5 min,10 min,20 min,30min and the fluorescence intensity peaked at 10min(p<0.01);(5)Immunohistochemistry and western blotting both showed the distribution of the BN receptor GRPR and NMBR in the hypothalamus;(6)Western blotting results showed that,compared to the vehicle group,c-fos protein expression in the hypothalamus was significantly increased after i.c.v.administration of BN 4μg(p<0.01).Immunofluorescence showed an increased expression of c-fos protein in the hypothalamus and c-fos positive cells were abundant in the dorsomedial nucleus of the hypothalamus;(7)There was no significant difference between the number of scratching induced by i.c.v.administration of BN and i.s.i.administration of BN.Compared to the i.c.v.administration group,latency of scratching was significantly reduced for i.s.i.administration(p<0.01);(8)The RNA-seq results indicate that the c AMP-associated PKA / CREB signaling pathway is involved in the BN-mediated biological effects,western blotting results showed that,compared to the vehicle group,PKA was significantly upregulated at 10 min(p<0.05)and 20min(p<0.01)after i.c.v.administration of BN,the phosphorylated CREB was significantly upregulated at 30min(p<0.05);(9)Compared to the vehicle pretreatment group,After pretreatment with PD168368(0.15-1.5μg)or RC3095(0.01-1μg),there was no significant difference in the number of scratching induced by BN.Compared to the vehicle pretreatment group,the number of scratching induced by BN was significantly reduced after i.s.i.administration of0.02μg Rp-cAMPS(p <0.01)and 0.2μg Rp-cAMPS(p <0.001).Conclusion: BN(i.c.v.)induced strong scratching behavior in mice,suggesting that the center modulation mechanism are involved BN-induced itch.There was no injection side bias in scratching behavior induced by BN.BN-induced itch has a different central mechanism from histamine-and chloroquine-induced itch.the tracer results showed that BN was distributed diffusely in the hypothalamus after i.c.v.administration and there were BN receptors in the hypothalamus;Consistently,the expression of GRPR and NMBR and c-fos protein increased significantly in the hypothalamus after i.c.v.administration of BN,the expression of PKA/CREB protein was up-regulated after treatment with BN,confirmed PKA/CREB signaling pathway is involved in the hypothalamic regulation of BN-mediated itch.