Neurobiological Mechanisms Of Electroacupuncture Inhibiting Chronic Itch And GRPR Receptor Expression By Endocannabinoid System

Chronic pruritus is defined as pruritus symptom lasting for more than six weeks.It is reported that the prevalence of chronic pruritus in the general population is 8.4% to 13.9%.There are many reasons for chronic itching,including dermatology,infectious diseases,systemic diseases,mental diseases,neurological diseases and psychological diseases.It is well known that the quality of life and social mental health are negatively correlated with the severity of pruritus.Psychological disorders associated with painful symptoms include sleep interruption,depression,emotional agitation,anxiety,and transformation in eating habits,low self-esteem and inattention.With the increasing prevalence of chronic itching,clinicians pay more and more attention to the mechanism of itching.Gastrin releasing peptide(GRP)is a neuropeptide associated with itching sensation.Intradermal injection of GRP can cause scratching in mice.GRP is released by neurons of dorsal root ganglion(DRG).Once released,GRP activates the neurons expressing GRP receptor(GRPR)in the spinal dorsal horn,and then transmits itch signals to brain.Studies have shown that ablation of spinal cord neurons expressing GRPR significantly reduces the scratching behavior of the mice induced by itching stimuli.In addition,it was found that GRPR and its ligand GRP were highly expressed in the spinal cord and skin of rhesus monkeys with chronic spontaneous pruritus,which is the first report of this kind of research in primate models.Therefore,GRPR can be used as an important index to measure the extent of itching,which provides a new theoretical basis for studying the mechanism of EA on inhibiting chronic itching.Cannabinoids have been known for their analgesic effects over the last few centuries.However,the probabilities of their abuse and harmful central effects,including tolerance and physical dependence,have prevented their effective use in clinical practice.Cannabinoid drugs have only recently been approved for medical purposes,mainly for neuropathic pain,multiple sclerosis and so on.Recent experimental studies have also shown that cannabinoids have antipruritic effects.It is well known that down-regulation of 5-hydroxytryptamine(5-HT)and norepinephrine can relieve pain.In addition,the descend inhibitory pathway can regulate the anti-injury effects of cannabinoids.Some studies have shown that,like chronic pain,the mechanism of chronic itching includes the brainstem regulation mechanism of itch signaling pathway.5-HT neurons participate in the brainstem descending analgesia system,which can aggravate pruritus by activating 5-HT1 A receptors on GRPR neurons.Studies have shown that electroacupuncture(EA)can significantly activate CB1 receptors in the midbrain,inhibit the release of gammaaminobutyric acid(GABA),weaken the inhibition of GABA on 5-HT neurons in the medulla,and promote the release of 5-HT,thereby strengthening the function of the descend inhibition system and exerting the analgesic effect.Whether EA can inhibit the activation of CB1 receptor and the release of 5-HT,thereby inhibiting the activation of 5-HT1A-GRPR receptor coupling and inhibiting the sensitization of chronic pruritus central circuit remains unknown.It provides a new opportunity for the study of EA inhibiting chronic itch.In conclusion,in the first part of this paper,we determine whether EA can improve chronic itching,and select the best EA parameters for the treatment on chronic itching.In the second part of this paper,we determine whether EA can alleviate chronic itching by down-regulating and activating CB1 receptor in the midbrain,promoting GABA releasing and inhibiting 5-HT releasing in the medulla in the descend pathway;At the same time,by using chemogenetics method we determine whether activating or inhibiting GABA neurons on l PAG can affect the formation of chronic itching.In the third part of this paper,we determine whether EA can alleviate chronic itching by downregulating and activating peripheral CB1 receptors,inhibiting GRP expression in skin and DRG,and reducing mast cell aggregation and degranulation in skin.PART? Electroacupuncture inhibits GRPR expression and chronic itch through Kappa opioid receptorsObjective(1)We aim to determine whether EA has a benign regulating effect on chronic itching,and formulate the best treatment plan of EA relieving itching,so as to solve the clinical problem that itching is difficult to be cured.(2)To study the neurobiological mechanism of DYN-A and GRPR participating in EA antipruritic,and to provide new theoretical basis and therapeutic strategies for EA antipruritic.Methods(1)Establishment of chronic pruritus model: Chronic itching was induced by applying a mixture of acetone and ether(1:1)of equal volume ratio(AEW)to the neck of mice.(2)EA group and sham EA group: Current was delivered with a Han's acupoint nerve stimulator(LH202,China).On the first day of modeling with acetone and ether,EA was given at Quchi(LI11)and Hegu(LI4)on the left side of mice,once every other day for 30 minutes,five times altogether.The EA intensity is 1 m A,the frequency is 2 Hz,15 Hz and 100 Hz,respectively,and the wave width is 0.3 ms.In sham EA group,the needle was placed under the skin of the acupoint for 30 minutes without electricity.(3)Video observation system was used to detect the basic pruritus behavior of mice in each group before acetone and ether modeling and the number of scratch bouts on the 9th day after modeling to evaluate the degree of pruritus.(4)The selective kappa opioid receptor(KOR)antagonist Binaltorphimine dihydrochloride(nor-BNI)was injected intraperitoneally 18-20 hours before the EA therapy.(5)Western blotting was used to detect the effect of EA at different frequencies on the expression of GRPR and DYN-A protein in cervical cord.(6)The immunoreactivity of GRPR and DYN-A in cervical dorsal horn was detected by immunofluorescence histochemistry.Results(1)After 9 days of modeling with acetone and ether,compared with control group,the number of scratch bouts in chronic itch group was significantly increased.The left Quchi acupoint and Hegu acupoint in mice were given EA with intensity of 1 m A and frequency of 2 Hz,15 Hz or 100 Hz respectively.After five times of EA treatment,compared with sham EA group,15 Hz or 100 Hz EA effectively alleviated chronic itching.EA at 100 Hz has better anti-pruritus effect,while low frequency 2Hz EA has no significant alleviating effect on chronic itch.Intraperitoneal injection of nor-BNI,an antagonist of KOR,significantly antagonized the effect of 100 Hz EA on relieving chronic itch.(2)Western blotting showed that the expression of GRPR protein in the cervical cord of chronic itch mice was significantly higher than that of control group.Compared with chronic itch group,the protein expression of GRPR in cervical cord was significantly reduced in 100 Hz EA group,but there is no significant effect in 2Hz?15Hz EA or sham EA group.It was suggested that 100 Hz EA could inhibit the protein expression of GRPR.(3)Western blotting showed that the expression of DYN-A protein in the cervical cord of chronic itch mice was significantly decreased than that of control group.Compared with chronic itch group,the protein expression of DYN-A in cervical cord was significantly increased in 100 Hz EA group,but there is no significant effect in 2Hz?15Hz EA or sham EA group.It was suggested that 100 Hz EA could promote the protein expression of DYN-A.(4)Western blotting showed that the expression of GRPR protein in the cervical cord of chronic itch mice was significantly higher than that of control group.Compared with chronic itch group,the protein expression of GRPR in cervical cord was significantly reduced in EA group,but there is no significant effect in sham EA group.Intraperitoneal injection of KOR antagonist nor-BNI significantly reversed the effect of EA on reducing the expression of GRPR in cervical cord.Immunofluorescence histochemistry showed that the positive area of GRPR immunoreaction in the cervical dorsal horn of chronic itch mice was significantly higher than that of control group;Compared with chronic itch group,the positive area of GRPR immunoreaction in the cervical dorsal horn was significant reduced in EA group,but there is no significant effect in sham EA group.Intraperitoneal injection of KOR antagonist nor-BNI can significantly reversed the effect of EA on reducing the positive area of GRPR immunoreaction in cervical dorsal horn.Conclusion:EA at 100 Hz may be the effecitive frequency of inhibiting chronic itch and GRPR expression by promoting the synthesis and release of DYN-A which activates KOR.PART? Endogenous cannabinoid CB1 receptor participates in the central mechanism of electroacupuncture(EA)alleviating chronic itchObjective(1)We aim to study whether EA can affect the chronic itch behavior and the expression of itch-specific receptor GRPR in mice,and screen out the optimal treatment for EA to improve chronic itch;(2)To study the effect of EA on the expression of transmitters and receptors in the DYNKOR-GRPR pathway,and the key role of KOR in relieving itch of EA and inhibition of GRPR expression;(3)To study the effect of EA on the expression of transmitters and receptors in the endocannabinoid CB1R-GABA-5-HT-GRPR/DYN pathway;(4)To study the central role of central endocannabinoid CB1 receptors in the activation of DYN-KOR-GRPR itch pathway by EA.Method(1)Video observation system was used to detect the basic itch behavior of mice in each group before acetone and ether modeling and the number of scratch bouts on the 9th day after modeling to evaluate the degree of pruritus.(2)EA group and sham EA group: the current was delivered with a Han's Acupoint Nerve Stimulator(LH202,China).On the first day of modeling with acetone and ether,EA was given at Quchi(LI11)and Hegu(LI4)acupoints,Xuehai(SP10)and Shousanli(LI10)acupoints for 30 minutes once every other day for 5 times.The intensity of EA were 1 m A and 3 m A,respectively,and the frequencies were all 100 Hz,and the wave width was 0.3 ms.The sham EA group was to subcutaneously place the needles to Quchi(LI11)and Hegu(LI4)with no electricity,and the needles were left for 30 minutes.(3)Western blotting was used to detect the effect of EA on the expression of GRPR and DYN-A in cervical cord,and the effect of EA on the expression of CB1 and CB2 receptors in the midbrain.(4)The concentration of 5-hydroxytryptamine(5-HT)in medulla was measured by fluorescence spectrophotometry.(5)The immunoreactivity of GRPR and DYN-A in the cervical dorsal horn was shown by double-immunofluorescence labeling.(6)Chemogenetics was used to excite or inhibit GABAergic neurons in the lateral periaqueductal gray(l PAG)and detect the effect on itch behavior.Result(1)After 9 days of modeling with acetone and diethyl ether,compared with control group,the number of scratch bouts was significantly increased in chronic itch group.EA(1 m A and 3 m A,100 Hz,0.3 ms)was applied to the left Quchi and Hegu acupoints of mice,as well as the Xuehai and Zusanli acupoints,respectively.Compared with chronic itch group,the number of scratch bouts of the group of Quchi and Hegu with 1 m A EA,the group of Quchi and Hegu with 3 m A EA,the group of Xuehai and Zusanli with 1 m A EA and the group of Xuehai and Zusanli with 3 m A EA was significantly reduced.The group of Quchi and Hegu with 3 m A EA has the best therapeutic effect.(2)After 9 days of modeling with acetone and diethyl ether,both western blotting and immunofluorescence results were found that compared with control group,the protein expression and positive reaction area of GRPR in cervical cord of chronic itch group were significantly increased;compared with chronic itch group,the protein expression and positive reaction area of GRPR in cervical cord of the group of Quchi and Hegu with 1 m A EA,the group of Quchi and Hegu with 3 m A EA,the group of Xuehai and Zusanli with 1 m A EA and the group of Xuehai and Zusanli with 3 m A EA was significantly reduced.Among them,the effect of group of Quchi and Hegu with 3 m A EA was most obviously.(3)After 9 days of modeling with acetone and diethyl ether,western blotting showed that the protein expression of DYN-A in cervical cord of chronic itch group was significantly reduced compared with control group;compared with chronic itch group,the expression of DYN-A in cervical cord of the group of Quchi and Hegu with 1 m A EA,the group of Quchi and Hegu with 3 m A EA,the group of Xuehai and Zusanli with 1 m A EA and the group of Xuehai and Zusanli with 3 m A EA was significantly increased.Among them,the effect of group of Quchi and Hegu with 3m A EA was most obviously.Immunofluorescence results showed that the immunoreactive positive area of DYN-A in cervical dorsal horn of chronic itch group was significantly reduced compared with control group.There was no significant difference among the group of Xuehai and Zusanli with 1 m A EA,the group of Xuehai and Zusanli with 3 m A EA and chronic itch group.The immunoreactive positive area of DYN-A in cervical dorsal horn of the group of Quchi and Hegu with 1 m A EA and the group of Quchi and Hegu with 3m A EA were significantly reduced compared with chronic itch group.It is suggested that the Quchi and Hegu with 1 m A and 3 m A EA promoted the release of DYN-A in the cervical dorsal horn.The effect of the group of Quchi and Hegu with 3m A EA was most obviously.(4)After 9 days of modeling with acetone and diethyl ether,the number of scratch bouts was significantly increased in chronic itch group.EA(3 m A,100 Hz,0.3 ms)was administered to the left Quchi and Hegu of the mouse,which reduced the number of scratch bouts in chronic itch mice,while the sham EA group didn't significantly improve the chronic itch behavior in mice.(5)After 9 days of modeling with acetone and diethyl ether,the protein expression of CB1 receptor in the midbrain tissue of chronic itch group was significantly higher than that of control group;compared with chronic itch group,the protein expression of CB1 receptor in midbrain of the group of Quchi and Hegu with 3 m A EA was significantly reduced,while the protein expression of CB1 receptor in midbrain of sham EA group had no significant difference.In addition,there was no significant difference in protein expression of CB2 receptor in midbrain tissue of each group.(6)After 9 days of modeling with acetone and diethyl ether,the 5-HT content in medulla tissue of chronic itch group was significantly decreased compared with control group,suggesting the release of 5-HT was increased;compared with chronic itch group,the content of 5-HT in medulla tissue of mice of the group of Quchi and Hegu with 3 m A EA was significantly increased,while the content of 5-HT in medulla of mice in sham EA group showed no significant change,suggesting that EA inhibited the release of 5-HT in medulla of chronic itch mice.(7)Modeling with acetone and diethyl ether,intraperitoneal injection of CB1 receptor antagonist AM251 was performed 10 minutes before EA.Behavioral results on the 9th day after modeling found that the number of scratch bouts in chronic itch group was significantly increased.Compared with chronic itch group,there was no significant difference in the number of scratch bouts in chronic itch with AM251 injection group,while the scratching behavior in EA group and EA with AM251 injection group was significantly reduced.The number of scratching of the mice in EA with AM251 injection group was significantly higher than that in EA group.(8)After 9 days of modeling with acetone and diethyl ether,the 5-HT content in the medulla tissue of chronic itch group was significantly decreased compared with control group,suggesting the release of 5-HT was increased;compared with chronic itch group,the content of 5-HT in medulla of mice of the EA group of Quchi and Hegu with 3 m A was significantly increased,suggesting that EA inhibits the release of 5-HT in medulla of chronic itch mice.Intraperitoneal injection of CB1 receptor antagonist AM251 significantly reversed the effect of EA on increasing the content of 5-HT in medulla,suggesting that CB1 receptor may be involved in the effect of EA inhibiting the release of 5-HT in medulla.(9)After injecting a cocktail of r AAV-m DIx-Cre-WPRE-p A and r AAV-Ef1?-DIOm Cherry-WPRE-p A no-load virus at a volume ratio of 1:3 into l PAG for 21 days,modeling was carried out with acetone and diethylether,and after 9 days,1 hour before the detection of behavioral test,intraperitoneal injection of CNO did not activate GABAergic neurons on l PAG.After modeling with acetone and diethylether,the number of scratch bouts in chronic itch group was significantly higher than that in control group;l PAG was injected with a cocktail of r AAV-m DIx-Cre-WPRE-p A and rAAV-h Syn-DIO-h M3D(Gq)-m Cherry-WPRE-p A virus in a volume ratio of 1:3 for 21 days before modeling,and then modeled with acetone and diethyl ether.After 9 days,1 hour before the behavior test,intraperitoneal injection of CNO activated GABAergic neurons on l PAG,and found that there was no significant difference in the number of scratches in chronic itch group compared with control group.(10)After injecting a cocktail of r AAV-m DIx-Cre-WPRE-p A and r AAV-Ef1?-DIO-h M4D(Gi)-m Cherry-WPREs-p A virus at a volume ratio of 1:3 into l PAG for 21 days,modeling was carried out with acetone and diethyl ether.After 9 days,1 hour before the detection of behavioral test,intraperitoneal injection of CNO inhibited GABAergic neurons on l PAG,and we found that the number of scratch bouts in chronic itch group was significantly higher than that in control group;compared with chronic itch group,EA group and sham EA group had no significant difference in the number of scratch bouts.Conclusion EA inhibits the expression of CB1 receptor in midbrain,weakens the inhibitory effect on GABAergic neurons,and reduces the release of 5-HT in medulla,thereby improving chronic itch.GABAergic neurons on l PAG are involved in the regulation of EA in chronic itch.PART? Endogenous cannabinoid CB1 receptor participates in the peripheral mechanism of electroacupuncture(EA)alleviating chronic itchingObjective To study the mechanism of EA on inhibiting peripheral GRP expression and mast cell proliferation through endocannabinoid CB1 receptor.Method(1)EA group and sham EA group: Current was delivered with a Han's acupoint nerve stimulator(LH202,China).EA was given at Quchi(LI11)and Hegu(LI4)on the left side of mice on the first day of modeling with acetone and ether,once every other day for 30 minutes,five times in total.The intensity of EA is 3 m A,the frequency is 100 Hz,and the wave width is 0.3 ms.In sham EA group,the needles were shallowly pricked at Quchi(LI11)and Hegu(LI4)acupoints subcutaneously without electricity,and the needles were kept for 30 minutes.(2)The number of mast cells in mouse cervical skin was detected by toluidine blue staining.(3)Immunofluorescence histochemistry was used to detect the expression of CB1 and GRP in the cervical dermis and DRG tissues of mice.(4)Western blotting was used to detect the effect of EA on the expression of CB1 protein in cervical skin of mice.Result(1)The number of mast cells in the cervical skin of mice in chronic itch group was significantly increased compared with that in control group after 9 days of modeling with acetone and ether;compared with chronic itch group,the number of mast cells in the cervical skin was significantly decreased in EA group;while the number of mast cells in the cervical skin of mice in sham EA group did not significantly change.(2)After 9 days of modeling with acetone and ether,the immunofluorescence results showed that the expression and positive area of GRP in cervical dermis and DRG of mice in chronic itch group was significantly increased compared with that in control group;compared with chronic itching model group,the expression and positive area of GRP in cervical dermis and DRG of mice were significantly decreased in EA group,while the expression and positive area of GRP in cervical dermis and DRG of mice in sham EA group did not significantly change.(3)After 9 days of modeling with acetone and ether,western blotting and immunofluorescence results showed that the expression and positive area of CB1 R protein in cervical skin and DRG of mice in chronic itch group was significantly increased compared with that in control group;compared with chronic itch group,the expression and positive area of CB1 R protein in cervical skin and DRG of mice was significantly decreased in EA group,and the expression and positive area of CB1R receptor didn't significantly change in cervical skin and DRG of mice in sham EA group.(4)Compared with control group,the number of mast cells and the expression and positive area of GRP in skin and DRG significantly increased in the chronic itch group after 9 days of modeling with acetone and ether.Compared with chronic itch group,the number of mast cells in skin tissue and the expression and positive area of GRP in dermis and DRG tissue of mice were significantly decreased in EA group;and intraperitoneal injection of CB1 receptor antagonist AM251 significantly reversed the effect of EA.Conclusion EA can reduce the number of mast cells in skin and the expression and positive area of GRP in dermis and DRG through downregulating the expression and positive area of CB1 receptor in skin and DRG,and then alleviate chronic itching.