Mechanism Of Electroacupuncture Analgesia In Rats With Acute Faflammatory Pain:Effecting On The Expression Of P-ERK1/2,p-CREB,NPY And Its Receptors In The Dorsal Horn Of Spinal Cord

RESEARCH BACKGROUND:Extracellular signal-regulated kinase（ERK）in spinal dorsal horn is activated in pain early and participates in the production and maintenance of pain.The activated extracellular signal-regulated kinase 1 and 2（ERK1/2）regulates the expression of target genes by regulating the activity of downstream transcription factorcAMP response element binding protein（CREB）.Spinal dorsal horn NPY and its two receptors（Y1R and Y2R）are located in the shallow dorsal horn of the spinal cord,which is a key area for nociceptive gating and regulation.In addition,NPY,its receptor and CREB can also regulate each other.OBJECTIVE:Observe the effects of electroacupuncture EA on the expression of p-ERK1/2,p-CREB,NPY and its receptors（Y1R and Y2R）in the spinal dorsal horn of acute inflammatory pain model rats,and explore the mechanism of treating acute inflammation pain with electroacupuncture"Sanli（ST36）+Yanglingquan（YC）".METHODS:Forty-eight male rats were randomly divided into a blank group（Saline group）,a model group（CFA group）,a fake electroacupuncture group（CFA+sham EA group）,and an electroacupuncture group（CFA+EA group）,with 6 rats in each group.Acute inflammatory pain model was established by injecting complete Freund’s adjuvant（CFA）into hind paws of the experimental rats.On the first day after successful modeling,electroacupuncture“ST36+YC”was started with a frequency of 2 Hz,an amplitude of 1 mA,a duration of 20 minutes,and a continuous wave once a day,Continuous treatment for 7 days.Thermal pain threshold（TWL）and mechanical pain threshold（PWTs）were calculated by thermal radiation test and von Frey test before modeling（T0）,and during modeling 1-7 day（T1-T7）after electroacupuncture therapy.The 3^rdd and 7^th day（T3 and T7）after electroacupuncture,the expression changes of NPY,NPY1R and NPY2R were examined by immunohistochemistry respectively and Western blot was used to detect the expression of ERK1/2,p-ERK1/2,CREB,p-CREB,NPY,NPY1R and NPY2R in the spinal dorsal horn.RESULTS:（1）At T1,the TWL and PWTs of the experimental rats after CFA injection were significantly reduced,and the difference was statistically significant（P<0.001）.Compared with the Saline group,the TWL and PWTs of the experimental rats in the CFA group and the CFA+sham EA group gradually decreased.By T3,the TWL and PWTs of the experimental rats were reduced to a minimum,and the difference was statistically significant（P<0.001）,and then gradually increased;there is no significant difference between the CFA+EA group and the Saline group.Compared with the CFA group,the PWTs in the CFA+EA group were significantly increased in the first 4 days,and the difference was statistically significant（P<0.001）.After T4,the PWTs were still significantly increased,and the difference was statistically significant（P<0.01）,but the TWL in the CFA+EA group continued to be normal,and the difference was statistically significant（P<0.001）;there was no significant difference between the CFA+sham EA group and it.（2）About p-ERK1/2and p-CREB:The results of Western blot showed that there was no significant difference in the expression of ERK1/2 and CREB proteins in the spinal dorsal horn of the experimental rats at T3 and T7.Compared with the Saline group,p-ERK1/ERK1,p-ERK2/ERK2,p-CREB/CREB increased in the spinal dorsal horn of the experimental rats in the CFA group and the CFA+sham EA group at T3,and the difference was statistically significant（P<0.05）.Them were significantly increased at T7,and the difference was statistically significant（P<0.01）;there were no significant differences between the CFA+EA group and it were at T3 and T7.Compared with the CFA group,p-ERK1/ERK1,p-ERK2/ERK2,and p-CREB/CREB were reduced in the spinal dorsal horn of the experimental rats in the CFA+EA group at T3,and the difference was statistically significant（P<0.05）.Them were significantly reduced At T7,and the difference was statistically significant（P<0.01）.（3）About NPY:immunohistochemical results showed that compared with the Saline group,the NPY in the spinal dorsal horn of the CFA group and the CFA+sham EA group was significantly increased at T3,and the difference was statistically significant（P<0.01）,Increased by T7,the difference was statistically significant（P<0.05）.The CFA+EA group increased significantly at T3 and T7,and the difference was statistically significant（P<0.001）.Compared with the CFA group,the NPY in the spinal dorsal horn of the CFA+EA group was significantly increased at T3,and the difference was statistically significant（P<0.01）.It continued to increase significantly at T7,and the difference was statistically significant（P<0.001）.Western blot results showed that compared with the Saline group,the NPY levels in the spinal dorsal horn of the CFA group,CFA+sham EA group and CFA+EA group were significantly increased at T3and T7（P<0.001）.Compared with the CFA group,the NPY in the spinal dorsal horn of the CFA+EA group was significantly increased at T3 and T7,and the difference was statistically significant（P<0.001）.（4）About Y1R and Y2R:Immunohistochemical results showed that compared with the Saline group,Y1R and Y2R were significantly increased in the spinal dorsal horn of the CFA group and CFA+sham EA group at T3,and the difference was statistically significant（P<0.01）).Them were significantly increased at T7,the difference was statistically significant（P<0.001）.There was no significant difference between the CFA+EA group and it at T3,and the CFA+EA group increased at T7,the difference was statistically significant（P<0.05）.Compared with the CFA group,Y1R and Y2R in the spinal dorsal horn of the CFA+EA group were reduced at T3,and the difference was statistically significant（P<0.05）,and the difference remained statistically significant until T7（P<0.05）.Western blot results showed that compared with the Saline group,Y1R was significantly increased in the spinal dorsal horn of the CFA group and CFA+sham EA group at T3 and T7,and the difference was statistically significant（P<0.001）.There was a statistically significant difference（P<0.001,P<0.01）about Y1R in the dorsal horn of the spinal cord between the CFA+EA group and it at T3 and T7.Compared with the CFA group,Y1R in the spinal dorsal horn of the CFA+EA group was significantly reduced at T3,and the difference was statistically significant（P<0.001）.it was still significantly reduced at T7,and the difference was statistically significant（P<0.01）.Compared with the Saline group,Y2R in the spinal dorsal horn of the CFA group and CFA+sham EA group was significantly increased at T3,and the difference was statistically significant（P<0.01）.At T7,them were significantly increased,and the difference was statistically significant（P<0.001）.At T3 and T7,the difference about Y2R in the spinal dorsal horn between the CFA+EA group and it was statistically significant（P<0.05,P<0.01）.Compared with the CFA group,the Y2R in the spinal dorsal horn of the CFA+EA group was reduced at T3,and the difference was statistically significant（P<0.05）.it was still significantly reduced at T7,the difference was statistical significance（P<0.01）.CONCLUSION:（1）Electroacupuncture can effectively relieve thermal hyperalgesia and mechanical hypersensitivity in CFA inflammatory pain model rats.（2）The ERK1/2-CREB signaling pathway may be involved in the formation of acute inflammatory pain.The mechanism of electroacupuncture for alleviating acute inflammatory pain may be related to the down-regulation of the ERK1/2-CREB signaling pathway in the spinal dorsal horn.（3）Electroacupuncture may exert anti-nociceptive effects by inducing a large amount of NPY expression and inhibiting the expression of Y1R and Y2R receptors in the spinal dorsal horn of acute inflammatory pain model rats.（4）Electroacupuncture may regulate the expression of NPY and its receptors through the ERK1/2-CREB signaling pathway.