Data Availability StatementThe datasets used and/or analyzed during the current research are available through the corresponding writer on reasonable demand. at baseline (one day before IT administration) with times 1, Cinnarizine 3, 5 and 7 after IT administration. Rats received IT regular saline, IT morphine or IT morphine + EA at ST36-GB34. The proteins degrees of ERK1/2, phosphorylated (p)-ERK1/2 and CB1 in the spinal-cord had been assayed by traditional western blotting. Furthermore, the result of IT shot from the CB1 agonist WIN 55,212-2 as well as the CB1 antagonist SR141716 in the antinociceptive aftereffect of EA in rats with MIH was looked into. Nociceptive ERK1/2 and behavior, phosphorylated (p)-ERK1/2 and CB1 proteins levels had been evaluated as stated above. The outcomes revealed that persistent IT shots of morphine induced a substantial decrease Cinnarizine in mechanised drawback threshold (MWT) and thermal drawback latency (TWL) followed with exceptional upregulation of p-ERK1/2 in the spinal-cord, which could end up being attenuated by EA on the ST36-GB34 acupoints. In the Cinnarizine rat style of MIH, IT shot of WIN 55,212-2 coupled with EA induced a substantial increase in MWT and TWL accompanied with a significant decrease in p-ERK1/2 and a significant increase in CB1 protein level compared with EA alone, while SR141716 induced the opposite results. The present study suggests that EA alleviates hyperalgesia induced by IT injection of morphine partially through the inhibition of ERK1/2 activation. Activation of the CB1 receptor enhances the antinociceptive effect of EA in rats with MIH partly through the regulation of the spinal CB1-ERK1/2 signaling pathway. (18). Briefly, rats without any anesthetic drug received acupuncture with two pairs of stainless acupuncture needles connected to two pairs of electrodes. Each pair of needles was inserted perpendicularly ~6 mm into the ipsilateral acupoints around the hind legs of the rats. Acupoints were located according to the Zusanli (ST36) and Yanglingquan (GB34) acupoints in humans. In rats, ST36 is located at the proximal 1/5 point on the line from the Cinnarizine depressive disorder lateral to the patella ligament, while GB34 is located at the depressive disorder anterior and inferior to the fibular head (19). A total of 2 non-acupoints were located 0.5 cm horizontal and lateral to the ST36 and GB34 acupoints, respectively, at non-meridian points. A constant electronic pulse (2 Hz, 1.5 mA) was administered by an electroacupuncture stimulator (SDZ-II; Suzhou Medical Equipment Factory, Suzhou, China) which was connected to the other end of the electrodes. When the EA was starting, the current was stimulating from one acupoint (ST36 or GB34) to another nonacupoint. Rats in the control group (n=16) received the acupuncture needles at the same points as the rats in the EA group but without EA treatment. These rats were kept in tubular acrylic holders for 30 min as served as controls. Experimental protocol Experiment 1: Effects of EA on MIH The animals were randomly divided into 3 groups (n=16 rats/group): The control group (C); the chronic morphine group (M); and the morphine + EA at ST36-GB34 group (ME). Animals in the M and ME groups were IT administrated twice with 15 g (10 l) morphine at 8 am and 6 pm daily for 8 days. Animals in the C group were IT treated daily with 10 l saline at the same time as the M group for 8 days. The animals in the ME group received EA activation (2 Hz, 1.5 mA, 30 min, two times/day) at the Zusanli-Yanglingquan acupoints (ST36-GB34) 20 min after morphine or saline administration every day. The mechanical withdrawal threshold (MWT; n=8 rats/group) and thermal withdrawal latency (TWL; n=8 rats/group) were decided at baseline (24 h before IT administration, day-1) and at the same time following the second treatment on days 1, 3, 5 and Cinnarizine 7 after drug administration. Around the 8th day after drug administration, randomized selecting of 6 rats in each group to collect the L4-6 segments of the spinal cord for determination of the levels of ERK1/2, p-ERK1/2 and CB1 in the intumescentia lumbalis of the HOXA2 spinal cord (as shown in Fig. 1). Open in a separate window Physique 1. Experimental design. Male Sprague-Dawley rats (weighing 24020 g each) randomly received C, M or ME treatment. Animals in the M and ME groups were IT.