The Study Of Pain Regulation Of GalR1 By Inhibiting ERK1/2 Transduction Pathway In Rats NAc With Neuropathic Pain

Objective:It’s reported that galanin (Gal) and galanin receptor 1 (GalR1) were involved in the modulation and transmission of algesthesia in the central nervous system, and the effects of Gal and GalR1 were related to extracellular signal-regulated kinasel/2 (ERK1/2) signal transduction pathway. But the specific molecular mechanism remained indistinct. In present study, the pain behavior test was employed to detect the Hindpaw Withdraw Latencies (HWLs) of neuropathic pain rats to noxious thermal and mechanical stimulation, and Western blot was used to test the expression of Gal, GalR1 and p-ERK1/2 in the NAc of rats with mononeuropathy, then intral-NAc administration of GalR1 agnoist M617 or antagonist M35 was to observe the aforementioned indexes. Above all, the study was aimed to research the antinociception of GalR1 in the NAc of rats with neuralgia, and to investigate the mechanism how the analgesia of GalRl acts via ERK1/2 signal transduction pathway in the NAc of rats with neuropathic pain.Methods:1. The animal model of neuropathic pain was duplicated by ligating the left sciatic nerve of Sprague-Dawley (SD) rats. The rats were divided into the sham-operation group and experimental group including the rats with left sciatic nerve ligation for 7 days,14 days and 28 days respectively.2. The nociceptive test was utilized to respectively measure the HWLs response to noxious thermal and mechanical stimulation by the hot plate and the Randal Selitto in rats with sham operation and with left sciatic nerve ligation for 7 days,14 days,28 days. The HWLs was considered as the index of hyperalgesia.3. The Western blot was used to test the expression of Gal, GalR1 and p-ERKl/2 in the NAc of rats with sham operation and left sciatic nerve ligation for 7 days,14 days,28 days respectively.4. Catheter implantation into the NAc of rats. The catheter was implanted in to the NAc of rats at 5 day after left sciatic nerve ligation.5. At 28 day after left sciatic nerve ligation, the rats were respectively received intra-NAc administration of:①1μl of sterilized 0.9% saline as control, ②1μl of sterilized 0.9% saline with lnmol of M617, ③1μl of sterllized 0.9% saline with lnmol of M35. The HWLs of rats with left sciatic nerve ligation 28 days to noxious thermal and mechanical stimulation was respectively measured after 1h followed the administration.6. The Western Blot was employed to detect the expression of p-ERK1/2 in the NAc of rats with left sciatic nerve ligation for 28 days after the intra-NAc administration.Results:1. Ligating the left sciatic nerve decreased the HWLs of rats to noxious thermal and mechanical stimulation. Compared with the sham operation group, at 14 day (n=6; hot-plate test:qleft=6.77, p<0.001, qright=7.04, p<0.001 Randall Selitto test: qleft=7.37, p<0.001, qright=6.66, p<0.001) and 28 day (n=6; hot-plate test:qleft=5.09, p<0.01, qright=4.76, p<0.05; Randall Selitto test:qleft=6.85, p<0.001, qright=4.92, p<0.05) after left sciatic nerve ligation, the HWLs of rats to noxious thermal and mechanical stimulation significantly decreased, indicating that the increase of hyperalgesia. In addition, the decreased left HWLs to noxious mechanical stimulation were observed at 7 day after left sciatic nerve ligation (n=6; hot-plate test:qleft=2.17, p>0.05, qrigght=1.76, p>0.05; Randall Selitto test:qleft=4.31, p<0.05, qright=1.73, p>0.05). 2. At 28 day after left sciatic nerve ligation, intra-NAc injection of lnmol of M617 significantly increased the HWLs to noxious thermal and mechanical stimulations compared with the 1ul sailne-treated control group (hot-plate test:qleft=9.01, p<0.001, qright=5.02, p<0.01; Randall Selitto test:qleft=11.37, p<0.001, qright= 11.34, p<0.001), but intra-NAc injection of lnmol of M35 impacted nonsignificant on the HWLs to noxious thermal and mechanical stimulation compared with the lul sailne-treated control group(hot-plate test:qieft=1.27, p>0.05, qright=0.48, p>0.05; Randall Selitto test: qleft=1.25, p>0.05, qright=0.43, p>0.05).3. The results from Western Blot showed that:① Gal:At 7 day (n=4; q=5.630; p<0.05),14 day (n=4; q=6.906; p<0.01) and 28 day (n=4; q=10.01; p<0.001), after left sciatic nerve ligation, the expression of Gal in the NAc of rats was gradually up-regulated compared with the sham operation group. ② GalR1l:At 14 day (n=4; q=6.144; p< 0.05) and 28 day (n=4, q=8.360; p<0.01) after left sciatic nerve ligation, the expression level of GalR1 increased significantly in the NAc of rats compared with the sham operation group, and the expression of GalR1 showed non-significant difference at 7 day (n=4; q=2.489; p>0.05) after left sciatic nerve ligation compared with the sham operation group. ③ p-ERK1/2:The expression of p-ERK1/2 increased at 28 day (n=4; q=6.264; p<0.01) after left sciatic nerve ligation compared with the sham operation group. At 7 day (n=4; q=3.74, p>0.05) and 14 day (n=4; q=2.209, p>0.05) after left sciatic nerve ligation, the expression of p-ERK1/2 showed non-significant difference compared with the sham operation group.④p-ERK1/2:At 28 day after left sciatic nerve ligation, the expression of p-ERKl/2 were significantly reduced in the NAc of rats after intra-NAc administration of lnmol M617 (n=4; q=4.671; p<0.05) compared with the 1ul sailne-treated control group. Otherwise, intra-NAc administration of lnmol M35 impacted non-significant difference (n=4; q=0.1459; p>0.05) compared with the 1ul sailne-treated control group.Conclusion:1. The operation of sciatic nerve ligation decreased the painful threshold of rats to noxious thermal and mechanical stimulation, inducing the neuropathic pain. 2. The operation of sciatic nerve ligation of rats up-regulated the expression of Gal and GalR1 in the NAc of rats, indicating that the involvement of Gal and GalR1 in the modulation of algesthesia in the NAc of rats with neuropathic pain.3. The operation of sciatic nerve ligation of rats up-regulated the p-ERK1/2 expression, suggesting that ERK1/2 signal transduction pathway involved in the transmission of algesia in the NAc of rats with neuropathic pain.4. Intra-NAc administration of GalR1 agonist M617 down-regulated the p-ERK1/2 expression in the neuropathic pain rats, intimating that the antinociceptive effect of GalRl could be achieved by inhibiting the ERK1/2 signaling transduction pathway in the NAc of rats with mononeuropathy.