Roles Of GalR1 In Curing Neuropathic Pain Of Rats Suffered From CCI Through DREAM Silencing Mediated By Lentivirus

Objective:The galanin receptor-1(GalR1) belongs to a G-protein-coupled receptor.The signal pathway of galanin plays an important role in pain modulation.To elaborate the mechanism of DREAM modulation pain further,our study approaches the roles of the galanin signal pathway in dorsal horn in the therapy of neuropathic pain in rats suffered from CCI through DREAM silencing mediated by lentivirus.Method:Twenty four male and healthy rats were divided into four groups(n=6,each):Group RNAi,Group blank vector,Group CCI and Group normal.Paw withdraw thermal latency(PWTL) and paw withdraw mechanical threshold(PWMT) were measured before inserting intrathecal cather,CCI model and every 2days after CCI 2d,4d,6d,8d,10d,12d and 14d.The rats in Group RNAi and Group blank vector were received pKCSHR-Puro/GFP-DREAM-LV(1×10⁹ IU/ml)10μl+NS5μl or blank vector 10μl+NS5μl via intrathecal injection once a day for 3 days from 7^th after CCI.After 14th day CCI,the spinal cords of L4-5 were took out and a part of them was took into paraffin sections,for observing the expressions of green fluorescent protein(GFP) via fluorescence microscope.Another part of them was quickly put into freezing tube and then conserved in liquid nitrogen for determing the expressions of mRNA of DREAM gene and GalR1 gene by Real-time PCR and the expressions of DREAM protein and GalR1 protein by Western-Blot method.Result:1.PWTL and PWMT:there were no differences among 4 groups with basic pain threshold in the days before inserting intrathecal cather and CCI model(P＞0.05).The PWTL and PWMT from the two days after CCI in Group RNAi,Group blank vector and Group CCI of every time point were much lower than Group normal(P＜0.01).The pain threshold after cured was higher than that at six days after CCI in Group RNAi(P＜0.01).However,the pain threshold after cured in the same time point in Group RNAi were also higher than that in Group blank vector and Group CCI(P＜0.01).There were no differences in the opposite side of operation(the right side)(P＞0.05).2.After the 14th day CCI,there are a lot of green fluorescent in dorsal horn in Group RNAi,which denoted expressions of GFP.It suggested that transfection of LV vector was success in dorsal bore in Group RNAi.3.The results of Real-time PCR:the DREAM gene expressions of mRNA in dorsal horn in the 14^th day after CCI in Group RNAi is lower than that in other Groups(P＜0.01).And there were no differences in the other groups(P＞0.05).The GalR1 gene expressions of mRNA in dorsal horn in Group RNAi were lower than that in Group blank vector and Group CCI(P＜0.01),but which were higher than that in Group normal (P＜0.01).And there were no differences in Group blank vector and Group CCI(P＞0.05).4.The results of Western-Blot method:the expressions of DREAM protein were down-regulated in Group RNAi.And there were no differences in other group(Group blank vector,Group CCI and Group normal).The expressions of GalR1 protein in Group RNAi,Group blank vector and Group CCI were up-regulated significancely compared with that in Group normal(P＜0.05).The expressions of GalR1 protein in Group RNAi were down-regulated compared with that in Group blank vector and Group CCI(P＜0.05).And there were no differences in Group blank vector and Group CCI(P＞0.05).Conclusion:1.DREAM may take part in the regulation of expressions of GalR1 gene and protein.2.GalR1 plays an important role in the treatment of neuropathic pain in rats suffered from CCI through the DREAM silencing mediated by lentivirus.