| With the further research of NR2B, the NR2B subunit is limited distributed in the central nervous system. Researchers expect to be able to develop more drugs specially targeted on the NR2B subunit in the processing and regulation of the pain related aversion, which are finer, more accurate adjustment to the targets and less side effects of non-selective NMDA receptor antagonist such as hallucinations, irritable and so on. In recent years,a large number of studies turned to this direction. For example, the gentiopicroside could not only effectively inhibit inflammation pain-induced allodynia, but also down-regulate the expression of the NR2B subunit in the ACC, indicated that the analgesic effect of gentiopicroside was closely related with the stimulation and active reactions of the glutamatergic synaptic transmission. Ifenprodil, a selective NR2B receptor antagonist, could highly act on the NR2B subunit, inhibit the expression of NR2B subunit and relieve the pain-induced by the compression injury in the dorsal root ganglion. Ifenprodil was already used to assess the role of the NR2B subunit in the brain function and disease, which maybe provide a therapeutic stragety in pain, mood disorders and neurodegenerative diseases。Small RNA interference technology, with the advantages of highly effective, specific, stable, short cycle and easy operation, is commonly used in genetic research recently to down-regulate or silence the target gene. Construct the siRNA-recombinant vector in vitro and then transfer it into the cells to generate the siRNA, which can not only increase the types of the effective tansfection cells, but also stably express. These siRNA could the expression of target gene for a long time. Tan and his colleagues found that down-regulation the expression of the NR2B subunit with intrathecal NR2B shRNA targeting NR2B contribute to inhibit the formalin-induced nociceptive aversion [4]In this study, we construct the siRNA-recombinant vector firstly and selected lentivirus-mediated RNAi vectors targeting the NR2B subunit. Then we observe the therapeutic effect on the bone caner-induced pain aversion with intrathecal NR2B shRNA targeting the NR2B subunit.Methods and Results1Effect of the ifenprodil administered into the rACC on the pain-related aversion in rats with bone-cancer pain.Methods:Totally thirty male wistar rats without place preference were randomly divided into three groups:control group (C-group, n=10), bone-cancer pain group (P-group, m=10) and ifenprodil group (Ifen-group, n=10). A total volume of3-μl MADB-106cells (4.8×109/mL) were inoculated into right tibia bone marrow cavity in P-group and Ifen-group, rats of C-group given NS at the same dose as a control. A total volume of1.2μl ifenprodil, a selective NR2B antagonist, was administered into the rACC on the14th day after operation while the C-group and the P-group given NS at the same dose as a control. The diversity of the mechanical stimulation withdrawal threshold were measured using Von Frey stimulation on the first day before the operation and the third,7th,10th,12th and14th day after the operation. And on the14th day after the injection with the ifenprodil, observe the pain-related aversion m rats.Results:After the operation, the mechanical stimulation withdrawal threshold decreased obviously in rats of the P-group and Ifen-group from the10th day to the14th day (P<0.05). The group comparison showed that the pain threshold to mechanical stimulation of rats in P-group and Ifen-group decreased significantly compared with C-group by day10th,12th and14th after inoculation with the tumor cells (P<0.05). The percentage time within the chamber A was (30±4)%in P-group, lower than the (52±5)%in C-group (P<0.05). After ifenprodil treatment, the percentage time within the chamber A of Ifen-group was (42±5)%, higher than P-group, but still lower than C-group (P<0.05). 2Intra-rACC lentiviral-mediated RNA interference targeting NR2B decrease the bone cancer pain-induced aversion in rats.Methods:Forty five rats were divided into three groups randomly, including the LV-NR2B group, LV-NC group and NS group. A total volume of3-μl MADB-106cells (4.8×109/mL) were inoculated into right tibia bone marrow cavity in rats. Secured the animals in the stereotaxic apparatus with placing bregma and injected NR2B/siRNAi recombinant lentivirus into rACC in rats (LV-NR2B group-recombinant lentivirus NR2B, LV-NC group-empty lentivirus containing no inserted sequence and NS group-injected with the same volume saline). The diversity of the mechanical stimulation withdrawal threshold were measured using Von Frey stimulation on the first day preoperative and the third,7th,14th and21st day postoperative. And on the14th day after the injection with the ifenprodil, observe the pain-related aversion in rats. After the behavioral detections, put to death all rats of different groups. Then detect the NR2B mRNA and NR2B protein expression respectively with RT-PCR, Western Blotting and immunofluorescence techniques.Results:Following the intra-ACC microinjection, we detected the behavior of the rats to probe preferably the therapeutic effect for3weeks. The group subjected to LV-NR2B vectors exhibited statistically significant ease of allodynia when compared with the NS or LV-NC groups (both P less than0.05). Accordingly, no differences were found between the NS and LV-NC groups. Interestingly, a statistically significant increase within the A room was observed in LV-NR2B groups compared with the NS or LV-NC groups (P less than0.05). LV-NR2B resulted in59.2%reduction of NR2B mRNA compared with the LV-NC and NS groups by RT-PCR (P less than0.05). Consistent with the observation of the down-regulation of NR2B mRNA by the RT-PCR, western blot analysis displayed a decrease up to49.6%compared with the LV-NC and NS groups (P less than0.05). In frozen and adjacent sections of the rACC, NR2B protein labeling by immunofluerence revealed a significant down-regulation in the NR2B group, while no obvious inhibition of NR2B protein was observed in the NS control group and LV-NC group. ConclusionIfenprodil, a selective NR2B antagonist, administers into the rACC, can effectively alleviate the pain-related aversion in rats with the tibial bone cancer. Besides, intra-rACC delivery of lentiviral-mediated RNAi targeting NR2B could effectively attenuate bone caner-induced mechanical allodynia and pain-related aversion in rats. The present study suggests that NR2B is a feasible RNAi target for bone cancer pain. And lentiviral vector delivery strategies are a promising novel approach for the treatment of bone cancer pain and for future studies on gene functions of NR2B. |
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