The Analgesic Effect Of Risedronate On Neuropathic Pain In Rats And Its Possible Molecular Mechanism

BackgroundRisedronate(RIS)is the third generation of nitrogen-containing bisphosphonates which is mainly used for osteoporosis,bone cancer pain and Paget's disease.Previous studies have shown that RIS mainly produces analgesic effects by modulating bone metabolic disorders,but whether RIS can produce analgesic effects on neuropathic pain has not been reported.Neuropathic pain is a debilitating disease often caused by central or peripheral nerve injury.It can result in an increased response to noxious stimuli(hyperalgesia)and stimuli that do not normally provoke pain(allodynia).It has been shown that the activation of glial cells and p38 MAPK signaling pathway are involved in the occurrence and development of NP,which can secrete a variety of pro-inflammatory cytokines,such as L-1?,IL-6 and TNF-?,etc.It's known that both osteoclasts and microglial cells are members of the monocyte phagocytic system and derived from hematopoietic stem cells,and Service et al.found that bisphosphonates attenuated tumor-induced peripheral and central sensitization markers,such as the astrocyte marker GFAP,in bone cancer pain model.We hypothesize that RIS may reduce neuropathic pain by inhibiting the activation of glial cells and p38 MAPK pathway and down-regulating the expression of pro-inflammatory cytokines.ObjectiveIn this study,spinal nerve ligation model was used to simulate neuropathic pain.RIS was used to investigate the analgesic effect and molecular mechanism of NP.Firstly,subcutaneous injection of RIS was used to observe the changes of pain behavior in rats and to investigate the analgesic effect of RIS on NP.The expression changes of microglia marker OX42,astrocyte marker GFAP,and p38 proteins were examined to investigate the roles of glial cells and p38 MAPK signaling pathway in RIS inhibition of NP.The expression of pro-inflammatory cytokines L-1?,IL-6,and TNF-? was examined to investigate the effect of these factors on NP inhibition in RIS.Methods1 Male SD rats were randomly divided into 6 groups(n=8): Sham group,surgery(SNL)group,RIS low dose treatment group [R(L)group],RIS mid dose treatment group [R(M)Group],RIS high-dose treatment group [R(H)group],and control group(Control group);RIS(0.1,0.5,1 mg/kg)or sterile saline subcutaneously,started at 30 mins before surgery,was administered for 7 days.The mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were measured on preoperative day 1 and postoperative 1,3,5,7,10,and 14 days.According to the above experimental results,another group of rats was randomized.Divided into 2 groups(n=8): Surgical(SNL)group and RIS optimal dose treatment group [R(B)group].Administration time is 7d to 21 d after surgery,and the pain behavior tests were performed on rats in preoperative day 1 and postoperative 1,3,7,8,10,12,14,16,21 days.2 Male SD rats were randomly divided into 3 groups(n=3): Sham group,SNL group,and R(M)group.RIS injection dose was selected according to the above-mentioned pain behavior test results.The time was 30 minutes before surgery to 7 days after surgery.At the 7th day after operation,all rats in each group were selected and the expression levels of p-p38?OX42 and GFAP were detected by immunofluorescence staining.3 Male SD rats were also randomly divided into 4 groups(n=4): Sham group,SNL group,R(M)group and Control group.The time of administration was 30 minutes before surgery and 7 days after surgery.At the 7th day after operation,all rats in each group were selected and the expression of p-p38 in the spinal cord of each group was detected by Western Blot technique.4 Male SD rats were randomly divided into 4 groups(n=6): Sham group,SNL group,R(M)group and Control group.The time of administration was 30 minutes before surgery and 7 days after surgery.At 7 days after operation,all rats in each group were selected for enzyme linked immunosorbent assay(ELISA)to measure the changes in expression of the pro-inflammatory cytokines IL-1??IL-6 and TNF-? in spinal cord(SP)and dorsal root ganglion(DRG).Results 1 RIS can reduce hyperalgesia and allodynia in rats after nerve injury.There was no significant difference in MWT and TWL at 1d before operation(P>0.05)and also in Control groups(P > 0.05).Compared with Sham group,the MWT and TWL levels were down-regulated at each time point in the SNL group,R(L)group,R(M)group and R(H)group(P < 0.05),but in R(L)group?R(M)group and the R(H)group,all indicators were better than the SNL group on postoperative 5,7,10,and 14 days(P < 0.05).And between 5?7?10?14d,compared with R(M)group,the R(L)group and R(H)group had lower indexes(P <0.05).The above results show that RIS can inhibit mechanical hyperalgesia and thermal allodynia after nerve injury in rats.2 RIS can inhibit the activation of microglia in spinal dorsal horn of rats after nerve injury.At 7 days after operation,the expression levels of p-p38,OX42 and GFAP proteins in the spinal dorsal horn of the SNL group and R(M)group were significantly higher than those of the Sham group(P<0.05).Compared with the SNL group,the expression of p-p38 and OX42 proteins were decreased in R(M)groups(P<0.05).Simultaneous immunofluorescence double-label results showed that p-p38 only co-standarded with OX42 but not with GFAP and NeuN.These results suggest that RIS may have analgesic effect on neuropathic pain by inhibiting the activation of microglia and the activation of p-p38 MAPK signaling pathway.3 RIS mediates neuropathic pain through the p38 MAPK signal transduction pathway.At 7 days after operation,in SNL group the expression of p-p38 was increased in the spinal cord compared with Sham group(P<0.05),also there was no significant difference in p-p38 expression of R(M)group(P>0.05).Compared with the SNL group,the expression of p-p38 was decreased in R(M)group(P<0.05).The above results prove once again that RIS may inhibit the activation of p-p38 MAPK signaling pathway and produce analgesic effect on neuropathic pain.4 RIS can down-regulate the expression of pro-inflammatory cytokines in rat spinal cord and dorsal root ganglion following nerve injury.After continuous administration for 7 days,the expressions of pro-inflammatory cytokines IL-1?,IL-6 and TNF-? in the spinal cord(SP)and dorsal root ganglion(DRG)were significantly upregulated in the SNL group(P < 0.05).But in the R(M)group,the expression of pro-inflammatory cytokines in the spinal cord(SP)and dorsal root ganglion(DRG)was significantly lower than that in the SNL group(P <0.05).This shows that RIS may produce anti-inflammatory and analgesic effects by inhibiting pro-inflammatory cytokines secreted by glial cells.ConclusionThe above results demonstrate that RIS can reduce neuropathic pain by inhibiting the activation of microglia,p38 MAPK signal transduction pathway,and down-regulating the expression of pro-inflammatory cytokines.