Inhibiting The JNK Signaling Pathway Reverses Hyperalgesia And Anxiety-Like Behavior In A Non-Specific Chronic Low Back Pain Model

Objective: Low back pain(LBP)has become a leading cause of disability worldwide.Astrocyte activation in the spinal cord plays an important role in chronic pain,spinal c-Jun N-terminal kinase(JNK)participates in astrocyte activation and induces the production of inflammatory cytokines.However,the role of the JNK signaling pathway in modulating the behavior of LBP model rats and its mechanism has not been elucidated.This study was to investigate the potential mechanism of the JNK signaling pathway on hyperalgesia and anxiety-like behavior in male non-specific LBP model rats.Methods: LBP was produced by two injections(day 0 and day 5)of NGF(nerve growth factor)into the multifidus muscle in the low back of male rats.The research was mainly divided into two parts:The first part was to study whether the NGF-induced LBP model presented with hyperalgesia to different noxious stimuli and anxiety-like behavior.The influence of NGF repetitive injections on astrocyte activation and chemokine expression in the L2 segment of the spinal cord was studied.Male SD rats were randomly divided into model(NGF)group and control(PBS)group.On day-1,9,12 and 15,mechanical pain threshold in the low backs or hindpaws,hot pain threshold and cold pain threshold in hindpaws were examined.On day 9,12 and 15,the open field test(OFT)was performed.Anxiety-like behavior was evaluated by the open field test,the inner zone distance percentage(ID%)and the percentage of inner zone time(IT%)were calculated.The density of GFAP positive cells at the ipsilateral side of the superficial laminae in the L2 segment of the spinal cord was observed by immunofluorescence.The protein expression of p-JNK,GFAP(glial fibrillary acidic protein),c-fos and chemokines(MCP-1 and CXCL1)in the L2 segment of the spinal cord were detected by Western blot.The second part was to explore whether JNK inhibitor interfered with hyperalgesia and anxiety-like behavior in the NGF-induced LBP model.The influence of the JNK inhibitor on astrocyte activation and chemokine expression in the L2 segment of the spinal cord was studied.The JNK inhibitor SP600125 was intrathecally administrated in rats from day 10 to day 12.Male SD rats were randomly divided into four groups:PBS+Vehicle(Veh.)group,NGF+Veh.group,NGF+SP600125 group,PBS+SP600125group.On day 12,mechanical pain threshold in the low backs or hindpaws,hot pain threshold and cold pain threshold in hindpaws were examined.Anxiety-like behavior was evaluated by the OFT and the ID% and IT% were calculated.The density of GFAP positive cells at the ipsilateral side of the superficial laminae in the L2 segment of the spinal cord were observed by immunofluorescence.The protein expression of p-JNK,GFAP,c-fos and chemokines(MCP-1 and CXCL1)in the L2 segment of the spinal cord were detected by Western blot.Results:Result 1: On day 9,12 and 15,the results of behavior showed that,compared with PBS group,rats in NGF group presented with decreased back pressure pain threshold(PPT)and paw withdrawal threshold(PWT)in the ipsilateral side(Ps<0.001),decreased paw withdrawal latency(PWL)(Ps<0.05)of the hot plate in the hindpaw,increased the number of responses(Ps<0.05)to the cold plate in the hindpaw.The results of OFT showed that,compared with PBS group,rats in NGF group presented with decreased IT% and ID%(Ps<0.01).The results of immunofluorescence showed that,compared with PBS group,rats in NGF group presented with increased density of GFAP positive cells at the ipsilateral side of the superficial laminae(Ps<0.001).The results of Western blot showed that,compared with PBS group,rats in NGF group presented increased protein expression of GFAP,c-fos,p-JNK,MCP-1 and CXCL1 in the L2 segment(Ps<0.01).Result 2: On day 12,the results of behavior showed that,compared with NGF+Veh.group,rats in NGF+SP600125 group presented with increased back pressure pain threshold(PPT)and paw withdrawal threshold(PWT)in the ipsilateral side(Ps<0.001),increased paw withdrawal latency(PWL)(P<0.01)of the hot plate in the hindpaw,decreased the number of responses(P<0.05)to the cold plate in the hindpaw.The results of OFT showed that,compared with NGF+Veh.group,rats in NGF+SP600125group presented with increased IT%(P<0.01).The results of immunofluorescence showed that,compared with NGF+Veh.group,rats in NGF+SP600125 group presented with decreased density of GFAP positive cells at the ipsilateral side of the superficial laminae(P<0.001).The results of Western blot showed that,compared with NGF+Veh.group,rats in NGF+SP600125 group presented with decreased protein expression of GFAP,p-JNK,MCP-1 and CXCL1 in the L2 segment(Ps<0.05),and a significant trend(P = 0.067)was found in the decrease of c-fos expression in NGF+SP600125 group.Conclusion: Hyperalgesia and anxiety-like behavior induced by repetitive NGF injections in LBP model rats could be reversed by the JNK inhibitor,meanwhile,SP600125 decreased astrocyte activation and the expression of c-fos,MCP-1,and CXCL1 proteins.The results indicate that intervening with the JNK signaling pathway in the spinal cord might be an effective therapeutic approach to alleviating LBP.