| BackgroundNeuropathic pain is one of the most common chronic pains.Neuropathic pain is mainly caused by nerve injury or inflammation,continuous pain symptoms after the wound healing is prominent feature of neuropathic pain,which is intense and stubborn and causes great pain to the patient.The exact mechanisms of neuropathic pain are still not clear.Poly(ADP-ribose)ation(PAR)is catalyzed by poly(ADP-ribose)polymerase(PARP)using nicotinamide adenine dinucleotide(NAD~+)provides ADP-ribose and combine with related nuclear proteins to form poly ADP-ribosylated nuclear proteins.PAR is considered to be a form of epigenetics.Studies implied that activation of PARP-1 promotes inflammation-related gene expression,which plays an important role in the development of inflammatory diseases.Recent studies demonstrated that nucleoprotein poly ADP-ribosylation catalyzed by PARP-1participates in long-term synaptic plasticity in brain learning and memory by regulating gene transcription.The plasticity of synaptic transmission at the level of the spinal cord induced by peripheral nerve injury or inflammation is considered to be the basis for the central sensitization of pathological pain formation and maintenance.However,the roles of PARP-1 in neuropathic pain have not been further studied and reported systematically.In this study,Lumbar 5 Spinal Nerve Ligation(L5 SNL)pain model,in combination with pain behavior tests and molecular biology techniques such as Western blot,RT-PCR,and qPCR,were used to observe that the role of ribozyme PARP-1 in neuropathic pain induced by L5 SNL and the mechanism of PARP-1activation in regulating neuropathic pain.The results will provide a new theoretical basis for further elucidating the mechanism of neuropathic pain,and may also provide new targets for the treatment of clinical pathological pain.Results1.Expression of PARP-1 and its catalytic product PAR,pro-inflammatory cytokines IL-1?and TNF-?in DRG and spinal cord after rat L5 SNL.(1)Establishment of L5 SNL model Male SD rats were randomly divided into experimental group(L5 SNL group,n=10)and control group(sham group,n=8).L5 SNL was performed according to the methods reported in the literature.The Paw withdrawal threshold(PWT)and Paw withdrawal latency(PWL)were detected on postoperative 1,3,5,7,10,14,21 and 28 days.After 1 day,PWT and PWL in the hind-limb of the rats began to decrease significantly(***P<0.001,**P<0.01compared with the sham group)and reached a minimum on the 7th day after operation(compared with the sham group,***P<0.001,***P<0.001),but no significant changes on the contralateral side.(2)Expression changes of PARP-1,PAR and IL-1?and TNF-?in rat DRG and spinal dorsal horn after L5 SNL The L4-5 DRG and L4-5 spinal cords were examined by RT-PCR and qPCR at 0,1,3,7,10,and 14 days after operation to detect PARP-1 mRNA levels,and the contralateral L4-5 DRG was taken.QPCR was used to detect the changes of PARP-1 m RNA levels in the L4-5 spinal cord expression if PARP-1 and its downstream product PAR,proinflammatory cytokines IL-1?and TNF–?were detected by Western blot in L4-5 DRG and L4-5 spinal cords.Results of RT-PCR and qPCR showed that L5 SNL induced improvement of the PARP-1mRNA level in the spinal L4-5 DRG and L4-5 spinal cords,and reached the highest level on the 7th or 10th postoperative day(compared with the control group,*P<0.05,**P<0.01,***P<0.001,n=3).Nevertheless,contralateral qPCR results showed that there is no significant difference in PARP-1 mRNA levels.Western blot results showed that the expression levels of PARP-1and its catalytic product PAR,IL-1?and TNF-?were maintained at a high level after L5 SNL,and reached highest on the 7th day compared to control group,(*P<0.05,**P<0.01,***P<0.001,n=3).These above results indicated that L5 SNL induced PARP-1 and its catalytic product PAR,as well as the expression of pro-inflammatory cytokines IL-1?and TNF-?in the spinal L4-5 DRG and L4-5 spinal cord.2.Cell Types of PARP-1 Expression in DRG and Spinal Dorsal Horn after Rat L5 SNL.The L4-5 DRG and spinal cord of L5 SNL and normal rats were stained by using immunofluorescence histochemical technique.We observed that L5 SNL induced the expression of PARP-1 in DRG and spinal dorsal horn increasing.Immunofluorescence double staining technique was further used to observe the cell types of PARP-1 expressed in DRG and spinal cord.The results showed that PARP-1is colocalized with large and medium-sized neuronal cells and small neurons in DRG and colocalized with neuronal cells and satellite-like glial cells in the spinal cord.3.Intrathecal injection of PARP-1 inhibitor Tiq-A can partly inhibit the neuropathic pain caused by L5 SNL,and it can also down-regulate the expression of PAR and pro-inflammatory cytokines IL-1?and TNF-?.After L5 SNL,PARP-1 inhibitor Tiq-A(5,25,50?g/10?l)was intrathecal injected once a day for 5 consecutive days in rats,and pain behavior changes were observed.The results showed that intrathecal injection of PWT and PWL in the hind-limb of the Tiq-A group showed a dose-dependent increase(1 d,P<0.001;3 d,P<0.001;5 d,P<0.001;7 d,P<0.001;10 d,P<0.001;14 d,P<0.001,n=8),compared with the solvent group(i.t.10?L 10%DMSO normal saline).Western blot results showed that the expression of PAR,IL-1?,and TNF-?decreased in the L4-5DRG and L4-5 spinal cords at the 7th day after operation in the high-dose group(i.t.50?g/10?l)(*P<0.05,**P<0.01,***P<0.001,n=3),compared with L5 SNL+Vehicle group.This indicates that inhibition of PARP-1 activity partially inhibit the development of neuropathic pain.Meanwhile,the expression of PAR,IL-1?and TNF-?induced by L5 SNL in the L4-5 DRG and L4-5 spinal cords of rats were also inhibited.4.After the development of neuropathic pain,intrathecal PARP-1inhibitor Tiq-A can partially reverse the symptoms of neuropathic pain.Rats were intrathecally injected with Tiq-A 50?g/10?l per day for 4 consecutive days at the 7th day after L5 SNL.Rats'hind limb PWT and PWL were measured.Our data showed that the PWT and PWL of the hind-limb of the rat increase significantly after the injection of the drug,and continued increase until the 14th day after the drug withdrawal(7 d,P<0.001;10 d,P<0.001;14 d,P<0.001,n=7).This indicates that inhibition of PARP-1 activity after neuropathic pain build-up can partially reverse the neuropathic pain caused by L5 SNL.5.Intrathecal injection of PARP-1 siRNA can partially inhibit the formation of neuropathic pain.The rats were randomly divided into sham+Transfection Regant(TR)group,L5SNL+PARP-1 siRNA group,L5 SNL+Scramble siRNA group,L5 SNL+TR group,8 in each group,and immediately injected drugs after the surgery.The siRNA group was injected with 1 nmol siRNA per day,and the transfection reagent group was injected with 10?l transfection reagents per day for 5 consecutive days.Meanwhile,changes in pain behavior of the rats were observed.The results showed that the intraoperative PARP-1 siRNA group and the L5 SNL+TR group showed that the PWT in the hind-limb of the rats began to increase significantly at 3 days after operation(***P<0.001),and continued until the 7th day after surgery(***P<0.001).The PWL of the operative side of the hindlimb was significantly elevated on the 1st postoperative day(**P<0.01),and it increase persistently until the 7th day postoperatively(***P<0.001).There was no statistical difference between Scramble siRNA group and L5 SNL+TR group.Western blot results showed that intrathecal injection of PAPR-1 siRNA reduced the expression of PARP-1,PAR,and IL-1?and TNF-?in spinal cord L4-5 DRG and L4-5 spinal cord(compared with sham+TR group,#P<0.05,##P<0.01,n=3).6.Motor function tests found that administration did not affect the motor function of the experimental rats.Rats were tested for motor function after testing the withdrawal threshold of mechanical stimulation and latent period of withdrawal after thermal stimulation.the tests of reflex,grasping reflex and righting reflex were performed,and the rats dosed showed no motor dysfunction..ConclusionUpregulation of PARP-1 contributes to the development and maintenance of neuropathic pain via regulating IL-1?and TNF-?expression in DRG and spinal cord following L5 SNL in rats. |
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