| BackgroundNerve injury or inflammation often causes neuropathic pain,mainly manifested as allodynia,hyperalgesia and spontaneous pain pain.At present,the mechanism is still not very clear,and the efficacy is not good,causing great pain to patients.T-LAK cell originated protein kinase(TOPK)is a newly discovered serine-threonine kinase that participates invarious biological processes such as tumorigenesis and development,cell proliferation and apoptosis,and inflammation.Previous studies have found that TOPK is expressed in testicular germ cells and several fetal tissues(including neural stem cells);recently it has been reported that TOPK is also expressed in the brain of ischemia-reperfusion rats.TOPK belongs to the upstream kinase of the Mitogen-activated protein kinase(MAPK)family.Previous studies have shown that activation of the MAPK signaling pathway is involved in neuropathic pain.Therefore,we speculate that TOPK may be related to the occurrence of pathological pain.However,the expression of TOPK in DRG and SP and its role in neuropathic pain have not been reported today.In the experiment,Lumbar 5 Spinal Nerve Ligation(L5 SNL)was used as a model for inducing neuropathic pain,and then the rats were tested for mechanical stimuli withdrawal threshold and heat contraction foot latency to observe the pain behavioral changes of rats.Through immunofluorescence histochemistry,Western blot,RT-PCR and qPCR and other molecular biology techniques,observe the expression of TOPK in the DRG and spinal dorsal horn of L5 SNL rats,combined with intrathecal microinjection of TOPK inhibitor and TOPK siRNA to explore TOPK Role in neuropathic pain in rats.The experimental results further clarified the mechanism of neuropathic pain and provided a new theoretical basis for it,and also provided a new drug target for the prevention and treatment of neuropathic pain.Results1.Changes in the expression of total TOPK(t-TOPK)and phosphorylated TOPK(p-TOPK)in DRG and spinal dorsal horn after SNLRandomly divide the rats into a control group(sham group,n=8)and an experimental group(L5 SNL group,n=8)and perform L5 SNL surgery,based on PWT and PWL 3 days and 1 day before surgery,Then the PWT and PWL of rats were detected 1,3,5,7,10,14,21 and 28 days after SNL,respectively.The results showed that the PWT and PWL of rats began to decrease from 1 day after surgery,and fell to the lowest on the 7th day,and the significant difference was maintained until 28 days after surgery(compared to the sham group,***P<0.001,one-way ANOVA).Compared with the preoperative basic value,there was no obvious change in the contralateral PWT and PWL,indicating that the model was successfully established.After L5 SNL in rats,the rats were acutely killed 1,3,7,14 and 21 days after surgery,respectively,and then the rat L4/L5 DRG and spinal dorsal horn were taken to detect the protein content changes of t-TOPK and p-TOPK And t-TOPK mRNA expression changes.Western blot results showed that there was no significant change in the amount of protein in the DRG and spinal dorsal horn of the spinal cord t-TOPK after L5 SNL,while the amount of protein expression in the dorsal root ganglion and spinal dorsal horn of the spinal cord was increased.It rose to the peak on the third day(compared to control,**P<0.01,**P<0.01,one-way ANOVA).RT-PCR and qPCR results showed that the expression of t-TOPK mRNA in DRG and dorsal horn of spinal cord increased after L5 SNL,and peaked on the third day(compared to control,*P<0.05,***P<0.001,one-way ANOVA).The above results indicate that rat L5 SNL caused the upregulation of p-TOPK protein expression in DRG and spinal dorsal horn and up-regulation of t-TOPK at the mRNA level.2.Cell types of t-TOPK and p-TOPK expressed in the DRG and spinal dorsal horn after L5 SNL in ratsIt was verified by immunofluorescence histochemistry.Immunofluorescence single staining results showed that the expression of t-TOPK in the dorsal root ganglion and spinal dorsal horn of the surgical side of the rat at 3 days after L5 SNL was unchanged compared with the sham group,while L5 SNL The expression level of p-TOPK in the dorsal root ganglion and spinal dorsal horn of rats was increased compared with the sham group 3 days after surgery.Then,using immunofluorescence double-staining technology,we found that in DRG,p-TOPK coexists with class A neuronal marker NF-200 and small-diameter non-peptide neuronal marker IB4,but with satellite-like glial cell marker GFAP No coexistence.In the dorsal horn of the spinal cord,p-TOPK coexists with the neuronal marker NeuN and astrocyte GFAP,but it is not expressed in microglia.3.Prior intrathecal injection of TOPK inhibitor HI-TOPK-032 alleviated neuropathic pain and inhibited p38 MAPK activation in the DRG and spinal dorsal horn following SNLAfter intrathecal injection of different doses of TOPK inhibitor HI-TOPK-032,the changes in pain behavior of rats after L5 SNL were observed.The results showed that the PWT and PWL of rats in the HI-TOPK-032 group after intrathecal injection of L5 SNL increased in a dose-dependent manner.Compared with the L5 SNL+Vehicle group,the PWT of the L5 SNL+HI-TOPK-032 100 ?g/10 ?l group began to increase on the first postoperative day until the fifth day(***P<0.001,one-way ANOVA);compared with the L5 SNL+Vehicle group,the PWL of the L5 SNL+HI-TOPK-032 100 ?g/10 ?l group began to increase on the 3rd postoperative day until the 7th day(**P<0.01,***P<0.001,one-way ANOVA).The PWT and PWL of normal rats after intrathecal injection of HI-TOPK-032(100 ?g/10 ?l)were unchanged from the baseline values.The experiment further observed the effect of TOPK inhibition on p38 MAPK.The experiment was divided into four groups:Naive group,L5 SNL+Vehicle group,L5 SNL+HI-TOPK-032 group,and Naive+HI-TOPK-032 group.The DRG and spinal dorsal horn of the operation side of rats in different groups.Western blot results showed that after intrathecal injection of HI-TOPK-032,the protein content of t-TOPK and t-p38 downstream substrate of DRG and spinal dorsal horn were not significantly different between the four groups;however,compared with the control group,L5 SNL+Vehicle The protein contents of p-TOPK and p-p38 in the group were significantly increased in the dorsal root ganglion and dorsal horn of the spinal cord(*P<0.05,**P<0.01,one-way ANOVA);and this change can be sheathed Intra-microinjection of HI-TOPK-032 inhibited(SNL+HI-TOPK-032 group compared with SNL+Vehicle group:*P<0.05,**P<0.01,one-way ANOVA).From the above results,it can be seen that the inhibition of TOPK activity can partially inhibit the formation of neuropathic pain,and also can inhibit the upregulation of p-p38 protein expression in dorsal root ganglion and spinal dorsal horn caused by L5 SNL.4.Intrathecal injection of HI-TOPK-032 on day 7 after SNL impaired the established neuropathic painEstablish L5 SNL model,conduct behavioral test,test until 7 days after operation,then intrathecal injection of TOPK inhibitor HI-TOP K-032 100 ? g/10 ?1 for 3 consecutive days,repeat the above operation,observe the changes of PWT and PWL in rats.The results showed that after HI-TOPK-032 treatment,the rat PWT increased slowly until the 12th postoperative day(SNL+HI-TOPK-032 compared with SNL+Vehicle group,12 days,*P<0.05,student t test);PWL of rats slowly increases from 7 days after surgery and continues until 12 days after surgery(SNL+HI-TOPK-032 compared with SNL+Vehicle group,7 d,*P<0.05;9 d,***P<0.001;12 days,***P<0.001;student t test).The above results indicate that intrathecal injection of the TOPK inhibitor HI-TOPK-032 can partially reverse the established neuropathic pain.5.Prior intrathecal injection of TOPK siRNA attenuated neuropathic pain following L5 SNLIn order to rule out the non-specific effects of drugs,the experiment also used intrathecal injection of TOPK siRNA to inhibit the synthesis of TOPK,and test the effects of PWT and PWL on the pain behavior of rats after SNL.The results showed that compared with the L5 SNL+Vehicle group,the PWT of rats after intrathecal injection of TOPK siRNA began to increase on the first day after surgery and continued until the fifth day(1 d,*P<0.05;3 d,***P<0.001;5 days,*P<0.05,one-way ANOVA);PWL of rats on the surgical side after intrathecal injection of TOPK siRNA also increased significantly on the first day after surgery,and continued until the postoperative period Day 3(1 d,**P<0.01;3d,*P<0.05;one-way ANOVA).However,there was no statistical difference between the intrathecal Scramble siRNA group and the L5 SNL+Vehicle group.Then Western blot was used to observe the effect of intrathecal injection of TOPK siRNA on the expression of TOPK and p-p38 in rat spinal dorsal horn and DRG.The experiment was divided into five groups:Naive group,L5 SNL+Vehicle group,L5 SNL+TOPK siRNA group,SNL+scRNA group and Naive+TOPK siRNA group,respectively,the DRG and spinal dorsal horn of different groups of rats were taken from the surgical side.The results showed that the protein content of t-p38,the downstream substrate of TOPK,did not change in dorsal root ganglion and spinal dorsal horn(L5 SNL+TOPK siRNA compared with L5 SNL+Vehicle group and SNL+scRNA group);but t-TOPK and The protein content of p-p38 was significantly reduced in the dorsal root ganglion and spinal dorsal horn(L5 SNL+TOPK siRNA compared with L5 SNL+Vehicle group and SNL+scRNA group,*P<0.05,***<0.01,***P<0.001,one-way ANOVA).It can be seen from the above that intrathecal injection of TOPK siRNA can partially inhibit the formation of neuropathic pain caused by L5 SNL,and also inhibit the protein expression of p-p3 8 caused by L5 SNL in the rat dorsal root ganglion and spinal dorsal horn Volume up.6.Intrathecal injection of TOPK siRNA on day 7 after SNL partially reversed mechanical allodynia and thermal hyperalgesia after SNLTOPK siRNA started intrathecal injection on the seventh day of model establishment,and the injection was continuous for three days.The results showed that after intrathecal injection of TOPK siRNA,rat PWT increased slowly until 9 days after surgery(L5 SNL+TOPK siRNA compared with L5 SNL+Vehicle group,9 d,*P<0.05,student t test);PWL also increased slowly on the surgical side of rats until 9 days after surgery(L5 SNL+TOPK siRNA compared with L5 SNL+Vehicle group,7 d,*P<0.05;9 d,***P<0.001;student t test).The above results indicate that intrathecal injection of TOPK siRNA can also partially reverse the established neuropathic pain.ConclusionSpinal nerve injury-evoked TOPK/p38 activation in the DRG and spinal dorsal horn contributed to the development and maintenance of neuropathic pain in rats. |
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