| BackgroundPain,as a protective mechanism,alerts the body to damage or potential injury.However,pathologic pain caused by nerve injury not only lose its’ normol function,but also seriously affects the quality of patients’ life due to its intense and persistent.Neuropathic pain has become a public health problem all over the world.Nevertheless,the exact mechanism is still unclear and there is no effective treatment in clinic.Double stranded RNA dependent protein kinase(PKR)is typically activated by double-stranded RNA during viral infection and inhibits intracellular protein synthesis by catalyzing the phosphorylation of the eukaryotic initiation factor alpha-subunit.In addition to its classic antiviral effects,recently study have shown that the activation of PKR is involved in inflammatory response and immune regulation in vivo.Cytokines,bacterial lipopolysaccharides and DNA damage can mediate inflammatory response by activating PKR.Neuroinflammation in the brain mediated by PKR is respond to the occurrence of diseases such as Parkinson’s disease and Alzheimer’s disease.Neuroinflammation plays an important role in the development of neuropathic pain,but until now,the expression and activation of PKR in Dorsal root ganglia(DRG)and spinal Dorsal horn after sciatic nerve injury,as well as its function in neuropathic pain are unclear.Therefore,the objectives of this study are as following:(1)observe the expression and activation of PKR in DRG and spinal dorsal horn after sciatic nerve injury;(2)To reveal the role of PKR activation in neuropathic pain;(3)Preliminary study on the mechanism of PKR’s involvement in neuropathic pain.MethodsIn this study,the neuropathic pain model induced by sciatic nerve injury was established by Lumbar 5 spinal nerve ligation(L5 SNL).Paw withdrawal threshold(PWT)and Paw withdrawal latency(PWL)are used to evaluate the degree of pain in rats.The expression and activation of PKR in DRG and spinal dorsal horn after SNL are tested by Western blot,q PCR and immunohistochemistry.Intrathecal injection of PKR inhibitor 2-AP and PKR si RNA was used to observe the role of PKR in the formation and maintenance of neuropathic pain after SNL,as well as its’ downstream molecular mechanism.Results1.Expression and cell types of total PKR as well as phosphorylated PKR(p-PKR)in DRG and spinal cord after L5 SNL in rats1.1 L5 SNL model constructionThere are two groups: SNL group and sham group.The rats were detected paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)according to schedule which drew lessons from research literature.The hind limb of PWT and PWL in L5 SNL group began to decrease at 1 day after surgery(compared with sham group,** P < 0.01,*** P < 0.001,one-way ANOVA),and reached the minimum on 7th day after operation(compared with sham group,*** P < 0.001,one-way ANOVA).The downward trend continued until 28 days after operation.That is to say,neuropathic pain induced by SNL surgery are successfully found,and the pain can maintain for a long time.1.2 Changes of total PKR and p-PKR expression in DRG and spinal dorsal horn at different points after SNLThe L4/5 DRGs and L4-5 spinal cord of normal rats and rats at different points after SNL surgery were obtained.The expression of PKR were detected at m RNA level and protein level by q PCR and Western blot,and the p-PKR was detected by anti-phosphorylated PKR antibody.Total PKR in DRG and the spinal cord have no significant change after L5 SNL surgery,while p-PKR are increased after L5 SNL surgery obviously,and reached the top in DRG at 3 days after surgery(compared with control group,** P < 0.01,one-way ANOVA),in spinal dorsal horn at 7 days after surgery(compared with control group,** P < 0.01,one-way ANOVA).1.3 Cell types of PKR expression in DRG and spinal dorsal hornStaining L4-5 DRG and spinal cord of normal rats and SNL rats at 7th day after operation by immunofluorescence single staining,It can be seen that the expression of p-PKR was increased visibly after SNL at L4-5 DRG and spinal cord,while the expression of total PKR have no significant change.Immunofluorescence double staining showed that PKR was co-localized with type A neurons and type C neurons at DRG,and satellite-like glial cells,astrocytes and neurons at the spinal cord.2.The role of PKR activation in the formation and maintenance of neuropathic pain after L5 SNL2.1 Effect of intrathecal injection of PKR inhibitor 2-AP on neuropathic pain after SNLBeginning on the day of surgery,rats in SNL + 2-AP group were intrathecal injected of 2-AP PKR inhibitors(0.5 to 6.8 mu mu mol/l,0.25 to 6.8 mu mu mol/l)for five days.Compared with solvent group(i.t.6.8 mu l 0.5% glacial acetic acid 0.01Μ PBS),group 2-AP art side of rats hind leg pain hypersensitivity caused by sciatic nerve injury has obvious relief,and sustainable to 7 days.The effect of high dose group was more significant than that of low dose group(*P < 0.05,** P < 0.01,***P < 0.001,one-way ANOVA).Molecular experiments showed that compared with SNL + Vehicle group,the levels of L4/5 DRGs and p-PKR in L4-5 spinal dorsal horn were significantly decreased in SNL + 2-AP group.In other words,the inhibitor 2-AP can reduce the activation of PKR and alleviate some neuropathic pain.To prove PKR activation has already established in the role of mental derangement rational pain,experiments on postoperative day 7 inhibitor 2-AP intrathecal injection,daily dosage of 0.5 mu per rats mol,for three consecutive days,beginning on the day of dosing,visible compared with solvent group(SNL + Vehicle),the inhibitor group(SNL + 2-AP)technique of the hind legs of rats ease pain hypersensitivity,and differences continue until 9 days after surgery(* P < 0.05,P <0.001,one-way ANOVA).This suggests that reduction of PKR activation by the inhibitor 2-AP also has an effect on established postoperative neuropathic pain.2.2 Effect of intrathecal injection of PKR si RNA on neuropathic pain in rats after SNLFrom the day of operation,si RNA of PKR was injected intraperitoneally into the rats after SNL operation,each of which was 1 nmole,and then once a day for 5consecutive days.The change of pain threshold of rats was observed behaviorally.Compared with the SNL + Vehicle and SNL + sc RNA groups,the SNL + PKR si RNA group significantly improved the pain hypersensitivity of the posterior limb after surgical measurement(** P < 0.01,*** P < 0.001,one-way ANOVA).The changes of protein expression after sampling in rats of different treatment groups showed that PKR si RNA could effectively silence PKR and significantly decrease the expression of p-PKR.To observe the effect of PKR knockdown on neuropathic pain in the maintenance stage of SNL,PKR si RNA was injected intraperitoneally on the 6th day after L5 SNL operation,once a day for 3 days.Before administration,hypersensitivity of pain was observed in the operative hind limbs of rats in both groups.From 7 days after administration,the hypersensitivity of pain in the operative hind limbs of rats in the PKR si RNA group was significantly alleviated compared with that in the sc RNA group(* P < 0.05,** P < 0.01,*** P < 0.001,one-way ANOVA).In other words,PKR si RNA interference with PKR expression can effectively alleviate hypersensitivity of pain caused by ligation of peripheral spinal nerves in rats.3.Molecular mechanism of PKR activation in neuropathic pain mediated by SNL3.1 Expression of IL-1β in DRG and spinal dorsal horn after SNL and the effect of PKR inhibition on the production of IL-1βTo test the expression of IL-1β in normal rats and postoperative rats,it shows that IL-1β has a visible rising tendency in L4/5 DRG and spinal cord of rats after SNL,and reach the top at 3th days after operation in DRG(* P < 0.05,** P < 0.01,one-way ANOVA),while in the spinal dorsal at 7th day after operation(* P < 0.05,**P < 0.001,one-way ANOVA).Inhibition of PKR by PKR inhibitor 2-AP and PKR si RNA also decrease the expression and activation of IL-1β in DRG and spinal dorsal horn of rats after SNL.3.2 The expression of IκB-α and IκB-β in rats’ DRG and spinal dorsal when PKR activated after SNL.To detect the expression of IκB-α and IκB-β of rats in different groups: normal rats,SNL + Vehicle,SNL + PKR si RNA,and SNL + sc RNA group,it can be seen that the expression of IκB-α and IκB-β in rats at SNL + Vehicle group and SNL + sc RNA group are significantly decreased after surgery,whereas they increase after using PKR si RNA.In other words,the inhibition of PKR can prevent the degradation of IκB-αand IκB-β(* P < 0.05,** P < 0.01,*** P < 0.001,one-way ANOVA)ConclusionThe activation of PKR in DRG and spinal dorsal horn after L5 SNL is involved in the formation and maintenance of neuropathic pain,which may mediate the degradation of IκB-α and IκB-β to promote the expression of IL-1β. |
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