The Mechanism Of NNOS Phosphorylation On Different Sites In Neuropathic Pain

Objective:Neuropathic pain(NP)is a common clinical pain disease with many causes and complicated mechanisms.NP can be caused by a variety of diseases,different from the acute pain caused by tissue damage,the degree is heavy,the course of disease is long,and the current treatment methods are still difficult to achieve the desired effect,so the research on its pathogenesis has been an important task for pain researchers.During the process of pain,neuronal nitric oxide synthase(nNOS),calcium ion/calmodulin-dependent protein kinase ?(CaMK?),and post-synaptic protein 95(PSD-95)are involved in the process of pain.In this study,nNOS was used as an entry point to explore the role of nNOS phosphorylation in the pathogenesis of neuropathic pain,and its role in the pathogenesis of neuropathic pain.It was also investigated whether CaMK? and PSD-95 are involved in the process of nNOS phosphorylation.Method: All experimental animals(110 male Sprague-Dawley rats)were randomly divided into 13 groups: non-operative group(Naive)(n=5),sham operation group(Sham)(n=20),and SNI group 1 day after surgery.(D1)(n=10),3 days after SNI(D4)(n=10),7 days after SNI(D7)(n=10),10 days after SNI(D10)(n =20),14 days after SNI surgery(D14)(n=10),DMSO+SNI surgery group(DMSO)(intrathecal administration: 10ul/rat,n=10),KN93+SNI surgery group(KN93)(30 uM/10 ul/rat,n=10),Wortmannin+SNI surgery group(Wortmannin)(40 uM/10 ul/rat,n=5).All experimental animals were required to measure the paw withdrawal threshold(PWT)before and after surgery.The experimental rats requiring surgery were pre-made with a sheath-in-tube model,and the rats were restored to a sciatic nerve injury(SNI)model 7 days after recovery.PWT was collected daily after surgery,and intrathecal administration was started on the first day after surgery,and continued until the 10 th day after surgery.The CaMK?Thr286 autophosphorylation level(CaMK?-T286),CaMK? protein expression and PSD-95 expression in the spinal cord were detected at each corresponding time point.The nNOS in the spinal cord was purified by2`-5`-ADP-agarose and the nNOS Ser1417 phosphorylation level(nNOS-NP1417),nNOS Ser847 phosphorylation level(nNOS-NP847),and CaMK? Thr286 in nNOSprecipitated samples were detected.Phosphorylation level(CaMK?-T286),CaMK? protein expression,and PSD-95 expression.Results: Part ?: To test whether the phosphorylation of different sites of nNOS is expressed,and to verify whether nNOS phosphorylation and the phosphorylation of different sites and time changes in NP process by KN-93 and Wortmannin inhibitors..The following results were obtained after the experiment: SNI can reduce the mechanical pain threshold of rats(P<0.05).Figure 1.Both nNOS Ser847 and Ser1417 are phosphorylated during pain.Figure 2,but the highest expression time points,after the KN-93 and Wortmannin were given in the sheath,the corresponding nNOS Ser847 and Ser1417 phosphorylation was significantly reduced(P<0.05).Part ?: Whether the occurrence of pain is related to the activity of CaMK? by detecting the expression level of CaMK? phosphorylation in the spinal cord and combining with animal morphological changes.The co-precipitation of nNOS by Agaros was used to detect the expression of CaMK? and PSD-95 in the nNOS precipitated samples,and to determine whether these related proteins were combined during the NP ogenesis.After the experiment,the following results were obtained:during the process of NP,CaMK? undergoes Th 286 autophosphorylation and is activated to participate in the pain process,and the expression level is significantly increased(P<0.05).Figure 5,its trend and pain behavior trend Consistent.PWT was significantly elevated(P< 0.05)after administration of the CaMK? activity inhibitor KN-93.CaMK?,CaMK? autophosphorylation,and PSD-95 expression were detected in nNOS precipitated samples.And by calculation results,it was found that the expression of CaMK? autophosphorylation is similar to the PWT trend,as shown in Fig.6.The expression trends of PSD-95 and CaMK? are also similar to those of pain behavior.When intrathecal KN-93 was injected,the expression of CaMK? and PSD-95 in nNOS precipitated samples was significantly decreased(P<0.05).Figure 7.The results showed that CaMK? binds to PSD-95 and nNOS during NP production.State and participate in the entire process together.And related to CaMK? activity.Conclusion: 1.In the rat model of neuropathic pain,nNOS Ser847 and Ser1417 were phosphorylated by CaMK? and PKB/Akt,respectively,which down-regulated andup-regulated nNOS activity,resulting in changes in NO synthesis and different regulation the effect of neuropathic pain.2.In the rat neuropathic pain model,CaMK?,PSD-95 and nNOS have a binding state and participate in the pain process.3.In the process of participating in pain,the activity of CaMK? may be the key to regulating this binding.