Sirt2 In The Spinal Cord Regulates Chronic Neuropathic Pain Through Nrf2-mediated Oxidative Stress Pathway In Rats And Its Mechanism

Objective:The latest definition of neuropathic pain(NP)is "pain caused by lesion or disease of the somatosensory system",which was established by the neuropathic pain special interest group(Neu PSIG)of the International association for the study of pain(IASP)in 2008.Chronic neuropathic pain is persistent and severe,which often leads to loss of appetite,sleep quality and ability to work.It even increases the incidence rate of emotional disorders such as depression and anxiety,and there is no effective treatment in clinic at present.Therefore,the research on its pathogenesis and treatment is a scientific problem to be solved urgently.At present,the pathogenesis of neuropathic pain is mainly divided into central sensitization and peripheral sensitization,in which central sensitization plays a key role.Recently,many studies suggest that oxidative stress plays an important role in central sensitization.Excessive reactive oxygen species(ROS)has a deleterious effect on organelles,antioxidant defenses and other biomolecules,leading to mitochondrial dysfunction,glial activation and inflammatory response.This adverse environment is ultimately responsible for the typical painful symptoms of NP.Nuclear factor erythroid derived-2-related factor 2(Nrf2)is an important transcription factor and master regulator of many antioxidant genes.Nrf2 pathway has been considered as a critical cellular defense mechanism against oxidative stress.Accumulating evidence shows that Nrf2 pathway is involved in the pathogenesis of NP,but the mechanism that mediates Nrf2 pathway in NP is unclear.The sirtuins(sirts)are a family of nicotinamide adenine dinucleotide(NAD)-dependent histone deacetylases(HDACs)that play important roles in many cellular functions,including histone deacetylation,protein acylation,and deacetylation.In addition,sirtuins have protective properties,antioxidant-promoting actions and ROS-suppressive effects in mammalian cells.A recent in vitro study revealed that Sirt2,a member of the sirtuin family of proteins,affects antioxidant capacity by modulating Nrf2 activity.The aim of this study was to investigate the effect of Nrf2 pathway activation on neuropathic pain in rats.Here we examined whether central Sirt2 regulates NP through Nrf2-mediated oxidative stress pathway.The purpose of this study is to explore whether Sirt2 can alleviate chronic neuropathic pain in rats through Nrf2 mediated oxidative stress pathway,so as to provide a new direction for the treatment of neuropathic pain.Method:Part ?: To examine the time course of changes in pain behaviors and expression of Nrf2 and NQO1 in the spinal cord,rats were assigned to sham(n = 5)and SNI(n = 30)groups.The SNI group underwent SNI surgery,while sham group received sham operation.Paw withdrawal threshold(PWT)to mechanical stimulation and paw withdrawal latency(PWL)to thermal stimulation were assessed at the ipsilateral hind paws 24 hours prior to SNI and day 1,3,7,10 and 14 post SNI.Five SNI rats were sacrificed after PWT and PWL measurement at each time point and sham rats were sacrificed at final time point.The total protein and nuclear protein was extracted from L4-6 spinal cord of injured side for molecular studies,and expression of Nrf2 and NQO1 in the whole tissue lysates and expression of Nrf2 in the nuclear fractions were detected by Western blot.Part ?: To examine the role of Nrf2 in the spinal cord in regulation of neuropathic pain,rats were divided into 5 experimental groups(n = 4 for each group): sham;SNI without treatment;SNI treated with DMSO(vehicle);SNI treated with Nrf2 agonist t BHQ at 1?M;and SNI treated with t BHQ at 10 ?M.Lumbar intrathecal catheter implantation was conducted 7 days prior to SNI.DMSO and t BHQ(dissolved in 10?L DMSO)was intrathecally injected once a day for 7 days,starting from day 1 after SNI.PWT and PWL were measured daily and animals were sacrificed at the end of the protocol to collect spinal cord tissues for molecular studies.The total protein and nuclear protein was extracted from L4-6 spinal cord of injured side for molecular studies,and expression of Nrf2 and NQO1 in the whole tissue lysates and expression of Nrf2 in the nuclear fractions were detected by Western blot.The level of 8-hydroxy-2 '-deoxyguanosine(8-OHd G)was detected by ELISA,and the level of superoxide dismutase(SOD)was detected by kit.Additional SNI rats treated with DMSO and t BHQ at 10 ?M(n = 3 for each group)were used for immunofluorescent study at the end of the protocol.Part ?: First,in order to observe the changes of Sirt2 expression in spinal cord of SNI rats,the rats were divided into sham operation group(sham)(n = 5)and SNI(n = 30)groups.The SNI group underwent SNI surgery,while sham group received sham operation.Paw withdrawal threshold(PWT)to mechanical stimulation and paw withdrawal latency(PWL)to thermal stimulation were assessed at the ipsilateral hind paws 24 hours prior to SNI and day 1,3,7,10 and 14 post SNI.Five SNI rats were sacrificed after PWT and PWL measurement at each time point and sham rats were sacrificed at final time point.The total protein was extracted from L4-6 spinal cord of injured side for molecular studies,and the expressions of Sirt2 were detected by Western blot.To determine the role of Sirt2 in regulation of Nrf2 pathway,rats were divided into 4experimental groups(n = 5 for each group): sham;SNI without treatment;SNI treated with a recombinant adenovirus expressing control and GFP(Ad-control);and SNI treated with a recombinant adenovirus expressing Sirt2 and GFP(Ad-Sirt2,1×108 PFU in 10 ?L saline).Ad-control and Ad-Sirt2 were intrathecally injected 24 hours prior to SNI.PWT and PWL were measured daily and animals were sacrificed at the end of the protocol to collect spinal cord tissues for molecular studies.The total protein and nuclear protein was extracted from L4-6 spinal cord of injured side for molecular studies,and the expressions of Nrf2 and NQO1 were detected by Western blot.The level of 8-hydroxy-2 '-deoxyguanosine(8-OHd G)was detected by ELISA,and the level of superoxide dismutase(SOD)was detected by kit.Additional SNI rats treated with Ad-control and Ad-Sirt2(n = 3 for each group)were used for immunofluorescent study at the end of the protocol.Results:Part ?: Time course of changes in pain behaviors and expression of Nrf2 and its downstream target in the spinal cord following SNI.SNI induced significant mechanical allodynia as indicated by decreased PWT and thermal hyperalgesia as evidenced by reduced PWL within 1 day and lasting up to 14 days compared to sham controls and baseline.Western blot analysis revealed that the expression of Nrf2 and NQO1 in the whole tissue lysates of SNI group tended to be higher at day 1,but gradually decreased from day 7 following SNI,compared with baseline or sham group.Expression of Nrf2 in the nuclear fractions in SNI group was significantly increased at day 1,but then started to gradually decrease in the following day.Significant reduction in expression of Nrf2 in the nuclear fractions of SNI group was observed from day 7 as compared to baseline or sham group.Part ?: Activation of Nrf2 pathway in the spinal cord ameliorates neuropathic pain.The mechanical allodynia and thermal hyperalgesia in SNI rats,compared with SNI rats without treatment,were attenuated by intrathecal t BHQ at both doses from day 4 for PWT and day 2 for PWL,respectively.Intrathecal injection of DMSO had no effects on mechanical allodynia and thermal hyperalgesia in SNI rats.Intrathecal t BHQ at either dose,but not DMSO,increased expression of Nrf2 and NQO1 in the whole tissue lysates and expression of Nrf2 in the nuclear fractions.Immunofluorescent study showed that SNI rats treated with intrathecal t BHQ at a dose of 10 ?M exhibited abundant Nrf2 immunoreactivity in the spinal cord,particularly in the nucleus,compared with SNI rats treated with intrathecal DMSO.Consistent with expression of Nrf2 and NQO1,the levels of SOD were markedly decreased,whereas the levels of 8-OHd G were increased in SNI rats without treatment.Intrathecal t BHQ at both doses completely reversed SNI-induced changes in SOD and 8-OHd G of note,intrathecal DMSO did not alter SNI-induced changes in SOD and 8-OHd G.Part ?: Upregulation of Sirt2 in the spinal cord alleviates neuropathic pain,which is associated with activation of Nrf2 pathway and decreased oxidative stress.The expression of Sirt2 in SNI rats was not altered at day 1 and day 3,but it was significantly decreased from day 7 and remained lower till the end of the experiment compared to baseline or sham rats.SNI rats that received intrathecal Ad-Sirt2,but not Ad-control,had significantly reduced mechanical allodynia and thermal hyperalgesia as compared to SNI rats without treatment.Intrathecal Ad-Sirt2 restored expression of Sirt2 in the spinal cord of SNI rats to similar level as in sham group.Importantly,we found that expression of Nrf2 and NQO1 in the whole tissue lysates and expression of Nrf2 in the nuclear fractions in SNI rats treated with intrathecal Ad-Sirt2 were normalized to those observed in sham rats.Moreover,intrathecal Ad-Sirt2 restored SNI-induced changes in SOD and 8-OHd G in the spinal cord.Intrathecal Ad-control had no effect on any of these measured parameters.Conclusion:1.Nrf2 activity and its downstream target NQO1 expression in the spinal cord are downregulated in rats with SNI-induced chronic neuropathic pain.2.Upregulation of Nrf2 activity restores NQO1 expression and prevents oxidative stress in the spinal cord,ameliorating mechanical allodynia and thermal hyperalgesia in rats with SNI-induced chronic NP.3.Sirt2 expression in the spinal cord is reduced in rats with SNI-induced chronic neuropathic pain.4.Overexpression of Sirt2 prevents reductions in Nrf2 activity and NQO1 expression as well as increase in oxidative stress in the spinal cord,leading to improvement of thermal hyperalgesia and mechanical allodynia.