Intrathecal Injection Of Resveratrol Attenuates Burn Injury Pain By Activating Spinal Sirtuin 1

There are more than 11 million patients suffering from varying degrees of burns around the world each year,of which 96%of the patients with acute pains,52%patients complained of persistent pains in or after the healing period,66%of them thought the pain disturbing the physical rehabilitation,and 55%thought the pain seriously affected daily life;more than 60%of the patients seek"family therapy" or "transferation of attention" as the pain management,and the chronic pain after burns lack of specific therapy,mainly for opioid analgesic drugs,many patients have drug abuse problems.Chronic pain after burn not only occurs in the lesion area,but also in non_heating skins showed allodynia and sensory abnormalities,the chronic pain after heat injury is a kind of neuropathic pain,the formation process of a central sensitization component,this clinical phenomenon generally occurs in the large area lesion of Ⅱ° and Ⅲ° burn patients,superficial I。burn patients generally do not have chronic pain.Moreover,the study found that chronic pain after thermal injury was mainly manifested by the decrease of tactile pain threshold,and its temperature and pain threshold were less affected.Neuropathic pain after thermal injury is mainly caused by poor healing and formation of nerve tissue in the skin,resulting in central sensitization and neuropathic pain.The chromosome is composed of DNA and Histone combined with continuous spiral through the formation of ionic bond combination,histones can influence gene transcription activity,histone acetylation is an important mechanism for the regulation of gene transcription,histone deacetylase(Histone Acetylases,HAT)through catalytic histone acetylation,activation of gene transcription,while histone deacetylase(Histone Deacetylase,HDAC)by histone deacetylase,inhibition gene transcription.Silent information regulatory proteins(Sirtuin)belong to class HDAC III.There are 7 classes of Sirtuin(SIRT1-7)in the human genome.SIRT1 exists in the nucleus,acting on histones in the nucleus,and regulating gene expression.Recent studies have shown that the imbalance of SIRT1,HDAC and histone acetylation is associated with neuropathic pain,but its specific role is controversial.Early view,in a rat model of neuropathic pain in the spinal cord,the expression level of SIRT1 decreased,the deacetylation activity decreased,whereas intrathecal administration of HDAC agonists Resveratrol(RSV)can produce analgesic effect.Some recent studies pointed out that HDAC inhibitor sodium butyrate,Trichostatin A(TsA),Valproic Acid(VPA),can be through improving the level of histone acetylation,promote gene transcription(such as metabotropic glutamate receptor 2(MgluR2),δ_opoid receptor),which leads to the analgesic effect.On this dispute,there are many explanations:one is the analgesic effect of HDAC inhibitors generally increased receptor expression,so it can be used as adjuvant to opioid drugs;two is now found there are 4 types of HDAC,SIRT1 belong to the third class,RSV is only applied to the SIRT1 without affecting the he HDAC while VPA and TsA inhibited first and second kind of HDAC inhibitors.The study of Bai et al that distant local thermal damage and injury parts of SIRT1 increased,inhibition of apoptosis,tissue repair,spinal cord and SIRT1 after heat injury is still not reported,there is no research focuses on the relationship between thermal damage after chronic pain and spinal SIRT1.RSV is a polyphenolic substance found in natural plants,and RSV has some analgesic effects on pain,neuropathic pain and inflammatory pain resulting from herniated nucleus pulposus.In neuropathic pain analgesia,Yin et al.Proved that in chronic constriction injury(CCI)formed immediately after the intrathecal injection of RSV can delay the neuropathic pain,in the CCI 4-7 days after intrathecal injection of RSV can reason the degree of suppression of neuropathic pain.The study of Shao et al,CCI 1 hours before,intrathecal injection of RSV can delay the development of neuropathic pain,indicating that RSV is a preventive effect on the formation of neuropathic pain,and the above research that the analgesic effect of RSV is related with SIRT1.Sharma and others believe that RSV has therapeutic effects on diabetic peripheral neuropathy,but the mechanism is achieved by means other than SIRT1.The therapeutic effect of RSV on Burn Injury Pain(BIP)rats and its mechanism of action are still not reported.Based on the above background,we carried out this research,which is divided into two parts,each part has two experiments.Chapter One Analgesic Effect of Intrathecal Injection of RSV on BIP Rats and Its Influence on Spinal Cord SIRT1/Ac_H3Objective:to establish the BIP rat model to verifγ the analgesic effect of intrathecal injection of RSV on BIP rats and to detect the changes of SIRT1/Ac_H3 in the spinal cord of rats.Methods:(1)the rats were randomly divided into 3 groups:Naive group,sham group,and BIP group,with 85 + 0.5℃,keep the pressure of 10G,the animal model of accurate control method of continuous heating 15 seconds of BIP rats;using Von Frey method to measure the rats in heat injury before and after thermal injury in first,third,fifth,7,9,11,14 and 21d of the mechanical withdrawal threshold(PMWT);the expression of Western in Blot were detected in third,seventh,fourteenth,after heat injury of spinal cord SIRT1/Ac_H3 21d.(2)the rats were randomly divided into 7 groups:sham group,sham+DMSO group,sham+400 gRSV group,BIP group,BIP+DMSO group,BIP+200 gRSV group and BIP+400 gRSV group,BIP modeling method according to the experimental one,the thermal damage 7-9d after continuous intrathecal administration with 3D(sham+DMSO group and the group BIP+DMSO intrathecal contrast agent DMSO,BIP+200 gRSV group,intrathecal injection of 200 gRSV,sham+400 gRSV group and BIP+400 gRSV group,intrathecal injection of 400 gRSV),using Von Frey method to measure the rats injury before and after thermal injury in first,third,seventh,9,14 and 21d PMWT in the heat;Western Blot detected the 5 group(sham group,sham+DMSO group,BIP group,BIP+DMSO group and BIP+400 gRSV group)rats in the expression of the 9D SIRT1/Ac_H3 in the spinal cord after thermal injury.Results:(1)1 day after burn injury,rats in BIP group(PMWT lower limbs continued to heat injury after 21d),there was significant difference compared with Naive group(P<0.05);7d after burn injury,the rats of BIP group decreased PMWT(contralateral limb until after thermal injury 21d),there was significant difference compared with Na group(P<0.05);her ve after heat injury seventh,fourteenth,21d,BIP in spinal cord of rats in SIRT1/Ac_H3 group was increased,there was significant difference compared with her Naive group(P<0.05).(2)the thermal damage of 7-9d after intrathecal administration of BIP+200,gRSV group and BIP+400 gRSV group compared with BIP group,rats of PMWT increased,the difference was statistically significant(P<0.05);BIP+DMSO group compared with BIP group,there was no significant difference in PMWT rats(P>0.05);BIP+200 gRSV group BIP+400 and gRSV two groups of rats had no significant difference between PMWT(P>0.05).After thermal injury fourteenth,21d,BIP+200 mu gRSV group and BIP+400 mu gRSV group two rats PMWT decreased,and compared with group BIP,there was no statistical difference(P>0.05).The thermal damage after 9D,BIP+400 gRSV group,SIRT1 expression of spinal cord increased,Ac_H3 decreased,there was significant difference compared with BIP group(P<0.05);BIP+DMSO group compared with BIP group,the expression of SIRT1/Ac_H3 in spinal cord,no significant difference(P>0.05).Conclusion:RSV has analgesic effect on BIP rats,and its mechanism may be achieved by activating spinal cord SIRT1.Therefore,RSV may be one of the drugs for the treatment of BIP.Chapter Two The Effect of Intrathecal Injection of SIRT1 Inhibitors on PMWT in Naive Rats and the Effect on RSV AnalgesiaObjective:to determine whether SIRT1,a highly selective inhibitor of EX527,affects PMWT in rats and whether it can affect the antinociceptive effects of RSV on BIP rats.Methods:(1)the rats were randomly divided into 6 groups,Na group,DMSO group,ve her EX527 6.4 g group,EX527 group,EX527 8 g 10 g 12.5 g group and EX527 group,EX527 rats in sheath injection of different doses of Von,with Frey method were measured.Rat before catheter,administered before and after administration of 30,60,90,120,150,180min PMWT(2)the rats were randomly divided into 5 groups,sham+DMSO+DMSO group,BIP+DMSO+DMSO group,BIP+EX527+DMSO group,BIP+DMSO +RSV group,BIP+EX527+RSV group,BIP model method in the first part,the thermal damage after 7d,BIP+EX527+DMSO group and BIP+EX527+RSV group,intrathecal EX527 8 g pretreatment,1H group BIP+EX527+RSV after intrathecal RSV 400 g,continuous administration of 3D,using Von Frey method to measure the rats injury before and after thermal injury in first,third,seventh,9,14 and 21d PMWT in the heat.Results:(1)the EX527 10 g group and 12.5 EX527 group G,PMWT thirtieth,sixtieth,was reduced in rats after administration of 90min,was statistically significant compared with her Na ve group(P<0.05);and after administration of 120th,150th,180min,PMWT and Na of rats in VE group compared to her was not statistically significant(P>0.05),there was no significant difference between the EX527 group and 6.4 g EX527 8 g in PMWT group and Na ve group(P>0.05)her.(2)The thermal damage after 7d,9D,BIP+DMSO,+RSV group and BIP+EX527+RSV group rats PMWT were increased,there was significant difference compared with BIP+DMSO+DMSO group(P<0.05);BIP+EX527+RSV group compared with BIP+DMSO group +RSV rats,PMWT decreased,the difference was statistically significant(P<0.05).Conclusion:the SIRT1 selective inhibitor EX527 pretreatment can reduce the analgesic effect of RSV on BIP rats,and the analgesic effect of RSV on BIP rats is at least partly achieved by activating SIRT1.