The Research Of LvOn-siTR4Regulated By Tetracycline Relieving Neuropathic Pain In Rats Of CCI

Neuropathic pain, characterized by hyperalgesia, allodynia and spontaneous pain,often occurs as a result of injuries to the peripheral nerve, dorsal root ganglion (DRG),spinal cord or brain. Currently, treatment ofneuropathic pain,which is mainly treated withopiod drugs in clinic, is still a challenge.As what is known,activated glial cells in the dorsalspinal cord participate in thedevelopment and maintenance of pain after peripheralnerveinjury. Toll-like-receptor4(TLR4) is responsible for the initiate of glial activationand plays an important role in the development of central painsensitization. However, thereare currently no specific TLR4inhibitors available. RNA interference provides a powerfulmethod for the exploration of genefunction, and lentiviral delivery systems have beenapproved for human.Long-term downregulation of gene expressionhas been achieved witha lentiviral-mediated short hairpin RNA (shRNA).Lentiviral vector can infectnondividingcells.In this present study, we developed a highly efficientmethod of lentiviral-mediateddelivery of short hairpin RNAs, which can silence TLR4in thespinal cord of rats Asanapproach for the treatment of neuropathic pain, the lentivirus can be regulated bydeoxycycline (DOX) to prevent excessive inhibition of TLR4expression in spinal cord.The lentivirus can express when DOX exists.Neuropathic painwas induced by chronic constriction injury(CCI) in rats andLvOn-siTLR4was administrated intrathecally. The study consists of two parts:1. LvOn-siTLR4which can be switched by DOX was constructed.2. Effects of LvOn-siTLR4, administrated intrathecally, onneuropathic pain wereevaluated;mRNA and protein expression level of TLR4in spinal cord.was researched. Inthis part,male Sprague-Dawley rats were randomly divided into six groups(n=7), shamgroup(sham operation+intrathecal normal saline， ITNS); CCIgroup(CCI+ITNS);LvOn-siTLR4+DOX group(CCI+IT LvOn-siTLR4+DOX p.o.);LvOn-siTLR4group(CCI+IT LvOn-siTLR4); Lv-mismatchshRNAgroup（CCI+ITLv-mismatch siRNA）, CCI+DOX group(CCI+IT NS+DOX p.o.).Sciatic nervedeligation and intrathecal injection of LvOn-siTLR4are operated in one anesthesiaMechanical and thermal paw withdrawal threshold were assessed todetermine the analgesic effects of LvOn-siTLR4On the seventh day after operation,The lumbarspinal cordsegments were isolatedExpression of TLR4mRNA andprotein was determined by reversetranscriptase-polymerase chain reactionand western blottinganalysis, respectively.The main results are as follows：1.Lentivirus that containsTLR4shRNAand can beswitched by DOX weresuccessfully constructed and transducedin vitro.2.①Compared with the sham group, rats in CCI group had significantly lowermechanical and thermal pain thresholds andhigher expression of TLR4mRNA andproteinin the spinal cord (P＜0．05);②Rats in the LvOn-siTLR4+DOX group hadsignificantly higher mechanical and thermal painthresholds (at1，3，5and7days afterligation，P＜0．05) and significantly lower expression of TLR4mRNA and protein in thespinal cord compared with those in CCI group, LvOn-siTLR4groupand Lv-mismatchsiRNA group(P＜0．05);③There was no significant difference in mechanical and thermalpain thresholds as well as in the expression level of TLR4in spinal cord betweenCCIgroup and CCI+DOX group.Conclusions:1. The lentivirus carryingTLR4-shRNA which can be regulated by DOX wassuccessfully constructed.2. Intrathecal injection of LvOn-siTLR4with DOX orally can decrease the expressionof TLR4mRNA and protein in the spinal cord on rats.In addition to this, theneuropathicpain induced by CCI was relieved apparently.