Role of Toll-like receptor 4/NF-κB signal transduction pathway in development and maintenance of bone cancer pain in rats

Abstract

Objective To evaluate the role of Toll-like receptor 4 (TLR4)/NF-κB signal transduction pathway in development and maintenance of bone cancer pain (BCP) in rats.Methods Eighty-one female Sprague-Dawley rats,aged 5-6 weeks,weighing 150-180 g,were randomly divided into 8 groups:control group (group C),BCP group,vehicle 1 group (Ⅴ1 group),TLR4 small interfering RNA (siRNA) 1 group (TLR4siRNA1 group),TLR4 mismatch siRNA (msiRNA) 1 group (TLR4msiRNA1 group),Ⅴ2 group,TLR4siRNA2 group and TLR4msiRNA2 group.There were 18 rats in group BCP and 9 rats in each group of the left 7 groups.BCP was induced by intra-tibial inoculation of 1 × 105 Walker 256 breast cancer cells.In Ⅴ1,TLR4siRNA1 and TL-R4msiRNA1 groups,vehicle,TLR4siRNA and TLR4msiRNA were intrathecally injected,respectively,on 4th day after inoculation (pre-injection).In Ⅴ2,TLR4siRNA2 and TLR4msiRNA2 groups,vehicle,TLR4siRNA and TLR4msiRNA were intrathecally injected,respectively,on 9th day after inoculation (post-injection).TLR4siRNA and TLR4msiRNA 2 μg were diluted with 5％ glucose and jetPEITM to 5 μl,and then given once a day for 3 consecutive days,and the rats were sacrificed at 24 h after the last injection.The rats in group BCP were sacrificed ondays 7 and 12 after inoculation.The lumbar segments (L4-6) of the spinal cord were removed for determination of the expression of NF-κBp65 mRNA,IL-6 mRNA and TNF-α mRNA (by real-time PCR) and NF-κBp65 (by Western blot and immuno-histochemistry).Results Pre-injection of TLR4siRNA could completely inhibit up-regulation of the expression of NF-κBp65 mRNA,NF-κBp65 and TNF-α mRNA in the spinal cord tissues,however,post-injection of TLR4siRNA could partially inhibit up-regulation of the expression of NF-κBp65 mRNA,NF-κBp65,IL-6 mRNA and TNF-α mRNA in the spinal cord tissues (P ＜ 0.05 or 0.01).Conclusion TLR4/NF-κB signaling pathway mediates the development and maintenance of BCP in rats. Key words: Toll-like receptor 4 ; NF-kappa B ; Bone neoplasms; Pain