The Role Of The Immune Response Mediated By TLR4 Signal Tansduction Pathway In A Rat Model Of Bone Cancer Pain

Part one:Establish and evaluation a rat model of bone cancer painObjective To establish and evaluate the rat model of tibial bone cancer pain using behavioral tests, X-ray scan and tibia HE staining and other methods.Methods A rat model of bone cancer pain was developed by intr-tibial inoculations of Walker256 breast cancer cells in female Sprague-Dawley rats(weight 150-180g).The mechanical allodynia (paw withdrawal threshold, PWT) and spontaneous pain (ambulatory score) were observed on the day 2, 4, 6, 9, 12, 14, 18 and 21 after inoculation(AI).The development of the bone tumor and structural damage in the bone were assessed by X-ray scan and HE stainning on the 6dayAI, 12dayAI and 18dayAI.Results①Rats with tibia tumors after Walker 256 cells inoculation displayed both tactile allodynia and spontaneous pain on the 6dayAI. Subsequently, the bone cancer pain was progressive severe.②Radiography of the left proximal epiphysis of the tibiae in the tumor-bearing group showed that there were bone density reduction, and moth-eaten-like small defects on the 6dayAI,significant bone destruction on the 12dayAI,and bilateral cortical bone damage and large bone defects on the 18dayAI③Histological studies showed that the trabecular bone was destroyed in varying degrees on the 6dayAI and 12dayAI;the bone marrow cavity was full of tumor cells,and the cortical bone was destroyed on the 18dayAI.Conclusion The present study successfully established a female rat model of bone pain from metastatic bone cancer. Part two: The expression of TLR4 and its downstream cytokine in spinal cord of the rat model of bone cancer painObjective To observe the expression of TLR4 and its downstream cytokine and the activation of microglia in spinal cord of the rat model of bone cancer pain.Methods The expression of TLR4 in spinal cord of nomal,sham and bone cancer pain group was detected with immunohistochemistry,western-blot, real-time-PCR on the 6dayAI, 12dayAI and 18dayAI. The expression of the microglial activation markers(CD11b and CD14) as well as cytokines (IL-1β, IL-6, TNF-αand IFN-β) was detected with real time-PCR in the same part and at the same time.Results The expression of TLR4mRNA and protein in spinal cord of the rat model of bone cancer pain significantly increased (P<0.05) on the 6dayAI, and further increase on the 12dayAI and 18dayAI (P<0.01); The expression of CD11b and CD14mRNA was significant increase on the 6dayAI in the bone cancer pain group compared with that in normal and sham group(P<0.01); The expression of IL-1βand TNF-αmRNA was also significant increase in the bone cancer pain group compared with that in normal and sham group on the 6dayAI (P<0.01), while the expression IL-6 and IFN-βmRNA was significantl increase on the 12dayAI (P<0.01).Conclusion In spinal cord of the rat model of bone cancer pain, the microglia were activated at the early stage of bone cancer pain,the expression of TLR4 was upregulation ,and the expression of its downstream cytokine was progressive increase, which suggested that the TLR4 signal transduction pathway minght be involved in the development of bone cancer pain.Part three: SiRNA inhibited the expression of TLR4 on the microgliaObjective To select the most effective siRNA which could inhibit TLR4 gene expression, siRNAs chemically synthesized in vitro were transfected the microglia (HAPI cell line) to induce TLR4 gene silencing.Methods Four different siRNA duplexes(siRNA312, siRNA439, siRNA1495 and siRNA2062) targeting the S-D rat TLR4 were selected using standard siRNA design rules. Additionally, a scrambled sequence was designed as a mismatch control. All siRNA duplexes were chemically synthesized in vitro. SiRNAs transfected the HAPI cells with LipofectamineTM2000. Forty-eight hours after transfection, the cells were harvested for western blot analysis of the level of knockdown proteins by the siRNAs. For targeting and detection of the mRNA levels, the cells were harvested after 24hr of transfection.Results 2μg TLR4 siRNA could reduce TLR4 mRNA by 83% (siRNA439), 61% (siRNA312), 55% (siRNA1495) and 53% (siRNA2062). Results from several western-blot test also showed that The most effective siRNA for knocking down TLR4 expression was siRNA439 (P<0.001).Conclusion Transfection the microglia by siRNA could effectively inhibit the TLR4 gene expression. siRNA439 was the best one.Part four: Intrathecal injection of TLR4siRNA could attenuate bone cancer pain in the rat modelObjective To observe the effects of intrathecal TLR4siRNA on bone cancer pain in the rat model at different time points.Methods Rats were intrathecally catheterizated. TLR4siRNA (2μg/10μl), negative control siRNA (2μg/10μl) or vehicle(in vivo jetPEITM)10μl were injected intrathecally to rats on the 4dayAI or 9dayAI. Injections were given daily for three consecutive days. Nociceptive testing and tissue harvesting were carried out 24 hr after the last injection. The expression of TLR4 was detected with western-blot and real time-PCR, and the expression of CD11b,CD14,IL-1β, IL-6, TNF-αand IFN–βwas detected with real time-PCR. Results Intrathecal injection of TLR4 siRNA on the 4dayAI could prevent the initial development of bone cancer pain.However,intrathecal injection of TLR4 siRNA on the 9dayAI could alleviate, but not reverse, well-established bone cancer pain. Intrathecal injection of TLR4siRNA could significantly reduce the expression of TLR4mRNA and protein(P<0.01) in the spinal cord of the rat model.The expression of CD11b, CD14, IL-1β, IL-6, TNF-αand IFN-βmRNA also significant decreased(P<0.01). Conclusion Intrathecal injection of TLR4siRNA could effectively inhibit the expression of TLR4 and its downstream cytokines, inhibit the activation of microglia and attenuate bone cancer pain in the rat model.Part five: The role of TLR4/NF-κB signaling pathway on the bone cancer pain of the rat modelObjective To observe the effects of intrathecal NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC) and TLR4siRNA on bone cancer pain in the rat model .Methods PDTC (20μg/10μl ) or saline 10μl was injected intrathecally to rats on the 9dayAI. Injections were given twice a day for three consecutive days. Nociceptive testing and tissue harvesting were carried out 6hr after the last injection(ALI). Combined the experiment samples in the quartus section,the expression of NF-κBp65 was detected with immunohistochemistry,western-blot and real time-PCR, and the expression of IL-6 and TNF-αwas detected with real time-PCR.Results Intrathecal PDTC could alleviate rats'mechanical allodynia and spontaneous pain, and significantly reduce the expression of NF-κB p65 mRNA and protein in spinal cord of the rat model (P<0.01).Aat the same time,the expression of IL-6 and TNF-αmRNA also significantly decreased(P<0.01). Intrathecal TLR4siRNA also could significantly reduce the expression of NF-κB p65 ,IL-6 and TNF-α.Conclusion TLR4/NF-κB signaling pathway mingt be involved in the bone cancer pain of the rat model. Part six: The role of TLR4/p38MAPK signaling pathway on the bone cancer pain of the rat modelObjective To observe the effects of intrathecal p38MAPK inhibitor SB203580 and TLR4siRNA on bone cancer pain in the rat model .Methods SB203580 (2μg/10μl ) or 5%DMSO 10μl was injected intrathecally to rats on the 9dayAI. Injections were given twice a day for three consecutive days. Nociceptive testing and tissue harvesting were carried out 6hALI. Combined the experiment samples in the quartus section, the expression of p38MAPK was detected with immunohistochemistry, western-blot and real time-PCR, and the expression of IL-1βand TNF-αwas detected with real time-PCR.Results Intrathecal SB203580 could alleviate rats'mechanical allodynia and spontaneous pain, and significantly reduce the expression of p38MAPK mRNA and protein in spinal cord of the rat model (P<0.01).At the same time,the expression of IL-1βand TNF-αmRNA also significantly decreased(P<0.01). Intrathecal TLR4siRNA aslo could significantly reduce the expression of p38MAPK , IL-1βand TNF-α.Conclusion TLR4/ p38MAPK signaling pathway may be involved in the bone cancer pain of the rat model.