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Down-regulation of Toll-like receptor 4 gene expression by short interfering RNA attenuates bone cancer pain in a rat model.

Lan LS, Ping YJ, Na WL, Miao J, Cheng QQ, Ni MZ, Lei L, Fang LC, Guang RC, Jin Z, Wei L - Mol Pain (2010)

Bottom Line: We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls.Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.

ABSTRACT

Background: This study demonstrates a critical role in CNS innate immunity of the microglial Toll-like receptor 4 (TLR4) in the induction and maintenance of behavioral hypersensitivity in a rat model of bone cancer pain with the technique of RNA interference (RNAi). We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.

Results: We assessed tactile allodynia and spontaneous pain in female Sprague-Dawley (SD) rats after intramedullary injection of Walker 256 cells into the tibia. In a complementary study, TLR4 small interfering RNA(siRNA) was administered intrathecally to bone cancer pain rats to reduce the expression of spinal TLR4. The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls. Only intrathecal injection of TRL4 siRNA at post-inoculation day 4 could prevent initial development of bone cancer pain; intrathecal injection of TRL4 siRNA at post-inoculation day 9 could attenuate, but not completely block, well-established bone cancer pain.

Conclusions: TLR4 might be the main mediator in the induction of bone cancer pain. Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

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Molecular down-regulation of TLR4 mRNA and protein upon treatment with siRNA. (B) TLR4 mRNA levels in microglial cells line (HAPI), normalized to β-actin, were significantly reduced in TLR4 siRNA (SITLR4) versus mismatch siRNA- and vehicle-treated cells 24 h after transfection. siRNA439 was the most effective at knocking down TLR4 expression(n = 4, ANOVA1w, P < 0.001, post hoc Dunnett testing, df = 3, F = 1.08). (A) The TLR4 protein levels detected by western blotting analysis in HAPI 48 h after transfection, normalized to β-actin. (C) TRL4 protein levels were significantly reduced following treatment with TLR4 siRNA (SITLR4) compared with vehicle or mismatch siRNA treatment. Each data point represents at least three independent experiments. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated cells; ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001 vs. mismatch siRNA-treated group.
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Figure 4: Molecular down-regulation of TLR4 mRNA and protein upon treatment with siRNA. (B) TLR4 mRNA levels in microglial cells line (HAPI), normalized to β-actin, were significantly reduced in TLR4 siRNA (SITLR4) versus mismatch siRNA- and vehicle-treated cells 24 h after transfection. siRNA439 was the most effective at knocking down TLR4 expression(n = 4, ANOVA1w, P < 0.001, post hoc Dunnett testing, df = 3, F = 1.08). (A) The TLR4 protein levels detected by western blotting analysis in HAPI 48 h after transfection, normalized to β-actin. (C) TRL4 protein levels were significantly reduced following treatment with TLR4 siRNA (SITLR4) compared with vehicle or mismatch siRNA treatment. Each data point represents at least three independent experiments. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated cells; ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001 vs. mismatch siRNA-treated group.

Mentions: To assure the effectiveness of the siRNA sequences for intrathecal injection, the ability of these siRNAs to knockdown TLR4 mRNA was determined in vitro. The TLR4 siRNA pool (2 μg) reduced TLR4 mRNA by 83% (siRNA439), 61% (siRNA312), 55% (siRNA1495), and 53% (siRNA2062), respectively, compared with a vehicle-treated group, 24 h after transfection in a microglial cells line (HAPI). This effect was not seen with scrambled duplexes (Fig. 4B). In addition to measuring the effect of the siRNA treatment on TLR4 mRNA, we also determined their effect TLR4 protein expression 48 h after transfection, by western blot analysis. TLR4 protein expression was significantly reduced following treatment with TLR4 siRNA compared with mismatched siRNA treatment (Fig. 4A,C). The most effective siRNA for knocking down TLR4 expression was siRNA439(n = 4, ANOVA1w, P < 0.001, post hoc Dunnett testing).


Down-regulation of Toll-like receptor 4 gene expression by short interfering RNA attenuates bone cancer pain in a rat model.

Lan LS, Ping YJ, Na WL, Miao J, Cheng QQ, Ni MZ, Lei L, Fang LC, Guang RC, Jin Z, Wei L - Mol Pain (2010)

Molecular down-regulation of TLR4 mRNA and protein upon treatment with siRNA. (B) TLR4 mRNA levels in microglial cells line (HAPI), normalized to β-actin, were significantly reduced in TLR4 siRNA (SITLR4) versus mismatch siRNA- and vehicle-treated cells 24 h after transfection. siRNA439 was the most effective at knocking down TLR4 expression(n = 4, ANOVA1w, P < 0.001, post hoc Dunnett testing, df = 3, F = 1.08). (A) The TLR4 protein levels detected by western blotting analysis in HAPI 48 h after transfection, normalized to β-actin. (C) TRL4 protein levels were significantly reduced following treatment with TLR4 siRNA (SITLR4) compared with vehicle or mismatch siRNA treatment. Each data point represents at least three independent experiments. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated cells; ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001 vs. mismatch siRNA-treated group.
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Figure 4: Molecular down-regulation of TLR4 mRNA and protein upon treatment with siRNA. (B) TLR4 mRNA levels in microglial cells line (HAPI), normalized to β-actin, were significantly reduced in TLR4 siRNA (SITLR4) versus mismatch siRNA- and vehicle-treated cells 24 h after transfection. siRNA439 was the most effective at knocking down TLR4 expression(n = 4, ANOVA1w, P < 0.001, post hoc Dunnett testing, df = 3, F = 1.08). (A) The TLR4 protein levels detected by western blotting analysis in HAPI 48 h after transfection, normalized to β-actin. (C) TRL4 protein levels were significantly reduced following treatment with TLR4 siRNA (SITLR4) compared with vehicle or mismatch siRNA treatment. Each data point represents at least three independent experiments. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated cells; ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001 vs. mismatch siRNA-treated group.
Mentions: To assure the effectiveness of the siRNA sequences for intrathecal injection, the ability of these siRNAs to knockdown TLR4 mRNA was determined in vitro. The TLR4 siRNA pool (2 μg) reduced TLR4 mRNA by 83% (siRNA439), 61% (siRNA312), 55% (siRNA1495), and 53% (siRNA2062), respectively, compared with a vehicle-treated group, 24 h after transfection in a microglial cells line (HAPI). This effect was not seen with scrambled duplexes (Fig. 4B). In addition to measuring the effect of the siRNA treatment on TLR4 mRNA, we also determined their effect TLR4 protein expression 48 h after transfection, by western blot analysis. TLR4 protein expression was significantly reduced following treatment with TLR4 siRNA compared with mismatched siRNA treatment (Fig. 4A,C). The most effective siRNA for knocking down TLR4 expression was siRNA439(n = 4, ANOVA1w, P < 0.001, post hoc Dunnett testing).

Bottom Line: We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls.Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China.

ABSTRACT

Background: This study demonstrates a critical role in CNS innate immunity of the microglial Toll-like receptor 4 (TLR4) in the induction and maintenance of behavioral hypersensitivity in a rat model of bone cancer pain with the technique of RNA interference (RNAi). We hypothesized that after intramedullary injection of Walker 256 cells (a breast cancer cell line) into the tibia, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4.

Results: We assessed tactile allodynia and spontaneous pain in female Sprague-Dawley (SD) rats after intramedullary injection of Walker 256 cells into the tibia. In a complementary study, TLR4 small interfering RNA(siRNA) was administered intrathecally to bone cancer pain rats to reduce the expression of spinal TLR4. The bone cancer pain rats treated with TLR4 siRNA displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines compared with controls. Only intrathecal injection of TRL4 siRNA at post-inoculation day 4 could prevent initial development of bone cancer pain; intrathecal injection of TRL4 siRNA at post-inoculation day 9 could attenuate, but not completely block, well-established bone cancer pain.

Conclusions: TLR4 might be the main mediator in the induction of bone cancer pain. Further study of this early, specific, and innate CNS/microglial response, and how it leads to sustained glial/neuronal hypersensitivity, might lead to new therapies for the prevention and treatment of bone cancer pain syndromes.

Show MeSH
Related in: MedlinePlus