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Abstract：
OBJECTIVE: Cancer-induced bone pain (CIBP) is a kind of pain with complex pathophysiology. Proteinase-activated receptor 2 (PAR-2) is involved in CIBP. This study explored the effects of PAR-2 on CIBP rats.
METHODS: CIBP rat model was established by injecting Walker 256 rat breast cancer cells into the left tibia of female Sprague-Dawley rats and verified by tibial morphology observation, HE staining, and mechanical hyperalgesia assay. CIBP rats were injected with PAR-2 inhibitor, ERK activator, and CREB inhibitor through the spinal cord sheath on the 13th day after operation. CIBP behaviors were measured by mechanical hyperalgesia assay. On the 14th day after operation, L4-5 spinal cord tissues were obtained. PAR-2 expression, co-expression of PAR-2 and astrocyte marker GFAP, GFAP mRNA and protein levels and the ERK pathway-related protein levels were detected by Western blot, immunofluorescence double staining, RT-qPCR, and Western blot.
RESULTS: CIBP rats had obvious mechanical hyperalgesia and thermal hyperalgesia from the 7th day after modeling; mechanical hyperalgesia threshold and thermal threshold were decreased; PAR-2 was increased in spinal cord tissues and was co-expressed with GFAP. PAR-2 silencing alleviated rat CIBP by inhibiting astrocyte activation. p-ERK/t-ERK and p-CREB/t-CREB levels in CIBP spinal cord were elevated, the ERK/CREB pathway was activated, while the ERK/CREB pathway was inhibited by PAR-2 silencing. The alleviating effect of PAR-2 inhibitor on hyperalgesia behaviors in CIBP rats were weakened by ERK activator, while were partially restored by CREB inhibitor.
CONCLUSIONS: PAR-2 knockdown inhibited the ERK/CREB pathway activation and astrocyte activation, thus alleviating CIBP in rats.
METHODS: CIBP rat model was established by injecting Walker 256 rat breast cancer cells into the left tibia of female Sprague-Dawley rats and verified by tibial morphology observation, HE staining, and mechanical hyperalgesia assay. CIBP rats were injected with PAR-2 inhibitor, ERK activator, and CREB inhibitor through the spinal cord sheath on the 13th day after operation. CIBP behaviors were measured by mechanical hyperalgesia assay. On the 14th day after operation, L4-5 spinal cord tissues were obtained. PAR-2 expression, co-expression of PAR-2 and astrocyte marker GFAP, GFAP mRNA and protein levels and the ERK pathway-related protein levels were detected by Western blot, immunofluorescence double staining, RT-qPCR, and Western blot.
RESULTS: CIBP rats had obvious mechanical hyperalgesia and thermal hyperalgesia from the 7th day after modeling; mechanical hyperalgesia threshold and thermal threshold were decreased; PAR-2 was increased in spinal cord tissues and was co-expressed with GFAP. PAR-2 silencing alleviated rat CIBP by inhibiting astrocyte activation. p-ERK/t-ERK and p-CREB/t-CREB levels in CIBP spinal cord were elevated, the ERK/CREB pathway was activated, while the ERK/CREB pathway was inhibited by PAR-2 silencing. The alleviating effect of PAR-2 inhibitor on hyperalgesia behaviors in CIBP rats were weakened by ERK activator, while were partially restored by CREB inhibitor.
CONCLUSIONS: PAR-2 knockdown inhibited the ERK/CREB pathway activation and astrocyte activation, thus alleviating CIBP in rats.
摘要：
目的：癌性骨痛（CIBP）是一种病理生理复杂的疼痛。蛋白酶激活受体2（PAR-2）参与CIBP。本研究探讨了PAR-2对BP大鼠CI的影响。
方法：将Walker256大鼠乳腺癌细胞注入雌性Sprague-Dawley大鼠左胫骨建立CIBP大鼠模型，并通过胫骨形态学观察进行验证。HE染色,和机械痛觉过敏试验。CIBP大鼠注射PAR-2抑制剂，ERK激活剂,和CREB抑制剂在术后第13天通过脊髓鞘。通过机械痛觉过敏测定法测量CIBP行为。手术后的第14天，获得L4-5脊髓组织。PAR-2表达，PAR-2和星形胶质细胞标志物GFAP的共表达，Westernblot检测GFAPmRNA和蛋白水平及ERK通路相关蛋白水平,免疫荧光双重染色，RT-qPCR,和Westernblot。
结果：CIBP大鼠从造模后第7天开始出现明显的机械性痛觉过敏和热痛觉过敏；机械性痛觉过敏阈值和热阈值降低；PAR-2在脊髓组织中升高，并与GFAP共表达。PAR-2沉默通过抑制星形胶质细胞活化减轻ratCIBP。P-ERK/t-ERK和P-CREB/t-CREB在CI血压脊髓水平升高,ERK/CREB通路被激活，而ERK/CREB通路被PAR-2沉默所抑制。ERK激活剂减弱PAR-2抑制剂对CIBP大鼠痛觉过敏行为的缓解作用,而通过CREB抑制剂部分恢复。
结论：PAR-2敲低抑制ERK/CREB通路激活和星形胶质细胞激活，从而减轻大鼠的CIBP。
方法：将Walker256大鼠乳腺癌细胞注入雌性Sprague-Dawley大鼠左胫骨建立CIBP大鼠模型，并通过胫骨形态学观察进行验证。HE染色,和机械痛觉过敏试验。CIBP大鼠注射PAR-2抑制剂，ERK激活剂,和CREB抑制剂在术后第13天通过脊髓鞘。通过机械痛觉过敏测定法测量CIBP行为。手术后的第14天，获得L4-5脊髓组织。PAR-2表达，PAR-2和星形胶质细胞标志物GFAP的共表达，Westernblot检测GFAPmRNA和蛋白水平及ERK通路相关蛋白水平,免疫荧光双重染色，RT-qPCR,和Westernblot。
结果：CIBP大鼠从造模后第7天开始出现明显的机械性痛觉过敏和热痛觉过敏；机械性痛觉过敏阈值和热阈值降低；PAR-2在脊髓组织中升高，并与GFAP共表达。PAR-2沉默通过抑制星形胶质细胞活化减轻ratCIBP。P-ERK/t-ERK和P-CREB/t-CREB在CI血压脊髓水平升高,ERK/CREB通路被激活，而ERK/CREB通路被PAR-2沉默所抑制。ERK激活剂减弱PAR-2抑制剂对CIBP大鼠痛觉过敏行为的缓解作用,而通过CREB抑制剂部分恢复。
结论：PAR-2敲低抑制ERK/CREB通路激活和星形胶质细胞激活，从而减轻大鼠的CIBP。
