Abstract：
BACKGROUND: Alpha 7 nicotinic acetylcholine receptor (α7nAChR)-mediated astrocytic activation is closely related to central sensitization of chronic migraine (CM). Xiongzhi Dilong decoction (XZDL), originated from Xiongzhi Shigao decoction of Yi-zong-jin-jian, has been confirmed to relieve CM in experiment and clinic. However, its underlying mechanism for treating CM has not been elucidated.
OBJECTIVE: To reveal the underlying mechanisms of XZDL to alleviate CM in vivo focusing mainly on α7nAChR-mediated astrocytic activation and central sensitization in TNC.
METHODS: CM rat model was established by subcutaneous injection of nitroglycerin (NTG) recurrently, and treated with XZDL simultaneously. Migraine-like behaviors of rats (ear redness, head scratching, and cage climbing) and pain-related reactions (mechanical hind-paw withdrawal threshold) of rats were evaluated before and after NTG injection and XZDL administration at different points in time for nine days. The immunofluorescence single and double staining were applied to detect the levels of CGRP, c-Fos, GFAP and α7nAChR in NTG-induced CM rats. ELISA kits were employed to quantify levels of TNF-α, IL-1β, and IL-6 in medulla oblongata of CM rats. The expression levels of target proteins were examined using western blotting. Finally, methyllycaconitine citrate (MLA, a specific antagonist of α7nAChR) was applied to further validate the mechanisms of XZDL in vivo.
RESULTS: XZDL significantly attenuated the pain-related behaviors of the NTG-induced CM rats, manifesting as constraints of aberrant migraine-like behaviors including elongated latency of ear redness and decreased numbers of head scratching and cage climbing, and increment of mechanical withdrawal threshold. Moreover, XZDL markedly lowered levels of CGRP and c-Fos, as well as inflammatory cytokines (IL-1β, IL-6 and TNF-α) in CM rats. Furthermore, XZDL significantly enhanced α7nAChR expression and its co-localization with GFAP, while markedly inhibited the expression of GFAP and the activation of JAK2/STAT3/NF-κB pathway in the TNC of CM rats. Finally, blocking α7nAChR with MLA reversed the effects of XZDL on astrocytic activation, central sensitization, and the pain-related behaviors in vivo.
CONCLUSIONS: XZDL inhibited astrocytic activation and central sensitization in NTG-induced CM rats by facilitating α7nAChR expression and suppressing JAK2/STAT3/NF-κB pathway, implying that the regulation of α7nAChR-mediated astrocytic activation represents a novel mechanism of XZDL for relieving CM.
OBJECTIVE: To reveal the underlying mechanisms of XZDL to alleviate CM in vivo focusing mainly on α7nAChR-mediated astrocytic activation and central sensitization in TNC.
METHODS: CM rat model was established by subcutaneous injection of nitroglycerin (NTG) recurrently, and treated with XZDL simultaneously. Migraine-like behaviors of rats (ear redness, head scratching, and cage climbing) and pain-related reactions (mechanical hind-paw withdrawal threshold) of rats were evaluated before and after NTG injection and XZDL administration at different points in time for nine days. The immunofluorescence single and double staining were applied to detect the levels of CGRP, c-Fos, GFAP and α7nAChR in NTG-induced CM rats. ELISA kits were employed to quantify levels of TNF-α, IL-1β, and IL-6 in medulla oblongata of CM rats. The expression levels of target proteins were examined using western blotting. Finally, methyllycaconitine citrate (MLA, a specific antagonist of α7nAChR) was applied to further validate the mechanisms of XZDL in vivo.
RESULTS: XZDL significantly attenuated the pain-related behaviors of the NTG-induced CM rats, manifesting as constraints of aberrant migraine-like behaviors including elongated latency of ear redness and decreased numbers of head scratching and cage climbing, and increment of mechanical withdrawal threshold. Moreover, XZDL markedly lowered levels of CGRP and c-Fos, as well as inflammatory cytokines (IL-1β, IL-6 and TNF-α) in CM rats. Furthermore, XZDL significantly enhanced α7nAChR expression and its co-localization with GFAP, while markedly inhibited the expression of GFAP and the activation of JAK2/STAT3/NF-κB pathway in the TNC of CM rats. Finally, blocking α7nAChR with MLA reversed the effects of XZDL on astrocytic activation, central sensitization, and the pain-related behaviors in vivo.
CONCLUSIONS: XZDL inhibited astrocytic activation and central sensitization in NTG-induced CM rats by facilitating α7nAChR expression and suppressing JAK2/STAT3/NF-κB pathway, implying that the regulation of α7nAChR-mediated astrocytic activation represents a novel mechanism of XZDL for relieving CM.
摘要：
背景：α7烟碱乙酰胆碱受体（α7nAChR）介导的星形胶质细胞活化与慢性偏头痛（CM）的中枢敏化密切相关。雄脂地龙汤（XZDL），起源于益宗金剑的雄枝石高汤，已在实验和临床上证实可缓解CM。然而,其治疗CM的潜在机制尚未阐明。
目的：揭示XZDL在体内减轻CM的潜在机制，主要集中在α7nAChR介导的星形细胞活化和TNC的中枢敏化。
方法：反复皮下注射硝酸甘油（NTG）建立CM大鼠模型，同时用XZDL处理。大鼠的偏头痛样行为(耳朵发红，头部刮伤,在NTG注射和XZDL给药之前和之后，在不同时间点进行了9天的评估，并评估了大鼠的疼痛相关反应（机械后爪退缩阈值）。免疫荧光单双染色检测CGRP的水平,c-Fos,NTG诱导的CM大鼠的GFAP和α7nAChR。ELISA试剂盒用于定量TNF-α的水平，IL-1β,和IL-6在CM大鼠延髓中的表达。使用蛋白质印迹法检查靶蛋白的表达水平。最后,枸橼酸甲酯（MLA，α7nAChR的特异性拮抗剂）用于进一步验证XZDL的体内机制。
结果：XZDL显著减轻NTG诱导的CM大鼠的疼痛相关行为，表现为异常偏头痛样行为的约束，包括耳朵发红的潜伏期延长，头部抓挠和笼子攀爬的次数减少，和机械退出阈值的增量。此外，XZDL显著降低CGRP和c-Fos的水平，以及炎性细胞因子(IL-1β，IL-6和TNF-α)在CM大鼠中的表达。此外，XZDL显著增强α7nAChR的表达及其与GFAP的共定位,同时显著抑制CM大鼠TNC中GFAP的表达和JAK2/STAT3/NF-κB通路的激活。最后,用MLA阻断α7nAChR逆转了XZDL对星形细胞活化的影响，中央敏化，和体内疼痛相关行为。
结论：XZDL通过促进α7nAChR的表达和抑制JAK2/STAT3/NF-κB通路抑制NTG诱导的CM大鼠星形细胞活化和中枢增敏,暗示α7nAChR介导的星形细胞活化的调节代表了XZDL缓解CM的新机制。
目的：揭示XZDL在体内减轻CM的潜在机制，主要集中在α7nAChR介导的星形细胞活化和TNC的中枢敏化。
方法：反复皮下注射硝酸甘油（NTG）建立CM大鼠模型，同时用XZDL处理。大鼠的偏头痛样行为(耳朵发红，头部刮伤,在NTG注射和XZDL给药之前和之后，在不同时间点进行了9天的评估，并评估了大鼠的疼痛相关反应（机械后爪退缩阈值）。免疫荧光单双染色检测CGRP的水平,c-Fos,NTG诱导的CM大鼠的GFAP和α7nAChR。ELISA试剂盒用于定量TNF-α的水平，IL-1β,和IL-6在CM大鼠延髓中的表达。使用蛋白质印迹法检查靶蛋白的表达水平。最后,枸橼酸甲酯（MLA，α7nAChR的特异性拮抗剂）用于进一步验证XZDL的体内机制。
结果：XZDL显著减轻NTG诱导的CM大鼠的疼痛相关行为，表现为异常偏头痛样行为的约束，包括耳朵发红的潜伏期延长，头部抓挠和笼子攀爬的次数减少，和机械退出阈值的增量。此外，XZDL显著降低CGRP和c-Fos的水平，以及炎性细胞因子(IL-1β，IL-6和TNF-α)在CM大鼠中的表达。此外，XZDL显著增强α7nAChR的表达及其与GFAP的共定位,同时显著抑制CM大鼠TNC中GFAP的表达和JAK2/STAT3/NF-κB通路的激活。最后,用MLA阻断α7nAChR逆转了XZDL对星形细胞活化的影响，中央敏化，和体内疼痛相关行为。
结论：XZDL通过促进α7nAChR的表达和抑制JAK2/STAT3/NF-κB通路抑制NTG诱导的CM大鼠星形细胞活化和中枢增敏,暗示α7nAChR介导的星形细胞活化的调节代表了XZDL缓解CM的新机制。
