二甲双胍抑制JNK改善缝隙连接功能缓解骨癌痛的实验研究
Experimental study of metformin in relieving bone cancer pain by inhibiting JNK and improving gap junction function
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摘要: 探究星形胶质细胞中c-Jun氨基末端激酶(JNK)-缝隙连接功能在骨癌痛形成中的作用，并考察5′-腺嘌呤核苷酸(AMP)依赖的蛋白激酶(AMPK)激动剂二甲双胍能否通过调节该功能缓解骨癌痛。制备肿瘤细胞植入(TCI)引起的大鼠骨癌痛模型，鞘内注射二甲双胍(50、100 μg)、JNK抑制剂SP600125(10 μg)、缝隙连接抑制剂(carbenoxolone，CBX)(10 μg)、AMPK抑制剂Compound C(CC)(10 μg)各20 μL。利用Von Frey丝检测大鼠机械痛阈值；用免疫荧光方法检测大鼠脊髓水平胶质纤维酸性蛋白(GFAP)，钙离子绑定衔接分子1(IBA-1)和缝隙连接蛋白(Cx43)的变化；Western blot法检测大鼠脊髓水平p-JNK的变化情况。本研究发现，JNK抑制剂能显著缓解TCI引起的骨癌痛，而CBX则能抑制该效果。二甲双胍处理抑制脊髓水平p-JNK水平的升高。鞘内注射不同浓度的二甲双胍(50、100 μg)，能够显著改善TCI引起的机械性痛觉异常，同样该作用也被CBX抑制。本研究表明，二甲双胍可通过激活AMPK，抑制JNK来调节缝隙连接功能，进而改善骨癌痛。Abstract: This study aims to explore the involvement of c-Jun N-terminal kinase(JNK)-Gap junction regulation in the rat model of bone cancer pain and figure out whether adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK )activator metformin could attenuate bone cancer pain through this mechanism. Tumor cell implantation(TCI)induced bone cancer pain model in rats was established. The rats were administered, respectively, with 20 μL of metformin(50, 100 μg), JNK inhibitor SP600125(10 μg), gap junction inhibitor(carbenoxolone, CBX)(10 μg)and AMPK inhibitor Compound C(CC)(10 μg). The Von Frey Assay was applied to test the mechanical pain threshold. The activity of Glial fibrillary acidic protein(GFAP), ionized calcium-binding adaptor molecule 1(IBA-1)and Connexin 43(Cx43)in spinal cord was evaluated by immunohistochemistry. Changes of p-JNK expression were detected by Western blot. JNK inhibitor SP600125 relieved TCI-induced bone cancer pain significantly in rats, while this analgesic effect was almost canceled by the blocker of gap junction carbenoxolone(CBX). Various concentration of metformin(50, 100 μg, i. t. )significantly inhibited TCI-induced mechanical allodynia and the changes of p-JNK and p-Cx43 expression were also reversed in spinal cord in rats. Together, these data suggested that activation of AMPK with metformin attenuated TCI-induced bone cancer pain via regulating the function of JNK-Gap junction in rats.
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Keywords:
- metformin /
- astrocytes /
- bone cancer pain /
- JNK /
- gap junction
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