二甲双胍对吗啡耐受的影响及机制研究

发布时间：2018-04-26 20:57

本文选题：二甲双胍 + 吗啡耐受　；参考：《南京医科大学》2015年博士论文

【摘要】：吗啡(morphine)为阿片受体激动剂,因其强大的镇痛作用成为临床广泛应用的强效镇痛药。然而,吗啡耐受及其副作用严重限制了临床应用。长期应用吗啡后其镇痛作用逐渐减弱,必须增加剂量才能得到原来的反应,即产生吗啡耐受(Morphine tolerance)。吗啡耐受迫使患者增加药量,进而更增加了吗啡的副作用。因此探讨吗啡耐受的机制,有效地防治吗啡耐受是基础和临床研究共同面临的挑战。近年来研究以及我们前期的研究发现,胶质细胞介导的神经炎症在吗啡耐受过程中发挥了重要的作用。小胶质细胞(microglia)和星形胶质细胞(astrocyte)的激活是神经炎症的主要特征,吗啡可通过直接或间接地方式激活胶质细胞。星形胶质细胞和小胶质细胞上都有u,δ和K阿片类受体表达,但是关于吗啡激活胶质细胞是否直接通过这些受体尚不明确。在一些体外实验中发现,吗啡可以直接影响胶质细胞的形态或功能。也有研究发现,吗啡可以通过TLR4直接激活胶质细胞。且小胶质细胞的抑制剂米诺环素可以显著抑制吗啡耐受,鉴于米诺环素有很严重的副作用,无法用于临床,因此找到一个安全有效的抑制小胶质细胞异常活化药物是治疗吗啡耐受的关键。二甲双胍(Metformin)为双胍类口服降血糖药,临床应用有着良好的安全性。二甲双胍起效的分子机制与AMPK信号转导系统有关。AMPK是一个位于真核细胞中的丝氨酸-苏氨酸激酶,是细胞中感受能量应激并调节物质代谢的关键分子。而最近的研究发现,AMPK的激动剂白藜芦醇可以显著抑制吗啡诱导的神经炎症,并改善吗啡耐受。由此可见,AMPK与吗啡耐受有密切关系,但是,二甲双胍是否是通过激活AMPK从而通过调节小胶质细胞的活性以及相关神经炎症来减轻吗啡耐受,尚未见文献报道。因此,我们通过体外培养BV-2、bEND3和在体吗啡耐受模型,对二甲双胍在吗啡耐受中的作用机制进行研究。第一部分二甲双胍抑制吗啡诱导体外细胞活化的胞内信号机制目的：研究二甲双胍抑制吗啡诱导bEND3内皮细胞、BV2小胶质细胞活化的胞内信号机制方法：培养内皮细胞系bEND3细胞,利用QPCR的方法,在细胞水平上考察吗啡对于前炎症因子IL-1β、 IL-6、TNF-α释放,以及TLR4和趋化因子CCL2的影响,并考察二甲双胍对这些变化的影响；培养小胶质细胞系BV-2细胞,利用QPCR的方法,在细胞水平上考察吗啡对于前炎症因子IL-1β、 IL-6、TNF-α释放的影响,并考察二甲双胍对这些变化的影响；利用免疫印迹的方法考察吗啡对p38磷酸化水平的影响,并考察二甲双胍对其的影响；利用细胞荧光染色考察吗啡对NF-κB核转位的影响,并考察二甲双胍对于其的作用；QPCR法检测二甲双胍对吗啡诱导的BV-2细胞TLR4 mRNA水平上升的影响。结果：二甲双胍可以显著抑制吗啡诱导的bEND细胞IL-6的异常升高,但是吗啡及二甲双胍对于TNF-α、IL-1β、TLR4和CCL2无显著影响。吗啡能诱导BV-2细胞强烈活化,其活化的表征为p38 MAPK磷酸化水平升高、NF-κB核移位和前炎症细胞因子(包括IL-1β、IL-6和TNF-α) mRNA表达增加,而二甲双胍可以显著抑制这些改变,且AMPK抑制剂可以取消二甲双胍的作用；同时二甲双胍可以抑制吗啡诱导的BV-2细胞中TLR4 mRNA水平的上升。第二部分二甲双胍对吗啡耐受小鼠行为学、脊髓神经化学及小胶质细胞活化的影响目的：研究二甲双胍对吗啡耐受行为学的影响及其作用机制方法：测定经典的疼痛行为学指标——小鼠甩尾潜伏期,考察二甲双胍对吗啡耐受行为学的影响；利用免疫印迹法检测二甲双胍对于吗啡慢性耐受标志性分子指标NR1、PKCγ、 p38的影响：利用免疫印迹法检测二甲双胍对慢性耐受模型脊髓前炎症因子IL-1β和TNF-α的影响；利用免疫印记和免疫荧光法检测二甲双胍对于脊髓小胶质细胞活化程度的影响。结果：二甲双胍能缓解慢性吗啡耐受的发展,并能抑制吗啡诱导的脊髓小胶质细胞活化和p38 MAPK磷酸化水平升高；二甲双胍能抑制吗啡慢性耐受引起的脊髓前炎症因子IL-1p和TNF-α的异常升高；二甲双胍能抑制吗啡慢性耐受引起的NP1和PKCγ磷酸化水平的异常升高。结论：二甲双胍能通过抑制p38 MAPK信号通路,进而抑制吗啡诱导的小胶质细胞活化,使得其能缓解吗啡慢性耐受。二甲双胍可能成为预防、最小化甚至逆转吗啡诱导的耐受和其它副作用的潜在临床靶点,促进阿片类药物的临床应用。
[Abstract]:morphine ( morphine ) is an opioid receptor agonist , which is a potent analgesic for clinical application because of its strong analgesic effect . However , morphine tolerance and its side effects severely limit the clinical application . After long - term use of morphine , its analgesic effect is gradually weakened , and the dose can be increased to get the original response , i . e . morphine tolerance . In recent years , it is found that morphine can directly or indirectly activate glial cells .
The effects of morphine on the release of IL - 1尾 , IL - 6 and TNF - 伪 were investigated by QPCR , and the effects of metformin on these changes were investigated .
The effect of morphine on p38 phosphorylation was investigated by Western blot .
The effect of morphine on the nuclear translocation of NF - 魏B was investigated by means of cell fluorescence staining , and the effect of metformin on NF - 魏B nuclear translocation was investigated .
The results showed that metformin could significantly inhibit the increase of IL - 6 in BEND cells induced by morphine , but morphine and metformin had no significant effect on the levels of TNF - 伪 , IL - 1尾 , TLR 4 and CCL2 . The activation of morphine and metformin increased the expression of p38 MAPK phosphorylation , and the expression of NF - 魏B nuclear translocation and pro - inflammatory cytokines ( including IL - 1尾 , IL - 6 and TNF - 伪 ) mRNA increased , while metformin could significantly inhibit these changes and AMPK inhibitors could abolish metformin .
The effects of metformin on morphine tolerance behavior , spinal neurochemistry and activation of microglial cells were studied . The effects of metformin on morphine tolerance behavior and its mechanism were studied .
The effect of metformin on the molecular markers NR1 , PKC纬 and p38 in chronic morphine tolerance was investigated by Western blotting . The effects of metformin on the levels of IL - 1尾 and TNF - 伪 were detected by Western blotting .
The results showed that metformin could alleviate the development of chronic morphine tolerance and inhibit the activation of morphine - induced microglial cells and the increase of p38 MAPK phosphorylation .
Metformin inhibited the abnormal elevation of IL - 1p and TNF - 伪 in the spinal cord induced by chronic morphine tolerance .
Conclusion : Metformin can inhibit the activation of morphine - induced microglial cells by inhibiting p38 MAPK signaling pathway , thus inhibiting the activation of morphine - induced microglial cells . Metformin may be a potential clinical target to prevent , minimize or even reverse morphine - induced tolerance and other side effects , and to promote clinical use of opioids .

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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