姜黄素对大鼠坐骨神经损伤后神经再生过程中细胞自噬的影响

Effect of curcumin on autophagy during nerve regeneration after sciatic nerve injury in rats

目的研究姜黄素对大鼠实验性坐骨神经损伤后神经再生过程中细胞自噬的影响及可能机制。方法建立大鼠实验性坐骨神经损伤模型,实验分为姜黄素组、U0126+姜黄素组、YL294002+姜黄素组、阴性对照组和正常组。姜黄素组腹腔注射姜黄素100 mg·kg^(-1),连续注射60 d; U0126+姜黄素组在姜黄素治疗前1 h腹腔注射MEK抑制剂U0126(30 mg·kg^(-1)·d-1); YL294002+姜黄素组在姜黄素治疗前30 min,用AKT信号通路激活物YL294002 (0. 5mg·kg^(-1)·d-1)预处理;阴性对照组于切断后直接缝合皮肤,不作神经修复术处理,给予等量二甲基亚砜治疗;正常组大鼠不做任何处理。药物干预处理7 d后,用Western blot法检测LC3-Ⅰ/Ⅱ、Beclin 1和p62的表达及ERK1/2和Akt的磷酸化情况。治疗60 d后,用反转录实时荧光定量-聚合酶链式反应(qRTPCR)和Western blot法检测PMP22、纤维蛋白和S100的表达来评估坐骨神经损伤后髓鞘再生情况。结果与正常组比较,阴性对照组LC3-Ⅱ和Beclin 1水平分别升高1. 7倍和2. 1倍,p-62水平降低0. 7倍,p-ERK1/2和p-Akt的表达分别上调1. 7倍和1. 9倍;姜黄素治疗后p-ERK1/2的表达增加1. 4倍,pAkt的表达降低0. 6倍。U0126+姜黄素组和YL294002+姜黄素组与姜黄素组相比,姜黄素激活ERK1/2通路的作用减弱、抑制Akt通路的作用增强。qRTPCR和Western blot分析显示,与阴性对照组比较,姜黄素对坐骨神经S100、PMP22 mRNA的表达分别增加2. 9倍和2. 1倍,蛋白的表达分别增加1. 7倍和1. 4倍,纤维蛋白沉积下降;而姜黄素对髓鞘再生和轴突再生的促进作用被ERK 1/2通路抑制剂和Akt通路激动剂逆转。结论姜黄素可诱导大鼠坐骨神经损伤后的细胞自噬,促进髓鞘再生,可能与调节MEK/ERK和PI3K/Akt信号通路有关。

Objective To explore the effect for cell autophagy and corresponding mechanisms of curcumin on the repair of sciatic nerve injury in rats. Methods After surgery induced sciatic nerve injury,the model rats were divided into five groups and treated with curcumin( intraperitoneal injection of curcumin 100 mg·kg-1,for 60 d),curcumin +U0126 [intraperitoneal injection of MEK inhibitor U0126( 30 mg·kg-1·d-1) 1 h before curcumin treatment] and curcumin +YL294002 cintraperitoneal injection of AKT signal pathway activator YL294002( 0. 5 mg·kg-1·d-1) 30 min before curcumin treatment]respectively for 7 d,and negative control group( direct suture of skin after transection,without nerve repair,with equal amount of DMSO).Normal group was given no treatment. The phosphorylation of Erk1/2 and Akt,and the expression of LC3-Ⅱ,Beclin1 and p62 were measured by Western blotting. After treatment for 60 d,myelination of the injured sciatic nerve was evaluated by the expression of PMP22,Fibrin and S100 were determined using quantitative real-time polymerase chain reation( qRT-PCR) and Western blot. Results Compared with normal group,the levels of LC3-Ⅱ and Beclin 1 in negative control group increased 1. 7 times and 2. 1 times,and p-62 decreased 0. 7 times. The expression of p-ERK1/2 and p-Akt was up-regulated 1. 7 times and 1. 9 times after sciatic nerve injury in rats. curcumin could significantly increase the expression of p-ERK1/2 1. 4 times and decrease the expression of p-Akt 0. 6 times after curcumin treatment. However,after treatment with U0126 and YL294002 before curcumin treatment,Curcumin reduced the activation of ERK1/2 pathway and enhanced the inhibition of Akt pathway compared with curcumin group.qRT-PCR and Western blot analysis showed that compared with negative control group,curcumin increased the expression of S100 and PMP22 mRNA in sciatic nerve by 2. 9 times and 2. 1 times,and the expression of protein increased 1. 7 times and 1. 4 times,respectively,and the deposition of fibrin decreased. The effect of curcumin on myelin regeneration and axon regeneration was reversed by ERK1/2 pathway inhibitor and Akt pathway agonist.Conclusion Curcumin can induce autophagy and promote myelin regeneration after sciatic nerve injury in rats,which may be related to the regulation of MEK/ERK and PI3K/Akt signaling pathway.