褪黑素调控磷脂酰肌醇3激酶/蛋白激酶B信号通路在脊髓损伤小鼠中神经保护作用的机制

Mechanism of melatonin regulating phosphatidylinositol 3 kinase/inhibited protein kinase B signaling pathway for neuroprotection in mice with spinal cord injury

目的探讨褪黑素(Mel)通过调控磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路对脊髓损伤小鼠发挥神经保护作用的机制。方法使用Allen's击打法制备脊髓损伤模型并按随机数字表法分为假手术组(Sham组)、脊髓损伤组(SCI组)以及褪黑素治疗组(Mel组),通过BMS评分观察小鼠的运动功能恢复情况,悬尾实验和糖水偏嗜实验评价小鼠的抑郁及焦虑状态,使用实时荧光定量聚合酶链反应(PCR)检测致炎因子白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)和抑炎因子IL-4的基因表达,免疫荧光染色观察神经元的数量,蛋白质印迹法(Westernblot)分析PI3K/Akt信号通路相关蛋白的激活。组间比较采用t检验和单因素方差分析。结果与SCI组小鼠比较,Mel组小鼠伤后21d(3.17±0.75比2.17±0.75)和伤后28d的BMS评分(3.83±0.75比2.50±0.55)高于SCI组(t=-2.301、-3.508,P<0.05)。Mel组的悬尾实验(126.82±21.43比174.36±14.91)和糖水偏嗜实验(70.39±6.94比58.36±7.47)与SCI组比较,显示Mel可改善小鼠的抑郁及焦虑状态(t=4.461、-2.886,P<0.05)。免疫荧光结果显示Mel组小鼠损伤脊髓组织中存活神经元数量(177.67±9.80比54.83±22.25)明显高于SCI组(t=-12.378,P<0.05)。Mel组小鼠的致炎因子IL-1β、TNF-α的基因表达水平分别为4.90±1.16比27.07±2.73、1.72±0.46比9.64±1.98,明显低于SCI组(t=18.299、9.571,P<0.05),抑炎因子IL-4的基因表达水平为0.64±0.13比0.22±0.09,明显高于SCI组(t=-6.564,P<0.05)。Westernblot结果显示Mel组的p-PI3K、p-Akt蛋白表达水平均显著高于SCI组(1.05±0.09比0.81±0.09、4.38±1.05比2.39±0.16,t=-4.623、-4.602,P<0.05)。结论褪黑素可以激活PI3K/Akt信号通路,抑制炎性反应,增加存活细胞数量,改善脊髓损伤小鼠的预后。

Objective To explore the neuroprotective role of melatonin(Mel)in spinal cord injured mice through the regulation of the phosphatidylinositol 3 kinase/inhibited protein kinase B(PI3K/Akt)signaling pathway.Methods The spinal cord injury(SCI)model was prepared using Allen's Impactor method,and the mice were randomly divided into a sham group(Sham group),SCI group,and Mel group.The motor function recovery was observed using the Basso Mouse Scale(BMS)locomotor rating scale.The tail suspension test and sucrose preference test were conducted to evaluate the depressive and anx-iety states of mice.Real-time fluorescence quantitative polymerase chain reaction(PCR)was used to detect the gene expression of pro-inflammatory factors interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),and anti-inflammatory factor IL-4.Immunofluorescence staining was performed to observe the number of neurons,and Western blotting analysis was carried out to assess the activation of proteins related to the PI3K/Akt sig-naling pathway.Statistical comparisons between groups were performed using t-tests and one-way analysis of variance(ANOVA).ResultsCompared to the SCI group,the BMS scores in the Mel group were increased at post-injury day 21(3.17±0.75 vs.2.17±0.75)and post-injury day 28(3.83±0.75 vs.2.50±0.55,t=-2.301,-3.508,P<0.05).The tail suspension test(126.82±21.43vs.174.36±14.91)and sucrose preference test(70.39±6.94 vs.58.36±7.47)of the Mel group showed improve-ment in the depressive and anxiety states of the mice compared to the SCI group(t=4.461,-2.886,P<0.05).Immunofluorescence staining showed a significant increase in the number of surviving neurons in the injured spinal cord tissue of the Mel group compared to the SCI group(177.67±9.80 vs.54.83±22.25,t=-12.378,P<0.05).The gene expression levels of IL-1βand TNF-αin the Mel group were significantly decreased compared to the SCI group(4.90±1.16 vs.27.07±2.73,1.72±0.46 vs.9.64±1.98),and the gene expression level of IL-4 in the Mel group was significantly increased compared to the SCI group(0.64±0.13 vs.0.22±0.09,t=18.299,9.571,-6.564,P<0.05).Western blotting results showed higher expression levels of p-PI3K and p-Akt proteins in the Mel group than the SCI group(1.05±0.09 vs.0.81±0.09,4.38±1.05 vs.2.39±0.16,t=4.623,-4.602,P<0.05).Conclusion This study demonstrates that Mel can activate the PI3K/Akt signaling pathway,inhibit inflammatory responses,increase the number of surviving cells,and improve the prognosis of spinal cord injured mice.