Bisperoxovanadium[bpV(pic)]对大鼠脊髓损伤中自噬的影响及机制研究

Effect and mechanism of Bisperoxovanadium[bpV(pic)]on autophagy in rats with spinal cord injury

目的探讨Bisperoxovanadium[bpV(pic)]对脊髓损伤(SCI)后自噬的影响及其机制。方法将20只SD大鼠购自右江民族医学院动物中心按随机数字表法分为假手术组、SCI组、bpV(pic)组;假手术组4只,另外两组每组8只。假手术组只行椎板切除术,不造成SCI;SCI组按Allen’s法制做SCI模型,不进行其他干预处理;bpV(pic)组在SCI模型基础上予bpV(pic)干预处理。伤后6 h、1、3、7、14 d对各组大鼠进行运动功能Basso-Beattie-Bresnahan(BBB)评分。伤后2周对SCI组和bpV(pic)组SCI部位进行切片后苏木精-伊红(HE)染色和尼氏染色分析病理变化;免疫荧光分析SCI组和bpV(pic)组的LC3B蛋白变化,免疫印迹分析LC3B、Beclin1和磷酸化第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因(PTEN)及磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的蛋白变化。2周后通过免疫组织化学进一步验证SCI组与bpv(pic)组磷酸化PTEN的变化。采用两独立样本的t检验。结果假手术组的大鼠并没有出现运动功能的障碍,BBB评分一直稳定在21分左右。在SCI手术对照组,大鼠出现严重的运动功能失调,第7天,SCI手术对照组BBB评分为(6.02±1.02)分,bpv(pic)治疗组为[(7.82±0.11)分,t=3.039,P<0.05],第14天SCI手术对照组评分为(8.87±1.29)分,bpv(pic)治疗组为[(12.02±1.12)分,t=4.211,P<0.05],尼氏染色结果显示SCI手术组阳性率(12.45±1.90)%,bpv(pic)治疗组为(24.14±2.09)%,t=7.154,P<0.05],差异均有统计学意义。LC3B免疫荧光结果显示,SCI手术组阳性率为(7.71±2.08)%,bpv(pic)治疗组为(21.14±1.97)%(t=9.352,P<0.05),免疫印迹染色,LC3B SCI手术组相对表达量为0.32±0.12,bpv(pic)治疗组为2.67±0.21(t=17.110,P<0.05);Beclin1,SCI手术组相对表达量为0.86±0.22,bpv(pic)治疗组为1.91±0.31(t=4.784,P<0.05),差异均有统计学意义。免疫印迹法结果显示,SCI手术组p-pten相对表达量为0.31±0.05,bpv(pic)治疗组为0.19±0.04(t=3.246,P<0.05);SCI手术组p-ERK1/2相对表达量为0.03±0.01,bpv(pic)治疗组为0.95±0.20(t=7.785,P<0.05);免疫荧光,p-ERK1/2 SCI手术组阳性率(19.91±1.18)%,bpv(pic)治疗组为(52.43±3.97)%(t=17.620,P<0.05),差异均有统计学意义。结论bpV(pic)能明显改善大鼠SCI后的神经修复及运动功能恢复,其可能机制是bpV(pic)通过抑制PTEN的磷酸化活性,进而激活ERK1/2信号,增强神经细胞的自噬,进而促进损伤修复。

Objective To investigate the effect of Bisperoxovanadium[bpV(pic)]on autophagy after spinal cord injury(SCI)and its underlying mechanism.Methods A total of 20 SD rats purchased from the Animal Center of Youjiang Medical University for Nationalities were randomly divided into sham operation group,SCI group and bpV(pic)treatment group,with 4 in sham operation group and 8 rats each in other two groups.In sham operation group,laminectomy was performed without SCI,and in SCI group,SCI model was made according to Allen’s method and no other intervention was given.Rats in bpV(pic)treatment group were treated with bpV(pic)on the basis of SCI model.The motor function of rats in each group was evaluated by Basso-Beattie-Bresnahan(BBB)motor function score system at 6 h,1 d,3 d,7 d and 14 d after injury.Two weeks after injury,the pathological changes of SCI in SCI group and bpV(pic)treatment group were analyzed by hematoxylin and eosin(HE)staining and Nissl staining,and the changes of LC3B protein in SCI group and bpV(pic)treatment group were analyzed by immunofluorescence.The protein changes of phosphorylated phosphatase and tensin homologue deleted on chromosome ten(PTEN)and phosphorylated extracellular signal-regulated kinase1/2(ERK1/2),LC3B and Beclinl were detected by Western blotting.Two weeks later,the changes of phosphorylated ERK1/2 in SCI group and bpV(pic)group were further verified by immunohistochemistry.Results Rats in the sham operation group showed no motor dysfunction,and the BBB score remained stable at about 21 points.In the SCI group,severe motor dysfunction was observed.BBB scores were significantly lower at 6 h,1 day,3 days,7 days and 14 days after operation.However,BBB scores were significantly increased after bpV(pic)treatment compared with the SCI group from 1 day after operation,and there was a statistically significant difference from day 7[Scores in SCI group,6.02±1.02,Scores in bpV(pic)treatment group,7.82±0.11,t=3.309,P<0.05],and the improvement was more apparent after 14 days[Scores in SCI group,8.87±1.29,Scores in bpV(pic)treatment group,12.02±1.12,t=4.211,P<0.05].Results of nissl staining showed the positive rate in SCI group was(12.45±1.90)%,and(24.14±2.09)%in bpV(pic)treatment group,t=9.352,P<0.05.Immunofluorescence assay showed that the expression of autophagy protein LC3B was significantly increased after treatment with bpV(pic)at 2 weeks after operation compared with SCI group[LC3B positive cells in SCI control group,7.71±2.08;LC3B positive cells in bpV(pic)treatment group,21.14±1.97,t=9.352,P<0.05].Western blotting showed that bpV(pic)significantly increased the expression of autophagic proteins LC3B[relative expression in SCI group,0.32±0.12;relative expression in bpV(pic)treatment group,2.67±0.21,t=17.110,P<0.05]and Beclinl[relative expression in SCI group,0.86±0.22;relative expression in bpV(pic)treatment group,1.91±0.31,t=4.784,P<0.05].Western blotting showed that bpV(pic)significantly inhibited the phosphorylation activity of PTEN[relative expression in SCI group,0.31±0.05;relative expression in bpV(pic)treatment group,0.19±0.04,t=3.246,P<0.05]and increased the expression of phosphorylated ERK1/2[relative expression in SCI group,0.03±0.01;relative expression in bpV(pic)treatment group,0.95±0.20,t=7.785,P<0.05].The phosphorylated ERK1/2 was increased with immunohistochemistry in the spinal cords[positive cells in SCI group,19.91±1.18,positive cells in bpV(pic)treatment group,52.43±3.97,t=17.620,P<0.05].Conclusion bpV(pic)can significantly improve the repair of injured nerve and the recovery of motor function after SCI,possibly by activating ERK1/2 signal and enhancing autophagy of nerve cells through inhibiting the phosphorylation activity of PTEN.