大鼠脊髓损伤后线粒体自噬和凋亡相关蛋白的表达变化及其发生机制

Changes and related mechanism of mitophagy-and apoptosis-related protein expressions after spinal cord injury in rats

目的探讨大鼠脊髓损伤后线粒体自噬和凋亡相关蛋白的表达变化及其发生机制。方法将96只健康雌性SD大鼠按随机数字表法分为假手术组(48只)和脊髓损伤组(48只),每组分为1、3、7、14、21、28 d共6个时间点,各时间点8只。假手术组将T8~9棘突及椎板切除但不损伤脊髓,脊髓损伤组采用Allen法建立脊髓损伤模型。采用BBB评分评估两组伤后各时间点运动功能;HE染色观察两组伤后各时间点脊髓组织病理学改变;免疫荧光观察两组伤后各时间点电压依赖性阴离子通道蛋白1(VDAC1)分别与微管相关蛋白1轻链3(LC3)Ⅱ、E3泛素连接酶(Parkin)、多泛素结合蛋白(p62)共定位阳性细胞情况;Western blot法检测两组各时间点线粒体LC3Ⅱ/LC3Ⅰ、细胞质Parkin、线粒体Parkin、细胞质p62、线粒体p62、细胞质细胞凋亡蛋白(Bax)、线粒体Bax、细胞质细胞色素C(Cyt C)、线粒体Cyt C的表达情况。结果(1)假手术组伤后各时间点BBB评分均为(21.00±0.00)分,脊髓损伤组伤后1、3、7、14、21、28 d BBB评分分别为(0.94±0.50)分、(1.69±0.70)分、(4.13±0.99)分、(11.81±1.03)分、(15.06±1.12)分、(18.38±0.83)分(P<0.01)。(2)与假手术组比较,脊髓损伤组伤后1、3 d脊髓组织结构明显破坏,可见大量出血灶,炎性细胞浸润,神经元肿胀,空洞形成;伤后7、14 d出血灶较前明显减少,神经元胞体肿胀,炎性细胞浸润,存在大量空洞;伤后21、28 d可见大量细胞参与修复,细胞排列紊乱。(3)与假手术组比较,脊髓损伤组伤后1、3 d VDAC1分别与LC3Ⅱ、Parkin、p62共定位阳性细胞数明显增多,此后共定位阳性细胞数逐渐减少。(4)假手术组和脊髓损伤组伤后1、3、7、14、21、28 d线粒体LC3Ⅱ/LC3Ⅰ表达量分别为0.56±0.05和1.00±0.05、1.19±0.11、0.86±0.05、0.80±0.08、0.66±0.13、0.51±0.11,细胞质Parkin表达量分别为0.80±0.13和0.47±0.08、0.29±0.06、0.57±0.07、0.70±0.05、0.97±0.09、0.88±0.12,线粒体Parkin表达量分别为0.67±0.09和1.07±0.18、1.27±0.15、0.82±0.12、0.59±0.09、0.53±0.13、0.57±0.14,细胞质p62表达量分别为1.25±0.08和1.04±0.04、0.94±0.05、1.09±0.05、1.19±0.06、1.20±0.04、1.27±0.05,线粒体p62表达量分别为0.61±0.06和0.88±0.07、1.09±0.09、0.98±0.07、0.70±0.08、0.68±0.08、0.60±0.09,细胞质Bax表达量分别为0.92±0.08和0.67±0.07、0.36±0.08、0.48±0.08、0.69±0.06、0.88±0.11、0.94±0.08,线粒体Bax表达量分别为0.57±0.04和0.74±0.04、0.91±0.05、0.76±0.05、0.63±0.08、0.61±0.05、0.57±0.05,细胞质Cyt C表达量分别为0.28±0.05和0.81±0.07、1.12±0.08、0.64±0.07、0.67±0.13、0.60±0.11、0.37±0.06,线粒体Cyt C表达量分别为1.02±0.07和0.91±0.14、0.37±0.07、0.73±0.06、0.91±0.11、0.95±0.13、1.10±0.15。与假手术组比较,脊髓损伤组线粒体LC3Ⅱ/LC3Ⅰ在伤后3 d表达最高,细胞质Parkin在伤后3 d表达最低,线粒体Parkin在伤后3 d表达最高,细胞质p62在伤后3 d表达最低,线粒体p62在伤后3 d表达最高,细胞质Bax在伤后3 d表达最低,线粒体Bax在伤后3 d表达最高,细胞质Cyt C在伤后3 d表达最高,线粒体Cyt C在伤后3 d表达最低。结论大鼠脊髓损伤后线粒体自噬及凋亡均增强,其发生机制可能是由于细胞质Parkin、p62转移至受损线粒体以增强线粒体自噬,而Bax从细胞质转移至受损线粒体内,促使线粒体中大量释放Cyt C以增强细胞凋亡。

Objective To explore the changes of mitophagy-and apoptosis-related protein expressions after spinal cord injury in rats and its related mechanism.Methods Ninety-six healthy female SD rats were divided into sham surgery group(n=48)and spinal cord injury group(n=48)according to the random number table.Each group was divided into six time points of 1,3,7,14,21 and 28 days with 8 rats at each time point.In the sham surgery group,the T8-9 spinous processes and vertebral plates were removed without damage to the spinal cord;in the spinal cord injury group,the spinal cord injury model was established using Allen′s method.At each post-injury time point in two groups,BBB score was used to evaluate the motor function of the rats;HE staining was used to observe the histopathological changes of the spinal cord;immunofluorescence was used to observe the co-localized positive cells of the voltage-dependent anion channel protein 1(VDAC1)with microtubule-associated protein 1 light chain 3(LC3)II,E3 ubiquitin ligase(Parkin),and polyubiquitin-binding protein(p62);Western blotting was used to observe expressions of the mitochondrial LC3 II/LC3 I,cytoplasmic Parkin,mitochondrial Parkin,cytoplasmic p62,mitochondrial p62,cytoplasmic apoptotic proteins(Bax),mitochondrial Bax,cytoplasmic cytochrome C(Cyt C),and mitochondrial Cyt C.Results(1)Compared with the sham surgery group[(21.00±0.00)points at all time points],the BBB scores in the spinal cord injury group were(0.94±0.50)points,(1.69±0.70)points,(4.13±0.99)points,(11.81±1.03)points,(15.06±1.12)points and(18.38±0.83)points at 1,3,7,14,21 and 28 days after injury,respectively(P<0.01).(2)Compared with the sham surgery group,the structure in the spinal cord injury group was significantly disrupted at 1 and 3 days after injury,when a large number of hemorrhagic foci,inflammatory cell infiltration,neuronal swelling,and cavity formation were observed.At 7 and 14 days after injury,the number of hemorrhagic foci was markedly reduced compared with the earlier period,when swollen neuronal cytosols,inflammatory cell infiltration and a large number of cavities were found.At 21 and 28 days after injury,a large number of cells were seen to be involved in the repair and the arrangement of cells was disorganized.(3)Compared with the sham surgery group,the number of co-localized positive cells of VDAC1 with LC3 II,Parkin and p62 separately in the spinal cord injury group,markedly increased at 1 and 3 days after surgery,and gradually decreased after that.(4)In the sham surgery group and at 1,3,7,14,21 and 28 days after injury in the spinal cord injury group,the mitochondrial LC3 II/LC3 I expression levels were 0.56±0.05,1.00±0.05,1.19±0.11,0.86±0.05,0.80±0.08,0.66±0.13 and 0.51±0.11,respectively;the cytoplasmic Parkin expression levels were 0.80±0.13,0.47±0.08,0.29±0.06,0.57±0.07,0.70±0.05,0.97±0.09 and 0.88±0.12,respectively;the mitochondrial Parkin expression levels were 0.67±0.09,1.07±0.18,1.27±0.15,0.82±0.12,0.59±0.09,0.53±0.13 and 0.57±0.14,respectively;the cytoplasmic p62 expression levels were 1.25±0.08,1.04±0.04,0.94±0.05,1.09±0.05,1.19±0.06,1.20±0.04 and 1.27±0.05,respectively;the mitochondrial p62 expression levels were 0.61±0.06,0.88±0.07,1.09±0.09,0.98±0.07,0.70±0.08,0.68±0.08 and 0.60±0.09,respectively;the cytoplasmic Bax expression levels were 0.92±0.08,0.67±0.07,0.36±0.08,0.48±0.08,0.69±0.06,0.88±0.11 and 0.94±0.08,respectively;the mitochondrial Bax expression levels were 0.57±0.04,0.74±0.04,0.91±0.05,0.76±0.05,0.63±0.08,0.61±0.05 and 0.57±0.05,respectively;the cytoplasmic Cyt C expression levels were 0.28±0.05,0.81±0.07,1.12±0.08,0.64±0.07,0.67±0.13,0.60±0.11 and 0.37±0.06,respectively;and the mitochondrial Cyt C expression levels were 1.02±0.07,0.91±0.14,0.37±0.07,0.73±0.06,0.91±0.11,0.95±0.13 and 1.10±0.15,respectively.Compared with the sham surgery group,in the spinal cord injury group mitochondrial LC3II/LC3I had the highest expression level at 3 days after injury;cytoplasmic Parkin had the lowest expression level at 3 days after injury;mitochondrial Parkin had the highest expression level at 3 days after injury;cytoplasmic p62 had the lowest expression level at 3 days after injury;mitochondrial p62 had the highest expression level at 3 days after injury;cytoplasmic Bax had the lowest expression level at 3 days after injury;mitochondrial Bax had the highest expression level at 3 days after injury;cytoplasmic Cyt C had the highest expression level at 3 days after injury;and mitochondrial Cyt C had the lowest expression level at 3 days after injury.Conclusion Mitochondrial autophagy and apoptosis are enhanced after spinal cord injury in rats,and the potential mechanism may be associated with the transfer of Parkin and P62 from the cytoplasm to the damaged mitochondria for enhanced mitophagy and the release of a large amount of Cyt C from the mitochondria caused by the transfer of Bax from the cytoplasm to the damaged mitochondria for enhanced apoptosis.