Expression of B-cell lymphoma-2 protein multisite phosphorylation in autophagy after spinal cord injury in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WENG Fengbiao ¹ , ZHU Lifan ¹ , YANG Liwen ¹ , LI Yong ¹ , LIU Rong ¹ , FAN Jin ² ,  ZHOU Jianxin ¹

1. Department of Orthopaedics, First People’s Hospital of Wujiang District in Suzhou City, Suzhou Jiangsu, 215200, P.R.China;
2. Department of Spinal Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing Jiangsu, 210000, P.R.China;

Corresponding author：

ZHOU Jianxin, Email: rechard007@163.com

Keywords：

Spinal cord injury; autophagy; apoptosis; B-cell lymphoma-2 protein; phosphorylation; rat

DOI：

10.7507/1002-1892.201812064

Video：

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Objective To investigate the changes of autophagy after spinal cord injury (SCI) in rats and its relationship with multisite phosphorylation of B-cell lymphoma-2 (Bcl-2) protein. Methods Forty male Sprague-Dawley rats aged 8 weeks were used to prepare SCI models by modified Allen method, and the SCI model were prepared successfully in 36 rats. The 36 SCI models were randomly divided into SCI group, autophagy inhibitor group, and autophagy promoter group, with 12 rats in each group. Another 12 rats were selected as sham operation group with only laminectomy and no spinal cord injury. At the end of modeling, the autophagy inhibitor group and the autophagy promoter group were intrathecally injected with 20 μL of 600 nmol/L 3-methyladenine and 25 nmol/L rapamycin, respectively, once a day for 4 weeks. The sham operation group and the SCI group were injected with only 20 μL of normal saline at the same time point. The motor function of rat in each group was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and 1, 2, 4 weeks after modeling. The rats in each group were sacrificed at 24 hours after the last injection and the spinal cord tissues were taken. ELISA assay was used to detect the levels of inflammatory factors in spinal cord tissues, including myeloperoxidase (MPO), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β); the morphological changes of spinal cord were observed by HE staining; the autophagy of mitochondria in spinal cord tissues was observed by transmission electron microscopy; the expressions of Beclin1 and microtubule-associated protein light chain 3 (LC3) were detected by immunofluorescence staining; neuronal apoptosis in spinal cord tissues were observed by TUNEL staining; LC3/TUNEL positive cells were calculated by immunofluorescence double staining; the expressions of Bcl-2 associated X protein (Bax), Bcl-2, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) were detected by Western blot. Results Compared with sham operation group, BBB score of SCI group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; peripheral space of nerve cells enlarged, cells swelled, vacuoles appeared, and autophagic bodies appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins, and apoptotic rate of neurons significantly increased; the LC3/TUNEL positive cells significantly increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased; all showing significant differences (P<0.05). Compared with SCI group, BBB score in autophagy inhibitor group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; a few autophagic vesicles appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins decreased and the apoptotic rate of neurons increased significantly; the LC3 positive cells decreased and the TUNEL positive cells increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased. The results of autophagy promoter group were opposite to those of autophagy inhibitor group; all showing significant differences between groups (P<0.05). Conclusion Induction of autophagy after SCI in rats can reduce neuronal apoptosis and protect spinal cord function, which may be related to the inhibition of Bcl-2 protein multisite phosphorylation.

Citation： WENG Fengbiao, ZHU Lifan, YANG Liwen, LI Yong, LIU Rong, FAN Jin, ZHOU Jianxin. Expression of B-cell lymphoma-2 protein multisite phosphorylation in autophagy after spinal cord injury in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(5): 618-627. doi: 10.7507/1002-1892.201812064 Copy

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11.	Borghi SM, Pinho-Ribeiro FA, Fattori V, et al. Quercetin inhibits peripheral and spinal cord nociceptive mechanisms to reduce intense acute swimming-induced muscle pain in mice. PLoS One, 2016, 11(9): e162267.
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13.	Zhang D, Xuan J, Zheng BB, et al. Metformin improves functional recovery after spinal cord injury via autophagy flux stimulation. Mol Neurobiol, 2017, 54(5): 3327-3341.
14.	Frudd K, Burgoyne T, Burgoyne JR. Oxidation of Atg3 and Atg7 mediates inhibition of autophagy. Nat Commun, 2018, 9(1): 95-106.
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16.	Xu R, Liu S, Chen H, et al. MicroRNA-30a downregulation contributes to chemoresistance of osteosarcoma cells through activating Beclin-1-mediated autophagy. Oncol Rep, 2016, 35(3): 1757-1763.
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18.	Zhang H, Tang J, Li C, et al. MiR-22 regulates 5-FU sensitivity by inhibiting autophagy and promoting apoptosis in colorectal cancer cells. Cancer Lett, 2015, 356(2 Pt B): 781-790.
19.	Liu J, Wang GH, Duan YH, et al. Modulation of the Nur77-Bcl-2 apoptotic pathway by p38α MAPK. Oncotarget, 2017, 8(41): 69731-69745.
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1. Mortezaee K, Khanlarkhani N, Beyer C, et al. Inflammasome: its role in traumatic brain and spinal cord injury. J Cell Physiol, 2018, 233(7): 5160-5169.
2. Inskip JA, Lucci VM, Mcgrath MS, et al. A community perspective on bowel management and quality of life after spinal cord injury: the influence of autonomic dysreflexia. J Neurotrauma, 2018, 35(9): 1091-1105.
3. Colin DJ, Hain KO, Allan LA, et al. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins. Biol Open, 2015, 5(3): 140156.
4. Liu D, Huang Y, Jia C, et al. Administration of antagomir-223 inhibits apoptosis, promotes angiogenesis and functional recovery in rats with spinal cord injury. Cell Mol Neurobiol, 2015, 35(4): 483-491.
5. 潘文明. 大鼠脊髓损伤后细胞自噬的表达及作用的实验研究. 苏州: 苏州大学, 2010.
6. 孙树凯, 马磊, 张海红, 等. Rap1a 高表达对 SNL 大鼠神经病理性疼痛及星形胶质细胞自噬的影响. 第三军医大学学报, 2016, 38(21): 2335-2339.
7. 任宪盛, 丁巍, 杨小玉. 虾青素对大鼠脊髓损伤后细胞凋亡的影响. 中国修复重建外科杂志, 2018, 32(5): 548-553.
8. Park S, Yang EJ. Modulation of neuroinflammation by Taklisodok-um in a spinal cord injury model. Neuro Immuno Modulation, 2018, 25(2): 1-7.
9. Namjoo Z, Moradi F, Aryanpour R, et al. Combined effects of rat Schwann cells and 17β-estradiol in a spinal cord injury model. Metab Brain Dis, 2018, 33(4): 1229-1242.
10. 李锦春, 钱传云, 蔡乙明, 等. 微生态制剂联合肠内营养对急性重症胰腺炎患者全身炎症反应、细菌移位以及免疫功能的影响. 中国现代医学杂志, 2018, 28(6): 85-89.
11. Borghi SM, Pinho-Ribeiro FA, Fattori V, et al. Quercetin inhibits peripheral and spinal cord nociceptive mechanisms to reduce intense acute swimming-induced muscle pain in mice. PLoS One, 2016, 11(9): e162267.
12. 姜凌, 常诚. 自噬与神经退行性疾病. 卒中与神经疾病, 2014, 21(2): 125-128.
13. Zhang D, Xuan J, Zheng BB, et al. Metformin improves functional recovery after spinal cord injury via autophagy flux stimulation. Mol Neurobiol, 2017, 54(5): 3327-3341.
14. Frudd K, Burgoyne T, Burgoyne JR. Oxidation of Atg3 and Atg7 mediates inhibition of autophagy. Nat Commun, 2018, 9(1): 95-106.
15. Fletcher K, Ulferts R, Jacquin E, et al. The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes. EMBO J, 2018, 37(4): 97840-97857.
16. Xu R, Liu S, Chen H, et al. MicroRNA-30a downregulation contributes to chemoresistance of osteosarcoma cells through activating Beclin-1-mediated autophagy. Oncol Rep, 2016, 35(3): 1757-1763.
17. Lalaoui N, Lindqvist LM, Sandow JJ, et al. The molecular relationships between apoptosis, autophagy and necroptosis. Semin Cell Dev Biol, 2015, 39(1): 63-69.
18. Zhang H, Tang J, Li C, et al. MiR-22 regulates 5-FU sensitivity by inhibiting autophagy and promoting apoptosis in colorectal cancer cells. Cancer Lett, 2015, 356(2 Pt B): 781-790.
19. Liu J, Wang GH, Duan YH, et al. Modulation of the Nur77-Bcl-2 apoptotic pathway by p38α MAPK. Oncotarget, 2017, 8(41): 69731-69745.
20. Yuan J, Zhao X, Hu Y, et al. Autophagy regulates the degeneration of the auditory cortex through the AMPK-mTOR-ULK1 signaling pathway. Int J Mol Med, 2018, 41(4): 2086-2098.
21. Pécot J, Maillet L, Le Pen J, et al. Tight sequestration of BH3 proteins by BCL-xL at subcellular membranes contributes to apoptotic resistance. Cell Rep, 2016, 17(12): 3347-3358.

Chinese Journal of Reparative and Reconstructive Surgery

Expression of B-cell lymphoma-2 protein multisite phosphorylation in autophagy after spinal cord injury in rats

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