miR-196b-5p减轻大鼠脊髓损伤后组织水肿和星形胶质细胞活化机制探讨

doi:10.11958/20220282

天津医药 ›› 2022, Vol. 50 ›› Issue (11): 1153-1157.doi: 10.11958/20220282

miR-196b-5p减轻大鼠脊髓损伤后组织水肿和星形胶质细胞活化机制探讨

孔俊东¹(), 李坚¹, 张强强¹, 李刚², 蔡家骏¹, 范仲凯¹^,^△()

1 锦州医科大学附属第一医院骨科（邮编121000）
2 山东大学齐鲁医院（青岛）骨科

收稿日期:2022-02-28 修回日期:2022-05-17 出版日期:2022-11-15 发布日期:2022-11-11
通讯作者: 范仲凯 E-mail:18369610687@163.com;fanzk_ln@163.com
作者简介:孔俊东（1995），男，硕士在读，主要从事脊髓损伤方面研究。E-mail：18369610687@163.com
基金资助:
2019年辽宁省教育厅科学技术研究项目(JYTJCZR201911);2020年辽宁省自然基金资助计划项目(2020-MS-298)

Mechanism of miR-196b-5p in alleviating secondary edema and astrocyte activation after spinal cord injury in rats

KONG Jundong¹(), LI Jian¹, ZHANG Qiangqiang¹, LI Gang², CAI Jiajun¹, FAN Zhongkai¹^,^△()

1 Department of Orthopedics, the First Affiliated Hospital of Jinzhou Medical University, Jinzhou 121000, China
2 Department of Orthopedics, Qilu Hospital (Qingdao), Shandong University

Received:2022-02-28 Revised:2022-05-17 Published:2022-11-15 Online:2022-11-11
Contact: FAN Zhongkai E-mail:18369610687@163.com;fanzk_ln@163.com

孔俊东, 李坚, 张强强, 李刚, 蔡家骏, 范仲凯. miR-196b-5p减轻大鼠脊髓损伤后组织水肿和星形胶质细胞活化机制探讨[J]. 天津医药, 2022, 50(11): 1153-1157.

KONG Jundong, LI Jian, ZHANG Qiangqiang, LI Gang, CAI Jiajun, FAN Zhongkai. Mechanism of miR-196b-5p in alleviating secondary edema and astrocyte activation after spinal cord injury in rats[J]. Tianjin Medical Journal, 2022, 50(11): 1153-1157.

摘要/Abstract

摘要：

目的探讨miR-196b-5p靶向水通道蛋白4（AQP4）促进大鼠脊髓损伤（SCI）后组织修复和神经元轴突再生的机制。方法双荧光素酶报告检测miR-196b-5p与AQP4靶向关系；实时定量聚合酶链反应（RT-qPCR）检测SCI后1 d、2 d、3 d、5 d和7 d miR-196b-5p与AQP4变化；RT-qPCR检测假手术（Sham）组、单纯脊髓损伤（SCI）组、agomiR-196b-5p干预（miRNA）组和miR-196b-5p阴性对照（NC）组脊髓miR-196b-5p与AQP4 mRNA相对表达水平，Western blot检测各组脊髓AQP4、胶质纤维酸性蛋白（GFAP）、增殖细胞核抗原（PCNA）和生长相关蛋白43（GAP-43）蛋白相对表达水平，干湿质量法和苏木精-伊红（HE）染色检测脊髓含水量及脊髓空洞大小。结果双荧光素酶报告显示AQP4是miR-196b-5p的靶基因。与Sham组比较，SCI后各时间点miR-196b-5p表达水平降低（P<0.05），而AQP4 mRNA表达升高并在2 d时达到高峰，随后逐渐降低。与Sham组比较，SCI组、miRNA组及NC组AQP4 mRNA及蛋白，GFAP、PCNA和GAP-43蛋白相对表达水平及脊髓含水量升高，而SCI组及NC组miR-196b-5p相对表达水平降低（均P<0.05）。miRNA组AQP4 mRNA，AQP4、GFAP、PCNA蛋白相对表达水平及脊髓含水量较SCI组及NC组降低，脊髓空洞面积减小，而miR-196b-5p和GAP-43相对表达水平升高（均P<0.05）。结论 miR-196b-5p通过靶向AQP4抑制SCI后继发性水肿、星形胶质细胞活化增生和胶质瘢痕的形成，最终促进神经元轴突再生和脊髓修复。

关键词: 脊髓损伤, 水肿, 水通道蛋白4, 星形胶质细胞, 胶质瘢痕, miR-195b-5p

Abstract:

Objective To explore the mechanism of miR-196b-5p in promoting tissue repair and neuronal axon regeneration by targeting AQP4 after spinal cord injury (SCI) in rats. Methods The targeting relationship between miR-196b-5p and AQP4 was detected by Dual-Luciferase reporter assay. Changes of miR-196b-5p and AQP4 at 1 d, 2 d, 3 d, 5 d and 7 d after SCI were detected by real time quantitative polymerase reaction (RT-qPCR). The relative expression levels of miR-196b-5p and AQP4 mRNA were detected by RT-qPCR in the Sham group, the SCI group, the miR-196b-5p overexpression (miRNA) group and the miR-196b-5p negative control (NC) group, respectively. The relative expression levels of AQP4, GFAP, PCNA and GAP-43 protein in spinal cord were detect by Western blot assay. Dry-wet weight method and hematoxylin eosin (HE) staining were used to detect water content and size of spinal cord cavity. Results Dual-Luciferase reporter assay showed that AQP4 was the target gene of miR-196b-5p. Compared with the Sham group, the expression of miR-196b-5p decreased at each time point after SCI, while the expression of AQP4 mRNA increased, peaked at 2 days and then decreased gradually (P<0.05). The relative expression levels of AQP4 mRNA, AQP4 protein, GFAP protein, PCNA protein, GAP-43 protein and spinal cord water content increased in the SCI group, the miRNA group and the NC group compared with those of the Sham group (P<0.05), while the relative expression levels of miR-196b-5p decreased in the SCI group and the NC group (P<0.05). The relative expression levels of AQP4 mRNA, AQP4 protein, GFAP protein, PCNA protein and spinal cord water content were lower in the miRNA group than those of the SCI group and the NC group. the area of spinal cord cavity decreased (P<0.05), while the relative expression levels of miR-196b-5p and GAP-43 protein increased (P<0.05). Conclusion miR-196b-5p can inhibit SCI induced secondary edema, astrocyte activation glial scar formation and ultimately promote axon regeneration and spinal cord repair by targeting AQP4.

Key words: spinal cord injury, edema, aquaporin 4, astrocytes, glial scar, miR-195b-5p

中图分类号:

R651.2

图/表 7

表1 RT-qPCP引物序列

Tab.1 The sequence of primer for RT-qPCR

基因名称	引物序列（5'→3'）	产物大小（bp）
AQP4	上游：CACGAAAGATCAGCATCGCC 下游：TTTCCATGAACCGTGGTGACT	216
GAPDH	上游：CTGGAGAAACCTGCCAAGTATG 下游：GGTGGAAGAATGGGAGTTGCT	138

图1 AQP4与miR-196b-5p靶向关系的预测与验证 A：miR-196b-5p与AQP4 mRNA的结合位点；B：双荧光素酶实验证实AQP4是miR-196b-5p的靶基因。

Fig.1 Prediction and validation of the targeting relationship between AQP4 and miR-196b-5p

表2 SCI后各组大鼠miR-196b-5p与AQP4表达变化 (n=3, $\bar{x}\pm s$)

Tab.2 Expression levels of miR-196b-5p and AQP4 in groups of rats after SCI

组别	miR-196b-5p	AQP4 mRNA
Sham组	1.00±0.15	1.00±0.18
SCI 1 d组	0.01±0.00^a	13.96±1.30^a
SCI 2 d组	0.02±0.00^a	25.61±2.04^ab
SCI 3 d组	0.07±0.01^a	16.15±0.78^ac
SCI 5 d组	0.11±0.02^a	10.79±1.49^ac
SCI 7 d组	0.11±0.02^a	3.84±0.64^c
F	119.063^＊＊	156.183^＊＊

表3 干预后各组大鼠miR-196b-5p、AQP4及脊髓含水量变化(n=3, $\bar{x}\pm s$)

Tab.3 Changes of miR-196b-5p, AQP4 and water content after treatment in four groups of rats

组别	miR-196b-5p	AQP4 mRNA	AQP4	脊髓含水量（%）
Sham组	1.00±0.15	1.00±0.31	0.78±0.16	70.17±0.18
SCI组	0.15±0.02^a	24.12±2.26^a	2.67±0.37^a	74.18±0.23^a
miRNA组	0.86±0.03^b	9.66±0.30^ab	1.86±0.26^ab	72.31±0.17^ab
NC组	0.25±0.06^ac	25.53±4.14^ac	2.68±0.30^ac	74.38±0.20^ac
F	80.655^＊＊	74.657^＊＊	30.444^＊＊	299.531^＊＊

图2 各组大鼠SCI后对AQP4、GFAP、PCNA及GAP-43蛋白表达水平的影响 A：SCI 3 d各组AQP4的表达水平；B：SCI 3 d各组GFAP和PCNA表达水平；C：SCI 4周各组GAP-43表达水平。

Fig.2 Effects of AQP4, GFAP, PCNA and GAP-43 protein expression levels after SCI in four groups of rats

表4 各组大鼠GFAP、PCNA及GAP-43蛋白相对表达水平比较(n=3, $\bar{x}\pm s$)

Tab.4 Comparison of relative expression levels of GFAP, PCNA and GAP-43 protein between the four groups of rats

组别	GFAP	PCNA	GAP-43
Sham组	1.00±0.12	1.00±0.18	1.00±0.03
SCI组	1.99±0.21^a	16.95±1.42^a	1.72±0.16^a
miRNA组	1.48±0.01^ab	11.29±1.02^ab	2.78±0.06^ab
NC组	1.88±0.11^ac	16.18±0.91^ac	1.81±0.04^ac
F	34.792^＊＊	164.079^＊＊	194.353^＊＊

图3 各组大鼠SCI后脊髓损伤修复结果（HE，×50）

Fig.3 Results of spinal cord repair after SCI in four groups of rats (HE, ×50)

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miR-196b-5p减轻大鼠脊髓损伤后组织水肿和星形胶质细胞活化机制探讨

Mechanism of miR-196b-5p in alleviating secondary edema and astrocyte activation after spinal cord injury in rats

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