头孢曲松对蛛网膜下腔出血大鼠海马神经细胞的保护作用及其机制

doi:10.13481/j.1671-587x.20190517

摘要/Abstract

摘要： 目的：探讨头孢曲松对蛛网膜下腔出血（SAH）大鼠的治疗作用，并阐明其作用机制。方法： 48只成年雄性SD大鼠随机分为假手术组、SAH组、3-甲基腺嘌呤（3-MA）组和头孢曲松（CEF）组，每组12只。血管内穿刺法建立SAH模型，腹腔注射给药，3-MA给药剂量为15 mg·kg^-1，CEF给药剂量为500 mg·kg^-1。造模后24h处死大鼠，HE染色观察大鼠脑组织病理表现，TUNEL染色法检测神经细胞凋亡情况，免疫组织化学Western blotting法检测各组大鼠海马组织中自噬相关蛋白Beclin-1、LC3-Ⅱ阳性细胞数和蛋白表达水平及凋亡因子Caspase-3蛋白表达水平。结果：与SAH组比较，CEF组大鼠神经功能评分明显升高（P<0.01）；HE染色检测，SAH组大鼠海马区神经细胞肿胀，胞质疏松，核明显固缩、碎裂和溶解；与SAH组比较，CEF组大鼠神经变性有所逆转，出现较多正常神经组织形态；3-MA组神经细胞损伤更为严重，满视野死亡神经细胞，细胞层次紊乱。定量分析显示，与SAH组比较，CEF组坏死神经细胞数明显降低（P<0.05），3-MA组坏死神经细胞数明显升高（P<0.05）。免疫组织化学法检测，与SAH组比较，CEF组大鼠海马组织中Beclin-1和LC3-Ⅱ阳性细胞数明显升高（P<0.05），3-MA组大鼠海马组织中Beclin-1和LC3-Ⅱ阳性细胞数明显降低（P<0.05）。TUNEL染色法检测，与SAH组比较，CEF组凋亡细胞数明显减少（P<0.05），3-MA组凋亡细胞数明显升高（P<0.05）。Western blotting法检测，与SAH组比较，CEF组大鼠海马组织中Caspase-3蛋白表达水平明显降低（P<0.05），3-MA组Caspase-3蛋白表达水平明显升高（P<0.05）。结论：头孢曲松对蛛网膜下腔出血大鼠神经损伤具有保护作用，其作用机制可能与上调神经细胞自噬和减少细胞凋亡有关。

关键词: 蛛网膜下腔出血, 头孢曲松, 细胞自噬, 细胞凋亡

Abstract: Objective:To investigate the therapeutic effect of ceftriaxone in the rats with subarachnoid hemorrhage(SAH), and to clarify its mechanism. Methods:A total of 48 adult male SD rats were randomly divided into sham operation group, SAH group, 3-methyl adenine (3-MA) group and ceftriaxone (CEF) group; there were 12 rats in each group. The SAH models were established by intravascular puncture;the rats were administered intraperitoneally;the dose of 3-MA was 15 mg·kg^-1, and the dose of CEF was 500 mg·kg^-1. The rats were sacrificed 24 h after model establishment, and the pathology of brain tissue of the rats was observed by HE staining. The apoptosis of neurons was detected by TUNEL staining. The number of autophagy-related proteins Beclin-1 and LC3-Ⅱ and the apoptosis factor Caspase-3 protein in hippocampus tissue of the rats in various groups were detected by immunohistochemistry and Western blotting methods. Results:Compared with SAH group, the neurological score of the rats in CEF group was significantly increased (P<0.01). The HE staining results showed the nerve cells in the hippocampus area of the rats in SAH group were swollen with loose cytoplasm and had obvious nuclear condensation,fragmentation and dissolution;compared with SAH group,the neural degeneration of the rats in CEF group was improved, and more normal nerve tissue morphology appeared; while the nerve cell damage in 3-MA group was more serious, the dead nerve cells were in full filed, and the cell layers were disordered. The quantitative analysis results showed that the number of necrotic nerve cells in CEF group was significantly lower than that in SAH,and the number of necrotic nerve cells in 3-MA group was significantly higher than that in SAH group (P<0.05). The immunohistochemical staining results showed that the number of Beclin-1 and LC3-Ⅱ posivive cells in hippocampus tissue of the rats in CEF group was significantly higher than that in SAH group (P<0.05); the number of Beclin-1 and LC3-Ⅱ positive cells in 3-MA group was significantly lower than that in SAH group (P<0.05). The TUNEL staining resluts showed that the number of apoptotic nerve cells in CEF group was significantly lower than that in SAH group (P<0.05),and the number of apoptotic nerve cells in 3-MA group was higher than that in SAH group(P<0.05). The Western blotting results showed that the expression level of Caspase-3 protein in hippocampus tissue of the rats in CEF group was significantly lower than that in SAH group (P<0.05), while the expression level of Caspase-3 protein in 3-MA group was significantly higher than that in SAH group (P<0.05). Conclusion:CEF has the protective effect on the nerve injury of the rats with SAH, and its mechanism may be related to up-regulation of autophagy and reduction of apoptosis of the neurons.

Key words: subarachnoid hemorrhage, ceftriaxone, cell autophagy, apoptosis

中图分类号:

R743.35

刘俊杰, 杜鹃, 杨雅菊, 王雪, 孙玉婷, 刘瑶, 赵雅宁, 李建民. 头孢曲松对蛛网膜下腔出血大鼠海马神经细胞的保护作用及其机制[J]. 吉林大学学报(医学版), 2019, 45(05): 1069-1074.

LIU Junjie, DU Juan, YANG Yaju, WANG Xue, SUN Yuting, LIU Yao, ZHAO Yaning, LI Jianmin. Protective effect of ceftriaxone on hippocampal neurons in subarachnoid hemorrhage rats and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1069-1074.

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