王 双,林 斌,陈志达,史吉胜,曾宇哲,吴松松.抑制TNFR/RIPK信号通路对大鼠急性脊髓损伤后神经功能的影响[J].中国脊柱脊髓杂志,2017,(4):353-360.
抑制TNFR/RIPK信号通路对大鼠急性脊髓损伤后神经功能的影响
中文关键词:  脊髓损伤  RIPK1  RIPK3  Bcl-2  坏死性凋亡  功能恢复
中文摘要:
  【摘要】 目的:探讨应用坏死性凋亡抑制剂Necrostatin-1(Nec-1)抑制TNFR/RIPK介导的坏死性凋亡信号通路对大鼠急性脊髓损伤(spinal cord injury,SCI)的作用。方法:72只雄性SPF级SD大鼠,体重0.25~0.30kg,随机分为4组:假手术组(Sham组,A组)、假手术+Necrostatin-1组(Sham+Nec-1组,B组)、SCI+二甲基亚砜(DMSO)(DMSO组,C组)、SCI+Nec-1组(Nec-1组,D组)。C、D组采用钳夹法制作大鼠急性SCI模型。所有大鼠硬膜下置管,A组不给药,B组和D组造模后30min经导管注射1μl Nec-1(25μg/μl),C组注射等量DMSO,1次/d,至取材时间点。各组分别于造模后12h、24h和3d三个时间点每组取6只大鼠,先行Basso/Beattie/Bresnahan(BBB)评分,再处死动物取脊髓组织。12h时间点动物处死前1h腹腔注射碘化丙啶(propidine iodide,PI)1mg/kg,取材检测脊髓组织PI红染细胞数;24h时取材采用苏木素-伊红(HE)染色观察脊髓损伤情况、尼氏(Nissl)染色观察神经元存活数目、Western Blot(WB)检测Bcl-2、坏死性凋亡蛋白RIPK1及RIPK3的表达水平;3d时取材行Tunel染色观察细胞凋亡情况。结果:造模后A组和B组各时间点的BBB评分均正常,C、D组各时间点均显著性低于A、B组,D组各时间点的BBB评分均显著高于同时间点C组(P<0.05)。造模后12h,D组PI红染细胞较C组明显减少,神经元崩解减轻(P<0.05)。造模后24h,A组和B组脊髓组织HE和Nissl染色正常,D组脊髓组织损伤程度和存活神经元数量均优于C组,差异有统计学意义(P<0.05);A组和B组Bcl-2、RIPK1及RIPK3均低水平表达,C组RIPK1及RIPK3表达显著性升高,D组Bcl-2表达较C组上调,RIPK1及RIPK3表达显著性下降,C、D两组比较差异均有统计学意义(P<0.05)。造模后3d,A组和B组可见少量凋亡小体,C组和D组明显增多,但D组凋亡小体数量较C组明显减少(P<0.05)。结论:抑制TNFR/RIPK信号通路可以减轻大鼠急性SCI后的病理变化,改善行为学评分,促进脊髓神经功能恢复。
Inhibition of TNFR/RIPK signaling pathway mitigate acute spinal cord injury in rats
英文关键词:Spinal cord injury  RIPK1  RIPK3  Bcl-2  Necroptosis  Functional recovery
英文摘要:
  【Abstract】 Objectives: To investigate the protective effect of inhibition of TNFR/RIPK signaling pathway by Necrostatin-1 after spinal cord injury(SCI), and to provide new reference for the treatment of SCI. Methods: 72 SPF level male S-D rats weighing 0.25-0.30kg were randomized equally into four groups: sham group(group A), sham+Nec-1 group(group B), DMSO group(group C), Nec-1 group(group D). Spinal cord clamp compression method was used to make rat acute spinal cord injury model in group C and D. After subdural catheterization, group A had no treatment, group B and D were injected with 1μl Nec-1(25μg/μl) by using a micro-syringe at 30 minutes after SCI, group C was injected with the same amount of DMSO solution, once a day until the time point of collection tissue. Each group was divided into three subgroups: 12h, 24h and 3d after SCI, and each subgroup included six rats. Rats conducted BBB score in each group to evaluate functional recovery. Six rats were intraperitoneally injected with propidine iodide(PI) 1h before sacrificed to detection PI positive cells at 12h in each group. Six rats were sacrificed in each group at 24h after modeling, then the removed spinal cords were taken HE staining to detect the nerve tissue pathological changes, Nissl staining to observe survivor number of nerve cells, western blot to detect Bcl-2, RIPK1 and RIPK3 protein expression level. And six rats were sacrificed at 3d in each group after modeling to take Tunel staining for the detection of apoptosis of nerve tissue. Results: After modeling, BBB scores were normal in group A and B, but in group C and D were significantly higher than those in group A and B. And the scores in group D were higher than those in group C in each time point(P<0.05). HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation. The degree of SCI and the number of neuronal survival in group D were better than those in group C, the difference was statistically significant at 24h(P<0.05). The expression levels of Bcl-2, RIPK1 and RIPK3 protein were stable low in group A and B, and RIPK1 and RIPK3 expression upregulated in group C, in group D the expression level of Bcl-2 upregulated, but RIPK1 and RIPK3 expression downregulated, the difference was statistically significant(P<0.05). 3d after SCI, there were seldom apoptotic bodies in group A and B, but the numbers of apoptotic body in group C and D was significantly higher than those in group A and B. And the number in group D was lower than that in group C(P<0.05). Conclusions: Inhibition of TNFR/RIPK signal pathway after rats SCI could reduce the pathological changes of spinal cord, increase the number of surviving neurons, promote nerve functional recovery and improve BBB scores.
投稿时间:2016-08-08  修订日期:2017-02-06
DOI:
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作者单位
王 双 解放军第175医院骨科医院脊柱外科 363000 福建省漳州市 
林 斌 解放军第175医院骨科医院脊柱外科 363000 福建省漳州市 
陈志达 解放军第175医院骨科医院脊柱外科 363000 福建省漳州市 
史吉胜  
曾宇哲  
吴松松  
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