目的:探讨紫草素对大鼠急性脊髓损伤后神经功能恢复的影响。
方法:96只雄性SD大鼠随机分为4组:假手术组(A组),假手术+紫草素组(B组),SCI+DMSO(C组),SCI+紫草组(D组)。C、D组用钳夹法制作年夜鼠急性SCI模子。硬膜下插管后,A组不给药,B组和D组大鼠在造模后30min通过导管注射紫草素100mg·kg-1,C组大鼠给予等量的DMSO,一天一次,直到收集组织的时间点。分别于莫来灵后6、12和3d对每组8只大鼠进行Basso-Beattie-Bresnahan(BBB)评分,在建模后的1、3、7和14天进行了斜板测试,然后收集脊髓组织。每组8只大鼠于处死前1h腹腔注射碘化丙啶(PI),24h检测PI阳性细胞。每组8只大鼠于造模后24h处死,HE染色观察脊髓损伤情况。Nissl染色观察神经细胞的存活数。采用Western-blot技术检测Bcl-2蛋白和凋亡相关蛋白RIPK1的表达水平。
结果:建模后,A、B组BBB评分正常,C、D组明显高于A、B组,且D组各时间点评分均高于C组(P<0.05)。建模后12小时,与C组相比,D组PI红染色细胞明显减少,神经元解体减轻(P<0.05)。HE、Nissl染色显示A、B组术后24h神经细胞形态正常。D组的SCI程度和神经元存活数均优于C组,24h时差异有统计学意义(P<0.05)。A组和B组Bcl-2和RIPK1蛋白表达很低;C组RIPK1表达明显升高,D组明显下降,差异有统计学意义(P<0.05);D组Bcl-2蛋白表达明显高于C组(P<0.05)。
结论:紫草素能减轻大鼠急性SCI后的病理变化,提高行为得分,促进脊髓神经功能的恢复。其具体机制可能与抑制TNFR/RIPK1信号通路介导的坏死性细胞凋亡有关。
OBJECTIVE: To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI) in rats.
METHODS: 96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group (Group A), sham operation+shikonin group (Group B), SCI+ DMSO(Group C), SCI+shikonin group (Group D).The acute SCI model of rats was made by clamp method in groups C and D . After subdural catheterization, no drug was given in group A. rats in groups B and D were injected with 100 mg·kg-1 of shikonin through catheter 30 min after modeling, and rats in group C were given with the same amount of DMSO, once a day until the time point of collection tissue. Basso-Beattie-Bresnahan(BBB) scores were performed on 8 rats in each group at 6, 12, and 3 d after moneling, and oblique plate tests were performed on 1, 3, 7 and 14 d after modeling, and then spinal cord tissues were collected. Eight rats were intraperitoneally injected with propidine iodide(PI) 1 h before sacrificed to detection PI positive cells at 24 h in each group. Eight rats were sacrificed in each group at 24 h after modeling, the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells. Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1.
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). At 12 h after modeling, the PI red stained cells in group D were significantly reduced compared with that in group C, and the disintegration of neurons was alleviated(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 of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D, with a statistically significant difference (P<0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C (P<0.05).
CONCLUSIONS: Shikonin can alleviate the pathological changes after acute SCI in rats, improve the behavioral score, and promote the recovery of spinal nerve function. The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.