基于TNFR/RIPK1通路探索紫草素对大鼠脊髓损伤的作用及机制  

Mechanism of Shikonin on spinal cord injury in rats based on TNFR/RIPK1 pathway

作　　者：史吉胜 钱吉泽 王金广 林斌[2] 庞同涛 SHI Ji-sheng;QINA Ji-ze;WANG Jin-guang;LIN Bin;PANG Tong-tao(Department of Orthopedics,Qilu Hospital of Shandong University Dezhou Hospital,Dezhou 263000,Shandong,China;Department of Orthopedics,the 909th Hospital of PLA,Zhangzhou 363000,Fujian,China)

机构地区：[1]山东大学齐鲁医院德州医院骨科,山东德州253600 [2]中国人民解放军第909医院骨科,福建漳州363000

出　　处：《中国骨伤》2024年第1期61-68,共8页China Journal of Orthopaedics and Traumatology

基　　金：山东省中医药科技项目(编号:2020Q119)。

摘　　要：目的:初步探讨紫草素对大鼠急性脊髓损伤(spinal cord injury,SCI)后神经功能恢复的影响及作用机制。方法:将96只Sprague-Dawley(SD)雄性大鼠分为4组:假手术组,即A组;假手术+紫草素组,即B组;脊髓损伤+二甲基亚砜(dimethyl sulfoxide,DMSO)组,即C组;脊髓损伤+紫草素组,即D组;每组24只。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30 min经导管注射紫草素100 mg·kg^(-1),C组注射等量DMSO,每日1次,至取材时间点。各组分别于造模后6、12 h和3 d每组取8只大鼠,行Basso-Beattie-Bresnahan(BBB)评分及造模后1、3、7、14、21d行斜板实验,再处死动物取脊髓组织。造模后1h每组大鼠腹腔注射碘化丙啶(propidine iodide,PI)1 mg·kg^(-1),术后24 h取材检测脊髓组织PI红染细胞数;24 h时取材采用苏木素-伊红(haematoxylin eosin,HE)染色观察脊髓损伤情况,尼氏(Nissl)染色观察神经元存活数量,使用Western-blot技术检测B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)蛋白及调亡相关蛋白受体相互作用蛋白激酶1(receptor-interacting protein kinase1,RIPK1)的表达水平。结果:造模后A组和B组各时间点的BBB评分均正常,C、D组各时间点均低于A、B组,D组造模后12 h和3d的BBB评分高于同时间点C组(P<0.05)。造模后12 h,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(P<0.05)。Bcl-2、RIPK1蛋白在A组、B组表达很低;RIPK1在C组表达明显增高,在D组表达明显下降,差异有统计学意义(P<0.05);Bcl-2蛋白在D组表达高于C组(P<0.05)。结论:紫草素可减轻大鼠急性SCI后的病理变化,改善行为学评分,促进脊髓神经功能恢复。其具体机制可能与抑制TNFR/RIPK1信号通路介导的坏死性凋亡有关。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

关 键 词：脊髓损伤 紫草素 功能恢复 

分 类 号：R605[医药卫生—外科学]