低剂量甲氨蝶呤激活Notch-1/Hey-1通路促进大鼠脊髓损伤神经功能恢复  

Low-dose methotrexate activates the Notch-1/Hey-1 pathway to promote neural function recovery in spinal cord-injured rats

作　　者：刘思航 陶周善 杨民 程昌锦 谢加兵 朱劲松 Liu Sihang;Tao Zhoushan;Yang Min;Cheng Changjin;Xie Jiabing;Zhu Jinsong(Department of Traumatology and Orthopedics,Yijishan Hospital Affiliated to Wannan Medical College,Wuhu,Anhui Province 241000,China)

机构地区：[1]皖南医学院第一附属医院(弋矶山医院)创伤骨科,安徽芜湖241000

出　　处：《创伤外科杂志》2025年第4期292-300,307,共10页Journal of Traumatic Surgery

基　　金：芜湖市科技成果转化项目(2021cg16);皖南医学院重点科研培育基金(WK2020ZF20);安徽省临床医学研究转化研究项目(202304295107020007)。

摘　　要：目的 探讨低剂量甲氨蝶呤(MTX)对大鼠脊髓损伤(SCI)后神经功能恢复的影响及作用机制。方法 36只SD大鼠随机分为假手术组、模型组、MTX组和甲基强的松龙(MP)组,各9只,使用环扎法(实现70%的截面压缩)构建大鼠SCI模型。所有大鼠术后24 h内首次给药,MTX组和MP组分别给予MTX(0.5mg/kg、qw)和MP(30mg/kg、qw)治疗,模型组及假手术组均予以磷酸盐缓冲液(200μl、qw)注射。术后3、14、21 d使用BBB评分和斜板评分评估大鼠后肢运动功能,HE染色评估脊髓组织病理改变,TUNEL检测观察SCI区域脊髓组织的凋亡细胞变化;免疫组化(IHC)检测大鼠SCI区域Notch-1、Bcl-2表达,蛋白免疫印迹法(WB)、反转录定量聚合酶链反应(RT-qPCR)检测SCI区域组织相关蛋白和基因Notch-1/Hey-1和Bcl-2表达。结果 MP组与模型组BBB评分比较,大鼠减压术后3 d[(7.2±0.2)分vs.(3.6±0.2)]分、14 d[(12.3±0.4)分vs.(5.1±0.3)分]、21 d[(15.3±0.7)分vs.(7.1±0.5)分]评分均升高(P<0.05);MTX组与模型组BBB评分比较,大鼠减压术后3 d[(6.4±0.4)分vs.(3.6±0.2)分]、14 d[(11.7±0.4)分vs.(5.1±0.3)分]、21 d[(15.4±0.5)分vs.(7.1±0.5)分]评分均升高,差异有统计学意义(P<0.05)。斜板评分与BBB评分显示出相同的趋势。与模型组比较,HE组织切片显示MP组和MTX组的SCI区域组织坏死较少。TUNEL结果显示MP组和MTX组的SCI区域组织细胞凋亡数量较模型组在各个时间段均出现明显降低,差异有统计学意义(P<0.05);IHC结果显示,与模型组相比,在术后3、14、21 d SCI大鼠脊髓组织样本中,MP组及MTX组中Bcl-2、Notch-1阳性细胞数量均显著升高,差异有统计学意义(P<0.05)。Western Blot和PCR检测结果发现,同模型组相比,MP组和MTX组在各个时间段Notch-1、Hey-1、Bcl-2 mRNA和蛋白表达均显著升高,差异有统计学意义(P<0.05)。结论 低剂量的MTX使用对SCI后大鼠神经功能起到保护作用,初步发现这种作用可能与激活Notch-1/Hey-1通路有�Objective To investigate the effects of low-dose methotrexate(MTX)on the functional recovery of rats after spinal cord injury(SCI)and its underlying mechanism.Methods Thirty-six Sprague Dawley rats were randomly divided into four groups:a sham operation group,a model group,a methylprednisolone(MP)group,and a methotrexate(MTX)group(n=9 for each group).The sham operation group only had the vertebral plate removed and the dura mater exposed,while the other three groups underwent SCI modeling via 70%cross-sectional compression for 8-12 h and all succeeded in SCI modeling.All rats received their first dose within 24 h after modeling:the MTX group was treated with intraperitoneal injection of 0.5 mg/kg MTX(once a week),MP group with intravenous injection of 30 mg/kg MP(once a week),and the model and sham operation groups with intraperitoneal injection of 200μL PBS(once a week).Motor function was assessed using the BBB score and inclined plane score at 3,14,and 21 d after modeling(n=3 for each group at each time point).After scoring,the rats were euthanized,and the spinal cord tissue around the ligation line was harvested for subsequent tests:HE staining was used to assess histological changes,TUNEL assay to observe the changes in apoptotic cells,immunohistochemistry(IHC)to detect the expression of Notch-1 and Bcl-2,and WB and RT-qPCR to further detect the expression of related proteins and genes(Notch-1/Hey-1 and Bcl-2)at the SCI area.Results MP and MTX groups showed significantly higher BBB scores than the model group at 3 d(7.2±0.2 and 6.4±0.4 vs.3.6±0.2),14 d(12.3±0.4 and 11.7±0.4 vs.5.1±0.3)and 21 d(15.3±0.7 and 15.4±0.5 vs.7.1±0.5)after decompression(all P<0.05).The inclined plane scores revealed the same trend.Compared with the model group,the MP and MTX groups revealed much less severe bleeding and swelling of the tissue at the SCI area by HE staining.Comparison between the MP and MTX groups also showed no significant difference.TUNEL assay showed that the number of apoptosis cells at the SCI area in t

关 键 词：脊髓损伤 甲氨蝶呤 甲泼尼龙 治疗机制 分子通路 

分 类 号：R651.2[医药卫生—外科学]