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

Liu Sihang; Tao Zhoushan; Yang Min; Cheng Changjin; Xie Jiabing; Zhu Jinsong

doi:10.3969/j.issn.1009-4237.2025.04.009

Abstract：Objective To investigate the effects of low-dose methotrexate(MTX)on the functional recov-ery 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 re-moved 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 model-ing:the MTX group was treated with intraperitoneal injection of 0.5 mg/kg MTX(once a week),MP group with in-travenous injection of 30 mg/kg MP(once a week),and the model and sham operation groups with intraperitoneal injection of 200 µL 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 at3d(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 the MP and MTX groups were significantly smaller than that in the model group at all time points(all P＜0.05).IHC results found that compared to the model group,the MP and MTX groups showed much more immunoreactive cells of Notch-1,Hey-1,Bcl-2 at each time point;WB and RT-qPCR further revealed significantly higher expression of their mRNA and proteins(all P＜0.05).Conclusion Low-dose MTX can reduce apoptosis in the spinal cord tis-sue of rats after SCI and promote the recovery of neural function.The positive effect of MTX on the neural function of rats after SCI may be related to the upregulation of Bcl-2 expression and the activation of the Notch-1/Hey-1 path-way.