Monotropein improves motor function of mice with spinal cord injury by inhibiting the PI3K/AKT signaling pathway to suppress neuronal apoptosis

doi:10.12122/j.issn.1673-4254.2025.04.13

Abstract

Abstract:

Objective To investigate the effect of monotropein on motor function recovery of mice with spinal cord injury (SCI) and explore the underlying mechanism. Methods Forty-five adult female C57BL/6 mice were randomized equally into sham operation group, SCI group, and SCI group with daily intraperitoneal monotropein injection. The mice in the former two groups received daily saline injections. Motor function of the mice was evaluated using BMS scores, slant plate test, and footprint analyses. Pathological changes and neuronal counts in the spinal cord were observed using HE, LFB, and Nissl staining. The biological functions of monotropein were explored using GO and KEGG enrichment analyses. NeuN/cleaved caspase-3 immunofluorescence assay and Western blotting were used to detect neuronal apoptosis in the spinal cord of the mice. In cultured HT22 cells, the effect of monotropein on TNF-α-induced cell apoptosis was evaluated using TUNEL staining and Western blotting. In monotropein-treated HT22 cells and SCI mice, the changes in the PI3K/AKT pathway were examined, and the effect of a PI3K/AKT pathway activator (IGF-1) on HT22 cell apoptosis and motor function recovery of SCI mice were observed. Results SCI mice with monotropein treatment showed significantly improved motor functions with reduced SCI areas and increased myelin retention and neuron counts in the spinal cord. Bioinformatics analysis suggested a role of PI3K/AKT signaling pathway in mediating the anti-apoptotic effects of monotropein. In SCI mice, monotropein obviously reduced apoptotic neurons, decreased expressions of cleaved caspase-3 and Bax and increased Bcl-2 expression in the spinal cord. In HT22 cells, monotropein significantly inhibited TNF-α-induced apoptosis and PI3K/AKT pathway activation. Treatment with IGF-1 obviously increased apoptosis of HT22 cells and exacerbated locomotor dysfunction in SCI mice. Conclusion Monotropein promotes motor function recovery in SCI mice by reducing neuronal apoptosis possibly by inhibiting the PI3K/AKT signaling pathway.

Key words: monotropein, spinal cord injury, neurons, apoptosis, PI3K/AKT

Yue CHEN, Linyu XIAO, Lü REN, Xue SONG, Jing LI, Jianguo HU. Monotropein improves motor function of mice with spinal cord injury by inhibiting the PI3K/AKT signaling pathway to suppress neuronal apoptosis[J]. Journal of Southern Medical University, 2025, 45(4): 774-784.

Figures/Tables 8

Fig.1 Assessment of motor function in monotropein (Mon)-treated mice with spinal cord injury (SCI). A: BMS Scoring. B: Inclined plane test. *P<0.05, **P<0.01 vs SCI. C,D: Footprint analysis 28 days after injury. blak arrows indicate normal hind leg tracks. (n=5). *P<0.05, **P<0.01.

Fig.2 Monotropein reduces lesion area, increases residual myelin, and inhibits motor neuron loss in SCI mice. A: HE staining of mouse spinal cord cross-sections at the injury site in the 3 groups. B: Quantitative analysis of lesion area at the injury site. C: LFB staining of mouse spinal cord cross-sections at the injury site in the 3 groups. D: Quantitative analysis of residual myelin. E: Nissl staining of neurons of mouse spinal cord cross-sections from 0.5 mm rostral to injury site center in the 3 groups. F: Quantitative analysis of residual number of motor neurons (n=5). **P<0.01.

Fig.3 GO functional enrichment and KEGG pathway enrichment analyses of the function and mechanism of monotropein. A: Prediction of the target proteins of monotropein using network pharmacology analysis. B: GO enrichment analysis. C: KEGG enrichment analysis.

Fig.4 Monotropein ameliorates neuronal apoptosis in SCI mice. A: NeuN (green) and cleaved caspase-3 (red) fluorescence costaining in mouse spinal cord tissue from the 3 groups. B: Quantification of the number of apoptotic neurons. C: Expression of apoptotic proteins in the spinal cord of the mice detected by Western blotting. D-F: Quantitative analysis of apoptotic protein expressions (n=5). *P<0.05, **P<0.01.

Fig.5 Monotropein ameliorates HT22 cell apoptosis induced by TNF-α. A: TUNEL staining for analyzing apoptosis in HT22 cells. B: Quantitative analysis of the percentage of TUNEL staining-positive cells. C: Western blotting for detection of apoptotic protein expression. D-F: Quantification of cleaved caspase-3, Bax, and Bcl-2 protein expression (n=3). *P<0.05, **P<0.01.

Fig.6 Effect of monotropein on expressions of PI3K/AKT signaling pathway proteins in the spinal cord tissue of SCI mice and in HT22 cells detected using Western blotting. A: Protein bands in the 3 groups. B, C: Quantitative analysis of p-PI3K and p-AKT protein expressions in SCI mice. D: Western blotting of PI3K/AKT signaling pathway protein expressions in HT22 cells. E, F: Quantitative analysis of p-PI3K and p-AKT protein expressions in HT22 cells (n=3). **P<0.01.

Fig.7 Monotropein alleviates neuronal apoptosis by inhibiting PI3K/AKT signaling pathway. A: Western blotting for assessment of PI3K/AKT signaling pathway protein expressions in HT22 cells. B, C: Quantitative evaluation of p-PI3K and p-AKT protein levels in HT22 cells. D: TUNEL staining of HT22 cells. E: Quantitative analysis of the percentage of TUNEL staining positive cells. F: Expression of apoptotic protein in HT22 cells. G-I: Quantitative analysis of cleaved caspase-3, Bax, and Bcl-2 protein levels in HT22 cells (n=3). *P<0.05, **P<0.01.

Fig.8 Monotropein promotes motor function recovery in SCI mice by inhibiting the PI3K/AKT signaling pathway. A: BMS scoring. B: Inclined plane test. **P<0.01 vs SCI+Mon+IGF-1. C, D: Footprint analysis 28 days after injury (n=5). **P<0.01.

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