Felodipine Promotes the Recovery of Mice With Spinal Cord Injury by Activating Macrolipophagy Through the AMPK-mTOR Pathway

Abstract

Spinal cord injury (SCI) is a serious clinical condition characterised by extensive mechanical damage that compromises the tissue structure and microenvironment of the affected area. This damage leads to the formation of fibrotic blood vessels and impaired energy metabolism, both of which hinder recovery. Felodipine, a clinically approved antihypertensive drug, acts as a selective calcium antagonist, primarily inhibiting extracellular calcium influx in arteriolar smooth muscle and selectively dilating arterioles. Additionally, felodipine has been demonstrated to induce autophagy. Considering these properties collectively, we hypothesised that felodipine could modulate the microenvironment of the injured spinal cord. In this study, we employed immunofluorescence and Western blot analyses to evaluate the effects of felodipine on microenvironment repair and neuroprotection, both in vitro and in vivo. Particular attention was given to its regulatory role in AMPK-mTOR pathway-mediated macrolipophagy. Our results demonstrated that felodipine effectively improved the injured spinal cord microenvironment by activating macrolipophagy, facilitating the clearance of myelin debris. Furthermore, felodipine promoted the restoration of endothelial cell tight junctions, thereby enhancing the integrity of the blood-spinal cord barrier. This attenuation of barrier disruption after SCI contributed to improved neuronal survival. These findings expanded the clinical application prospect of felodipine and presented new therapeutic avenues for treating SCI.

Keywords: AMPK; Felodipine; macrolipophagy; microenvironment; spinal cord injury.

FIGURE 1

Autophagy activation could accelerate lipid metabolism in VECs following SCI. (A and B) Immunofluorescence staining of Bodipy (green), myelin basic protein (MBP) (red), CD31 (white) and DAPI (blue) in each group following SCI. The white boxes indicated the enlarged areas. The part conducted by the white arrow indicated the presence of large amounts of myelin debris (MBP) and lipids (Bodipy) in VECs (CD31). Magnification: ×20; scale bar: 100 μm.

FIGURE 2

Felodipine could attenuate lipid accumulation‐induced fibrosis in HBMECs through the AMPK‐mTOR pathway in vitro. (A) The chemical structure formula of felodipine. (B) Adopting CCK‐8 to measure relative cell viability, the optimal concentration of felodipine was 100 nM in HBMECs. (C) Western blot indicates the expression of pAMPK, AMPK, pmTOR and mTOR in HBMECs. (D and E) Quantitative analysis of pAMPK/AMPK and pmTOR/mTOR protein expression. *Represents p < 0.05, **represents p < 0.01 versus the control group and ^##represents p < 0.01. The data are the mean ± SD (n = 3). (F) Immunofluorescence staining of Bodipy (green), collagen I (red) and DAPI (blue) in HBMECs. Scale bar: 100 μm. (G) The mean fluorescence intensity of Bodipy in HBMECs. (H) Quantification of the proportion of Bodipy⁺ and collagen I⁺ cells.

FIGURE 3

Felodipine could induce autophagy in HBMECs through the AMPK‐mTOR pathway in vitro. (A) Immunofluorescence staining of LC3 (green) and DAPI (blue) in HBMECs. Scale bar: 100 μm. (B) The mean fluorescence intensity of LC3 in HBMECs. (C) Western blot indicating the expression of LC3 and P62 in HBMECs. (D and E) Quantitative analysis of LC3II/LC3I and P62 protein expression. (F) Immunofluorescence staining of Bodipy (green) and LC3 (red) in HBMECs. Scale bar: 50 μm. (G and H) The mean fluorescence intensity of Bodipy and LC3 in HBMECs. N.S. (not significant), *represents p < 0.05, **represents p < 0.01 versus the control group and ^##represents p < 0.01. The data are the mean ± SD (n = 3).

FIGURE 4

Felodipine promoted tissue repair and behavioural recovery of the injured spinal cord. (A) The H&E and Nissl staining spinal cord sections following SCI. Scale bar: 100 μm. (B) Counting analysis of Nissl staining positive neurons. (C) The BMS score of mice at the corresponding time points. **Represents p < 0.01 versus the sham group. ^##Represents p < 0.01 versus the sham group.

FIGURE 5

Felodipine could inhibit VEC fibrosis by activating autophagy in vivo. (A) Immunofluorescence staining of collagen I (green), CD31 (red) and DAPI (blue) in each group following SCI. Scale bar: 100 μm. (B) The Masson staining spinal cord sections following SCI. Scale bar: 100 μm. (C) Counting analysis collagen per aera by Masson staining. (D) Immunofluorescence staining of CD31 (red), LC3 (white) and DAPI (blue) in each group following SCI. Scale bar: 100 μm. (E and F) The mean fluorescence intensity of collagen I and CD31 in each group. (G and H) The mean fluorescence intensity of LC3 in each group. N.S. (not significant), **represents p < 0.01 versus the sham group and ^##represents p < 0.01 versus the sham group. The data are the mean ± SD (n = 3).

FIGURE 6

Felodipine promoted the restoration of vascular tight junction and protected neurons in vivo. (A) Western blot indicates the expression of collagen I, zonula occludens protein 1 (ZO1) and occludin in each group following SCI. (B‐D) Quantitative analysis of collagen I, ZO1 and occludin protein expression. (E) Immunofluorescence staining of occludin (green) and DAPI (blue) in each group following SCI. Scale bar: 100 μm. (F) Immunofluorescence staining of ZO1 (green) and DAPI (blue) in each group following SCI. Scale bar: 50 μm. (G) Immunofluorescence staining of Neuronal nuclei antigen (NeuN) (green) and DAPI (blue) in each group following SCI. Scale bar: 50 μm. (H–J) The mean fluorescence intensity of occludin, ZO1 and NeuN in each group. **Represents p < 0.01 versus the sham group. ^##Represents p < 0.01 versus the sham group. The data are the mean ± SD (n = 3).

FIGURE 7

Felodipine promoted the recovery of mice with SCI by activating macrolipophagy. Schematic diagram indicating how felodipine promoted SCI recovery.