Curcumin/pEGCG-encapsulated nanoparticles enhance spinal cord injury recovery by regulating CD74 to alleviate oxidative stress and inflammation

Abstract

Spinal cord injury (SCI) often accompanies impairment of motor function, yet there is currently no highly effective treatment method specifically for this condition. Oxidative stress and inflammation are pivotal factors contributing to severe neurological deficits after SCI. In this study, a type of curcumin (Cur) nanoparticle (HA-CurNPs) was developed to address this challenge by alleviating oxidative stress and inflammation. Through non-covalent interactions, curcumin (Cur) and poly (-)-epigallocatechin-3-gallate (pEGCG) are co-encapsulated within hyaluronic acid (HA), resulting in nanoparticles termed HA-CurNPs. These nanoparticles gradually release curcumin and pEGCG at the SCI site. The released pEGCG and curcumin not only scavenge reactive oxygen species (ROS) and prevents apoptosis, thereby improving the neuronal microenvironment, but also regulate CD74 to promote microglial polarization toward an M2 phenotype, and inhibits M1 polarization, thereby suppressing the inflammatory response and fostering neuronal regeneration. Moreover, in vivo experiments on SCI mice demonstrate that HA-CurNPs effectively protect neuronal cells and myelin, reduce glial scar formation, thereby facilitating the repair of damaged spinal cord tissues, restoring electrical signaling at the injury site, and improving motor functions. Overall, this study demonstrates that HA-CurNPs significantly reduce oxidative stress and inflammation following SCI, markedly improving motor function in SCI mice. This provides a promising therapeutic approach for the treatment of SCI.

Keywords: CD74; Curcumin; Mice; ROS; SCI.

Scheme 1

Schematic illustration of the mechanism by which HA-CurNPs promote spinal cord injury repair by alleviating oxidative stress and inflammation

Fig. 1

Synthesis and characterization of HA-CurNPs. (A) Representative TEM images of CurNPs and HA-CurNPs (scale bar = 100 nm). (B) Particle size of pEGCG, CurNPs and HA-CurNPs determined using dynamic light scattering (DLS). (C) Zeta potential of pEGCG, Cur, CurNPs, and HA-CurNPs. (D) UV-visible absorption spectrum of pEGCG, Cur, CurNPs, and HA-CurNPs measured using a UV-visible spectrophotometer. (E) Fourier transform infrared spectroscopy (FTIR) of pEGCG, Cur, CurNPs, and HA-CurNPs. (F) The release rates of pEGCG and Cur from HA-CurNPs in environments with and without H₂O₂. (G) Quantitative analysis of the scavenging rate of ABTS⁺ radicals by Cur, CurNPs, and HA-CurNPs. (H) Quantitative analysis of the scavenging rate of DPPH radicals by Cur, CurNPs, and HA-CurNPs. (I) Scavenging ability of Cur against reactive oxygen species measured at different concentrations and times through the ABTS⁺ radical scavenging assay. (J) Scavenging ability of CurNPs against reactive oxygen species measured at different concentrations and times through the ABTS⁺ radical scavenging assay. (K) Scavenging ability of HA-CurNPs against reactive oxygen species measured at different concentrations and times through the ABTS⁺ radical scavenging assay

Fig. 2

Evaluation of the antioxidant stress efficacy of HA-CurNPs. (A) Cytotoxic effects of Cur, CurNPs, and HA-CurNPs on HT22 cells. (B) Flow cytometry analysis of the ROS scavenging ability of Cur, CurNPs, and HA-CurNPs in cells. (C) Quantitative analysis of ROS levels using flow cytometry (n = 3). (D) Measurement of intracellular ROS levels in cells treated with Cur, CurNPs, and HA-CurNPs using DCFH-DA (scale bar = 50 μm). (E) Measurement of intracellular ROS levels in cells treated with Cur, CurNPs, and HA-CurNPs using DHE (scale bar = 50 μm). (F) Evaluation of ROS levels at the site of spinal cord injury in mice treated with Cur, CurNPs, and HA-CurNPs using the L-012 chemiluminescence probe. (G) Quantitative evaluation of DCF fluorescence intensity in cells from different groups (n = 3). (H) Quantitative evaluation of DHE fluorescence intensity in cells from different groups (n = 3). (I) Quantitative evaluation of ROS fluorescence levels at the site of spinal cord injury in mice treated with Cur, CurNPs, and HA-CurNPs. All data are presented as the mean ± SEM (ns, not statistically significant; **, P < 0.01; ***, P < 0.001 vs. Injury)

Fig. 3

Effects of HA-CurNPs on microglial polarization and injured neuron repair. (A) Immunocytochemistry staining with anti-CD80 and anti-CD206 antibodies was performed to investigate HA-CurNPs’ effects on microglial polarization (scale bar = 200 μm). (B) Quantitative evaluation of CD206 fluorescence intensity in Fig. A (n = 3). (C) Quantitative evaluation of CD80 fluorescence intensity in Fig. A (n = 3). (D) Western blot analysis with anti-CD80 and anti-CD206 antibodies to explore HA-CurNPs’ effects on microglial polarization. (E) Quantitative evaluation of the relative gray value of CD206 in Fig. D (n = 3). (F) Quantitative evaluation of the relative gray value of CD80 in Fig. D (n = 3). (G) Representative images of primary hippocampal neurons after oxidative stress induced by glutamate, treated with Cur, CurNPs, and HA-CurNPs in different groups (scale bar = 20 μm). (H) Statistical evaluation of the length of total branches (LTB) in hippocampal neurons (n = 30). (I) Statistical evaluation of the length of primary branches (LPB) in hippocampal neurons (n = 30). (J) Statistical evaluation of the number of total branches (NTB) in hippocampal neurons (n = 30). (K) Statistical evaluation of the number of primary branches (NPB) in hippocampal neurons (n = 30). All data are presented as the mean ± SEM (ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. LPS/Injury)

Fig. 4

Evaluation of HA-CurNPs on motor function recovery in SCI mice. (A) Appearance of mice standing. (B) Methods for evaluating motor function in SCI mice. (C) Changes in BMS scores of SCI mice over two months. (D) Inclined plane test to assess motor ability and balance in SCI mice. (E) Footprint analysis to assess hind limb motor function in mice. (F) Statistical analysis of stride length in (E) (n = 5). (G) Statistical analysis of stride width in (E) (n = 5). (H) CatWalk system analysis of the regularity index of motor function in SCI mice. (I) CatWalk system analysis of the average ground contact intensity and area for the right hind paw in SCI mice. (J) Quantitative evaluation of the regularity index of motor function in SCI mice (n = 5). (K) Quantitative evaluation of the average ground contact intensity for the right hind paw in SCI mice (n = 5). (L) Quantitative evaluation of the average ground contact area for the right hind paw in SCI mice (n = 5). All data are presented as the mean ± SEM (ns, not statistically significant; **, P < 0.01; ***, P < 0.001 vs. Injury)

Fig. 5

Evaluation of HA-CurNPs on hind limb joint activity and neural electrical conductivity in SCI mice. (A) Color-coded stick figures of joint activity during mice locomotion. (B) Analysis of knee and ankle joint activity in each group. (C) Representative electrophysiological images at different stimulation sites. (D) Quantitative evaluation of motor evoked potential (MEP) amplitudes (n = 3). (E) Quantitative evaluation of potential amplitudes at 0.5 mm rostral to the injury site (n = 3). (F) Quantitative evaluation of potential amplitudes at 2 mm caudal to the injury site (n = 3). (G) Quantitative evaluation of potential amplitudes at 5 mm caudal to the injury site (n = 3). All data are presented as the mean ± SEM (ns, not statistically significant; ***, P < 0.001 vs. Injury)

Fig. 6

The impact of HA-CurNPs on spinal cord tissue repair. (A) Gross morphology of spinal cord tissue (scale bar = 1 cm). (B) HE staining of spinal cord tissue, with the dashed lines indicate the site of spinal cord injury. (C) LFB staining of spinal cord tissue, with the dashed lines indicate the site of spinal cord injury. (D) Immunohistochemical staining of spinal cord tissue with anti-NFH (red) and anti-GFAP (green) (scale bar = 200 μm). (E) Immunohistochemical staining of spinal cord tissue with anti-BrdU (red) (scale bar = 200 μm)

Fig. 7

Mechanism of HA-CurNPs in promoting M2 polarization of microglia. (A) The number of differentially expressed proteins between groups. (B) Circos plot showing enriched pathways and proteins. (C) Heatmap of protein expression identified by enrichment analysis. (D) Western blot analysis of CD74 expression in different groups after LPS and HA-CurNPs treatment. (E) Western blot analysis of CD74 expression in spinal cord protein from different groups of mice after spinal cord injury and HA-CurNPs treatment. (F) Quantitative evaluation of CD74 relative gray value in Fig. D (n = 3). (G) Quantitative evaluation of CD74 relative gray value in Fig. E (n = 3). (H) Immunocytochemical detection of CD80 (red) and CD206 (green) expression in BV2 cells after LPS treatment and transfection with si-NC and si-CD74. (I) Quantitative evaluation of CD206 fluorescence intensity in Fig. H (n = 3). (J) Quantitative evaluation of CD80 fluorescence intensity in Fig. H (n = 3). (K) Western blot analysis of CD80 and CD206 expression in different groups of BV2 cells after LPS treatment and transfection with si-NC and si-CD74. (L) Quantitative evaluation of CD206 relative gray value in Fig. K (n = 3). (M) Quantitative evaluation of CD80 relative gray value in Fig. K (n = 3). All data are presented as the mean ± SEM (ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. LPS/Injury)