Does combined therapy of curcumin and epigallocatechin gallate have a synergistic neuroprotective effect against spinal cord injury?

Abstract

Systematic inflammatory response after spinal cord injury (SCI) is one of the factors leading to lesion development and a profound degree of functional loss. Anti-inflammatory compounds, such as curcumin and epigallocatechin gallate (EGCG) are known for their neuroprotective effects. In this study, we investigated the effect of combined therapy of curcumin and EGCG in a rat model of acute SCI induced by balloon compression. Immediately after SCI, rats received curcumin, EGCG, curcumin + EGCG or saline [daily intraperitoneal doses (curcumin, 6 mg/kg; EGCG 17 mg/kg)] and weekly intramuscular doses (curcumin, 60 mg/kg; EGCG 17 mg/kg)] for 28 days. Rats were evaluated using behavioral tests (the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test, flat beam test). Spinal cord tissue was analyzed using histological methods (Luxol Blue-cresyl violet staining) and immunohistochemistry (anti-glial fibrillary acidic protein, anti-growth associated protein 43). Cytokine levels (interleukin-1β, interleukin-4, interleukin-2, interleukin-6, macrophage inflammatory protein 1-alpha, and RANTES) were measured using Luminex assay. Quantitative polymerase chain reaction was performed to determine the relative expression of genes (Sort1, Fgf2, Irf5, Mrc1, Olig2, Casp3, Gap43, Gfap, Vegf, NfκB, Cntf) related to regenerative processes in injured spinal cord. We found that all treatments displayed significant behavioral recovery, with no obvious synergistic effect after combined therapy of curcumin and ECGC. Curcumin and EGCG alone or in combination increased axonal sprouting, decreased glial scar formation, and altered the levels of macrophage inflammatory protein 1-alpha, interleukin-1β, interleukin-4 and interleukin-6 cytokines. These results imply that although the expected synergistic response of this combined therapy was less obvious, aspects of tissue regeneration and immune responses in severe SCI were evident.

Keywords: curcumin; epigallocatechin gallate; inflammatory response; neural regeneration; spinal cord injury.

Figure 1

Locomotor and sensory recovery in rats following curcumin and epigallocatechin gallate (EGCG) application after spinal cord injury. (A–C) The effect of curcumin, EGCG, and their combination on locomotor recovery after spinal cord injury was evaluated using the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test (A), flat beam test (B), and flat beam time score (C). Animals treated with combined therapy of curcumin and EGCG, or curcumin alone, performed significantly better in the BBB open-field locomotor test than animals treated with saline. In the flat beam test, EGCG treated animals showed the best recovery, followed by curcumin + EGCG treated animals. (D) The thermal nociception was evaluated using the plantar test. No additional hyperalgesia was found after application of any of the used drugs. Two-way repeated measures analysis of variance with the Student-Newman-Keuls post hoc test was used to determine statistical significance. *P < 0.05 (The color of asterisks indicates the statistically different group(s)).

Figure 2

Histological and immunohistochemical analysis results of injured spinal cords. The amount of spared white (A) and grey matter (B) was determined 9 weeks after spinal cord injury (SCI) in rats treated with saline, curcumin, EGCG, or their combination. No statistically significant effect of applied drugs on tissue preservation was observed. In addition, the volume of white matter and gray matter across the measured regions revealed no significant difference (G). However, a strong trend toward smaller cavity size was found (F). The effect of the application of curcumin, EGCG, or their combination on glial scar formation (C), and the average number of protoplasmic astrocytes per section (D) around the central lesion cavity was measured 9 weeks after SCI. The combined application of curcumin and EGCG had a suppressive effect on glial scar formation at the area of the lesion epicenter. In the central region of the injury, all treatments showed a positive effect on decreasing the number of protoplasmic astrocytes. The distribution of the effect on X axis is measured as distance in mm from the lesion center (set as 0) (A–D). The effect of applied curcumin, EGCG, or their combination on axonal sprouting. (E) The combination of curcumin and EGCG had a synergistic effect on axonal sprouting, when compared to saline treated animals. Two-way repeated measures analysis of variance (ANOVA) with Student-Newman-Keuls test was used to determine statistical significance (A–D). One-way ANOVA with Student-Newman-Keuls test was used to determine the level of statistical significance (E–G). *P < 0.05 (the color of asterisk indicates the statistically different group(s)). Symbol of # is used when trend toward significance has reached statistical value P = 0.05–0.06. n = 5 per group.

Figure 3

Gene expression changes in injured spinal cords after application of curcumin, epigallocatechin gallate (EGCG) or their combination. Relative gene expression of factors related to the recovery process in response to application of curcumin, EGCG or their combination 9 weeks after spinal cord injury. The expression level of saline treated animals was set as zero value. One-way analysis of variance with Student-Newman-Keuls post hoc test was used to determine the level of statistical significance. *P < 0.05(the color of asterisk indicates the statistically different group(s)). n = 4 per group.

Figure 4

Effect of curcumin, EGCG, or their combination on cytokine levels in injured spinal cords. The levels of cytokines and chemokines are presented after application of curcumin, EGCG or their combination at 1 (A), 3 (B), 7 (C) and 10 days after spinal cord injury (D). The levels measured in animals with no lesion were set as 100%. Two-way analysis of variance with Student-Newman-Keuls post hoc test was used to determine statistical significance. *P < 0.05 (The color of asterisks indicates the statistically different group(s)). n = 5/group per time point. MIP-1α: Macrophage inflammatory protein 1-alpha; IL-4: interleukin-4; IL-1β: interleukin-1beta; IL-2: interleukin-2; IL-6: interleukin-6; RANTES: regulated on activation, normal T cell expressed and secreted; EGCG: epigallocatechin gallate.