Trehalose-Carnosine Prevents the Effects of Spinal Cord Injury Through Regulating Acute Inflammation and Zinc(II) Ion Homeostasis

Abstract

Spinal cord injury (SCI) leads to long-term and permanent motor dysfunctions, and nervous system abnormalities. Injury to the spinal cord triggers a signaling cascade that results in activation of the inflammatory cascade, apoptosis, and Zn(II) ion homeostasis. Trehalose (Tre), a nonreducing disaccharide, and L-carnosine (Car), (β-alanyl-L-histidine), one of the endogenous histidine dipeptides have been recognized to suppress early inflammatory effects, oxidative stress and to possess neuroprotective effects. We report on the effects of the conjugation of Tre with Car (Tre-car) in reducing inflammation in in vitro and in vivo models. The in vitro study was performed using rat pheochromocytoma cells (PC12 cell line). After 24 h, Tre-car, Car, Tre, and Tre + Car mixture treatments, cells were collected and used to investigate Zn²⁺ homeostasis. The in vivo model of SCI was induced by extradural compression of the spinal cord at the T6-T8 levels. After treatments with Tre, Car and Tre-Car conjugate 1 and 6 h after SCI, spinal cord tissue was collected for analysis. In vitro results demonstrated the ionophore effect and chelating features of L-carnosine and its conjugate. In vivo, the Tre-car conjugate treatment counteracted the activation of the early inflammatory cascade, oxidative stress and apoptosis after SCI. The Tre-car conjugate stimulated neurotrophic factors release, and influenced Zn²⁺ homeostasis. We demonstrated that Tre-car, Tre and Car treatments improved tissue recovery after SCI. Tre-car decreased proinflammatory, oxidative stress mediators release, upregulated neurotrophic factors and restored Zn²⁺ homeostasis, suggesting that Tre-car may represent a promising therapeutic agent for counteracting the consequences of SCI.

Keywords: Apoptosis; Inflammation; Ion homeostasis; Neurotrophic factors; Spinal cord injury.

Scheme 1

Synthesis of trehalose–carnosine

Fig. 1

A, B Effects of Tre–car on the cytoplasmic pool of labile zinc in PC12 cells. Average intensity values for the FluoZin-3 emission corresponding to the Zn²⁺ content in the cytoplasm for control untreated cells and treated 20 h with Tre, Car, Tre–car or Tre + Car mixture in RPMI1640 complete medium with 1% HS and 0.5% FBS; 20 µM zinc (Magri et al. 2016) or 50 µM membrane-impermeable zinc chelator DPA treatment were used as a positive and negative control, respectively. The effect of Tre–car, Car or Tre + Car mixture treatment analysed by fluorescence images of cells incubated with zinc probe (A) and quantification of fluorescent intensity normalized to the number of cells presented in each field (B) confirms the significantly increased zinc concentration in PC12 cells. As a baseline to exclude cell auto-fluorescence, PC12 cells were treated only with Hoechst33342 without FluoZin-3. Scale bars are 42 μm. All values are mean ± SD of three independent experiments of 6–8 randomly chosen fields. Significant differences between treatments were determined using one-way ANOVA method ^#p ≤ 0.05, ^##p ≤ 0.01, ^###p ≤ 0.001 versus untreated control cells. C Tre, Car, Tre–car or Tre + Car mixture affected ZnT1 expression. Starved PC12 cells were stimulated for 24 h with 5 mM Tre, Car, Tre–car, Tre + Car, 20 µM zinc or 50 µM membrane-impermeable zinc chelator DPA in RPMI11640 complete medium with 1% HS and 0.5% FBS. The expression level of ZnT1 is reported as a ratio to that of GAPDH. Treatment with Car, Tre–car or Tre + Car mixture significantly increased ZnT1 expression. All values are mean ± SD ^#p < 0.01, ^##p < 0.001 versus untreated control cells (samples n = 4, three individual experiments)

Fig. 2

Tre, Car or Tre–car differentially affected ZnT1 and ZnT3 expression after SCI. The expression level of zinc transporters is reported as a ratio to actin. A representative blot of lysates is shown, and densitometry analysis is reported. Data are expressed as SD

Fig. 3

The severity of tissue damage following SCI is decreased in Tre–car-treated mice. Extensive damage to the spinal cord was observed in the SCI mouse group (B) compared to the sham mouse group (A). C, D Tre and Car treatments after SCI. E Tre–car treatment significantly reduces the SCI lesion score. F Relative histological score. ***p < 0.001 versus sham group; ^#p < 0.05 versus SCI group; ^##P < 0.01 versus SCI group. ^###p < 0.001 versus SCI group. (samples n = 10, three individual experiment). Data are expressed as SD

Fig. 4

Western blot analysis of spinal cord samples. A NF-kB expression level evaluation and relative densitometric analysis shown in A1. B IκB-α expression level evaluation and relative densitometric analysis shown in B1. ***p < 0.001 versus sham group; ^#p < 0.05 versus SCI group; ^##p < 0.01 versus SCI group. ^###p < 0.001 versus SCI group (samples n = 10, three individual experiments)

Fig. 5

Effects of Tre–car on Nt and PARP. A substantial increase in Nt-positive staining was observed in spinal cord tissues collected from mice 24 h after SCI compared to sham mice (B, A). Treatment with Tre and Car reduced the positive staining of Nt (C, D). Tre–car treatment protects tissue after SCI (E). A substantial increase in PAR-positive staining was observed in spinal cord tissues of SCI mice compared to sham mice (H, G). Treatment with Tre and Car reduced the positive staining of PARP (I, J). Tre–car treatment protects tissue after SCI (K). Quantitative panels of Nt and PARP, respectively (F, L). 8-hydroxy-2-deoxyguanosine (8-OHdG) content in serum (M). ***p < 0.001 versus sham; ^#p < 0.05 versus SCI; ^##p < 0.01 versus SCI; ^###p < 0.001 versus SCI; ND not detectable. (samples n = 10, three individual experiments). Data are expressed as SD

Fig. 6

Effects of Tre–car on apoptosis levels. Western blot analysis showing the expression of Bax (A) and Bcl-2 (B) 24 h after SCI with densitometric analysis shown A1 and B1. A substantial increase in Bax-positive staining was observed in spinal cord tissues collected from mice 24 h after SCI compared to sham mice (D, C) (samples n = 10, three individual experiments). Treatment with Tre and Car reduced the positive staining of Bax (E, F). Tre–car treatment protects tissues after SCI (G). A substantial increase in Bcl-2-positive staining was observed in sham mouse spinal cord tissues compared to SCI mice (I, J). Treatment with Tre and Car increased the positive staining of Bcl-2 (K, L). Tre–car treatment protects after SCI (M). Quantitative panels of Bax and Bcl-2 (H, N). p53 and Caspase-3 protein expression increased 24 h post SCI, compared with sham group, while Tre, Car and Conjugate treatments significantly both p53 and Caspase-3 expression levels (O, P, see densitometric analysis O1 and P1). ***p < 0.001 versus sham, ^#p < 0.05 versus SCI; ^##p < 0.01 and ^###p < 0.001 versus SCI. ND not detectable. (samples n = 10, three individual experiments). Data are expressed as SD

Fig. 7

Western blot analysis. Expression level evaluation of PI3K (A), p-Akt (B), p-ERK (C) and p-CREB (D) 24 h after SCI. Densitometric analysis Panels A1, B1, C1 and D1. ***p < 0.001 versus sham; ^#p < 0.05 versus SCI; ^##p < 0.01 versus SCI. ^###p < 0.001 versus SCI. (samples n = 10, three individual experiments). Data are expressed as SD

Fig. 8

Effects of Tre–car on BDNF and GDNF. Western blot analysis showing the expression of BDNF (A) and GDNF (B) 24 h after SCI. Densitometric analysis is shown in A1 and B1. A substantial decrease in BDNF-positive staining was observed in spinal cord tissues collected from mice 24 h after SCI compared to sham mice (C, D). Treatment with Tre and Car increased the positive staining of BDNF (E, F). (samples n = 10, three individual experiments). Tre–car treatment further increased the positive staining of BDNF after SCI (G). Basal GDNF-positive staining was observed in sham mouse spinal cord tissues compared to the decreased value found in SCI mice (I, J). Treatment with Tre and Car slightly increased the positive staining of GDNF (K, L). Tre–car treatment induces a significant increase in positive staining of GDNF after SCI (M). Quantitative panels of BDNF and GDNF (H, N). ***p < 0.001 versus sham; ^##p < 0.01 versus SCI. ^###p < 0.001 versus SCI. (samples n = 10, three individual experiments). Data are expressed as SD