The effects of combined low-intensity exercise with naringenin on regenerating protein family in vivo and in vitro after sciatic nerve injury

Abstract

The purpose of this study was to investigate the effect of combining low-intensity treadmill exercise with naringenin treatment on the expression of axonal regrowth-related proteins following sciatic nerve injury (SNI). The extracts were evaluated for cytotoxicity and cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the effects of the extracts were analyzed in vitro using primary cultured Schwann cells and dorsal root ganglion neurons (DRGs). In vivo, axonal regrowth-related protein expression levels and neurite outgrowth were assessed through Western blot and immunofluorescence staining, respectively. The results indicated that neither extract exhibited cytotoxicity. In primary cultured Schwann cells, 10 μM naringin and 10 μM/50 μM naringenin significantly increased growth associated protein-43 (GAP-43) expression, while in DRGs, both naringin and naringenin treatments resulted in increased neurite length. For in vivo experiment, all animals were divided into the vehicle group, the naringin-treated group post-SNI (Gin), the naringenin-treated group post-SNI (Genin), the naringin and exercise group post-SNI (Gin+Ex), and the naringenin and exercise group post-SNI (Genin+Ex). Naringenin treatment after early SNI enhanced GAP-43 expression. Following 14 days of exercise combined with treatment, both GAP-43 and phosphorylated extracellular signal-regulated kinase levels were significantly increased in the Genin and the Genin+Ex groups, whereas phosphorylated-protein kinase B significantly increased only in the Genin+Ex group. Our findings suggest that naringenin, when used in conjunction with low-intensity treadmill exercise, may effectively promote the expression of axonal growth-related proteins following SNI.

Keywords: Axonal regrowth; Dorsal root ganglion; Low-intensity exercise; Naringenin; Naringin; Sciatic nerve injury.

Fig. 1

Role of naringin and naringenin on cytotoxicity and cell viability in SH-SY5Y cells. (A, B) Quantitative graph of cell proliferation after treatment with naringin and naringenin. (C, D) Effect of naringin and naringenin on cytotoxicity in H₂O₂-exposed SH-SY5Y cell. *P<0.05, **P<0.01, ***P<0.001 vs. nontreated group. ^##P<0.01, ^###P<0.001 vs. only H₂O₂ treated group.

Fig. 2

Naringin and naringenin increased GAP-43 expression in Schwann cells and neurite outgrowth of DRG neurons in vitro. (A) Expression level of GAP-43 in the harvested Schwann cells. (B) Mean neurite length of cultured DRG neurons. GAP-43, growth associated protein-43; DRG, dorsal root ganglion; Veh, vehicle; Gin, naringin; Genin, naringenin. *P<0.05, **P<0.01, ***P<0.001 vs. Veh.

Fig. 3

Naringenin increased GAP-43 expression level in the injured sciatic nerve. (A) Quantitative analysis of GAP-43/actin at one day post crush. (B) Quantitative analysis of GAP-43/actin at 3 days post crush. GAP-43, growth associated protein-43; Veh, vehicle; Gin, naringin; Genin, naringenin. *P<0.05, ***P<0.001 vs. Veh.

Fig. 4

Effects of combined exercise with naringin or naringenin on in vivo regenerative proteins. (A) Representative Western blot images of GAP-43 and p-AKT and p-ERK1/2 at 14 days after SNI. (B–D) Quantitative analysis of GAP-43/actin, p-AKT/actin and p-ERK1/2/actin ratio. GAP-43, growth associated protein-43; p-AKT, phosphorylated-protein kinase B; p-ERK1/2, phosphorylated extracellular signal-regulated kinase; Veh, vehicle; Gin, naringin; Genin, naringenin. *P<0.05, ***P<0.001 vs. Veh.