Citrus Naringenin Increases Neuron Survival in Optic Nerve Crush Injury Model by Inhibiting JNK-JUN Pathway

Abstract

Traumatic nerve injury activates cell stress pathways, resulting in neuronal death and loss of vital neural functions. To date, there are no available neuroprotectants for the treatment of traumatic neural injuries. Here, we studied three important flavanones of citrus components, in vitro and in vivo, to reveal their roles in inhibiting the JNK (c-Jun N-terminal kinase)-JUN pathway and their neuroprotective effects in the optic nerve crush injury model, a kind of traumatic nerve injury in the central nervous system. Results showed that both neural injury in vivo and cell stress in vitro activated the JNK-JUN pathway and increased JUN phosphorylation. We also demonstrated that naringenin treatment completely inhibited stress-induced JUN phosphorylation in cultured cells, whereas nobiletin and hesperidin only partially inhibited JUN phosphorylation. Neuroprotection studies in optic nerve crush injury mouse models revealed that naringenin treatment increased the survival of retinal ganglion cells after traumatic optic nerve injury, while the other two components had no neuroprotective effect. The neuroprotection effect of naringenin was due to the inhibition of JUN phosphorylation in crush-injured retinal ganglion cells. Therefore, the citrus component naringenin provides neuroprotection through the inhibition of the JNK-JUN pathway by inhibiting JUN phosphorylation, indicating the potential application of citrus chemical components in the clinical therapy of traumatic optic nerve injuries.

Keywords: JNK-JUN pathway; JUN phosphorylation; naringenin; neuroprotection; optic nerve crush; retinal ganglion cells; traumatic nerve injury.

Figure 1

ONC injury increases JUN phosphorylation and results in RGC death. (A) Representative images of whole retina co-immunostained with antibodies against phospho-JUN and βIII tubulin (Tuj1), scale bar: 20 μm; (B) Representative images of whole retina co-immunostained with antibodies against RBPMS (RGC marker) and βIII tubulin (Tuj1), scale bar: 50 μm; (C) Statistical analysis of RGC survival 14 days after ONC injury. Data are represented as mean ± SEM, n = 9; *** p < 0.001; 3 dpc: 3 days post-crush; 14 dpc: 14 days post-crush; ONC, optic nerve crush; RGCs, retinal ganglion cells.

Figure 2

Inhibition of stress-induced pro-apoptotic JNK-JUN pathway by citrus components in HEK293T cells. (A) Western blot results of treatments of different concentrations of naringenin, after pre-incubation with sorbitol to activate the JNK-JUN pathway in HEK293T cells. (B) Statistical analysis of phosphorylation level of JUN relative to GAPDH loading control after treatments of different concentrations of naringenin. (C) Western blot results of treatments of different concentrations of nobiletin, after pre-incubation with sorbitol to activate the JNK-JUN pathway in HEK293T cells. (D) Statistical analysis of phosphorylation level of JUN relative to GAPDH loading control after treatments of different concentrations of nobiletin. (E) Western blot results of treatments of different concentrations of hesperidin, after pre-incubation with sorbitol to activate JNK-JUN pathway in HEK293T cells. (F) Statistical analysis of phosphorylation level of JUN relative to GAPDH loading control after treatments of different concentrations of hesperidin. Rabbit anti-phospho-JUN antibody was used to detect JUN phosphorylation. Mouse anti-GAPDH antibody was used to detect loading control-GAPDH. Data are represented as mean ± SEM of 4 replicates; ** p < 0.01; *** p < 0.001. (G) Relative cell viability analysis of naringenin in HEK 293T cells. (H) Relative cell viability analysis of nobiletin in HEK 293T cells. Each point is represented as mean ± SEM of 6 replicates.

Figure 3

Neuroprotection effects of RGCs in traumatic optic nerve injury model after treatment with citrus components. (A) Representative images of naïve retina, 14 dpc retina, and 14 dpc retina after treatment with naringenin, nobiletin or hesperidin, respectively. Retinas were co-immunostained with rabbit antibody against RGC specific marker-RBPMS and mouse antibody against neuron specific marker-βIII tubulin (Tuj1). Scale bar: 50 μm. (B) Statistical analysis of percentage of survived RGCs in groups treated with compounds relative to naive control retina. Data are represented as mean ± SEM, n = 7; *** p < 0.001. (C) The chronogram of the mouse experiments for RGC survival study. 14 dpc: 14 days post-crush; RGCs, retinal ganglion cells.

Figure 4

Naringenin promotes RGC survival by inhibiting JNK-JUN pathway. (A) Representative images of WT retina, ONC retina treated with PBS control, and ONC retina treated with naringenin. Retinas were immunostained with antibody against phospho-JUN, scale bar: 50 μm. (B) Statistical analysis of p-JUN positive RGC number in naringenin treatment group relative to the control retina of PBS treatment. Data are represented as mean ± SEM, n = 8; * p < 0.05. (C) The chronogram of the mouse experiments for studying the inhibition of JNK-JUN pathway. 3 dpc: 3 days post-crush; RGCs, retinal ganglion cells.