Polydatin Attenuates OGD/R-Induced Neuronal Injury and Spinal Cord Ischemia/Reperfusion Injury by Protecting Mitochondrial Function via Nrf2/ARE Signaling Pathway

Abstract

Spinal cord ischemia/reperfusion injury (SCII) is a devastating complication of spinal or thoracic surgical procedures and can lead to paraplegia or quadriplegia. Neuronal cell damage involving mitochondrial dysfunction plays an important role in the pathogenesis of SCII. Despite the availability of various treatment options, there are currently no mitochondria-targeting drugs that have proven effective against SCII. Polydatin (PD), a glucoside of resveratrol, is known to preserve mitochondrial function in central nervous system (CNS) diseases. The aim of the present study was to explore the neuro- and mito-protective functions of PD and its underlying mechanisms. An in vitro model of SCII was established by exposing spinal cord motor neurons (SMNs) to oxygen-glucose-deprivation/reperfusion (OGD/R), and the cells were treated with different dosages of PD for varying durations. PD improved neuronal viability and protected against OGD/R-induced apoptosis and mitochondrial injury in a dose-dependent manner. In addition, PD restored the activity of neuronal mitochondria in terms of mitochondrial membrane potential (MMP), intracellular calcium levels, mitochondrial permeability transition pore (mPTP) opening, generation of reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels. Mechanistically, PD downregulated Keap1 and upregulated Nrf2, NQO-1, and HO-1 in the OGD/R-treated SMNs. Likewise, PD treatment also reversed the neuronal and mitochondrial damage induced by SCII in a mouse model. Furthermore, the protective effects of PD were partially blocked by the Nrf2 inhibitor. Taken together, PD relieves mitochondrial dysfunction-induced neuronal cell damage by activating the Nrf2/ARE pathway and is a suitable therapeutic option for SCII.

Figure 1

Effects of PD on the viability and morphology of SMNs exposed to OGD/R. (a) The structure of PD. (b) Percentage of viable SMNs exposed to OGD/R conditions for varying durations (8-24 h). (c) Percentage of viable SMNs with/out PD (3.75, 7.5, 15, 30, or 60 μM) treatment during hypoxic injury. (d) Extracellular LDH levels in the indicated groups. (e) Representative images showing the morphology changes in the primary neurons of indicated group and the (f) number of normal neurons and (g) average neurite length. Scale bars = 50 μm. ^∗P < 0.05 vs. Control; #P < 0.05 vs. OGD; ΔP < 0.05 vs. OGD + PD (15 μM).

Figure 2

Isolation, culture, and identification of primary SMNs. (a) Spinal cords were dissected from the 14-day-old mouse embryos. Scale bars = 50 mm. Representative images of SMNs on (b) Day 3, (c) Day 5, and (d) Day 7. Primary SMNs were labeled with (e) Tuj1, (f) MAP-2, (g) ChAT, and (h) GFAP. Scale bars = 50 μm.

Figure 3

PD attenuates OGD/R-induced damage in SMNs. Cells were divided into the Control, PD (15 μM), PD (30 μM), OGD/R, OGD/R + PD (15 μM), and OGD/R + PD (30 μM) groups. (a) Representative images showing Annexin V-FITC/PI stained cells. (b) Representative images showing TUNEL-positive cells. Scale bars = 100 μm. Quantification of the apoptotic cells in (c) Annexin V-FITC/PI and (d) TUNEL assays. (e) Immunoblot showing Bcl-2, Bax, and c-Caspase-3 protein levels in the neurons of the indicated groups. (f–h) Quantification of the relative protein levels. ^∗P < 0.05 vs. Control; #P < 0.05 vs. OGD; ΔP < 0.05 vs. OGD + PD (15 μM).

Figure 4

PD restores mitochondrial functions in OGD/R-injured neurons. (a) [Ca²⁺]_i levels in cells exposed to normoxia or hypoxia-reperfusion in the indicated groups. (b) Normalized relative fluorescence units (NRFU) of calcein indicating mPTP opening in the differentially treated cells. (c) The ratios of polymeric (red) and monomeric (green) forms of JC-1 corresponding to the MMP in the indicated groups. Scale bars = 100 μm. (d) MitoSOX fluorescence intensity indicative of ROS levels. Data are presented as fold change over the Control group. Scale bars = 10 μm. (e) Neuronal ATP release (nmol/mg protein) as measured by a luciferase-based assay. ^∗P < 0.05 vs. Control; #P < 0.05 vs. OGD; ΔP < 0.05 vs. OGD + PD (15 μM).

Figure 5

PD exerts its antiapoptotic effects by activating the Nrf2/ARE pathway. (a) Immunoblot showing levels of Keap1, Nrf2, NQO-1, and HO-1 protein in the neurons of different groups. ^∗P < 0.05 vs. Control; #P < 0.05 vs. OGD; ΔP < 0.05 vs. OGD + PD (15 μM). Then, cells were divided into the Control, OGD/R, OGD/R + PD, and OGD/R + PD + Brusatol groups. (b) Percentage of apoptotic cells as detected by Annexin V-FITC/PI assay. (c) Percentage of TUNEL-positive apoptotic cells. (d) Immunoblot showing Bcl-2, Bax, and c-Caspase-3 protein levels in each group. ^∗P < 0.05 vs. Control; #P < 0.05 vs. OGD; ΔP < 0.05 vs. OGD + PD.

Figure 6

PD-mediated mito-protective depends on the Nrf2/ARE pathway. (a) [Ca²⁺]_i levels in cells exposed to normoxia or hypoxia-reperfusion in the indicated groups. (b) NRFU of calcein indicating mPTP opening in the differentially treated cells. (c) The ratios of polymeric (red) and monomeric (green) forms of JC-1 corresponding to the MMP in the indicated groups. (d) MitoSOX fluorescence intensity indicative of ROS levels. Data are presented as fold change over the Control group. Scale bars = 10 μm. (e) Neuronal ATP release (nmol/mg protein) as measured by a luciferase-based assay. ^∗P < 0.05 vs. Control; #P < 0.05 vs. OGD; ΔP < 0.05 vs. OGD + PD.

Figure 7

PD treatment protects SMNs from SCII-induced apoptosis. (a, b) Immunofluorescence images showing colocalization of NeuN (green) and c-Caspase-3 (red) in the spinal cord of the indicated groups. Scale bar = 50 μm. (c) Quantified expression of Bcl-2, Bax, and c-Caspase-3. (d) Representative images of Nissl staining at 7 dpi and the number of VMNs. Scale bars = 50 μm. (e) TEM images showing microstructures of neurons. Black arrows indicate peripheral edema and disintegrated organelles. Scale bars = 2 μm. ^∗P < 0.05 vs. Sham; #P < 0.05 vs. SCII; ΔP < 0.05 vs. PD.

Figure 8

PD alleviates oxidative stress and mitochondrial dysfunction caused by SCII. (a) MDA content, (b) SOD activity, and (c) GSH levels in the spinal cords at 7 dpi. (d) TEM images showing mitochondrial ultrastructure. Arrows indicate swollen mitochondria. Scale bars = 500 nm. (e) Relative Cyt-c protein expression levels in each group. ^∗P < 0.05 vs. Sham; #P < 0.05 vs. SCII; ΔP < 0.05 vs. PD.

Figure 9

Mechanistic basis of the neuroprotective action of PD in SCII. PD promotes the nuclear translocation of Nrf2 and activates Nrf2/ARE singling, thereby inducing the ARE-driven genes NQO-1 and HO-1 that attenuate mitochondrial dysfunction. It also restores the Bcl-2/Bax balance and blocks Cyt-c-initiated Caspase cascade, which prevents neuronal damage and apoptosis.