The Zinc Ion Chelating Agent TPEN Attenuates Neuronal Death/apoptosis Caused by Hypoxia/ischemia Via Mediating the Pathophysiological Cascade Including Excitotoxicity, Oxidative Stress, and Inflammation

Abstract

Aims: We aim to determine the significant effect of TPEN, a Zn(2+) chelator, in mediating the pathophysiological cascade in neuron death/apoptosis induced by hypoxia/ischemia.

Methods: We conducted both in vivo and in vitro experiments in this study. PC12 cells were used to establish hypoxia/ischemia model by applying oxygen-glucose deprivation (OGD). SHR-SP rats were used to establish an acute ischemic model by electrocoagulating middle cerebral artery occlusion. The effect of TPEN on neuron death/apoptosis was evaluated. In addition, the relative biomarks of excitotoxicity, oxidative stress, and inflammation reactions in hypoxia/ischemia PC12 cell model as well as in SHR-SP rat hypoxia/ischemia model were also assessed.

Results: TPEN significantly attenuates the neurological deficit, reduced the cerebral infarction area and the ratio of apoptotic neurons, and increased the expression of GluR2 in the rat hypoxia/ischemia brain. TPEN also increased blood SOD activity, decreased blood NOS activity and blood MDA and IL-6 contents in rats under hypoxia/ischemia. In addition, TPEN significantly inhibited the death and apoptosis of cells and attenuated the alteration of GluR2 and NR2 expression caused by OGD or OGD plus high Zn(2+) treatments.

Conclusions: Zn(2+) is involved in neural cell apoptosis and/or death caused by hypoxia/ischemia via mediating excitotoxicity, oxidative stress, and inflammation.

Keywords: Excitotoxicity; Free Zn2+; Hypoxia/ischemia; Inflammation; Oxidative stress; TPEN.

Figure 1

The effect of TPEN on neurological deficits and brain infarction area in acutely ischemic rat brain. (A) The coagulated points of middle cerebral artery. (B) Comparison of the neurological deficit scores (n = 10), **P < 0.01, compared with control group (no TPEN treatment for acute ischemic rat). (C) Typical TTC staining map every coronal section,Scale Bar = 10 mm. (D) Infarction percent of every coronal section (n = 10), *P < 0.05 **P < 0.01, compared with control group. (E) Average infarction percent of each treatment (n = 10), *P < 0.05, compared with control group.

Figure 2

TPEN inhibiting neuron apoptosis of acute ischemic rat. (A) Representative TUNEL staining map of each treatment, Scale Bar = 50 μm. (B) Average percent of TUNEL‐positive neuron in each treated rat (n = 10), *P < 0.05, compared with the control group.

Figure 3

The effect of TPEN on GluR2 expression in acutely ischemic rat brains. (A) Representative Western blotting bands from the two groups. (B) Average relative expression levels of brain GluR2 (n = 10), *P < 0.05, compared to the control group.

Figure 4

The effect of TPEN on blood MDA, SOD, NOS, and IL‐6 levels in acutely ischemic rats. (A) MDA concentration (n = 10). (B) SOD activity (n = 10). (C) Total NOS activity (n = 10). (D) Inducible NOS activity (n = 10). (E) IL‐6 concentration (n = 10), *P < 0.05, compared to the control group in each panel.

Figure 5

Hypoxia/ischemia induces death or apoptosis in PC12 cells, and TPEN reverses death or apoptosis induced by hypoxia. (A) Results of cell survival status obtained applying AO/EB staining method (green cells were alive, and red cells were death), Scale Bar = 50 μm. (B) Summarized results of relative cell survival ratio obtained applying AO/EB staining method (n = 5, *P < 0.05, **P < 0.01 compared with OGD; ##P < 0.01 compared with OGD+Zn). (C) Immunofluorescence images showed apoptotic PC12 (green nuclei) as detected by terminal deoxynucleotidyl transferase (Tdt)‐mediated dUTP nick end labeling (TUNEL) in different treatment groups (the experiment was repeated three times), Scale Bar = 50 μm.

Figure 6

The level of intracellular GluR2 and NR2 expression. (A) Immunofluorescence images showing GluR2 expression, Scale Bar = 100 μm. (B) Histogram of immunofluorescence density of GluR2 expression (n = 3) and NR2 expression. (C) Immunofluorescence images showing NR2 expression, Scale Bar = 100 μm. (D) Histogram of immunofluorescence density of NR2 expression (n = 3) in PC12 cells under hypoxia/ischemia induced by OGD treatment, high Zn²⁺, and TPEN separately or combined treatments for 3 and 6 h. *P < 0.05, **P < 0.01, compared with Control, #P < 0.05, ##P < 0.01 compared with OGD, ^^P < 0.01 compared with ODG+Zn (n = 6).