c‑Jun N‑terminal kinase inhibition attenuates early brain injury induced neuronal apoptosis via decreasing p53 phosphorylation and mitochondrial apoptotic pathway activation in subarachnoid hemorrhage rats

Abstract

Early brain injury (EBI)‑induced neuronal apoptosis is primarily responsible for the subsequent complications of aneurysmal subarachnoid hemorrhage (aSAH), which may increase the risk of mortality in patients with aSAH. c‑Jun N‑terminal kinase (JNK) has been demonstrated to be a promoter of EBI‑induced cell apoptosis, although the mechanism has yet to be fully elucidated. The present study aimed to explore whether the role of JNK1 is associated with tumor protein p53 (p53), which is one of the most important factor that triggers cell apoptosis. JNK1 expression was downregulated via in vivo small interfering RNA transfection in an aSAH rat model in order to assess differences in the behavior, survival times, morphology and genetics of the experimental animals. The results revealed that JNK1 inhibition improved the neurological scores and survival times of SAH rats by interrupting cascaded neuronal apoptosis. The interruption of EBI‑induced neuronal apoptosis may originate from a decrease in the level of p53 phosphorylation and deactivation of the downstream mitochondrial apoptotic pathway. Taken together, these results suggest that JNK1 may be a promising target for improving the prognosis of patients with aSAH.

Keywords: aneurysmal subarachnoid hemorrhage; early brain injury; apoptosis; c-Jun N-terminal kinase; tumor protein 53.

Figure 1.

(A) JNK1 transcript levels in the three groups of transfected rat hippocampus tissues, as determined by reverse transcription quantitative polymerase chain reaction. (B) The JNK1 transcript levels in the siRNA group were significantly decreased compared with those in the scramble group. *P<0.05. JNK1, c-Jun terminal kinase 1; siRNA, small interfering RNA.

Figure 2.

Representative images of rat brain tissue. (Left image) In the sham-operated group, no blood was identified on the surface of the brain. (Right image) In the subarachnoid hemorrhage group, the inferior basal temporal lobe was stained with blood. The hippocampus areas of brain (depicted by the area of black ovals, which were most sensitive to apoptotic stimulation) were obtained for subsequent analysis.

Figure 3.

Neurological scores and survival curve analysis of SAH rats following the different treatments. (A) The neurological scores assay revealed that the siR group exhibited a significantly decreased score compared with the Scr group at 72 h following SAH. The score of the siR group at 72 h was also decreased compared with that at 24 h. (B) The survival assay revealed that the average survival time of rats in the siR group was significantly increased compared with that in the Scr group. SAH, subarachnoid hemorrhage. *P<0.05. Con, control; siR, siRNA; Scr, scramble.

Figure 4.

Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assays revealed the apoptotic neurons in the hippocampus areas of rat brains. Images are at magnification, ×400. The apoptotic rate in the Scr group was significantly increased compared with that in the siR group at the same time point. JNK1 inhibition resulted in an inhibition of the increasing apoptotic rate observed between 24 and 72 h. *P<0.05. Con, control; siR, siRNA; Scr, scramble; SAH, subarachnoid hemorrhage.

Figure 5.

Nissl staining of the hippocampus areas of rat brains reveal the living neurons. (A) Images are at magnification, ×400. (B) The number of living neurons in the siR group was significantly increased compared with that in the Scr group at the 24 and 72 h time points following SAH. JNK1 inhibition inhibited continuously death of the neurons between 24 and 72 h. *P<0.05. JNK1, c-Jun terminal kinase 1. Con, control; siR, siRNA; Scr, scramble; SAH, subarachnoid hemorrhage.

Figure 6.

Effects of JNK1 inhibition on transcription levels of apoptosis-associated genes as determined by reverse transcription quantitative polymerase chain reaction. JNK1 inhibition led to an increase in the Bcl-2 transcript level and a decrease in the Bax transcript level, but no significant effect on any other genes was observed. The transcript levels for (A) c-Jun terminal kinase 1, (B) tumor protein 53, (C) Bax, (D) Bcl-2, (E) mitogen-activated protein kinase 11, (F) nuclear factor κ-light-chain-enhancer of activated B-cells, and (G) caspase-3 are presented. *P<0.05 vs. Con group; ^#P<0.05 vs. the Scr 24 h group; ^&P<0.05 vs. the Scr 72 h group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Con, control; siR, siRNA; Scr, scramble.

Figure 7.

Effects of JNK1 inhibition on the protein expression levels of apoptosis-associated genes as determined by western blot analysis. (A) Representative bands are presented. JNK1 inhibition led to an increase in Bcl-2 expression and a decrease in the protein expression levels of p-p53, Bax and cleaved caspase-3, but had no effect on the expression of p-p38 or NF-κB. Data are presented for (B) p-JNK1, (C) p-p53, (D) Bax, (E) Bcl-2, (F) p-p38, (G) NF-κB, and (H) Cleaved caspase-3. *P<0.05 vs. the Con group, ^#P<0.05 vs. the Scr 24 h group; ^&P<0.05 vs. the Scr 72 h group. p, phosphorylated; p38, mitogen-activated protein kinase 11; p53, tumor protein 53; JNK1, c-Jun terminal kinase 1; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; NF-κB, nuclear factor κ-light-chain-enhancer of activated B-cells; Con, control; siR, siRNA; Scr, scramble.