Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats

Abstract

Ischemic stroke is a common disease with high mortality and morbidity worldwide. One of the important pathophysiological effects of ischemic stroke is apoptosis. A neuroprotective effect is defined as the inhibition of neuronal apoptosis to rescue or delay the infarction in the surviving ischemic penumbra. Resveratrol is a natural polyphenol that reportedly prevents cerebral ischemia injury by regulating the expression of PI3K/AKT/mTOR. Therefore, this study aimed to elucidate the neuroprotective effect of resveratrol on cerebral ischemia/reperfusion injury and to investigate the signaling pathways and mechanisms through which resveratrol regulates apoptosis in the ischemic penumbra. Rats were subjected to middle cerebral artery occlusion for 2 h followed by 24 h reperfusion. Cerebral infarct volume was measured using 2% TTC staining. TUNEL staining was conducted to evaluate neuronal apoptosis. Western blotting and immunohistochemistry were used to detect the proteins involved in the JAK2/STAT3/PI3K/AKT/mTOR pathway. The results suggested that resveratrol significantly improved neurological function, reduced cerebral infarct volume, decreased neuronal damage, and markedly attenuated neuronal apoptosis; these effects were attenuated by the inhibition of PI3K/AKT with LY294002 and JAK2/STAT3 with AG490. We also found that resveratrol significantly upregulated the expression of p-JAK2, p-STAT3, p-AKT, p-mTOR, and BCL-2 and downregulated expression of cleaved caspase-3 and BAX, which was partially reversed by LY294002 and AG490. These results suggested that resveratrol provides a neuroprotective effect against cerebral ischemia/reperfusion injury, which is partially mediated by the activation of JAK2/STAT3 and PI3K/AKT/mTOR. Resveratrol may indirectly upregulate the PI3K/AKT/mTOR pathway by activating JAK2/STAT3.

Keywords: AKT; Ischemic penumbra; Resveratrol; STAT3; Stroke; mTOR.

Figure 1

Resveratrol exerts a neuroprotective effect 24 h after I/R. (A) The neurological deficit score in the resveratrol group was significantly decreased compared with the vehicle group. However, it was significantly increased after administration of 6 μl (20 nM/ml) AG490 in the Res + AG490 group compared with the Res group. (B–C) Infarct volumes in the resveratrol group were significantly decreased compared with the vehicle group. The infarct volumes were significantly increased after administration of 6 μl (20 nM/ml) AG490 in the Res + AG490 group compared with the Res group. Red represents normal tissue and white represents infarct tissue. (D) Morphology and structure of brain cells in Veh group rats. Data are presented as mean ± SEM. (*P < 0.05 and **P < 0.01; n = 5 in each group).

Figure 2

Effects of resveratrol on protein expression of JAK2, STAT3, AKT, mTOR, p-JAK2, p-STAT3, p-AKT, p-mTOR, BCL-2, BAX, and cleaved caspase-3 at 24 h after reperfusion. (A) The protein expression of p-AKT was increased 1.66-fold in the resveratrol group compared to the vehicle group, which was partially reversed by LY294002 (PI3K inhibitor). (B) The protein expression of p-mTOR was increased 1.66-fold in the resveratrol group compared to the vehicle group, which was also partially reversed by LY294002. (C) JAK2 inhibitor AG490 significantly decreased the protein expression of p-AKT compared with the resveratrol group. (D) The effect of PI3K inhibitor LY294002 on p-JAK2 after resveratrol treatment was not significant. (E) JAK2 inhibitor AG490 significantly decreased the protein expression of p-STAT3 compared with the resveratrol group. (F) Western blot indicated significantly higher BLC-2 and lower BAX and cleaved caspase-3 in the resveratrol group than in the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as mean ± SEM. (*P < 0.05; n = 5 in each group).

Figure 3

Effects of resveratrol on mRNA and protein expression of BCL-2, BAX, and cleaved caspase-3 after 24 h of cerebral I/R. (A) RT-qPCR analysis showed significantly higher BLC-2 and lower BAX and cleaved caspase-3 in the resveratrol group than in the vehicle group. These effects were partially reversed by LY294002 and AG490. (B) Typical immunohistochemical photographs of BCL-2, BAX, and caspase-3. (C) Immunohistochemical staining showed more BLC-2-positive and fewer BAX- and cleaved caspase-3-positive cells in the resveratrol group compared to the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as the mean ± SEM. (*P < 0.05; n = 5 in each group).

Figure 4

Effects of resveratrol on inhibition of neuronal apoptosis in the ischemic penumbra after 24 h of cerebral I/R and the mode of action for resveratrol's neuroprotection role in ischemic stroke. (A) TUNEL results showed that the TUNEL-positive cells in the resveratrol group were decreased compared to the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as mean ± SD (*P < 0.05; n = 5 in each group). (B) The diagram of the JAK2/STAT3 and PI3K/AKT signaling pathway, which was involved in the neuroprotective effect of resveratrol against I/R injury. Resveratrol activated the JAK2/STAT3 and then activated the PI3K/AKT/mTOR signaling pathway, which in turn led to increased BLC-2 and decreased BAX and cleaved caspase-3, resulting in inhibition of apoptosis; this was reversed by the PI3K inhibitor LY294002 and JAK2 inhibitor AG490. However, reperfusion after ischemic stroke prevented these effects to induce apoptosis.