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Aging and disease    2019, Vol. 10 Issue (4) : 807-817     DOI: 10.14336/AD.2018.0728
Orginal Article |
Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling
Xu-Xu Deng1,2, Shan-Shan Li1,2, Feng-Yan Sun1,2,3,*
1Department of Neurobiology, School of Basic Medical Sciences and National Clinical Research Center for Aging and Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
2Institute for Basic Research on Aging and Medicine, the State Key Laboratory of Medical Neurobiology, School of Basic Medical Sciences, Fudan University, Shanghai, China
3Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital, Fudan University, Shanghai, China
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Abstract  

Pharmacological studies have indirectly shown that necroptosis participates in ischemic neuronal death. However, its mechanism has yet to be elucidated in the ischemic brain. TNFα-triggered RIPK1 kinase activation could initiate RIPK3/MLKL-mediated necroptosis under inhibition of caspase-8. In the present study, we performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia in rats and used immunoblotting and immunostaining combined with pharmacological analysis to study the mechanism of necroptosis in ischemic brains. In the ipsilateral hemisphere, we found that ischemia induced the increase of (i) RIPK1 phosphorylation at the Ser166 residue (p-RIPK1), representing active RIPK1 kinase and (ii) the number of cells that were double stained with P-RIPK1 (Ser166) (p-RIPK1+) and TUNEL, a label of DNA double-strand breaks, indicating cell death. Furthermore, ischemia induced activation of downstream signaling factors of RIPK1, RIPK3 and MLKL, as well as the formation of mature interleukin-1β (IL-1β). Treatment with necrostatin-1 (Nec-1), an inhibitor of necroptosis, significantly decreased ischemia-induced increase of p-RIPK1 expression and p-RIPK1+ neurons, which showed protection from brain damage. Meanwhile, Nec-1 reduced RIPK3, MLKL and p-MLKL expression levels and mature IL-1β formation in Nec-1 treated ischemic brains. Our results clearly demonstrated that phosphorylation of RIPK1 at the Ser166 residue was involved in the pathogenesis of necroptosis in the brains after ischemic injury. Nec-1 treatment protected brains against ischemic necroptosis by reducing the activation of RIPK1 and inhibiting its downstream signaling pathways. These results provide direct in vivo evidence that phosphorylated RIPK1 (Ser 166) plays an important role in the initiation of RIPK3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain.

Keywords RIPK1      necroptosis      necrostatin-1 (Nec-1)      cerebral stroke      inflammation      neuroprotection     
Corresponding Authors: Sun Feng-Yan   
About author:

These authors contributed equally to this work.

Just Accepted Date: 04 September 2018   Issue Date: 01 August 2019
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Xu-Xu Deng
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Xu-Xu Deng,Shan-Shan Li,Feng-Yan Sun. Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling[J]. Aging and disease, 2019, 10(4): 807-817.
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http://www.aginganddisease.org/EN/10.14336/AD.2018.0728     OR     http://www.aginganddisease.org/EN/Y2019/V10/I4/807
Figure 1.  Expression and identification of phosphorylated RIPK1 in rat brains after ischemic injury

(A) Expression levels of RIPK1 in sham-operated (Sham) and ipsilateral (IPSI)/contralateral (CON) striatum at 24 h after MCAO. Expression levels of total RIPK1 (B) and low molecular weight RIPK1 (C) increased in the ipsilateral striatum compared with the contralateral striatum and sham-operated rats (n≥6). *p<0.05. (D) De-phosphorylation of RIPK1 in the ipsilateral striatum at 24 h after MCAO by calf intestinal alkaline phosphatase (CIP) treatment. (E) Expression levels of RIPK1 in the nuclear and cytoplasmic compartments of cells in the ipsilateral striatum, H3 was used as a specific nuclear marker and GAPDH as a specific cytoplasmic marker. (F) L-RIPK1 immuno-reacted with p-RIPK1 (Ser166) monoclonal antibody.

Figure 2.  Distribution of phosphorylated RIPK1 (Ser166) in the ischemic rat brain

(A) Morphology of p-RIPK1 positive (p-RIPK1+) cells in the contralateral (CON) and the ipsilateral (IPSI) striatum at 24 h after MCAO. (B) p-RIPK1+ (red) signals co-localized with DAPI (blue) in the ipsilateral striatum of rats at 24 h after MCAO. (C) p-RIPK1 and NeuN double positive (p-RIPK1+-NeuN+) neurons in the ipsilateral striatum at 24 h after MCAO. (D) The number of p-RIPK1+-NeuN+ neurons increased in the ipsilateral (IPSI) compared with the contralateral (CON) striatum at 24 h after MCAO (n=5). *p<0.05. (E) p-RIPK1+ signals co-localized with TUNEL stain (green) in the ipsilateral striatum at 24 h after MCAO. Both black and white scale bars in all figures represent 50 μm.

Figure 3.  Necrostatin-1 treatment protected brains against ischemic injury

(A) Experimental protocol of necrostatin-1 (Nec-1) treatment in MCAO rats. Nec-1/inactive necrostatin-1 (iNec) were stereotaxically injected to the right lateral ventricle 30 min before MCAO surgery. Rat brains were subjected to reperfusion at 30 min after occlusion of the MCA. Neurological evaluations were performed at 1 h before MCAO (pre-) and 12, 24, 72 h after MCAO. (B) Schematic of the brain shows the injection position of Nec-1/iNec (contralateral ventricle) and the areas of the infarct core (dotted line) after ischemic injury. (C-D) Nec-1 reduced infarct volume at 24 h after MCAO compared with iNec group (n=6-7). (E-F) Nec-1 reduced neurological impairments after cerebral ischemia. The graphs show neurological scores (E) and forelimb placing scores (F) of rats treated with Nec-1 (black bar) or iNec (white bar). **p<0.01.

Figure 4.  Necrostatin-1 treatment prevented phosphorylation of RIPK1 at Ser166 in neurons after cerebral ischemia

(A-B) Nec-1 treatment significantly inhibited ischemia-induced increase of p-RIPK1 expression in the ipsilateral striatum (IPSI) at 24 h after MCAO (compared with iNec treatment, p<0.05, n≥10). (C-D) Nec-1 reduced the number of p-RIPK1-NeuN double positive (p-RIPK1+-NeuN+) neurons in the ipsilateral striatum (IPSI) at 24 h after MCAO (compared with iNec treatment, p<0.05, n=5). CON=contralateral striatum. Scale bars indicate 50 μm.

Figure 5.  Necrostatin-1 treatment inhibited ischemia-induced expression of RIPK3/MLKL and formation of IL-1β in rat brains after MCAO

Expression levels of RIPK3, MLKL, p-MLKL and IL-1β (A) in the ipsilateral (IPSI) and contralateral (CON) striatum at 24 h after MCAO. Statistical analysis showed that Nec-1 treatment significantly suppressed RIPK3 (B), MLKL (C), p-MLKL (D) and mature IL-1β (E) expression levels in the ipsilateral striatum of MCAO rats compared with iNec-1 treatment (CON), n=4, *p<0.05.

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