RIP₁k Is Involved In The Neuronal Necroptosis Induced By Ischemic Stroke Via Activation Of The Autophagic-lysosomal Signaling Pathway

Aim: Our lab previous studies have investigated that the RIP1K specific inhibitorNecrostatin-1(Nec-1) and RIP1K knockdown can significantly reduce infarction volumeand improve neurological symptoms in permanent middle cerebral artery occlusion(pMCAO) induced rats and mice. In this paper, we further investigate whether RIP1K isinvolved in the ischemic stroke induced neuronal necroptosis via activation of theautophagic-lysosomal signaling pathway.Methods: In vivo, permanent middle cerebral artery occlusion (pMCAO) was usedto establish focal cerebral ischemia in rats or mice. The morphology of neurons inischemic cortex was examined with transmission electron microscopy (TEM); Nec-1orlentivirus was administrated by intracerebroventricular injection. In vitro,oxygen-glucose deprivation (OGD) was used to establish cerebral ischemia cell model.The morphological changes of neurons induced by OGD was examined by lightmicroscope; Cell viability of the injured neurons induced by OGD was tested by LDHassay; RIP1K konckdown was established in the fifth day of cultured neurons bylentivirus transfection. RIP1K mediated autophagic-lysosomal signaling pathway relatedproteins were detected by western blot analysis.Results: Transmission electron microscopy (TEM) revealed that cerebral cortexneurons showed necrotic morphology after pMCAO insult, and Nec-1could efficientlyinhibited necrotic death of neurons. The light microscope results showed that thenumber of necrotic cells of neurons were significantly increased after OGD treatment,co-treatment with Nec-1significantly improved the number and morphology of neurons.LDH assay showed that the LDH leakage was markedly increased after OGD (P<0.01),and Nec-1(1,10,100μM) significantly decreased the neuronal LDH leakage (P0.05,P0.01). Western blot analysis showed that the protein level of RIP1K was markedlyincreased (P0.01) and MAP2was decreased (P0.01) in the ischemic neurons both in vivo and in vitro. In contrast, Nec-1treatment or RIP1K knockdown markedlydown-regulated RIP1K protein level (P0.01) and up-regulated MAP2protein level(P0.01). Western blot analysis also indicated that autophagic-lysosomal signalingpathway related proteins LC3, Cathepsin B and Cathepsin L were up-regulated inOGD-induced neurons, and Nec-1(1,10,100μM) or RIP1K konckdown down-regulatedthe expression of LC3, Cathepsin B and Cathepsin L (P0.05, P0.01).Conclusion:1. The current findings suggest that Nec-1or RIP1K knockdown has asignificant protection on pMCAO or OGD-induced neuronal injury.2. The protective effects of Nec-1or RIP1K knockdown on ischemic strokeinduced neuronal necroptosis is associated with inhibiting RIP1K mediatedautophagic-lysosomal signaling pathway.