RIP1 Kinase Participates In The Formation Of Astrogliosis And Glial Scar Induced By Ischemic Stroke

Aim: RIP1 kinase(RIP1K) is the key kinase of necroptosis. This study was to find the role of RIP1 K in the formation of astrogliosis and glial scar induced by ischemia, and to investigate its mechanisms associating with the VEGFD-VEGFR3 signaling pathway.Method: In vivo, transient middle cerebral artery occlusion(t MCAO) was used to establish glial scar model in rat. Lentivirally-delivered sh RNA against RIP1K(sh RNA RIP1K) or scr sh RNA RIP1K(control lentivirus) was administrated by intracerebroventricular injection five days before the ischemia. The recovery of neurological function was analyzed by assessing brain atrophy and behavioral symptoms. Primary astrocyes were exposed to oxygen-glucose deprivation(OGD) /reperfusion to form in vitro glial scar model. The neurite growth of neurons was detected in astrocyte and neuron co-culture system. The injury of astrocytes induced by OGD/reperfusion was detected with LDH test. The protein level of glial scar associated proteins GFAP, neurocan and phosphacan, and RIP1 K, VEGFD and VEGFR3 were determined by western blot and Immunohistochemical.Result: In t MCAO rats, when lentivirus was administrated five days before the onset of ischemia/reperfusion. RIP1 K knockdown significantly diminished the brain astrophy(P<0.01) and ameliorate behavioral symptoms at 28 days after ischemia(P<0.01). Western Blot analysis revealed that RIP1 K and glial scar associated protein GFAP, neurocan and phosphacan were significantly increased after t MCAO(P<0.01). RIP1 K knockdown could markedly decreased the protein level of glial scar associated protein, GFAP, neurocan and phosphacan, and VEGFD and VEGFR3. In an in vitro glia scar model, Western Blot analysis revealed that the protein level of glial scar associated protein, GFAP, neurocan and phosphacan, and RIP1 K, VEGFD and VEGFR3 was significantly increased after OGD/reperfusion, but decreased after RIP1 K knockdown. Elisa results showed that the protein levels of VEGFD were increased both in astrocytes and in medium. In an astrocyte exposed to OGD/reperfusion conditions and neuron co-culture system, RIP1 K knockdown of astrocyte could reduce the LDH leakage, and promote neuronal axon extension.Conclusion: 1. Inhibition of RIP1 K promote the recovery of neural function after ischemic stroke, and the effect may be related to RIP1 K knockdown-mediated inhibition of the astrogliosis and glial scar.2. The inhibitory effect of RIP1 K knockdown on glial scar is associated with inbibiting VEGFD-VEGFR3 signaling pathway.