RIP1K Participates In Ischemic Stroke-induced Astrogliosis And Glial Scar Formation By Regulating VEGFD-VEGFR3 Signaling Pathway

Objective:RIP1K(receptor interacting protein 1 kinase)plays an important role in necroptosis.Previous studies have shown that RIP1K knockdown reduced astrogliosis and glial scar formation after cerebral ischemia,and its mechanism may be related to the VEGFD(vascular endothelial growth factor D)-VEGFR3(vascular endothelial growth factor receptor 3)pathway.Therefore,this subject further investigated RIP1K participates in the regulation of glial scar formation after ischemic stroke by regulating the expression of VEGFD gene and activating VEGFD-VEGFR3 pathway;the effect of inhibiting RIP1K to reduce glial scar formation does not depend on its neuroprotective effect;its downstream pathway VEGFD-VEGFR3 plays a role in the formation of glial scar after ischemic stroke.Methods:This study established the transient middle cerebral artery occlusion(tMCAO)model and oxygen-glucose deprivation and reoxygenation(OGD/Re)model.Lentiviruses with short hairpin RNA targeting RIP1K(shRNA RIP1K)were administered intracerebroventricularly for RIP1K konckdown,and Necrostatin-1(Nec-1)was used to block the RIP1K activity pharmacologically.Axitinib and SAR131675 were administered to inhibit VEGFR3 activity.EdU was used to label the proliferation of astrocytes after OGD/Re.Immunohistochemistry was used to detect the expression of glial scar markers GFAP(glial fibrillary acidic protein),neurocan and phosphacan after cerebral ischemia/reperfusion(I/R).Immunofluorescence was used to detect the proliferation of astrocytes after OGD/Re.Western Blotting was performed to further confirm the expression level of GFAP,neurocan,phosphacan,VEGFD and VEGFR3.Gene chip analysis was used to detect the effect of RIP1K knockdown on VEGFD gene expression.The medium containing VEGFD recombinant protein(400 ng/ml)was used to induce the formation of glial scars in primary astrocytes.Lactate dehydrogenase(LDH)leakage detection kit was used to detect OGD/Re-induced astrocytes injury.Co-culture of astrocytes and neurons was used to detect the extension of neuronal axons in OGD/Re-induced astrocytes.Results:(1)RIP1K knockdown was successful in rat cerebral cortex.RIP1K expression is increased in tMCAO-induced reactive astrocytes and glial scars in rat cerebral cortex.Immunohistochemistry results showed that RIP1K knockdown reduced the increase of glial scar markers such as GFAP,neurocan and phosphacan induced by tMC AO in rats.EdU staining showed that RIP1K knockdown inhibited OGD/Re-induced proliferation of reactive astrocytes.Co-culture of astrocytes and neurons showed that RIP1K knockdown contributed to the extension of neuronal axons after cerebral ischemia and reperfusion.(2)Delayed administration of Nec-1 after tMC AO significantly reduced the expression of glial scar marker GFAP,neurocan and phosphacan after 7 days and the thickness of the glial scar after 14 days.(3)The results of gene chip analysis showed that the expression of VEGFD gene Figf was decreased after RIP 1K knockdown.Nec-1 could reduce the expression of VEGFD in OGD/Re-induced astrocytes.(4)Further research found that the expression of VEGFD,VEGFR3 and glial scar marker proteins in the cerebral cortex of tMCAO rats increased synchronously.The results of double-labeled neurons(Map-2)and VEGFR3 showed that the co-localization of VEGFR3 and neurons did not overlap significantly.(5)VEGFD recombinant protein(400 ng/ml)induces the formation of glial scars in primary astrocytes.Axitinib and SAR131675 as the inhibitors of VEGFR3 reduced the formation of glial scar in tMCAO-induced rat cerebral cortex and OGD/Re-induced astrocytes.Axitinib reduced the volume of cerebral infarction in the acute phase after tMCAO in mice,improved behavioral indicators.SAR131675 decreased the LDH leakage in OGD/Re-induced astrocytes.Conclusion:(1)RIP1K is up-regulated in tMCAO-induced reactive astrocytes in rat cerebral cortex.(2)RIP1K promotes astrogliosis and the formation of glial scars after cerebral ischemia by regulating the expression of VEGFD gene and activating the VEGFD-VEGFR3 pathway.(3)RIP1K knockdown or Nec-1 delayed administration inhibits the proliferation of reactive astrocytes and the formation of glial scars after cerebral ischemia,and promotes the extension of neuronal axons.(4)Inhibiting the activity of VEGFR3 inhibits the proliferation of reactive astrocytes and the formation of glial scars after cerebral ischemia.