The Roles Of Matrilin-3 In Glial Scar Formation Induced By Ischemic Brain Injury And Its Autophagy Dependent Mechanisms

Aim:Cerebral ischemia induces reactive activation of astrocytes in penumbra region,and the proliferation of reactive astrocytes participates in the formation of glial scar.On the one hand,glial scar restricts the further spread of injury area;on the other hand,glial scar,a molecular and physical barrier,inhibits the regeneration of axons and is not beneficial to brain functional repair.Matrilin-3,the extracellular matrix protein,plays a key role in cartilage development,proliferation and hypertrophy of chondrocytes and osteoarthritis.Our previous study showed that matrilin-3 existed in brain and protected astrocytes injury induced by oxygen and glucose deprivation and reoxygenation(OGD/Re).However,the roles and mechanisms of matrilin-3 in reactive astrogliosis and glial scar formation remain unknown.Therefore,this study aimed to investigate the roles of matrilin-3 in the formation of glial scar induced by ischemic brain injury and its autophagy-dependent mechanism.Methods:In vivo,transient middle cerebral artery occlusion(tMCAO)was established in Male Sprague-Dawley(SD)rats.Overexpression of matrilin-3 was performed with lentivirus of MATN-3-flag overexpression(LV-MATN-3).LV-MATN-3 or LV-CON(control)were administrated intracerebroventricularly 5 days before tMCAO in rats.At 7 days after reperfusion,the effects of matrilin-3 overexpression on glial scar-related proteins GFAP,phosphacan,and neurocan were detected by Western Blotting and immunohistochemistry.The thickness of glial scar was measured at 14 days after reperfusion,and brain atrophy volume was measured at 28 days after reperfusion.And the behavior tests were performed at 1,3,7,14,21,28 days after tMCAO.In vitro,primary astrocytes were cultured and oxygen and glucose deprivation(OGD)-reoxygenation(OGD/Re)model was established.We used lentivirus of shRNA atg5 to knockdown atg5 in primary astrocytes.The effects of knockdown of atg5,autophagy inhibitor wortmannin or autophagy activator rapamycin on the protein levels of matrilin-3 and glial scar proteins GFAP,phosphacan and neurocan were detected by Western Blotting and Immunofluorescence;We detected the effects of rapamycin on the lysosomal-related proteins such as Lamp1,Cathepsin B and Cathepsin L at OGD-6h/Re-24h.At the same time,the effects of rapamycin on OGD-6h/Re-24h induced astrocyte lysosomal membrane permeability were detected by Acridine Orange(AO)staining.Results:In a rat model of transient middle cerebral artery occlusion and reperfusion(tMCAO),we found that overexpression of matrilin-3 significantly reduced the volume of brain atrophy and improved neurobehavioral symptoms.Overexpression of matrilin-3 inhibited glial scar protein levels of GFAP,phosphacan,and neurocan at 7 days after reperfusion and reduced glial scar thickness at 14 days after reperfusion.In an OGD-reoxygenation of astrocytes,knockdown of atg5 down-regulated the protein level of matrilin-3,but had no significance effects on the protein levels of GFAP,phosphacan and neurocan.After reoxygenation,rapamycin or wortmannin was treated immediately,we found that rapamycin increased the protein level of matrilin-3,but significantly decreased the levels of glial scar-related proteins GFAP,phosphacan,and neurocan;In contrast,wortmanin increased the phosphacan and neurocan protein levels after OGD-6h/Re-24h.Furthermore,rapamycin reduced the protein levels of Lampl,active-Cathepsin B,and active-Cathepsin L after OGD-6h/Re-24h.Acridine Orange(AO)results showed that rapamycin improved the lysosomal membrane permeability at OGD-6h/Re-24h.Conclusion:(i)Overexpression of matrilin-3 significantly improves neurological deficits and promoted brain functional recovery induced by tMCAO;(ii)Overexpression of matrilin-3 inhibits glial scar formation induced by tMCAO;(iii)Inhibition of autophagy down-regulates the protein level of matrilin-3,while activation of autophagy up-regulates the protein level of matrilin-3,indicating that autophagy regulates the level of matrilin-3 in astrocytes.(iv)The activation of autophagy inhibits the formation of glial scar and inhibition of autophagy enhances glial scar formation after OGD/Re.(v)Overexpression of matrilin-3 inhibits glial scar formation via an autophagy dependent manner.