The Roles And Mechanisms Of Matrilin-3 In Nerve Cell Death Induced By Ischemic Brain Damage

Aim: According to previous reports, the tissue distribution of matrilin-3 was more restricted to skeletal tissues and not detected in brain, but our recent research suggested that matrilin-3 was extensively expressed in neurons, astrocytes and different brain area. Therefore, this study is aim to further explore the changes of matrilin-3 in cerebral ischemia, and the roles and mechanisms of matrilin-3 in nerve cell death induced by cerebral ischemia, and the relationship between matrilin-3 and glial scar.Methods: In vivo, the transient middle cerebral artery occlusion(tMCAO) model was performed. In vitro, primary neurons and astrocytes were cultured, and oxygen and glucose deprivation(OGD) or oxygen glucose deprivation and reperfusion(OGD-reperfusion) model was performed. Western blotting and immunofluorescence were used to detect the expression of matrilin-3. After knockdown or overexpression of matrilin-3, lactate dehydrogenase(LDH) assay was used to detect the injury of astrocytes or neurons induced by OGD or OGD-reperfusion. Western blot and immunofluorescence was performed to detect the protein levels of GFAP, neurocan and phosphacan, the marker proteins of glial scar, in astrocytes after overexpression of matrilin-3. In atg5-/- MEF and atg5 knockdown astrocytes, western blot was used to detect the expression of matrilin-3.Results: In a rat model of transient middle cerebral artery occlusion(tMCAO), we found that the protein levels of matrilin-3 were down-regulated in the ischemic core of cortex or striatum after day 1 or day 3 of reperfusion, respectively, while in the peri-infarct area of glia scar formation, matrilin-3 was significantly up-regulated in astrocytes. Consistent with the in vivo model, in an OGD-reperfusion-induced glia scar formation model, the matrilin-3 protein levels were obviously up-regulated. However, there was no significant change in the matrilin-3 protein levels in an OGD-reperfusion-induced neuronal cell death. Furthermore, matrilin-3 knockdown could inhibit astrocytes proliferation and induce the morphological changes of astrocytes showing hypertrophy and irregular, and finally induced a large number of cells death after OGD 6 h, and knockdown of matrilin-3 significantly increased LDH leakage, further aggravating the injury in astrocytes induced by OGD, but in neurons, the LDH results showed that knockdown of matrilin-3 showed either no protection or no further damage to the neuronal cell injury induced by OGD. These results suggesting that matrilin-3 functions differently in neurons and astrocytes upon ischemia insult. However, overexpression of matrilin-3 decreased the astrocytic injury induced by OGD-reperfusion, and reduced the protein levels of GFAP, neurocan and phosphacan, suggesting that this protective effect might be achieved by inhibiting the formation of glial scar. Moreover, atg5-/- and atg5 knockdown significantly decreased the expression of matrilin-3 in fibroblast cells and astrocytes, respectively.Conclusion: Our results indicate that matrilin-3 may function differently in astrocytes and neurons upon ischemia insult. Matrilin-3 may played a key role in proliferation and development of astrocytes, and knockdown of matrilin-3 further aggravated the injury in astrocytes induced by OGD, but overexpression of matrilin-3 decreased the injury induced by OGD-reperfusion in astrocytes, and this protective effect might be achieved by inhibiting the formation of glial scar. Moreover, autophagy was involved in the regulation of matrilin-3.