Matrilin-3 Reduces The Formation Of Glial Scar Induced By Cerebral Ischemia Through Inhibition Of BMP-2/Smad Pathway In A RIP1 Dependent Manner

Aim:Matrilins is an extracellular matrix protein,which belongs to the vWFA domain family.Matrillin-3 is the third and the smallest member of matrilins family.Our previous results revealed the presence of matrilin-3 in the brain of rat and human for the first time.In addition,overexpression of matrilin-3 can reduce the formation of glial scar and protect brain damage caused by ischemic stroke,but the mechanism of matrilin-3 is still unclear.Therefore,this study aimed to investigate the mechanism of matrilin-3 in glial scar formation induced by focal cerebral ischemia.Methods:The transient middle cerebral artery occlusion(tMCAO)model was established in rats,the middle cerebral artery of rats was occluded for 90 minutes following reperfusion for 1 d,3 d,7 d and 14 d,Western Blotting and/or Immunohistochemistry analysis were used to detect the expressions of BMP-2/Smad pathway-related proteins(BMP-2,Smad1,5,9 and p-Smad1,5,9)and necroptosis-related proteins(RIP1,p-RIP1,RIP3,p-RIP3,MLKL and p-MLKL).5 days before tMCAO,Lentiviruses with LV-CON and LV-MATN-3 were injected intracerebroventricularly(ICV)in rats.10 minutes before tMCAO,the RIP1 inhibitor,Necrostatin-1(Nec-1),was injected intracerebroventricularly(ICV)in rats.On the 7th day of ischemia/reperfusion(I/R)after matrilin-3 overexpressing lentivirus and Nec-1 treatment,Western Blotting and/or Immunohistochemistry analysis were used to detect the expressions of BMP-2/Smad pathway and necroptosis-related proteins.The model of Oxygen and Glucose Deprivation/Reoxygenation(OGD/Re)was established,astrocytes were exposed to OGD for 6 h following reoxygenation for 12 h and 24 h,the expressions of BMP-2/Smad pathway and necroptosis-related proteins were detected with Western Blotting analysis.Lentiviruses with LV-CON and LV-MATN-3 were transfected to primary cultured astrocytes.Nec-1 was added to the glucose-free medium 10 min before OGD,and then reoxygenated and continued to treat with Nec-1 for 24 h.Western Blotting and/or Immunofluorescence analysis were used to detect the expressions of BMP-2/Smad pathway,necroptosis and glial scar-related proteins.Results:In the ischemic marginal zone of cerebral cortex in rats on the 7th day of ischemia/reperfusion,the expressions of BMP-2/Smad pathway-related proteins,BMP-2 and p-Smad1,5,9 were increased,while the expression of total protein Smad1,5,9 was not affected.At the same time,the nuclear translocatin of p-Smad1,5,9 was significantly increased.Moreover,the expressions of necroptosis-related proteins(RIP1,p-RIP1,RIP3,p-RIP3,MLKL and p-MLKL)were also significantly increased.After overexpression of matrilin-3,nuclear translocatin of p-Smad1,5,9 can be inhibited.What’s more,Nec-1 inhibited the expressions of BMP-2 and p-Smad1,5,9 and also inhibited the expressions of the glial scar-marker proteins GFAP,Phosphacan and Neurocan.The expressions of BMP-2/Smad pathway and necroptosis-related proteins were increased at 24 h reperfusion of the astrocytes after OGD 6 h,and overexpression of matrilin-3 and Nec-1 can not only inhibit the expressions of two pathway-related proteins and the nuclear translocation of p-Smad1,5,9,but also inhibit the expressions of GFAP,Phosphacan and Neurocan.Conclusion:BMP-2/Smad pathway and necroptosis-related proteins are involved in the formation of glial scar induced by focal cerebral ischemia;Overexpression of matrilin-3 can inhibit the formation of glial scar by inhibiting the activation of BMP-2/Smad pathway,and necroptosis-related proteins may be involved in the regulation of BMP-2/Smad pathway by matrilin-3;Nec-1 can inhibit the formation of glial scar induced by focal cerebral ischemia,which has a protective effect on ischemic brain injury.