Matrilin-3 Overexpression Reduces The Formation Of Glial Scar Induced By Focal Cerebral Ischemia By Inhibiting Inflammatory Signals Mediated By RIP1K And GSK3?

Aim:Matrilin-3 is an extracellular non-collagen matrix protein that is an important component of cartilage development.Matrilin-3 is closely related to the occurrence of osteoarthritis.We previously demonstrated that matrilin-3 exists in human and rat brain tissues and matrilin-3 overexpression can reduce the the glial scar formation induced by ischemic stroke,but the mechanism has not been studied.Therefore,this study was aimed to explore the mechanism of matrilin-3 overexpression-mediated reduction in glial scar formation induced by focal cerebral ischemia through inhibiting inflammatory pathway.Methods:In vivo,SD rats were subjected to the transient middle cerebral occlusion(tMCAO).After cerebral ischemia for 90 min following reperfusion for 1,3,7,14 d(day),the levels of inflammatory related cytokines,such as IL-1?(interleukin 1 beta),IL-6(interleukin 6),TNF-a,(tumor necrosis factor-a),IL-1Ra(interleukin 1 receptor antagonist)and GSK3?(Glycogen synthase kinase 3)related proteins were detected with Western blotting analysis at 1,3,7,14 d after reperfusion.The effect of Necrostatin-1(Nec-1),a RIP 1K inhibitor and SB216763,a GSK3? inhibitor on the glial scar-related proteins,such as GFAP,phosphacan,neurocan were detected at 7 d after reperfusion.In vitro,the primary astrocytes were subjected to oxygen glucose deprivation(OGD)for 6 h following reoxygenation for 12 h,24 h(OGD/Re).Astrocytes were cultured to third passage or second passage and transfected with the lentiviruses of MATN-3-flag overexpression(LV-MATN-3).Elisa(Enzyme-Linked ImmunoSorbent Assay)was applied to detect the levels of inflammatory related cytokines in the medium.Western Blotting and immunofluorescence analysis were used to detect the expression of p-GSK3? after matrilin-3 overexpression and Nec-1 treatment.Nec-1 or/and SB216763 was added in astrocytes when OGD and reperfusion for 24 h.The lactate dehydrogenase(LDH)leakage detection kit was used to detect the protection of SB216763 after OGD/Re injury in astrocyte.The necrosis of cells after Nec-1 and SB216763 treatment was observed with propidium iodide(PI)staining.Immunoprecipitation(IP)was used to detect the binding of RIP1K to RIP3K.Results:In vivo,the levels of inflammatory related cytokines,such as IL-1?,IL-6,TNF-?,IL-1Ra and p-GSK3? were increased in the peri-infarct region of ischemic cerebral cortex after ischemia/reperdusion(I/R)for 7 d,while the expression of t-GSK3? didn't change.In vitro,Western Blotting and Elisa analysis showed that the inflammatory related cytokines,such as IL-1?,IL-6,TNF-?,IL-1Ra and p-GSK3? were increased at OGD6h-Re24h in astrocytes and cell medium.Matrilin-3 overexpression decreased the levels of inflammatory related cytokines,such as IL-1?,IL-6,and TNF-a in astrocytes,and increased IL-1Ra in cell medium.Simultaneously,matrilin-3 overexpression can reduce the expression of necroptosis related proteins,such as RIP1K,p-RIP1K,RIP3K,p-RIP3K,MLKL,p-MLKL and p-GSK3? in astrocytes.Nec-1 and SB216763 can decrease the number of PI-positive cell,the levels of inflammatory related cytokines,such as IL-1?,IL-6,TNF-a and glial scar-related proteins GFAP,neurocan,phosphacan.Nec-1 can reduce the expression of p-GSK3? and SB216763 can reduce the expression of necroptosis related protein.Compared with Nec-1(10 ?M)and SB216763(1 ?M)alone,combination Nec-1 and SB216763 reduced glial scar markers,such as GFAP and neurocan.Conclusion:Matrilin-3 overexpression can reduce the levels of inflammatory related cytokines,associating with its inhibition of the programmed necrosis related proteins,such as RIP1K,p-RIP1K,RIP3K,p-RIP3K,MLKL,p-MLKL and p-GSK3?.Both Nec-1 and SB216763 can reduce the levels of inflammatory related cytokines and glial scar-related proteins.RIP IK and p-GSK3? may interact to modulate the inflammatory related cytokines and glial scar-related proteins.