The Mechanism Of Nrf2-SRA Pathway In Hematoma Clearance By Regulating Astrocyte Polarization After Vitro Intracerebral Hemorrhage

Objective:Astrocytes are the most numerous and largest glial cells in the central nervous system.Erythrocyte and its metabolites are one of the critical factors leading to secondary brain injury after intracerebral hemorrhage.Recent studies have shown that reactive astrocytes under pathological conditions can be divided into two subtypes,A1 type with neurotoxic effect and A2 type with protective effect.Our previous animal experiments showed that nuclear factor-E2 related factor 2(Nrf2)can promote hematoma clearance after intracerebral hemorrhage by regulating the phenotypic and functional transformation of astrocytes.The scavenger receptor class A(SRA)is expressed in astrocytes,and Nrf2 can regulate the functional transformation of astrocyte by affecting the expression of SRA,thereby promoting hematoma clearance after intracerebral hemorrhage.This article aims to verify whether the Nrf2-SRA pathway can regulate the phenotypic transformation of astrocytes and its direct effect on erythrocytes after intracerebral hemorrhage in vitro experiment.Methods:C8D1A astrocytes were cultured in vitro,and the astrocytes were divided into three groups: normal control group,Nrf2 inhibitory group(astrocytes + Nrf2-siRNA),Nrf2 agonist group(Astrocytes + Xuezhikang)were cultured for 72 hours.The expression site and protein content of Nrf2,SRA,C3(A1 astrocyte marker)and S100A10(A2 astrocyte marker)were detected by immunofluorescence double-labeling method and Western blot.The positive cells of C3 and S100A10 were detected by flow cytometry to verify the phenotype of astrocyte.The morphology of astrocytes in each group were observed under the microscope.Astrocytes in each group were co-cultured with isolated erythrocytes to simulate an intracerebral hemorrhage model in vitro,and observed the effect of Nrf2 on the interaction between astrocytes and erythrocytes.Results:Compared with the normal control group,the morphology of astrocytes in the Nrf2-siRNA group did not change significantly,while the astrocytes in the Xuezhikang group had more cytoplasmic granules,increased cell synapses,and increased cell-to-cell connections.At the protein level,the Nrf2-siRNA group reduced the expression of Nrf2,SRA,and S100A10 in astrocytes,while the expression of C3 increased.And Xuezhikang group up-regulated the expression of Nrf2,SRA,and S100A10 in astrocytes,but decreased the expression of C3.Flow cytometry showed no significant difference among the three groups.The co-culture of astrocytes and erythrocytes showed that different from the direct phagocytosis of erythrocytes by microglia previously discovered by our research group,the Nrf2 agonist-Xuezhikang can promote the adhesion between astrocytes and erythrocytes.Conclusion:Nrf2-siRNA can promote the transformation of astrocytes to the A1 phenotype.As an agonist of Nrf2,Xuezhikang can promote the transformation of astrocytes to the A2 phenotype by regulating the expression of SRA.By enhancing the adhesion between astrocytes and erythrocytes in vitro,as a bridge for information communication,it contributes to the directed phagocytosis of microglia,and maybe play an important role in the removal of endogenous hematoma after intracerebral hemorrhage.