Neuregulin 1 Regulates Microglia Polarization By Inhibiting The NF-κB Pathway Through ErbB4

BackgroundThe pathological features of Alzheimer’s disease（AD）are the production and abnormal deposition of Aβ,neurofibrillary entanglement and excessive neuroinflammatory response.The inflammatory response caused by microglia in the central nervous system（CNS）plays an important role in Alzheimer’s disease（AD）.Neuregulin-1（NRG1）is widely distributed in the nervous system and participates in a variety of neural development processes.NRG1 is a member of the neuregulin family and has been demonstrated to have anti-inflammatory properties.Our previous studies have shown that NRG1 has a significant neuroprotective effect on AD mice,but the relationship between NRG1,microglia phenotype and neuroinflammation remains unclear.ObjectivesThe purpose of this study was to investigate whether NRG1 through NRG1-Erb B4-NF-κB signaling pathway regulates the phenotypic reversal or improvement of microglia Aβ_1-42 induced neuroinflammatory response.Methods1.BV2 microglia cell lines were cultured in vitro,and over-NRG1 BV2 cell lines（OVER-N）and Er BB4-knockdown BV2 cell lines(ER4^KD)were constructed by lentivirus transfection technique.The experimental groups were as follows:Control group:BV2 cells without any treatment.Aβgroup:BV2 cells were treated with Aβ_1-42for 4 h.NRG1 group:BV2 cells were treated with exogenous NRG1 for 2 h.Aβ+NRG1 group:BV2 cells were treated with Aβ_1-42 for 4 h,and then treated with exogenous NRG1 for 2 h.Aβ+OVER-NRG1 group:OVER-N BV2 cells were treated with Aβ_1-42 for 4 h.Aβ+OVER-NRG1 NC group:Negative control BV2 cells were treated with Aβ_1-42 for 4 h.Aβ+NRG1+ER4^KD group:ErbB4 Knockdown BV2cells were treated with Aβ_1-42 for 4 h,and then treated with exogenous NRG1 for 2 h.Aβ+NRG1+ER4^KD NC group:Erb B4 Knockdown BV2 negative control cells were treated with Aβ_1-42 for 4 h and then treated with exogenous NRG1 for 2 h.2.Cell viability of BV2 cells in each group was analyzed by the Cell Counting Kit-8 method.3.A scratch experiment was designed to test whether NRG1 could improve the migration ability of BV2 cells.4.The content of i NOS and TNF-αin the supernatants of BV2 cells in each group was measured with a sandwich enzyme-linked immunosorbent assay kit（ELISA）.5.We used flow cytometry to assess the expression of the M1 microglial surface markers i NOS and CD120 and the M2 microglial surface markers CD206 and Arg-1in BV2 cells by fluorescence-activated cell sorting.6.The supernatants of cultured BV2 cells were collected after different treatments.SH-SY5Y cells were incubated in conditioned medium for 24 h.The effect of NRG1 on Bax was tested using Western blotting and immunofluorescence staining.Results1.The results of the CCK-8 assay showed that the cell activity of the Aβ-treated BV2 cells was significantly reduced compared with the control group（P<0.01）.The cell viability of BV2 cells treated with NRG1 was increased compared with the Aβ-treated group（P<0.01）.2.Compared with the control group,the relative cell migration rate of the Aβ-treated group was significantly reduced（P<0.01）.Compared with the Aβ-treated group,the relative cell migration rate of the Aβ+NRG1 group and the Aβ+OVER-NRG1 group was significantly increased（P<0.01）.3.Compared with BV2 cells in the control group,the contents of TNF-αand i NOS in the Aβ-treated group were significantly increased（P<0.01）.Compared with BV2 cells in the Aβ-treated group,the levels of TNF-αand i NOS in the Aβ+N group and the Aβ+OVER-N group were significantly reduced（P<0.01）.4.Compared with SH-SY5Y cells in the control group,the expression of Bax in the Aβ-treated group was increased（P<0.01）.Compared with that in the Aβ-treated group,the expression of Bax in the Aβ+N group and the Aβ+OVER-N group was decreased（P<0.01）.5.The results of FACS phenotype analysis showed that compared with those in the control group,the percentages of cells positive for CD120 and i NOS in the Aβ-treated group were higher（P<0.01）.Compared with those in the Aβ-treated group,the percentages of cells positive for CD120 and i NOS in the Aβ+NRG1 group and the Aβ+OVER-N group were lower.In addition,the expression of Arg-1 and CD206increased in the Aβ+N group and the Aβ+OVER-N group（P<0.05）.6.Compared with BV2 cells in control group,the expression of NF-κB p65protein in the nucleus of BV2 cells in Aβgroup was increased（P<0.01）and the expression of P-Erb B4 protein was decreased（P<0.01）.Compared with BV2 cells in Aβgroup,the expression of NF-κB p65 protein in the nucleus of BV2 cells in Aβ+NRG1 and Aβ+over-NRG1 groups was decreased（P<0.01）,the expression of NF-κB P65 protein in the cytoplasm was increased（P<0.01）,and the expression of P-Erb B4 protein was increased（P<0.01）.7.Erbb4-knockout BV2(ER4^KD BV2)cells were constructed by transfection of BV2 cells with lentivirus.Consistent with this blockade of Erb B4 expression,downregulation of the expression of the inflammatory factors TNF-αand i NOS（P<0.01）and the M1 microglia markers CD120 and i NOS was abolished（P<0.05）.We found that the downregulation of NF-κB-p65（P<0.05）subunit expression in the nuclei of NRG1-treated BV2 cells was also blocked by Erb B4si RNA transfection.ConclusionsNRG1 inhibited the neuroinflammatory response mediated by microglia and related to inhibition of the NF-κB signaling pathway through activation the Erb B4, which promotes the transformation of microglia from the proinfammatory M1 phenotype to the anti-infammatory M2 phenotype.