PRMT2 Inhibits M1-type Polarization Of Microglia Induced By LPS And Its Mechanism

[Background and Objective] A large number of studies have shown that the chronic inflammatory response characterized by the activation of microglia in the brain is an important sign of neurodegenerative diseases.M1-type polarized microglia have the effect of promoting neuroinflammation and neurotoxicity,while M2-type polarization has the effect of anti-inflammatory and tissue repair.The balance between microglia M1 and M2 types plays an important role in the progression of neuroinflammation-related diseases.Therefore,it will be of great significance for the treatment of these diseases to explore the conditions under which microglia can change from M1-type proinflammatory state to M2-type anti-inflammatory state.Our previous research found that PRMT2 may be involved in LPS-induced microglial neuroinflammation,but the mechanism is unclear.In this study,by observing the effect of PRMT2 on LPS-induced polarization of N9 microglia,and to explore its possible mechanism,providing new ideas and clues for the prevention and treatment of neuroinflammation.[Methods] Mouse microglia cell lines N9 microglia was cultured to construct a PRMT2 lentiviral vector LV-PRMT2(4952-1),which was transfected into mouse N9 microglia and overexpressed PRMT2.Negative control was transfected with an empty lentiviral vector.Microglia were pretreated with autophagy inhibitor 3-MA for 0.5 h.N9 microglia were treated with LPS(2 μg/mL)for 6 h to construct an in vitro inflammation model.The experiment was divided into the following groups: Control group,LV-empty group,LV-PRMT2 group,Control + LPS group,LV-empty + LPS group,LV-PRMT2 + LPS group.MTT assay was used to detect cell viability.Western Blot was used to detect the expression of PRMT2,iNOS,TNF-α,and IL-6,Arg1,IL-4,IL-10,TFEB,intraructal TFEB,plasmonic TFEB,autophagy related proteins Beclin-1,LC3-II / LC3-I and p62;The number of autophagosomes in cells was observed by transmission electron microscope.[Results]1.LPS down-regulated PRMT2 expression in microgliaCompared with the control group,the protein expression of PRMT2 in microglia was significantly reduced after LPS treatment(P < 0.05).2.Construction of N9 microglia overexpressing PRMT2Compared with the control group,N9 microglial successfully transfected with lentiviral vector showed green fluorescence;Compared with the LV-empty group,the PRMT2 protein in the LV-PRMT2 group was significantly increased(P < 0.05).3.Transfection of lentiviral vector and LPS treatment had no effect on the viability of N9 microgliaThere was no significant difference in cell viability among the three groups of Control,LV-empty,and LV-PRMT2(P > 0.05).The Control,LV-empty,and LV-PRMT2 groups were treated with LPS(2 μg/mL)for 6 h,and there was no significant difference in cell viability in each group(P > 0.05).4.Overexpression of PRMT2 regulates the polarization of microglia treated by LPS4.1 Compared with the LV-empty group,the levels of iNOS,TNF-α,and IL-6 proteins in the LV-empty + LPS group were significantly increased(P < 0.05);Compared with LV-empty + LPS group,the levels of iNOS,TNF-α,and IL-6 proteins in LV-PRMT2 + LPS group were significantly decreased(P < 0.05).4.2 Compared with the LV-empty group,the levels of Arg1,IL-4 and IL-10 in the LV-empty + LPS group were significantly decreased(P < 0.05).Compared with the LV-empty + LPS group,the levels of Arg1,IL-4 and IL-10 in the LV-PRMT2 + LPS group were significantly higher(P < 0.05).5.Overexpression of PRMT2 promotes the level of autophagy in lps-treated microglia5.1 Compared with the LV-empty group,the levels of LC3-II / LC3-I in the LV-empty + LPS group were significantly reduced(P < 0.05),while p62 levels increased significantly(P < 0.05);Compared with LV-empty + LPS group,the levels of LC3-II / LC3-I in the LV-PRMT2 + LPS group increased significantly(P < 0.05),while the level of p62 decreased significantly(P < 0.05).5.2 Compared with the LV-empty group,autophagosome levels in the LV-empty + LPS group were reduced;Compared with the LV-empty + LPS group,autophagosome levels in the LV-PRMT2 + LPS group were increased.6.Overexpression of PRMT2 regulates the polarization of microglia treated by LPS,and autophagy inhibitor 3-MA reverses this process6.1 Compared with the LV-PRMT2 + LPS group,the levels of iNOS,TNF-α and IL-6 protein in the LV-PRMT2 + LPS + 3-MA group were significantly increased(P < 0.05).6.2 Compared with the LV-PRMT2 + LPS group,the levels of Arg1,IL-4 and IL-10 protein in the LV-PRMT2 + LPS + 3-MA group were significantly decreased(P < 0.05).7.Overexpression of PRMT2 promotes TFEB nuclear translocation in microglia7.1 There is no interaction between TFEB and PRMT2.7.2 Compared with the LV-empty group,the cytoplasmic TFEB protein level in the LV-empty + LPS group was significantly increased(P < 0.05),and the nuclear TFEB protein level was significantly reduced(P < 0.05);Compared with the LV-empty + LPS group,the cytoplasmic TFEB protein level in the LV-PRMT2 + LPS group was significantly reduced(P < 0.05),and the nuclear TFEB protein level was significantly increased(P < 0.05).[Conclusions] PRMT2 inhibits the M1-type polarization of N9 microglia induced by LPS.The mechanism may involve the TFEB-autophagy pathway.