Study On Role Of Toll-like Receptors-peli1 Signaling Pathway In Lanthanum-induced Microglial Neuroinflammation

Objective:The most typical and widely used REEs is Lanthanum（La）.Lahas neurotoxicity and cytotoxicity at certain doses.In normal circumstances,the main function of Microglia（MG）is immune surveillance and host defense.When inflammatory stimuli arise,MG is rapidly activated and releases a lot of proinflammatory factors.Studies have shown that MG expresses multiple Toll-like receptors（TLRs）proteins.Peli1 protein is highly expressed in MG,which demonstrated the key role of Peli1 in neuroinflammation.This study investigated whether LaCl₃acted on the TLRs-Peli1 signaling pathway to induce the release of pro-inflammatory cytokines such as iNOS,IL-6,TNF-αand CCL2 in MG,and then produced neurotoxic damaging effects through in vitro cell experiments.These results provide new clues and laboratory data for further elucidating the mechanism of Ladamage to the nervous system.Methods:In this study,mouse MG series BV2 cell was used.And the survival rate of BV2 cells infected with Lawas detected by CCK-8 to determine the dose and time.The effect of Laon microglia cell morphology was observed under microscope.The expression of Iba1 was observed by immunofluorescence method to determine the effect of Laexposure on microglia activation.The release of TNF-α,IL-6 and CCL2 were detected by ELISA.The expressions of TLRs-Peli1 signaling pathway related proteins were detected by Western Blot.Peli1 was silenced and the release of TNF-α,IL-6 and CCL2 was detected by ELISA.The expression of Peli1 downstream related proteins was detected by Western Blot.Results:1.After treatment with 0.025,0.05 and 0.1 mmol/L LaCl₃for 24 hours,BV2cells showed an activated state,the cell body became round,the refractive index around was enhanced,and the filamentous pseudopodia increased and lengthened.With the increase of LaCl₃concentration,the activated morphology became more obvious.2.With the increase of LaCl₃concentration,the green fluorescence intensity in cytoplasm increased and the cell body was enlarged.3.The contents of TNF-α,IL-6 and CCL2 in cell culture medium increased gradually with the increase of LaCl₃concentration.4.The expression levels of TLR2,TLR3,TLR4,My D88,TRIF,TRAF6,RIP1,Peli1,P-IKK,P-NF-κB p65,P-p38 MAPK,iNOS,TNF-α,IL-6 and CCL2 proteins in LaCl₃treatment group increased gradually compared with the control group.The difference was statistically significant.The expression levels of IKK,NF-κB p65 and p38 MAPK proteins showed no significant difference compared with the control group.5.After silencing Peli1,the contents of TNF-α,IL-6 and CCL2 in cell culture medium of LaCl₃treatment group were significantly decreased.The expression levels of P-IKK,P-NF-κB p65,P-p38 MAPK,iNOS,TNF-α,IL-6 and CCL2 protein in siPeli1+LaCl₃treatment group were significantly decreased compared with NC+LaCl₃treatment group.But the total protein expression levels of IKK,NF-κB p65 and p38 MAPK showed no significant difference among the four groups.Conclusion:1.Lainduces activation of microglia cell line BV2 cells and activates TLRs-Peli1 signaling pathway,and causes the expression of related proteins in this signaling pathway to increase,resulting in excessive release of inflammatory factors.2.Silencing Peli1 has an antagonistic effect on the excessive release of inflammatory factors in BV2 cells induced by Laand the increase of downstream Peli1 protein expression in TLRs-Peli1 signaling pathway.