The Mechanism Study On Taxifolin's Protection To Chlorpyrifos Induced BV2 Cells Neurotoxicity

Objective To investigate the protective mechanism of Taxifolin's protection to chlorpyrifos induced BV2 cells neurotoxicity,and to provide new ideas for the prevention and treatment of children's nervous system diseases.Methods(1)BV2 cells were randomly divided into 4 groups: DMSO group : containing0.1% DMSO RPMI 1640 complete medium treated cells,according to the experimental requirements set at different time points(CPF and Tax are fat soluble,easily soluble in DMSO,it is provided with a concentration of 0.1% DMSO RPMI 1640 complete medium treatment group as control cells group);CPF group: 200 ?mol/L CPF containing RPMI1640 complete medium processing cells,time is 6 h;Tax group: containing Tax RPMI1640 complete medium pretreatment of cells according to the experimental requirements set different concentrations and different time points;Tax+CPF group: 100 ?mol/L Tax containing RPMI 1640 complete medium pre treatment of cells,time is 6 h;and then with200 ?mol/L RPMI 1640 CPF complete medium treated cells,time is 6 h.(2)Effect of detection of Tax CCK-8 method on BV2 cell viability;cell morphological changes were observed under light microscope.(3)Fluorescent staining to detect reactive oxygen species,(ROS)level of BV2 cells.(4)Enzyme linked immunosorbent assay(ELISA)to detect TNF-? and IFN-? protein level of cell supernatant;(5)Immunofluorescence staining;(IF)to detect autophagy marker protein LC3 II expression;(6)Western blot detection of p-AMPK,AMPK,Nrf2,HO-1,p62 protein expression level and after Conpound C blocking AMPK pathway,Western blot detected the protein expression level again.Result(1)Under the light microscope: CPF induced BV2 cells became round,cell synaptic atrophy,and adherent cell aggregation becomes worse,floating.Tax,Tax+CPF groups of BV2 cell morphology is more close to the DMSO group,the wall is better than that of CPF group,no obvious aggregation phenomenon.(2)Under the fluorescence microscope,the green fluorescence of the DMSO group was less and the brightness was weak.The green fluorescence of CPF group was more and the brightness was enhanced,while the green fluorescence of Tax and Tax+CPF cells decreased and the fluorescence intensity decreased.Compared with the DMSO group,the ROS in the CPF group was significantly increased(P<0.001).ROS of Tax and Tax+CPF group had no significant increase(P>0.05).compared with the CPF group,Tax,Tax+CPF group significantly reduced,and the difference was statistically significant(P<0.05).(3)Detection of TNF-?and IFN-? levels in the supernatant with ELISA were shown: compared with DMSO group,CPF group and Tax+CPF cells TNF-? and IFN-? levels were significantly increased,and the difference was statistically significant(P<0.05).Tax group TNF-?levels have increased,but the difference was not statistically significant(P>0.05),IFN-?the level increased significantly,the difference was statistically significant(P<0.05).Compared with CPF group,TNF-? cells and IFN-? level of both Tax and Tax+CPF group decreased significantly,and the difference was statistically significant(P<0.05).(4)Immunofluorescence staining.Results showed that LC3 II expressed mainly in the cytoplasm.Compared with DMSO group,the LC3 II expression of CPF group cells in the cytoplasm was increased less.The expression of LC3 II of Tax and Tax+CPF groups in the cytoplasm increased more.At the same time,Western blot results show: Compared with DMSO group,p62 protein expression in CPF group had no significant difference(P>0.05).p62 protein expression increased in Tax group(P<0.05).p62 protein expression increased in Tax+CPF group decreased,but the difference was not statistically significant(P>0.05)(5)Western blot results showed: compared with DMSO group,AMPK,p-AMPK,Nrf2,HO-1 protein expression in CPF group had no significant change(P>0.05).There was no significant changes in the expression of p-AMPK protein in Tax group(P>0.05),AMPK,Nrf2,HO-1 protein expression decreased in Tax group,but the difference was not statistically significant(P>0.05).There was no significant changes of AMPK protein in Tax+CPF group,p-AMPK protein expression decreased(P<0.05).There was no significant increase of Nrf2,HO-1 protein expression(P>0.05).Compared with CPF group,AMPK,p-AMPK,Nrf2,HO-1 protein expression was of no significant change in Tax group(P>0.05).In Tax+CPF group p-AMPK protein expression reduced(P<0.05).Nrf2 and HO-1 expression protein increased,but the difference had no significant difference(P>0.05).Treatment four groups BV2 cells with Conpound C after 1 h.Compared with DMSO group,the AMPK protein expression in CPF group decreased significantly(P<0.05).AMPK and Nrf2 protein expression in Tax group decreased significantly(P<0.05).p-AMPK and HO-1 protein expression decreased,but the difference was not statistically significant(P>0.05).In Tax+CPF group AMPK and p-AMPK were significantly decreased(P<0.05).Nrf2,HO-1 protein expression had no significant difference(P>0.05).Compared with CPF group,AMPK in Tax group was significantly increased(P<0.05).p-AMPK,Nrf2,HO-1 protein expression had no significant difference(P>0.05).There was no significant difference of p-AMPK, AMPK,Nrf2,HO-1 protein expression in Tax+CPF group(P>0.05).Conclusion(1)CPF has toxic effect on BV2 cells,which is characterized by the BV2 cell body becomes round,the cell synapse is atrophic,and the adhesion is poor,and the cell aggregation is floating.(2)Tax can improve the BV2 cell activity,and it has a protective effect on CPF induced neurotoxicity.Tax downregulation of ROS,TNF-?,IFN-? and p62 level and increase the level of LC3 II,suggesting that Tax can reduce inflammation and oxidative stress reaction,promote autophagy,playing a protective effect on the CPF induced BV2 cells cytotoxicity.(3)Tax downregulation of p-AMPK and upregulation of Nrf2 and HO-1,suggesting that the protective mechanism of Tax on CPF induced BV2 cells is the downregulation of p-AMPK and activation of Nrf2/HO-1 signaling pathway.