Study Of The Nrf2 Pathway On The Blood Cerebrospinal Fluid Barrier Dysfunction Induced By Lanthanum

Objectives In this study,rat choroid plexus epithelial cell line-Z310 cells were used for establishing the blood cerebrospinal fluid barrier（BCSFB）in vitro.Meanwhile,a variety of experimental means including biochemistry,cell and molecular biology etc.were employed to explore the key role of the nuclear factor erythroid 2-related factor 2（Nrf2）pathway in resisting oxidative stress and maintaining the function of BCSFB under lanthanum exposure.The finding will provide a novel theoretical basis for the study of the role of lanthanum in disrupting the brain barrier system leading to central nervous system dysfunction.Methods 1 Z310 cells were treated with 0（control）,0.125,0.25,0.5 mmol/L lanthanum chloride（LaCl₃）and cotreated with 40μmol/L the Nrf2 activator tert-butylhydroquinone（tBHQ）.2 A CCK-8 kit and a cell necrosis test kit were used to detect the viability rate of Z310 cells and the necrosis rate,respectively.And the morphological changes of Z310cells were observed with inverted microscope and nucleus staining assay.3 Z310 cells were used to construct the BCSFB culture model in vitro.Changes of the transepithelial electrical resistance（TEER）of BCSFB mode in vitro were detected with an epithelial volt/Ωmeter EVOM2.4 Flow cytometry were used to detect the changes of reactive oxygen species（ROS）,and the biochemical methods were applied to detect the changes of CAT and GSH-Px activities.5 The proteins distribution of Nrf2,xCT,occludin,etc.in Z310 cells were detected by immunofluorescence assay.6 Western blot was used for measuring the proteins expression of Nrf2,NQO1,occludin,etc.and the inflammatory factor expression of IL-1βand TNF-αin Z310 cells.7 Real time-PCR was employed for detecting the mRNA expression of Nrf2 and its downstream proteins including Nqo1,Ho-1.Results 1 Effect of oxidative stress on the BCSFB mode dysfunction in vitro induced by lanthanum:1)Compared with the control group,the TEER of BCSFB mode in vitro gradually reduced,with the differences being statistically significant in the LaCl₃-treated groups,in which the transepithelial cell resistance in the 0.5 mmol/L LaCl₃-treated group was reduced by 62.84%.Compared with the control group,the viability rate of Z310 cells evidently decreased,the necrosis rate increased and the cell morphology changed after LaCl₃ treatment.2)Effect of LaCl₃ treatment on the antioxidant capacity of Z310 cells:Compared with the control group,the level of ROS in Z310 cells markedly increased as the LaCl₃ concentration increased;the antioxidant enzymes CAT and GSH-Px activities in Z310 cells were reduced in the LaCl₃-treated groups,in which CAT and GSH-Px activities in the 0.5 mmol/L LaCl₃-treated group decreased by 56.02%and 72.04%,respectively.3)Compared with the control group,the mRNA and proteins expression of Nrf2,NQO1 and HO-1 were significantly decreased after treatment with different concentrations of LaCl₃（P<0.05）.2 The effect of tBHQ antagonize lanthanum-induced oxidative damage in Z310cells:1)Compared with the LaCl₃-treated groups,the expression of Nrf2 in Z310 cells was significantly increased in the tBHQ prevention groups,which in the tBHQ+0.5 mmol/L LaCl₃-treated group was 2.54 folds higher than that in the 0.5 mmol/L LaCl₃-treated group;in addition,Nrf2 downstream proteins such as HO-1,NQO1,xCT,etc.were increased,with the differences being statistically significant.2)Compared with the LaCl₃-treated groups,the viability of Z310 cells was increased,the necrosis rate decreased after the tBHQ prevention.And compared with the LaCl₃-treated groups,the TEER of BCSFB in vitro was markedly increased in tBHQ prevention groups,in which the tBHQ+0.5 mmol/L LaCl₃-treated group was 26.93%higher than that in the 0.5 mmol/L LaCl₃-treated group.3The role of the Nrf2 pathway on the expression changes of tight junction proteins of BCSFB induced by lanthanum:1)Compared with the LaCl₃-treated groups,Nrf2 was significantly activated in tBHQ prevention groups,and the level of ROS was markedly reduced（P<0.05）.2)Compared with the control group,the expression of MMP9 protein in Z310 cells was observably increased,the expression of TIMP1 protein was reduced,while tight junction proteins such as occludin,ZO-1,JAM1,etc.were significantly decreased in LaCl₃-treated groups（P<0.05）.3)Nrf2 activation could reduced the increase of MMP9 protein levels induced by LaCl₃in Z310 cells,and tight junction proteins including occludin,ZO-1,JAM1,etc.were significantly increased.Conclusions Lanthanum can downregulate the expression of Nrf2 pathway,thus decreased the antioxidant capacity of Z310 cells along with the increase of ROS level,which caused Z310 cells damaged.Besides,lanthanum increased the expression of MMP9following the excessive degradation of the tight junction proteins including occludin,ZO-1,JAM1,etc..Ultimately,TEER was decreased,which can be considered that La can cause BCSFB mode dysfunction.Figure 32;Table 6;Reference 162...