The Role Of Wnt/β-catenin And Endoplasmic Reticulum Stress Pathway In PFOS-induced L02 Cells Injury And Relevant Mechanism

Perfluorooctane sulfonic acid(PFOS)is a C8 fluorocarbon compound composed of perfluorinated and polyfluorinated alkyl substances.PFOS is chemically stable,and is widely present in various environmental media such as land and water.PFOS can also enter the organism through a variety of pathways and accumulate through the food chain.In addition,PFOS is carried by bile into the intestine and then transported back to the liver via the enterohepatic circulation,which may be one of the reasons for the high PFOS concentration in the liver.PFOS entering the organism is preferentially distributed and accumulated in the liver,and therefore the liver is the main target organ for PFOS damage.An increasing number of studies suggest that the Wnt/β-catenin signaling pathway and endoplasmic reticulum stress pathway play an important role in liver injury.In this study,human normal hepatocytes(L02 cells)were used to investigate the role of Wnt/β-catenin pathway and endoplasmic reticulum stress pathway in PFOS-induced L02 cell injury,and what is the significance.Objective:To clarify the effect of PFOS on L02 cell injury and the mechanism of Wnt/β-catenin signaling pathway and endoplasmic reticulum stress pathway in PFOS-induced L02 cell injury.Method:Cell culture and grouping: Human normal liver cell line(L02 cells)was cultured in vitro in RPMI 1640 medium containing 10% fetal bovine serum.CCK-8 method to detect cell survival,CCK-8 method to determine the concentration and duration of action of PFOS and DKK1.The experimental grouping and treatment were: The experiment was divided into four groups: Control Group(DMSO Group),PFOS group,PFOS + DKK1 group,PFOS + 4-PBA group.The control group L02 cells was treated with 0.1% DMSO for 12 h,DMSO group;the PFOS group L02 cells was treated with 200 μM PFOS for 12 h;the PFOS+DKK1(Wnt pathway inhibitor)group L02 cells was pretreated with 50 n M Dickkopf-related protein 1(DKK1)for 2 h and 200 μM PFOS for 12 h.The PFOS+4-PBA(endoplasmic reticulum stress inhibitor)group L02 cells was pretreated with 2 m M4-phenylbutyric acid(4-PBA)for 2 h,followed by 200 μM PFOS for 12 h.Cell morphology was observed by inverted microscopy;apoptosis was observed by DAPI staining;cell ROS production and apoptosis levels were detected by flow cytometry;Wnt pathway-related proteins(Wnt1,β-catenin,GSK3β),endoplasmic reticulum stress-related proteins(GRP78,PERK,ATF6),apoptosis-related proteins(Bax,Caspase3,Caspase12)expression.The experimental data were analyzed by IBM SPSS 24.0,and the normal distributed data were expressed as x±s.The analysis of variance(One-Way ANOVA)method was used to compare the differences between multiple groups,and the LSD method was used for the comparison between two groups.The test standard was set at α = 0.05.Results:1.Effect of PFOS on cell viabilityThe cell survival rate decreased significantly with the increase of PFOS concentration.the cell survival rate was 57.07±1.48% at the PFOS concentration of 200μM,which was statistically different from all other groups(P<0.05).In this experiment,200 μM was chosen as the exposure concentration of PFOS.2.Morphological changes of each group of cellsIn the DMSO group,the cell outline was clear,the morphology was regular,round or oval,the cell membrane rupture was less,the cell density was uniform,the living cells density did not change apparently,and the growth was good.In the PFOS+4-PBA group,the cell outline was blurred,some cells had cell membrane rupture,and the morphology changed to elongated shape;compared with the PFOS group,tthe living cells density did not change apparently.3.Occurrence of apoptosis in each groupIn the DMSO group,after DAPI staining,the cells adhered well,the cell membrane was intact and round or ovoid,the nuclei were evenly stained,and the cell density was uniform.Compared with the DMSO group,the adherent L02 cells in the PFOS group was reduced,the L02 cell density was greatly decreased,the cell gap became larger,the cell membrane was damaged,the L02 cell nuclear fragmentation was serious,and the apoptotic features were obvious,and apoptotic vesicles were visible.Compared with the PFOS group,cells in the PFOS+4-PBA and PFOS+DKK1 groups had partially damaged cells,significantly improved nuclear fragmentation and reduced number of apoptotic vesicles.4.Levels of ROS production in each group of cellsCells in the PFOS group produced significantly higher levels of ROS compared with the DMSO group(P < 0.05);cells in the PFOS+4-PBA and PFOS+DKK1 groups produced significantly lower levels of ROS compared with the PFOS group(P < 0.05).5.Wnt/β-catenin pathway-related protein expression levelsWnt1,β-Catenin,and GSK3β proteins expression levels were significantly higher in cells of the PFOS group compared with the DMSO group(P < 0.05).Compared with the PFOS group,the expression levels of Wnt1 and GSK3β proteins in cells of the PFOS+DKK1 and PFOS+4-PBA groups were significantly lower(P < 0.05).β-Catenin protein expression levels were not found to be statistically significant between the PFOS group,PFOS+DKK1 group and PFOS+4-PBA group.differences.6.Expression levels of proteins related to the endoplasmic reticulum stress pathwayThe expression levels of GRP78 and PERK proteins were significantly higher in cells of PFOS group compared with DMSO group,and the differences were statistically significant(P < 0.05).The expression levels of GRP78 and PERK proteins in cells of PFOS+DKK1 and PFOS+4-PBA groups were lower than the PFOS group(P < 0.05).The expression level of ATF6 protein was decreased in the PFOS group compared with the DMSO group(P < 0.05).Compared with the PFOS group.The PFOS+4-PBA group and PFOS+DKK1 group the ATF6 protein expression levels have no statistically differences.7.Apoptosis and apoptosis-related protein expression levelsThe level of apoptosis was significantly higher in the PFOS group compared with the DMSO group(P < 0.05).The levels of apoptosis were significantly lower in the PFOS+4-PBA group and PFOS+DKK1 group compared with the PFOS group(P < 0.05).The expression levels of Bax,Caspase3 and Caspase12 proteins were significantly higher in the PFOS group compared with the DMSO group(P < 0.05).The expression levels of Bax,Caspase3 and Caspase12 proteins were significantly lower in the PFOS+DKK1 group and PFOS+4-PBA group compared with the PFOS group(P < 0.05).Conclusion:1.PFOS can reduce the survival rate of L02 cells,disrupt cell morphology,lead to oxidative stress and increase the level of apoptosis in L02 cells.2.PFOS can activate the Wnt/β-catenin signaling pathway in L02 cells,and inhibition of this signaling pathway can alleviate PFOS-induced oxidative stress and apoptosis,thus alleviating cell injury.3.PFOS can lead to endoplasmic reticulum stress in L02 cells,and the endoplasmic reticulum stress inhibition will ease oxidative stress and apoptosis which is induced by PFOS,thus alleviate cell injury.4.PFOS can lead to apoptosis in L02 cells by a mechanism that may be mediated by endoplasmic reticulum stress.