In Vivo And In Vitro Studies On Nonylphenol Induced Inflammatory Responses In Hippocampal Microglia Of Offspring Rats And Microglial Cell Lines

Objective: Nonylphenol(NP)is a common industrial surfactants,widely used in the production of detergents and cosmetics industries.After use,it is discharged into the environment and accumulated in large quantities.NP is also an endocrine-disrupting chemical(EDC),which molecular structure is similar to the structure of estradiol(E2)in human body.It can mimic E2 and simulate estrogen receptor(ER)in the body,which interferes with the homeostasis and regulation of the body.Recently,the effects of NP exposure on the central nervous system(CNS)of the fetus and child has received increasing attention,because it may result in impairments in the CNS and may interfere with CNS development.Microglia(MG)are resident immune cells located in the CNS and are vital to CNS homeostasis and defense against exogenous chemicals and physical changes.They are very likely to become a potential target of NP.In the present study,we selected the mitogen-activated protein kinase(MAPK)signaling and ER,which are closely related to the inflammatory activation of MG.The results of this study will provide a scientific basis for the possible mechanism of NP induced inflammatory responses in hippocampal microglia of offspring rats and microglial cell lines.Materials and Methods:1.In vivo experimentsThe in vivo experiments were carried out by maternal gavage with different concentrations of NP from gestational day(GD)0 to postnatal day(PND)21 to construct the maternal NP-exposed rat model.On PND 7 and PND 21,the pups were analyzed.The levels of pro-inflammatory cytokines in the hippocampus of pups were detected by ELISA.MG in hippocampus was observed by immunofluorescence.The changes of Akt/MAPK/AP-1 signaling pathways were analyzed by Western blot and immunofluorescence.2.In vitro experimentsWe established NP exposure model in BV2 microglial cell line.MTS was used to detect BV2 cell tolerance to NP.Light microscopy was used to observe changes in BV2 cells after NP exposure.ELISA was used to detect the pro-inflammatory cytokines in culture supernatants.The changes of ER/Akt/MAPK signaling were detected by Western blot and immunofluorescence.The binding activity of AP-1 to DNA was detected by EMSA.After pretreatment with ER-specific antagonists in BV2 cells,ELISA was used to detect changes in pro-inflammatory cytokines in culture supernatants.Results:1.Effects of Maternal Exposure to NP during Pregnancy and Lactation on Body Weights of DamsOn GD 14,the mean weights of dams for the 10,50,and 100 mg/kg groups were 99.5%,96.1%,and 91.2% of the control group,respectively.On GD 21,the mean weights of these dams were 98.2%,95.8%,and 89.9% of the control group,respectively.The survival of the control,10,50,and 100 mg/kg groups were 86.7%,81.3%,77.8%,and 73.3%.The above results indicated that NP did not exert obvious toxicity to the body weights of dams.2.Effects of Maternal Exposure to NP during Pregnancy and Lactation on Body Weights of OffspringWe did not observe any treatment-related effects on the body weights or hippocampus weights of pups subjected to maternal NP exposure during pregnancy and lactation.The above results indicated that NP did not exert obvious toxicity to the body weights of pups and weights of offspring hippocampus.3.Maternal Exposure to NP Induced Pro-Inflammatory Cytokine Production in the Hippocampus of PupsThe level of TNF-α was significantly increased on PND 7 and 21 in the 10 mg/kg NP group.The level of TNF-α in the 50 mg/kg NP group was significantly increased on PND 7.IL-1β production exhibited a similar pattern to that of TNF-α on PND 7 and 21.The level of secreted IL-6 remained stable on PND 7,but on PND 21,peak levels were achieved when dams were treated with a dose of 10 mg/kg NP.Secreted IL-6 remained 1.9-fold higher than control at 50 mg/kg NP.No significant differences in TNF-α,IL-1β,and IL-6 were observed in the 100 mg/kg NP group over all time points and groups.4.Maternal Exposure to NP Increased Microglia and Akt Activation in the Hippocampus of PupsBy analyzing CD11 b immunoreactive cells in offspring hippocampus,we found the number of MG was significantly increased to 3.8-and 3.3-fold of control when dams were treated with the 10 mg/kg dose of NP on PND 7 and 21.In the 50 mg/kg NP group,the number of MG in offspring hippocampus was 3.5-and 2.4-fold of control on PND 7 and 21.These results suggest that maternal exposure to NP increases the number of MG in offspring hippocampus.Our results showed that maternal exposure to 10 mg/kg NP significantly increased p-Akt levels in the hippocampus on PND 7 and 21.Importantly,the strong immunofluorescence staining of p-Akt was found to co-localize with the CD11 b,a MG marker.Western blotting results also confirmed that maternal exposure to 10 mg/kg NP elevated the level of p-Akt in offspring hippocampus significantly,but the expression of total Akt remained stable.5.Maternal Exposure to NP Increased MAPK Signaling Activation in Hippocampus of PupsOn PND 7,our results indicate the level of p-p38 started to increase when dams received either 10 or 50 mg/kg NP.The level of p-p38 was significantly increased when dams received 100 mg/kg NP dose(2.2-fold).The level of p-p38 was significantly increased on PND 21 when dams received either 10 or 50 mg/kg NP(3.0-and 2.5-fold,respectively).The expression of p-JNK was highest when dams received a dose of 10 mg/kg NP on PND 7.On PND 21,the levels of p-JNK in hippocampus of pups was significantly increased when dams received either the 10 or the 50 mg/kg NP dose(2.9-and 1.9-fold,respectively);however,the levels of p38,JNK,p-ERK,and ERK remained steady on both PND 7 and 21.These results suggested that maternal NP exposure increased the levels of p-p38 and p-JNK in the offspring hippocampus.6.Maternal Exposure to NP Increased Phosphorylation of c-Jun in the Hippocampus of PupsOur data showed that the expression of c-Jun and c-Fos remained steady on both PND 7 and 21;yet,maternal exposure to 10 mg/kg NP significantly increased the phosphorylation of c-Jun(p-c-Jun)on both PND 7 and 21 in offspring hippocampus.The level of p-c-Jun was significantly increased to 1.3-and 1.4-fold of control in the 50 mg/kg NP group on PND 7 and 21.7.Effects of NP on Cell Viability of BV2 MGNP,at concentrations of 0.01-50 μM,did not noticeably affect cell viability.However,80 μM NP led to an approximately 30% reduction in cell viability compared to the control group.Furthermore,100 μM NP caused an 80% reduction in viability.To rule out interference of NP-induced cell death,we selected 30,50,and 70 μM NP for subsequent experiments.8.Effects of NP on Pro-Inflammatory Cytokine Secretion by BV2 MGAfter NP treatment,BV2 MG were in an activated state with increased cytoplasm and fewer dendritic extensions.The ELISA results showed that as the NP concentration increased,the level of secreted IL-6 reached a peak at 50 μM NP,which decreased at 70 μM but was still 3.5-fold of the control group.NP also increased IL-1β secretion significantly.In contrast,TNF-α secretion declined slightly with increasing NP concentration.9.Effects of NP on Akt Activation in BV2 MGWe found that the p-Akt level increased above the control levels following treatment with 30 or 50 μM NP but returned to a normal level when BV2 MG were treated with 70 μM NP.Western blot analysis demonstrated no significant difference in the level of Akt between the control and NP-treated groups.However,the levels of p-Akt increased with 30 μM NP,achieved a 1.6-fold peak increase with 50 μM and returned to a normal level by 70 μM.10.Effects of NP on MAPK Signaling Activation in BV2 MGResults showed that NP treatment increased p-JNK expression significantly.70 μM NP significantly increased the level of p-p38.In contrast,a 1.3-fold increase in p-p38 expression was observed with 70 μM NP.Furthermore,p-ERK expression significantly decreased with increasing NP concentration.11.Time-course Effects of NP on Akt/MAPK signaling in BV2 MGThe time-course study showed that 50 μM NP significantly increased the level of p-Akt from 0.5 to 12 h.The level of p-Akt achieved a peak at 0.5 h and 1 h,and remained at approximately 1.5-fold of the 0 h value at 12 h.The level of p-JNK reached a peak at 0.5 and 1 h,and remained at 1.7-fold of the 0 h value at 12 h.The level of p-p38 significantly increased from 0.5 to 3 h and then gradually returned to a level,which was higher than the 0 h value.At all time points(0.5-12 h),50 μM NP decreased the level of p-ERK.Downregulation of p-ERK was significant at 12 h.12.Effects of NP on AP-1 Activation in BV2 MGThe EMSA results demonstrated that this interaction was enhanced by 30 μM NP,reached its peak at 50 μM and remained higher than that of the control group at 70 μM.13.Effects of NP on ER in BV2 MGIn BV2 MG,ERα was not detected,and ERβ was stably expressed in BV2 cells.The results of immunofluorescence showed that ERβ was mainly expressed in the nucleus of BV2,and there was no significant change in the level of ERβ immunofluorescence when the dose of NP was increased.Western blot results showed that ERβ levels did not change significantly with increasing dose of NP.14.Effect of ERβ Antagonist on Secretion of IL-6 in BV2 Cells after NP ExposureAfter treatment with 50 μM NP,the level of IL-6 increased significantly to 5.3 times that of the control group.After using ERβ antagonists,the level of IL-6 in the antagonist group significantly decreased to 33% in the NP-treated group.The level of IL-6 in the ERβ antagonist group were not statistically different from that in the control group.The results showed that treatment with ERβ antagonists did not cause an increase in IL-6 but effectively inhibited NP-induced IL-6 secretion.Conclusion:1.Maternal exposure to NP during pregnancy and lactation led to increases in both activation and number of MG and production of IL-1β,IL-6,and TNF-α were observed in offspring hippocampus.2.The Akt/MAPK/AP-1 signaling was involved in this activation of MG and increased production of pro-inflammatory cytokines in pups subjected to maternal exposure to NP.3.Moderate and low concentration of NP exposure can lead to inflammatory activation and increased production of pro-inflammatory cytokines in BV2 MG.4.The Akt/MAPK/AP-1 signaling may be involved in this activation of MG and increased production of pro-inflammatory cytokines by exposure to NP in vitro.5.The ERβ may be involved in the increased production of pro-inflammatory cytokines by exposure to NP in vitro.