Nonylphenol Induced Anxiety Behavior In Rats And Regulated BDNF-TrkB Signaling Network

Part Ⅰ Nonylphenol chronic exposure-induced anxiety behavior in rats and its effect on the expression of BDNF/TrkBObjective: To determine whether anxiety-like behavior can be induced after chronic NP-exposure,and NP’s effects on BDNF-Trk B-signaling-network in hippocampus of rats.Methods: 40 male 28-day-old Sprague-Dawley rats were randomly assigned into four groups: control group（C,corn oil）,low group（L-NP 0.4 mg/kg）,medium group（M-NP4mg/kg）and high group（H-NP 40mg/kg）,and 10 rats in each group.At 90th and 180th days after NP exposure,the anxiety-like performances of SD rats was detected by elevated cross maze together with light-dark box;High performance liquid chromatography（HPLC）was used to detect NP content;Morphological changes of hippocampus was observed by HE staining;The expression of BDNF in the rat`s hippocampus was detected via immunofluorescence;Western blot was perfromed to detect the protein expression levels of BDNF-Trk B signaling network（BDNF,CREB,P-CREB,P-Trk B,Trk B,PSD-95,and Synapsin1）;Reverse transcription-polymerase chain reaction was perfromed to detect the mRNA expression of BDNF/Trk B network（CREB,BDNF,Trk B,PSD-95,and Synapsin1）.Results: 1.The nonylphenol accumulation in rats` hippocampus increased with the rising of its dose（P = 0.01）.2.Compared with the corn oil group,with the increase of both exposure dose and time of NP,the residence times in the closed arm（P = 0.023）,and in the dark areas（P = 0.016）increased in NP groups.3.Nerve cells in M-NP and H-NP groups are arranged irregularly.4.The protein expressions of CREB,P-CREB,P-Trk B and Trk B decreased in H-NP group（P < 0.01）;5.Compare with the control,the mRNAs level of BDNF,CREB,Trk B,PSD-95 and Synapsin1 in the NP-exposure groups（P < 0.01）decreased.The changes of the expressions of BDNF,CREB,Trk B,PSD-95 and Synapsin1 mRNA at 6^th months were greater than those of at 3^th months after NP exposure.Conclusion: Chronic exposure to 4mg/kg NP could induce anxiety-like behavior and decrease the expression of BDNF-Trk B-network related proteins/genes in hippocampus,and the longer the NP-exposure time,the more obvious the toxicity of nonylphenol.Part Ⅱ Effects of nonylphenol on BDNF-TrkB signaling network in N2a cellsObjective: Using Silencing-BDNF-N2a cells to study the effects of nonylphenol on the signaling network of BDNF-Trk B.Methods: The N2a cell’s absorbance value was detected via CCK-8,and the IC50 and the minimum concentration of nonylphenol’s poisonic effect on the cells were valued accordingly,so as to determine the NP exposure concentration in N2a cells.The N2a cells was divided into six groups: blank,NP（NP,40 μM）,control interference vector（NC si RNA）,control interference vector + NP（NC si RNA + NP）,BDNF interference vector（BDNF si RNA）,and BDNF interference vector + NP（BDNF si RNA + NP）groups.The N2a cell apoptotic rate was detected by Flow cytometry.Immunofluorescence assay was performed to observe the BDNF expression of the cells.The proteins expression of BDNF-Trk B-signaling-network related indexs（BDNF,CREB,Trk B,PSD-95,and Synapsin1）were detected by western blot.RT-PCR was explored to detect the expression mRNA levels of BDNF-Trk B-signaling-network related indexs（BDNF,CREB,Trk B,PSD-95,and Synapsin1）.Results: 1.NP exposure inhibited the growth of N2a cells.The lowest toxic concentration of NP for N2a cells was 40 μM.2.NP-exposed N2a cells lead to shrinkage.3.There was more the number of apoptotic N2a cells in NP exposure group than blank group（P < 0.01）.4.The fluorescence expressions of BDNF decreased in the NP,NC si RNA + NP,BDNF si RNA and BDNF si RNA + NP groups compared with the control group（P < 0.01）.5.Compare with the control,the protein expression levels of BDNF,CREB,P-CREB,P-Trk B,Trk B,PSD-95 and Synapsin1 in the NP,NP +NC si RNA,BDNF si RNA and NP + BDNF si RNA groups decreased（P = 0.001）.The protein expression levels of BDNF,CREB,P-CREB,P-Trk B,Trk B,PSD-95 and Synapsin1 in the BDNF si RNA + NP group was lower than those in the BDNF si RNA group（P = 0.021）.6.Compare with the control,the expression levels of BDNF,CREB,Trk B,PSD-95 and Synapsin1 mRNA in the NP,NP +NC si RNA,BDNF si RNA and BDNF si RNA + NP groups decreased（P = 0.036）.The expression levels of BDNF,CREB,Trk B,PSD-95 and Synapsin1 mRNA in the BDNF si RNA + NP group was lower than those in the BDNF si RNA group（P = 0.048）.Conclusion: NP down-regulates PSD-95 and Synapsin1 expression in N2a cells through the activation of BDNF-Trk B signaling pathway.