| Objective:To explore the role of miR-134/BDNF/TrkB/CREB pathway in the depressionlike behavior induced by B[a]P in mice using animal experiment and cell culture,and to provide a scientific basis for the study on the mechanism of B[a]P's neurotoxicity.Methods:In vivo: Forty adult male SPF ICR mice were randomly divided into 4 groups based on their body weights,including the solvent(peanut oil)control group,B[a]P treated groups at three doses of low(0.5 mg/kg),medium(2.0 mg/kg)and high(10.0 mg/kg),10 mice per group.The mice were intraperitoneally injected with B[a]P solution for 30 times,once every two day.The solvent control group was injected with the same volume of peanut oil(2.5 mL/kg).At the end of the treatment,mice were tested the depression-like behavior using sucrose preference test,emotional behavior score,hole plate experiment and forced swimming test.The cerebral cortical tissue of mice were sectioned,stained with hematoxylin-eosin(HE),and observed the morphological changes under a microscope.Cortical neuronal apoptosis was detected using the flow cytometry(Annexin V-FITC/PI staining).Gene levels of miR-10 b,miR-124,miR-134,miR-155 and BDNF were determined using the real-time fluorescent quantitative polymerase chain reaction(QPCR)in the cerebral cortex of mice.The cortical protein levels of BDNF,TrkB,p-TrkB,CREB,p-CREB in mice were determined using Western blotting methods.In vitro: Hippocampal neuron HT22 cells derived from mice were cultured in DMEM complete medium containing 10% fetal bovine serum.The same batch of logarithmic growth phase HT22 cells(about 80% of adherent area)were randomly divided into 4 groups,including solvent control group,B[a]P treated groups at doses of low(0.2 ?M),medium(2.0 ?M),and high(20 ?M).The medium was replaced with low serum(3%)and treated with different doses of B[a]P for 48 hours.The solvent group was only treated with DMSO(0.5% DMSO).At the end of the treatment,cells were observed the morphological changes under an inverted microscope,measured the cell viability by CCK-8,and detected the leakage rate of lactate dehydrogenase(LDH)using a microplate reader and the neuronal apoptosis using flow cytometry(Annexin V-FITC/PI).We sequenced miRNA profile using small RNA resequencing,and verified the differential miR-134 expression using QPCR.In order to verify whether miR-134 regulates the BDNF/TrkB/CREB pathway in the neuronal damage following B[a]P treatment in HT22 cells,we co-treated neurons with 20?M B[a]P and miR-134 inhibitor(100 nM)for 48 h.Then we observed the morphological changes,detected the cell viability and neuronal apoptosis using the above mentioned methods,and determined the protein expressions of BDNF,TrkB,pTrkB,CREB and p-CREB proteins using Western Blotting.Result:1.In vivo,the depression-like behavior of mice was significantly increased with the increasing dose of B[a]P,as demonstrated by the significant decreased sucrose partiality in the high-dose group,the decreased emotional behavior score in all treated groups,and the decreased frequency and duration of the Cave in high-dose group compared to the solvent control group(P < 0.05).The cortical neurons were swollen,degenerated,and necrotic in the B[a]P treated groups,the damage severity were dependently on the doses of B[a]P.The neuronal early apoptosis rate in cerebral cortex was significantly increased in all the treated groups compare to the solvent control group(P < 0.05).Compared to the solvent control group,miR-10 b gene levels was significantly increased in the middle-dose and high-dose B[a]P groups(P < 0.05),miR-124,miR-134 and miR-155 were significantly higher in the high-dose B[a]P group.Pearson correlation analysis showed that the neuronal apoptosis was positively correlated with the levels of miR-10 b,miR-124,miR-134 and miR-155 in the cerebral cortex in mice,and the correlation coefficients were 0.59,0.80,0.79 and 0.76,respectively.Compared to the solvent control group,the levels of BDNF mRNA in all the treated groups were significantly decreased,and the expressions of BDNF protein were significantly decreased in the middle and high-dose B[a]P groups(P < 0.05),the expressions of TrkB and p-TrkB protein in each exposed group were decreased(P < 0.05);the expression of CREB protein was decreased in the middle and high-dose B[a]P groups(P < 0.05),and the expression of p-CREB protein in each exposed group was significantly decreased(P < 0.05).2.In vitro,the severity of cell damage was significantly increased with the increasing dose of B[a]P,the number of cells were decreased,the fragments were increased,cells congregated into groups,synapses became thicker and shorter,and cell junctions was broken.Compared to the solvent control group,the cell viability were decreased in all treated group(P < 0.05),and the LDH leakage rate in the supernatant were significantly increased in the medium and high-dose B[a]P groups(P < 0.05).The early apoptotic rate of cells increased significantly in all treated groups(P < 0.05).Compared to the solvent control group,there are 125,61 and 56 miRNAs were upregulated,and there are 360,73 and 112 miRNAs were down-regulated,respectively.There were 151,39 and 45 differentially expressed microRNAs targeting BDNF,respectively.Among them,miR-10 b was decreased in all B[a]P treated groups,miR-124 were decreased in the low-dose and high-dose B[a]P groups,and it's increased in the middle-dose B[a]P treated group,miR-134 were increased in all B[a]P treated groups,miR-155 were decreased in all B[a]P treated groups.Compared to the solvent control group,the level of miR-134 was decreased in the low-dose B[a]P group(P > 0.05),and the levels were increased in the middle-dose and high-dose B[a]P treated groups(P < 0.05),which was consistent with the in vivo results,therefore,miRNA-134 is the target of further study in vitro.3.Intervention experiment results showed that,compared to the solvent control group,the expressions of BDNF,TrkB,p-TrkB,and p-CREB protein were significantly decreased in the B[a]P treated group(P < 0.05),the expressions of CREB were no significantly changed(P > 0.05).Compared to the B[a]P treated group,intervened with inhibitors of miR-134 improved the morphology of cells,the viability of cells were significantly increased(P < 0.05),the early apoptotic rates were decreased(P < 0.05),and the expressions of BDNF,TrkB,p-TrkB and p-CREB protein were significantly increased(P < 0.05),while no significant difference was found in CREB of neurons pretreated with miR-134 inhibitor compared to the only B[a]P treated group(P > 0.05).Conclusion:B[a]P can cause the depression-like behavior and impaired the neuron cells in the cerebral cortex in mice,and the miR-134/BDNF/TrkB/CREB pathway is possibly the potential mechanism underlying the neuronal impairment induced by B[a]P. |
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