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Background Benzo[a]pyrene (BaP) has neurotoxicity, which can induce the loss of hippocampal neurons in humans and animals and lead to spatial learning and memory dysfunction, but its mechanism is still unclear. Objective To observe the ferroptosis of mouse hippocampal neuron HT22 cells induced by 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), an active metabolite of BaP, and to explore its potential mechanism, so as to provide a basis for the study of BaP neurotoxicity mechanism. Method Mouse hippocampal neuron HT22 cells were selected and divided into four groups: solvent control group and low, medium, and high concentration BPDE exposure groups (0.25, 0.50, and 0.75 μmol·L−1). Cell survival was detected by CCK8 method. Cell morphology and ultrastructure were observed under light and electron microscopes. The levels of reactive oxygen species (ROS) and Fe2+ were detected by fluorescence probe method. Iron, 4-hydroxynonenoic acid (4-HNE), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) levels were detected with commercial kits. The expression levels of acyl-CoA synthase long chain family member 4 (ACSL4), cyclooxygenase 2 (COX2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were detected by Western blotting. After interventions with ferroptosis inhibitors 20 μmol·L−1 deferoxamine (DFO) and 10 μmol·L−1 ethyl 3-amino-4-cyclohexylaminobenzoate (Fer-1), the cell survival rate of each BPDE exposure group and the changes of the ferroptosis characteristic indicators and protein expression levels were observed. Results With the increase of BPDE concentration, the survival rate of HT22 cells decreased gradually, and the survival rate of each BPDE group was significantly lower than that of the solvent control group (P<0.01). Under light microscope, the number of cells in the high concentration BPDE group was significantly reduced, and atrophic cells and reduced synapses were recorded. Under electron microscope, the HT22 cells in the high concentration BPDE group showed mitochondrial shrinkage, decreased crista, and increased mitochondrial membrane density. Compared with the solvent control group, the levels of intracellular lipid ROS, Fe2+, 4-HNE, and MDA significantly increased in the high concentration group (P<0.01), the GSH and GSH-PX levels were significantly decreased (P<0.01), the protein expression levels of ASCL4 and COX2 were significantly increased (P<0.01), and the protein expression levels of SCL7A11 and GPX4 were significantly decreased (P<0.01). The ferroptosis inhibitors DFO and Fer-1 significantly reversed the cell survival rate (P<0.01), the ferroptosis characteristic indicators (ROS, Fe2+, 4-HNE, MDA, GSH, and GSH-PX levels) (P<0.01), and the expression levels of ferroptosis-related proteins (ACSL4, COX2, SLC7A11, and GPX4) (P<0.01) in the high concentration BPDE group. Conclusion BPDE can induce ferroptosis in mouse hippocampal neuron HT22 cells, which may be related to the inhibition of SLC7A11/GSH/GPX4 axis and the induction of iron metabolism disorder.
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Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of β amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aβ25-35 induction(Aβ) group, hepcidin-siRNA(siRNA) group, Aβ25-35 + hepcidin-siRNA(Aβ + siRNA) group, Aβ25-35+YHG(Aβ+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aβ25-35+hepcidin-siRNA+YHG(Aβ+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aβ group, siRNA group, and Aβ+siRNA group than in the control group(P<0.05) and the expression was lower in the Aβ+siRNA group(P<0.05) and higher in the Aβ+YHG group(P<0.05) than in the Aβ group. Moreover, the ADAM17 protein expression was lower in the Aβ+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aβ+siRNA+YHG group than in the Aβ+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.
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Humans , ADAM17 Protein , Alzheimer Disease/genetics , Amyloid beta-Peptides , Drugs, Chinese Herbal/pharmacology , Hepcidins/genetics , PuerariaABSTRACT
Objective: To detect ceramide(Cer) signaling pathway-related proteins expression levels in HT-22 with Gandou decoction (GDD), in order to explore its molecular targets and mechanism in regulating Cer signaling pathway. Method: The experiment was divided into normal group (normal HT-22 cultured by 10%blank rabbit serum), model group (HT-22 cells incubated with CuSO4), and GDD group (HT-22 cells incubated with CuSO4, continuously cultured by rabbit serum containing10%, 15%, 20%GDD). HT-22 cells were incubated with different concentrations of CuSO4.The cell growth and proliferation were assessed using methyl thiazolyl tetrazolium(MTT) method; flow cytometry was used to analyze the expression of reactive oxygen species (ROS); Western blot was used to detect relevant protein expression of Cer signaling pathway. Result: The results of MTT showed that CuSO4 inhibited the growth and proliferation of HT-22 cells in a time and concentration-dependent manner; flow cytometry results showed that the model group increased the release of ROS compared with the normal group (PPPPConclusion: High copper can induce oxidative stress and deactivate Cer signaling pathway, which led to hippocampal neuron injury. These findings suggest that GDD reduces neurotoxicity induced by copper overload by increasing the copper excretion that inhibits the expressions of ASM, Cer, p38 MAPK, Cyt C, Caspase-9, Caspase-3.GDD reduces neurotoxicity induced by copper overload by decreasing copper levels in brain and then regulating Cer signaling pathway.
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OBJECTIVE To explore whether different concentrations of potassium bisperoxo (1,10-phenanthroline) oxovanadate [BPV (phen)) affect cell cycle by regulating the expression of DNA meth-yltranferases (DNMT) and cell cycle-related genes. METHODS HT22 cells were treated with BPV (phen) 0.3 and 3 μ mol • L-1 for 24 h. The cell viability was detected by MTS assay, cell cycle was detected by flow cytometry, the activity of DNMT was detected by ELISA, the mRNA expressions of p21, DNMT1, DNMT3A and DNMT3B were measured with real-time quantitative PCR, while the levels of corresponding proteins were measured by Western blotting. RESULTS Compared with the DMSO control group, BPV(phen) 0.3 μ mol• L-1 did not affect cell viability, but the cell viability of BPV(phen) 3 pmol-L-1 increased signifi-cantly (P<0.05). There was no significant difference in cell cycle between DMSO control and BPV(phen) 0.3 nmol-L-1 group, but the proportion of cells in S phase increased((76.0±1.6)%)(P<0.05) and in G2 phase decreased ((2.1 ±1.5)%) (P<0.05) of BPV(phen) 3.0 μ mol • L-1 group. The intracellular DNMT activity of BPV(phen) 3.0 Mmol • L-1 group was significantly increased compared with the DMSO control group (F<0.05). There was no significant difference in the mRNA expressions of p21, DNMT1, DNMT3A and DNMT3B between DMSO control and BPV(phen) 0.3 μ mol • L-1 group, but all increased in BPV(phen) 3.0 μ mol • L-1 group (P<0.05, P<0.01). Western blotting results showed that there was no significant difference in the protein expressions of P21, DNMT1, DNMT3A and DNMT3B between DMSO control and BPV (phen) 0.3 μ mol • L-1 group, but only the protein expressions of DNMT3B and P21 of BPV (phen) 3.0 μ mol • L-1 group increased significantly (F<0.05). CONCLUSION BPV(phen) can regulate the expression of downstream and cell cycle-related genes by altering the expression of DNMTs, which in turn affects the growth and proliferation of HT22 cells.