ABSTRACT
Background Benzo[a]pyrene (BaP) is neurotoxic and can cause neuronal damage by oxidative stress. Proanthocyanidin (PC) has antioxidant activity, and its mechanism may related to nuclear factor-erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway. Objective To explore potential protective effect of PC on hippocampal neuron injury induced by BaP oxidative stress. Methods Hippocampal neurons of neonatal SD rats delivered within 24 h were isolated and cultured, and cell activity was detected by cell counting kit-8 (CCK-8) method. According to the pre-experimental results, a control group and three BaP groups of 10, 20 and 40 µmol·L−1 were set up for Stage I experiment. The length of neurites and number of branches of hippocampal neurons in each group were observed by immunofluorescence method. Reactive oxygen species (ROS) fluorescence probe method was used to measure ROS levels in cells. Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression of Nrf2, Kelch-like epichlorohydrin associated protein-1 (Keap1), HO-1, B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) in hippocampal neurons of each group, respectively. According to the results of Stage I experiment, three group were set up, including control group, BaP alone treatment group (BaP 20 µmol·L−1), and PC intervention group (BaP 20 µmol·L−1 + PC 2.5 µg·mL−1) for Stage II experiment, with the same protocol as Stage I. Results For Stage I experiment, compared with the control group, the 10, 20, and 40 µmol·L−1 BaP groups showed gradually shortened length of neurites [(177.60±3.49), (142.40±6.52), and (100.50±19.40) µm] (P<0.05) and decreased number of branches (8.00±1.00, 6.33±1.53, 4.33± 0.58) of hippocampal neurons (P<0.05); increased ROS production (2.38±0.33, 8.08±0.26, 9.86±0.19) (P<0.05); the qRT-PCR results showed that the mRNA expression levels of Nrf2 (0.35±0.03, 0.25±0.01, 0.13±0.03), Keap1 (0.70±0.01, 0.47±0.03, 0.15±0.02), HO-1 (0.77±0.02, 0.60±0.02, 0.32±0.01), and Bcl-2 (0.65±0.03, 0.47±0.02, 0.18±0.02) gradually decreased, and the mRNA expression level of Bax gradually increased (1.24±0.01, 2.25±0.15, 4.98±0.30) (P<0.05); the Western blotting results showed that the protein expression trends of Nrf2, Keap1, HO-1, Bcl-2, and Bax were consistent with the mRNA results. For Stage II experiment, compared with the BaP alone treatment group, the length of neurites in the PC intervention group became longer, (149.90±3.01) μm vs (202.00±4.45) μm (P<0.05), the number of branches increased, (4.67±0.58) vs (8.33±0.58) (P<0.05); the ROS production reduced, (10.81±0.63) vs (7.31±0.70) (P<0.05); the mRNA expression levels of Nrf2, Keap1, HO-1, and Bcl-2 increased (P<0.05), and the mRNA expression levels of Bax decreased (P<0.05); the Nrf2, Keap1, HO-1, and Bcl-2 protein expression levels increased (P<0.05), and Bax protein expression level decreased (P<0.05). Conclusion PC may exert neuroprotective effects by activating the Nrf2-HO-1 signaling pathway, inhibiting BaP-induced oxidative stress in neuronal cells, and reducing cytotoxicity.
ABSTRACT
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.
ABSTRACT
Objective: To investigate the neuroprotective effect of modified Yuejuwan and Ganmai Dazao Tang (YJGZ) on glutamate-induced cell injury of mouse hippocampal neuron cell line (HT22). Method: The HT22 cell was cultured aseptically, and the cell injury model was established with high concentration of glutamate acid, YJGZ aqueous extract and drug-containing serum were prepared. Then experiment was divided into normal group, model group, YJGZ drug-containing serum groups (1%, 5%, 10%), YJGZ aqueous extract group (166 mg·L-1), and nimodipine group (100 μmol·L-1). Methye thiazolye telrazlium (MTT) was used to detect the cell survival rate of each group, the level of lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the protein expression of hippocampal N-methyl-D-aspartate receptor subunit 2B protein (NR2B), cyclic adenosine response element binding protein (CREB), phosphorylated cyclic adenosine response element binding protein (p-CREB), extracellular regulated protein kinase (ERK),and phosphorylated extracellular regulated protein kinase (p-ERK). Result: As compared with normal group, the cell survival rate was significantly decreased in model group (PPPPPPPPPPConclusion: YJGZ aqueous extract has a significant protective effect on glutamate-induced HT22 cell injury.