Yi-Nao-Jie-Yu Prescription Relieves Post-Stroke Depression by Mitigating Ferroptosis in Hippocampal Neurons Via Activating the Nrf2/GPX4/SLC7A11 Pathway

Abstract

Post-stroke depression (PSD) poses a serious impact on patients' life quality. Effective drugs to treat this annoying disease are still being sought. Yi-nao-jie-yu (YNJY) prescription has been found to relieve PSD; however, the underlying mechanisms remain unelucidated. This work elucidated the therapeutic effects and mechanisms underlying YNJY prescription in PSD. PSD rat model was treated with YNJY prescription and ML385. Depression-like behaviors of rats was appraised. Hematoxylin-eosin, Nissl, and NeuN immunofluorescence staining were performed to observe hippocampal neuronal damage. Transmission electron microscopy was used to assess hippocampal mitochondrial damage. Commercial kits and western blotting were adopted to research ferroptosis-related factors and Nrf2/GPX4/SLC7A11 signals. In vitro experiments were performed using rat hippocampal neurons to explore the mechanism by which YNJY prescription relieves PSD. In PSD rats, YNJY prescription relieved depression-like behaviors, attenuated hippocampal neuronal damage, mitigated hippocampal ferroptosis and mitochondrial damage, and activated hippocampal Nrf2/GPX4/SLC7A11 pathway. By in vitro experiments, erastin treatment exacerbated hippocampal neuronal damage, ferroptosis, mitochondrial damage, and lipid peroxidation; however, YNJY prescription abolished these erastin-induced effects. In the erastin-treated hippocampal neuronal model of PSD, ML385 treatment increased ferroptosis, hippocampal neuronal damage, and lipid peroxidation; however, YNJY prescription counteracted these ML385-induced effects. By in vivo study, ML385 reversed the relief of YNJY prescription on depressive-like behaviors of PSD rats, and the inhibition on ferroptosis in PSD rats' hippocampus. YNJY prescription relieves PSD by blocking ferroptosis via activating the Nrf2/GPX4/SLC7A11 pathway. It may be a promising agent for treating PSD clinically.

Keywords: Ferroptosis; Hippocampal neurons; Nrf2/GPX4/SLC7A11; Post-stroke depression; Yi-nao-jie-yu prescription.

Fig. 1

YNJY prescription relieved the depression-like behaviors of rats with PSD. (A) Design of the animal experiment. (B) Body weight of the rats at the end of the experiment. N = 10 per group. (C) Effect of YNJY prescription on the sucrose preference of rats in the sucrose preference test. N = 10 per group. (D) Effect of YNJY prescription on the immobility time of rats among the five groups in TST. N = 10 per group. (E) Effect of YNJY prescription on the immobility time of rats in FST. N = 10 per group. (F and G) Effect of YNJY prescription on the rearing and crossing counts of rats in OFT. N = 10 per group. * P < 0.05, ** P < 0.01, *** P < 0.001, and ***** P < 0.0001. The symbol “ns” indicated that the difference was not statistically significant

Fig. 2

YNJY prescription weakened neuronal damage in the hippocampus of rats with PSD. (A) Hematoxylin and eosin staining demonstrating hippocampal damage in the rats in the five groups. The red arrows indicated the damaged neurons. N = 3 per group. (B) Nissl staining showing hippocampal neuron loss in the rats in the five groups. The red arrows indicated the damaged neurons. N = 3 per group. (C) NeuN immunofluorescence staining showing hippocampal neuronal damage in the rats in the five groups. N = 3 per group. * P < 0.05 and ** P < 0.01. The symbol “ns” indicated that the difference was not statistically significant

Fig. 3

YNJY prescription attenuated ferroptosis in the hippocampus of rats with PSD. (A–E) Levels of ferroptosis-related factors in rat hippocampus tested using commercial kits. N = 6 per group. (F) TEM showing mitochondrial damage in rat hippocampus. N = 6 per group.* P < 0.05 and ** P < 0.01

Fig. 4

YNJY prescription activated the Nrf2/GPX4/SLC7A11 pathway in the hippocampus of rats with PSD. (A–D) Western blotting detecting the protein levels of Nrf2, GPX4, and SLC7A11 in rat hippocampus. N = 3 per group. (E–G) qRT-PCR showing the expression of mRNA for Nrf2, GPX4, and SLC7A11 in rat hippocampus. N = 3 per group. (H and I) NeuN/Nrf2 immunofluorescence staining of rat hippocampus. N = 3 per group. * P < 0.05 and ** P < 0.01

Fig. 5

YNJY prescription counteracted erastin-induced hippocampal neuronal damage and ferroptosis. (A) NeuN/MAP-2 immunofluorescence staining showing the hippocampal neurons isolated from rats. (B and C) CCK-8 assay showing the safe and effective concentration of the YNJY prescription. N = 6 per group. (D) LDH activity detection in the hippocampal neurons using a commercial kit. N = 3 per group. (E) TUNEL staining showing the apoptosis of hippocampal neurons. N = 3 per group. (F–I) Levels of ferroptosis-related factors were determined using commercial kits. N = 3 per group. (J) TEM showing mitochondrial damage in the hippocampal neurons. N = 3 per group. * P < 0.05 and ** P < 0.01

Fig. 6

YNJY prescription reversed erastin-induced mitochondrial damage. (A and B) JC-1 staining of the hippocampal neurons to evaluate mitochondrial membrane potential. N = 3 per group. (C and D) C11-BODIPY staining of the hippocampal neurons to evaluate lipid peroxidation. N = 3 per group. (E and F) Mito-Tracker Red staining of the hippocampal neurons to investigate mitochondria length. N = 3 per group. ** P < 0.01. The symbol “ns” indicated that the difference was not statistically significant

Fig. 7

YNJY prescription mitigated erastin-induced ferroptosis in the hippocampal neurons by activating the Nrf2/GPX4/SLC7A11 pathway. (A) CCK-8 assay measuring the viability of the hippocampal neurons in each group. N = 5 per group. (B–E) Levels of the ferroptosis-related factors in the hippocampal neurons of each group were detected using commercial kits. N = 3 per group. (F–I) Western blotting showing the activity of the Nrf2/GPX4/SLC7A11 pathway in the hippocampal neurons of each group. N = 3 per group. (J and K) C11-BODIPY staining of the hippocampal neurons in each group to explore the degree of lipid peroxidation. N = 3 per group. * P < 0.05 and ** P < 0.01

Fig. 8

YNJY prescription might mitigate the depression-like behaviors of PSD rats by alleviating hippocampal ferroptosis via activating the Nrf2/GPX4/SLC7A11 pathway. (A) Sucrose preference of rats to appraise the depression-like behaviors. (B) TST of rats to observe the immobility time. N = 10 per group. (C) FST of rats to monitor the immobility time. N = 10 per group. (D-E) OFT of rats to count rearing and crossing counts. N = 10 per group. (F-I) The contents of ferroptosis-related factors in the hippocampus of rats were detected by commercial kits. N = 6 per group.* P < 0.05 and ** P < 0.01. The symbol “ns” indicated that the difference was not statistically significant