Carthamin yellow attenuates brain injury in a neonatal rat model of ischemic–hypoxic encephalopathy by inhibiting neuronal ferroptosis in the hippocampus

2.1 Animals

SPF-grade mother rats and neonatal Sprague–Dawley rats on postnatal day 7 (P7) were obtained from the Charles River Laboratories. The neonatal Sprague–Dawley rats were randomly divided into the Sham group, HIBD + vehicle group, and HIBD + CY group. All rats were housed in an air-conditioned room with a 12 h light/dark cycle and were provided with adequate food and water. Efforts were made to reduce the number of animals used and to minimize rats suffering.

2.2 The HIBD model and CY administration

The neonatal rat HIBD model was established based on the study conducted by Rice et al. and Gou et al. [14,15]. Postnatal day 7 (P7) rats were anesthetized with isoflurane (induction concentration: 4%, maintenance concentration: 2%), and then the left common carotid artery was exposed and incised through the middle after double ligation with 6-0 sutures. Afterward, the rats were allowed to recover in a constant-temperature dam (37.0°C) for half an hour. Subsequently, the rats were placed in a hypoxic chamber (8% O₂ + 9.2% N₂ mixture (1.5 L/min)) for 2.5 h. In the Sham group, the common carotid artery exposure was without ligation or hypoxia. CY was obtained from Sigma Aldrich (Merck KGaA). CY was administered intraperitoneally to neonatal rats once daily for 2 weeks.

2.3 Behavioral test

In this study, we used the wire hang test for behavioral tests with reference to Chang et al. and Oliván et al. [16,17] on postnatal day 21. Briefly, a wire mesh grid (15 cm × 25 cm) was used. The rats were placed on the wire mesh grid 40 cm above a foam cushion. Then, the mesh was inverted 180°, and the rats were forced to grasp the wire using their four limbs. The hanging time was recorded as the duration the rats remained hanging before falling on the cushion. This test was repeated 5 times with an interval of 3 min between trials.

2.4 Immunofluorescence staining

Paraffin sections were dewaxed to water, and tissue sections were placed in a repair box filled with the EDTA antigen repair buffer for antigen repair in a microwave oven, medium heat for 8 min to boiling, cease-fire for 8 min, and then turned to medium-low heat for 7 min and cooled naturally. Then, the slides were placed in PBS (5 min × 3 times). BSA (3%) was used for sealing; the sealing liquid was thrown off and anti-NeuN (Abcam, ab177487, 1:200) was applied. The sections were incubated overnight at 4°C in a wet box, washed with PBS (5 min × 3 times), dried, and dropped with FITC-conjugated goat anti-rabbit IgG (1:200, Thermo Fisher, America) to cover the tissues, and incubated for 50 min at room temperature with DAPI at room temperature for 10 min with PBS, and then washed with PBS for 5 minand with water for 10 min. The slices were washed with PBS (5 min × 3 times) and then an autofluorescence quencher was added for 5 min, rinsed with running water for 10 min, and sealed with an anti-fluorescence quenching sealer. The slices were observed under a fluorescence microscope (BX53, OLYMPUS, Japan) under the same conditions.

2.5 Western blotting

Neonatal rat hippocampal tissues were extracted and total protein was obtained using RIPA buffer containing PMSF. The protein concentration was determined using the BCA assay kit (Beyotime, China) on postnatal day 21. Subsequently, the protein loading buffer was added and the samples were denatured at 95°C for 10 min. After that, the proteins were isolated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to an activated PVDF membrane. Following blocking, the membrane was incubated overnight at 4°C with the appropriate concentration of primary antibody. Subsequently, the fluorescently labeled secondary antibody (IRDye700 and IRDye800, goat anti-rabbit) was incubated for 1 h at 37°C. Color rendering was performed using a chemiluminometer (Licor, America). The primary antibodies used were as follows: anti-NeuN (ab177487, abcam), anti-GPX4 (ab125066, abcam), anti-FTH1 (ybs-5907R, YOYOBIO), anti-PTGS2 (ab226869, abcam), anti-ACSL4 (PA5-27137, Invitrogen), anti-Nrf2 (PA5-27882, Invitrogen), and anti-Keap-1(ab227828, abcam).

2.6 Quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was extracted and cDNA was synthesized following the instructions of the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, America) on postnatal day 21. Real-time PCR was performed on the Applied Biosystems 7300/7500 real-time quantitative PCR instrument (Applied Biosystems, Thermo Fisher Scientific, America). GAPDH was used as an internal control. The primer sequence is shown in Table 1.

2.7 ELISA

Hippocampus tissues were homogenized in PBS, and the supernatant was collected after centrifugation on postnatal day 21. GPX4 (No. JK-E4590; Shanghai Jing Anti-Bio Co., Ltd.; Shanghai, China), FTH1 (No. CSB-EL009030MO; Huamei Bio; Wuhan, China), PTGS2 (ZY-PTGS2-Mu; Shanghai ZeYe Biotechnology Co., Ltd; Shanghai, China), ACSL4 (HB-P9S2303X; Shanghai Chemical Biotechnology Co., Ltd; Shanghai, China), GSH (Item No. E-EL-0026c; Elab Biotechnology Co., Ltd; Wuhan, China), and rat ELISA kits were used according to the guidelines of the kits. Absorbance was measured by an enzyme marker (Bio-Rad). The levels of markers were determined from the standard curve and the levels were expressed as the calculated concentration/total protein.

2.8 Detection of malondialdehyde (MDA) and superoxide dismutase (SOD)

To assess the oxidative stress, hippocampal samples were collected and examined for SOD activity and MDA concentration. SOD activity was measured using the SOD (HP-E10001, HEPENGBIO) assay kit, and MDA concentration was determined using the MDA assay kit (S0131, Beyotime Biotechnology). The analysis was conducted on postnatal day 21.

2.9 Measurement of iron concentrations

Iron concentrations of hippocampal tissues and serum were measured using an atomic absorption spectrometer (model 180–80; Hitachi; Japan) after nitric acid digestion on postnatal day 21.

2.10 Statistical analysis

Statistical analysis was performed using Prism software (GraphPad 9.0, San Diego, CA, USA) and SPSS 26.0 (IBM, USA). All data were expressed as mean ± SD. The normality of the distribution was evaluated using the Kolmogorov–Smirnov test, and statistical analyses were conducted using one-way ANOVA with Tukey’s multiple comparison test used for the statistical analyses. P < 0.05 was considered statistically significant.

1. Ethical approval: The research related to animals’ use complied with all the relevant national regulations and institutional policies for the care and use of animals. All experimental procedures and animal care were approved by the Laboratory Animal Ethics Committee of Xiangnan University and were performed in accordance with the National Institutes of Health guidelines for the care and use of animals (Ethical approval number: no. 21C1786).

3.1 CY ameliorates behavioral deficits and reduces hippocampal neuronal death in a neonatal rat HIBD model

To investigate the therapeutic effect of CY on the HIBD model, we performed a wire hang test on neonatal rats after CY treatment (on postnatal day 21). Our results demonstrated that the HIBD + vehicle group exhibited significant behavioral deficits compared with the Sham group. However, these deficits were significantly improved when CY (40 mg/kg) was administered (Figure 1c). Moreover, we examined the effect of CY on hippocampal neurons in the neonatal rat HIBD model by staining with hippocampal NeuN with immunofluorescence. We observed that the mean fluorescence intensity of the hippocampus in the HIBD + vehicle group was significantly lower than that in the Sham group, and the number of hippocampal neurons was effectively improved after CY (40 mg/kg) treatment (Figure 1a and b). Furthermore, we analyzed the protein expression of NeuN in the hippocampal tissue. The results indicated that the expression of NeuN was decreased in the HIBD + vehicle group compared with the Sham group, whereas the expression of NeuN was significantly increased in the HIBD + CY group compared with the HIBD + vehicle group (Figure 1d–f). These findings suggest that CY can effectively ameliorate behavioral deficits and reduce hippocampal neuronal death in the neonatal rat HIBD model.

Figure 1

CY reduces hippocampal neuronal death in a neonatal rat HIBD model. (a) Immunofluorescence of NeuN in the hippocampus of a HIBD model; scale bar: 100, 50 μm. (b) Mean fluorescence intensity of NeuN in the hippocampus of a HIBD model. (c) Behavioral test of the HIBD model in neonatal rats. (d) Western blot of NeuN in the hippocampus. (e) Expression statistics of the NeuN protein (WB). (f) Relative mRNA expression of NeuN (qRT-PCR) **P < 0.01 and *** P < 0.001, n = 5.

3.2 CY inhibits ferroptosis in hippocampal neurons of the neonatal rat HIBD model

To explore the specific mechanism of CY on hippocampal neuronal death in vivo, we administered CY (40 mg/kg) to HIBD rats. Subsequently, we conducted a semi-quantitative analysis to detect GPX4, FTH1, PTGS2, and ACSL4. Also, we detected Fe²⁺ in hippocampal tissues, as well. The results showed that GPX4 and FTH1 were significantly decreased and PTGS2 and ACSL4 were significantly increased in the HIBD + vehicle group compared with the Sham group. In contrast, the expression of GPX4 and FTH1 was significantly increased and the expression of PTGS2 and ACSL4 was significantly decreased in the HIBD + CY group compared with the HIBD + vehicle group (Figure 2a–i). In addition, our results of Fe²⁺ detection in the hippocampal tissue and serum of the HIBD model suggested that Fe²⁺ was significantly increased in the HIBD + vehicle group compared with the Sham group. The Fe²⁺ was significantly decreased after the treatment with CY (Figure 2j and k). These results suggest that CY has the effect of inhibiting the process of ferroptosis in hippocampal neurons.

Figure 2

CY inhibits ferroptosis in hippocampal neurons of the neonatal rat HIBD model. (a) Western blot of GPTX4, FTH1, PTGS2, and ACSL4. (b) Expression statistics of the GPTX4 protein (WB). (c) Expression statistics of the FTH1 protein (WB). (d) Expression statistics of the PTGS2 protein (WB). (e) Expression statistics of the ACSL4 protein (WB). (f) Relative mRNA expression of GPX4 (qRT-PCR). (g) Relative mRNA expression of FTH1 (qRT-PCR). (h) Relative mRNA expression of PTGS2 (qRT-PCR). (i) Relative mRNA expression of ACSL4 (qRT-PCR). (j) Fe²⁺ content in serum. (k) Fe²⁺ content in the hippocampus. *P < 0.05; **P < 0.01; and ***P < 0.001, n = 5.

3.3 CY suppresses lipid peroxidation and improves antioxidant capacity

In order to further investigate the effect of CY on lipid peroxidation capacity and antioxidant capacity in the HIBD model, we examined the expression levels of MDA, SOD, and GSH in hippocampal tissues. The results showed that compared with the HIBD + vehicle group, the HIBD + CY group effectively reduced the expression of MDA in the neonatal rat HIBD model (Figure 3b). In addition, CY also effectively improved the expression of SOD and GSH (Figure 3a and c). These results indicate that CY treatment can effectively inhibit the lipid peroxidation process and improve the antioxidant capacity in the neonatal rat HIBD model.

Figure 3

CY suppresses lipid peroxidation and improves antioxidant capacity. (a) GSH content in the hippocampus. (b) MDA content in the hippocampus. (c) SOD content in the hippocampus. *P < 0.05; **P < 0.01; and ***P < 0.001, n = 5.

3.4 CY represses the activation of the Nrf2/Keap-1 signaling pathway

To investigate the deeper mechanism of CY in attenuating ferroptosis, we examined the relative mRNA levels and protein expression levels of Nrf2 and Keap-1. We observed that the relative mRNA expression and protein expression levels of Nrf2 and Keap-1 were higher in the HIBD + vehicle group compared with the Sham group. When CY treatment was given, the relative mRNA expression and protein expression levels of Nrf2 and Keap-1 decreased (Figure 4a–g). These findings suggest that CY not only affects the protein expression of Nrf2 and Keap-1 but also influences their gene expression.

Figure 4

CY represses Nrf2/Keap-1 signaling pathway activation. (a) Western blots of Nrf2 and Keap-1. (b) Expression statistics of Nrf2 protein (WB). (c) Expression statistics of the Keap-1 protein (WB). (d) Relative mRNA expression of Nrf2 (qRT-PCR). (e) Relative mRNA expression of Keap-1 (qRT-PCR). (f) Expression of Nrf2 in the hippocampus (ELISA). (g) Expression of Keap-1 in the hippocampus (ELISA). *P < 0.05; **P < 0.01; and *** P < 0.001, n = 5.