Carthamin yellow (CY), a flavonoid compound extracted from safflower, has been reported to attenuate cardiac ischemia and reperfusion injury. However, whether CY could ameliorate ischemic stroke is not completely understood. In the present study, the preventive effects of CY on experimental ischemic stroke were investigated using middle cerebral artery occlusion (MCAO) model rats. Neurological scores, brain edema, infarct area and microtubule-associated protein 2 (MAP-2) immunoreactivity were assessed to evaluate the effects of CY on ischemic brain injury. The involvement of inflammation and ferroptosis were examined to investigate the mechanism underlying the effects of CY. The results demonstrated that 2-week CY treatment attenuated the neurological deficit score, brain water content and infarct area, and increased MAP-2 immunoreactivity in the cortex in MCAO model rats. CY administration also deactivated the cortex NF-κB/NLR family pyrin domain containing 3 inflammasome signaling pathway, and decreased serum TNF-α, IL-1β and IL-6 concentrations. Moreover, CY treatment inhibited Fe2+ and reactive oxygen species accumulation, and reversed acyl-CoA synthetase long-chain family member 4, transferrin receptor 1, glutathione peroxidase 4 and ferritin heavy chain 1 protein expression levels in the brain. The levels of glutathione, superoxide dismutase and malondialdehyde in the serum were also reversed by CY treatment. Collectively, the results of the present study demonstrated that CY protected rats against ischemic stroke, which was associated with mitigation of inflammation and ferroptosis.
Ischemic stroke, which accounts for 85% of all stroke cases, is the leading cause of destructive cerebrovascular disease, and displays high mortality and morbidity rates (
The NLR family pyrin domain containing 3 (NLRP3) inflammasome, which consists of NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1, is an important component of the innate immune system (
Ferroptosis is a novel type of cell death driven by iron-dependent accumulation of lipid-based reactive oxygen species (ROS), which is regulated by the inactivation of glutathione peroxidase 4 (GPX4) that can reduce lipid peroxides at the expense of glutathione (GSH) (
Carthamin yellow (CY) is a flavonoid compound isolated from safflower. In Traditional Chinese medicine, it is considered that CY improves blood circulation and alleviates pain; thus, CY is used for the treatment of coronary heart disease, cerebrovascular disease and angiitis in China (
A total of 32 male Sprague-Dawley rats (aged 6-8 weeks; 250-280 g) were purchased from Shanghai Sipper-BK Lab Animal Co., Ltd. Animals were housed in an animal center at a controlled temperature (23±1°C) and humidity (60±2%) with 12-h light/dark cycles, and free access to food and water. All experiments were conducted in compliance with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation and approved by the Institutional Animal Care and Use Ethics Committee at Nanjing University of Chinese Medicine (approval no. 201907A544).
CY was obtained from Sigma-Aldrich (Merck KGaA;
Animals were randomly divided into the following four groups (n=8 per group): i) Sham; ii) MCAO; iii) CY (20 mg/kg); and iv) CY (40 mg/kg). CY was administered intragastrically to rats once daily for 2 weeks. At 60 min after the last administration, MCAO surgery was performed as previously described (
For the evaluation of functional recovery, neurological scores were assessed at 24 h post-MCAO induction by an observer blinded to the treatments as previously reported (
Rats were sacrificed and the brains were rapidly removed. Coronal sections were cut into 2-mm thick slices and stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich; Merck KGaA) for 30 min at 37°C followed by fixation with 10% paraformaldehyde for 10 min. Infarction size was assessed using ImageJ software (version 1.52; National Institutes of Health). The size of infarct regions was calculated using the following equation: Infarct rate (%) = Infarct volume/total volume ×100.
The wet-dry method was applied to determine brain edema. The brains were immediately removed and weighed to obtain the wet weight. Following drying in an oven at 100°C for >24 h, the brains were weighed to obtain the dry weight. The percentage of brain water content was calculated using the following formula: Brain water content (%) = [(wet weight)−(dry weight)]/(wet weight) ×100.
Immunofluorescence staining was performed to detect microtubule-associated protein 2 (MAP-2) and NF-κB expression as previously described (
Western blotting was performed as previously described (
The levels of TNF-α (TNF-α ELISA Kit; cat. no. PT516), IL-1β (IL-1β ELISA Kit; cat. no. PI303), IL-6 (IL-6 ELISA Kit; cat. no. PI328), MDA (MDA detection kit; cat. no. S0131), GSH (GSH detection kit; cat. no. S0052) and SOD (SOD detection kit; cat. no. S0101) in serum were detected using different detection kits (Beyotime Institute of Biotechnology) according to the manufacturer's protocol.
ROS content was assayed using 2,7-dichlorofluorescin diacetate (DCF), a ROS detection kit (cat. no. H131224; Shanghai Aladdin Bio-Chem Technology Co., Ltd.) according to the manufacturer's protocol.
Iron concentration was assessed using an Iron Assay kit (cat. no. MAK025-1KT; Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions.
Statistical analysis was performed using GraphPad Prism software (version 6; GraphPad Software, Inc.). Data are presented as the mean ± SEM. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
To determine whether CY induced neurological function recovery in MCAO model rats, neurological scores were evaluated at 24 h post-reperfusion (
MCAO induced a noticeable increase in brain water content in the ischemic hemisphere at 24 h post-reperfusion, which was reduced following CY treatment (
To examine the protective effects of CY on neurons, immunofluorescence staining for MAP2 was performed. Obvious MAP-2 immunostaining was observed in the cortex of Sham rats (
Subsequently, immunofluorescence staining, western blotting and ELISAs were performed to investigate the effects of CY on inflammation. The results demonstrated that in the cortex of MCAO model rats, p-NF-κB p65 expression detected via immunofluorescence staining, as well as the protein expression levels of p-NF-κB p65, p-IκBα, NLRP3, caspase-1 and IL-1β detected via western blotting were significantly increased compared with the Sham group (
To determine the involvement of ferroptosis in MCAO model rats, iron and ROS accumulation, lipid peroxidation levels and the expression levels of ferroptosis-related proteins were assessed. MCAO model rats displayed obvious ROS and iron accumulation in the cortex compared with Sham rats (
In the present study, CY-mediated effects on experimental ischemic stroke were investigated. CY improved neurological deficit scores, brain water content, infarct area and MAP-2 expression in MCAO model rats. CY also deactivated the NF-κB/NLRP3 inflammasome signaling pathway in the cortex. In addition, CY treatment decreased the serum concentrations of TNF-α, IL-1β and IL-6. CY also alleviated MCAO-induced ferroptosis, as demonstrated by reduced iron and ROS accumulation, reduced lipid peroxidation level and restored ferroptosis-related protein expression levels.
The MCAO model is one of the models that most closely simulates human ischemic stroke (
Ischemic stroke has been reported to damage neurons (
Evidence suggests that the post-ischemic inflammatory responses alter the outcome of ischemic stroke (
Ferroptosis is a newly defined cell death process that is characterized by iron-dependent accumulation of lipid peroxides to lethal levels (
Collectively, the results of the present study suggested that CY treatment protected rats against ischemia-reperfusion injury by alleviating inflammation and ferroptosis (
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HG, CB and JL were involved in the design of the study. HG, LZ and PT performed the experiments. DC analyzed the data. YL interpretated the data and drafted the manuscript with the support of all authors. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
The study was approved by the Animal Care and Use Ethics Committee of Nanjing University of Chinese Medicine (Nanjing, China) (approval no. 201907A544).
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
Chemical structure of carthamin yellow.
Effects of CY on neurological deficit scores, brain water content and infarct area. (A) Neurological deficit score. (B) Brain water content. (C and D) Infarct area. Data are presented as the mean ± SEM. ##P<0.01 vs. Sham; *P<0.05 and **P<0.01 vs. MCAO. CY, carthamin yellow; MCAO, middle cerebral artery occlusion.
MAP-2 expression in the cortex of MCAO model rats. (A) Representative images of immunofluorescence staining for MAP-2. Scale bar, 50
p-NF-κB expression in the cortex of MCAO model rats. (A) Representative images of immunofluorescence staining for p-NF-κB. Scale bar, 50
Effects of CY on the NF-κB/NLRP3 inflammasome signaling pathway in the cortex. NF-κB/NLRP3 inflammasome signaling pathway-related protein expression levels were (A) determined via western blotting and (B) semi-quantified. Data are presented as the mean ± SEM. ##P<0.01 vs. Sham; *P<0.05 and **P<0.01 vs. MCAO. CY, carthamin yellow; NLRP3, NLR family pyrin domain containing 3; MCAO, middle cerebral artery occlusion; p, phosphorylated.
TNF-α, IL-1β and IL-6 concentrations in serum. (A) TNF-α, (B) IL-1β and (C) IL-6. Data are presented as the mean ± SEM. ##P<0.01 vs. Sham; *P<0.05 and **P<0.01 vs. MCAO. TNF, tumor-necrosis factor; IL, interleukin; MCAO, middle cerebral artery occlusion; CY, carthamin yellow.
ROS and iron accumulation in the cortex. (A and B) ROS generation. (C) Fe2+ production. Data are presented as the mean ± SEM. ##P<0.01 vs. Sham; **P<0.01 vs. MCAO. ROS, reactive oxygen species; MCAO, middle cerebral artery occlusion; CY, carthamin yellow; DCF, 2,7-dichlorofluorescin diacetate.
Lipid peroxidation level in serum. (A) GSH, (B) SOD and (C) MDA. Data are presented as the mean ± SEM. ##P<0.01 vs. Sham; *P<0.05 and **P<0.01 vs. MCAO. GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; MCAO, middle cerebral artery occlusion; CY, carthamin yellow.
Expression levels of ferroptosis-related proteins in the cortex. Ferroptosis-related protein expression levels were (A) determined via western blotting and (B) semi-quantified. Data are presented as the mean ± SEM. ##P<0.01 vs. Sham; *P<0.05 and **P<0.01 vs. MCAO. MCAO, middle cerebral artery occlusion; CY, carthamin yellow; ACSL4, acyl-CoA synthetase long-chain family member 4; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; TFR1, transferrin receptor.
Schematic illustration of the proposed mechanism underlying CY-mediated protection against cerebral ischemia-reperfusion injury. CY, carthamin yellow; ACSL4, acyl-CoA synthetase long-chain family member 4; NLRP3, NLR family pyrin domain containing 3; PUFA, polyunsaturated fatty acids; PL, phospholipid.