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INTRODUCTION
Including ischemic and hemorrhagic strokes, a stroke (cerebral stroke), as well recognized as cerebral vascular accident, is an acute cerebrovascular disease brought on via the rapid rupture of a blood vessel in the brain or a blood vessel obstruction that restricts blood flow to the brain [1]. Epidemiological survey studies have shown that hypertension, heart disease, diabetes mellitus, and hyperlipidemia are the main risk factors for cerebrovascular disease, while age, gender, race, smoking, and alcoholism are listed as risk factors associated with cerebrovascular disease [2].
Approximately 60% to 70% of all strokes are ischemic strokes, which occur more frequently than hemorrhagic strokes. Brain tissue is damaged by ischemia, resulting in damage to its structure and function. Sometimes, ischemia followed by reperfusion not only fails to restore structure and function but exacerbates the damage, resulting from an overabundance of free radicals destroying cells in this tissue’s regaining of blood supply, an injury called cerebral ischemia/reperfusion injury (CI/RI) [3]. The molecular mechanisms of brain tissue damage caused by CI/RI include inflammatory responses, excitatory amino acid toxicity, intracellular calcium overburden, impaired energy metabolism, free radical damage, and apoptosis.
During the occurrence of CI/RI, reactive oxygen species (ROS) are released in huge amounts and scavenged inadequately; the imbalance between oxidative and antioxidant effects causes oxidative damage to cells and tissues. Therefore, oxidative stress is an essential aspect of CI/RI [4]. Nuclear factor E2-related factor 2 (Nrf2) is an essential transcription factor that controls endogenous antioxidant stress [5]; studies have shown that its mediated signaling pathway can attenuate CI/RI through multiple pathways, such as inhibiting oxidative stress, reducing inflammation [6], controlling mitochondrial fission and apoptosis [7], and protecting the blood–brain barrier [8]. In addition, brain tissue is highly oxygenated and rich in unsaturated fatty acids that are easily oxidized, and its ability to resist peroxidation and scavenge free radicals is significantly lower than that of other tissues. Hence, the generation of huge quantities of free radicals during CI/RI induces oxidative stress and damage to brain tissue. Increased Nrf2 expression after CI/RI induces the production of various endogenous antioxidant enzymes, including quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), superoxide dismutase (SOD), glutathione sulfotransferase, and glutathione peroxidase (GSH-Px), which reduce or scavenge oxygen radicals and improve the antioxidant ability of cells and tissues [9]. Treatment that included an Nrf2 activator increased Nrf2, HO-1, and NQO1 expression in addition to actively increasing SODs and GSH-Px activity in brain tissues, resulting in decreased ROS activity and malondialdehyde (MDA) content. Notably, the above protection properties were suppressed once Nrf2 was knocked down. Collectively, our findings showed that activating the Nrf2 signaling pathway could decrease CI/RI by reducing oxidative stress.
Naturally, isoprene-substituted benzoic acids produce amorfrutin A (AA), which has anticancer activity. It was extracted from the fruits of Amorpha fruticosa L. (bastard indigo), a plant grown in northern Shanxi, China [10]. This natural active substance not only has hypoglycemic [11], anti-inflammatory, and anti-tumor effects but is also well tolerated [12]. Studies have shown that AA demonstrates a limited antibacterial action coverage to Gram-positive and acid-resistant microorganisms. The nuclear transcription factor of nuclear factor kappa-B (NF-κB) has an essential part in controlling genes of apoptosis, the immune system, tumor cell proliferation, as well as tissue differentiation [13]. AA suppresses the tumor necrosis factor-α (TNF-α)-triggered expression of several inflammatory genes by inhibiting NF-κB and PPARγ activity. In addition, AA improves insulin resistance when the nuclear receptor PPAR is bound [14], in addition to improving different metabolic and inflammatory factors without causing fatty liver accumulation and other adverse events, including hepatotoxicity.
The current main clinical treatments for acute ischemic stroke currently include intravenous thrombolysis and arterial thrombus retrieval, but some patients still experience varying degrees of neurological injury after aggressive reperfusion therapy. Therefore, novel therapeutic strategies and drugs are required to minimize ischemic brain damage and enhance brain function restoration after CI/RI. There is still much to learn about the anti-inflammatory and antioxidant capacity of AA in stroke. Accordingly, the present investigation sought to examine the probable impacts and molecular mechanisms of AA on ischemia-induced brain injury and find new neuroprotective drugs for CI/RI development [15].
In this study, we investigated the protective effects of AA in ischemic stroke by establishing the middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen glucose deprivation/reoxygenation (OGD/R) model in vitro and in vivo, respectively, and further examined the signaling pathways related to the antioxidant and anti-inflammatory effects exerted by AA. Our findings provide a basis for developing AA as a therapeutic drug for ischemic stroke.
METHODS
Experimental medications
AA (C21H24O4, molecular weight = 340.41), which chemical structural formula shown in Fig. 1A, was synthesized in-house in our laboratory (purity > 98%). Edaravone (Eda) was purchased from Macklin (P816062).
Cell culture
Mouse BV2 cells and rat PC12 cells were purchased from Wuhan Pricella Biotechnology Co., Ltd. BV2 cells were cultured in high glucose Dulbecco’s modified eagle medium (DMEM) (Thermo Fisher Scientific, Inc.) media enriched with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Pricella Biotechnology Co., Ltd.) at 37°C under a humidified atmosphere of 5% CO2 and 95% air. PC12 cells were cultured under the same conditions.
OGD/R and drug treatment
BV2 cells were cultured at a 2 × 104 density with normal medium in a 96-well plate for 24 h. To establish the OGD/R model, the normal medium was replaced with glucose-free DMEM, and the whole plate was placed in a 2.5 L anaerobic tank together with an anaerobic production bag for 4 h. After 4 h of OGD, the cells were subsequently returned to an incubator under normoxic conditions with different concentrations of drug for 3 h. The same operation was carried out on PC12 cells.
Assessment of cell viability
The original medium was removed and the cells were washed three times with phosphate buffer saline (PBS), followed by addition of 100 µl of medium containing 10% CCK-8 reagent (MedChemExpress). The cells were then incubated for 1 h at 37°C and the absorbance values (OD) were determined at 450 nm on a microplate reader. The OD value ratios between each group and the blank control were used to calculate the cell activity.
Determination of ROS level in BV2 cells and PC12 cells
ROS production was detected with 2´,7´-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe as described in the instructions of the Reactive Oxygen Assay Kit (Beyotime Biotechnology). DCFH-DA was diluted in serum-free medium at 1:1,000 to a final concentration of 10 µM. After 3 h of drug treatment, the cell culture medium was removed and appropriate volume of diluted DCFH-DA was added to cover the cells. The cells were incubated at 37°C for 20 min, then washed three times with serum-free cell culture medium to fully remove DCFH-DA that did not enter the cells. The fluorescence intensity was detected with the excitation wavelength of 488 nm and the emission wavelength of 525 nm by a Fluorescent enzyme labeling instrument (Gene Company Ltd.). The fluorescence intensity reflected the amount of ROS generated.
Determination of nitric oxide (NO) generation in BV2 cells and PC12 cells
BV2 cells and PC12 cells were used to prepare the OGD/R model respectively, and 50 µl of supernatant was carefully aspirated after 3 h of drug treatment. After adding 50 µl of Reagent I and Reagent II in sequence, the absorbance value (OD) was determined at 540 nm on an enzyme standard meter, and the concentration of NO was calculated using the standard tune prepared in advance.
Determination of TNF-α level in BV2 cells
BV2 cells were medicated with OGD/R model preparation, and after 3 h of complex glucose and oxygen administration, 50 µl of the supernatant was carefully aspirated, and biotin antigen and affinity-HRP were supplied, respectively. In addition, after conducting the reaction at 37°C for 30 min, the addition of color developer and termination solution was conducted, the absorbance value (OD) was determined at 450 nm on an enzyme marker, and the concentration of TNF-α was calculated using the standard tune prepared in advance.
Experimental animals
Seventy-two male Sprague Dawley rats, SPF grade, aged 7–8 weeks, weighed 210–230 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd. All rats were kept in a well-ventilated animal house (22°C ± 3°C, 60% ± 5% humidity) under a 12 h light–dark cycle with free access to water and food. After one week of adaptive feeding, the animals were randomly divided into 6 groups (n = 12): sham, MCAO/R, MCAO/R + AA (5 mg/kg), MCAO/R + AA (10 mg/kg), MCAO/R + AA (20 mg/kg), and MCAO/R + Eda (10 mg/kg) group. All animal experiments were approved by the Institutional Animal Care and Use Committee of Guizhou Medical University (approval number: 2304461).
MCAO/R and drug treatment
The rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg, Sigma Chemical Company), the left middle cerebral artery was exposed and occluded via modified Longa-Zea method [16]. In brief, a nylon suture (Yangzhou Fuda Medical Equipment Co., Ltd.) with a diameter of 0.24 mm was carefully inserted from the incision into the internal carotid artery until it blocked the origin of the middle cerebral artery. After 90 min of CI, the sutures were carefully withdrawn to restore blood flow. The sham group performed a similar surgery but did not insert the suture. All drugs were prepared as clear injections (4 mg/ml) using a solvent mixture (N, N-dimethylacetamide:PEG-400:saline = 1:6:3). After 2 h of reperfusion, rats were injected intraperitoneally with different concentrations of drugs.
Neurobehavioral score
Neurobehavioral evaluation was performed using the Longa method 2 h after reperfusion and 24 h after administration. The results were scored on a 5-point scale: 0 for rats without deficits, 1 for rats with contralateral forelimb extension difficulties, 2 for rats with slight contralateral forelimb rotation, 3 for rats with severe contralateral rotation, 4 for rats with no spontaneous motor activity, and 5 for rats that died.
Assessments of cerebral infarct volume
To calculate the infarct volume, 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Chemical Company) staining was utilized. Following 24 h of drug treatment, brain samples were harvested and sliced to a thickness of 2 mm. The rats brain sections were incubated in 1% TTC solution for 20 min at 37°C in the dark, and the sections were soaked in 4% paraformaldehyde overnight. Normal areas of the brain are red, while infarct areas appear white. Cerebral infarct volume was measured using Image J software (Version 1.8.0, National Institutes of Health). The percentage infarct volume was computed as (cerebral infarct area / total brain area) × 100%.
Hematoxylin-eosin (H&E) staining
At 24 h after drug administration, rats were executed. Whole brains were extracted and fixed overnight in 4% paraformaldehyde. After processed with different concentrations of ethanol and xylene, the brain tissue was paraffin-embedded. The paraffin sections were cut into 10 µm thick slices, stained with H&E, and observed and photographed under a light microscope (BX51; Olympus).
Measurement of inflammatory variables and oxidative stress-related markers
Following cervical dislocation and the execution of rats following 24 h of reperfusion, the left reperfusion-injured brains were quickly excised and homogenized with PBS buffer, brain tissue (mg): 1% PBS (ml) = 1:9, prepared as 10% homogenized tissue, and the supernatant was removed after 15 min of being centrifuged at 3,500 rpm, and the protein content was estimated utilizing BCA protein quantification. The kit’s instructions were followed while measuring the amounts of SOD, GSH-Px, and MDA in the brain tissue. The supernatant was taken, and interleukin-1β (IL-1β) and TNF-α were measured in the rat brain tissue according to the ELISA kit instructions.
Western blot
Rat ischemic brain tissues were taken and homogenized on ice with RIPA lysis buffer (Beyotime Biotechnology) containing phosphatase inhibitor and protease inhibitor. After 30 min, the supernatant was collected after centrifugation at 13,500 rpm at 4°C for 20 min. The quantification of the protein concentration in the brain tissue was conducted using BCA Protein Assay Kit (Solarbio Science & Technology Co., Ltd.). The protein samples were separated using 10% SDS-PAGE gels (Shanghai Genefist Life Science Co., LTD), and then transmitted into PVDF membranes. The membranes were blocked with 5% skim milk for 2 h prior to being conjugated overnight at 4°C with the following primary antibodies:Keap1 (1:1,000, 8047S, Cell Signaling Technology), Nrf2 (1:1,000, ab92946, Abcam), HO-1 (1:1,000, ab68477, Abcam), NQO1 (1:1,000, ab80588, Abcam), and β-actin (1:2,000, Cat No. 81115-1-RR, Proteintech). Lastly, the membrane was conjugated with secondary antibodies: goat anti-rabbit IgG (H + L) and incubated for 2 h at room temperature. The proteins were visualized by an enhanced chemiluminescence detection kit (Applygen) using the Tanon 5200 Chemiluminescence Image Analysis System (Tanon).
Statistical analysis
Our findings are demonstrated as the mean ± standard deviation and were examined or plotted utilizing GraphPad Prism 8.0. Using the t-test, data analysis between the two groups was undertaken. One-way ANOVA was employed to examine the data among the groups. p < 0.05 revealed a statistically significant variation.
RESULTS
AA reduced OGD/R-induced BV2 cells injury
In order to assess the cytotoxicity of the compounds and determine the appropriate drug concentration, the BV2 cells induced by OGD/R were treated with different concentrations of AA (0, 5, 10, 20, 25, 40, 50, 80 and 100 µM) for 3 h respectively. The effect of AA on OGD/R-induced injury in BV2 cells is shown in Fig. 1B, cell viability showed a significant decrease in the OGD/R group as compared to the control group (p < 0.01). While treatment with AA (5–25 µM) markedly increased cell viability as compared to the OGD/R group (p < 0.01), and the cell viability was highest in the OGD/R + AA (20 µM) group (p < 0.01). After OGD/R injury, AA 10 µM, 20 µM significantly increased OGD/R-induced BV2 cell viability (0.40 ± 0.017, 0.44 ± 0.13) greater than the OGD/R group (0.34 ± 0.013) (p < 0.01, p < 0.05, Fig. 1C). Therefore, these results suggested that AA conferred protective effects on BV2 cells after OGD/R injury, and doses of 20 µM and 10 µM were identified as the optimal drug doses for further experiments.
To further investigate the potential protective role of AA in OGD/R-induced cellular damage, intracellular levels of ROS and NO were quantitatively analyzed in BV2 treated with varying concentrations of AA following OGD/R exposure. As shown in Fig. 1D, E, the levels of ROS and NO were significantly increased in BV2 cells subjected to OGD/R as compared to the control group (p < 0.01). In contrast, ROS and NO were significantly reduced in BV2 cells after AA intervention (p < 0.05, p < 0.01), and the inhibition was enhanced in a dose-dependent manner. To investigate whether the protective effects of AA against OGD/R-induced injury in BV2 cells are associated with its anti-inflammatory properties, we further examined the secretion of TNF-α in AA-treated BV2 cells following OGD/R exposure. The results demonstrated that AA treatment significantly suppressed OGD/R-induced upregulation of TNF-α (Fig. 1F, p < 0.01). These findings suggest that the protective effects of AA are mediated, at least in part, through the inhibition of neuroinflammatory responses. These data indicated that AA could inhibit OGD/R-induced oxidative stress injury and inflammatory response in BV2 cells.
AA reduced OGD/R-induced PC12 cells injury
Additional experiments were conducted using the PC12 neuronal cell line, a widely accepted model for studying neuronal responses to ischemic injury. The effect of AA on OGD/R-induced injury in PC12 cells is illustrated in Fig. 1G, the cell viability in the OGD/R group was significantly lower than that in the control group (p < 0.01). Treatment with AA at different concentrations resulted in a increase in cell viability compared to the model group (p < 0.01). Notably, the most pronounced protective effect was observed at concentrations of 20 µM and 25 µM. These results demonstrate that AA exerts a protective effect against OGD/R-induced injury in PC12 cells, and this protective effect exhibits a dose-dependent manner within the concentration range of 5 µM–20 µM.
Similarly, the levels of ROS and NO were measured in PC12 cells (Fig. 1H, I). Treatment with AA at concentrations of 10 µM and 20 µM significantly suppressed the OGD/R-induced upregulation of ROS and NO (p < 0.01). These results further support the antioxidative and anti-inflammatory properties of AA in neuronal cells under ischemic conditions.
AA treatment decreased cerebral infarct volume and improved neurological function in MCAO/R rats
Infarct volume and neurobehavioral scores are specific markers for assessing the extent of brain damage. TTC staining was employed to identify the cerebral infarct area following 24 h of administration. As shown in Fig. 2A, B, no cerebral infarcts were observed in the sham group, and large infarcts were seen in the MCAO/R group (27.31 ± 5.44%). The cerebral infarct volume in AA 5 mg/kg, 10 mg/kg and 20 mg/kg treated rats were 18.06 ± 4.16%, 16.82 ± 2.75% and 8.11 ± 1.21%, respectively, which was significantly lower than that in the MCAO/R group (p < 0.01).
The neurobehavior of rats following 24 h of administration was evaluated by the Longa Scale. The rats in the sham group did not exhibit any neurobehavioral deficits and the neurological deficit score was significantly increased in the MCAO/R group as compared to the sham group (2.68 ± 0.52, Fig. 2C), indicating successful induction of neurological injury. In contrast, treatment with Eda significantly improved neurological function, with scores reduced to 1.33 ± 0.52. The scores treated with AA at doses of 5 mg/kg, 10 mg/kg and 20 mg/kg decreased to 2.33 ± 0.82, 1.67 ± 0.52 and 1.33 ± 0.52 respectively. These findings suggest that AA exerts dose-dependent neuroprotective effects in alleviating neurological deficits.
AA treatment ameliorated pathological brain damage in MCAO/R rats
The neuronal cells in the sham group were well-structured and arranged in an orderly fashion, with large and round nuclei, as revealed by H&E staining (Fig. 2D). In the MCAO/R group, the cells were significantly reduced, the cell structure was severely damaged, the nuclei were deeply stained and shrunken, the arrangement between the cells was disorganized, the gaps were widened, and the structure was vacuolated. The administration of 5 mg/kg and 10 mg/kg showed no significant improvement, with edema still evident and severe damage to cellular structures. After a therapeutic dose of 20 mg/kg, the cytoarchitecture of the cerebral cortex recovered, edema was reduced, and the degree of necrosis was reduced, which was comparable to the influence of Eda therapy.
AA treatment reduced the levels of inflammatory factors in MCAO/R rats
TNF-α and IL-1β levels were identified in the ischemic area of MCAO/R rats. As indicated in Fig. 3A, B, the TNF-α and IL-1β values were significantly elevated in the MCAO/R group as compared to the sham group, with 107.63 ± 11.87 pg/ml and 22.26 ± 1.10 pg/ml, respectively (p < 0.01). The TNF-α and IL-1β values were 61.22 ± 7.31 pg/ml and 17.07 ± 1.20 pg/ml in the MCAO/R + AA 10 mg/kg group, and 44.26 ± 8.07 pg/ml and 10.36 ± 2.11 pg/ml in the MCAO/R + AA 20 mg/kg group. Thus, AA could effectively suppress TNF-α and IL-1β release in the brain tissue of MCAO/R rats. These results suggest that AA may exert an anti-inflammatory effect on MCAO/R rats.
AA treatment attenuated oxidative stress in MCAO/R rats
As shown in Fig. 3C, D, SOD and GSH-Px activities were significantly reduced in the brain tissue of MCAO/R rats as compared to the sham group (458.65 ± 22.76 U/mgprot) (42.44 ± 9.25 U/mgprot, p < 0.01). AA (20 mg/kg) significantly elevated SOD and GSH-Px activities in the ischemic region of MCAO/R rat brains (727.23 ± 73.69 U/mgprot, 94.83 ± 1.56 U/mgprot, p < 0.01), and Eda had similar effects (655.72 ± 61.96 U/mgprot, 44.20 ± 3.45 U/mgprot). Fig. 3E revealed that the MDA content of ischemic brain tissue was significantly increased in the MCAO/R group as compared to the sham group (1.99 ± 0.22 nmol/mgprot, p < 0.01), and that Eda, AA (10 mg/kg, 20 mg/kg) significantly reduced MDA production (1.19 ± 0.048 nmol/mgprot, 1.41 ± 0.014 nmol/mgprot, 1.11 ± 0.068 nmol/mgprot, p < 0.05, p < 0.01). These results suggest that AA may perform an antioxidant function in MCAO/R rats.
AA treatment promoted the activation of the Nrf2/HO-1 signaling pathway in cerebral MCAO/R rats
Using Western blot analysis, Nrf2, HO-1, and NQO1 were discovered to examine the influence of AA on oxidative stress-induced CI/RI. The protein expression of Kelch-like ECH-associated protein 1 (Keap1) in the 20 mg/kg group (42.67 ± 4.21) was significantly reduced compared to the MCAO/R group (85.64 ± 8.49) (Fig. 4A). The Nrf2, HO-1, and NQO1 protein expression patterns in the MCAO/R group were 49.68 ± 5.14, 72.60 ± 7.48, and 92.79 ± 4.19, respectively. The protein expressions of Nrf2, HO-1, and NQO1 showed a significant increase in the 20 mg/kg treatment (87.55 ± 4.99, 97.59 ± 1.79 and 111.44 ± 5.02), with statistically significant variations (p < 0.01, Fig. 4B–D). According to our results, AA was able to improve the antioxidant ability of tissues and enhance the antioxidant defense of tissues through the Nrf2/HO-1 pathway.
DISCUSSION
The present study aims to elucidate the protective role of AA in CI/RI and explore its potential molecular mechanisms. After ischemic stroke, the level of oxidative stress and inflammation in ischemic brain tissue were significantly increased. AA improves oxidative stress damage by activating Nrf2/HO-1 signaling pathway. In addition, AA can also exert anti-neuroinflammatory activity by inhibiting the release of inflammatory factors.
The current treatment principle for acute ischemic stroke is to rapidly recover blood flow reperfusion via surgical thrombosis or intravenous thrombolysis [17]. According to several investigations, the pathogenesis of CI/RI is complex and diverse, involving disorders of brain tissue energy metabolism, excess oxygen free radicals and inflammatory mediators, cell autophagy, and apoptosis, but no ideal drug has been found for prevention and treatment. AA was found to be promising in the treatment of inflammatory disorders and malignancies caused by the abnormal activation of NF-κB and STAT3 [18]. Nevertheless, the function and pathway of AA in CI/RI are completely unknown.
The mechanisms underlying the pathogenesis of CI/RI are complex, involving various factors such as the disruption of energy metabolism in brain tissue [19], excess oxygen free radicals, and inflammatory mediators [20]. Impaired neurological function in the brain is the result of a post-ischemic inflammatory response, and the microglia have a unique part in the overall pathological process [21]. Microglia has a function in developing and maintaining homeostasis in the central nervous system and activates in response to neural tissue damage, promoting neuroinflammation and neurodegeneration. After CI/reperfusion, activated glial cells produce large amounts of inflammatory cytokines (IL-1β, TNF-α, etc.), NO, and ROS [22]. These inflammatory mediators cause a complex cascade of intra- and extra-cellular and inter-tissue signaling responses, leading to an excessive inflammatory response [23], which in turn leads to neuronal death. It can be seen that the activated microglia-mediated inflammatory responses have a major part in the pathophysiology of CI/RI [24]. In this study, an OGD/R injury model of BV2 cells was constructed to simulate the activation state of microglia after CI. It was found that the administration of AA at concentrations of 10 µM and 20 µM significantly enhanced the survival rate of BV2 cells following exposure to OGD for 4 h and reoxygenation for 3 h. After OGD/R injury, ROS, NO, and TNF-α were significantly elevated in BV2 cells, while AA could markedly reduce the secretion of oxidative stress-related molecules and inflammatory factors. Similarly, an OGD/R model was established in PC12 neuronal cells using the same experimental protocol. Cell viability, ROS, and NO levels were quantitatively analyzed. Consistent with the findings in BV2 cells, AA treatment increased cell viability and suppressed ROS and NO overproduction in PC12 cells. Under the experimental conditions of this study, after the OGD/R model was established, the expression levels of inflammatory factors in PC12 cells did not show significant differences compared to the control group, thus this part of the data was not presented. Combined with the effect of AA on cell survival after OGD/R injury, it can be preliminatively concluded that AA may play a neuroprotective role by inhibiting the release of ROS, NO, and inflammatory cytokines in cells. These findings provide a solid basis for conducting subsequent in vivo experiments.
The MCAO/R model in rats was employed to simulate the process of CI reperfusion, aiming to further investigate the role and underlying mechanism of AA in brain protection. The MCAO/R model in rats was employed to simulate the process of CI reperfusion, aiming to further investigate the role and underlying mechanism of AA in brain protection. The analysis of TTC staining revealed that AA (10, 20 mg/kg) effectively reduced cerebral infarct volume in MCAO/R rats. Moreover, AA could alleviate the neurological deficits caused by ischemia in rats. The H&E staining indicated that the presence of brain tissue edema, damage to the ischemic cortex, and a significant occurrence of apoptotic and necrotic cells at the site of ischemia in MCAO/R rats. The intraperitoneal administration of AA in MCAO/R rats could enhance cerebral edema resolution, mitigate the extent of histopathological injury and cortical apoptosis, thereby exerting a neuroprotective effect. Since the 5 mg/kg group did not show a better therapeutic effect, this dose was not set up for the subsequent experiments. In addition, prior research has shown that inflammatory reactions are a key aspect of MCAO/R [25]. Secondary brain injury occurs during local tissue reperfusion in CI, which is important because of microvascular dysfunction mediated by the inflammatory response [26]. Early in CI, astrocytes are activated and produce huge quantities of inflammatory cytokines, including IL-1β and TNF-α, which can stimulate the body’s inflammatory reaction, activate endothelial cells, increase tissue factor levels, and subsequently increase the release of oxygen radicals and NO radicals, causing damage to surrounding tissues, indirectly promoting apoptosis [27]. In vitro experiments preliminarily verified that AA could inhibit the excessive production of inflammatory factors in BV2 cells. Therefore, we also tested it in MCAO/R rats, and measured the levels of IL-1β and TNF-α in the ischemic tissue of rats using ELISA. The results showed that AA could significantly reduce the production of inflammatory cytokines. The inhibition of the inflammatory response may be one of the crucial mechanisms by which AA improves brain injury.
Considering the essential function of oxidative stress in the pathogenesis processes involved in ischemic stroke [28], this experiment also focused on whether AA had an effect on the oxidative stress response in the brain tissue of MCAO/R rats. Oxidative stress is a process in which the production of oxygen free radicals in the body is greater than the antioxidant capacity, resulting in oxidative damage to tissues [29]. Under normal conditions, the scavenging of oxygen radicals in the body is mainly via the scavenging of free radical detoxifying enzymes, such as SOD, GSH-Px, and glutathione reductase, thus maintaining relatively balanced scavenging and production of free radicals. If the body’s capacity to produce free radicals is greater than its scavenging ability, it results in pathological changes in the body [30]. SOD and GSH-Px are common endogenous antioxidant enzymes that perform a crucial role in lowering oxidative stress damage in the body; MDA is a lipid peroxidation byproduct that serves as a measure of lipid peroxidation in tissue cells [31]. SOD achieves anti-aging mainly by scavenging excess free radicals in the body and thus protecting biofilm systems and biological macromolecules, such as nucleic acids. GSH-Px maintains the structure and function of the intact cell membrane by eliminating the products of the body’s oxygen metabolism so that glutathione activity is maintained. SOD and GSH-Px activity in brain tissue directly reflects the brain’s ability to resist oxidative stress. MDA is one of the metabolites of oxidative stress. By evaluating the amount of MDA in brain tissue, we can understand the lipid peroxidation value in the body and, indirectly, the severity of cellular damage. In this experiment, SOD and GSH-Px activity, in addition to MDA content, were measured at the site of cerebral infarction in MCAO/R rats. Based on our findings, MCAO/R rats showed significant oxidative damage in cortical tissues, with a significant reduction in SOD and GSH-Px activity and increased MDA levels in comparison to the sham group. After treatment with AA, the activity of SOD and GSH-Px antioxidant enzymes increased, and the MDA level decreased, indicating that CI–reperfusion caused severe oxidative damage in ischemic brain tissues and that treatment with AA could reduce this damage.
The activation of the Nrf2/HO-1 pathway has been demonstrated in numerous studies to play a pivotal role in antioxidant stress [32,33]. As a crucial transcription factor, Nrf2 predominantly interacts with Keap1 in the cytoplasm to form the Keap1-Nrf2 complex, thereby impeding the nuclear translocation of Nrf2 [34]. Upon ROS attack, the configuration of Keap1 undergoes alterations, leading to the dissociation and translocation of Nrf2 into the nucleus. Subsequently, Nrf2 binds to antioxidant response elements and modulates the expression of genes encoding antioxidant factors such as NQO1, HO-1, and SOD [35]. HO-1 facilitates the breakdown of heme into ferrous iron, CO, and biliverdin. Among these products, both biliverdin and its reduced form, bilirubin, exhibit potent ROS scavenging activity [36]. NQO1 inhibits the production of ROS and plays an important role in protecting endogenous antioxidants by maintaining the reduced forms of ubiquinone and alpha-tocopherol quinone [37]. Our results showed that AA could down-regulate Keap1, up-regulate Nrf2, NQO1, and HO-1 protein expression, promote the activation of Nrf2/HO-1 signaling pathway, and improve the anti-oxidative stress ability of ischemic brain tissue. In conclusion, AA can activate the Nrf2/HO-1 signaling pathway in the brain of stroke rats, which may be the internal mechanism of AA playing a role in the treatment of stroke.
In summary, the present work provides preliminary evidence that AA was effective in alleviating OGD/R- and MCAO/R-induced injury and highlights that its probable mode of action is linked to activating the Nrf2/HO-1 pathway. AA improved CI/RI by regulating the expression of anti-inflammatory cytokines and antioxidant enzymes and upregulating Nrf2 expression, which accelerates enzymatic reactions by inducing the production of antioxidant and detoxifying enzymes in neuronal cells while promoting the expression of GSH, SOD, and other antioxidants, which have neuroprotective effects. It is conceivable from the results of this study that AA has the potential to be utilized as a neuroprotective agent for ischemic stroke.
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