Cerebral ischemia reperfusion injury is the restoration of blood flow after vascular occlusion, which results in further damage to the brain tissues. In this study, we used the transient middle cerebral artery occlusion (tMCAO) model to simulate cerebral ischemia reperfusion injury. Figure 8
summarizes the effects of thymoquinone on small-molecule metabolism. We found that thymoquinone had a protective effect on cerebral ischemia–reperfusion injury by decreasing the neurological deficit scores, reducing the brain infarct size, alleviating brain edema damage, improving cell morphology damage, and increasing the number of normal neurons. MALDI-MSI analysis indicated that thymoquinone was able to regulate the abnormal metabolism in the areas with brain damage by promoting glucose aerobic oxidation, regulating intracellular energy metabolism, improving phospholipid molecular levels, increasing the content of antioxidant small molecules, and balancing the homeostasis of sodium and potassium ions. These regulatory effects of thymoquinone ultimately reduced the damage in the brain tissue.
In this study, the common and recognized rats model of cerebral injury was established by ischemia for 2 h and reperfusion for 24 h [5
]. The time of ischemia and reperfusion is different in different animal species. The duration of ischemia and reperfusion was mainly based on the physiological characteristics of rats. The purpose of this study was to investigate the effect of cerebral injury on small molecule metabolism, so samples were collected after 2 h of ischemia and 24 h of reperfusion. However, this sampling timeline may affect the determination of some small molecules. With the prolonged reperfusion time after ischemia, the animal’s physiopathological state may enter the recovery period, and some small metabolic molecules may return to baseline levels. Therefore, we took the samples immediately after 24 h reperfusion to reduce the bias in metabolite determination.
As the main active ingredient of Nigella sativa
, thymoquinone has a wide range of neuropharmacological functions with reported protective effects against various central nervous system diseases. Thymoquinone was found to interact with Aβ1
to prevent its accumulation and slow the development of Alzheimer’s disease [7
]. In a rotenone-induced Parkinson’s disease model, thymoquinone was shown to alleviate mitochondrial dysfunction and oxidative stress to protect the dopaminergic neurons [8
]. Thymoquinone also enhanced the memory and exerted antipsychotic effects via the reduction of dopamine levels and acetylcholinesterase activity and the increase of glutathione levels [9
]. In addition, in a model of transient forebrain ischemia, thymoquinone-loaded nanoparticles were shown to alleviate hippocampal damage caused by ischemia [8
]. Thymoquinone-loaded PLGA–chitosan nanoparticles via nose-to-brain administration were able to reduce the infarct volume in rat brains after cerebral ischemia reperfusion, and the locomotory activity and forelimb grip strength were also subsequently increased. Thymoquinone reduced lipid peroxidation in the brain after middle cerebral artery occlusion and increased the glutathione level and the activity of enzymes such as catalase and superoxide dismutase [10
]. Similarly, Hosseinzadeh et al. [11
] used a model of transient global cerebral ischemia induced by a four-vessel-occlusion method to show that thymoquinone played a protective role by reducing the levels of malondialdehyde in the hippocampus after cerebral ischemia reperfusion and inhibiting lipid peroxidation.
This study showed that prophylactic administration of 5 mg/kg thymoquinone significantly improved the neurobehavioral scores and decreased the percentage of infarction and water content in the brain after ischemia–reperfusion injury. Thymoquinone was able to improve ischemic penumbra by reducing the number of degenerated cells, increasing the number of Nissl-positive neurons, decreasing the infarct area, and exerting neuroprotective effects. Compared with edaravone, thymoquinone displayed a better neuroprotective effect in improving neurobehavioral scores and increasing the number of Nissl-positive neurons. The lower dose of 2.5 mg/kg led to an improved performance in the neurological deficit test but limited effects on the other indicators.
Based on the neuroprotective functions, we further investigated the effects of thymoquinone on endogenous small-molecule metabolism in the brain tissues after cerebral ischemia reperfusion injury using mass spectrometry imaging combined with a 1,5-DAN hydrochloride matrix.
The aerobic oxidation of glucose is the primary means by which most cells derive energy and include the glycolytic pathway, pyruvate oxidative decarboxylation, the TCA cycle, and oxidative phosphorylation. As the main energy-consuming organ, the brain is highly sensitive to ischemia and hypoxia. Occlusion of the middle cerebral artery can result in a scarce supply of oxygen and glucose, leading to the destruction of the aerobic oxidation process [12
]. Mass spectrometry imaging results in this study showed that glucose, pyruvate, citric acid, and succinate were abnormally accumulated in the damaged brain after cerebral ischemia reperfusion injury, which is consistent with the findings by Huang et al. [13
]. However, in their study, the level of succinate, the TCA cycle intermediate, was significantly lower in the permanent middle cerebral artery occlusion (pMCAO) group than in the sham group. This finding was contradictory to our result obtained by MSI, and this may have been due to differences between permanent and transient MCAO. We found that the content of glucose, citric acid, and succinate in the damaged brain area increased after thymoquinone administration, and this improvement was also observed in edaravonetreatment group. Currently, there are no reports on the effect of thymoquinone on aerobic oxidation process. We used mass spectrometry imaging for the first time to reveal the correlation between thymoquinone and aerobic oxidation-related molecules. Thanks to the antioxidant biological activity, thymoquinone can act as a redox mediator to taking the “excess” of electrons from the respiratory complexes during reperfusion.
The TCA cycle is an important pathway that links glucose and amino acids. The TCA cycle intermediate α-ketoglutarate can be converted into glutamate, and the mutual conversion between the two neurotransmitters is achieved through the glutamate–glutamine cycle. As an important neurotransmitter in the central nervous system, excess extracellular glutamate can overstimulate glutamate receptors, leading to excitotoxicity [14
]. Excitotoxicity has been described in various neurological diseases, such as Alzheimer’s disease, Parkinson’s disease, cerebral ischemia, and epilepsy.
N-acetyl-L-aspartate, a small amino acid found in high amounts in the brain, is mainly synthesized and stored by neurons and regulated by oligodendrocytes and can be enzymatically interconverted into aspartate. Upon occlusion of the middle cerebral artery, the glutamate and N-acetyl-L-aspartate levels in the affected brain areas decreased, whereas the glutamine level was significantly increased [13
]. Proton nuclear magnetic resonance spectroscopy revealed that N-acetyl-L-aspartate was present in the neurons in normal brain tissue at a higher concentration but that was significantly reduced in pathological conditions such as impaired cellular metabolism and neuronal damage [15
]. Magnetic resonance spectroscopy showed a significant reduction in N-acetyl-L-aspartate levels in the infarct region of patients after ischemic stroke [17
]. Consistent with the findings of these studies, we found a decrease in glutamate, N-acetyl-L-aspartate, and aspartate levels after cerebral ischemia–reperfusion injury by MSI.
There are few reports on the regulation of the four excitatory amino acids by thymoquinone. Thymoquinone was shown to modulate glutamate-mediated neurotoxicity and inhibit cell apoptosis and Aβ formation through neuromodulation [18
]. This study was the first to use mass spectrometry imaging to explore the function of thymoquinone on excitatory amino acids after cerebral ischemia–reperfusion injury. Compared with edaravone, thymoquinone could not only increase the levels of glutamine and N-acetyl-L-aspartate after injury but also increase the levels of glutamate and aspartate, and this increased the understanding of its protective mechanism in cerebral ischemia–reperfusion injury.
Mitochondria are the regulatory centers of cellular energy metabolism and oxidative stress. Mitochondrial oxidative phosphorylation is the main pathway for ATP production. The creatine–phosphocreatine system plays a significant role in the transport, storage, and utilization of high-energy phosphate bonds in the body. After cerebral ischemia reperfusion injury, the normal electron transport processes in the mitochondrial oxidative respiratory chain are inhibited, hindering oxidative phosphorylation in the mitochondria [19
]. Cerebral ischemia reperfusion injury can also lead to the excessive accumulation of ROS, which can further aggravate mitochondrial damage and degrade cellular energy metabolism [20
]. In this study, mass spectrometry imaging was used to investigate the changes in energy-related small molecules in the areas with brain damage. Except for xanthine, most of the downstream ATP metabolites and creatine in the damaged brain were decreased. Farooqui et al. [21
] previously found that thymoquinone could regulate the metabolism of carbohydrates in the kidneys by improving the activity of enzymes and also alleviate the effect of cisplatin-induced toxicity on the intracellular energy metabolism. Similarly, Shahid et al. [22
] showed the effect of thymoquinone on cisplatin-induced energy depletion in intestinal cells. There is no report on the regulation of ATP and related endogenous small molecules by thymoquinone, and our results showed that thymoquinone and positive control (edaravone group) possess the comparable ability in regulating energy-related small molecules. This study showed for the first time that thymoquinone can regulate energy-related small molecules after cerebral ischemia reperfusion injury.
Phospholipids are the basic structural components of neuronal cell membranes and play an important role in the maintenance of normal cell morphology and physiological functions. Shanta et al. [23
] used MALDI MS to show the differential expression of various phospholipid molecules in both the ischemic and normal regions of the brain in an ischemic model. They identified 11 upregulated phospholipids, including lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and sodiated forms of sphingomyelin (SM) and PCs, as well as 7 downregulated phospholipids in the areas with ischemic damage. Nielsen et al. [24
] used desorption electrospray ionization and MALDI to study the expression of different phospholipid molecules in the brain at different time points after pMCAO. In this study, 1,5-DAN hydrochloride solution was used as a matrix, and mass spectrometry imaging was used to investigate changes in four phospholipid molecules (PE, PA, PI, and PS) in the ischemic brain. Cerebral ischemia–reperfusion injury reduced the levels of PE (16:0/22:6), PE (p-18:0/22:6), PE (18:0/22:6), PA (18:0)/22:6), PI (18:0/20:4), and PS (18:0/22:6) and increased the levels of PE (18:0) and PI (18:0). Thymoquinone increased the levels of PE (16:0/22:6), PE (p-18:0/22:6), PE (18:0/22:6), PI (18:0/20:4), and PS (18:0/22:6) and decreased the level of PE (18:0), thereby regulating phospholipid levels in areas with brain damage. Thymoquinone and edaravone had their own characteristics in the regulation of phospholipid molecules, and better improvement was observed in PE (18:0/22:6), PI (18:0/20:4), and PS (18:0/22:6) in thymoquinone-treatment group. We showed for the first time the protective effect of thymoquinone on cerebral ischemia–reperfusion injury through regulation of phospholipid metabolism.
Sodium and potassium ions ensure the homeostasis of the intracellular environment under physiological conditions and maintain the normal electrophysiological function of neurons. The energy supply to the brain after ischemia is converted from aerobic to anaerobic glycolysis. Excessive accumulation of lactic acid causes lactic acidosis, and accumulation of lactate/H+
activates the Na+
exchanger and increases the amount of Na+
in the cells [13
]. Liu et al. [25
] used mass spectrometry imaging to investigate the effect of butylphthalide after cerebral ischemia infarction and found that the Na+
content in the injured hemisphere was increased, whereas the K+
content was decreased after pMCAO. This data is consistent with our findings after cerebral ischemia–reperfusion injury. However, studies on the regulation of metal ions by thymoquinone have not been reported. We found that thymoquinone had a better effect in homeostasis. Thymoquinone reduced the content of sodium ions and increased the content of potassium ions, while edaravone only improved the potassium ions.
Oxidative stress caused by abnormal accumulation of ROS is another major pathological process of cerebral ischemia–reperfusion injury. Cerebral ischemia reperfusion injury increases the electron leakage in the oxidative respiratory chain and promotes the production of ROS. Excess ROS in mitochondria can consume in vivo antioxidants and inhibit the endogenous antioxidant defense system. In this study, mass spectrometry imaging was used to detect the mass spectrometric signals of three endogenous antioxidants: Taurine, ascorbic acid, and reduced glutathione. The levels of these antioxidants were significantly reduced in the areas with brain damage after cerebral ischemia–reperfusion injury. Reduced glutathione and ascorbic acid are important antioxidants in the body that directly remove ROS from the brain. Taurine exerts an antioxidant effect by inhibiting ROS production and the inflammatory response through the mitochondria electron transport chain [26
]. Studies have reported the antioxidative effects of thymoquinone. Thymoquinone was shown to reduce the levels of malondialdehyde and protect against lipid peroxidation damage caused by cerebral ischemia–reperfusion [11
]. Xiao et al. [10
] showed that thymoquinone could reduce lipid peroxidation in the brain after middle cerebral artery occlusion and increase the glutathione level and the activity of catalase and superoxide dismutase. However, the effects of thymoquinone on taurine and ascorbic acid have not yet been reported. This study shed light on the regulation of reduced glutathione by thymoquinone and found that thymoquinone could increase the level of these two antioxidant molecules in the ischemic areas of the brain.
Finally, mass spectrometry imaging showed changes in arachidonic acid, pantetheine 4′-phosphate, glycerol 3-phosphate, and hippuric acid. Thymoquinone increased the level of arachidonic acid and reduced the level of pantetheine 4′-phosphate but had no effect on glycerol 3-phosphate and hippuric acid levels, while edaravone could only improve the level of glycerol 3-phosphate. Arachidonic acid is a major component of the biomembrane structure and is released from the membrane structure and converted into various physiologically active lipid molecules under catalysis by phospholipase A2. In addition, arachidonic acid plays an active role in synaptic plasticity, membrane fluidity, and neurogenesis [27
]. We hypothesize that the regulation of arachidonic acid by thymoquinone may be related to the anti-oxidant and anti-inflammatory effects of thymoquinone by regulating the amount of ROS to reduce lipid peroxidation damage by ROS in the biomembrane structure. At present, there are few reports on the relationship between pantetheine 4′-phosphate and nerve damage. Our results suggest that cerebral ischemia–reperfusion injury leads to an increase in pantetheine 4′-phosphate levels, but the specific mechanisms by which this occurs requires further research.