Thymoquinone is one of the main components present in Nigella sativa seeds and is known to have various biological functions in inflammation, oxidative stress, tumors, aging, and in lowering blood glucose levels. Few studies have focused on its neuroprotective effects and its regulation of small-molecule metabolites during cerebral ischemia reperfusion injury. In this study, transient middle cerebral occlusion (tMCAO) was used to establish the rat model of cerebral ischemia reperfusion injury. We investigated the effects of thymoquinone using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in a model of ischemia reperfusion injury to explore the changes in small-molecule metabolites in the brain. We found that that thymoquinone significantly improved neurobehavioral scores, reduced the cerebral infarct area, alleviated brain edema, and increased the number of normal neurons following injury. MALDI-MSI revealed that thymoquinone reduced abnormal accumulations of glucose, citric acid, succinate and potassium ions. Thymoquinone also increased the amount of energy-related molecules such as ADP, AMP, GMP, and creatine, antioxidants such as glutathione, ascorbic acid, and taurine, and other metabolism-related molecules such as glutamate, glutamine, aspartate, N-acetyl-L-aspartate, and sodium ions in damaged areas of the brain following cerebral ischemia reperfusion injury. In summary, based on the neuroprotective effect of thymoquinone on cerebral ischemia reperfusion injury, this study revealed the regulation of thymoquinone on energy metabolism and small-molecule substance metabolism.

Dl-3-n-butylphthalide (NBP) is a drug that is used in the treatment of ischaemic stroke. However, to the best of our knowledge, there are no systematic studies investigating the effects of dl-3-n-butylphtalide on the brain metabolism of small molecules. In this study, we first investigated the effects of dl-3-n-butylphthalide on the spatial distribution of small molecules in the brains of rats with permanent middle cerebral artery occlusion (pMCAO) using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF–MS) imaging. After pMCAO modelling or a sham operation, rats were given four mg/kg of dl-3-n-butylphthalide through the caudal vein or saline once a day for nine days. The degree of neurological deficit in rats was evaluated using the modified neurological severity score (mNSS). MALDI–TOF–MS imaging was used to observe the content and distribution of small molecules related to metabolism during focal cerebral ischaemia. Multiple reaction monitoring (MRM) mode with liquid chromatography tandem mass spectrometry (LC–MS/MS) was used to verify the results obtained from MALDI–TOF–MS imaging. These small molecules were found to be involved in glucose metabolism, ATP metabolism, the glutamate–glutamine cycle, malate aspartate shuttle, oxidative stress, and inorganic ion homeostasis. Of the 13 metabolites identified by MALDI–TOF–MS imaging, seven compounds, ATP, ADP, AMP, GMP, N-acetylaspartic acid, ascorbic acid and glutathione, were further validated by LC–MS/MS. Taken together, these results indicate that dl-3-n-butylphthalide significantly improved ATP metabolism, level of antioxidants, and sodium-potassium ion balance in a rat model of pMCAO.

The extracellular space (ECS) is the space between the neurons and the capillaries in the brain. The volume fraction (α) and the tortuosity (λ) are the main parameters used to describe its characteristics. Thymoquinone has been proved to possess anti-oxidant and anti-inflammatory activity. In this study, we used a gadolinium-diethylenetriaminepentacetate (Gd-DTPA)-enhanced magnetic resonance imaging (MRI) system to determine the effects of thymoquinone on ECS parameters in transient middle cerebral artery occlusion rats (tMCAO) to prove the neuroprotective effect of thymoquinone on brain tissue damage caused by ischemic stroke. Neurological examinations, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylineosin (H&E) staining and assaying of ECS parameters using MRI were performed 24 h after surgery. We found that thymoquinone could improve the behavioural performance by neurological examinations. TTC staining indicated that thymoquinone significantly decreased the percentage of hemi-cerebral infarction. Also, H&E staining showed that thymoquinone could inhibit the neuron necrosis in the hippocampal CA1 region. We found that thymoquinone treatment could inhibit the changes in ECS diffusion parameters, which might prove that thymoquinone might protect brain tissue damage caused by ischemic stroke. Thymoquinone can protect the brain against cerebral ischemia-reperfusion injury, effectively ameliorate abnormalities in characteristics of ECS and decrease cerebral infarction in tMCAO rats.