Study on the treatment of ischemic stroke rats with two-dimensional graphite carbon nitride nanosheets loaded with Senkyunolide I

Objective: To investigate the therapeutic effect of two-dimensional graphite phase carbon nitride (gC₃N₄) nanosheets loaded with Senkyunolide I (SEI) on ischemic stroke rats. Methods: The layered g-C₃N₄ was formed by thermal reaction of melamine, and the thin layer g-C₃N₄ nanosheets were obtained and characterized after thermal stripping and acid peeling. SEI was loaded onto g-C₃N₄ nanosheets, the SEI in the supernatant was determined by UV-vis absorption spectrum, the mass ratio of SEI loaded with g-C₃N₄ was calculated, and the drug release of SEI-g-C₃N₄ composite nanosheets was determined at 1 h, 3 h, 6h, 12 h, 24 h and 48 h. The SD rats with middle cerebral artery occlusion (MCAO) were injected with SEI-g-C₃N₄ composite nanosheets 48 h before the establishment of the model. Neurobehavioral score was performed 24 h after the establishment of the model. After the score, the samples were stained with TTC, TUNEL and HE, respectively. Results: Through the characterization experiment, the prepared g-C₃N₄ nanosheets had the advantages of small size (about 100 nm), large specific surface area, good dispersion and positive surface charge (33.3 mV). The results of UV-vis absorption spectra showed that g-C₃N₄ had successfully loaded SEI and the mass ratio of SEI/g-C₃N₄ was 15.06. The drug release results showed that the release rate of SEI was close to 50% at 24 h. The neurological function score of MCAO group was significantly higher than that of sham group (P＜0.0001), indicating that the modeling was successful. Compared with MCAO group, the neurological function score of MCAO+SEI group was decreased (P＞0.05), and that of MCAO+SEI-g-C₃N₄ group was decreased (P＜0.001). Compared with MCAO+SEI group, the neurological function score of MCAO + SEI-g-C₃N₄ group was decreased (P＜0.01). The results of TTC staining showed that compared with sham group, the cerebral infarction size of MCAO group was increased significantly (P＜0.0001); compared with MCAO group, the cerebral infarction size of MCAO+SEI group was decreased (P＜ 0.0001), and that of MCAO+SEI-g-C₃N₄ group was decreased significantly (P＜0.0001). Compared with MCAO+SEI group, the cerebral infarction size of MCAO+SEI-g-C₃N₄ group was decreased (P＜0.001). The results of TUNEL staining showed that the apoptosis rate of MCAO group was higher than that of sham group (P＜ 0.0001), indicating that the modeling was successful, that of MCAO+SEI-g-C₃N₄ group was higher than that of sham group (P＞0.05), and that of MCAO+SEI-g-C₃N₄ group was lower than that of MCAO group (P＜0.001). The results of in vivo biosafety experiment showed that compared with sham group, there was no significant difference in body weight between SEI group and SEI-g-C₃N₄ group, and no obvious pathological changes in cortical brain structure and cell morphology. Conclusion: The SEI-g-C₃N₄ composite nanosheets with good biosafety are successfully prepared, and they can reduce the cerebral infarction size, inhibit apoptosis, reduce the pathological changes of brain tissue, and improve the neurobehavioral function of MCAO rats, indicating that SEI-g-C₃N₄ composite nanosheets can be used in the treatment of ischemic stroke rats.