Objective To investigate the mechanism of chrysophanol in treating acute cerebral ischemia reperfusion injury (CIRI) based on the receptor⁃interacting protein kinase (RIP)1/RIP3/mixed lineage kinase domain⁃like protein (MLKL) signaling pathway. Methods Seventy⁃two rats were randomly divided into sham operation group, CIRI model group, 50 µg/kg chrysophanol group, 100 µg/kg chrysophanol group, 150 µg/kg chrysophanol group, or 150 µg/kg chrysophanol+8 µg/kg RIP1 group, with 12 rats in each group. Except for the sham operation group, the CIRI model was established using the arterial embolization method in the remaining groups. Rats in the groups with various doses of chrysophanol were intraperitoneally injected with corresponding concentrations of chrysophanol, the 150 µg/kg chrysophanol+8 µg/kg RIP1 group received successively intraperitoneal injection of chrysophanol and received tail vein injection of RIP1, while rats in the sham operation group and CIRI model group were intraperitoneally injected with an equal volume of normal saline for 14 consecutive intervention days. Before the first intraperitoneal injection and after the last intraperitoneal injection, the cranial nerve injuries of rats in each group was assessed using the Longa score. After the last intraperitoneal injection, brain tissue was collected from each group. Cerebral infarction was evaluated using triphenyltetrazolium chloride staining, pathological morphological changes in brain tissue were assessed using HE staining, and neuronal apoptosis in brain tissue was observed using TUNEL staining. The relative mRNA expressions of Caspase⁃8, tumor necrosis factor α (TNF⁃α), interleukin 1β (IL⁃1β), and IL⁃6 in brain tissue were measured using quantitative real⁃time PCR. The protein expressions of RIP1, RIP3, phosphorylated MLKL (p⁃MLKL), and MLKL in brain tissue were detected using Western blot. Results After the last intraperitoneal injection, compared with the sham operation group, the CIRI model group exhibited increases in Longa score, cerebral infarction area, neuronal cell apoptosis rate, and up⁃regulated mRNA expressions of TNF⁃α, IL⁃1β, and IL⁃6 in brain tissue, elevated protein expressions of RIP1 and RIP3, and elevated value of the p⁃MLKL/MLKL ratio, while the mRNA expression of Caspase⁃8 was decreased (P<0.05). Additionally, neuronal cells in the brain tissue of the CIRI model group exhibited deformation and necrosis, with a large number of inflammatory cells attached. Compared with the CIRI model group, the 50 µg/kg, 100 µg/kg, and 150 µg/kg chrysophanol groups depicted decreases in Longa score, cerebral infarction area, neuronal cell apoptosis rate, and down⁃regulated mRNA expressions of TNF⁃α, IL⁃1β, and IL⁃6, decreased protein expressions of RIP1 and RIP3, and decreased value of the p⁃MLKL/MLKL ratio, while the mRNA expression of Caspase⁃8 was increased (P<0.05). The brain tissue structure in these groups was intact, neuronal cells were neatly arranged, and only a small number of inflammatory cells were present. Compared with the 150 µg/kg chrysophanol group, the 150 µg/kg chrysophanol + 8 µg/kg RIP1 group expressed increases in Longa score, cerebral infarction area, neuronal cell apoptosis rate, and up⁃regulated mRNA expressions of TNF⁃α, IL⁃1β, and IL⁃6, elevated protein expressions of RIP1 and RIP3, and elevated value of the p⁃MLKL/MLKL ratio, while the mRNA expression of Caspase⁃8 was decreased (P<0.05). In this group, neuronal cells in the brain tissue were loosely arranged with vacuolar deformation, and inflammatory cell infiltration was evident. Conclusion Chrysophanol may alleviate the inflammatory response, inhibit neuronal apoptosis, and protect damaged neurons in brain tissue of an acute CIRI rat model by inhibiting RIP1/RIP3/MLKL signaling pathway.  

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