| Objective: The therapeutic effect and underlying mechanism of icariin(ICA)on cerebral ischemia/reperfusion(I/R)injury in rats were explored.Methods: The male SD rats(250-280 g)were divided into 7 groups at random: Sham group,model group,ICA low-dose group(10 mg/kg ICA),ICA medium-dose group(20mg/kg ICA),ICA high-dose group(40 mg/kg ICA),positive control group(12 mg/kg nimodipine)and endoplasmic reticulum stress induction group(0.16 mg/kg tunicamycin).The model of cerebral ischemia-reperfusion injury in the rats,including 2 h ischemia and24 h reperfusion,was accomplished by applying the method of transient middle cerebral artery occlusion(MCAO).At 24 h reperfusion,rats were evaluated for neurological deficit using and m NSS method.The rats were then sacrificed,and their brain tissues were collected.The water content of the brain was tested by dry-wet weight method.Hematoxylin-eosin staining was operated to survey the histomorphological changes in the peripheral area of rat brain tissue.Double immunofluorescence staining was explored to detect neuron marker Neu N and microglia marker Iba1 expression in ischemic peripheral area.Immunohistochemistry(IHC)staining was used to determine the expression of interleukin 1β(IL-1β),interleukin 6(IL-6)and tumor necrosis factor-α(TNF-α).Molecular docking analysis was conducted using Molecular Operating Environment 2019 software to explore the mechanism of ICA on the inflammation after ischemic stroke in rats.Immunofluorescence staining was used to examine the expression of endoplasmic reticulum stress(ERS)marker glucose-regulated protein 78(GRP78).Western blot was operated to detect expression of inositol requiring enzyme-1 α(IRE1α),phospho-IRE1α(p-IRE1α),protein kinase RNA-like ER kinase(PERK),phospho-PERK(p-PERK),spliced XBP1(XBP1s),unspliced XBP1(XBP1u),thioredoxin-interacting protein(TXNIP),NOD-like receptor thermal protein domain associated protein 3(NLRP3),cysteinyl aspartate specific proteinase 1(Caspase-1),IL-1β,IL-6 and TNF-α in the ischemic peripheral area of rat brain tissue.Results: 1.ICA protected against cerebral I/R injury: Compared with the model group,the histopathological damage in the ICA groups were significantly reduced.The neurological function scores were decreased in the ICA groups,and the differences were statistically significant.2.ICA suppressed the inflammatory injury induced by I/R:Compared with the model group,the brain water content and neuronal death were significantly improved,whereas the microglial activation was notably reduced in the ICA groups.The expression of IL-1β,IL-6 and TNF-α in the ICA groups were decreased,and the differences were statistically significant.3.ICA regulated inflammation through ERS signaling pathway: The result of molecular docking showed that the free energy of ICA to GRP78,IRE1α and PERK were less than-5 kcal/mol,which can be stably combined.In addition,compared with the model group,the expression of ERS downstream inflammation-related targets XBP1 s,XBP1u,TXNIP,NLRP3,and Caspase-1 were decreased in the ICA groups,and the differences were statistically significant.Conclusion: 1.ICA has protective effects on I/R injury by intervening in the ischemic peripheral area and inhibiting the inflammatory response.2.ICA inhibits the NLRP3 inflammasome through the IRE1α-XBP1 and IRE1α/PERK-TXNIP signaling pathways,thereby regulating the activation of microglia,and protecting I/R injury. |
