The Role Of RIP3/CaMKⅡ/Pyk2 Pathway In Programmed Cell Death In Cerebral Infarction

Cerebral infarction is a serious threat to human health,but effective treatment measures are very limited.Therefore,innovative treatment methods based on the pathogenesis of cerebral infarction are of great significance to reduce the nerve damage after cerebral infarction.Programmed cell death mainly includes apoptosis,necroptosis,etc.,which is widely involved in the pathogenesis of a variety of diseases,and is also an important cause of nerve damage after cerebral infarction,and has important pathophysiological effects.The pathogenesis of cerebral infarction cell apoptosis signaling pathway in cerebral infarction has been extensively studied for many years,but the molecular mechanism of necroptosis pathway is unclear.The classical necroptosis pathway is triggered and activated by tumour necrosis factor alpha(TNF-α),and receptor-interacting protein 1(RIP1)and receptor-interacting protein 3(RIP3)phosphorylate each other to form "necrosome".Further phosphorylation of the mixed lineage kinase domain-like protein(MLKL),which ultimately leads to cell death.RIP1,RIP3,and MLKL are commonly used as molecular markers for necroptosis.A recent study found that RIP1 and RIP3 deletions can reduce brain injury following ischemia/reperfusion(MCAO/R)in mice by reducing necroptosis,but the specific mechanism has not been elucidated.A study of a mouse myocardial ischemia/reperfusion model found that RIP3 mediates the activation of calmodulin-dependent kinase II(CaMKⅡ)in the atypical necroptosis signaling pathway,ultimately leading to necroptosis and apoptosis.However,it is unclear whether the RIP3/CaMKⅡ pathway is involved in cell death in cerebral infarction models.Our team’s previous research found that the expression of proline-rich tyrosine kinase 2(Pyk2)and mitochondrial calcium uniporter(MCU)was upregulated in rat cerebral infarction models,accompanied by mitochondrial damage,increased calcium influx,reactive oxygen species production and increased apoptosis.Another study found that the CaMKⅡ/Pyk2 signaling cascade in chondrocytes promoted chondrocytes proliferation and matrix synthesis.These studies suggest that the RIP3/CaMKⅡ/Pyk2 signaling pathway may play an important role in the occurrence and development of cerebral infarction,but its molecular regulatory mechanism needs to be further explored.In this study,in the first part,RIP3 knockout mice were used to explore whether the RIP3/CaMKⅡ/Pyk2 pathway was involved in programmed cell death in mouse cerebral infarction models,and the molecular regulatory mechanism was discussed.In the second part,primary cortical neurons were cultured to establish an in vitro neuronal hypoxia model,in which the effect of RIP3/CaMKⅡ/Pyk2 pathway on primary neurons after hypoxia was verified.In the third part,plasma from hospitalized patients with acute cerebral infarction and healthy control groups was collected,and the expression levels of plasma necroptosis markers RIP1,RIP3 and MLKL were compared between the acute massive cerebral infarction group,the acute non-massive cerebral infarction group and the control group,so as to find feasible peripheral blood indicators for the diagnosis,treatment and prognosis assessment of acute cerebral infarction.Part one The RIP3/CaMKⅡ/Pyk2 pathway is involved in the mechanism of programmed cell death in mouse d MCAO modelsObjective: To clarify the effects of RIP3 knockout on cerebral infarction volume and motor dysfunction and neuronal ultrastructure in mouse cerebral infarction models.To analyze the effects of RIP3 knockout on mitochondrial membrane potential decrease,calcium influx increase,reactive oxygen species production,and programmed cell death.To investigate whether the RIP3/CaMKⅡ/Pyk2 cascade is involved in necroptosis and apoptosis in mouse cerebral infarction models.Methods: WT and RIP3 knockout mice were treated with distal middle cerebral artery occlusion(d MCAO),and the differences in cerebral infarction volume and motor dysfunction between the two groups were compared.Electron microscopy was used to observe the ultrastructural damage of neurons after cerebral infarction in two groups.Western blot was used to detect the expression of RIP3,CaMKⅡ,Pyk2 and other classical necroptosis marker proteins and apoptosis proteins in cerebral infarction tissues.Flow cytometry was used to analyze the changes of mitochondrial membrane potential,calcium influx and reactive oxygen species in cerebral infarction tissues of the two groups.Results: RIP3 knockout reduced the volume of cerebral infarction in mouse cerebral infarction models,alleviated motor dysfunction,and improved neuronal ultrastructural damage after cerebral infarction.RIP3 knockout inhibits decreased mitochondrial membrane potential,increased cellular calcium influx and reactive oxygen species production after cerebral infarction.RIP3 knockout inhibits CaMKⅡ/Pyk2 cascade activation and reduces necroptosis and apoptosis in mouse cerebral infarction models.Conclusion:1.The volume of cerebral infarction,motor dysfunction and neuronal ultrastructural damage of RIP3 knockout mouse cerebral infarction model were significantly reduced.RIP3 knockout inhibits a decrease in mitochondrial membrane potential,an increase in intracellular calcium influx,and reactive oxygen species production after cerebral infarction.2.RIP3 knockout can inhibit the activation of CaMKⅡ/Pyk2 cascade reaction,reduce the expression of necroptosis and apoptosis proteins,and ultimately reduce necroptosis and apoptosis of neurons.Part two Expression changes of RIP3/CaMKⅡ/Pyk2 pathway in primary cortical neuronal oxygen sugar deprivation/reoxygenation modelObjective: Culture primary cortical neurons in vitro to verify the role of RIP3/CaMKⅡ/Pyk2 pathway in programmed cell death in an oxygen sugar deprivation/reoxygenation(OGD/R)model.Methods: Primary cortical neurons were extracted and cultured in WT and RIP3 knockout fetal mice,and microtubule-associated protein 2(MAP2)stained positive cells by laser confocal microscopy to identify primary cortical neurons.CCK-8 was used to detect cell viability after OGD/R in two groups of primary cortical neurons.Western blot detected the expression levels of RIP3/CaMKⅡ/Pyk2 pathway signaling proteins in two groups of primary cortical neurons.Immunofluorescence staining compared calcium influx levels after OGD/R in two groups of primary cortical neurons.Results: In the OGD/R model,the viability of primary cortical neurons of RIP3 knockout fetal mice was significantly higher than that of primary cortical neurons in WT group.RIP3 knockout inhibited the expression of CaMKⅡ and Pyk2 after OGD/R in primary cortical neurons.RIP3 knockout inhibited the increase in calcium influx after OGD/R in primary cortical neurons.Conclusion: This section verifies the effect of RIP3/CaMKⅡ/Pyk2 pathway on programmed cell death at the cellular level.In the OGD/R model of primary cortical neurons,RIP3 knockout can improve cell viability and reduce programmed cell death by inhibiting the CaMKⅡ/Pyk2 signaling pathway and inhibiting calcium influx.Part three Detection of levels of markers of necroptosis in plasma in patients with acute cerebral infarctionObjective: To investigate the relationship between the levels of RIP1,RIP3 and MLKL,the marker proteins of necroptosis in plasma of patients with acute cerebral infarction,and to seek simple and easy methods for the diagnosis,treatment and prognosis assessment of acute cerebral infarction.Methods: The clinical data and plasma samples of 120 patients diagnosed with acute anterior circulation cerebral infarction who were hospitalized in the Department of Neurology of the Second Hospital of Hebei Medical University from March 2020 to June 2023 were enrolled,including80 patients with acute massive cerebral infarction and 40 patients with acute non-massive cerebral infarction.At the same time,40 healthy adults in the physical examination center of our hospital were collected as a healthy control group.Collect clinical data of patients: including gender,age,Body mass index(BMI),previous underlying diseases,smoking and drinking history,family history of stroke,National Institutes of Health Stroke Scale(NIHSS)score at admission,etc.All enrolled patients were prepared with plasma samples drawn from elbow venous blood on the day of admission.Plasma RIP1,RIP3 and MLKL levels were measured by enzyme-linked immunosorbent assay(ELISA).All participants voluntarily agreed to join the study.Results: Compared with the healthy control group,the plasma levels of RIP1,RIP3 and MLKL in the acute massive cerebral infarction group and the acute and acute non-massive cerebral infarction group were significantly increased,and the acute massive cerebral infarction group was significantly higher than that in the acute non-massive cerebral infarction group.However,there was no significant correlation between plasma RIP1,RIP3,and MLKL levels and NIHSS scores.Conclusion:1.Compared with the healthy control group,the level of plasma necroptosis markers in patients with acute massive cerebral infarction group and acute non-massive cerebral infarction group was significantly increased.2.There was no significant correlation between plasma necroptosis marker levels in patients with acute cerebral infarction and the severity of acute cerebral infarction symptoms.