The Role And Mechanism Of IL-1R1 In Modulating Secondary Injury After Intracerebral Hemorrhage

Hemorrhagic stroke is a common nervous system disease devastating neurological injury,accounting for about 15% of all strokes annually.The most prevalent type of hemorrhagic stroke is intracerebral hemorrhage(ICH),which associated with high morbidity and mortality throughout the world.Although the primary lesion was cleared,40% of the patients with intracranial hemorrhage still had progressive neurological damage,which indicated that secondary injury mediated by thrombin cascade reaction,hemoglobin degradation and inflammatory reaction played an important role in craniocerebral injury.At present,there is no effective intervention for primary injury,and only early prevention can be adopted.Therefore,to clarify the mechanism of secondary injury after intracranial hemorrhage has important guiding significance for clinical and scientific research,and has become an important research direction in the field of stroke.Multiple studies have shown that inflammation is a major factor in the secondary injury of neurons after intracranial hemorrhage in craniocerebral trauma and hemorrhagic stroke.After intracranial hemorrhage,peripheral neutrophils and mononuclear macrophages migrate to hematoma,which can induce neuronal inflammatory injury.After intracranial hemorrhage,RBC metabolites,such as hemoglobin and Hemin or Heme,are also important factors inducing secondary neuronal inflammatory injury.It has been reported that Hemin can cause the activation of microglial cells,releasing inflammatory factors such as TNF-?,IL-6 and IL-1 to further induce inflammatory neuronal damage.Hemin itself is a lipid soluble molecule,on the one hand,it can integrate into the cell membrane,causing oxidative damage to the cell membrane;On the other hand,hemin could be decomposed by heme oxygenase(HO)to generate iron ions,CO and bilirubin.Excessive iron ions and bilirubin could induce neuron cell death.At present,most studies only focus on the neurotoxicity of hemin metabolites and the inflammatory injury to neurons caused by microglia activation.The types of neuronal death can be divided into three types: cell necrosis,cell apoptosis and procedural necrosis.Cell necrosis is considered as a sudden,passive and irreversible cell death mode,while both apoptosis and programmed necrosis are cell programmed death.Therefore,by regulating apoptosis or programmed necrosis,it is an important way to reduce the secondary brain damage after intracranial hemorrhage.Several studies have shown that programmed necrosis in the secondary damage of neurons than the more important role in cell apoptosis,when necroptosis initialed,RIP1 and RIP3(Receptor interacting protein 1/3),forming complexes and further phosphorylation mixed lineage kinase domain-like protein(MLKL),this compound is called "necrosome".Current studies mainly focus on the apoptosis and pro-inflammatory mechanism of neuroglia caused by hemin,however,the sensitivity of neurons to external stimuli is much higher than that of glial cells.Therefore,we speculate that hemin may directly induce neuronal death after intracranial hemorrhage.Recent studies have found that interleukin-1 receptor antagonist(IL-1RA)or interleukin-1 antibody can reduce neuron death in animal models of ischemic and excitatory brain injury.However,in the ICH process,whether IL-1R1(IL-1 receptor,IL-1R1)is involved in the necroptosis of neurons and thereby affecting the survival of neurons has not been reported.In order to further study the effect of hemin on neurons after intracranial hemorrhage,clinical ICH specimens were firstly observed the cellular expression and localization in haematoma areas,to determine whether neuron necroptosis was activated in clinical ICH patients.Then,using animal models of intracranial hemorrhage in corpora and in vitro,we further validation of the clinically phenomena we observed,and further explored the expression pattern of necrosome after ICH in animals and its role in neuron cell death.Then we use the IL-1R1 as the breakthrough point,from the perspective of receptor signal transduction pathways,further study of the regulatory mechanism in hemin induced neuron necroptosis.Finally,we use IL-1R1 gene knockout mice to further verify the cytological mechanism by living proof.Part one: activated neuronal necroptosis in ICH patientsGeneral clinical and cranial CT data and related clinical specimens were collected from 28 patients with intracranial hemorrhage,and the IGCS(coma index)was also evaluated,results indicated that iGCS score was significantly increased after intracranial hemorrhage.MRI combined with HE staining was used to detect the pathological reaction and results showed that the brain tissue of the patient presented hyperpigmentation/iron pigmentation,liquefactive necrosis and inflammatory cell migration.The expression of ho-1 and necrosome was detected by immune staining,and showed that the HO-1(marker of hemin)was expressed only in neurons,indicating that hemin could specifically target neurons and to further induce neuron injury.We also found that although intracranial hemorrhage could induce astrocytes activation,astrocytes were not involved in the activation of programmed necrosis,and necrosome were expressed in neurons specially.Further,the expression of caspase 3 was further detected by TUNEL staining and immune staining,and the results showed that caspase3-mediated apoptosis was also involved in intracranial hemorrhagic induced neuronal death.The above results indicates us that multiple death patterns were involved in intracranial hemorrhage induced neuronal death and hemin can target neurons directly to trigger neuron injury after intracranial hemorrhage.Part two: hemin could induce to neuronal necroptosis by triggering necrosome activationHemin was injected into the skull to build the model of intracranial hemorrhage of mice,and brain tissues were obtained after surgery.Immunohistochemistry and Western blot were used to confirm that whether hemin could specifically induce the expression of ho-1 and necrosome activation in neurons,results showed that after hemin treatment,HO-1 was mainly expressed in hippocampal neurons,which was consistent with the clinical results and the protein levels of HO-1 and necrosome were significantly increased after hemin treatment.We next tested the secretion of inflammatory factors TNF-? excretion,IL-6 and IL-1? in brain tissue by the enzyme linked immunosorbent assay(ELISA),and found that after hemin treatment,the secretion of inflammatory cytokines TNF-?,IL-6 and IL-1? were dramatically higher than the control groups.The above results indicates us that hemin could induce the expression of HO-1 in hippocampus neurons,and it can reflect the target cells of hemin and two mechanisms were involved in hemin induced neuronal injury: inflammatory injury and necroptosis.Part three: IL-1R1 is involved in hemin induced neuronal necroptosisWe first isolated mouse hippocampal neurons for in vitro culture,and the neurotoxicity of hemin was detected by LDH,results showed indicated us that the number of apoptotic/necrotic cells was significantly increased after hemin treatment in cultured neurons when compared with the control groups.Specifically,LDH release was significantly higher,and the number of apoptosis and necrosis neurons was also significantly increased after hemin treatment.Protein expression levels of necrosome and IL-1R1 were detected by Western blot after hemin treatment,and found that hemin could induce the expression of necrosome and IL-1R1 in neurons.We further tested the secretion of the inflammatory cytokine IL-1? in the cerebrospinal fluid/cell supernatant was detected by ELISA,and found that The secretion of IL-1? in cerebrospinal fluid of ICH patients and in cell supernatant were dramatically increased.Protein levels of IL-1R1 in clinical brain tissue were determined by immune staining,and the expression of IL-1R1 was further verified by Western blot and immune staining in the animal model,results showed that The expression of IL-1R1 in animal model and in clinical samples were up-regulated and were only expressed in neurons.Finally,immunoprecipitation was used to determine the relationship between IL-1R1 and necrosome in primary hippocampal neurons,and the co-localization of IL-1R1 and necrosome was further verified in clinical brain samples by immunofluorescence staining.Immunoprecipitation results showed that,hemin could induce the coupling of IL-1R1 with necrosome,and IL-1RA could disrupt the coupling.The fluorescence double-labeling results indicated that IL-1R1 and necrosome occurred synthetically after intracranial hemorrhage.Taken together,the above results indicated us: firstly,after intracranial hemorrhage in the animals,glial cells can secrete inflammatory mediators to induce the secondary injury of neurons.This study for the first time indicated that neurons also can secrete inflammatory mediators and express inflammatory receptors,thus inducing the inflammatory injury of neurons themselves.Secondly,hemin triggered neuronal death via two mechanisms: IL-1? mediated inflammatory injury and necrosome depended necroptosis.Thirdly,IL-1R1 and necrosome coupling is the key point in initiating hemin induced neuron necroptosis.Part four: inhibition of IL-1R1 could relieve hemin induced neuronal necroptosisIL-1RA was pre-treated in the hippocampal neurons before hemin insult,LDH release was detected,expressions of necrosome were detected by Western blot and immunofluorescence,and cell membrane integrity was detected by Annexin V-PI staining,results showed that the number of cell death was significantly reduced when pre-treated with IL-1RA in neuron cultures,and the expression and activation of necrosome were also down-regulated.IL-1RA could protect cell membrane integrity after hemin treatment.When IL-1RA co-treated with hemin,IL-RA was still capable of inhibiting necrosome expression and alleviating hemin induced neuronal injury.Finally,immunoprecipitation was used to detect whether IL-1RA could affect the coupling between IL-1R1 and necrosome,results showed that IL-1RA could disrupt the coupling between IL-1R1 and necrosome,and hence inhibit necrosome activation to further protect hemin induced neuron injury.The above results indicated us that IL-1RA could alleviate hemin induced neuronal necroptosis by inhibiting IL-1R1.Part five: genetic knockout IL-1R1 reduces hemin induced neuronal necroptosis and inflammatory responseThe experiment was divided into two parts.In the first part,the intracranial hemorrhage model was constructed.IL-1RA(5?g)or vehicle(PBS)was treated after 6h of hemin treatment.In the second part wild-type mice and IL-1R1 knockout mice were injected with hemin,and the expression of necrosome in brain tissue was determined by Western blot and immunofluorescence,results showd us that The expression of necrosome was significantly decreased on the first day after IL-1RA treatment,and was slightly upregulated on the third day and then gradually decreased on the fifth day.Similarly,the neurological function score of the mice was slightly increased on the third day,indicating that the therapeutic window of IL-1RA was 24 h.Intracranial injection with hemin could induce acute ventricular dilatation,brain edema and neuronal degeneration injury,and the gene knockout IL-1R1 could reduce expression of necrosome and decrease hydrocephalus and alleviate neurological defects.The water content,inflammatory factor secretion and neurological dysfunction of the mice were also tested,and results showed that intracranial injection with hemin could induce acute ventricular dilatation,brain edema and neuronal degeneration injury,and the gene knockout IL-1R1 could reduce expression of necrosome and decrease hydrocephalus and alleviate neurological defects.Taken together,our study demonstrated us that hemin could directly induce neuron necroptosis and IL-1R1 expression.The coupling between IL-1R1 and necrosome is the key point in activating RIP1-RIP3-MLKL mediated neuron necroptosis.Neuronal necroptosis,inflammatory response in brain tissues and acute brain edema can be reduced by inhibiting the expression of IL-1R1.Disrupt the coupling between of IL-1R1 and necrosome is an important mechanism and therapeutic target to control hemin induced neuron injury after ICH.