Application of Ikzf1 as therapeutic target of ICH

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of Ikzf1 as a therapeutic target of ICH (Infectious Cells). The inventor finds that inflammatory response and scorch symptom are inhibited in lipopolysaccharide (LPS)-induced BV-2 cells and ICH rats by regulating the Ikzf1, the Ikzf1 in microglial cells is inhibited, secretion of totipotent interferon (Ptn) is increased, and apoptosis is inhibited; therefore, Ikzf1 is a key target for regulating the differentiation direction of microglial cells to influence secondary injury under ICH conditions. The invention also provides an application of the Ikzf1 in preparation of a medicine for treating ICH. The Ikzf1 provided by the invention is used as a therapeutic target of ICH, and is used for regulating the conversion of microglial cells from a proinflammatory state to a neural restoration promoting state, and a new therapeutic choice is provided, so that secondary brain injury after ICH is relieved.

Description

Technical Field

[0001] The patent of this invention relates to the field of biomedicine technology, and specifically, to the application of Ikzf1 as a therapeutic target for ICH. Background Art

[0002] Intracranial hemorrhage (ICH) is a clinical syndrome in which blood vessels in the brain are damaged for different reasons and rupture and bleed, resulting in blood accumulation in the brain parenchyma. It is a type of stroke. Considering that most ICH survivors experience neurological damage and are prone to recurrent stroke, cognitive decline, and systemic vascular diseases, the study of ICH in the field of neuropathology is of great significance. During the progression of ICH, intracerebral hemorrhage causes tissue damage, which triggers a series of secondary lesions characterized by excessive production of proinflammatory cytokines, neuronal loss, and brain edema. Microglia may be activated and enriched at the site of injury within a few hours. Therefore, neuroinflammation is the main cause of continued injury after ICH.

[0003] Traditionally, microglial activation is believed to play a harmful role in ICH because activated microglia become inflammatory as ICH progresses, and inhibiting microglial activation can alleviate ischemia-induced brain damage. Existing research evidence also shows that inhibiting microglial activation plays a key role in alleviating neuronal apoptosis, promoting neurogenesis, and promoting functional recovery after ICH. The differential polarization of microglia may explain the biphasic role of microglia in ICH. In addition, the interaction between microglia and neurons may also play a key role, but its specific mechanism is still not very clear. Therefore, regulating the transition of microglia from a pro-inflammatory state to a state that promotes neural repair is a potential therapeutic option to alleviate secondary brain injury after ICH.

[0004] With the development of regenerative medicine, exosome (Exo) therapy has great potential as a neuroprotective treatment for ICH, as it can significantly improve learning and memory effects in ICH rats by regulating dysphagia and apoptosis. However, the dynamic changes of cells in the damaged microenvironment after ICH treatment with human mesenchymal stem cells (hMSCs)-derived Exo have not been fully understood. In addition, the regulatory mechanisms involved in the transition of microglial states in ICH treatment need to be deeply understood.

[0005] Single-cell RNA sequencing (scRNA-Seq) and single-cell spatial transcriptome (ST) technologies reveal the spatial and developmental heterogeneity of brain microglia and provide a high-resolution view of the transcriptional landscape of microglia during neurodevelopment and disease progression. To further decipher the regulation of microglia under ICH conditions, the inventors of the present application integrated scRNA-seq and ST technologies in a rat model of ICH induced by type IV collagenase. By comparing the transcriptional dynamics between ICH and exosome (Exo) treatment conditions (ICH+Exo), the inventors of the present application found that the ankylosing mediator Ikaros (Ikzf1) is a key trigger of secondary injury in ICH in the MIkzf1 microglial subtype (highly expressing Ikzf1). Ikzf1 encodes a transcription factor belonging to the zinc finger DNA binding protein family. Alterations in Ikzf1 levels in the adult hippocampus are sufficient to induce synaptic plasticity and memory deficits by altering the state and function of microglia.

[0006] However, there is no related report on the role of Ikzf1 in microglia after ICH in the prior art. Therefore, the inventors of the present application provide an application of Ikzf1 as a therapeutic target for ICH through research. Summary of the invention

[0007] The purpose of the present invention is to solve the problem mentioned in the background art that there is no relevant report on the role of Ikzf1 in microglial cells after ICH in the prior art, and to provide an application of Ikzf1 as a therapeutic target for ICH.

[0008] The above-mentioned purpose of the present invention is achieved like this:

[0009] The present invention provides application of Ikzf1 as a therapeutic target for ICH.

[0010] Furthermore, the application method is: by inhibiting Ikzf1 in microglial cells, the secretion of omnipotent interferon is increased and apoptosis is inhibited.

[0011] The present invention also provides application of Ikzf1 in preparing medicine for treating ICH.

[0012] The present invention also provides the use of the Ikzf1 inhibitor in preparing a medicine for treating ICH.

[0013] The difficulty and significance of the technical problem solved by the present invention are:

[0014] Research evidence in the prior art shows that microglial activation plays a key role in alleviating neuronal apoptosis, promoting neurogenesis, and promoting functional recovery after ICH. The differential polarization of microglial cells may explain the biphasic role of microglial cells in ICH. In addition, the interaction between microglial cells and neurons may also play a key role, but its specific mechanism is still not very clear in existing research reports. To this end, the inventors of the present application found that both inflammatory response and anxiety were inhibited in lipopolysaccharide (LPS)-induced BV-2 cells and ICH rats by regulating Ikzf1. By inhibiting Ikzf1 in microglial cells, the secretion of omnipotent interferon (Ptn) was increased and apoptosis was inhibited. It was confirmed that Ikzf1 is a key target for regulating the differentiation direction of microglial cells to affect secondary injury under ICH conditions. The inventors of the present application revealed the central role of Ikzf1 in coordinating the transcriptional program of microglia in response to ICH, highlighting Ikzf1 as a potential therapeutic target for ICH and providing a new treatment option to alleviate secondary brain injury after ICH.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides for the first time the application of Ikzf1 as a therapeutic target for ICH. By regulating and inhibiting Ikzf1 in microglial cells, the secretion of pan-interferon (Ptn) is increased and apoptosis is inhibited, and the microglial cells are regulated from a pro-inflammatory state to a state that promotes neural repair. The present invention provides a new treatment option that can reduce secondary brain damage after ICH and provides a new idea for the development of ICH therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1: This is a spatial transcriptomic analysis of Exo treatment after ICH in an embodiment of the present invention ((A) Annotated brain cryosections are displayed on the spatial transcriptomic (ST) slide, highlighting the cerebral cortex (green), brain nuclei (red), fiber bundles (yellow), hippocampus (light blue), perihematoma area (purple), hematoma area (dark green) and thalamus (pink) and other regions; (B) Bubble map shows the characteristic markers of these brain regions; (C) Expression map of genes Stx1a, Penk, Plp1, Hpca, Il18, Spp1 and Prkcd across ST slides; (D) Monocle 2 Analysis showing the changes in the developmental trajectory of cells in the perihematoma area in the ICH and ICH+Exo groups over time (after 3-14 days); (E) Heat map representing the dynamic expression of genes that showed significantly different patterns between the ICH and ICH+Exo groups, divided into five clusters by hierarchical clustering; (F) Comparative analysis of KEGG pathway activity between different ST samples was evaluated by using gene set variation analysis (GSVA), pathway scores were normalized, and significance was determined when the adjusted P value was <0.05);

[0018] Figure 2 The dynamic changes of the cellular microenvironment after ICH in the embodiment of the present invention ((A) scRNA-seq data were visualized by UMAP, and 12 different cell types were identified: Astro-Epen (astrocytes and ependymal cells), CUN-LGEGABA (GABAergic cells from the cerebral nucleus and lateral striae glial processes), CTX-CGE GABA (GABAergic cells from the cerebral cortex and caudal ganglia), CTX-MGE GABA (GABAergic cells from the cerebral cortex and central ganglia), DG-IMNGlut (glutamatergic immature neurons from granular gyrus neurons), Immune (immune cells), IT-ET Glu (glutamatergic cells from the intraparietal and extraparietal areas), LSX GABA (GABAergic cells from the lateral brain complex), NP-CTX-L6bGlut (glutamatergic cells from layer 6b of the near-projection cerebral cortex), OPC-Oligo (oligodendrocyte precursors and oligodendrocytes), TH- Glut (glutamatergic cells from the thalamus) and blood vessels; (B) Bar graphs show the proportion of each cell type in the ICH and ICH+Exo groups at different time points; Key KEGG pathway terms associated with significantly enriched genes identified by GSEA were found in different groups; (D) Representative GO biological process pathway terms associated with significantly enriched genes identified by GSEA; (E) Cell types identified by scRNA-seq and regions defined by spatial transcriptomics (ST) were mapped temporally and spatially (3–14 dpi); the fan plots in ST show the expression ratios between different cell types);

[0019] Figure 3 is the characteristics and functional analysis of microglial subtypes in the embodiment of the present invention ((A) TSNE diagram showing four immune subtypes; (B) dot plot of the standardized average expression of characteristic genes of each immune subtype; TSNE plot showing the four microglial subclasses; (D) TSNE plot showing the temporal changes and proportions of microglial subclasses; (E) Violin plots of the normalized mean expression of homeostatic genes and disease-associated microglial (DAM) genes for each microglial subclass; (F) Representative GO biological process pathway activities scored for each subtype by GSEA; (G) Double immunostaining of Iba1 and Ptn was performed in the area near the hematoma and quantitatively analyzed for Sham, ICH, and ICH+Exo groups (3 dpi), n = 3 per group Rats; (H) Double immunostaining of Iba1 and Ikzf1 was performed near the hematoma area and quantitatively analyzed in Sham, ICH and ICH+Exo groups (3 dpi), n=3 rats in each group; (I) The proportion of microglial subclass conversion in the rat brain obtained from Sham, ICH and ICH+Exo groups (3 dpi) was analyzed by flow cytometry, n≥6 rats in each group; data are presented as mean ± SD; normal distribution was checked by Shapiro-Wilk test, P>0.05);

[0020] Figure 4 Regulators and trajectory analysis of microglia subclasses in an embodiment of the present invention ((A) SCENIC regulator analysis shows key regulators in microglia subclasses; (B) Ikzf1 gene regulatory network (GRN) diagram, describing the interaction between key regulators and high-scoring target genes; (C) GSEA analysis results of high-scoring target genes of representative GO biological processes, applicable to Ikzf1; (D) Developmental trajectories of microglia subclasses inferred using Monocle2, and colored according to subclass classification; (E) Developmental trajectories of microglia visualized using Monocle2, colored according to pseudo-time scores; (F) Heat map showing distinct gene expression patterns on two developmental trajectories (branch 1 and branch 2) (derived from the MResident subclass); Hierarchical clustering divides genes into four clusters, and GSEA-enriched biological process pathways are shown on the right; (G) Monocle2-derived fitting curves showing the expression trends of specific pathways in branch 1; (H) AUCELL scores of specific pathways displayed on the tSNE map of microglia);

[0021] Figure 5The results of the present invention show that Exo improves the cognitive function of ICH rats, improves ICH pathology and microglial pyrolysis ((A, C) The cognitive function of rats in the sham, ICH and ICH+Exo groups was evaluated by MWM test; (B) The linear graph shows the escape latency; (D) The bar graph shows the time ratio of staying in the target quadrant / total time ratio (n≥4); (E) The brains of rats in the ICH and ICH+Exo groups, protein expression was detected by WB; (F) The bar graph shows the expression of Nlrp3 / β in the brain -actin, Asc / β-actin and caspase-1(P20) / Pro-Caspase-1 ratio (n=3); (G) Brain tissues of sham, ICH and ICH+Exo groups (3-7dpi), protein expression was detected by ELISA (n=6); (H) Representative electron micrographs showing the ultrastructure of the brains of rats in sham, ICH and ICH+Exo groups (3dpi); (I) Bar graph showing the ratio of damaged myelin sheath (n=3);

[0022] Figure 6 is a schematic diagram of the workflow of the entire study in the embodiment of the present invention (including sample acquisition, sequencing and integrated analysis);

[0023] Figure 7 It is the injection site and sequencing sampling site of collagenase in the ICH model in the embodiment of the present invention;

[0024] Figure 8 are the characteristic genes of 12 cell types and the changing trends detected by scRNA-seq in the embodiment of the present invention ((A) the heat map shows the characteristic genes of 12 cell types detected by scRNA-seq; (B) the line graph represents the changing trends of the cell types in the ICH and ICH+Exo groups);

[0025] Fig. 9 is the subtype analysis in the embodiment of the present invention;

[0026] Fig.10 are characteristic genes of the four microglial subclasses in the embodiments of the present invention;

[0027] Fig.11 is the GO enrichment of the transcriptional gene network in the embodiment of the present invention;

[0028] Fig.12 The regulatory pathways and key target genes of miRNA in Exo in the embodiment of the present invention ((A) bar graph shows the distribution of small RNA length; (B) Ven diagram shows the co-expression of miRNA; (C) Ven diagram shows the co-expression of target genes; (D) representative GO (biological process) pathways in target genes by GSEA; the significance threshold was set to adjusted P value <0.05). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0031] With reference to the accompanying drawings of the specification, the following are preferred embodiments provided by the present invention.

[0032] Example: The embodiment of the present invention provides the application of Ikzf1 as a therapeutic target for ICH. The application method is: by inhibiting Ikzf1 in microglial cells, the secretion of omnipotent interferon is increased and apoptosis is inhibited.

[0033] The embodiment of the present invention also provides the use of Ikzf1 in preparing a drug for treating ICH.

[0034] The embodiment of the present invention also provides the use of an Ikzf1 inhibitor in the preparation of a drug for treating ICH. The Ikzf1 inhibitor is, for example, Lenalidomide (Leprol).

[0035] The following is an experiment of the implementation scheme of the present invention:

[0036] Spatial transcriptional profile characteristics of Exo treatment after cerebral hemorrhage: The inventors of this application used a rat model of cerebral hemorrhage induced by type IV collagenase to conduct experiments, and treated rats with cerebral hemorrhage with Exo by intracranial stereotactic injection. Then, the results of the Morris water maze (MWM) test showed that after Exo treatment, the cognitive impairment of rats with cerebral hemorrhage was significantly improved, and spatial learning and memory performance were improved (such as Figure 5 (as shown in AD in the figure). Existing studies have shown that after cerebral hemorrhage, Nlrp3 inflammasome plays an important role in cognitive decline, and the accumulation of Nlrp3 inflammasome can induce cell pyroptosis and accelerate brain degeneration. Therefore, in order to explore whether Exo can inhibit the expression of Nlrp3 inflammasome in the rat model of cerebral hemorrhage, the inventors of the present application detected Nlrp3 inflammasome-related proteins including Nlrp3, Asc and caspase-1 by immunoblotting (WB). The results showed that Exo reduced the expression of Nlrp3, Asc and caspase-1 in rats with cerebral hemorrhage (as shown in AD in the figure). Figure 5 At the same time, the results of ELISA also showed that Exo reduced the levels of Il1β, Il18 and Nlrp3 in the brains of rats with cerebral hemorrhage (as shown in Figure 5). Figure 5In addition, electron microscopy images showed that Exo improved the demyelination damage of myelin sheath of nerve cells after intracerebral hemorrhage (as shown in G). Figure 5 The above experimental results indicate that Exo improves cognitive function and reduces inflammatory pathology and neural damage in rats with cerebral hemorrhage.

[0037] In order to reveal the time and ST changes of brain events after ICH and Exo treatment, the inventors of the present application processed time series of frozen rat brain tissue. After type IV collagenase-induced ICH, ICH+Exo-treated and sham-operated rat brain tissue (referred to as ICH group, ICH+Exo group and sham-operated group, respectively), the inventors of the present application collected rat brain tissue samples along the coronal plane at intervals of one millimeter before and after the hematoma area for subsequent standardized time series analysis (1, 3, 7 and 14 dpi) and ST analysis (e.g. Figure 6 and Figure 7 The inventors of the present application deconvoluted the ST dataset to infer the activity map after hematoxylin-eosin (H&E) staining and bright field imaging, and annotated representative STs according to tissue characteristics and marker genes in the Allen Brain Atlas (https: / / portal.brain-map.org). Seven major regions were defined in the rat brain: cerebral cortex, brain nuclei, nerve fiber bundles, hippocampus, perihematoma area, hematoma area, and thalamus ( Figure 1 However, the sham group did not appear to contain perihematoma or hematoma areas ( Figure 1 BC in Fig.

[0038] In order to interpret the transcriptome dynamics during the transition from ICH to the perihematoma region, the inventors of the present application used Monocle 2 to pseudo-time sequence brain spots and reconstruct their trajectories ( Figure 1 The spots close to the hematoma area were sorted at a relatively late stage on the pseudo-time trajectory. In the ICH and ICH+Exo groups, the genes significantly changed along their respective trajectories were clustered into five groups ( Figure 1(F in Figure 5). In the ICH and ICH+Exo groups, class I genes (represented by Axl, Ptn, Grm5, Nrxn1, and Nrxn3, which are associated with axon development) decreased expression near the hematoma area. Class II genes (represented by Tlr8, S100a9, and Il1b, which are associated with inflammatory factors) increased expression near the hematoma area. Class III genes (represented by Ptprc and Ctsz, which are associated with microglial activation, and Spp1, Gpnmb, and Ifit, which are associated with neurodegenerative diseases) increased expression in a subchronological order. Notably, genes associated with pyroptosis and inflammation (class V, represented by Gsdmd, Irf1, Irf8, and Stat3) showed different expression patterns between the ICH and ICH+Exo groups.

[0039] Gene set variation analysis (GSVA) ​​of ST data revealed enriched pathways in ICH and ICH+Exo groups ( Figure 1 F in Figure 3). After 3 days, the ICH group had higher enrichment scores in pathways related to cellular immune activation (such as FcεRI signaling pathway, B cell receptor signaling pathway, T cell receptor signaling pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway) compared with the ICH+Exo group. Correspondingly, at the same time point, the ICH group had higher enrichment scores in the expression of apoptosis pathways compared with the ICH+Exo group. However, after 7 and 14 days, the ICH+Exo group had higher enrichment scores in pathways related to synaptic plasticity (such as long-term potentiation, axon guidance, and wnt signaling pathways).

[0040] Dynamic changes of cellular microenvironment after intracerebral hemorrhage: In the ICH group, ICH+Exo group, and Sham group, rat brain tissue samples were collected at intervals of one millimeter before and after the hematoma area in the coronal plane for subsequent standardized time series analysis (1, 3, 7, 14, 21, and 28 dpi) for scRNA-seq analysis ( Figure 7 AB). After quality control, a total of 278,394 cells were described for data analysis. Twelve different cell types were identified by using the MapMyCells platform31, combining marker genes for each cell type, including astrocytes-Epen, CUN-LGE GABA, CTX-CGE GABA, CTX-MGE GABA, DG-IMN Glut, immune cells, IT-ET Glu, LSX GABA, NP-CTX-L6b Glut, OPC-Oligo, TH Glut, and vascular cells (e.g. Figure 2 A in Figure 8 A).

[0041] The inventors of the present application observed that the CUN-LGE GABA, LSX GABA and TH Glut cell populations were significantly reduced in the acute (3dpi) and subacute (7-14dpi) stages of ICH and ICH+Exo groups. It is worth noting that in the ICH+Exo group, the decrease in cell percentage was less obvious than in the ICH alone group. This suggests that Exo treatment helps to alleviate the loss of these specific cell types after injury. In addition, during the period of 3-7dpi, the immune cell counts in both groups increased significantly, while the increase in the ICH+Exo group was smaller. This indicates that the ICH+Exo group had a lower level of immune-mediated stress relative to the ICH group. Ast-Epen and OPC-Oli markers also showed changes after injury in both groups. However, these changes were more significant in the ICH+Exo group, indicating that the myelin damage, decomposition, repair and remodeling processes carried out in the central nervous system (CNS) were enhanced. During the treatment process, other cell types showed only slight changes ( Figure 2 B and Figure 8 (B) The overall pattern of cellular responses highlights the differential effects of Exo therapy on various neural and immune cell populations after ICH.

[0042] In order to investigate the changes in cellular pathways and biological functions in ICH and ICH+Exo tissues, the inventors of the present application performed gene set abundance analysis (GSEA) on the Kyoto genome and gene ontology biological process data from 3-14dpi, showing the most significant cellular changes. The results showed that the Sham group was only enriched in ligand-receptor, and this enrichment was also found in the ICH+Exo group during the 3-14dpi period. In addition, immune activation-related pathways (cell adhesion molecules, Rap1 signaling pathway, osteoblast differentiation, Fcgamma R-mediated phagocytosis, and platelet activation) were enriched in the 3-7dpi group. However, pathways related to neural repair and neural function, such as GABAergic synapses, axon guidance, and retrograde endocannabinoid signaling pathways, were enriched in the Exo 3-14dpi group (see Figure 2 GO biological process abundance analysis showed similar results. In the Sham group, ICH 14dpi group and ICH+Exo group (3-14dpi), pathways related to synapses and signal transduction were significantly enriched, such as chemical synaptic transmission regulation, transsynaptic signal transmission regulation, and motor behavior regulation. The ICH group (3-7dpi) was significantly enriched in bone marrow leukocyte activation, leukocyte activation involved in immune response, and leukocyte cell-cell adhesion (see Figure 2 D).

[0043] The inventors of this application performed a comprehensive analysis of gene sets from scRNA-seq data and applied a robust cell type decomposition (RCTD) to interpret the ST data. This approach enabled the inventors of this application to identify significant overlap between immune cells in ICH and ICH+Exo tissues and gene sets specific to the perihematoma region and hematoma region in the ST data (see Figure 2 E in ). scRNA-seq data helped identify microglia cells, and subsequent RCTD analysis showed their specific enrichment in the perihematoma and hematoma regions. Further analysis of the cerebral cortex showed a predominance of DG-IMN glutamate, IT-ET glutamate, and NP-CTX-L6P glutamate cells. In contrast, the thalamus was mainly enriched in CTX-MGE glutamate and TH glutamate cells. The cerebral nuclei were characterized by a high prevalence of CUN-LGE GABA cells, while the fiber regions were significantly enriched in OPC-Oli cells. The spatial distribution of these cell types is consistent with the results of recent studies on the whole-brain cell atlas of mice, providing insights into the cell composition inside and outside the hematoma region. This consistency with existing data emphasizes the relevance and accuracy of the inventors' research results in understanding the cellular landscape in the context of ICH.

[0044] Microglial subtype conversion during Exo treatment: The inventors of this application then performed cell subtype analysis on the scRNA-Seq data and identified 29 cell subtypes ( Fig. 9 The inventors of this application focused on the immune cells in the perihematoma and hematoma areas determined by ST, mainly focusing on MDM, DC, microglia and neutrophil subtypes ( Figure 3 AB in the brain). Among these four subtypes, microglia dominate in number. In addition, microglia are the main immune cells in the brain, so the inventors of this application chose to conduct a more in-depth study on them. Based on the gene expression profile, the inventors of this application further divided microglia into four subtypes: Mptn, MIkzf1, MResident and Mfew ( Figure 3 C and Fig.10 These subclasses all expressed the microglial markers Cst3 and Csf1r, confirming their identity ( Figure 3 In the inventors' analysis, the Mfew subclass was downgraded due to low expression levels and limited temporal presence.

[0045] In the acute (3 dpi) and subacute (7-14 dpi) stages of ICH, the MIkzf1 subtype was dominant, with high expression of Apoe, B2m, Il18, Il6r, Nlrp3, and Ikzf1, suggesting a role in inflammation and immune response ( Figure 3In contrast, in the ICH+Exo group, the MPtn subclass became dominant (62.31-73.14%), characterized by high levels of neuroprotective and mature disease-related markers such as Apoe, B2m, Cd9, Trem2, and Bdnf ( Figure 3 DE in ). The MResident subclass, which was dominant in the Sham group (62.52%), exhibited low levels of disease-related genes, suggesting a steady-state state. GSEA showed that the MResident and MPtn subclasses were significantly enriched in synaptic and neurodevelopmental pathways, consistent with their roles in synaptic pruning and neural support. The MIkzf1 subclass was enriched in immune regulatory pathways, supporting its involvement in immune responses after ICH ( Figure 3 F). By immunofluorescence and flow cytometry, the inventors of the present application analyzed the expression of Ptn and Ikzf1 in microglia in the rat brain after ICH. It was observed that the co-expression of Ptn and Iba1 increased significantly in the ICH+Exo group, while the co-expression of Ikzf1 and Iba1 increased in the ICH group ( Figure 3 Flow cytometry confirmed these findings, with significant differences in expression patterns between the ICH group and the ICH+Exo group ( Figure 3 I in.

[0046] To elucidate the main driver genes in the two specific microglial subtypes, the inventors of the present application used single-cell regulatory network inference and clustering (SCENIC) analysis. This analysis identified key transcripts in these subtypes: Srrm3, Zmat4, Bcl11a, Sox6, Klf9, Npas2, and Rfx3 dominated the MPtn subtype; Ikzf1, Runx1, Irf8, Nfkb1, and Pou2f2 were characteristic of the MIkzf1 subtype; Bmyc, Egr4, Gtf2f1, Ets2, Egr4, and Srebf1 were mainly present in the MResident subtype ( Figure 4 In order to further explore the role of key transcription factors in MIkzf1 isoforms, the inventors of the present application constructed a gene regulatory network (GRN) for the top five transcripts with Normalized Enrichment Scores (NES) exceeding 3.5 ( Figure 4 In addition, the inventors of the present application conducted GO enrichment analysis on the biological processes of each transcript and its regulatory targets ( Fig.11 ). Notably, Ikzf1 and its target genes are significantly associated with immune cell differentiation and activation, Fc-γ-mediated immune activation, and immune response activation signaling ( Figure 4C). These findings suggest that Ikzf1 plays a key role in microglia-mediated immune responses and regulation of inflammatory processes after ICH.

[0047] Furthermore, pseudotemporal analysis revealed developmental trajectories from MResident to MIkzf1 or MPtn, suggesting divergent roles in microglial transition after ICH ( Figure 4 DE in ). These findings delineate dynamic changes in microglial subtypes after ICH and identify distinct pathways and transcriptional regulators underlying their roles in immune responses and neuroprotection. MIkzf1 and MPtn subtypes represent distinct responses to ICH and have important implications for understanding microglial function and therapeutic targeting under neuroinflammatory conditions.

[0048] The inventors of the present application performed pseudo-time analysis to identify the changes in genes from the initial state to the two different cell branches. These genes were divided into four categories and visualized using heat maps. Subsequent enrichment analysis using the GO database focused on biological processes ( Figure 4 F in the figure). This analysis showed that the first group of genes associated with the first branch of the pseudo-timeline were significantly enriched in pathways related to lymphocyte differentiation, positive regulation of cell activation, and various immune response pathways. In contrast, the second group of genes associated with the second branch were enriched in pathways related to cognition, learning, memory, and neuroprotective functions, indicating a role for this branch in neuroprotection after ICH. Analysis of the third group showed the least difference from the starting point, enriched in pathways involved in motor behavior, synaptic assembly, and cell junction assembly, consistent with homeostatic microglial function. Further pathway analysis showed that the expression pathways along the first branch from MResident to the MIkzf1 isoform increased, including lymphocyte differentiation, myeloid cell differentiation, and positive regulation of cell activation, while the pathways along the second branch towards the MPtn isoform decreased ( Figure 4 The area under the curve (AUC) scores confirmed that these pathways were mainly enriched in the MIkzf1 isoform ( Figure 4 H in Figure 3). These results suggest that MIkzf1 and MPtn isoforms represent different trajectories of microglial differentiation after ICH, with the MIkzf1 isoform showing a stronger affinity to immune-related pathways, whereas the MPtn isoform is more consistent with neuronal function.

[0049] miRNA in Exo is used to regulate Ikzf1 expression in ICH treatment: In order to detect the regulatory pathways and key target genes involved in improving ICH prognosis in Exo therapy, the inventors of the present application conducted sequencing analysis of miRNA in Exo ( Fig.12 A total of 178 miRNAs were detected in all three replicates ( Fig.12 B), these miRNAs target a total of 346 overlapping genes ( Fig.12 B in the figure). The GO database focuses on the enrichment analysis of biological processes and shows that the enriched pathways can be divided into seven groups, including myeloid cell differentiation, positive protein kinase activity, intrinsic signaling pathway damage in the process of neuronal apoptosis and death, etc. ( Fig.12 D in Figure 4). Pathways associated with myeloid cell differentiation include the differentiation of microglia in the brain. Microglia are a type of myeloid cell. These results suggest that Exo regulates microglial subtype switching in myeloid cell differentiation function by regulating some key genes including Ikzf1.

[0050] In summary, the above studies by the inventors of the present application show that reducing Ikzf1 expression can prevent microglia from converting from the MResident subtype to the MIkzf1 subtype after ICH. This intervention promotes the conversion from the MResident to the MPtn subtype, rather than the MIkzf1 subtype, thereby reducing inflammation, cremation, and neuronal apoptosis in the affected microenvironment after ICH. Ikzf1 has been shown to be a promising therapeutic target for ICH and brain injury, providing a new perspective on the etiology and treatment of neuroinflammation and neuronal injury.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Ikzf1 as a therapeutic target for ICH.

2. The use according to claim 1, characterized in that: The application method is: by inhibiting Ikzf1 in microglial cells, the secretion of omnipotent interferon is increased and apoptosis is inhibited.

3. Application of Ikzf1 in the preparation of drugs for treating ICH.

4. Application of Ikzf1 inhibitors in the preparation of drugs for the treatment of ICH.

Images