Application of Si-Ikzf1 in preparation of products for inflammation treatment and neuroprotection

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of Si-Ikzf1 in preparation of products for inflammation treatment and neuroprotection, the Si-Ikzf1 provided in the scheme of the invention reduces formation of cell eschar by promoting microglial cell MResident subtype to be generated into MPtn subtype instead of MIkzf1, and improves post-ICH neurological functions by regulating Ikzf1-SIRT1 and downstream signal pathways thereof. The scheme of the invention reveals high expression of inflammatory bodies and scorch symptom related proteins in MIkzf1 subtype microglial cells, and Si-Ikzf1 can promote the conversion of MResident in ICH rats to MPtn instead of MIkzf1, and the MPtn represents an anti-inflammatory effect of promoting nerve remodeling, and the Si-Ikzf1 is capable of promoting the conversion of MResident in ICH rats to MPtn in ICH rats to MPtn in ICH rats to MPtn in ICH rats to MPtn in ICH rats. It is revealed that Si-Ikzf1 hinders MIkzf1 inflammation, scorch symptom and cell apoptosis, so that cognitive behavior and demyelination injury are improved; moreover, the scheme of the invention proves that the Si-Ikzf1 can reverse the reduction of SIRT1 expression in BV-2 cells and ICH rat brains after inflammation stimulation, and promote the up-regulation of Ptn in MPtn.

Description

Technical Field

[0001] The patent of this invention relates to the field of biomedicine technology, specifically, to the application of Si-Ikzf1 in the preparation of products for inflammation treatment and neuroprotection. Background Art

[0002] Intracranial hemorrhage (ICH) is a common neurological injury associated with a mortality rate of up to 50%. Since most ICH survivors experience neurological damage and are prone to recurrent stroke, cognitive decline, and systemic vascular diseases, the study of ICH is of great value in the field of neurological diseases. In the course of ICH, substantial bleeding in brain tissue causes tissue damage, which triggers a series of secondary lesions characterized by excessive production of proinflammatory cytokines, neuronal loss, and cerebral edema. Microglia may be activated and enriched at the site of injury within hours. Neuroinflammation is the main cause of subsequent damage in ICH.

[0003] Traditionally, microglial activation is thought to play a deleterious role in ICH because activated microglia undergo dysregulation as ICH progresses, and inhibiting microglial activation can alleviate ischemic brain injury. In recent years, increasing research evidence has shown that after ICH, microglial activation plays a key role in alleviating neuronal apoptosis, promoting neurogenesis, and promoting functional recovery. 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 not fully understood. Therefore, regulating the transition of microglia from a pro-inflammatory condition to a state that promotes neural repair is a potential therapeutic option to alleviate subsequent brain injury after ICH.

[0004] With the development of regenerative medicine, exosome therapy has become an important potential means of neuroprotective treatment for ICH, because it can significantly improve learning and memory effects in ICH rats by regulating dysphagia and apoptosis. However, the dynamic changes of cells after human mesenchymal stem cell (hMSCs)-derived exosome treatment after ICH injury have not been fully appreciated. In addition, it is necessary to understand the regulatory mechanism of microglial state transition involved in ICH treatment.

[0005] Single-cell RNA sequencing (scRNASeq) and single-cell spatiotemporal 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 type IV collagenase-induced ICH rat model. By comparing the transcriptional dynamics between ICH and exosome treatment conditions (ICH+Exo), the inventors of the present application found that in ICH, the inflammatory mediator Ikaros (Ikzf1) is a key trigger of ICH secondary injury in the MIkzf1 microglial subtype (highly expressing Ikzf1).

[0006] Ikzf1 encodes a transcription factor belonging to the zinc ring finger DNA binding protein family. Alterations in Ikzf1 levels in the adult hippocampus are sufficient to induce synaptic plasticity and memory deficits by altering microglial state and function. However, the mechanism of action of Ikzf1 in microglia after ICH is still unclear and has not been reported in the prior art.

[0007] Therefore, the inventors of the present application have confirmed through research and experiments that they provide the use of Si-Ikzf1 in the preparation of products for inflammatory treatment and neuroprotection to solve the above problems. Summary of the invention

[0008] The purpose of the present invention is to solve the technical problems raised in the background technology, and to provide the application of Si-Ikzf1 in the preparation of products for inflammation treatment and neuroprotection.

[0009] The above-mentioned purpose of the present invention is achieved like this:

[0010] The solution of the present invention provides the use of Si-Ikzf1 in preparing products for treating inflammation and protecting nerves.

[0011] Furthermore, the inflammation treatment and neuroprotection are treatments during the course of intracranial hemorrhage.

[0012] Furthermore, the product is a medicine.

[0013] Furthermore, Si-Ikzf1 reduces cell eschar formation by promoting the generation of microglial MResident subtype into MPtn subtype rather than MIkzf1, and improves neurological function after ICH by regulating Ikzf1-SIRT1 and its downstream signaling pathways.

[0014] The difficulty and significance of the technical problem solved by the present invention are:

[0015] Existing research evidence shows that after ICH, microglial activation plays a key role in reducing neuronal apoptosis, promoting neurogenesis and promoting functional recovery. The differential polarization of microglia may explain the biphasic effect of microglia in ICH. In addition, the interaction between microglia and neurons may also play a key role, but its specific mechanism is not yet fully understood. The inventors of this application provide the application of Si-Ikzf1 in the preparation of products for inflammatory treatment and neuroprotection through research, and in the experiment, by regulating Ikzf1, the inflammatory response and anxiety were inhibited in lipopolysaccharide (LPS)-induced BV-2 cells and ICH rats. After inhibiting Ikzf1, the secretion of pro-cell factors (Ptn) in microglia increased, and apoptosis was inhibited. Since Ptn is secreted by the main subtype MPtn in the ICH+Exo group, it is confirmed that it has an inhibitory effect on cell apoptosis. Therefore, the scheme of the present invention confirms that Ikzf1 is a key target for regulating the differentiation direction of microglia, affecting secondary damage under ICH conditions.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The scheme of the present invention revealed the high expression of inflammasome and pyrolysis-related proteins in MIkzf1 subtype microglial cells, and revealed that Si-Ikzf1 could promote the transformation of MResident to MPtn in ICH rats, but not MIkzf1, confirming that the latter exhibited anti-inflammatory effects that promoted neural remodeling. Si-Ikzf1 hindered MIkzf1 inflammation, pyrolysis and cell apoptosis, thereby improving cognitive behavior and demyelinating damage. Si-Ikzf1 could promote the upregulation of Ptn in MPtn.

[0018] 2. The application of Si-Ikzf1 provided by the present invention in the preparation of products for inflammation treatment and neuroprotection not only provides a new treatment option for neuroprotective treatment of ICH, but also provides a new idea for the development of ICH drugs for treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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);

[0020] 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);

[0021] 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);

[0022] Figure 4 Regulators and trajectory analysis of microglial subclasses in an embodiment of the present invention ((A) SCENIC regulator analysis shows key regulators in microglial subclasses; (B) Ikzf1 gene regulatory network (GRN) diagram, describing the interaction between key regulatory factors 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 microglial 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);

[0023] 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);

[0024] 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);

[0025] Figure 7 It is the injection site and sequencing sampling site of collagenase in the ICH model in the embodiment of the present invention;

[0026] 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);

[0027] Fig. 9 is the subtype analysis in the embodiment of the present invention;

[0028] Fig.10 are characteristic genes of the four microglial subclasses in the embodiments of the present invention;

[0029] Fig.11 is the GO enrichment of the transcriptional gene network in the embodiment of the present invention;

[0030] Fig.12 The regulatory pathways and key target genes of miRNA in Exo in the embodiment of the present invention ((A) bar graph showing the distribution of small RNA length; (B) Ven graph showing the co-expression of miRNA; (C) Ven graph showing the co-expression of target genes; (D) representative GO (biological process) pathways in target genes by GSEA; the significance threshold was set at an adjusted P value < 0.05);

[0031] Fig.13 In the present invention, Exo and Si-Ikzf1 regulate intercellular communication in MIkzf1 and MPtn through the Ptn-Ptprz1 axis ((A) Heat map shows the changes in cell communication weights in different regions of ST under different experimental conditions: ICH vs. Sham 3 days later, ICH + Exovs. ICH 3 days later, ICH + Exovs. ICH 7 days later; (B) By using the peripheral hematoma area as the signal source, the contribution weights of significant signal pathways are ranked, thereby affecting the overall information flow; (C) Bubble map describes the signal transduction intensity of all subtypes of the PTN pathway in the peripheral hematoma area and other areas; (D) In ​​Sham, ICH and ICH + Exo STs, the co-localization of Ptn and Ptprz1 expression is shown at 3 dpi, red indicates high Ptn expression, green indicates high Ptprz1 expression, and blue indicates =(E) Upper: pie chart predicts the point distribution of the peripheral hematoma area of ​​ST data; Lower: detailed view of the upper figure; (F) TSNE diagram shows the matching of the peripheral hematoma area and hematoma area subclasses; (G) By using MPtn as the signal source, the contribution weights of significant signal pathways are ranked to affect the overall information flow; (H) Bubble chart shows the signal transduction intensity of all PTN pathway subtypes between MPtn and nerve cells; (I) Double immunostaining and quantitative analysis of Ptn and Ptprz1 were performed on the peripheral hematoma area in Sham, ICH and ICH+Exo groups (3dpi), with 3 rats in each group; (J) Double immunostaining and quantitative analysis of Ptn and Ptprz1 were performed on the peripheral hematoma area in Sham, ICH+Si-Ikzf1 and ICH+Si-NC groups (3dpi), with 3 rats in each group);

[0032] Fig.14It is Si-Ikzf1 in the present invention that promotes the development of MResident to MPtn subtype, rather than MIkzf1 subtype, and reduces cell eschar-like death ((A) Double immunostaining of Ikzf1 and Iba1 in peripheral hematoma area in Sham, ICH+Si-Ikzf1 and ICH+Si-NC groups (3 dpi), 3 rats in each group; (B) Double immunostaining of Ptn and Iba1 in peripheral hematoma area in Sham, ICH+Si-Ikzf1 and ICH+Si-NC groups (3 dpi). (C) ELISA was used to detect the protein expressions of Ptn (upper) and Nlrp3 (lower) in BV-2 cells (Ctrl, LPS, LPS+Si-Ikzf1 and LPS+Si-NC groups), with 4 experiments in each group; (D) The protein expressions of Ptn (upper) and Nlrp3 (lower) were detected in the brains of rats in Sham, ICH, ICH+Si-Ikzf1 and ICH+Si-NC groups (3 dpi), with 6 rats in each group; (E) f1 and ICH+Si-NC groups (3dpi), double immunostaining and quantitative analysis of Gsdmd and Iba1 in the peripheral hematoma area were performed, with 3 rats in each group; (F) WB was used to detect the protein expression of BV-2 cells (Ctrl, LPS, LPS+Si-Ikzf1 and LPS+Si-NC groups); (G) The bar graph shows the expression of Nlrp3 / β-actin, Asc / β-actin, caspase-1 (P20 / Pro), IL-1β (P17 / Pro), IL-18 / β-actin in and Gsdmd (P30 / Pro), 3 experiments in each group; (H) WB was used to detect the protein expression in the brain of rats in Sham, ICH, ICH+Si-Ikzf1 and ICH+Si-NC groups (3 dpi); (I) The bar graphs represent the ratios of Nlrp3 / β-actin, Asc / β-actin, caspase-1 (P20 / Pro), IL-1β (P17 / Pro), IL-18 / β-actin and Gsdmd (P30 / Pro), 4 rats in each group);

[0033] Fig.15Inhibition of Ikzf1 in the present invention can prevent microglial eschar death and neuronal apoptosis in ICH rats ((A) Representative TEM images of BV-2 cells in Ctrl, LPS+Si-Ikzf1, and LPS+Si-NC groups; the bottom image shows an inset of the top image at a higher magnification; the bar graph shows the number of wells; magnification: ×10000, scale bar: 2 μm; 3 rats in each group; (B) Representative TEM images of microglia in Sham, ICH+Si-Ikzf1, and ICH+Si-NC groups (under electron microscopy, microglia were darkly stained with flat or jagged nuclear morphology); the bottom image shows the magnification Illustration of the upper top image; bar graph shows the number of wells; magnification: ×10000, scale bar: 2 μm; 3 rats per group; (C) Apoptosis of mouse primary neurons and Sh-Sy5y cells cultured in LPS-treated CM was evaluated by flow cytometry using the AnnexinV-PE apoptosis detection kit; (D) Bar graph shows apoptosis ratio, 3 rats per group; (E) Protein expression of mouse primary neurons and Sh-Sy5y cells cultured in LPS-treated CM was detected by WB; (F) Bar graph shows the expression of Bax / Bcl2, Caspase-3(P17) / Casp in Sh-Sy5y cells (G) Hoechst33342 / PI staining of mouse primary neurons cultured in CM treated with LPS; (H) Bar graph showing PI / Hoechst ratio, 3 rats in each group; (I) WB was used to detect protein expression in brain tissues of Sham, ICH+Si-NC and ICH+Si-Ikzf1 groups; (J) Bar graph showing Bax / Bcl2, Caspase-3(P17) / Caspase-9(Pro) / PARP1(Pro) ratios in BV-2 cells; (G) Hoechst33342 / PI staining of mouse primary neurons cultured in CM treated with LPS; (H) Bar graph showing PI / Hoechst ratios, 3 rats in each group; (I) WB was used to detect protein expression in brain tissues of Sham, ICH+Si-NC and ICH+Si-Ikzf1 groups; (J) Bar graph showing Bax / Bcl2, Caspase-3(P17) / PARP1(Pro) ratios in BV-2 cells / Caspase-3(Pro), Caspase-9(P39) / Caspase-9(Pro), and PARP1(P89) / PARP1(Pro) ratios, 4 rats in each group; (K) TUNEL staining of Sham, ICH+Si-NC, and ICH+Si-Ikzf1 groups (3 dpi); (L) Bar graph showing PI / Hoechst ratios; (M) Double immunostaining of Caspase-3(P17) and Map2 in the peripheral hematoma area and quantitative analysis of Sham, ICH+Si-Ikzf1, and ICH+Si-NC groups (3 dpi, 3 rats in each group);

[0034] Fig.16The present invention improves neurological function through the Ikzf1-SIRT1 axis mediated by Si-Ikzf1 ((A) MRIT2*WI images and three-dimensional modeling of the volume change of the peripheral hematoma area; the bar graph shows the percentage reduction of the peripheral hematoma area, 3 rats in each group; (BE) MWM test was used to evaluate the cognition of rats in the Sham, ICH+Si-Ikzf1 and ICH+Si-NC groups (n≥5); the escape latency is shown in The ratio of the residence time in the target quadrant to the total time is shown in (E); (F) TEM images show the integrity of myelin in Sham, ICH+Si-Ikzf1 and ICH+Si-NC groups; (G) The bar graph shows the percentage of damaged myelin (3 dpi), with 3 rats in each group; (H) TEM images show the integrity of myelin in Sham, ICH+Si-Ikzf1 and ICH+Si-NC groups; (I) The bar graph shows the percentage of damaged myelin (21 dpi), with 3 rats in each group; (J) The time course of the slope of the field postsynaptic potential (fEPSP) of CA3-CA1 long-term potentiation (LTP) in hippocampal slices of ICH+Si-NC and ICH+Si-Ikzf1 groups; the arrow indicates the high-frequency stimulation point; the bar graph in the frame shows Normalized 10-min average of fEPSP slope 60 min after high-frequency stimulation, relative to the baseline value of the last 10 min; (K) WB analysis of brain protein expression in Sham, ICH+Si-Ikzf1, and ICH+Si-NC groups; (L) Bar graph showing the expression ratios of Ikzf1 / β-actin, P-Ampk / Ampk, and SIRT1 / β-actin in ICH rats (3 dpi), 4 rats in each group; (M) WB analysis of protein expression in Ctrl, LPS, LPS+Si-Ikzf1, and LPS+Si-NC groups; (N) Bar graph showing the expression ratios of Ikzf1 / β-actin, P-Ampk / Ampk, and SIRT1 / β-actin in BV-2 cells, 3 rats in each group);

[0035] Fig.17 is the co-localization of Ptn-Ptprz1 in STs in the embodiment of the present invention (co-localization of Ptn and Ptprz1 in STs (3-14 dpi); red represents high expression of Ptn, green represents high expression of Ptprz1, and blue represents high expression of both Ptn and Ptprz1);

[0036] Fig.18It is that Si-Ikzf1 in the embodiment of the present invention improves the behavioral scores of ICH rats (the expression of Ptprz1 and the expression of Ptn in the microglial subtypes of Chst9 GABA, Folh1GABA, HPF Fzd5 Glut, Sema5 GABA, Sst GABA, STR-D1 GABA, STR-D2 GABA and Th PrkcdGrin2c-Glut);

[0037] Fig.19 The experimental procedures in the embodiments of the present invention involve infection of BV-2 cells with lentivirus and collection of conditioned medium for intervention with Sh-Sy5y and mouse primary cells;

[0038] Fig. 20 It is the procedure for infecting rat brain tissue with adenovirus and establishing an ICH model in the embodiment of the present invention;

[0039] Fig.21 The Si-Ikzf1 in the present invention reduces the protein expression of Il-1β and Il18 in bv-2 cells and brain tissues of ICH rats;

[0040] Fig. 22 It is that Si-Ikzf1 in the present invention improves the behavioral scores of ICH rats ((A) mNss was evaluated in ICH rats after 1-28 days, n≥5 rats in each group; (B) sensorimotor dysfunction in ICH rats after 1-28 days was evaluated using the foot stagger test, n≥5 rats in each group);

[0041] Fig.23 : The identification and absorption of exosomes in the examples of the present invention ((a) representative transmission electron microscopy image of exosomes; (b) schematic diagram of exosome particle size; (c) flow cytometry technology to detect the expression of exosome marker proteins; (d) immunoblotting to detect the expression of exosome marker proteins; (e) image of exosomes taken up by BV-2 cells; scatter plots illustrate the uptake of exosomes by BV-2 cells at different time points (2 hours, 4 hours, 6 hours), n = 6 in each group). DETAILED DESCRIPTION

[0042] 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.

[0043] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0044] With reference to the accompanying drawings of the specification, the following are preferred embodiments provided by the present invention.

[0045] Example: The embodiment of the present invention provides the use of Si-Ikzf1 in the preparation of products (drugs) for inflammation treatment and neuroprotection. The inflammation treatment and neuroprotection are treatments in the process of intracranial hemorrhage (ICH). Among them, Si-Ikzf1 promotes the generation of microglial MResident subtype into MPtn subtype instead of MIkzf1, reduces cell eschar formation, and improves neurological function after ICH by regulating Ikzf1-SIRT1 and its downstream signaling pathways.

[0046] The following experimental studies have shown that:

[0047] 1. 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 5 In 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.

[0048] 2. Revealing the time and ST changes of brain events after ICH and Exo treatment: The inventors of the present application processed frozen rat brain tissues in time series. After type IV collagenase-induced ICH, ICH+Exo-treated and sham-operated rat brain tissues (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.

[0049] 3. To interpret the transcriptome dynamics during the transition from ICH to the perihematoma region: The inventors of this application used Monocle2 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.

[0050] 4. Gene set variation analysis (GSVA) ​​of ST data revealed enriched pathways in ICH and ICH+Exo groups ( Figure 1G): 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).

[0051] 4. Dynamic changes of the 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 in ). After quality control (see Methods), 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).

[0052] 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 2B 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.

[0053] 5. Study 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).

[0054] 6. Comprehensive analysis of gene sets from scRNA-seq data: Robust cell type decomposition (RCTD) was applied to interpret ST data. This approach enabled the identification of significant overlap between immune cells in ICH and ICH+Exo tissues and specific gene sets in the perihematoma and hematoma regions in 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.

[0055] 7. 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 Among these four subtypes, microglia are dominant 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 analysis by the present inventors, the Mfew subclass was downgraded due to its low expression level and limited time existence.

[0056] 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 3 In 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.

[0057] 8. Single-cell regulatory network inference and clustering (SCENIC) analysis: To clarify the main driver genes in two specific microglial subtypes, the inventors of this application used single-cell regulatory network inference and clustering (SCENIC) analysis. This analysis identified the key transcripts in these subtypes: Srrm3, Zmat4, Bcl11a, Sox6, Klf9, Npas2 and Rfx3 are dominant in the MPtn subtype; Ikzf1, Runx1, Irf8, Nfkb1 and Pou2f2 are the characteristics of the MIkzf1 subtype; Bmyc, Egr4, Gtf2f1, Ets2, Egr4 and Srebf1 are 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 signal transduction ( Figure 4 C). These findings suggest that Ikzf1 plays a key role in microglia-mediated immune responses and regulation of inflammatory processes after ICH.

[0058] 9. Pseudo-temporal analysis: Pseudo-temporal analysis showed developmental trajectories from MResident to MIkzf1 or MPtn, indicating 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.

[0059] 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 4F 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.

[0060] 10. 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 this 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.

[0061] 11. Exosomes and Si-Ikzf1 regulate intercellular communication between MIkzf1 and MPtn through Ptn-Ptprz1: To elucidate the changes in the cellular microenvironment of the perihematoma and hematoma areas after ICH and the subsequent exosome therapy, the inventors of the present application used CellChat to analyze the cell communication in ST and scRNA-seq data. The results showed that the cell communication in these areas was significantly enhanced after ICH, especially in the ICH group (3dpi) compared with the sham control group ( Fig.13 Figure 4 (A). This increase is associated with the complex pathological response observed around the hematoma after ICH.

[0062] Further analysis showed that the perihematoma region acted as a key signaling hub, with significant pathway involvement including MK, PSAP, GAS, PTN, and GRN. Among these pathways, the PTN pathway was the most responsive, showing a significant increase in activity between 3-7 dpi in the ICH+Exo group compared to the ICH only group ( Fig.13 This is supported by detailed examination of PTN signaling interactions in different regions, identifying targets such as SDC1-4 and PTPRZ1 ( Fig.13 C). The effect of Ptn-Ptprz1 was mainly concentrated between the perihematoma and hematoma areas, which was associated with a significant increase in signal intensity between 3-14 dpi in the ICH+Exo group ( Fig.13 DE and Fig.17 In order to enhance the accuracy of cell microenvironment analysis, the inventors of the present application integrated the ST data of the perihematoma area and the hematoma area with the scRNA-seq data by convolution analysis ( Fig.13 E). ST data further confirmed the increased expression of Ptn and Ptprz1 in these regions, consistent with the distribution of MPtn subtypes.

[0063] Integration of ST data and scRNA-seq data enabled the identification of specific glutamatergic and GABAergic neuron subclasses, including HPF Fzd5 Glut, TH Prkcd Grin2c-Glut, Chst9 GABA, Folh1 GABA, Sema5GABA, STR-D1 GABA, STR-D2 GABA, Sst GABA, Pvalb GABA, and microglial subclasses including MPtn, MIkzf1, MResident, and Mfew ( Fig.13 F). Notably, the MPtn isoform was more prevalent in the ICH+Exo group, suggesting a role in enhanced cell communication. This was confirmed by further communication analysis, highlighting the PTN pathway as the component with the highest signal intensity and significantly increased activity in the ICH+Exo group between 3-14 dpi ( Fig.13 G), while the effect of Ptn-Ptprz1 mainly occurred between MPtn and neurons, which was associated with the significant increase in signal intensity between 3-7 dpi in the ICH+Exo group ( Fig.13 H and Fig.18 Finally, immunofluorescence staining showed that the co-expression of Ptn and Ptprz1 in the perihematoma area was significantly increased in the Si-Ikzf1 and Exo groups compared with the ICH group ( Fig.13 These results highlight the potential of Exo and Si-Ikzf1 therapeutics to promote intercellular communication via the Ptn-Ptprz1 axis and provide insights into their mechanisms of action in alleviating ICH damage.

[0064] 12. Si-Ikzf1 promotes the generation of MResident subtypes into MPtn subtypes rather than MIkzf1, reducing cell eschar formation: In order to study the effect of Ikzf1 on the microglial cell status in vivo (LPS group) and in vitro (ICH group) of ICH, the inventors of the present application used adenovirus to specifically silence the Ikzf1 gene of rat brain microglia (ICH+Si-Ikzf1 group) and used blank vector virus as a control (ICH+Si-NC group). The experimental design and virus information can be found in Fig.19 , while the Ikzf1 gene in Bv-2 cells was silenced by lentivirus (LPS+Si-Ikzf1 group), and blank vector virus was used as a control (LPS+Si-NC group). The experimental design and virus information can be found in Fig. 20 The inventors of the present application further studied the effect of Si-Ikzf1 on the transformation of microglial subtypes in the perihematoma region after ICH. Immunofluorescence analysis showed that compared with the ICH+Si-NC group, the colocalization of Ikzf1-Iba1 in the ICH+Si-Ikzf1 treatment group was significantly reduced, and the colocalization of Ptn-Iba1 was significantly increased ( Fig.14 AB in Figure 4). ELISA results further confirmed that the Ptn protein level was reversed in LPS-induced BV-2 cells and ICH brain tissues treated with Si-Ikzf1 ( Fig.14 These findings suggest that Ikzf1 could be a potential regulator of microglial differentiation from the MResident subtype to the MIkzf1 or MPtn subtype.

[0065] Activation of the Nlrp3 inflammasome in microglia led to the release of proinflammatory cytokines and eschar induction. Si-Ikzf1 treatment significantly reduced the Nlrp3 protein level in LPS-induced BV-2 cells and ICH brain tissues, as shown by ELISA ( Fig.14 CD in the microglia) and reduced the expression of Gsdmd in microglia by immunofluorescence ( Fig.14After treatment, a significant inhibition of the expression of Nlrp3 inflammasome-related proteins, including Asc and cleaved forms of caspase-1 and Gsdmd, was observed in BV-2 cells induced by LPS ( Fig.14 FG in ICH brain tissue ( Fig.14 These results suggest that Si-Ikzf1 inhibits the over-activation of Nlrp3 inflammasome, thereby alleviating the inflammatory response in the ICH model.

[0066] 13. Si-Ikzf1 reduces the release of inflammatory factors in the damaged microenvironment and helps improve neural injury: Previous studies have shown that ICH-induced microglial pyroptosis can lead to cell membrane perforation, thereby increasing inflammatory factors in the damaged microenvironment and inducing apoptosis of peripheral nerve cells. Transmission electron microscopy (TEM) analysis showed that compared with the Si-NC group, the Si-Ikzf1-treated group had less Gsdmd-N pore formation in BV-2 cells (Fig. 7A) and microglial cells around hematoma (Fig. 7B). At the same time, ELISA results showed that the levels of IL-1β and IL-18 were reduced in these models ( Fig.21 ).

[0067] To evaluate the effects of proinflammatory cytokines on neural cells, conditioned medium from LPS-treated BV-2 cells was applied to mouse principal neurons and Sh-Sy5y cells. The medium induced significant apoptosis in these cells, which was reversed by Si-Ikzf1 treatment ( Fig.15 CD in Fig. 7). Western blot analysis showed that Si-Ikzf1 treatment reduced the Bax / Bcl-2 ratio and the cleavage of caspase-3, -9, and PARP1 in Sh-Sy5y cells (Fig. 7E-F). Hoechst 33342 / PI apoptosis staining (Fig. 7G-H) further confirmed the reduction of apoptosis in mouse principal neurons. In addition, Western blot results showed that the levels of apoptosis-related proteins in the ICH+Si-Ikzf1 group were reduced (Fig. 7I-J). TUNEL staining results (Fig. 7K) and immunofluorescence co-staining results of Map2 and Caspase-3 (Fig. 7L-M) together indicated that compound Si-Ikzf1 alleviated cell apoptosis in the perihematoma area after ICH and effectively reduced the expression of apoptosis-related proteins in nerves. These data collectively indicate that targeted inhibition of Ikzf1 in microglia not only facilitates the transition of the MIkzf1 isoform to the MPtn isoform, but also significantly reduces neuronal apoptosis in an inflammatory setting, providing a potential therapeutic avenue for alleviating neural injury after ICH.

[0068] 14. Effects on neurological function through the Ikzf1-Sirtuin 1 (SIRT1) axis: The inventors' research results showed that Si-Ikzf1 alleviated pyroptosis and inflammatory responses after ICH. Magnetic resonance imaging (MRI) showed that the volume of the perihematoma area in the Si-Ikzf1 group was significantly reduced compared with the Si-NC group ( Fig.16 The modified Neurological Severity Score (mNSS) showed that Si-Ikzf1 partially reversed the sensorimotor deficits observed after 1 to 28 days, with significant improvements after 7 days ( Fig. 22 This recovery was confirmed by the plantar fault test, where Si-Ikzf1 treated rats showed a decrease in the number of faults compared to the Sham group after 7 days ( Fig. 22 In addition, the MWM test results confirmed that Si-Ikzf1 significantly alleviated the cognitive impairment of ICH rats, which was manifested by enhanced spatial learning and memory performance, reduced escape latency, increased residence time in the target quadrant, and increased frequent crossing of the platform ( Fig.16 Ultrastructural analysis using transmission electron microscopy showed that Si-Ikzf1 improved the demyelination of myelin sheaths in rat neurons after 3 days and 21 days ( Fig.16 FI in ). Electrophysiological assessment by patch clamp recording showed that Si-Ikzf1-treated ICH rats had significantly enhanced hippocampal CA3-CA1 long-term potentiation (LTP) 7 days later compared with Si-NC ICH rats ( Fig.16 in J).

[0069] Recent studies have highlighted a novel regulatory role for the Ikzf1-SIRT1 axis in macrophage activation via AMP-activated protein kinase (AMPK) signaling. To explore the function of this axis in microglia, the inventors of the present application evaluated the expression levels of Ikzf1 and SIRT1 and the phosphorylation of AMPK in microglia and ICH brain tissues. The inventors' results showed that Si-Ikzf1 treatment significantly reduced the expression of Ikzf1 and increased the expression of SIRT1 and the phosphorylation of AMPK in rat brain tissues (Fig. 8K-L) and BV-2 cells ( Fig.16 These results suggest that the regulatory effects of the Ikzf1-SIRT1 axis in microglia are mediated through AMPK signaling. Taken together, these analyses highlight the therapeutic potential of SI-Ikzf1 in improving neurological function after ICH by regulating the Ikzf1-SIRT1 axis and its downstream signaling pathways.

[0070] In summary: Since ICH is a severe form of stroke with high mortality and morbidity, a prominent feature of ICH is the presence of inflammation and neuronal damage. In multicellular organs, such as the brain under ICH conditions, cellular function and tissue homeostasis rely on interactions between adjacent individual cell types. Single-cell technology can describe the molecular heterogeneity of different cell types and changes during disease development. However, the lack of spatial context makes it unclear how these different cell types interact in space to coordinate tissue function. Previous studies have observed that intravenous injection of MSC-derived exosomes into ICH rats can improve the disease condition, but the mechanism of action of Exo treatment of ICH is not fully understood. Here, the inventors of this application integrated ST and scRNA-seq data, focusing on the molecular mechanisms of different cell types, cell-cell interactions, cell-cell communication and signaling networks, to provide a comprehensive map of the rat brain under ICH and Exo treatment conditions. The transcriptome analysis results of ST combined with scRNA-seq showed that Exo therapy reduced the expression of genes related to inflammation, pyrolysis and apoptosis in the injury microenvironment of ICH rats, and increased neurovascular remodeling, promoting neurological, behavioral and cognitive outcomes during recovery. These processes are mainly mediated by Ikzf1.

[0071] Microglia are involved in important brain functions, including development and plasticity. During central nervous system injury, microglia are responsible for phagocytosis and removal of microorganisms, dead cells and protein aggregates. In addition, microglia secrete many soluble factors, such as cytokines and neurotrophic factors, which participate in the immune response and tissue repair of the central nervous system. The inventors of the present application found that the MIkzf1 subclass microglia in the ICH group were enriched with inflammatory response factors. Through transcriptional prediction, pseudo-sequential analysis and experimental evidence, the inventors of the present application confirmed that Ikzf1 is a key switch factor that determines the transformation of resident microglia into the MIkzf1 subclass. Ikzf1 is considered to be a key regulator of macrophage activation. In the study of the inventors of the present application, it was found that Ikzf1 is specifically expressed in adult hippocampal microglia, and the lack of Ikzf1 in the resting state can lead to glial proliferation and impaired learning and memory ability in mice. However, the role of Ikzf1 in traumatic neurological diseases such as ICH has not been studied in the prior art.

[0072] Here, the inventors of the present application revealed how the expression level of Ikzf1 affects the response of microglia under ICH conditions. First, the inventors of the present application found high expression of inflammasome and pyrolysis-related proteins in MIkzf1 subtype microglia. Second, Si-Ikzf1 promoted the conversion of MResident to MPtn in ICH rats, but not MIkzf1; the latter showed anti-inflammatory effects that promoted neural remodeling. Moreover, Si-Ikzf1 hindered MIkzf1 inflammation, pyrolysis, and apoptosis, thereby improving cognitive behavior and demyelinating damage. Third, the regulation of inflammasomes and pyrolysis involves Ikzf1-SIRT1. Si-Ikzf1 reversed the decrease in SIRT1 expression in BV-2 cells and ICH rat brains after inflammatory stimulation. Fourth, Si-Ikzf1 promoted the upregulation of Ptn in MPtn. Consistent with this finding, the activity of the Ptn signaling pathway in MPtn was increased in the intercellular communication analysis of ST and scRNA-seq data. Ptn expression is upregulated in the brain with different neuropathological lesions, but little is known about the cell types that overexpress Ptn under these pathological conditions. In the study of the present application scheme, the inventors of the present application observed that Ptn expression was upregulated in MPn under Si-Ikzf1 conditions. In addition, the inventors of the present application found that Exo treatment can enhance the co-localization of Ptn and Iba1 (microglial marker) in the peritraumatic area. Reducing Ikzf1 expression also led to increased co-localization of Ptn and Iba1 after ICH, as well as increased Ptn levels in BV-2 cells and ICH rat brains. These results indicate that Ptn is an important neurotrophic factor involved in the repair of the central nervous system, neuronal survival and differentiation.

[0073] In summary, the inventors' studies have shown that reducing Ikzf1 expression can prevent microglia from switching from the MResident subtype to the MIkzf1 subtype after ICH. This intervention promoted the switch from the MResident to the MPtn subtype, rather than the MIkzf1 subtype, thereby reducing inflammation, dyscrasias, and neuronal apoptosis in the affected microenvironment after ICH. Ikzf1 is considered a promising therapeutic target for ICH and brain injury, providing a new perspective on the etiology and treatment of neuroinflammation and neuronal injury.

[0074] The following is the experimental method of this implementation scheme:

[0075] 1. Cell culture: Sh-Sy5y and BV-2 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). All cells were maintained in DMEM medium containing 10% FBS and 1% penicillin-streptomycin (Invitrogen, Scotland, UK) in a 5% CO2 incubator at 37°C.

[0076] 2. Isolation and culture of mouse primary neurons: In the study of the present invention, mouse primary neurons were obtained from the cerebral cortex of C57BL / 6 mice within five days after birth. The brain was carefully dissected under a stereomicroscope, and the meninges and blood vessels were removed to separate the cerebral hemispheres. The cerebral hemisphere tissue was then cut using microsurgical forceps and digested with 0.175% trypsin (50:1 DNA) for 10 minutes. The digestion was stopped by adding an equal volume of culture medium with 10% fetal bovine serum (FBS; Gibco). The cells were then suspended in a single cell in the culture medium by gentle pipetting and then filtered through a 40 μm filter. Cell subculture was performed every 5-7 days.

[0077] 3. Animals: In this study, adult male Sprague-Dawley rats weighing 250-300 g were obtained from Chongqing Enswell Biotechnology Co., Ltd. (China). Rats were housed in a specific pathogen-free environment and received feed and water ad libitum. All experimental procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Sichuan Provincial People's Hospital.

[0078] 4. Intracranial hemorrhage model and Exo injection: Rats were anesthetized with isoflurane gas, fixed in a stereotaxic apparatus after hair removal. After disinfection, the skin was shaved to expose the periosteum, and holes were made 0.1 mm behind the sagittal suture and 3 mm to the right of the midline. Then, 2 μL of collagenase IV (0.25 U / μL) was extracted from a vial and slowly injected into the needle to a depth of 5.5 mm. The needle was fixed in place for 10 minutes, the hole was closed with bone wax, the wound was sutured, and disinfected again.

[0079] At 48 hours after ICH, the inventors of the present application used the mNSS scoring system to evaluate the model and selected rats with an mNSS score of more than 10 for subsequent experiments. Then 2×1010 particles / rat of Exo were injected into the original hole through the extracerebral spinal cord. In order to reduce the effect of Exo on the hematoma area, the inventors of the present application selected the injection point at a depth of 4.5 mm. The sham group only punctured the brain tissue with a microinjection needle, while the ICH group was injected with saline instead of Exo 48 hours after ICH.

[0080] 5. Spatial transcriptome analysis and sequencing: Brain tissue was sectioned with 10 μm thickness using OCT embedding and mounted on Visium spatial gene expression slides (10x Genomics, #1000184; Pleasanton, CA, USA). Tissue was stained and microscopic images were taken with a 10× objective. Brain tissue was then permeabilized to allow RNA to diffuse and bind to the slide surface. Permeabilization was performed for 12 min using the spatial tissue optimization program (10x Genomics, #1000193). cDNA was then synthesized from the fixed RNA on the slide and cDNA was collected to generate sequencing libraries containing unique sample indexes and P5 and P7 primers for Illumina-compatible sequencing. Paired-end sequencing (2×100) was performed on an Illumina NovaSeq 6000 instrument (San Diego, CA, USA). Preparation of single-cell suspensions of rat brain tissue

[0081] After collecting the rat brain tissue, wash it three times with sterile PBS solution. Transfer up to 200 mg of brain tissue each time to a tube containing 4.9 ml of HEPES buffer, 10 μl of DNase I solution, and 100 μl of collagenase D solution (2 mg / ml), for every 150-450 mg of brain tissue. The cells were filtered through a mesh cell filter (70 μm) and collected in a 50 ml centrifuge tube. The mesh cell filter was rinsed with 5 ml of HEPES buffer to room temperature, and the resulting cell suspension was centrifuged at 250 × g for 15 minutes. After removing the supernatant, the cells were resuspended with the required volume of PEB buffer and prepared for single-cell sequencing.

[0082] 6. Single-cell sequencing library preparation and quality control: ScRNA-Seq libraries were prepared using the Single Cell 3'v2 chemistry. Next-generation sequencing was performed on an Illumina NextSeq500 with a transcript length of 150 base pairs. Sample demultiplexing, barcoding, and single-cell gene UMI counting were performed using CellRanger Count v3.0.2 (10×Genomics). Dimensionality reduction, clustering, and visualization were performed using Seurat v4. Cells with less than 200 detected genes were filtered out. Data were normalized and scaled using SCTransform and Find Variable Features. All samples were merged using the CCA method. Principal component analysis was performed using the RunPCA function of Seurat. UMAP visualization and Louvain clustering were performed using the first 20 principal components. Subclustering analysis was performed using CellMarker and Single. FindMarker was used to identify differentially expressed genes, based on a fold change cutoff of 0.2 and an adjusted P value of <0.05.

[0083] 7. Spatial transcriptome library preparation: The inventors of the present application used the 10×Genomics spatial RNA-seq Visium platform to conduct spatial transcriptome experiments. The oligomers at each point have a unique PCR handshake, a unique spatial barcode, a unique molecular identifier (UMI), and a poly-dT-VN tail for capturing the 3' end of the mRNA molecule. The spatially labeled cDNA library was constructed using the 10×Genomics Visium spatial gene expression 3' library construction V1 kit. The cDNA library was sequenced on the IlluminaNextSeq 500 / 550 using a 150 cycle high output kit. The Fluidigm (located in South San Francisco, California, USA) frame around the capture area on the Visium slide was manually aligned using the Loop Browser 4.0.0 software (10×Genomics), and the points covering the tissue were selected. Next, the sequencing data was aligned to the chicken reference genome using the Space Ranger 1.0.0 pipeline to generate a feature point barcode expression matrix.

[0084] 8. RNA sequencing: Human bone marrow-derived mesenchymal stem cell exosomes (HQExoA002L3001, Huamei Biotechnology Co., Ltd., Wuhan, China). Total RNA in exosomes was extracted using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, New York, NY, USA). The cDNA library was then sequenced on an Illumina HiSeq 2000. Raw reads were collected using Illumina analysis software, and exosome recognition and uptake data can be found in Fig.23 Found in.

[0085] 10. Cell trajectory analysis: Monocle2 was used to perform pseudo-time-based cell trajectory analysis. Genes that were significantly different between the two groups were used to run trajectory analysis, and the adjusted P value was < 0.05.

[0086] 11. Transcriptional regulatory network analysis: Infer TF regulatory networks using the SCENIC workflow. Use three R / Bioconductor packages, including GENIE3, RcisTarget, and AUCell, to identify potential TF targets and regulators, and then score the regulator activity on individual cells.

[0087] 12. GSEA and pathway enrichment analysis: GSEA enrichment analysis was performed using the molecular marker database GO gene set (C5) through the R package ClusterProfiler. The adjusted P value < 0.05 was used as a cutoff value. The differentially expressed genes were mapped to the human genome resource, including the KEGG pathway and the GO biological process pathway.

[0088] 13. Protein immunoblotting: Total protein content was extracted from cells or brain tissue using RIPA lysis buffer at 4°C. The lysate was centrifuged at 5200g for 15 min at 4°C. Subsequently, the supernatant was transferred to a new tube. The protein concentration was determined using the Bradford protein assay kit (Beijun Biotechnology Co., Ltd., Shanghai, China). The proteins were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane (PALL, New York, NY, USA). They were then blocked in QuickBlockWestern (Pulong Biotechnology, Wuhan, China) and incubated with primary antibodies (1:1000) at 4°C overnight, followed by incubation at 30°C for 2 h with secondary antibodies (1:1000). After washing with TBST, the membranes were stained with UltraSignal TM Bands were visualized using ECL Western Blotting Detection Reagent (4A Biotech Co., Ltd., Beijing, China) and imaged using a Tanon 4600 Imaging System (Tanon, Shanghai, China). Protein band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), representing relative expression levels.

[0089] 14. Flow cytometry analysis: Flow cytometry apoptosis detection experiment: Cell apoptosis was analyzed using the AnnexinV-FITC / PI apoptosis detection kit (4A Biotechnology Co., Ltd., Beijing, China). After treatment, the cells were collected and centrifuged at 1500 rpm for 8 minutes. After removing the supernatant, the cells were resuspended in 500 μL of a solution containing PI and AnnexinV-FITC solution. After incubation at room temperature for 15 minutes, the cells were analyzed using a NovoCyte Quanteon 4016 (Agilent Technologies, Santa Clara, CA, USA). Data acquisition and cell survival analysis were performed using FlowJo v10.0 software (Randall, Stanford University, CA, USA).

[0090] 15. Isolation of rat primary microglia and flow cytometry-based subpopulation identification experiment: In this study, primary microglia were obtained from the pseudohematoma and hematoma areas of rat brain slice tissue in the Sham, ICH and ICH+Exo groups. The tissue was then digested for 10 minutes using microsurgical forceps and 0.175% trypsin (50:1 DNase). Enzymatic digestion was stopped with culture medium containing 10% fetal bovine serum (Gibco). The culture medium was then gently aspirated to obtain a single cell suspension, which was then filtered through a 40 μm sieve. CD11b / c-expressing cells were isolated using MicroBeads, rat / CD11b / c (Miltenyi Biotec., Germany). Immunostaining of cell membrane proteins included CD45-Alexa Fluor 700 and CD11b / c-FITC antibodies. After incubation at 4°C for 30 minutes, the cells were fixed using a cell perforation and fixation kit and then filtered through Ptn- Plus 750 and Ikzf1- Intracellular protein staining was performed using Plus 405. Subsequently, after incubation for 45 min at 4°C, cells were analyzed using NovoCyte Quanteon 4016 (Agilent Technologies, Santa Clara, CA, USA). Data acquisition and cell viability analysis were performed using FlowJo v10.0 software (Randall, Stanford University, CA, USA).

[0091] 16. Transmission electron microscopy: BV-2 cells: BV-2 cells were fixed in 0.5% phosphate-buffered glutaraldehyde at 4°C overnight.

[0092] Rat brain tissue: Rat brain tissue was fixed in 2.5% phosphate-buffered glutaraldehyde at 4°C overnight, washed with PBS, fixed with 1% ruthenic acid, washed with mirin buffer, dehydrated with a gradually increasing ethanol series, exposed to propylene ether and embedded using the EMbed812 kit (Electron Microscopy Sciences, Hatfield, PA, USA). After polymerization, the Epon blocks were semi-thinly sectioned at 1 μm. Images were acquired using a transmission electron microscope (JEM-1400FLASH).

[0093] 17. Behavioral experiment: Morris water maze test: The MWM apparatus consists of a circular dark gray water tank with a diameter of 160 cm and a height of 80 cm, filled with water at 25℃±2. An escape platform is located 1 cm below the water surface in the center of the three quadrants. During the training period, each rat was trained twice a day for five days. After the five-day training period, cognitive function was evaluated as described previously. Rats were released into the water from the same quadrant, and their escape latency was recorded. At the same time, the number of rats entering the original platform quadrant was recorded using an animal behavior analysis system (Zhongshi Science&Technology Co., Ltd., Beijing, China).

[0094] 18. Modified Neurological Severity Score: mNSS score was assessed as previously reported62. It includes motor, sensory, reflex, and balance tests, as well as neurological function recordings after 1, 3, 7, 14, and 28 days.

[0095] 19. Foot Fault Test: The foot fault test was used to assess motor function as previously described63. Rats were placed on a grid and allowed to move along the grid. If the paw fell between the grids or slipped, a foot fault was recorded. The total number of steps taken by the rat to traverse the grid was counted, and the total number of foot faults for each forelimb was recorded. The percentage of forelimb faults was calculated with the total number of steps.

[0096] 20. Enzyme-linked immunosorbent assay: Rat Nlrp3, IL-1β, IL-18 (Ruixin Biotechnology Co., Ltd., Quanzhou, China) and Ptn quantitative ELISA kit (Cloud-Clone Corp Co, Ltd, Wuhan, China) were used to measure the amount of protein secreted into cell culture supernatant and rat brain tissue according to the manufacturer's instructions. The sample absorbance was measured at 450 nm within 30 minutes.

[0097] Hoechst 33342 and PI staining

[0098] After treatment, primary neuronal cells were fixed in 4% paraformaldehyde and stained with 5 mg / L Hoechst 33342 and 5 mg / L PI solution for 10 min. Then, the slides were taken out and dried, and the slides were stained with FluorSave TM The cells were encapsulated with encapsulation medium (Carbazine, San Diego, CA, USA). Representative images showing blue and red signals were taken by fluorescence microscopy. Cell death was analyzed by calculating the percentage of PI / Hoechst.

[0099] 21. Immunofluorescence staining: Apoptotic cells and the expression of Iba1, Ptn, Gsdmd, Caspas-3 (P17) and Map2 in rat brain tissue were detected by immunofluorescence and TUNEL staining. Briefly, the sections were blocked in 10% goat serum containing 0.1% TritonX-100 for 0.5 h. Then, the sections were incubated with primary antibodies at 4°C overnight, followed by incubation with fluorescent dye-conjugated secondary antibodies for 2 h. After incubation, the sections were stained with DAPI and photographed by Zeiss LSM 800 confocal microscope. The mean fluorescence intensity was measured by ImageJ software.

[0100] 22. Patch clamp technique: Experimental plan: (1) Dialyze the hippocampus and cut the tissue into 380 μm thick rat brain slices. (2) After culturing for 1-2 hours, start recording. (3) Record the process of CA3-CA1 LTP. The inventors of the present application recorded the baseline with a stimulation intensity that can cause 40%-50% of the maximum response amplitude, and regarded it as the basic stimulation intensity. After the response stabilizes, the recording for more than 30 minutes is used as the baseline. The fluctuation range of the baseline slope is required to be less than 10%. Run HFS immediately after inducing LTP baseline stimulation, running each beam at 100 Hz for 1 second. Stimulate three times with an interval of 10 seconds. The inventors of the present application used a single test pulse to record 60 minutes of post-induction responses.

[0101] 23. Data processing: fEPSP slope over time: For each LTP response, the slope of the fEPSP after stimulation was normalized to the mean (100%) of the baseline fEPSP slope. The inventors of the present application calculated the mean of the data at the same time point in the experiment. The degree of LTP was expressed as a percentage of the baseline fEPSP slope. In order to compare the long-term synaptic plasticity between different groups, for each LTP response, 20 signals before and after 10 minutes were used as a reference for standardization and the mean was taken. Then, the mean was taken for the same group.

[0102] 24. MRI data of rat brain images were obtained; anesthesia was induced with 5% isoflurane in a 2:1 mixture of nitrogen and oxygen (95% oxygen, 5% carbon dioxide) using a small animal anesthesia machine (Zhongshi Science & Technology Co., Ltd., Beijing, China). Rats were transferred to a stand and fixed with ear sticks and incisor bars. MRI data were collected according to the method described by Prior et al.

[0103] 25. Statistical analysis: The data of this study were obtained from at least three independent experiments. The values ​​are expressed as mean ± standard deviation and analyzed using GraphPadPrism 9.3 statistical software (San Diego, CA, USA). One-way ANOVA was used for inter-group comparisons, followed by Turkey post hoc test, and the significance level was set at P < 0.05.

[0104] 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 Si-Ikzf1 in the preparation of products for inflammation treatment and neuroprotection.

2. The use according to claim 1, characterized in that: The inflammation treatment and neuroprotection are treatments during the course of intracranial hemorrhage.

3. The use according to claim 2, characterized in that: The product described is a drug.

4. The use according to claim 3, characterized in that: The Si-Ikzf1 reduces cell eschar formation by promoting the generation of microglial MResident subtype into MPtn subtype rather than MIkzf1, and improves neurological function after ICH by regulating Ikzf1-SIRT1 and its downstream signaling pathways.

Images