Application of APOE as target to preparation of medicine for preventing and / or treating uveitis

Abstract

The invention discloses an application of APOE as a target in preparation of a medicine for preventing and / or treating uveitis, the medicine comprises an APOE inhibitor, and the APOE inhibitor is used for inhibiting expression or activity of APOE protein so as to reduce microglial cell mediated inflammatory response. The invention discloses a key effect of APOE in uveitis pathogenesis, especially in microglial cell activation, for the first time, and provides a brand new drug target for treatment of uveitis; the inhibitor aiming at APOE can intervene in abnormal activation of microglial cells from the source, and possibly has better specificity and fewer side effects than a traditional immunosuppressor; meanwhile, the invention lays a solid theoretical and experimental foundation for developing a new generation of drugs for targeted therapy of uveitis.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of APOE as a target point in preparation of a medicine for preventing and / or treating uveitis. BACKGROUND

[0002] Autoimmune uveitis (AU) is a blinding eye disease, and its clinical management mainly relies on corticosteroids and immunosuppressive agents, lacking specific targeted treatment means. Abnormal activation of microglia cells is confirmed to be a key link in the pathogenesis of uveitis. However, the upstream key molecules driving the activation of microglia cells and the specific mechanism thereof are not completely clear, which limits the precise treatment of uveitis.

[0003] Apolipoprotein E (APOE) is a pleiotropic protein known to play a role in lipid metabolism and nervous system diseases, but its specific function and mechanism in uveitis, especially in the pathological process related to microglial cell activation, have not been reported. SUMMARY

[0004] The technical problem to be solved by the application is to provide application of APOE as a target point in preparation of a medicine for preventing and / or treating uveitis, which first discloses that APOE is a key positive regulatory factor in the pathogenesis of microglia-mediated uveitis, and taking it as a drug target can provide a new strategy for the prevention and treatment of uveitis.

[0005] The technical problem to be solved by the application is solved by the following technical scheme: Application of apolipoprotein E (APOE) as a target point in preparation of a medicine for preventing and / or treating uveitis.

[0006] Preferably, in the above technical scheme, the medicine contains an APOE inhibitor, the APOE inhibitor is used to inhibit the expression or activity of the APOE protein, thereby reducing the inflammatory reaction mediated by microglia cells, and the inflammatory reaction includes abnormal proliferation, migration and release of inflammatory factors of microglia cells.

[0007] Preferably, in the above technical scheme, the APOE inhibitor is selected from one of an RNA interference molecule, an antisense nucleotide for the APOE gene, or a neutralizing antibody, a small molecule inhibitor for the APOE protein; the RNA interference molecule is a small hairpin RNA (shRNA) or a small interfering RNA (siRNA).

[0008] Preferably, in the above technical scheme, the medicine can reduce the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) in microglia cells.

[0009] Preferably, in the above technical solution, the uveitis is autoimmune uveitis.

[0010] A pharmaceutical composition for preventing and / or treating uveitis, comprising an effective dose of an APOE inhibitor and a pharmaceutically acceptable excipient.

[0011] Preferably, in the above technical solution, the pharmaceutical composition is an injection or eye drops.

[0012] Use of a reagent for detecting a biomarker, the biomarker being apolipoprotein E (APOE), in the preparation of a product for diagnosing uveitis, the reagent being used to detect the expression level or activity of APOE in a sample.

[0013] A kit for detecting a biomarker of uveitis, the kit comprising a reagent for detecting the expression level or activity of apolipoprotein E (APOE) in a sample.

[0014] The above technical solution of the present application has the following beneficial effects: (1) The present application first discloses the key role of APOE in the pathogenesis of uveitis, especially in the activation of microglial cells, providing a new drug target for the treatment of uveitis.

[0015] (2) The inhibitor targeting APOE can intervene in the abnormal activation of microglial cells from the source, and may have better specificity and fewer side effects than traditional immunosuppressive agents.

[0016] (3) The present application lays a solid theoretical and experimental foundation for the development of a new generation of targeted drugs for treating uveitis. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present application and, together with the description, serve to explain the principles of the present application.

[0018] Figure 1Figure 1 is a chart of transcriptomic analysis results. Among them, A. GO analysis of VKH patient transcriptome; B. Boxplot of differentially expressed genes of VKH patient transcriptome related to inflammation; C. Boxplot of differentially expressed genes of VKH patient transcriptome related to immune activity and lipid metabolism characteristics; D. Schematic diagram of mouse experimental design of single-cell transcriptome sequencing; E. Single-cell RNA-seq reveals retinal CD45+ cell populations from untreated mice and EAU mice; F. Subclusters of CD45+ cells, including monocytes, B cells, T cells, and microglia; G. t-SNE plot of microglia cells isolated from naive retinas and EAU retinas; H. Heatmap showing differentially expressed genes in microglia cells between naive mice and EAU mice; I. Volcano plot showing microglia gene expression patterns in EAU mice compared to untreated mice; J-L. Violin plots showing differential expression of P2ry12 (J), Tmem119 (K), and APOE (L) between untreated mice and EAU mice.

[0019] Figure 2 Figure 2 is a chart of experimental results for in vivo verification of the positive correlation between APOE expression and EAU severity and the alleviation of the disease by APOE gene deletion. Among them, A-D are charts of clinical and pathological scores of EAU mouse eye inflammation; E-K show up-regulation of APOE and inflammatory factor expression in the retinas of EAU mice; L-T show that APOE gene knockout can significantly reduce the clinical symptoms, pathological damage, and molecular inflammation level of EAU.

[0020] Figure 3 Figure 3 is a chart of in vitro functional verification of APOE regulating microglial cell activation. Among them, A: Western blot chart; B-E charts are quantitative analysis charts (inflammatory factor expression) corresponding to chart A; F-G: Transwell experiment and statistics (cell migration); H-I: EdU experiment and statistics (cell proliferation). J: sh-APOE transfected cells; K-O: Western blot chart and statistics; P-Q: Transwell experiment and statistics after transfection (cell migration); R-S: EdU experiment and statistics after transfection (cell proliferation).

[0021] Figure 4 Figure 4 is a chart of reactive oxygen species detection. Among them, A-B: fluorescence intensity and statistics of intracellular ROS level. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement, numerical expressions, and numerical values of the components and steps set forth in these embodiments do not limit the scope of the present application unless otherwise specifically stated.

[0023] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified. The equipment used in the experiments is well known to those skilled in the art unless otherwise specified.

[0024] Example 1: Confirmation of the association of APOE with uveitis

[0025] (1) Patient diagnosis and sample processing: The researchers randomly collected whole blood samples from 6 VKH patients and 6 age-matched healthy controls at Beijing Tongren Hospital. All VKH cases met the international diagnostic criteria and were excluded from ocular trauma or intraocular surgery history. The research protocol has been approved by the Institutional Review Board of Beijing Tongren Hospital (Protocol Number: TREC2024-KY106) and follows the Declaration of Helsinki. Written informed consent was obtained from the patients before inclusion in the study.

[0026] (2) Whole blood sample RNA sequencing: High-purity RNA was extracted from peripheral blood mononuclear cells (PBMCs) according to the instructions of the TRNzol Universal Kit (Tiangen, Cat No: DP424). Sequencing libraries were generated using the VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme) according to the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. The sequencing library was quantified using the Invitrogen High Sensitivity DNA Kit on the Qubit 4.0, and its integrity was detected using the Agilent Bioanalyzer 2100 system. Subsequently, the sequencing library was sequenced on the MGISEQ-T7 platform, generating 150-base pair double-end reads.

[0027] (3) Animal materials and processing: All animal-related experiments were conducted in accordance with the Statement on the Use of Animals in Ophthalmic and Vision Research of the American Society for Vision and Ophthalmology and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal research protocol was approved at Beijing Tongren Hospital (Affiliated to Capital Medical University). Six to eight-week-old C57BL / 6NCrl mice (Stock No: 027) were purchased from Suzhou SJA Biological Technology Co., Ltd., and APOE- / - mice (Stock No: C001067) were purchased from Suzhou SJA Biological Technology Co., Ltd. Mice were routinely fed in the animal facility of the Ophthalmology and Vision Research Institute at Beijing Tongren Hospital.

[0028] (4) Experimental autoimmune uveitis (EAU) induction: The method for inducing experimental autoimmune uveitis (EAU) is existing technology and will not be described in detail here. Human IRBP651-670 peptide (LAQGAYRTAVDLESLASQLT, Shanghai Sangon Biotech) was dissolved in sterile PBS and then mixed with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, stock no.: F5881). The mixture was manually mixed in an ice-cold syringe to obtain an emulsion. 500 μg of the emulsified IRBP651-670 was intraperitoneally injected with 1 μg of pertussis toxin (PTX; Sigma-Aldrich, catalog no. P7208), and the emulsified IRBP651-670 was injected subcutaneously. All immunized mice were evaluated by slit-lamp examination 14 days post-injection to ensure successful establishment of experimental autoimmune uveitis (EAU). Clinical and histopathological evaluations were performed by two-person blinded scoring to validate the results.

[0029] Results showed that transcriptomic analysis of peripheral blood mononuclear cells from VKH patients and GO analysis revealed a significant enrichment of inflammation-related pathways. Figure 1 Figure A). Classical inflammatory genes (such as CXCL2, JUN, DUSP8) were observed in VKH patients. Figure 1 The transcriptional level of the genes (Figure B) was significantly upregulated, and the levels of immune-related lipid metabolism genes (such as CYP2A6, APOE, SOSC3, Figure C) were also increased.

[0030] To investigate transcriptomic differences in microglia in EAU mice, researchers performed FACS sorting on CD45+ immune cells in the retina of immunized EAU mice and then performed single-cell RNA sequencing (Figure 1, D). A significant increase in CD45+ cells was observed in EAU mice (Figure 1, E). Further cluster analysis showed that most CD45+ cells were microglia (Figure 1, F), and their relative abundance in the EAU mouse retina was significantly increased. Figure 1 (G diagram). Furthermore, transcriptome comparisons of microglia from uninfected and experimentally autoimmune uveitis (EAU) mice revealed that apolipoprotein E (APOE) was one of the most differentially upregulated genes. Figure 1 The H and I plots (showing the characteristics of the cells) also indicate that this phenomenon was observed in the transcriptome of VKH patients. Notably, the violin plot shows that microglia-specific genes such as P2ry12 (…) are present in EAU mouse retinal microglia. Figure 1 J-graph) and Tmem119 ( Figure 1 The expression of APOE was downregulated (K-plot), while its expression was significantly upregulated (K-plot). Figure 1 (L diagram).

[0031] Example 2: In vivo level validation of the function of APOE

[0032] (1) Slit lamp examination for anterior segment evaluation: A slit lamp biomicroscope with integrated high-resolution digital camera (Model SLM-8E, S&T Company, Chongqing, China) was used. Mice were manually restrained and the eyes were gently exposed. Topical corneal surface anesthesia was performed using 0.5% proparacaine hydrochloride eye drops. Images were taken in a dark room to reduce glare, and camera settings were optimized for best resolution and exposure.

[0033] For experimental autoimmune uveitis (EAU) scoring, 0: no obvious signs of inflammation; 1: mild ciliary hyperemia; 2: obvious ciliary hyperemia; 3: severe ciliary hyperemia with iris adhesion; 4: eyeball atrophy or blindness.

[0034] (2) Western blotting: Retinas were obtained by surgery, and cells were digested with trypsin to prepare lysates for Western blotting. Briefly, tissues and cells were lysed and prepared using RIPA lysis buffer (Bi Yun Tian, P0013B).

[0035] Primary antibodies for Western blotting were as follows: anti-iNOS (Cell signaling Technology, 2982, dilution 1:1000); APOE (Cell signaling Technology, 49285, dilution 1:1000); IL-6 (Proteintech, 21865-1-AP, dilution 1:1000; Cell signaling Technology, 12912T, dilution 1:1000); TNF-a (Proteintech, 26405-1-AP, dilution 1:1000); anti-Flag (Cell signaling Technology, 14793, dilution 1:1000); anti-b-actin (Proteintech, 81115-1-RR, dilution 1:5000); secondary antibody: HRP-labeled goat anti-rabbit IgG (H+L) (Proteintech, SA00001-2, dilution 1:5000). Visualization of Western blotting was performed using an ECL kit (Advansta, CA, USA). Blot images were analyzed using ImageJ software.

[0036] (3) Hematoxylin-eosin (H&E) staining: After euthanizing the animal, the eyeball was surgically removed and immediately fixed with an eye fixative (Wuhan Cyagen Biosciences; catalog number: G1109) for at least 24 hours. The tissue was then embedded in paraffin, and sections were prepared to a thickness of 4–6 micrometers. The tissue sections were stained with H&E according to the manufacturer's instructions (Wuhan Cyagen Biosciences; catalog number: G1005).

[0037] The pathological scoring criteria are as follows: 0 points: no cell infiltration, no structural changes; 0.5 points: mild cell infiltration, no structural changes; 1 point: moderate cell infiltration, mild focal retinal folds; 2 points: moderate cell infiltration, significant retinal folds with focal photoreceptor cell damage; 3 points: severe cell infiltration, extensive retinal folds with retinal detachment, serous exudate and subretinal hemorrhage, moderate photoreceptor cell damage; 4: severe (or mild) cell infiltration, retinal atrophy, extensive photoreceptor cell damage.

[0038] (4) Immunofluorescence microscopy examination: Paraffin sections were dewaxed, antigen-retrieved, and blocked with 3% BSA for 30 minutes. They were then incubated overnight at 4°C with primary antibodies (anti-APOE, Cyagen Biosciences, GB112288, 1:200; anti-IBA1, Heguang Pure Chemicals, 019-19741-20, 1:3000). Subsequently, they were incubated at room temperature with different secondary antibodies for 1 hour. Stained slides were mounted with DAPI mounting medium (Abcam, AB104139). Images were captured using a fluorescence microscope (Leica DMi8, Germany), and quantitative analysis was performed using ImageJ software.

[0039] The results showed that EAU mice exhibited severe ocular inflammation and abnormalities, with iritis, anterior chamber opacity, and scleral vasodilation observed under slit lamp examination. Figure 2 (Figures A and B). Histological examination of the posterior segment revealed vitreous infiltration, retinitis, and choroiditis. Figure 2 Figures C and D). Further analysis of the retinal proteomic profile showed that the expression of apolipoprotein E (APOE) and several inflammation-related proteins, including inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), was upregulated (Figures E-I in Figure 2). To further investigate the relationship between retinal APOE expression and microglia, immunofluorescence staining of IBA1 and APOE was performed. The results showed that after immunization for experimental autoimmune uveitis (EAU), APOE expression in the retina was significantly upregulated, mainly mediated by IBA1+ microglia. Figure 2(J and K plots). These data indicate that the upregulation of APOE expression in the retina of EAU mice is mediated by microglia and is positively correlated with the degree of inflammation.

[0040] To investigate the role of APOE in the pathogenesis of early-onset inflammatory ulcer (EAU), wild-type (WT) and APOE knockout (APOE- / -) mice were used. Consistent with known associations between APOE, microglia activation, and inflammation, APOE- / - mice showed increased activity in the anterior segment (…). Figure 2 The EAU phenotype was significantly reduced in both the L and M plots of Figure 2 and the N and O plots of Figure 2. Simultaneously, the expression of inflammation-related molecules (including iNOS, TNF-α, and IL-6) was decreased in retinal tissue from APOE- / - mice. Figure 2 P-plots and T-plots). These findings support the key role of apolipoprotein E (APOE) in retinal inflammation in experimental autoimmune uveitis (EAU) immunized mice.

[0041] Example 3: Validating the effectiveness of targeting APOE at the cellular level

[0042] (1) Cell lines and cell culture: Human microglia HMC3 were purchased from Pronosai (Wuhan, China). HMC3 cells were typically cultured in minimum essential medium (BasalMedia, China) supplemented with 10% fetal bovine serum (Gibco, Australia) and 1% penicillin-streptomycin (ThermoFisher) at 37°C and 5% carbon dioxide.

[0043] (2) Inflammatory stimulation: Different concentrations of interleukin-17a (IL-17a) and interferon-γ (IFN-γ) were used to stimulate it. This combination of two cytokines is a typical combination that induces pro-inflammatory microglial responses.

[0044] (3) EdU proliferation assay: HMC3 cell proliferation was assessed by EdU incorporation assay according to the manufacturer’s instructions using the EdU cell proliferation kit (Beyotime, C0071S), and the number of EdU+ cells was quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland).

[0045] (4) Migration measurement: 1×10 4HMC3 cells were seeded into 8-micron pore size filters in the upper chamber of a 24-well Transwell (Corning, 3422) micrometer, without fetal bovine serum (FBS), while an appropriate amount of FBS-containing culture medium was added to the lower chamber. After 24 hours, the cells were fixed with 4% paraformaldehyde (Cybernett, Wuhan, China; catalog number: G1101) and stained with 1% crystal violet (Cybernett, Wuhan, China; catalog number: G1014). The migrating cells were then imaged using a fluorescence microscope (Leica DMi8, Germany), and the number of migrating cells was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

[0046] The results showed that the degree of inflammation and APOE expression in HMC3 cells were positively correlated with the stimulation concentration. Higher concentrations of IL-17a and IFN-γ stimulation upregulated the expression of APOE, inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) (Figures A-E in Figure 3). Migration experiments indicated that the migration ability of HMC3 cells was also correlated with the stimulation intensity (Figures F and G in Figure 3). Simultaneously, EdU incorporation experiments showed that the proliferation capacity of HMC3 cells increased with increasing inflammation level. Figure 3 (H and I plots). These data indicate that apolipoprotein E (APOE) is closely related to the inflammatory phenotype and severity of microglia.

[0047] Example 4: APOE knockdown (KD) reduces inflammation in microglia.

[0048] To knock out APOE, a lentiviral delivery system was used. Small hairpin RNA (shRNA) targeting APOE (sh-APOE) was cloned into a gfp-labeled lentiviral vector to mediate gene transduction. Figure 3 (J figure). After lentiviral transduction, HMC3 cells were stimulated with IL-17a and IFN-γ, and then harvested for protein analysis.

[0049] Lentiviral transduction: Lentivirals used for gene transduction were constructed, packaged, and purified by Shanghai Gemma Gene Chemical Co., Ltd. Typically, the APOE shRNA sequence was cloned into the pMagic7.1-Puro / GFP vector to deliver APOE shRNA. 293T cells were co-transfected with pCMV-dR8.91, pCMV-VSV-G6363, and the targeting vector to generate pseudoviral particles. The pseudoviral particles were then concentrated and purified by ultracentrifugation, and their titers were determined before use.

[0050] For retroviral transduction, in short, HMC3 cells are introduced at a density of 2 × 10⁶ cells per well.5 The density of BV-2 cells was seeded in 6-well plates for 24 hours, then transduced with lentivirus vector to knock down APOE. After 72 hours, the lentivirus transduction efficiency was evaluated using fluorescence microscope (Leica DMi8, Germany). Lentivirus transduced cells were selected by puromycin and neomycin for 2 days, and the survived cells were used for subsequent experiments.

[0051] Results show that APOE knockdown reduced the expression of inflammatory mediators iNOS, TNF-a and IL-6, as described above (Figures Figure 3 In addition, APOE knockdown also reduced cell migration (Figures Figure 3 P and Q) and cell proliferation (Figures Figure 3 R and S) accelerated after IL-17a and IFN-g stimulation.

[0052] Example 5 APOE is involved in microglial activation by regulating oxidative stress and mitochondrial function

[0053] Active oxygen detection: After treating the cells, the culture medium was replaced, followed by the addition of CellROX™ probe (Thermo, C10422) and NucBlue™ Live Cell Stain ReadyProbes™ reagent (Thermo, R37605). The cells were then incubated in a 37°C cell incubator for 30 minutes. After incubation, the cells were washed with PBS three times and observed under a fluorescence microscope (Leica DMi8, Germany).

[0054] Oxygen consumption rate measurement The determination of cell mitochondrial respiration was carried out using Seahorse XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA) and Seahorse XFe 24 Extracellular Flux Analyzer (Agilent) according to the manufacturer's protocol. Briefly, 2.5 x 1 cells were seeded into Seahorse XF 24 cell culture microplates per well. After recording the baseline measurement, 1.5 mM oligomycin, 1.5 mM FCCP and 1.0 mM rotenone-antimycin A were injected in turn. Data analysis was performed using Seahorse Wave 2.6.3 software package (Agilent), and OCR values were expressed in pmol / min.

[0055] Results: After APOE knockdown, ROS levels decreased (as shown in Figures Figure 4 A-B).

[0056] In the present study, patients diagnosed with Vogt-Koyanagi-Harada (VKH) disease, AU subcohort was recruited, and their peripheral blood mononuclear cells were harvested for transcriptomic study, we found that components of oxidative stress pathway were significantly enriched, and the expression of APOE was upregulated, and associated with classical inflammatory signatures. Meanwhile, using a mouse model of EAU, we studied the transcriptomic changes of microglia by single-cell RNA-seq, and noted the upregulation of APOE. Subsequent in vivo experiments using WT and APOE- / - mice and in vitro cell assays determined a positive correlation between APOE expression, microglial activation, and uveitis development. Overall, APOE is a key mediator of the activation of microglia and uveitis pathogenesis, thus expanding the overall understanding of uveitis pathophysiology and providing potential therapeutic strategies for uveitis.

[0057] Although the present application has been disclosed with reference to the embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and changes without departing from the spirit and scope of the present application, and the scope of protection of the present application is defined by the claims and their equivalents.

Claims

1. Use of apolipoprotein E (APOE) as a target in the preparation of a medicament for preventing and / or treating uveitis.

2. Use according to claim 1, characterized in that, The medicament comprises an APOE inhibitor for inhibiting the expression or activity of APOE protein, thereby reducing the microglial cell-mediated inflammatory response.

3. Use according to claim 2, characterized in that, The APOE inhibitor is selected from one of an RNA interference molecule, an antisense nucleotide against the APOE gene, or a neutralizing antibody, a small molecule inhibitor against the APOE protein; the RNA interference molecule is a small hairpin RNA (shRNA) or a small interfering RNA (siRNA).

4. Use according to claim 2, characterized in that, The medicament can reduce the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), or interleukin-6 (IL-6) in microglial cells.

5. Use according to claim 2, characterized in that, The uveitis is autoimmune uveitis.

6. A pharmaceutical composition for preventing and / or treating uveitis, characterized by, The medicament comprises an effective dose of the APOE inhibitor and pharmaceutically acceptable excipients.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutical composition is an injection or eye drops.

8. Use of a reagent for detecting a biomarker in the manufacture of a product for diagnosing uveitis, characterized in that, The biomarker is apolipoprotein E (APOE), and the reagent is used for detecting the expression level or activity of APOE in a sample.

9. A kit for detecting a biomarker of uveitis, characterized by, The kit comprises reagents for detecting the expression level or activity of apolipoprotein E (APOE) in a sample.

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