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

Abstract

The invention discloses an application of PPIF as a target in preparation of a medicine for preventing and / or treating uveitis, the medicine comprises a PPIF inhibitor, and the PPIF inhibitor is used for inhibiting expression or activity of PPIF protein so as to reduce microglial cell mediated inflammatory response. The invention discloses the key effect of PPIF in the uveitis pathogenesis, especially in microglial cell activation, for the first time, and provides a brand new drug target for treatment of uveitis; an inhibitor aiming at PPIF can intervene in abnormal activation of microglial cells from the source, and possibly has better specificity and fewer side effects compared with 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, and particularly relates to application of PPIF as a target in preparation of a drug for preventing and / or treating uveitis. BACKGROUND

[0002] Autoimmune uveitis (AU) is a serious blinding intraocular inflammatory disease, and its clinical treatment mainly depends on glucocorticoids and broad-spectrum immunosuppressants. Although these non-specific therapies can control inflammation to a certain extent, long-term use can cause cataract, glaucoma, opportunistic infection and other serious side effects, and the curative effect of some patients is poor or drug resistance is easily produced. Therefore, developing specific target drugs for key pathogenesis of the disease is a major challenge to be solved in the current ophthalmology field.

[0003] Microglia, as the resident immune cells in the central nervous system (including the retina), plays a core role in the occurrence and development of uveitis. In the pathological state, microglia is abnormally activated, releases a large amount of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6), chemokines and reactive oxygen species (ROS) and other mediators, destroys the blood-retinal barrier, and recruits peripheral immune cells to infiltrate, which jointly drives and amplifies the inflammatory cascade reaction in the eye. Therefore, inhibiting the overactivation of microglia has become an important strategy for targeted therapy of uveitis. However, the upstream key signaling pathways driving the activation of microglia have not been fully elucidated, which limits the precise treatment of uveitis.

[0004] In recent years, the role of oxidative stress in autoimmune diseases has been increasingly concerned. Reactive oxygen species (ROS) is not only a direct effector molecule causing tissue damage, but also an important inflammation signal amplifier. At present, the abnormal expression or activation of PPIF has been confirmed to be closely related to myocardial ischemia-reperfusion injury, neurodegenerative diseases, acute pancreatitis and other diseases, and its inhibitors (such as cyclosporine A and its non-immunosuppressive analogs) have shown therapeutic potential in some disease models.

[0005] However, whether PPIF plays a key role in autoimmune uveitis, especially in the pathological process related to the activation of microglia, has not been reported at home and abroad. Exploring the function of PPIF in uveitis not only helps to deepen the understanding of the disease mechanism, but also is expected to provide a direct theoretical basis and experimental basis for the development of a new treatment drug specifically targeting the mitochondrial function of microglia. SUMMARY

[0006] The technical problem solved by the present application is to provide an application of a peptide prolyl cis-trans isomerase F (PPIF) as a target in preparation of a medicine for preventing and / or treating uveitis, the present application first discloses that PPIF is a key positive regulatory factor in the pathogenesis of uveitis, and inhibition of PPIF can effectively alleviate the inflammatory response mediated by microglial cells, thereby providing a brand-new strategy for prevention and treatment of uveitis.

[0007] The technical problem solved by the present application is solved by the following technical solutions: An application of PPIF as a target in preparation of a medicine for preventing and / or treating uveitis.

[0008] Preferably, in the above technical solution, the medicine comprises a PPIF inhibitor, the PPIF inhibitor is used to inhibit the expression or activity of PPIF, and then reduce the inflammatory response mediated by microglial cells.

[0009] Preferably, in the above technical solution, the PPIF inhibitor is selected from one of an RNA interference molecule, an antisense nucleotide for a PPIF gene, or a neutralizing antibody, a small molecule inhibitor for a PPIF protein; the RNA interference molecule is a small hairpin RNA (shRNA) or a small interfering RNA (siRNA).

[0010] Preferably, in the above technical solution, the uveitis is autoimmune uveitis, Vogt-Koyanagi-Harada syndrome.

[0011] A pharmaceutical composition for preventing and / or treating uveitis comprises an effective dose of a PPIF inhibitor and a pharmaceutically acceptable adjuvant.

[0012] Preferably, in the above technical solution, the pharmaceutical composition is an injection or an eye drop.

[0013] An application of a reagent for detecting a biomarker in preparation of a product for diagnosing uveitis, the biomarker is PPIF, and the reagent is used to detect the expression level or activity of PPIF in a sample.

[0014] An application of a reagent for detecting a biomarker in preparation of a kit for detecting uveitis.

[0015] The above technical solution of the present application has the following beneficial effects: (1) The present application first discloses the key role of PPIF in the pathogenesis of uveitis, especially in the activation of microglial cells, and provides a brand-new drug target for the treatment of uveitis.

[0016] (2) The inhibitor of PPIF 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.

[0017] (3) The present application lays a solid theoretical and experimental foundation for the development of a new generation of drugs for targeted treatment of uveitis. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0019] Figure 1 For proteomics analysis, A is a volcano plot of differentially expressed proteins; B and C are KEGG pathway enrichment analysis charts; D is a key differential protein display chart; E is a comparison chart of the mRNA expression level of PPIF in peripheral blood mononuclear cells of VKH patients and healthy controls; F and G are Western Blot verification results and statistics.

[0020] Figure 2 For the effect of PPIF knockdown on microglial cell function, A and B are the results of reactive oxygen species detection, wherein A is a representative image of CellROX probe fluorescence staining; and B is the quantitative statistics of the average fluorescence intensity of ROS.

[0021] Figure 3 For in vivo verification of PPIF knockdown and overexpression in EAU mouse models, A-D are the results of PPIF knockdown reducing EAU phenotype. Wherein A-B are representative images of anterior segment and clinical score statistics; C-D are representative images of H&E staining of retinal tissue and statistics; E-H are the results of PPIF overexpression aggravating EAU phenotype. Wherein, E-F are representative images of anterior segment and clinical score statistics; G-H are representative images of H&E staining of retinal tissue and statistics. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. Note 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.

[0024] Unless otherwise specifically stated, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specifically stated. The equipment used in the experiments is well known to those skilled in the art unless otherwise specifically stated.

[0025] Example 1 demonstrates the relevance of PPIF to uveitis

[0026] (1) Proteomics screening revealed PPIF as a key differentially expressed protein: The human microglia cell line HMC3 was used. To simulate the inflammatory environment of uveitis, cells were stimulated with interleukin-17a (IL-17a, 50 ng / mL) and interferon-γ (IFN-γ, 100 ng / mL). Cell samples from inflammatory and unstimulated cells were collected, and total protein was extracted using RIPA lysis buffer.

[0027] Proteomics analysis was performed by Jingjie PTM Biolab (Hangzhou, China). In brief, HMC3 cells were collected and lysed, proteins were extracted and hydrolyzed with trypsin. Subsequently, peptides were dissolved in liquid chromatography mobile phase A and separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system (Thermo Fisher Scientific). Peptides were analyzed using an Orbitrap Exploris 480 mass spectrometer. Bioinformatics analysis was assisted by Jingjie PTM Biolab (Hangzhou, China). DIA data were analyzed using the DIA-NN search engine (v.1.8). The relative quantification of altered peptides was determined by standardizing the signal intensity levels of each sample. Differentially expressed proteins that showed significant changes under inflammatory stimulation were screened.

[0028] (2) Results and verification: 1) Inflammatory stimulation induces widespread changes in the microglial proteome: Quantitative proteomic analysis was performed on HMC3 cells under inflammatory and unstimulated conditions. For example... Figure 1 As shown in Figure A, it indicates that the inflammatory environment can broadly affect the protein expression profile of microglia.

[0029] 2) PPIF was identified as a key upregulated factor in the inflammatory response of uveitis: Based on the differentially expressed proteins, our KEGG pathway enrichment analysis showed that inflammatory stimulation significantly activated oxidative phosphorylation and reactive oxygen species (ROS) pathways. Figure 1 Figures B and C). Further analysis of differentially expressed proteins revealed that peptidyl prolyl cis-trans isomerase F (PPIF) is one of the proteins most significantly upregulated under inflammatory conditions. Figure 1 The D-plot suggests that it may play a key role in microglia-mediated inflammatory responses.

[0030] 3) Clinical samples confirm a strong correlation between PPIF and uveitis: To confirm the clinical significance of PPIF, we analyzed transcriptomic data from peripheral blood mononuclear cells (PBMCs) of patients with Vogt-Koyanagi-Harada (VKH) disease. For example... Figure 1As shown in Figure E, the mRNA expression level of PPIF in PBMCs of VKH patients was significantly increased compared with that of healthy controls. This result demonstrates the abnormally high expression of PPIF in human uveitis, highlighting its potential value as a disease biomarker and therapeutic target.

[0031] 4) Protein quantification assay to verify high PPIF expression: To directly verify PPIF expression, we performed a Western blot experiment. Western blot results ( Figure 1 The FH plot showed that PPIF was significantly upregulated in HMC3 cells after inflammatory stimulation, compared with the control group. This directly demonstrates that the increased expression level of PPIF is sufficient to drive the inflammatory response in microglia.

[0032] Conclusion: This embodiment, through unbiased proteomics screening, gain-of-function experiments, and clinical association analysis, jointly confirms that PPIF is a key pathogenic factor activated by the inflammatory environment in the pathogenesis of uveitis, providing a solid basis for its role as an independent therapeutic target.

[0033] Example 2: PPIF knockdown reduces inflammation in microglia and retinal inflammation in an EAU mouse model.

[0034] (1) Knock down sh-PPIF construction

[0035] To knock down PPIF, a lentiviral delivery system was used. A small hairpin RNA (shRNA) targeting PPIF (sh-PPIF) was cloned into a gfp-labeled lentiviral vector to mediate gene transduction. After lentiviral transduction, HMC3 cells were stimulated with IL-17a and IFN-γ, and then harvested for protein analysis.

[0036] Lentiviral transduction: Lentivirals used for gene transduction were constructed, packaged, and purified by Shanghai Gemma Gene Chemical Co., Ltd. Typically, the PPIF shRNA sequence was cloned into the pMagic7.1-Puro / GFP vector to deliver PPIF 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.

[0037] For retroviral transduction, in short, HMC3 cells are introduced at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density in 6-well plates and cultured for 24 hours, then transduced with a lentiviral vector to knock down PPIF. After 72 hours, the lentiviral transduction efficiency was assessed using a fluorescence microscope (Leica DMi8, Germany). Lentivirally transduced cells were screened for 2 days with puromycin and neomycin, and surviving cells were used for subsequent experiments.

[0038] (2) Cell model establishment: The human microglia cell line HMC3 was used, with an empty vector control group and PPIF knockdown. Cells were transduced using the pLVX-CMV-3FLAG-PPIF-CAG-mCherry lentiviral vector to establish a stable PPIF knockdown cell line.

[0039] The PPIF gene was cloned into the pLVX-CMV-3FLAG-PPIF-CAG-mCherry-T2A-neomycin-WPRE vector to deliver PPIF. 293T cells were co-transfected with pCMV-dR8.91, pCMV-VSV-G6363, and the targeting vector to generate pseudovirus particles. The pseudovirus particles were then concentrated and purified by ultracentrifugation, and their titers were determined before use.

[0040] For retroviral transduction, in short, HMC3 cells are introduced at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density in 6-well plates and cultured for 24 hours, then transduced with a lentiviral vector to knock down PPIF. After 72 hours, the lentiviral transduction efficiency was assessed using a fluorescence microscope (Leica DMi8, Germany). Lentivirally transduced cells were screened for 2 days with puromycin and neomycin; surviving cells were used for subsequent experiments.

[0041] (3) Detection of reactive oxygen species (ROS) levels: Intracellular ROS levels were detected using CellROX™ probes. Results are as follows: Figure 2 As shown in the AB diagram, under inflammatory stimulation, the ROS production of cells in the sh-PPIF group was significantly lower than that in the inflammatory stimulation group.

[0042] (4) In vivo experimental verification: PPIF knockdown reduces the clinical and pathological phenotypes of EAU: such as Figure 3 The AD diagram shows a representative H&E staining image of retinal tissue. Compared with the control group, mice in the sh-PPIF group exhibited milder retinal structural damage, inflammatory cell infiltration, and retinal folds. Corresponding clinical and pathological score charts further quantitatively confirmed the significance of this pathological reduction.

[0043] In summary, Example 2 demonstrates through in vitro experiments that knocking down PPIF in human microglia significantly reduces their response to inflammatory stimuli, manifested as a decrease in ROS levels. In vivo experiments confirm that knocking down PPIF in the EAU model significantly alleviates retinal inflammatory damage, validating at the whole-animal level that PPIF is an important factor driving the progression of uveitis.

[0044] Example 3: PPIF overexpression exacerbates inflammation in microglia and retinal inflammation in an EAU mouse model.

[0045] (1) Establishment of cell model: HMC3 cells were spaced at 2 × 10⁶ cells per well. 5 Cells were seeded at a density in 6-well plates and cultured for 24 hours, then transduced with a lentiviral vector to overexpress PPIF. After 72 hours, lentiviral transduction efficiency was assessed using a fluorescence microscope (Leica DMi8, Germany). Lentivirally transduced cells were screened for PPIF by puromycin and neomycin for 2 days, and surviving cells were used for subsequent experiments.

[0046] (2) Intravitreal injection of adenovirus: Six- to eight-week-old wild-type C57BL / 6J mice were randomly divided into two groups: an experimental group and a control group. The experimental group received the following treatment: Shanghai Jikai Gene Chemical Co., Ltd. constructs, packages, and purifies adenoviruses for gene transduction. Typically, the PPIF gene is cloned into the pAD-CAG-3FLAG-PPIF-P2A-mCherry-WPRE vector to deliver PPIF. Pseudoviral particles are generated by co-transfection of 293T cells with pCMV-dR8.91, pCMVVSV-G6363, and a targeting vector. The pseudoviral particles are then concentrated and purified by ultracentrifugation, and their titers are determined before use.

[0047] Intravitreal injection was performed using standard methods. In short, after general and ocular surface anesthesia and mydriasis in mice, adenovirus was injected into the vitreous cavity of WT mice using a microsyringe to overexpress PPIF in the retina.

[0048] For the control group mice, an equal dose of empty vector virus was injected under the same conditions.

[0049] Two weeks after the viral injection, all mice were immunized with EAU.

[0050] (3) Experimental autoimmune uveitis (EAU) induction

[0051] Induction of experimental autoimmune uveitis (EAU) was performed according to existing techniques. 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, 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 administered intraperitoneally with 1 μg of pertussis toxin (PTX; Sigma, catalog no. P7208), and the emulsified IRBP651-670 was administered subcutaneously.

[0052] 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 using a double-blind scoring method to validate the results.

[0053] (4) Results and Analysis: 1) Detection of reactive oxygen species (ROS) levels: Intracellular ROS levels were detected using CellROX™ probes. Results are as follows: Figure 2 As shown in the CD diagram, under inflammatory stimulation, the ROS production of cells in the PPIF overexpression group was significantly higher than that in the inflammatory stimulation group.

[0054] 2) PPIF overexpression exacerbates the clinical and pathological phenotypes of EAU: such as Figure 3 The EH map shows representative H&E staining images of retinal tissue. Compared with the EAU group, mice in the PPIF overexpression group exhibited more severe retinal structural damage, inflammatory cell infiltration, and retinal folds. Corresponding clinical and pathological score statistical plots further quantitatively confirmed the significance of this pathological exacerbation.

[0055] In summary, in vitro experiments demonstrated that overexpression of PPIF in microglia significantly increased their response to inflammatory stimuli, manifested as elevated ROS levels. In vivo experiments confirmed that overexpression of PPIF in the EAU model significantly exacerbated retinal inflammatory damage, validating PPIF as a crucial factor driving uveitis progression at the whole-animal level.

[0056] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. The use of PPIF as a target in the preparation of medicaments for the prevention and / or treatment of uveitis.

2. The application according to claim 1, characterized in that, The drug contains a PPIF inhibitor, which inhibits the expression or activity of PPIF, thereby reducing microglia-mediated inflammatory responses.

3. The application according to claim 2, characterized in that, The PPIF inhibitor is selected from one of the following: RNA interference molecules targeting the PPIF gene, antisense nucleotides, or neutralizing antibodies or small molecule inhibitors targeting the PPIF protein; the RNA interference molecule is a small hairpin RNA (shRNA) or a small interfering RNA (siRNA).

4. The application according to claim 3, characterized in that, The uveitis mentioned is autoimmune uveitis, Vogt-Koyanagi-Harada syndrome.

5. A pharmaceutical composition for the prevention and / or treatment of uveitis, characterized in that, This includes effective doses of PPIF inhibitors and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is an injection or eye drops.

7. The use of a reagent for detecting biomarkers in the preparation of products for diagnosing uveitis, characterized in that, The biomarker is PPIF, and the reagent is used to detect the expression level or activity of PPIF in the sample.

8. The use of the reagent according to claim 7 in the preparation of a kit for detecting uveitis.

Images