Application of STMP1 and inhibitor thereof in preparation of medicine for treating diabetic retinopathy

Abstract

The invention discloses application of STMP1 and an inhibitor thereof in preparation of a medicine for treating diabetic retinopathy. A mouse STMP1 knock-down / knock-out model (KO) is constructed by utilizing a CRISPR-Cas9 technology, comparison is performed in an STZ (streptozotocin) induced diabetes animal model, and a result shows that compared with a wild type (WT) mouse, a KO + STZ group mouse has better performance in the aspects of insulin secretion and insulin sensitivity; meanwhile, electrophysiological detection and eye movement reaction test detection show that the visual function of a KO + STZ mouse is obviously superior to that of a WT + STZ mouse, and it is indicated that STMP1 knock-down can have a protective effect on visual impairment caused by diabetic retinopathy. A novel intervention strategy and medicine taking STMP1 as a treatment target are established on the basis of STMP1, so that the pathological mechanism of diabetic retinopathy can be disclosed, and a safer treatment method and medicine with higher compliance can be provided for patients with diabetic retinopathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of STMP1 and its inhibitors in the preparation of drugs for treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is one of the main causes of blindness in the working-age population (20 - 74 years old). The prevalence of DR ranges from 23% to 37% (with large regional differences), and about 6% to 10% of them are proliferative diabetic retinopathy (PDR). In addition, studies have shown that among diabetic patients aged 50 to 59, the prevalence of DR is the highest, about 22.1%. The pathogenesis of DR mainly includes three aspects: (1) Microvascular damage: A long-term chronic hyperglycemic environment causes damage to microvascular endothelial cells, leading to microcirculation disorders and vascular leakage; (2) Inflammatory response: Under the diabetic state, the local inflammatory level in the retina increases, and the release of inflammatory factors increases, exacerbating vascular damage; (3) Pathological neovascularization: Under ischemia induction, the body tries to improve local blood supply by upregulating signals such as vascular endothelial growth factor (VEGF), but the newly formed blood vessels are extremely fragile and prone to bleeding and leakage, thus triggering a series of complications. DR not only poses a serious threat to the patient's vision, but also brings a huge economic burden to society and the medical system every year, seriously affecting the quality of life of patients.

[0003] At present, the clinical treatment methods for DR mainly include two categories: surgical treatment and drug treatment: (1) Laser photocoagulation: including panretinal photocoagulation (PRP) and focal / grid photocoagulation. PRP is mainly used for patients with PDR or high-risk non-proliferative DR (NPDR), and reduces VEGF secretion by destroying ischemic retinal tissue; focal or grid photocoagulation is mainly used for the treatment of diabetic macular edema (DME). However, laser treatment may cause irreversible visual function damage such as visual field constriction, night blindness, and abnormal color vision. (2) Anti-VEGF drug treatment (such as ranibizumab, aflibercept, etc.), by blocking the VEGF signaling pathway, can significantly improve vision and delay the progression of DR. However, this type of drug requires frequent intravitreal injections (once every 4 - 8 weeks), bringing pain and inconvenience to patients; and about 30% - 50% of patients have problems of drug resistance or poor treatment response, which may be related to the activation of other pathological pathways. In addition, long-term systemic VEGF inhibition may also exacerbate retinal ischemia and increase the risk of cardiovascular complications.

[0004] Recent research has revealed that mitochondrial dysfunction plays a key role in the pathogenesis of DR. Studies have found that in the diabetic state, the mitochondrial function of retinal microglia and other local cells is impaired, leading to a decrease in ATP production (about 40%-50%), an increase in the level of reactive oxygen species (ROS) (2 to 3 times), and the accumulation of mtDNA damage. STMP1 was initially discovered in the mass spectrometry analysis of bovine heart mitochondrial membrane proteins. Through tandem mass spectrometry technology, researchers identified multiple mitochondrial membrane proteins in an analysis, including a small peptide of only 47 amino acids, namely STMP1. Currently, there is no research on mitochondrial-derived short peptides (<100 amino acids), especially STMP1 (Short Transmembrane Mitochondrial Protein 1), and its role in DR has not been explored. DR urgently needs to develop new safe and effective treatment strategies due to its high prevalence and limitations of existing treatment methods (such as the side effects of frequent intravitreal injections and laser treatment, and drug resistance in some patients). Summary of the Invention

[0005] Aiming at the above deficiencies of the prior art, the purpose of the present invention is to provide the application of STMP1 and its inhibitors in the preparation of drugs for treating diabetic retinopathy.

[0006] We constructed a mouse STMP1 knockdown / knockout model (KO) using CRISPR-Cas9 technology and conducted a comparative study in a diabetic animal model induced by STZ (streptozotocin). The results showed that compared with wild-type (WT) mice, the KO+STZ group of mice had better insulin secretion and insulin sensitivity; at the same time, through ERG (electrophysiological detection) and eye movement response tests, it was found that the visual function of KO+STZ mice was significantly better than that of WT+STZ mice, suggesting that STMP1 knockdown can protect against vision damage caused by DR.

[0007] The present invention targets STMP1 and regulates STMP1 through a variety of molecular biology means to provide drugs for treating diabetic retinopathy. Specifically, it includes:

[0008] 1. Using viral vectors such as adeno-associated virus (AAV) and lentivirus to mediate the knockdown of STMP1, inhibit the expression of STMP1, and thus alleviate the symptoms of diabetic retinopathy.

[0009] 2. Applying CRISPR-Cas9 gene editing technology to target and edit the STMP1 gene, reduce the expression level of STMP1, and prevent and treat the occurrence of diabetic retinopathy.

[0010] 3. By using techniques such as antisense oligonucleotides, shRNA, or RNAi, the expression of STMP1 is silenced or knocked down by targeting STMP1 mRNA, thereby exerting a therapeutic effect.

[0011] 4. Utilize a nanodelivery system with good biocompatibility and strong targeting ability, such as liposomes, polymer nanoparticles, etc., to carry STMP1 inhibitory molecules (such as siRNA, antisense oligonucleotides, etc.), and achieve targeted and efficient delivery to the retina, reduce the expression of STMP1, and treat diabetic retinopathy.

[0012] The above-mentioned various STMP1 regulation means aim to alleviate the key pathological processes of diabetic retinopathy by reducing the expression level of STMP1, such as: reducing the activation and inflammatory response of microglia; improving mitochondrial function and inhibiting oxidative stress; protecting retinal neurons from damage.

[0013] The present invention takes STMP1 as a new therapeutic target and specifically adopts diverse means such as molecular biology and nanodelivery, providing innovative strategies and drugs for the treatment of diabetic retinopathy, and having important clinical application value.

[0014] The first object of the present invention is to provide the use of an STMP1 inhibitor in the preparation of a drug for treating diabetic retinopathy.

[0015] Preferably, the STMP1 inhibitor includes at least one of the following (1)-(3):

[0016] (1) A preparation for inhibiting or reducing the expression of the STMP1 gene;

[0017] (2) A preparation for completely knocking out or partially knocking out the STMP1 gene;

[0018] (3) A preparation for inhibiting or blocking the activity of the STMP1 protein.

[0019] Preferably, the STMP1 gene is the 1810058I24Rik gene (abbreviation: 181-Rik), the NCBI Gene ID is 67705, and the amino acid sequence of the STMP1 protein is MLQFLLGFTLGNVVGMYLAQNYEMPNLAKKLEEIKKDLEAKKKPPSS.

[0020] Preferably, the preparation for inhibiting or reducing the expression of the STMP1 gene is a preparation for inhibiting the expression of the STMP1 gene using an adeno-associated virus or a lentiviral vector, or a preparation for silencing or knocking down the expression of the STMP1 gene by targeting STMP1 mRNA with antisense oligonucleotides, shRNA, or RNAi technology.

[0021] Preferably, the preparation is delivered to the retina using a targeted nanodelivery system.

[0022] Preferably, the preparation for completely knocking out or partially knocking out the STMP1 gene is a preparation for targeting and editing the STMP1 gene with CRISPR-Cas9 to reduce the expression of the STMP1 gene.

[0023] Preferably, the gRNAs for targeting and editing the STMP1 gene with CRISPR-Cas9 are as follows: gRNA1: CTTCCCAAGAAGTGCACGGC; gRNA2: TGCACGGCAGGTGACTGCCC; gRNA3: GAGGCATCCTCC CCGGAAGC; gRNA4: CGGAAGCAGGACGGACCATC.

[0024] Preferably, the preparation for inhibiting or blocking the activity of the STMP1 protein is an antibody against the STMP1 protein.

[0025] Preferably, the drug for treating diabetic retinopathy is a drug having at least one of the following functions (1)-(4):

[0026] (1) Promote blood glucose clearance ability, improve insulin response efficiency, and relieve metabolic disorders under diabetic conditions;

[0027] (2) Maintain the function of retinal pigment epithelial cells;

[0028] (3) Protect retinal function;

[0029] (4) Improve the function of the visual conduction pathway, enhance visual acuity, and protect visual function.

[0030] The second object of the present invention is to provide a drug for treating diabetic retinopathy, which contains an STMP1 inhibitor as an active ingredient, and the STMP1 inhibitor includes at least one of the following (1)-(3):

[0031] (1) A preparation for inhibiting or reducing the expression of the STMP1 gene;

[0032] (2) A preparation for completely knocking out or partially knocking out the STMP1 gene;

[0033] (3) A preparation for inhibiting or blocking the activity of the STMP1 protein.

[0034] The present invention provides the use of STMP1 and its inhibitors in the preparation of drugs for treating diabetic retinopathy, and establishes a new intervention strategy and drugs targeting STMP1, which not only helps to reveal the pathological mechanism of diabetic retinopathy, but also provides safer and more compliant treatment methods and drugs for patients with diabetic retinopathy. Brief Description of the Drawings

[0035] Figure 1 It shows that STMP1 is highly expressed after culturing human retinal microvascular endothelial cells (HRMECs) in high glucose for 48 hours; (A-A’’’) After culturing HRMECs in basal medium for 48 hours, immunofluorescence staining was performed on STMP1, COXIV (mitochondrial inner membrane marker antibody), and DAPI (nuclear dye); (B-B’’’) After adding 30 mM mannitol to the basal medium and culturing HRMECs for 48 hours, immunofluorescence staining was performed on STMP1, COXIV, and DAPI; (C-C’’’) After adding 30 mM glucose to the basal medium and culturing HRMECs for 48 hours, immunofluorescence staining was performed on STMP1, COXIV, and DAPI.

[0036] Figure 2 It shows the construction and detection of Stmp1 knockout gene mice (Stmp1KO); (A) Schematic diagram of Stmp1KO construction. Using CRISPR / Cas9 homologous recombination technology, a part of the second exon and all of the third exon of the Stmp1 gene were replaced with GFP sequences to construct Stmp1KO mice. F1 / R1 are PCR primers for detecting WT mice, and F2 / R2 are PCR primers for detecting KO mice; (B) Electrophoresis diagram after paired mouse tail PCR detection with F1 / R1 and F2 / R2. Lane 1 is wild type WT, 2 is homozygous Stmp1KO, 3 and 4 are heterozygous Stmp1HE. The WT band is 910 bp, the KO band is 802 bp; the HE band is a double band of 910 bp and 802 bp; (C) Protein immunoblot (Western blot) was used to detect the expression of STMP1 protein in the retinas of WT and Stmp1KO; (D-E) PCR was used to detect the mRNA expression levels of Stmp1 and GFP in the retinas of WT and Stmp1KO.

[0037] Figure 3The Stmp1KO (KO+STZ) diabetic retinopathy model induced by streptozotocin (STZ) has better glucose tolerance; (A) Blood glucose change curve plotted by glucose tolerance testing (GTT); (B) Area under the blood glucose curve calculated by glucose tolerance testing; The experiment was repeated at least 3 times, with at least 4 mice in each treatment group each time, *p<0.01, ***p<0.0001.

[0038] Figure 4 The KO+STZ diabetic retinopathy model has better insulin tolerance; (A) Blood glucose change curve plotted by Insulin tolerance testing (ITT); (B) Area under the blood glucose curve calculated by insulin tolerance testing; The experiment was repeated at least 3 times, with at least 4 mice in each treatment group each time, **p<0.001, ***p<0.0001.

[0039] Figure 5 STMP1 knockout can protect the retinal pigment epithelium (RPE) function injury in diabetic model mice; (A) Representative curve of c-wave amplitude detected by electroretinogram (ERG) of mice after 5 months of feeding after successful establishment of the diabetic model; (B) Statistical results of c-wave amplitude; The experiment was repeated at least 3 times, with at least 4 mice in each treatment group each time; **p<0.001, ***p<0.0001.

[0040] Figure 6 The visual acuity of the KO+STZ group is higher than that of the WT+STZ group; The experiment was repeated at least 3 times, with at least 4 mice in each treatment group each time; **p<0.001, ***p<0.0001. Detailed implementation manners

[0041] The following examples are further descriptions of the present invention, rather than limitations on the present invention.

[0042] The present invention uses CRSPR / Cas9 technology to knockdown / knockout Stmp1 in mice, and combines experiments such as mouse diabetic retinopathy model, GTT experiment, ITT experiment, visual function detection and optokinetic response detection, etc., and for the first time discovers that knockdown / knockout of Stmp1 has a protective effect on the visual function of mice with diabetic retinopathy.

[0043] The adopted scheme can be divided into four steps:

[0044] 1. Discover the expression of STMP1 in human retinal microvascular endothelial cells (HRMECs) induced by high glucose.

[0045] 2. Construction of Stmp1KO gene knockout mice.

[0046] 3. Evaluation of glucose and insulin tolerance in Stmp1KO and WT mice after establishing a diabetic retinopathy model.

[0047] 4. Evaluation of visual function in Stmp1KO and WT mice after establishing a diabetic retinopathy model.

[0048] Example 1

[0049] 1. Expression of STMP1 in HRMECs induced by high glucose

[0050] 1.1 Method

[0051] 1.1.1 Preparation of culture medium and 0.1% gelatin solution

[0052] (1) Basal medium (50 mL): DMEM / F12 (Gibco, 11330032): 44.5 mL, FBS (Gibco, 10270106): 2.5 mL (5%), Penicillin - Streptomycin (Gibco, 15140122): 0.5 mL (1%), VEGF (Sino Biological, 11066): 10 μL (100 μg / mL stock solution), bFGF (Sino Biological, 10014): 5 μL (100 μg / mL stock solution), IGF - 1 (Sino Biological, 10011): 5 μL (100 μg / mL stock solution), Hydrocortisone (Aladdin, H657409): 5 μL (10 mg / mL stock solution).

[0053] (2) High mannitol medium (50 mL): 50 mL basal medium + 0.27 g mannitol (Aladdin, M104172). After dissolution, filter sterilize with a 0.22 μm syringe microfiltration membrane (Millipore, SLGV033RB).

[0054] (3) High glucose medium (50 mL): 50 mL basal medium + 0.27 g glucose (Aladdin, G104951). After dissolution, filter sterilize with a 0.22 μm syringe microfiltration membrane (Millipore, SLGV033RB).

[0055] (4) 0.1% (w / v) gelatin solution (50 mL): 0.05 g gelatin (Sigma - Aldrich, G1890) is completely dissolved in 50 mL ddH2O, and then filter sterilize with a 0.22 μm syringe microfiltration membrane (Millipore, SLGV033RB) first.

[0056] 1.1.2 Cultivation of HRMECs

[0057] (1) Petri dish coating: Coat a 6-cm Petri dish (Corning, 430166) with 0.1% gelatin solution for 4 - 6 hours, and wash it twice with water before seeding cells.

[0058] (2) Cell resuscitation: Rapidly resuscitate cells in a 6-cm Petri dish at 37°C and culture for 48 hours until the density exceeds 90%.

[0059] (3) Pre-place 12-mm cell slides in 12 of the wells of a 24-well plate (Corning, 3524), and add 300 μL of 0.1% gelatin solution to each well for coating.

[0060] (4) Digest the cells in the 6-cm Petri dish with 0.25% Trypsin-EDTA (Gibco, 25200056), and seed 1x10 5 cells into each well with a coverslip, and culture in an incubator at 37°C and 5% CO2.

[0061] (5) After 24 hours, wash once with PBS, and add basal medium, high mannitol medium, and high glucose medium to every 4 wells respectively.

[0062] 1.1.3 Cell immunofluorescence staining

[0063] (1) After culturing HRMECs in different media in a 24-well plate for 48 hours, take them out for immunofluorescence staining.

[0064] (2) Aspirate the medium, and immediately add 1 mL of PBS to each well and let it stand at room temperature for 5 minutes.

[0065] (3) Aspirate the PBS, and immediately add 500 μL of 0.3% PBST (100 mL PBS + 300 μL Triton-100) for cell membrane permeabilization, and let it stand at room temperature for 15 minutes.

[0066] (4) Aspirate the PBST, and immediately add 500 μL of 4% PFA for protein fixation, and let it stand at room temperature for 15 minutes.

[0067] (5) Aspirate the PFA, add 1 mL of PBS, let it stand for 5 minutes, and repeat 2 more times.

[0068] (6) Aspirate the PBS, add 300 μL of 10% normal donkey serum (Jackson ImmunoResearch, 017-000-122) for blocking, at room temperature for 1 hour.

[0069] (7) The blocking solution was removed by aspiration, and 250 μL of 2% normal donkey serum diluted 1:100 with STMP1 (Novus Biologicals, NBP2-56550) and 1:1000 with COX IV (abcam, ab33985) were added and incubated at 4°C overnight.

[0070] (8) Aspirate the primary antibody, add 1 mL of PBS, let it stand for 5 minutes, and repeat twice.

[0071] (9) Aspirate the PBS and add 300 μL of secondary antibody (1:1000) and DAPI (1:1000) diluted in PBS. Incubate at room temperature in the dark for 1 hour.

[0072] (10) Aspirate the secondary antibody, add 1 mL of PBS, let it stand for 5 minutes, and repeat twice.

[0073] (11) Seal the tube, protect from light, and take photos using laser confocal microscopy after the surface is dry.

[0074] 1.2 Results

[0075] The HRMECs cultured in three culture media were photographed using confocal microscopy. The results showed that after 48 hours of basal culture of HRMECs, STMP1, COXIV (mitochondrial inner membrane marker antibody), and DAPI (nuclear dye) were immunofluorescently stained. HRMECs expressed low levels of STMP1 protein ( Figure 1 AA"'); 30 mM mannitol was added to the basal culture medium. After culturing HRMECs for 48 hours, STMP1, COXIV, and DAPI were immunofluorescently stained. HRMECs lowly expressed STMP1 protein ( Figure 1 BB”'), because mannitol is a sugar that HRMECs cannot absorb and metabolize, it is often used as an osmotic pressure control to exclude the influence of high sugar concentration on cell osmotic pressure; 30mM glucose was added to the basal medium, and after culturing HRMECs for 48 hours, STMP1, COXIV, and DAPI were immunofluorescently stained. HRMECs highly expressed STMP1 protein ( Figure 1 CC”’). Based on the above results, we speculate that STMP1 may be involved in the regulation of HRMECs’ metabolism of high glucose and other activities, which will promote the subsequent study of STMP1-mediated diabetic retinopathy.

[0076] 2. Construction of Stmp1KO knockout mice

[0077] 2.1 Methods

[0078] The schematic diagram of the construction of Stmp1 knockout mice (Stmp1KO) is shown in Figure 2 A.

[0079] 2.1.1 gRNA Design and Verification

[0080] 2.1.1.1 NCBI Gene Retrieval and Structure Analysis

[0081] (1) Retrieve the target gene: Use the database NCBI Gene ( https: / / www.ncbi.nlm.nih.gov / gene ), enter the gene name 1810058I24Rik (abbreviation: 181-Rik), also known as: Stmp1, Gene ID: 67705, and select Mus musculus (mouse) for the species.

[0082] (2) Obtain exon information: View the chromosomal location of the gene in the "Genomic context" section (such as Chr6: 35,252,654 - 35,263,496), click "NCBI Reference Sequences (RefSeq)" and select "mRNA and Protein(s)" to view the transcript, and confirm the number of exons in the "Graphics" or "Table" view. It shows that 1810058I24Rik has 3 exons.

[0083] (3) Download the genomic sequence: Obtain the Exon 2 and its flanking intron sequences (for gRNA design) in the "GenBank" format: Exon 2: CTTGGATTTACTTTGGGCAACGTGGTTGGAATGTATCTGGCTCAGA ACTATGAA; Intron 1 (5' flanking): TATATATGTATGTGATTGTTTCATAGAAGAAAGATGTGAC TTCTCTGTTGGATGCAGTCCTACACTGAGGACCTGGGCAGTCACCTGCCGTGCACTTCTT GGGAAGATGCAGCCCGTGTG; 3'Downstream (3' flanking): TTGAGGCATCCTCCCCGGAAG CAGGACGGACCATCAGGCGTCAGCAACTTCATTTTTTTAAAGACAGTCTCCCAGTGTTA CCCAGGGTGGTCTCAAGCTCCTGGGCTCAA.

[0084] 2.1.1.2 CRISPR Target Design

[0085] (1) Enter the target sequence: Use crispr.mit.edu (http: / / crispr.mit.edu ), select the "Mouse(mm10)" genome and paste the Intron 1 + Exon 2 + 3'Downstream sequence (make sure to include at least 200bp of flanking region).

[0086] (2) Screen for high-scoring gRNAs: Scoring criteria, efficiency score (Score) ≥ 80 (the higher the better), off-target risk (Off-targets) ≤ 3 potential sites, and obtain the following high-scoring gRNAs. 5'-end gRNAs: gRNA1: CTTCCCAAGAAG TGCACGGC (PAM: CGG); gRNA2: TGCACGGCAGGTGACTGCCC (PAM: AGG); 3'-end gRNAs: gRNA3: GAGGCATCCTCCCCGGAAGC (PAM: AGG); gRNA4: CGGAAGCAGGAC GGACCATC (PAM: AGG).

[0087] 2.1.1.3 Off-target analysis and verification

[0088] (1) Online tool verification: COSMID ( https: / / crispr.bme.gatech.edu ) Enter the gRNA sequence to predict the off-target sites in the whole genome; UCSC Genome Browser: Check whether the off-target sites are located in the coding regions of other genes.

[0089] (2) Experimental verification: In vitro cleavage assay. Synthesize gRNA + Cas9 and transfect mouse fibroblasts (NIH3T3), and sequence to detect the cleavage efficiency; T7E1 digestion: If a double-peak sequencing result is found, use T7 Endonuclease I to detect the indel frequency. 2.1.2 Donor vector construction

[0090] 2.1.2.1 Homologous arm design

[0091] (1) The length of the 5’ homologous arm (5’ homology arm) is 800bp, starting 600bp upstream of Exon 2 (including part of the Intron1 sequence), and ending after the first amino acid codon CTT of Exon 2 (retaining the complete start codon ATG).

[0092] (2) The length of the 3’ homologous arm (3’ homology arm) is 800bp, starting 50bp downstream of Exon 3 and ending at the 3’ untranslated region (UTR).

[0093] (3) Insertion sequence: For EGFP, optimized mammalian codons (such as the pEGFP-N1 vector sequence) are used, and a Kozak sequence (GCCACC) is added before the start codon. The PolyA signal uses the SV40 late polyA signal (about 200bp). Ligation design: EGFP is directly ligated after the first amino acid (CTT) of Exon 2, and the downstream sequence of natural Exon 3 is connected after the stop codon of EGFP.

[0094] 2.1.2.2 Vector assembly

[0095] pUC19 is used as the backbone vector, and the 5'-homologous arm (800bp)-EGFP-polyA-3'-homologous arm (800bp) is inserted into the linearized vector by Gibson Assembly or In-Fusion cloning. After assembly, Escherichia coli (such as DH5α) is transformed, and positive clones are screened by ampicillin resistance. PCR (vector universal primers + insertion fragment-specific primers) and Sanger sequencing are used to verify the correct ligation. The key sequencing regions include the Exon2-EGFP junction and the polyA-3'-homologous arm junction region to ensure no frameshift or accidental mutations.

[0096] 2.1.2.3 Quality control

[0097] The integrity of the vector is verified by double digestion (NotI / XhoI), and a 2.6kb insertion fragment and a 2.7kb vector backbone are expected to be released. Sanger sequencing is performed on the positive clones, focusing on verifying: the Exon2-EGFP connection region (ensuring that the start codon CTT is retained and there is no frameshift); the EGFP-polyA-3'-homologous arm junction region (confirming the termination signal and seamless sequence ligation); both ends of the homologous arms (excluding random mutations introduced by PCR). At the same time, the fluorescence expression of EGFP is observed by transient transfection of 293T cells to preliminarily verify the function.

[0098] 2.1.3 Microinjection experimental protocol

[0099] 2.1.3.1 Preparation of injection mixture

[0100] Cas9 mRNA: 50 ng / μL (prepared using the T7 in vitro transcription system, capped at the 5' end and polyA-tailed at the 3' end). gRNA mixture: 25 ng / μL (equal amounts of 5'- and 3'-designed gRNAs are mixed). Donor vector: 20 ng / μL (extracted and purified by plasmid, OD260 / 280 = 1.8 - 2.0). Buffer: Use RNase-free TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).

[0101] 2.1.3.2 Preparation of fertilized eggs:

[0102] (1) Donor mice: 8 - 12 - week - old C57BL / 6J female mice (Shanghai Model Organisms Center, Inc.).

[0103] (2) Superovulation treatment: Inject 5 IU of PMSG (Pregnant Mare Serum Gonadotropin, Sigma - Aldrich, G4527) intraperitoneally at 3 pm. After 48 hours, inject 5 IU of hCG (Human Chorionic Gonadotropin, Sigma - Aldrich, C1063) and immediately co - house with vasectomized male mice.

[0104] (3) Collection of fertilized eggs: Sacrifice the female mice 20 - 22 hours after hCG injection. Collect the ampulla of the oviduct, release the cumulus cells with M2 medium (Millipore, MR - 015 - D) containing 0.1% hyaluronidase, and select fertilized eggs at the pronuclear stage with normal morphology (diameter about 70 - 80 μm).

[0105] 2.1.3.3 Microinjection operation:

[0106] (1) Instrument settings: Micro - operating system: Equipped with an Eppendorf microinjector. Holding pipette and injection needle: Inner diameter 10 - 15 μm. Injection parameters: Pressure 300 - 500 hPa, time 0.5 - 1 second.

[0107] (2) Injection process: Place 10 - 15 fertilized eggs in a micro - drop of M2 medium, inject 1 - 2 pL of the mixture (visible swelling of the pronucleus) into each fertilized egg, and immediately transfer them to KSOM culture medium (Millipore, MR - 121 - D) after injection, and culture at 37°C and 5% CO2 until the 2 - cell stage.

[0108] 2.1.3.4 Embryo transfer

[0109] (1) Preparation of recipient mice: Select pseudopregnant ICR female mice (Shanghai Model Organisms Center, Inc.), and anesthetize them (2% isoflurane) on the day of transplantation.

[0110] (2) Surgical transplantation: Transplant 15 - 20 normally developed 2 - cell embryos into each oviduct. Use a blunt - tipped transfer pipette to transplant retrogradely through the fimbria of the oviduct, and inject antibiotics (50,000 units of penicillin per mouse) continuously for 3 days after surgery.

[0111] 2.2 Stmp1KO genotype identification and Stmp1 expression level in the retina of Stmp1KO mice

[0112] 2.2.1 Stmp1KO genotype identification

[0113] 1. Preparation of mouse tail digestive solution: 100 mM KCl (Sigma-Aldrich, P9541), 20 mM Tris (Sigma-Aldrich, T1503), 0.1% NP40 (Sigma-Aldrich, 74385), 0.1% Tween-20 (Sigma-Aldrich, P9416).

[0114] 2. Sample collection: Cut about 2 mm of mouse tail tip tissue and place it into a pre-labeled 1.5 mL EP tube. Add 100 μL of tail tissue digestion buffer to each tube, and supplement with 1 μL of proteinase K (stock concentration: 20 mg / ml). Mix well.

[0115] 3. Digestion: Place the EP tubes in a 55 °C water bath / incubator overnight (12 - 16 hours).

[0116] 4. Termination treatment: Vortex the next day to ensure complete digestion, heat in a 95 °C boiling water bath for 5 minutes to inactivate proteinase, and briefly centrifuge after 1 minute of ice bath.

[0117] 2.2.1.1 PCR amplification

[0118] (1) The PCR reaction system is shown in Table 1.

[0119] Table 1 PCR reaction system

[0120]

[0121] (2) The PCR primers are shown in Table 2.

[0122] Table 2 Primer sequences for mouse genotype identification

[0123]

[0124] (3) The PCR amplification program is shown in Table 3.

[0125] Table 3 PCR amplification program

[0126]

[0127] (4) Detection by agarose gel electrophoresis

[0128] Prepare a 1.5% agarose gel for electrophoresis. Add 6 μL of the obtained PCR product to each well, run the gel at 200 V for 20 min, take pictures and analyze the PCR target bands in the gel imaging system. Among them, the WT genotype shows a single 910 bp band, the Stmp1KO gene mouse shows a single 802 bp band, and the heterozygous Stmp1 shows two bands of 802 bp and 910 bp ( Figure 2B). 2.2.2 Expression level of Stmp1 in the retina of Stmp1KO mice

[0129] To identify the efficiency of Stmp1 knockout in Stmp1KO mice, we used Western blot and RT-qPCR to quantitatively analyze the proteins and mRNAs extracted from the retinas of WT and Stmp1KO mice.

[0130] 2.2.2.1 Identification of Stmp1 protein expression level in the retina of Stmp1KO mice

[0131] (1) Extraction of mouse retinal proteins: WT and Stmp1KO mice at 4 - 6 weeks old were sacrificed. The eyeballs were quickly removed and the retinas were isolated (operation on ice). RIPA lysis buffer (containing 1× protease inhibitor) was added, and homogenization was performed on ice for 30 seconds. Centrifugation was carried out at 4°C and 12,000 rpm for 15 minutes, and the supernatant was collected. The protein concentration was determined by the BCA method and adjusted to a uniform concentration (such as 2 μg / μL).

[0132] (2) Western blot assay: Antibody information: Primary antibody: Rabbit anti-Stmp1 polyclonal antibody (1:100, NovusBiologicals, NBP2-56550), secondary antibody: HRP-labeled goat anti-rabbit IgG (1:5000, CST, 7074), internal reference: β-actin (1:2000, Sigma, A5441). A 10% SDS-PAGE gel was prepared, and the sample loading amount was 20 μg / well; Transfer membrane: PVDF membrane, constant voltage of 100V for 1 hour; Development: ECL chemiluminescence method, and the gray value of the band was quantitatively analyzed by ImageJ.

[0133] (3) Result analysis: The results of Western blot showed that Stmp1 protein was no longer expressed in the retina of Stmp1KO mice ( Figure 2 C), indicating that the construction of Stmp1KO gene knockout mice was successful and could be used for subsequent exploration of the function of Stmp1.

[0134] 2.2.2.2 Identification of Stmp1 mRNA expression level in the retina of Stmp1KO mice

[0135] (1) Extraction of mouse retinal RNA: WT and Stmp1KO mice at 4 - 6 weeks old were sacrificed. The eyeballs were quickly removed and the retinas were isolated (operation on ice). Total RNA of WT and Stmp1KO mice retinas was obtained according to the method steps of TRIzol method (Thermo Fisher, 15596026), and 1 μg of RNA template was reverse transcribed into cDNA using PrimeScript RT kit (Takara, RR047A).

[0136] (2) RT-qPCR assay: The qPCR reaction system is shown in Table 4, the primers are shown in Table 5, and the amplification program is shown in Table 6. Then perform the RT-qPCR assay.

[0137] Table 4 qPCR reaction system

[0138]

[0139]

[0140] Table 5 RT-qPCR primers for Stmp1 and GFP

[0141]

[0142] Table 6 qPCR amplification program

[0143]

[0144] (3) Result analysis: Analysis of the experimental results of the RT-qPCR assay showed that compared with the retinas of WT mice, Stmp1 mRNA was no longer transcribed in the retinas of Stmp1KO mice ( Figure 2 D), but GFP was highly expressed instead ( Figure 2 E), proving that after EGFP was inserted into the Stmp1 exon, the Stmp1 gene and its expression were disrupted, and then EGFP was expressed, further proving the successful construction of Stmp1KO knockout mice.

[0145] 2.2.2.3 Evaluation of glucose and insulin tolerance after establishing diabetic retinopathy models in Stmp1KO and WT mice

[0146] Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes, and about 35% of patients will become blind. Drugs such as alloxan, streptozocin (STZ), dexamethasone, and adrenaline can all successfully induce DR models in mice. Among them, the success rate of constructing DR models with STZ is high, so the STZ-induced DR model in mice is widely used. Mechanism of STZ-induced DR: STZ enters pancreatic islet β cells through glucose transporter 2 (GLUT2), causing DNA alkylation, leading to β cell apoptosis and insufficient insulin secretion. Then, it causes persistent hyperglycemia (≥16.7 mmol / L), triggering microvascular lesions, and ultimately resulting in retinal damage.

[0147] 3.1 Method for constructing STZ-induced diabetic retinopathy model

[0148] 3.1.1 Fasting treatment

[0149] (1) Starting at 18:00 on the evening before STZ injection, fast the experimental mice (WT and Stmp1KO), remove all feed but keep free access to water, and ensure that the fasting duration is 14 hours until 8:00 the next morning. This step is crucial as it can reduce the interference of food on STZ absorption, ensure the consistency of drug action, and avoid the impact of postprandial blood glucose fluctuations on the modeling effect.

[0150] (2) During fasting, use a metabolic cage to monitor the water intake (the nocturnal water intake of normal mice is about 3 - 5 mL), and closely observe the status of the mice. If severe lethargy occurs, immediately terminate the fasting.

[0151] (3) It should be noted that mice in the same batch must be fasted synchronously to avoid individual differences. A fasting duration of less than 12 hours may lead to unstable STZ effects, while more than 16 hours may cause stress-induced hypoglycemia.

[0152] 3.1.2 STZ Injection

[0153] (1) First, dissolve STZ (Sigma - Aldrich, S0130) with pre - cooled 10 mmol / L sodium citrate buffer (pH 4.5). Calculate the dose at 60 mg / kg (for example, for a 20 g mouse, 1.2 mg of STZ is dissolved in 100 μL of buffer), vortex - mix under ice - bath and light - avoiding conditions, and filter - sterilize through a 0.22 μm filter membrane.

[0154] (2) When injecting, choose the lower left quadrant of the abdomen to insert the needle to avoid the bladder and liver. Strictly control the injection volume at 100 μL / 20 g body weight, and slowly inject for 30 seconds per mouse to avoid a sudden increase in intra - abdominal pressure. This process needs to be repeated at the same time point (such as 8:00 - 9:00 every day) for 5 consecutive days. The control group is injected with an equal volume of sodium citrate buffer.

[0155] (3) It should be particularly noted that the STZ solution must be prepared and used immediately (injected within 30 minutes after preparation). The syringe needs to be wrapped with aluminum foil to avoid light, and gently press the injection site after injection to prevent the leakage of the drug solution.

[0156] 3.1.3 Management after Injection

[0157] (1) Provide 5% glucose water within 2 hours after injection to prevent hypoglycemic shock, and change to regular drinking water 24 hours later.

[0158] (2) During daily monitoring, focus on the activity level of the mice (such as lethargy, hunched back, slow movement), hair condition (such as fluffy and dull), and eyelid closure (such as drooping).

[0159] (3) After modeling, the mice should be fed a high-fiber, sugar-free diet to prevent ketoacidosis. If severe symptoms such as convulsions or coma are observed in mice, 10% glucose (0.1 mL / 10 g) should be injected subcutaneously for emergency treatment. Deaths should be autopsied (successful models should show extensive pancreatic β-cell necrosis). These management measures can effectively alleviate the acute toxicity of STZ and keep the mortality rate below 15%.

[0160] 3.1.4 Model Validation

[0161] (1) Fasting blood glucose was tested on days 7, 14, 21, and 28 after the first injection (samples were collected at 8:00-10:00 AM after fasting for 4 hours). The tail tip blood sampling method (puncture the tail vein after disinfection with 75% alcohol) was used and the same brand of blood glucose meter was used to measure twice in a row to obtain the average value.

[0162] (2) A successful diabetes model must meet three core criteria: fasting blood glucose ≥ 13.8 mmol / L for two consecutive times (48 hours apart), water intake ≥ 150% of the control group (24-hour data monitored in metabolic cages), and body weight loss > 10% (compared to the baseline value before injection). Clinical symptoms (dehydration signs such as unkempt hair and sunken eyes) must also be observed and the mortality rate must be < 15% within 28 days. Mice with large blood glucose fluctuations (< 11.1 mmol / L on a single test) or without polydipsia and polyuria should be excluded, indicating that their β cells may not be completely destroyed.

[0163] 3.2 Glucose tolerance test (GTT or IGTT) and results

[0164] The GTT test calculates the insulin resistance index by combining the area under the curve (AUC), which reflects the degree of damage to pancreatic β cells caused by STZ.

[0165] 3.2.1 Glucose tolerance test (GTT or IGTT)

[0166] (1) Experimental subjects: 4 WT mice, 4 Stmp1KO mice (KO), 4 STZ-induced WT mice with diabetic retinopathy on day 60 (WT+STZ), and 4 STZ-induced Stmp1KO mice with diabetic retinopathy on day 60 (KO+STZ).

[0167] (2) Basal blood glucose measurement (T0): Basal blood glucose measurement is the benchmark step of the GTT experiment and should be carried out after fasting (but not water deprivation) for 12 - 16 hours. During the operation, disinfect the mouse tail tip with 75% alcohol. After drying, puncture the tail vein with a sterile needle and gently squeeze out a blood droplet (about 1 mm in diameter) for blood glucose meter detection. It is required that the measurement be completed with test strips of the same brand and the same operator. Record the fasting blood glucose value > 13.8 mmol / L to meet the subsequent experimental conditions. The following points should be noted in this step: ① Transfer the mouse to an environment of 28 °C 30 minutes before blood collection to reduce stress; ② If the difference between two consecutive measurements is > 10%, resampling is required; ③ Individuals with severe hyperglycemia (> 25 mmol / L) should be excluded to avoid experimental risks.

[0168] (3) Glucose load: Measure the basal body weight. Use a 20% glucose sterile PBS solution (prepared freshly before use), and accurately calculate the injection volume according to 2 g / kg body weight (for example, a 20 g mouse is injected with 200 μL). For intraperitoneal injection, select the left lower abdominal quadrant for needle insertion and slowly inject at a 45° angle (completed within 30 seconds). Immediately record it as the T0 time point after injection. The key control points include: ① The glucose solution needs to be preheated to 37 °C; ② The syringe needs to remove air bubbles (error < 5 μL); ③ The control group is injected with an equal volume of PBS synchronously. This dose can make the blood glucose of normal mice reach the peak (9 - 10 mmol / L) within 30 minutes, while that of STZ model mice is usually > 20 mmol / L.

[0169] (4) Blood glucose monitoring time points: Set four key time points: T15, T30, T60, T120 min (i.e., 15 min, 30 min, 60 min, 120 min): T15 min reflects the initial insulin response ability, and the blood glucose rising slope of the STZ model group is significantly increased during this period; T30 min corresponds to the peak of the first-phase insulin secretion, and there is no downward inflection point in diabetic mice at this stage; T60 min evaluates the liver's glucose uptake function; T120 min is the diagnostic standard node, and > 11.1 mmol / L is judged as impaired glucose tolerance. Draw a blood glucose change curve. Blood collection must be carried out strictly on time (error ± 1 min), and the environmental temperature (22 ± 1 °C) and light should be kept constant throughout the experiment.

[0170] (5) Blood collection technical specifications: Adopt the alternating tail vein puncture method: The first time is at 1 cm from the tail tip, and then move 0.5 cm proximally each time. Use a 29G insulin needle to puncture obliquely (at a 30° angle), and the blood collection volume ≤ 10 μL per time (total blood collection < 50 μL to avoid anemia). The key techniques are: ① Immediately press with a nanofiber hemostatic membrane after blood collection; ② Let the blood droplet form naturally (avoid tissue fluid dilution caused by squeezing forcefully); ③ If coagulation occurs, collect with a heparinized capillary tube. It is recommended to use an automatic blood collection instrument (MedPharma BH - 100) to improve repeatability.

[0171] (6) Calculation of the area under the blood glucose curve (AUC): AUC = 0.25×(T0 + T30) / 2 + 0.5×(T30 + T60) / 2 + 0.5×(T60 + T120) / 2, with the unit of mmol / L·h. The typical value of the STZ model > 2000.

[0172] 3.2.2 Results

[0173] 3.2.2.1 Dynamic changes in blood glucose

[0174] Results of the glucose tolerance test ( Figure 3 A) showed that STZ-treated Stmp1KO mice (KO + STZ group) exhibited significant improvement in glucose metabolism. After intraperitoneal injection of a 1 g / kg glucose load, the increase in blood glucose levels in the KO + STZ group was significantly lower than that in the WT + STZ control group. This difference became apparent at 15 minutes and persisted until the 120-minute test endpoint. Notably, 120 minutes after the glucose load, the blood glucose level in the KO + STZ group dropped to (24.2 ± 1.5) mmol / L, which was significantly 9.02% lower than that in the WT + STZ group ((26.6 ± 2.1) mmol / L, p < 0.01), indicating that Stmp1 gene knockout significantly promoted the blood glucose clearance ability in the diabetic state. Throughout the test, the blood glucose curve of the KO + STZ group was consistently below that of the WT + STZ group, showing a more gentle trend, suggesting that Stmp1 deficiency might improve the dynamic balance of glucose metabolism through multiple mechanisms.

[0175] 3.2.2.2 Quantitative analysis

[0176] Through the quantitative analysis of the area under the blood glucose curve (AUC) ( Figure 3B), significant metabolic differences were observed among groups. The WT and KO mice in the untreated group showed normal glucose tolerance, with AUC values of 1600 ± 140 and 1580 ± 130 mmol / L·h, respectively, and there was no statistical difference between the two groups (p > 0.05). In the STZ-induced diabetic state, the AUC value of the WT+STZ group increased significantly to 3250 ± 150 mmol / L·h, an increase of approximately 103% compared to the untreated WT group (p < 0.0001), confirming the successful establishment of the diabetic model. Notably, the AUC value of the KO+STZ group was 2850 ± 120 mmol / L·h, which was higher than that of the untreated group but significantly decreased by 12.3% compared to the WT+STZ group (p < 0.0001). This difference indicates that the deletion of the Stmp1 gene can partially alleviate STZ-induced impaired glucose tolerance, reducing the AUC increase in diabetic mice from 103% in the WT+STZ group to 78% in the KO+STZ group, suggesting that the KO+STZ group has a better glucose tolerance ability than the WT+STZ group and a better insulin-secreting function, enabling better regulation of blood glucose. These data not only confirm the effectiveness of STZ treatment but also reveal the important role of Stmp1 in regulating glucose metabolism, providing an important theoretical basis for the development of new treatment strategies for diabetes and its complications.

[0177] 3.3 Insulin Tolerance Test (ITT or IPTT) and Results

[0178] The insulin sensitivity of the STZ-induced diabetic retinopathy model was systematically evaluated through the Insulin Tolerance Test (ITT), which reflects the body's resistance to peripheral insulin and complements the GTT test.

[0179] 3.3.1 Insulin Tolerance Test Method (ITT or IPTT)

[0180] (1) Experimental Subjects: 4 WT mice, 4 Stmp1 KO (KO) mice, 4 WT mice with diabetic retinopathy induced by STZ for 60 days (WT+STZ), and 4 KO mice with diabetic retinopathy induced by STZ for 60 days (KO+STZ).

[0181] (2) Fasting Treatment: Before the start of the experiment, all mice were subjected to standardized fasting treatment. The specific procedure was as follows: At 8:00 am on the experimental day, the feed was removed on time, and the mice were allowed to drink freely until 2:00 pm, ensuring a fasting duration of 6 hours. During fasting, the mice were housed separately in cages with no more than 5 mice per cage to reduce stress responses caused by overcrowding. The fasting environmental temperature was strictly controlled at 25 ± 1°C, and strong light irradiation was avoided. After fasting, the subsequent experimental operations were carried out immediately.

[0182] (3) Basal value determination (T0): Basal blood glucose determination is the key starting step of the experiment. First, accurately weigh each mouse (accurate to 0.1g), and then disinfect the skin at the first joint of the tail tip with a 75% alcohol cotton ball. After the alcohol evaporates, use a sterile 26G needle to lightly puncture the tail vein, allowing blood to flow out naturally to form a blood drop with a diameter of about 1mm. Use a pre-calibrated blood glucose meter for testing and record the basal blood glucose value (G0). During the measurement process, please note: ① Avoid squeezing the tail hard; ② If blood flow is not smooth, apply light pressure at the base of the tail; ③ Measure each mouse twice, and re-sample if the difference is greater than 10%.

[0183] (4) Insulin loading: Insulin injection must be strictly carried out according to the standard. The injection volume (0.75U / kg) is calculated according to the body weight after fasting, and the insulin solution pre-warmed to room temperature is drawn up using a special insulin syringe (29G needle). When injecting, fix the mouse in a head-low and tail-high position, select the lower left quadrant of the abdominal cavity (avoiding the midline and bladder area), insert the needle at a 45° angle, and slowly push the drug solution (complete within 30 seconds). After the injection is completed, maintain the position for 10 seconds to prevent the drug solution from reflux. Special attention: ① The syringe must be completely free of bubbles; ② Different groups of mice are injected alternately to avoid time deviation; ③ The actual injection time (accurate to seconds) is recorded as the T0 time point.

[0184] (5) Dynamic blood glucose monitoring: Blood glucose monitoring is carried out in a strict time sequence. At the four core time points of T15, T30, T60, and T120 min, blood is collected from the 3rd, 5th, and 7th joints of the tail tip in turn (rotation principle). A blood glucose change curve is drawn. Before each blood collection, the needle must be disinfected with alcohol and a new needle must be used for puncture. The blood collection volume must be controlled within 5 μL. Blood glucose testing must be completed within 30 seconds after blood collection, and the data must be recorded in real time. When the blood glucose value is <4 mmol / L, the experiment is terminated immediately, and 10% glucose solution (0.1 mL / 10 g) is injected subcutaneously for rescue. In special circumstances, the T120 min monitoring point can be added, but it must be clearly marked in the experimental record. All operations must be completed on a constant temperature table (25°C) to eliminate the interference of temperature on blood glucose metabolism.

[0185] (6) Blood glucose curve plotting: The dynamic blood glucose change curve was plotted using the professional analysis software GraphPad Prism 9.0. The specific process was as follows: First, the blood glucose measurement values at each time point (T0, T15, T30, T60, T120 min) were entered into the system according to the groups. The X-axis was set as time (0 - 120 min), and the Y-axis was set as blood glucose concentration (0 - 40 mmol / L). The original data was smoothed using the Sigmoidal fitting algorithm in non-linear regression to generate a continuous curve. When presenting the curve, the following elements should be included: ① The connecting line of the means of each group; ② The error bars of the standard error of the mean (SEM); ③ The significant difference markers (*p < 0.01, **p < 0.001, ***p < 0.0001). To highlight the key change stages, a vertical reference line should be added at the 30-min time point, and the average percentage of blood glucose reduction at this point should be marked.

[0186] (7) AUC calculation and analysis: The calculation of the area under the blood glucose curve (AUC) was standardized using the trapezoidal integration method. The specific calculation formula was: AUC = [(G0 + G15) / 2] × 0.25 + [(G15 + G30) / 2] × 0.25 + [(G30 + G60) / 2] × 0.5.

[0187] 3.3.2 Results

[0188] 3.3.2.1 Dynamic blood glucose response characteristics

[0189] Insulin tolerance test results ( Figure 4 A) It was found that the STZ-induced Stmp1 gene knockout diabetic mice (KO + STZ group) showed significant blood glucose regulation advantages. At 30 minutes after intraperitoneal injection of 0.75 U / kg insulin, the blood glucose level in the KO + STZ group decreased to (17 ± 3) mmol / L, significantly lower than that in the WT + STZ group, which was (24 ± 4) mmol / L (the reduction rate reached 29.2%, p < 0.01). This improvement trend was more obvious at 60 minutes. The blood glucose level in the KO + STZ group was maintained at a stable level of (15.5 ± 1.2) mmol / L, while the WT + STZ group was still at a relatively high level of (22.7 ± 2) mmol / L (the difference amplitude was 31.7%, p < 0.001). It was suggested that the deletion of Stmp1 not only enhanced insulin sensitivity. These dynamic characteristics together indicated that the knockout of the Stmp1 gene could significantly improve the insulin response efficiency in the diabetic state.

[0190] 3.3.2.2 Quantitative analysis results

[0191] Systematic analysis of the area under the blood glucose curve (AUC) revealed significant metabolic differences among the groups ( Figure 4B). In the untreated control group without STZ, the AUC value of WT mice was 460 ± 20 mmol / L·h, while that of Stmp1KO mice (KO group) was 475 ± 33 mmol / L·h. There was no statistical difference between the two groups (p > 0.05), indicating that the deletion of Stmp1 did not affect glucose metabolism under basal conditions. However, in the STZ-induced diabetic model, the AUC value of the WT+STZ group increased sharply to 2546 ± 153 mmol / L·h, a 453% increase compared to the untreated WT group (p < 0.0001), which confirmed the successful establishment of the diabetic model. Notably, the AUC value of the KO+STZ group was 1754 ± 130 mmol / L·h, which was higher than that of the normal control group but significantly decreased by 31.1% compared to the WT+STZ group (p < 0.001), indicating that the KO+STZ group had better insulin sensitivity than the WT+STZ group and the KO+STZ group could lower blood glucose faster. This improvement was equivalent to the therapeutic effect of clinical metformin (30% - 35% AUC reduction). Combining with the results of the GTT test ( Figure 3 ), it was suggested that the deletion of Stmp1 might relieve diabetic metabolic disorders by protecting pancreatic islet β-cell function and enhancing peripheral insulin sensitivity.

[0192] 4. Visual function assessment of Stmp1KO and WT mice after establishing a diabetic retinopathy model

[0193] 4.1 Visual function assessment method

[0194] 4.1.1 Electroretinogram (ERG) detection

[0195] (1) Electroretinogram (ERG), as the core electrophysiological technique for evaluating retinal function, records the comprehensive electrical activities of retinal neurons and epithelial cells triggered by light stimulation, providing important evidence for the diagnosis and mechanism research of retinal diseases. Each wave component has a clear localization indication value. The ERG waveform contains multiple waveforms, such as a wave, b wave, and c wave, which represent the functional markers of different retinal cells respectively. Among them, the c wave is the functional marker of the RPE. Since the early sign of STZ-induced diabetic retinopathy (DR) is the abnormal function of the RPE, therefore, the normality of the c wave of ERG can indirectly reflect the DR lesion.

[0196] (2) Experimental subjects: 4 WT mice, 4 Stmp1KO (KO) mice, 4 WT mice with diabetic retinopathy induced by STZ for 5 months (WT+STZ), and 4 Stmp1KO mice with diabetic retinopathy induced by STZ for 5 months (KO+STZ).

[0197] (3) Dark adaptation and anesthesia: Before the experiment, all mice need to adapt to a completely dark environment for 12 hours to eliminate the interference of light adaptation on retinal electrical activity. Anesthesia was induced with 1.5% isoflurane (mixed with 1 L / min of oxygen), and the anesthesia state was maintained through a nasal cone. The respiratory rate was monitored to be stable at 60 - 80 breaths per minute to ensure appropriate anesthesia depth. After the anesthesia took effect, 1% tropicamide eye drops were used to dilate the pupils (for 10 minutes) to expand the pupil diameter to ≥2 mm, ensuring that light stimuli fully entered the retina.

[0198] (4) Electrode placement: First, 0.3% methylcellulose gel was dropped on the corneal surface to avoid mechanical damage caused by direct contact of the electrode. Subsequently, a gold-ring corneal electrode (recording the c-wave) was placed, gently fitting to the center of the eyeball; the reference electrode (stainless steel needle electrode) was inserted subcutaneously into the forehead, and the ground electrode was fixed subcutaneously at the tail. The impedance of all electrodes needed to be <5 kΩ (the contact part was wiped with a physiological saline cotton ball to reduce the impedance) to ensure stable signal conduction.

[0199] (5) c-wave detection parameter settings: The slow potential was recorded using the DC coupling mode (0.1 - 30 Hz band-pass filtering), and the sampling rate was set to 100 Hz to capture the slow changes of the c-wave. Stimulation parameters: Full-field white light flash (intensity 2.0 log cd·s / m 2 , duration 5 ms), and the background light was turned off to avoid inhibiting the generation of the c-wave. After each stimulation, there was an interval of 5 minutes, and it was repeated 3 times to take the average value to reduce random noise interference. The baseline was synchronously recorded for 5 seconds (before the flash) and the response period for 30 seconds (covering the entire process of the c-wave rise and recovery).

[0200] (6) Data acquisition: After starting the recording, the real-time waveform was observed to ensure signal stability (baseline drift <±10 μV). If interference occurred (such as animal movement or 50 Hz noise), the recording was immediately paused and the electrode position was adjusted. After the detection of each group of mice was completed, the cornea was cleaned with 37°C physiological saline, and antibiotic eye ointment was dropped to prevent infection.

[0201] 4.1.2 Ocular motor response test detection

[0202] (1) The ocular motor response test detection is a key technique for evaluating the nerve function damage of diabetic retinopathy (DR). By quantitatively analyzing parameters such as saccadic movement (latency, velocity, accuracy), smooth pursuit gain, and optokinetic nystagmus, the damage of the visual pathway can be accurately located. Its detection results complement those of ERG, jointly revealing the multi-level damage mechanism of DR from the peripheral retina to the central nervous system.

[0203] (2) Experimental subjects: 4 wild-type (WT), 4 Stmp1 knockout (KO), 4 WT mice induced with diabetic retinopathy by STZ for 5 months (WT + STZ), and 4 Stmp1KO mice induced with diabetic retinopathy by STZ for 5 months (KO + STZ).

[0204] (3) Preparation before experiment: Systematic preparation is required before the eye movement response experiment. First, the experimental animals need to undergo adaptive training with a head fixation device 3 days in advance, 20 minutes per day to reduce stress interference. Second, use the EyeLink 1000 Plus eye tracker for system calibration to ensure that the spatial error is less than 0.1°. The key parameter settings include: sampling rate 1000 Hz, stimulus screen brightness 50 cd / m 2 , target size 0.5°, and background contrast 80%. All equipment needs to operate stably for more than 30 minutes in an environment with constant temperature (22 ± 1 °C) and constant humidity (50 ± 5% RH). Special attention should be paid to controlling the blood glucose of diabetic model mice within the range of 15 - 20 mmol / L, and 10% glucose injection should be prepared for emergency.

[0205] (4) Detection: The standardized detection process includes three core steps. First, head fixation (positioning coordinates AP - 3.0 mm, ML ± 2.0 mm) and corneal reflex calibration (error < 0.2°) are completed under light anesthesia, and detection starts 30 minutes after waking up. Saccade detection uses horizontal step targets with random jumps of 10° - 30°, and parameters such as latency, peak velocity, and endpoint accuracy are accurately recorded. Subsequently, 0.2 Hz sinusoidal smooth pursuit detection (±15° amplitude) is performed to calculate the eye velocity / target velocity gain value. Finally, optokinetic nystagmus is induced by rotating a black and white striped drum (5 - 50° / s gradient), and the slow-phase gain is recorded. The ambient light needs to be kept constant throughout the process, and a single detection is controlled within 25 minutes.

[0206] (5) Data acquisition: Data acquisition follows a strict standardized process. The original eye movement position data is continuously recorded at a frequency of 1000 Hz, and 50 Hz low-pass filtering is used to eliminate high-frequency noise. The EyeLink Data Viewer software collects data in real-time, and at the same time, precise stimulus-response synchronization is achieved through the Presentation system.

[0207] 4.2 Analysis of visual function test results

[0208] 4.2.1 Analysis of electroretinogram (ERG) results ( Figure 5 )

[0209] After successful establishment of the diabetic model, mice were raised for 5 months and detected with ERG. The c-wave was statistically plotted, and the results showed that the amplitude of the c-wave of the ERG in the KO + STZ group was higher than that in the WT + STZ group ( Figure 5A). Further statistical analysis showed that in the untreated group, the c-wave amplitude of wild-type (WT) mice was (420 ± 62) μV, while that of Stmp1 knockout (KO) mice was (384 ± 53) μV. There was no significant difference between the two groups (p > 0.05), indicating that the deletion of the Stmp1 gene itself did not affect the basic RPE function and was a safe site. However, in the STZ-induced diabetic model, the c-wave amplitude of the WT+STZ group decreased sharply to (84 ± 13) μV, with the smallest c-wave amplitude, indicating the most severe damage in these mice, which was 80% lower than that of the WT group (p < 0.0001). This result confirmed the severe RPE functional damage caused by diabetes. Notably, the c-wave amplitude of the KO+STZ group was maintained at (191 ± 31) μV. Although it was lower than the normal level, it was significantly increased by 127% compared with the WT+STZ group (p < 0.001), and recovered to 45% of the functional level of the WT group ( Figure 5 B). This result indicates that the deletion of Stmp1 itself has no significant effect on normal retinal function, but can protect the RPE from diabetic damage.

[0210] 4.2.2 Analysis of the results of the optokinetic response test

[0211] The optokinetic response can accurately reflect the visual function status of mice. The experimental data ( Figure 6 ) showed that the visual acuity of untreated wild-type (WT) mice was (0.49 ± 0.03) cycle / deg, and that of the Stmp1 knockout (KO) group was (0.46 ± 0.02) cycle / deg. There was no significant difference between the two groups (p > 0.05), indicating that gene knockout itself did not affect the basic visual function. In the STZ-induced diabetic model, the visual acuity of the WT+STZ group decreased significantly to (0.38 ± 0.08) cycle / deg, which was 22.4% lower than that of the WT group (p < 0.001), confirming the damage to the central visual pathway caused by diabetes. The visual acuity of the KO+STZ group was maintained at (0.42 ± 0.06) cycle / deg, which was significantly increased by 10.5% compared with the WT+STZ group (p < 0.01), and there was no significant difference from KO (p > 0.01). This result suggests that the deletion of Stmp1 may partially compensate for the visual function damage caused by diabetes by protecting the signal conduction efficiency of the retina-thalamus-cortex pathway, providing a new basis for the development of targeted treatment strategies.

[0212] In summary, by constructing a Stmp1 gene knockout mouse model, the present invention systematically reveals the key role of STMP1 in diabetic retinopathy. The experimental results show that Stmp1 knockout has no significant effect on the visual function of normal mice, but exhibits multiple protective effects under diabetic conditions: 1) significantly improving glucose tolerance and insulin sensitivity, suggesting that it may alleviate diabetic metabolic disorders by protecting pancreatic islet β-cell function and enhancing peripheral insulin sensitivity; 2) effectively maintaining the function of retinal pigment epithelial cells, increasing the ERG c-wave amplitude of diabetic model mice by 127% compared with the control group; 3) significantly improving the function of the visual conduction pathway, increasing the visual acuity of the optokinetic response by 10.5%. These findings indicate that STMP1 plays an important role in diabetic retinopathy, and its knockout can protect retinal function through multiple pathways, providing an important theoretical basis and potential targets for the development of novel treatment strategies for diabetic retinopathy.

Claims

1. Use of STMP1 inhibitor in the preparation of a medicament for treating diabetic retinopathy.

2. The application according to claim 1, characterized in that The STMP1 inhibitor includes at least one of the following (1)-(3): (1) A preparation for inhibiting or reducing the expression of STMP1 gene; (2) A preparation for completely knocking out or partially knocking out the STMP1 gene; (3) A preparation for inhibiting or blocking the activity of STMP1 protein.

3. The application according to claim 2, characterized in that, The STMP1 gene is the 1810058I24Rik gene, and the amino acid sequence of the STMP1 protein is MLQFLLGFTLGNVVGMYLAQNYEMPNLAKKLEEIKKDLEAKKKPPSS.

4. The application according to claim 2, wherein The preparation for inhibiting or reducing the expression of STMP1 gene is a preparation for inhibiting the expression of STMP1 gene using adeno-associated virus or lentiviral vector, or a preparation for silencing or knocking down the expression of STMP1 gene by targeting STMP1 mRNA with antisense oligonucleotide, shRNA or RNAi technology.

5. The application according to claim 2, characterized in that The preparation is delivered to the retina by a targeted nano-delivery system payload.

6. The application according to claim 2, characterized in that, The preparation for completely knocking out or partially knocking out the STMP1 gene is a preparation for targeting and editing the STMP1 gene with CRISPR-Cas9 to reduce the expression of STMP1 gene.

7. The application according to claim 6, wherein The gRNAs for targeting and editing the STMP1 gene with CRISPR-Cas9 are as follows: gRNA1: CTTCCCAAGAAGTGCACGGC; gRNA2: TGCACGGCAGGTGACTGCCC; gRNA3: GAGGCATCCTCCCCGGAAGC; gRNA4: CGGAAGCAGGACGGACCATC.

8. The application according to claim 2, wherein The preparation for inhibiting or blocking the activity of STMP1 protein is an antibody against STMP1 protein.

9. The application according to any one of claims 1-8, characterized in that, The medicament for treating diabetic retinopathy is a medicament having at least one of the following (1)-(4) functions: (1) Promoting blood glucose clearance ability, improving insulin response efficacy, and relieving metabolic disorders in the diabetic state; (2) Maintaining the function of retinal pigment epithelial cells; (3) Protecting retinal function; (4) Improving the function of visual conduction pathway, enhancing visual acuity, and protecting visual function.

10. A medicament for treating diabetic retinopathy, which contains an STMP1 inhibitor as an active ingredient, and the STMP1 inhibitor includes at least one of the following (1)-(3): (1) A preparation for inhibiting or reducing the expression of STMP1 gene; (2) A preparation for completely knocking out or partially knocking out the STMP1 gene; (3) A preparation for inhibiting or blocking the activity of STMP1 protein.

Images