Application of PKM2 / GLUT1 axis in T2DM cataract

Abstract

The invention discloses an application of a PKM2 / GLUT1 axis in T2DM cataract, belongs to the technical field of biological medicine, aims to solve the problem that the PKM2 / GLUT1 axis is unclear in action in cataract pathogenesis of a T2DM patient, and explores whether PKM2 / GLUT1 axis imbalance mediates mitochondrial dysfunction to play a role in the cataract of the T2DM patient through culture of a clinical sample and a human LECs cell line cultured in vitro. It is found that PKM2 / GLUT1 axis imbalance can promote occurrence and development of cataract of T2DM patients, and the mechanism may be related to mediated LECs mitochondrial dysfunction and apoptosis. By improving the activity of the PKM2, lens opacity in a high-glucose environment can be slowed down, so that a new strategy is provided for preventing and treating cataract of a T2DM patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biomedicine, and particularly relates to the use of the PKM2 / GLUT1 axis in T2DM-induced cataracts. Background Art

[0002] Cataract is one of the main causes of visual impairment and even blindness. DM (diabetes mellitus) is a systemic metabolic disease characterized by hyperglycemia, which is mainly divided into type 1 diabetes (T1DM) and type 2 diabetes (T2DM). T1DM is mainly caused by the destruction of pancreatic islet β cells, resulting in absolute insulin deficiency; T2DM, as the most common type, is usually associated with insulin resistance and relative insulin secretion deficiency. Previous studies have found that compared with non-diabetic patients, T2DM patients have a higher incidence of cataracts, and the onset age and the time of visual impairment are earlier. This finding has attracted extensive attention, suggesting that there may be an internal connection between two seemingly independent diseases. The persistent state of hyperglycemia not only affects the body's metabolic balance but may also, through multiple mechanisms such as increasing oxidative stress and inflammation, lead to the denaturation and aggregation of lens proteins, thus promoting the formation of cataracts. However, its pathogenesis has not been clearly defined and further research is needed.

[0003] Glucose metabolism is a key step in the body's metabolic process. Glucose metabolism can be divided into aerobic oxidation and anaerobic glycolysis. The lens is an avascular tissue with low oxygen content. Therefore, under normal circumstances, the lens mainly metabolizes through the anaerobic glycolysis pathway. In the state of hyperglycemia, a large amount of glucose enters the lens tissue through the aqueous humor, leading to abnormal glucose metabolism and ultimately resulting in the occurrence of cataracts.

[0004] Glucose uptake is the first rate-limiting step in glucose metabolism, and this process is tightly regulated. GLUT (glucose transporter) is a class of 12-transmembrane proteins with a unique function of facilitated diffusion for transporting glucose (from high concentration to low concentration). GLUT is widely distributed and there are a total of 14 subtypes. Lim et al. found that only GLUT1 is expressed in human LECs (lens epithelial cells), and this protein plays an important role in the normal development of the lens and maintaining the transparency of the lens. It is reported that clinically, patients with GLUT1 deficiency usually suffer from congenital cataracts, and lens opacity also occurs in GLUT1 gene knockout mice.

[0005] Pyruvate kinase (PK), the last rate-limiting enzyme in the anaerobic glycolysis pathway, has four subtypes (M1, M2, L, and R). Among them, PKM2 has attracted much attention because it can directly regulate glucose metabolism and has phosphokinase activity. PKM2 has multiple configurations. The tetrameric configuration of PKM2 located in the cytoplasm has high glycolytic activity and can catalyze the production of pyruvate. The monomer / dimer configuration of PKM2 is located in the nucleus, has low glycolytic activity, and is closely related to energy metabolism, substance synthesis, and gene transcription. Salani et al. found that insulin-like growth factor 1 can promote the nuclear translocation of PKM2 through the Akt pathway, increasing the expression of GLUT1. Another study found that after the activation of extracellular signal-regulated kinase, it can directly bind to PKM2 and phosphorylate PKM2 at the Ser37 site, promoting the nuclear translocation of PKM2, inducing the expression of the gene c-Myc, and then upregulating the expression of glycolytic genes such as GLUT1, promoting tumorigenesis. In addition, our research group has previously found that in the lens epithelial cells (LECs) of patients with type 2 diabetes mellitus (T2DM), the translocation of PKM2 to the nucleus increases, playing a role as a protein kinase and upregulating the expression of GLUT1. However, the role of the PKM2 / GLUT1 axis in the pathogenesis of cataracts in T2DM patients still needs further study. Summary of the Invention

[0006] In view of the above problems, the present application provides the use of the PKM2 / GLUT1 axis in T2DM-related cataracts. Using a variety of technical means such as transmission electron microscopy, immunohistochemistry, and Western blot, in clinical samples and the in vitro cultured human LEC cell line (SRA01 / 04), it is explored whether the dysregulation of the PKM2 / GLUT1 axis mediates mitochondrial dysfunction and plays a role in the cataracts of T2DM patients. At the same time, the effect of regulating the PKM2 / GLUT1 axis on the occurrence and development of cataracts in T2DM patients is explored, providing a research basis for its prevention and targeted treatment.

[0007] In the first aspect, the present invention provides the use of a PKM2 activator and / or a GLUT1 inhibitor in the preparation of a drug for preventing or treating T2DM-related cataracts.

[0008] In some preferred examples of this aspect, the manifestations of the T2DM-related cataracts are increased mitochondrial dysfunction and apoptosis of LECs.

[0009] In some preferred examples of this aspect, the mitochondrial dysfunction of the LECs is manifested as abnormal mitochondrial morphology and imbalanced mitochondrial dynamics in the LECs.

[0010] In some preferred examples of this aspect, the PKM2 activator is TEPP-46.

[0011] In some preferred examples in this regard, the GLUT1 inhibitor is Phloretin.

[0012] In a second aspect, the present invention provides the use of PKM2 and / or GLUT1 in the preparation of a product for diagnosing T2DM-related cataracts, wherein the expression of PKM2 is decreased in LECs of T2DM-related cataracts, and the expression of GLUT1 is increased in LECs of T2DM-related cataracts.

[0013] In a third aspect, the present invention provides the use of PKM2 and / or GLUT1 in the preparation of a product for evaluating the therapeutic effect of T2DM-related cataracts or judging the prognosis of T2DM-related cataracts.

[0014] In some preferred examples in this regard, the product includes a kit and a chip.

[0015] Among them, the detection includes the detection of protein levels, and the detection of protein levels includes immunoassay, Western blot, protein chip, chemiluminescence immunoassay, surface plasmon resonance (SPR) method, etc. To simplify the experimental procedure, the detection of protein levels also includes protein detection kits, such as ELISA detection kits, colloidal gold detection kits, immunoprecipitation kits, chemiluminescence kits, immunofluorescence kits, etc. The kit is generally equipped with corresponding instructions, and the instructions generally include company logo and name, kit name, kit composition, shelf life, application field, usage method, etc. Users can obtain satisfactory results with little or no optimization according to the instructions.

[0016] The immunoassay method is ELISA assay and / or colloidal gold detection.

[0017] The ELISA assay uses an ELISA detection kit, and the kit includes: a solid-phase carrier coated with PKM2 monoclonal antibody and / or GLUT1 monoclonal antibody, an enzyme-labeled antibody, a substrate of the enzyme, a protein standard, a negative control, a diluent, a washing solution, an enzyme reaction termination solution, etc.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] In the in vitro experiment of LECs samples from the anterior lens capsule of clinical patients, the present invention found that the imbalance of the PKM2 / GLUT1 axis can promote the occurrence and development of cataracts in T2DM patients, and its mechanism is related to mediating mitochondrial dysfunction and apoptosis of LECs. After applying the PKM2 activator TEPP-46 and the GLUT1 inhibitor Phloretin, the lens opacity degrees are 51.91±7.88 AU / pixel and 50.55±10.03 AU / pixel respectively, which are significantly lower than those in the high-glucose group.

[0020] The present invention discovers that by increasing the activity of PKM2, the lens opacity under high-glucose conditions can be alleviated, thus providing a new strategy for the prevention and treatment of cataracts in T2DM patients. Brief Description of the Drawings

[0021] Figure 1 It is a flow chart of qRT-PCR.

[0022] Figure 2 It shows the changes in the expression of PKM2 / GLUT1 in lens epithelial cells (LECs) of cataracts in T2DM patients; among them, A is the electrophoresis pattern of GLUT1 protein expression in LECs of each group of clinical samples (n = 5). B is the comparison of the relative expression levels of GLUT1 protein in each group. C is the immunofluorescence staining pattern of GLUT1 in LECs of each group of clinical samples (n = 5); scale bar = 50 μm. D is the electrophoresis pattern of PKM2 protein expression in LECs of each group of clinical samples (n = 5). E is the comparison of the relative expression levels of PKM2 protein in each group. F is the immunofluorescence staining pattern of PKM2 in LECs of each group of clinical samples (n = 5); scale bar = 50 μm. *P < 0.05; **P < 0.01. ARC: Age-related cataract group; T2DM: Cataract group of type 2 diabetes; DAPI: 4',6-diamidino-2-phenylindole; GLUT1: Glucose transporter 1; PKM2: Pyruvate kinase M2; β-actin: β-actin.

[0023] Figure 3 It shows the morphological and kinetic changes of mitochondria in LECs of cataracts in T2DM patients. Among them, A is the slit lamp and transmission electron microscope images of each group of clinical samples (n = 5); white arrow: mitochondrion; scale bar = 2 μm. B is the comparison of the relative mRNA expression levels of mitochondrial kinetic-related markers in LECs of each group of clinical samples (n = 10 in the ARC group, n = 10 in the T2DM group); Slit-lamp: Anterior segment slit lamp; TEM: Transmission electron microscope; Drp1: Dynamin-related protein 1; Mfn1: Mitofusin 1; Mfn2: Mitofusin 2.

[0024] Figure 4 It shows the changes in the expression of PKM2 / GLUT1 in LECs cultured in vitro under high glucose. Among them, A is the electrophoresis pattern of GLUT1 protein expression in each group of cell samples (n = 5). B is the comparison of the relative expression levels of GLUT1 protein in each group. C is the immunofluorescence staining pattern of GLUT1 in each group of cell samples (n = 5); scale bar = 20 μm. D is the electrophoresis pattern of PKM2 protein expression in each group of cell samples (n = 5). E is the comparison of the relative expression levels of PKM2 protein in each group. F is the immunofluorescence staining pattern of PKM2 in each group of cell samples (n = 5); white arrow: PKM2 enters the nucleus; scale bar = 20 μm.

[0025] Figure 5 Morphological changes of mitochondria in LECs cultured in vitro under high glucose conditions. Among them, A: Transmission electron microscopy images of cell samples in each group (n = 5); white arrows: mitochondria; scale bar = 1 μm. B: Representative photos of mitochondrial probes in cell samples of each group; scale bar = 10 μm. C: Electrophoresis patterns of the expression of markers related to mitochondrial dynamics and apoptotic marker proteins in cell samples of each group (n = 5). D: Comparison of the relative expression levels of markers related to mitochondrial dynamics and apoptotic marker proteins in cell samples of each group; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-associated X protein.

[0026] Figure 6 Mitochondrial function changes in LECs induced by high glucose. Among them, A: Comparison of the oxygen consumption rate of cell samples in each group (n = 5). B: Representative images of mitochondrial membrane potential and mitoSOX in cell samples of each group (n = 5); scale bar = 50 μm. C: Comparison of mitochondrial membrane potential in cell samples of each group. D: Comparison of the fluorescence intensity of mitoSOX in cell samples of each group. E: Flow cytometry diagrams of mitochondrial membrane potential in cell samples of each group (n = 5). F: Flow cytometry diagrams of mitochondrial ROS in cell samples of each group (n = 5). G: Flow cytometry diagrams of cell apoptosis in cell samples of each group (n = 5). JC-1 monomer: JC-1 monomeric form; JC-1 aggregate: JC-1 aggregated form; MitoSOX: Mitochondrial superoxide.

[0027] Figure 7 Effect of PKM2 on mitochondrial morphology and dynamics in high glucose-induced LECs. Among them, A: Representative photos of mitochondrial probes in cell samples of each group (n = 5); scale bar = 10 μm. B: Electrophoresis patterns of the expression of markers related to mitochondrial dynamics and apoptotic marker proteins in cell samples of each group (n = 5). C: Comparison of the relative expression levels of markers related to mitochondrial dynamics and apoptotic marker proteins in cell samples of each group. D: Electrophoresis patterns of the expression of markers related to mitochondrial dynamics and apoptotic marker proteins in cell samples of each group (n = 5). E: Comparison of the relative expression levels of markers related to mitochondrial dynamics and apoptotic marker proteins in cell samples of each group.

[0028] Figure 8 Effect of PKM2 on mitochondrial function in high glucose-induced LECs. Among them, A: Comparison of the oxygen consumption rate of cell samples in each group (n = 5). B: Representative images of mitochondrial membrane potential and mitoSOX in cell samples of each group (n = 5); scale bar = 50 μm. C: Comparison of mitochondrial membrane potential in cell samples of each group. D: Comparison of the fluorescence intensity of mitoSOX in cell samples of each group. E: Flow cytometry diagrams of mitochondrial membrane potential in cell samples of each group (n = 5). F: Flow cytometry diagrams of mitochondrial ROS in cell samples of each group (n = 5). G: Flow cytometry diagrams of cell apoptosis in cell samples of each group (n = 5).

[0029] Figure 9Effect of GLUT1 on mitochondrial morphology and dynamics of LECs induced by high glucose. Among them, A representative photos of mitochondrial probes of cell samples in each group (n = 5); scale bar = 10 μm. B Electrophoresis patterns of the expression of PKM2, GLUT1, mitochondrial dynamics-related markers and apoptosis marker proteins in cell samples in each group (n = 5). C Comparison of the relative expression levels of PKM2, GLUT1, mitochondrial dynamics-related markers and apoptosis marker proteins in cell samples in each group.

[0030] Figure 10 Effect of GLUT1 on mitochondrial function of LECs induced by high glucose. Among them, A Comparison of the oxygen consumption rate of cell samples in each group (n = 5). B Representative images of mitochondrial membrane potential and mitoSOX of cell samples in each group (n = 5); scale bar = 20 μm. C Comparison of mitochondrial membrane potential of cell samples in each group. D Comparison of mitoSOX fluorescence intensity of cell samples in each group. E Flow cytometry diagrams of mitochondrial membrane potential of cell samples in each group (n = 5). F Flow cytometry diagrams of mitochondrial ROS of cell samples in each group (n = 5). G Flow cytometry diagrams of cell apoptosis of cell samples in each group (n = 5)

[0031] Figure 11 Role of the PKM2 / GLUT1 axis in high-glucose-induced lens opacity. Among them, A Representative images of the lenses of SD rats in each group (n = 5 in the NC group, n = 5 in the HG group, n = 6 in the HG + DMSO group, n = 6 in the HG + IN-1 group; n = 6 in the HG + TEPP-46 group; n = 6 in the HG + Phloretin group). B Comparison of lens opacity in SD rats in each group. NC: normal group; HG: high-glucose group; HG + DMSO group: high glucose + 1 μL DMSO; HG + IN-1 group: high glucose + 1 μL IN-1; HG + TEPP-46 group: high glucose + 1 μL TEPP-46; HG + Phloretin group: high glucose + 1 μL phloretin. Detailed implementation manners

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and the drawings. It should be understood that the specific embodiments described are only for explaining the specific applications of the present invention, rather than limiting the scope of the present invention. Through these embodiments, it is intended to help understand the principles, operation methods and advantages of the present invention, but it does not exclude appropriate adjustments and adaptations during the implementation process of the present invention.

[0033] Next, various technical means such as transmission electron microscopy, immunohistochemistry, and Western blot will be used to explore whether the dysregulation of the PKM2 / GLUT1 axis mediates mitochondrial dysfunction and plays a role in cataracts in T2DM patients in clinical samples and the human LECs cell line (SRA01 / 04) cultured in vitro. At the same time, the effect of regulating the PKM2 / GLUT1 axis on the occurrence and development of cataracts in T2DM patients will be explored to provide a research basis for its prevention and targeted treatment.

[0034] 1. Materials and Methods

[0035] 1.1 Materials

[0036] 1.1.1 Subjects

[0037] It has been approved by the Ethics Committee of the Affiliated Hospital of Nantong University. Before the experiment, the content and purpose of this study were clarified to all enrolled subjects, and informed consent forms were signed. Before the experiment, all enrolled subjects received complete and comprehensive routine examinations and ophthalmic specialty examinations. Fifty-five patients (55 eyes) with T2DM cataracts in the Affiliated Hospital of Nantong University from September 2021 to January 2022 were selected (hereinafter referred to as the T2DM group). Fifty-five patients (55 eyes) with ARC (age-related cataract) were matched and selected according to age, gender, and lens opacity degree (based on the LOCSⅢ grading) (hereinafter referred to as the ARC group). The basic information of the patients is shown in Table 1.

[0038] Table 1 Basic Information of Patients

[0039]

[0040] 1) T2DM Group

[0041] Inclusion criteria: ① Diabetes diagnostic criteria: fasting blood glucose ≥ 7.0 mmol / L; 2-hour blood glucose in oral glucose tolerance test ≥ 11.1 mmol / L; postprandial blood glucose ≥ 11.1 mmol / L; random blood glucose ≥ 11.1 mmol / L, and accompanied by typical diabetic symptoms; HbA1c (glycosylated hemoglobin, type A1C) ≥ 6.5%

[29] ; ② Diagnosed with cataract; ③ Age ≥ 50 years old and best corrected visual acuity ≤ 0.5. Exclusion criteria: ① Other types of cataracts such as congenital and complicated cataracts; ② History of fundus diseases or eye surgeries; ③ History of other systemic diseases such as hypertension.

[0042] 2) ARC Group

[0043] ①Diagnosed as ARC; ②Age ≥ 50 years old and best corrected visual acuity ≤ 0.5. Exclusion criteria: ①Other types of cataracts such as congenital and complicated cataracts; ②History of fundus diseases or eye surgeries; ③History of other systemic diseases such as DM and hypertension.

[0044] 1.1.2 Experimental animals

[0045] SD (Sprague Dawley) rats, 8 weeks old, male, clean grade, purchased from the Experimental Animal Center of Nantong University, and examined without eye diseases. The animal experiments in this study comply with the guidelines for the use of experimental animals of the National Institutes of Health of the United States and are raised by professionals in a standard environment.

[0046] 1.2 Main instruments

[0047] Bio pipette (Eppendorf, Germany); High-pressure steam sterilizer (Sanyo, Japan); Confocal microscope (Nikon, Japan); Constant temperature water bath (Saiweier Biotechnology Co., Ltd., China); High-speed refrigerated centrifuge (Eppendorf, Germany); Western blot general-purpose basic electrophoresis instrument (Bio-Rad, USA); Microplate reader (GeneCompanyLimited, China); Flow cytometer (BD Falcon, USA); Miniature palm centrifuge (Aibensen Scientific Instruments Co., Ltd., China); 4°C, -20°C, -80°C experimental-grade refrigerators (Sanyo, Japan); Inverted fluorescence microscope (Leica, Germany); Multifunctional shaker (Qibei Instrument Manufacturing Co., Ltd., China); Micro weighing balance (Puchun Measuring Instruments Co., Ltd., China); Plastic film sealer (Kaiming Electronic Appliance Co., Ltd., China); Portable incubator (Furuijie Co., Ltd., China); Laminar flow hood (Haier Co., Ltd., China); SIM-F140AY65 ice maker (Sanyo Co., Ltd., Japan); 5% CO2 cell constant temperature incubator (Thermo Fisher, USA); Vortex mixer (Luxi Analytical Instrument Factory Co., Ltd., China); Electrothermal constant temperature drying oven (Hengchang Instrument Factory, China); Nanodrop2000 ultra-micro spectrophotometer (Thermo Fisher, USA); 7500 real-time fluorescence quantitative PCR instrument (ABI, USA); Transmission electron microscope (HITACHI, Tokyo, Japan).

[0048] 1.3 Main reagents

[0049] 1.3.1 Information on antibodies required for the experiment

[0050] Table 2 Information on antibodies required for the experiment

[0051]

[0052] 1.3.2 Primer sequences required for the experiment

[0053] Table 3 Primer sequences required for the experiment

[0054]

[0055] 1.3.3 Reagents and instruments required for animal experiments

[0056] (1) Disposable sterile syringe (1 mL) (Kendall Medical Instrument Co., Ltd., China)

[0057] (2) Isoflurane anesthetic (Reword Life Science Co., Ltd., China)

[0058] (3) Titanium alloy micro ophthalmic scissors (Suzhou Bili Medical Technology Co., Ltd., China)

[0059] (4) Titanium alloy micro ophthalmic non-toothed forceps (Suzhou Bili Medical Technology Co., Ltd., China)

[0060] (5) Titanium alloy micro ophthalmic toothed forceps (Suzhou Bili Medical Technology Co., Ltd., China)

[0061] (6) M199 medium (GibcoBRLLifeTechnologies, USA)

[0062] (7) Glucose solution (Beyotime, China)

[0063] (7) Phloretin (MCE, China)

[0064] 1.3.4 Reagents required for cell culture and subsequent experiments

[0065] (1) DMEM (GibcoBRLLifeTechnologies, USA)

[0066] (2) 10% FBS (fetal bovine serum) (Cellbox, China)

[0067] (3) 0.25% Trypsin-EDTA digestive solution (GibcoBRLLifeTechnologies, USA)

[0068] (4) Penicillin-streptomycin double antibody (Xinsemei Co., Ltd., China)

[0069] (5) Serum-free cell cryopreservation solution (Xinsemei Co., Ltd., China)

[0070] (6) Cell culture plates (6 / 12 / 24 / 96 wells) (Corning, USA)

[0071] (7) Cell culture flask (25 / 75 cm2) (Coring, USA)

[0072] (8) Cell culture dish (35 / 60 mm) (Coring, USA)

[0073] (9) Centrifuge tube (15 / 50 mL) (Coring, USA)

[0074] (10) 35 mm confocal culture dish (Coring, USA)

[0075] (11) Cell counting chamber (Qiujing Biochemical Reagent Instrument Co., Ltd., China)

[0076] (12) Empty plasmid (Suzhou Ribobio Co., Ltd., China)

[0077] (13) PKM2 overexpression plasmid (Suzhou Ribobio Co., Ltd., China)

[0078] (14) GLUT1 overexpression plasmid (Suzhou Ribobio Co., Ltd., China)

[0079] (15) Lipofectamine8000 transfection kit (Beyotime, China)

[0080] (16) PKM2 activator (TEPP-46) (MCE, China)

[0081] (17) PKM2 inhibitor (PKM2-IN-1) (MCE, China)

[0082] (18) Dimethyl sulfoxide (DMSO) (Beyotime, China)

[0083] (19) Enhanced JC-1 detection kit (Beyotime, China)

[0084] (20) Mitochondrial superoxide detection fluorescent dye (Dojindo Laboratories, Japan)

[0085] (21) Cell oxygen consumption rate detection kit (Bioscience, China)

[0086] (22) 0.25% Trypsin cell digestive solution (without EDTA) (GibcoBRLLifeTechnologies, USA)

[0087] (23) AnnexinV-FITC / PI Apoptosis Detection Kit (Dojindo Laboratories, Japan)

[0088] (24) Flow cytometry tube (BD Falcon, USA)

[0089] (25) 96-well cell culture plate (black frame, transparent flat bottom) (LABSELECT, China)

[0090] 1.3.5 Reagents required for qRT-PCR

[0091] (1) DEPC-treated water (Biosharp, China)

[0092] (2) TRIzol reagent (Invitrogen, USA)

[0093] (3) Chloroform (Lubo Chemical Reagent Co., Ltd., China)

[0094] (4) Isopropanol (Shenyuan Chemical Reagent Co., Ltd., China)

[0095] (5) Absolute ethanol (Shijitongda Chemical Co., Ltd., China)

[0096] (6) HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, China)

[0097] (7) ChamQ SYBR qPCR Master Mix (HighROX) (Vazyme, China)

[0098] (8) Primers (Sangon, China)

[0099] 1.3.6 Reagents required for WB

[0100] (1) RIPA lysis buffer (Beyotime, China)

[0101] (2) Protease inhibitor (Beyotime, China)

[0102] (3) 4× Protein loading buffer (Solarbio Science & Technology Co., Ltd., China)

[0103] (4) Cell scraper (Costar, USA)

[0104] (5) BCA Protein Quantification Kit (Thermo Scientific, USA)

[0105] (6) PAGE Gel Rapid Preparation Kit (Yamei Biotechnology, China)

[0106] (7) Tween-20 (Solarbio Science & Technology Co., Ltd., China)

[0107] (8) 1.0 mm glass plate and gel comb (Bio-Rad Laboratories, USA)

[0108] (9) Dual-color prestained protein marker (Yaenzyme Biotechnology, China)

[0109] (10) Methanol (Hushi Laboratory Equipment Co., Ltd., China)

[0110] (11) Sponge pad (Bio-Rad Laboratories, USA)

[0111] (12) Transfer filter paper (Beyotime, China)

[0112] (13) PVDF membrane (Roche, Switzerland)

[0113] (14) Skim milk powder (Mengniu Dairy Co., Ltd., China)

[0114] (15) Antibody diluent (Abbkine, China)

[0115] (16) ECL ultra-sensitive chemiluminescence solution (Abbkine, China)

[0116] (17) Tris-Glycine SDS-PAGE Running Buffer (Saiweier Biotechnology Co., Ltd., China)

[0117] (18) Tris-Glycine Transfer Buffer (Saiweier Biotechnology Co., Ltd., China)

[0118] 1.3.7 Reagents required for transmission electron microscopy and immunofluorescence

[0119] (1) Cell culture slides (Biosharp, China)

[0120] (2) 4% Paraformaldehyde fixative (Solarbio Science & Technology Co., Ltd., China)

[0121] (3) Goat serum (Solarbio Science & Technology Co., Ltd., China)

[0122] (4) Triton X-100 (Sigma-Aldrich, USA)

[0123] (5) Glycerol (Solarbio Science & Technology Co., Ltd., China)

[0124] (6) DAPI solution (1 mg / mL) (Solarbio Science & Technology Co., Ltd., China)

[0125] (7) Adhesive glass slides (Liusheng Laboratory Equipment Co., Ltd., China)

[0126] (8) Coverslip (Liusheng Laboratory Equipment Co., Ltd., China)

[0127] (9) Goat anti-rabbit / mouse IgG with fluorescent label (568 / 488) (Abcam, UK)

[0128] (10) Glutaraldehyde fixative (Solarbio Science & Technology Co., Ltd., China)

[0129] (11) Mitochondrial green fluorescent probe (Beyotime, China)

[0130] (12) Hoechst 33342 live cell staining solution (Beyotime, China)

[0131] 1.3.8 Formulations of main solutions

[0132] (1) Electrophoresis buffer: Add 800 mL of triple-distilled water to a beaker, pour in the powder, stir with a magnetic bar, and after thorough mixing, add triple-distilled water to make up to 1000 mL. Prepare and use immediately.

[0133] (2) Transfer buffer: First add 200 mL of absolute methanol to a beaker, pour in the powder, add 800 mL of triple-distilled water, stir with a magnetic bar, and after thorough mixing, pre-cool at 4 °C before use.

[0134] (3) TBST buffer: Add 800 mL of triple-distilled water to a beaker, pour in the powder, stir with a magnetic bar, and after thorough mixing, add triple-distilled water to make up to 1000 mL. Prepare and use immediately.

[0135] (4) Blocking solution (5% skim milk): According to the ratio of 5 g:100 mL, dissolve an appropriate amount of skim milk powder in TBST buffer, mix well and store at 4 °C for later use.

[0136] (5) 1×PBS buffer: Add 10×PBS buffer and triple-distilled water in a ratio of 1:9 and make up to 1 L, mix well and store at room temperature for later use.

[0137] (6) Hank’s solution: After preparing according to the standard formula, autoclave, cool to room temperature, and dispense into 50 mL centrifuge tubes under the laminar flow hood, then store at 4 °C for later use.

[0138] 2. Methods

[0139] 2.1 Clinical sample collection

[0140] Clinical samples (about 5 mm in the central area of the lens) were obtained by continuous circular capsulorhexis during cataract surgery. Samples for qRT-PCR experiments were placed in 1.5 mL Oxygenenzymefree EP tubes containing 1 mL of TRIzol reagent after being removed from the body. Samples for immunofluorescence experiments were placed in EP tubes containing 1 mL of 4% paraformaldehyde fixative after being removed from the body. Samples for transmission electron microscopy were placed in EP tubes containing 1 mL of glutaraldehyde solution and stored in the dark after being removed from the body. The remaining samples were placed in ordinary EP tubes for Western blot experiments and stored at -80 °C for later use.

[0141] 2.2 SRA01 / 04 cell culture and intervention treatment

[0142] 2.2.1 Cell culture

[0143] The SRA01 / 04 cell line was placed in a cell culture flask (75 cm2) pre-filled with complete medium, and the cells were placed in an incubator at 37 °C, 5% CO 2 and 95% O 2 for culture. Normally, the cell culture medium was changed once every 1 - 2 days. First, observe changes in the color and properties of the culture medium; then observe the growth of the cells under a microscope. When changing the cell culture medium, use a Pasteur pipette to suck out the original medium, then add pre-warmed Hank’s solution to wash the cells once, then use a pipette to suck out the waste liquid, and finally add an appropriate amount of pre-warmed complete medium.

[0144] 2.2.2 Transfection and treatment with activators and inhibitors

[0145] When the cell density in the 6-well plate reached 70% - 80%, according to the Lipofectamine 8000 instruction manual, pcDNA3.1-PKM2 / GLUT1 or the blank vector was transfected into SRA01 / 04 cells. After culturing for 12 hours, the cell culture medium was changed once, and total cell protein was extracted after culturing for 3 days. Treat the cells according to the experimental needs, and add the PKM2 activator TEPP-46 and inhibitor IN-1 to the corresponding groups respectively.

[0146] 2.2.3 Cell treatment and experimental grouping

[0147] Select SRA01 / 04 cells at passages 3 - 6 for the experiment. Inoculate 3×105 cells in a 6-well plate and let them adapt for 12 hours. Discard the original medium, wash the cells once with pre-warmed Hank’s solution, then add 2 mL of medium without FBS to starve the cells for several hours. After starvation, discard the original medium, then wash the cells once with pre-warmed Hank’s solution, and then perform the corresponding treatment.

[0148] The experimental groups were divided into: control group (5.5mM), high glucose group (25mM), high glucose + empty vector group, high glucose + PKM2 overexpression group, high glucose + PKM2 overexpression + TEPP-46 group, high glucose + PKM2 overexpression + IN-1 group, and high glucose + GLUT1 overexpression group.

[0149] 2.3 Western blot

[0150] 2.3.1 Protein extraction and concentration determination

[0151] The protein extraction of clinical samples adopts a mixed sample method. Eight samples in each group are placed in an EP tube, 80μLRIPA lysis buffer and protease inhibitors are added, and they are placed on ice and fully ground. Use a cell brush to scrape the cell sample and place it in an EP tube, add an appropriate amount of RIPA lysis buffer and protease inhibitors, and place it on ice and fully grind it. After the sample is fully ground, place the EP tube on a 4°C shaker and rotate for 30 minutes, and centrifuge at 14000rpm for 40 minutes. The supernatant is then collected and placed in a new EP tube, the group name is marked and the volume is recorded, and it is stored at -80°C for later use. Take a small amount of protein samples from each group, operate according to the instructions of the kit, and use an enzyme reader to detect the protein concentration and record it.

[0152] 2.3.2 SDS-PAGE gel electrophoresis (SDS-polyacrylamide gel electrophoresis)

[0153] About 20 μg of total protein from each sample was subjected to SDS-PAGE gel electrophoresis and transferred to a PVDF membrane, blocked with 0.5% skim milk at room temperature for 2 hours, washed with TBST, and the primary antibody of the target molecule was added respectively, incubated at 4°C overnight; washed thoroughly with TBST, added the corresponding secondary antibody, incubated at room temperature for 1 hour; washed thoroughly with TBST, and added chemiluminescent reagent for color development. The bands obtained by electrophoresis were analyzed using ImageJ software. The target protein used β-actin as an internal reference, and the relative expression of each target protein was calculated and normalized. Each group of experiments was repeated three times or more independently.

[0154] 2.4qRT-PCR

[0155] like Figure 1As shown in the figure, the following points should be noted when extracting RNA from a single clinical sample using the TRIzol method: After the sample is taken out of the body, it should be immediately placed in a 1.5 mL Axygen enzyme-free EP tube. If the sample contains blood, it needs to be gently rinsed with PBS until there is no blood left. The operation should be carried out on ice throughout the process. The sample should be fully homogenized and lysed. After adding chloroform and centrifuging, there may be floating capsular fragments on the surface of the upper liquid phase. Avoid inhaling them when aspirating the supernatant. After normal extraction, the RNA concentration is generally about 150 ng / μL, and the absorbance 260 / 280 is between 1.8 and 2.1. Reverse transcription is carried out with a total of 500 ng of RNA, and the cDNA is diluted with 10 - 20 μL of DEPC water before use.

[0156] 2.5 Immunohistochemistry

[0157] Seed 5×10 4 cells in a 24-well plate containing cell slides. Add 500 μL of medium to each well. After the cells adhere to the wall, wash them once with Hank’s solution and then perform the intervention, and culture for an appropriate time. Aspirate the medium with a Pasteur pipette and add Hank’s solution for washing. Add 500 μL of 4% paraformaldehyde fixative to each well and fix in the dark at room temperature for 30 minutes. Aspirate the liquid with a pipette gun, add an appropriate amount of PBS buffer along the wall of the tube, and wash on a low-speed shaker for 5 minutes. Repeat 3 times. Block with 5% goat serum at 37°C for 2 hours. Depending on whether the target molecule is a membrane protein, choose whether to permeabilize with 0.5% Triton X-100 at room temperature for 20 minutes. After blocking, aspirate the liquid with a pipette gun, wash with PBS buffer for 5 minutes. Repeat 3 times. Dilute the primary antibody (1:250) with 5% goat serum, add 200 μL to each well, and incubate overnight at 4°C. The next day, let it warm up to room temperature for 30 minutes. Recover the primary antibody, wash with PBS buffer for 5 minutes. Repeat 3 times. Dilute the Alexa Fluor 488 / 568 fluorescent secondary antibody (1:250) with 5% goat serum, add 200 μL to each well, and incubate in the dark on a low-speed shaker at room temperature for 2 hours. Wash with PBS buffer for 5 minutes. Repeat 3 times. Drop the appropriate concentration of DAPI according to the instructions and incubate in the dark for 5 minutes. Wash with PBS buffer for 5 minutes. Repeat 5 times. Use forceps to take out the cell slides, dry the liquid on the surface of the slides with filter paper, and mount the slides with glycerol. Observe and select fields of view under a confocal microscope, and take pictures to avoid repeating the fields of view.

[0158] Seed 5×10 4The cells were seeded in a confocal dish, 1 mL of culture medium was added, and the mixture was shaken well. After the cells adhered to the wall, they were washed once with Hank’s solution and then subjected to corresponding interventions for an appropriate incubation time. The culture medium was discarded, and the cells were washed once with Hank’s solution and the liquid was aspirated completely. The operation was carried out under light protection. The pre-prepared mitochondrial fluorescent probe and the working solution for staining the cell nucleus were added to the confocal dish and incubated in a 37 °C cell culture incubator for 30 minutes. The working solution was aspirated, and the cells were washed 3 times with Hank’s solution. Fresh culture medium was added, and the mitochondrial morphology was observed under a confocal microscope. The ImageJ software was used for semi-quantitative analysis of the results of the immunofluorescence experiment.

[0159] 2.6 Detection of mitochondrial morphology and function

[0160] 2.6.1 Transmission electron microscopy

[0161] Clinical specimens were immediately placed in glutaraldehyde solution for fixation under light protection after being taken out of the body. Cell samples were processed as needed. When the number of cells was more than 1×106, the culture medium was aspirated with a Pasteur pipette, an appropriate amount of pre-warmed PBS buffer was added for washing, and then trypsin was added for digestion followed by centrifugation. After washing with PBS buffer, the cells were transferred to a 1.5 mL EP tube and centrifuged again. The supernatant was removed, and an appropriate amount of glutaraldehyde solution was added for fixation under light protection.

[0162] After the two types of samples were fixed in glutaraldehyde solution, they were continuously fixed in 2% OsO4 solution for 60 minutes. After selecting the area of interest, ultra-thin sections were obtained and then stained with uranyl acetate and lead citrate. Finally, the sections were observed using a transmission electron microscope.

[0163] 2.6.2 Detection of cell oxygen consumption rate

[0164] According to the manufacturer's instructions, the BBoxiProbe TM R01 kit was used to measure the cell oxygen consumption rate. 1×10 4 cells were seeded in a 96-well plate and pre-operated according to the experimental requirements. 150 μL of complete medium containing the R01 fluorescent probe and 10 μL of oxygen fluorescent probe were added. Then the 96-well plate was placed in an enzyme-linked immunosorbent assay (ELISA) reader, and the fluorescence was detected every 2 minutes for 120 minutes. The cell oxygen consumption rate (%) = (the final fluorescence of the cells in the treatment group - the initial fluorescence of the cells in the treatment group) / (the final fluorescence of the cells in the control group - the initial fluorescence of the cells in the control group) × 100%.

[0165] 2.6.3 Detection of mitochondrial membrane potential

[0166] 5×10 4The cells were seeded in confocal dishes and intervened in advance according to the experimental requirements. The mitochondrial membrane potential of the cells was detected according to the manufacturer's instructions of the enhanced JC-1 detection kit. Confocal microscopy and flow cytometry were used for detection and analysis. ImageJ software was used to analyze the red fluorescence intensity and green fluorescence intensity, calculate the ratio of red fluorescence to green fluorescence, and perform normalization to represent the mitochondrial membrane potential level.

[0167] 2.6.4 Detection of mitochondrial ROS

[0168] Seed 5×10 4 The cells were seeded in confocal dishes and intervened in advance according to the experimental requirements. The mitoSOX far-red reagent was added to the cell culture medium and incubated in the dark at 37 °C for 30 minutes. Then the cells were rinsed with PBS to remove the reagent, and fresh complete medium was added. Immediately, detection was carried out on a confocal microscope. ImageJ software was used to analyze the fluorescence intensity and perform normalization.

[0169] 2.7 Detection of cell apoptosis

[0170] The apoptosis rate of the cells was determined using an Annexin-FITC / PI cell apoptosis detection kit according to the manufacturer's instructions. After treatment, the cells were harvested and resuspended in PBS buffer (1×106 cells / mL). Subsequently, AnnexinV-FITC and PI were added and incubated in the dark for 30 minutes. Detection was carried out using flow cytometry. The operation was gentle throughout to avoid cell damage. The apoptosis rate = early apoptosis rate + late apoptosis rate (Q2 + Q3).

[0171] 2.8 In vitro culture of the lens

[0172] A total of 25 Sprague-Dawley (SD) rats were used in this experiment. The SD rats were sacrificed, and their eyeballs were removed. The eyeballs were repeatedly rinsed with sterile PBS buffer containing penicillin-streptomycin double antibody, and the lenses were obtained under a microscope. Each lens was placed in a 24-well plate containing 1 mL of M199 culture medium. The 24-well plates were placed in a 37 °C cell culture incubator and cultured for 2 days, with the culture medium changed once a day. After two days, the turbid lenses caused by the operation were discarded, and the transparent lenses were taken for subsequent experiments (a total of 34 lenses were collected). The transparent lenses were randomly divided into a normal control group (NC group), a high glucose group (HG, 50 mmol / L glucose-M199), a high glucose + DMSO group (HG+DMSO, 50 mmol / L glucose-M199 + 1 μL DMSO), a high glucose + TEPP-46 group (HG+TEPP-46, 50 mmol / L glucose-M199 + 1 μL TEPP-46), a high glucose + IN-1 group (HG+IN-1, 50 mmol / L glucose-M199 + 1 μL IN-1), and a high glucose + phloretin group (HG+Phloretin, 50 mmol / L glucose-M199 + 1 μL phloretin). The culture medium was changed every 24 hours and cultured for 5 days. Subsequently, photos were taken under a dark field microscope. Using ImageJ software for calculation, the images were converted to grayscale, and each pixel was automatically assigned a grayscale level. The standard deviation (SD) and mean density (MD) of each pixel were calculated within a diameter of 1 mm. In this study, the maximum SD of the transparent lens was 49, and the maximum MD of the completely turbid lens was 255. Substituting into the calculation method reported by Lu et al.: Opacification (a.u.) = (1 - SD / 49) × MD / 255 × 100.

[0173] 2.9 Statistical analysis

[0174] Data analysis was performed using Graphpad Prism 8.0 software. Measurement data were confirmed to be normally distributed by the Shapiro-Wilk test, and the experimental data were expressed as mean ± standard deviation. Analysis of variance, Student's t-test, and one-way analysis of variance were used for inter-group comparison. A P value < 0.05 was considered statistically significant. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0175] 3. Results

[0176] 3.1 Changes in the expression of PKM2 / GLUT1 in lens epithelial cells (LECs) of cataracts in patients with type 2 diabetes mellitus (T2DM)

[0177] The relative expression level of GLUT1 protein in LECs of the T2DM group was 1.49 ± 0.10, which was increased compared with that of the ARC group (1.00 ± 0.17), and the difference was statistically significant (t = 4.256, P < 0.05)( Figure 2 A - B). The fluorescence intensity of GLUT1 in LECs of the T2DM group was higher, while that of the ARC group was lower( Figure 2 C). The relative expression level of PKM2 protein in the T2DM group was 0.61 ± 0.06, which was lower than that of the ARC group (1.00 ± 0.14), and the differences were all statistically significant (t = 5.436, P < 0.01)( Figure 2 D - E). The fluorescence intensity of PKM2 in the T2DM group was lower, while that of the ARC group was higher( Figure 2 F). The above results indicate that the PKM2 / GLUT1 axis is dysregulated in LECs of cataracts in T2DM patients.

[0178] 3.2 Changes in mitochondrial morphology and dynamics in LECs of cataracts in T2DM patients

[0179] Mitochondria in both the T2DM group and the ARC group were distributed around the nucleus and showed short rod shapes. Compared with the ARC group, the number of mitochondria in the T2DM group increased, with obvious swelling, significant reduction of internal cristae, and vacuolization changes( Figure 3 A). The relative mRNA expression level of Drp1 in the T2DM group was 1.44 ± 0.17, which was higher than that of the ARC group (1.00 ± 0.25); the relative mRNA expression level of Mfn1 in the T2DM group was 0.76 ± 0.13, which was lower than that of the ARC group (1.00 ± 0.19); the relative mRNA expression level of Mfn2 in the T2DM group was 0.74 ± 0.17, which was lower than that of the ARC group (1.00 ± 0.18), and the differences were all statistically significant (t = 4.711, 3.535, 2.517, all P < 0.05)( Figure 3 B). The above results indicate that the mitochondrial morphology is abnormal and mitochondrial dynamics is imbalanced in LECs of cataracts in T2DM patients.

[0180] 3.3 Changes in the expression of PKM2 / GLUT1 in LECs cultured in vitro under high glucose

[0181] The relative expression level of GLUT1 protein in the high - glucose group was 1.86 ± 0.25, which was significantly increased compared with that of the control group (1.00 ± 0.03) (t = 5.822, P < 0.01)( Figure 4 A - B). The fluorescence intensity of GLUT1 in LECs increased after high - glucose treatment( Figure 4 C). The relative expression level of PKM2 protein in the high - glucose group was 0.67 ± 0.13, which was lower than that of the control group (1.00 ± 0.21) (t = 2.282, P < 0.05)( Figure 4D - E). Immunofluorescence revealed that after high - glucose treatment, the fluorescence intensity of PKM2 in LECs decreased, and there was partial nuclear translocation of PKM2 ( Figure 4 F). The above results indicate the dysregulation of the PKM2 / GLUT1 axis in LECs induced by high glucose.

[0182] 3.4 Changes in mitochondrial morphology and function of LECs cultured in vitro under high - glucose conditions

[0183] Compared with the control group, in the high - glucose group, mitochondria showed aggregated distribution, increased in number, swelled, and most were spherical. Figure 5 A). Mitochondria in LECs treated with high glucose were in a fragmented state, with significantly shortened length and a spherical shape. Figure 5 B). The relative expression levels of Mfn1 and Mfn2 proteins in the high - glucose group were 0.69 ± 0.07 and 0.86 ± 0.05 respectively, lower than those in the control group (1.00 ± 0.16 and 1.00 ± 0.06), and the differences were statistically significant (t = 4.193, 2.896, both P < 0.05). Compared with the control group (1.00 ± 0.18), the relative expression level of Drp1 protein increased in the high - glucose group (2.36 ± 0.14) (t = 10.30, P < 0.001). The relative ratio of apoptosis - marker proteins Bax to Bcl - 2 in the high - glucose group was 1.56 ± 0.10, increased compared with the control group (1.00 ± 0.21) (t = 4.193, P < 0.05) ( Figure 5 C - D).

[0184] The relative cellular oxygen consumption rate of LECs after high - glucose treatment was (45.67 ± 7.09)%, lower than that of the control group (t = 13.26, P < 0.001) ( Figure 6 A). Observation under a confocal microscope showed that compared with the control group, the fluorescence intensity of mitochondrial monomers (green) in the high - glucose group was significantly enhanced, while the fluorescence intensity of mitochondrial polymers (red) decreased. The relative ratio of red fluorescence / green fluorescence intensity in the high - glucose group was 0.73 ± 0.08, decreased compared with the control group (1.00 ± 0.09) (t = 7.938, P < 0.001), indicating a decrease in mitochondrial membrane potential. At the same time, the fluorescence intensity of mitoSOX (purple) in the high - glucose group was 3.03 ± 0.40, increased compared with the control group (1.00 ± 0.09) (t = 8.583, P < 0.01), indicating an increase in ROS in mitochondria. Figure 6 B - D). The same results were obtained by flow cytometry experiments ( Figure 6 E - F). The apoptosis rate of LECs after high - glucose treatment was (29.64 ± 4.94)%, increased compared with the control group (15.93 ± 3.61)% (t = 3.880, P < 0.05) ( Figure 6G). The above results indicate that high glucose induces abnormal mitochondrial morphology, mitochondrial dynamics imbalance, and mitochondrial dysfunction in LECs.

[0185] 3.5 Effect of the PKM2 / GLUT1 axis on mitochondrial morphology and function of LECs induced by high glucose

[0186] 3.5.1 Effect of PKM2 on mitochondrial morphology and function of LECs induced by high glucose

[0187] Compared with the high glucose + empty vector group, after overexpressing PKM2, the mitochondrial vacuoles in LECs decreased, the significantly decreased short rod-shaped or spherical mitochondria, and most were elongated; the combined treatment with TEPP-46 made this phenomenon more obvious; while in the IN-1 group, the mitochondria were observed to be significantly shorter and the granules increased ( Figure 7 A). The average relative expression levels of Mfn1 and Mfn2 proteins in the high glucose + PKM2 overexpression group were 1.49 ± 0.07 and 1.56 ± 0.09 respectively, higher than 1.00 ± 0.11 and 1.00 ± 0.04 in the high glucose group, and the differences were statistically significant (F = 72.12, 134.9, both P < 0.0001). The average relative expression level of Drp1 protein in the high glucose + PKM2 overexpression group was 0.72 ± 0.07, lower than that in the high glucose group (1.00 ± 0.05) (F = 24.82, P < 0.01). The average relative ratio of Bax to Bcl-2 in the high glucose + PKM2 overexpression group was 0.61 ± 0.10, lower than that in the high glucose group (1.00 ± 0.13) (F = 21.46, P < 0.01). The relative expression levels of Mfn1 and Mfn2 proteins in the high glucose + PKM2 overexpression + IN-1 group were 1.05 ± 0.10 and 1.33 ± 0.11 respectively, lower than those in the high glucose + PKM2 overexpression group (1.50 ± 0.11, 1.60 ± 0.08) (F = 18.18, 41.81, both P < 0.05); the relative expression level of Drp1 protein was 0.93 ± 0.06, higher than that in the high glucose + PKM2 overexpression group (0.67 ± 0.05) (F = 1.39, P < 0.01); the relative ratio of Bax to Bcl-2 was 0.81 ± 0.04, higher than that in the high glucose + PKM2 overexpression group (0.57 ± 0.13) (F = 7.343, P < 0.05) ( Figure 7B-C). The relative expression levels of Mfn1 and Mfn2 proteins were 1.76±0.06 and 1.84±0.12 respectively in the high glucose + PKM2 overexpression + TEPP-46 group, which were higher than those in the high glucose + PKM2 overexpression group (1.49±0.05, 15.3±0.11) (F = 65.49, 62.79, both P < 0.05). The relative expression level of Drp1 protein was 0.65±0.08 in the high glucose + PKM2 overexpression + TEPP-46 group and 0.76±0.05 in the high glucose + PKM2 overexpression group; the relative ratio of Bax to Bcl-2 in the high glucose + PKM2 overexpression + TEPP-46 group was 0.39±0.08, which was lower than that in the high glucose + PKM2 overexpression group (0.65±0.05) (F = 29.42, P < 0.05)( Figure 7 D-E).

[0188] The relative cell oxygen consumption rates of the high glucose + PKM2 overexpression group, the high glucose + PKM2 overexpression + TEPP-46 group and the high glucose + PKM2 overexpression + IN-1 group were (203±21.03)%, (260±32.74)%, (64±11.58)% respectively. The overall comparison showed statistically significant differences (F = 60.32, all P < 0.05)( Figure 8 A). The fluorescence intensity of mitochondrial monomers (green) decreased significantly after treatment with overexpressed PKM2, while the fluorescence intensity of mitochondrial polymers (red) increased. The relative ratio of red fluorescence intensity to green fluorescence intensity in the high glucose + PKM2 overexpression group was 2.10±0.58, which was higher than that in the high glucose + empty vector group (1.00±0.20). On the basis of overexpressing PKM2, the activator TEPP-46 (3.91±0.46) made the above phenomenon more obvious. While applying the inhibitor IN-1, it was observed that the fluorescence intensity of mitochondrial monomers (green) increased significantly, the fluorescence intensity of mitochondrial polymers (red) decreased, and the relative ratio of red fluorescence intensity to green fluorescence intensity (0.67±0.07) decreased, with statistically significant differences (all P < 0.05) (F = 43.33, Figure 8 B-D). At the same time, compared with the high glucose + empty vector group (1.00±0.09), the relative fluorescence intensity of mitoSOX in the high glucose + PKM2 overexpression group (0.52±0.04) decreased. Applying the inhibitor IN-1 significantly increased the relative fluorescence intensity of mitoSOX (1.52±0.17), with statistically significant differences (F = 50.99, all P < 0.05). After applying the activator TEPP-46, a decreasing trend of the relative fluorescence intensity of mitoSOX (0.44±0.07) was observed( Figure 8 B-D). The same results were obtained by flow cytometry experiments Figure 8E-F). The apoptosis rates of the high glucose + empty vector group, high glucose + PKM2 overexpression group, high glucose + PKM2 overexpression + IN-1 group, and high glucose + PKM2 overexpression + TEPP-46 group were (38.16 ± 2.20)%, (28.8 ± 2.76)%, (46.27 ± 5.18)%, and (23.67 ± 3.08)%, respectively. The overall comparison showed statistically significant differences (F = 27.02, all P < 0.05). Figure 8 G). The above results indicate that overexpression of PKM2 can improve the mitochondrial dynamics imbalance and mitochondrial dysfunction induced by high glucose. Application of the PKM2 activator TEPP-46 to increase PKM2 activity further restores the mitochondrial dynamics balance and mitochondrial function of LECs, reduces apoptosis, while the application of the PKM2 inhibitor IN-1 inhibits the protective effect of overexpressed PKM2.

[0189] 3.5.2 Effect of GLUT1 on the mitochondrial morphology and function of LECs induced by high glucose

[0190] In the high glucose + empty vector group, part of the mitochondria was elongated and part was granular. After GLUT1 overexpression, most of the mitochondria were granular. Figure 9 A). Compared with the relative expression levels of GLUT1 protein in the high glucose group and high glucose + empty vector group (1.00 ± 0.12; 1.33 ± 0.23), the relative expression level of GLUT1 protein in the high glucose + GLUT1 overexpression group (2.14 ± 0.29) increased, with statistically significant differences (F = 20.98, P < 0.05). The relative expression level of PKM2 protein in the high glucose + GLUT1 overexpression group was 0.58 ± 0.05, which was lower than that in the high glucose group and high glucose + empty vector group (1.00 ± 0.21; 0.99 ± 0.05), with statistically significant differences (F = 11.00, P < 0.05). The relative expression levels of Mfn1 and Mfn2 proteins in the high glucose + GLUT1 overexpression group were 0.66 ± 0.16 and 0.64 ± 0.14, respectively, which were lower than those in the high glucose group (1.00 ± 0.09; 1.00 ± 0.05) and high glucose + empty vector group (1.01 ± 0.11; 1.00 ± 0.19), with statistically significant differences (F = 7.492, 8.762, both P < 0.05). The relative expression level of Drp1 protein in the high glucose + GLUT1 overexpression group was 1.47 ± 0.13, which was higher than that in the high glucose group (1.00 ± 0.05) and high glucose + empty vector group (1.01 ± 0.19); the relative ratio of Bax to Bcl-2 was 1.94 ± 0.11, which was higher than that in the high glucose group (1.00 ± 0.15) and high glucose + empty vector group (1.16 ± 0.24), with statistically significant differences (F = 12.26, 24.71, both P < 0.05). Figure 9 B-C).

[0191] The relative cell oxygen consumption rate in the high-glucose + GLUT1 overexpression group was (44.87 ± 6.87)%, which was lower than that in the high-glucose + empty vector group (t = 13.91, P < 0.001). Figure 10 A). The fluorescence intensity of mitochondrial monomers (green) in the high-glucose + GLUT1 overexpression group increased significantly, while the fluorescence intensity of mitochondrial polymers (red) decreased. The relative ratio of red fluorescence / green fluorescence intensity in the high-glucose + GLUT1 overexpression group was 0.51 ± 0.15, which was lower than that in the high-glucose + empty vector group (1.00 ± 0.17) (t = 3.708, P < 0.05). The relative fluorescence intensity of mitoSOX in the high-glucose + GLUT1 overexpression group was 1.46 ± 0.14, which was higher than that in the high-glucose + empty vector group (1.00 ± 0.05) (t = 5.447, P < 0.01). Figure 10 B - D). The same results were obtained from the flow cytometry experiment. Figure 10 E - F). The apoptosis rate of cells in the high-glucose + GLUT1 overexpression group was (47.06 ± 6.01)%, which was higher than that in the high-glucose + empty vector group (30.72 ± 1.95)% (t = 4.452, P < 0.05). Figure 10 G). These results indicate that the overexpression of GLUT1 in a high-glucose environment exacerbates the high-glucose-induced mitochondrial dynamics imbalance, leading to mitochondrial dysfunction and apoptosis of LECs.

[0192] 3.6 Role of the PKM2 / GLUT1 axis in high-glucose-induced lens opacity

[0193] After removing the lenses that became turbid due to the operation, a total of 34 lenses of SD rats were successfully obtained in this experiment. As Figure 11 shown, the lenses in the NC group (18.83 ± 2.85 AU / pixel) were transparent after 5 days of in vitro culture, and the lenses in the HG group (72.65 ± 3.80 AU / pixel) showed annular opacity starting from the equator. The degree of lens opacity in the HG + IN-1 group was 90.45 ± 4.20 AU / pixel, which was significantly more severe than that in the HG group; the degree of lens opacity in the HG + TEPP-46 group was 51.91 ± 7.88 AU / pixel, which was significantly lower than that in the HG group; the degree of lens opacity in the HG + Phloretin group was 50.55 ± 10.03 AU / pixel, which was significantly lower than that in the HG group, and the differences were statistically significant (F = 54.79, all P < 0.05).

[0194] 4. Conclusions

[0195] 1. The PKM2 / GLUT1 axis is dysregulated in the LECs of cataracts in T2DM patients, and the mitochondrial morphology and dynamics are abnormal.

[0196] 2. Under hyperglycemic conditions, the imbalance of the PKM2 / GLUT1 axis in LECs mediates the imbalance of mitochondrial dynamics, leading to mitochondrial dysfunction and cell apoptosis.

[0197] 3. Increasing the activity of PKM2 can alleviate hyperglycemic-induced mitochondrial dysfunction and cell apoptosis in LECs.

[0198] 4. Increasing the activity of PKM2 can delay hyperglycemic-induced lens opacity and may be a key potential therapeutic target for preventing or treating cataracts associated with T2DM.

[0199] As described above, these are only several embodiments of the present application and do not impose any form of limitation on the present application. Although the present application is disclosed above with preferred embodiments, it is not intended to limit the present application. Any person skilled in the art, without departing from the scope of the technical solution of the present application, making some changes or modifications using the disclosed technical content above is equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. Use of a PKM2 activator and / or a GLUT1 inhibitor in the preparation of a medicament for preventing or treating T2DM cataract.

2. The use according to claim 1, characterized in that The T2DM cataract is manifested by LECs mitochondrial dysfunction and increased LECs apoptosis.

3. The use according to claim 2, characterized in that The LECs mitochondrial dysfunction is manifested as abnormal mitochondrial morphology and imbalanced mitochondrial dynamics in LECs.

4. The use according to claim 3, characterized in that The PKM2 activator is TEPP-46.

5. The use according to claim 3, characterized in that The GLUT1 inhibitor is Phloretin.

6. Use of PKM2 and / or GLUT1 in the preparation of a product for diagnosing T2DM cataract, characterized in that: The expression of PKM2 is downregulated in LECs of T2DM cataract, and the expression of GLUT1 is upregulated in LECs of T2DM cataract.

7. Application of PKM2 and / or GLUT1 in the preparation of products for evaluating the therapeutic effect of T2DM cataract or determining the prognosis of T2DM cataract.

8. The use according to claim 6 or 7, characterized in that: The products include a test kit and a chip.

Images