Application of lactic acid modified histone H3 in preparation of products for treating and / or preventing lung microvascular endothelial cell dysfunction

Abstract

The invention provides an application of a lactic acid modified histone H3 in preparation of a product for treating and / or preventing lung microvascular endothelial cell dysfunction, and the lactic acid modified histone H3 is obtained by carrying out lactic acid modification on the 14th amino acid of histone, the lung microvascular endothelial cell dysfunction is sepsis mediated lung microvascular endothelial cell dysfunction. The invention provides a new idea for the treatment of sepsis-mediated lung microvascular endothelial cell dysfunction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of lactylated histone H3 in the preparation of a product for treating and / or preventing pulmonary microvascular endothelial cell dysfunction. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated immune response to infection. There are approximately 50 million cases of sepsis worldwide each year, with a mortality rate of up to 20%, and it is the leading cause of death and readmission in hospitalized patients. According to epidemiological surveys in my country, the incidence of sepsis in ICUs is approximately 20% to 30%, and the mortality rate is as high as 30% to 50%. Sepsis is often accompanied by severe immune imbalance and dysregulated systemic inflammatory response, leading to multiple organ dysfunction or even failure. Among them, acute respiratory distress syndrome (ARDS) is one of the most common and serious complications of sepsis, which is caused by a variety of intrapulmonary or extrapulmonary pathogenic factors, characterized by acute lung injury (ALI) and rapidly develops into acute progressive respiratory failure. Therefore, in-depth research on the pathogenesis of sepsis complicated with ARDS, identification of key pathogenic targets, and exploration of effective intervention and treatment methods are crucial to improving clinical prognosis and survival rate. Summary of the invention

[0003] In view of the defects in the prior art, the purpose of the present invention is to provide the use of lactylated histone H3 in the preparation of products for the treatment and / or prevention of pulmonary microvascular endothelial cell dysfunction, providing a new idea for the treatment of sepsis-mediated pulmonary microvascular endothelial cell dysfunction.

[0004] The first aspect of the present invention provides the use of lactylated histone H3 in the preparation of a product for treating and / or preventing pulmonary microvascular endothelial cell dysfunction.

[0005] In one embodiment of the present invention, the lactic acid-modified histone H3 is a histone whose 14th amino acid is lactic acid-modified.

[0006] In one embodiment of the present invention, the pulmonary microvascular endothelial cell dysfunction is sepsis-mediated pulmonary microvascular endothelial cell dysfunction.

[0007] The second invention of the present invention provides the use of lactylated histone H3 as a target for treating pulmonary microvascular endothelial cell dysfunction.

[0008] In one embodiment of the present invention, the target is a drug target.

[0009] The third aspect of the present invention provides the use of an inhibitor of histone H3 lactylation modification in the preparation of a product for treating and / or preventing pulmonary microvascular endothelial cell dysfunction.

[0010] In one embodiment of the present invention, the inhibitor can be at least one of 2-deoxyglucose (2-Deoxy-D-glucose, 2-DG) and oxamate sodium (OX); 2-deoxyglucose (2-Deoxy-D-glucose, 2-DG) is a non-metabolizable glucose analog and a competitive inhibitor of glycolysis, which inhibits endogenous lactic acid production by inhibiting glycolysis; oxamate sodium (OX) is a lactate dehydrogenase (LDH) inhibitor, which can inhibit the production of endogenous lactic acid from pyruvate. The above inhibitors can reduce the production of lactic acid through different mechanisms, thereby reducing the lactic acid modification of the 14th lysine in the production of H3 protein.

[0011] The inhibitor is a substance that acts on the lactylation modification of lysine at position 14 of the H3 protein.

[0012] In one embodiment of the present invention, the inhibitor comprises at least one of a glycolysis inhibitor and a LDHA inhibitor.

[0013] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: the use of the lactylated histone H3 provided by the present invention in the preparation of a product for diagnosing and / or treating and / or preventing pulmonary microvascular endothelial cell dysfunction provides a new idea for the treatment of sepsis-mediated pulmonary microvascular endothelial cell dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0015] Figure 1 The results are as follows: the level of histone H3K14 in lung tissue and microvascular endothelial cells of septic mice in Example 1 was significantly increased; wherein, Figure 1 A is the non-targeted metabolomics results of lung tissues of septic mice, which shows that the lactate level in the lung tissues of septic mice is significantly increased; Figure 2 B is the Western Blot result of panlactation (KLa) in mice with sepsis and control group, which shows that the level of lysine panlactation (Kla) in the sepsis group was significantly increased compared with that in the control group; Figure 1 C is the whole protein lactic acid modification results of lung tissues of septic mice, which showed that the 14th lysine of histone H3 was significantly lactic acid modified (H3K14la); Figure 1 D is the western blot result of lung tissue of septic mice, which further verified the result. Figure 1E is the result of immunofluorescence staining of lung tissue, which shows that lactylated H3K14 co-localizes with the vascular endothelial cell marker CD31, indicating the occurrence of lactylated modification of H3K14 in lung microvascular endothelial cells (indicated by the white tip);

[0016] Figure 2 In Example 2, inhibiting the production of lactic acid can significantly inhibit the level of H3K14la and improve the lung injury and inflammatory response of septic mice; wherein, Figure 2 A is the test result of plasma lactate level in mice. Figure 2 C is the plasma cytokine level of IL-6 in mice, Figure 2 A and C show that after mice were pretreated with glycolysis inhibitor 2-DG and LDHA inhibitor Oxamate Sodium (OX), LPS was intraperitoneally injected into the mice to induce a mouse sepsis model. The results showed that 2-DG and OX pretreatment significantly inhibited the plasma lactate level and cytokine IL-6 level. Figure 2 B is the Western blot result of mice. WB results showed that the levels of Kla and H3K14la in the lung tissues of mice treated with 2-DG and OX were significantly reduced; Figure 2 D is the result of H&E staining of mouse lung tissue, which shows that 2-DG and OX pretreatment significantly reduced the degree of lung injury in septic mice ( Figure 2 D); The results are expressed as mean ± standard error, **p<0.01, ***p<0.001 indicate significant differences;

[0017] Figure 3 In Example 2, inhibiting the production of lactic acid can significantly inhibit the level of H3K14la and improve the lung injury and inflammatory response of septic mice, and inhibiting the effect of lactic acid production on the function of microvascular endothelial cells in the lung tissue of septic mice; Figure 3 A is the Western blot result of mouse lung tissue. Figure 3 B is the result of the detection of Ang2 in mouse plasma. The figure shows that both 2DG and OX pretreatment significantly reduced the protein levels of VCAM1 and Ang2 in mouse lung tissue ( Figure 3 A) and the level of Ang2 in plasma ( Figure 3 B); Figure 3 C is the level of Evans blue dye in mouse lung tissue. The level of Evans blue dye leakage into lung tissue was significantly reduced in mice treated with 2DG and OX. Figure 3 D is the result of IHC staining of mouse lung tissue. The number of CD45-positive leukocytes adhering and infiltrating in lung tissue was significantly reduced. The results are expressed as mean ± standard error. **p<0.01 and ***p<0.001 indicate extremely significant differences.

[0018] Figure 4 In Example 3, H3K14 lactylation modification regulates sepsis-related vascular endothelial cell function; Figure 4 A is the result of lactate level in the cell supernatant of umbilical vein endothelial cells after LPS stimulation. Using LPS-stimulated umbilical vein endothelial cells (HUVEC) as an in vitro endothelial injury model, it was found that the lactate level in the cell supernatant increased significantly with the extension of LPS stimulation time; Figure 4 B is the Western blot result of umbilical vein endothelial cells after LPS stimulation. LPS stimulation leads to a significant increase in the levels of Kla and H3K14la in HUVECs; Figure 4 C is the Western blot result after HUVEC was stimulated with lactate, and it was found that the levels of Kla and H3K14la in endothelial cells were significantly increased; Figure 4 D is the immunofluorescence staining result of umbilical vein endothelium. Figure 4 E is the Western blot result after HUVEC was pretreated with glycolysis inhibitor 2-DG and LDHA inhibitor Oxamate Sodium, and after LPS stimulation of cells. The results showed that 2-DG and Oxamate Sodium significantly inhibited the level of H3K14la, and at the same time significantly downregulated the levels of inflammatory adhesion molecule VCAM1 and leakage marker Ang2 in endothelial cells; the results are expressed as mean ± standard error, **p<0.01, ***p<0.001 indicate significant differences;

[0019] Figure 5 This is a diagram showing the mechanism of action of lactic acid-modified histone H3 in the preparation of the treatment of pulmonary microvascular endothelial cell dysfunction in the present invention. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0021] In recent years, studies have shown that lactate, as a signaling molecule, participates in regulating multiple pathophysiological processes such as immune cell activation, differentiation and immune tolerance in sepsis, thereby affecting the course of the disease. Lactic acid has an important regulatory effect on endothelial cell function. In septic mice, lactate produced by bone marrow neutrophils promotes neutrophil migration and aggravates tissue damage by affecting endothelial cell permeability. It is worth noting that endothelial cells rely on aerobic glycolysis to provide 85% of their energy under physiological conditions. Under inflammatory stimulation, proinflammatory factors and HIF-1α increase the glycolysis level of endothelial cells, and the generated lactate can aggravate the inflammatory response by activating the NF-κB pathway. This suggests that lactate, as a metabolite of glycolysis, can directly regulate endothelial cell function through non-metabolic pathways and promote the occurrence of ARDS in sepsis. Therefore, clarifying its molecular mechanism is particularly important for the prevention and treatment of ARDS in sepsis.

[0022] Based on this, the inventors of the present application have found that histone Kla binds to lysine residues by adding lactoyl groups, and the main sites are located on H3 and H4 histones, such as H3K14la, H3K18la, etc. Similar to other acylation modifications, histone Kla activates gene expression and participates in various disease processes, directly regulates gene transcription in chromatin, and thus changes cell homeostasis. Through the analysis of whole protein lactylation modification of lung tissue of septic mice, the inventors of the present application found that the level of lactylation modification (H3K14la) of the 14th lysine residue of H3 was significantly increased. Pulmonary microvascular endothelial cell damage is an important pathophysiological mechanism of ARDS in sepsis. Lactic acid may mediate endothelial cell damage through H3K14la and promote the development of ARDS in sepsis. Previous studies have shown that lactic acid produced by glycolysis can regulate the immune function of macrophages and tumor development through lactylation modification (H3K18la) of the 18th lysine residue of H3, confirming that histone lactylation, as an epigenetic modification, is an important mechanism for lactic acid to exert non-metabolic functions. This application study discovered H3K14la as a new lactylation modification site and its specific role in sepsis-induced pulmonary microvascular endothelial cell dysfunction.

[0023] The present invention studies the use of histone H3K14 lactic acid modification in the prevention and treatment of pulmonary microvascular endothelial cell dysfunction in sepsis ARDS. Specifically, 1. The present invention detects the lactic acid level in mouse lung tissue by liquid chromatography tandem mass spectrometry (LC / MS / MS) non-targeted metabolomics and whole-protein lactic acid modification omics of lung tissue, and finds that the 14th lysine of histone H3 undergoes significant lactic acid modification (H3K14la). This discovery can provide a target for studying lactic acid modification in sepsis ARDS; 2. The present invention uses in vitro primary umbilical vein endothelial cells ( The present invention uses HUVEC) injury as a model, and through the intervention of glycolysis-derived lactic acid and exogenous lactic acid, clarifies the effect of changing H3K14la levels on the inflammatory response and barrier function of vascular endothelial cells; 3. The present invention detects the levels of lactic acid and lactate dehydrogenase LDH in the plasma of patients with sepsis and sepsis combined with ARDS, and the levels of soluble adhesion molecules sVCAM1 and Ang2, which are markers of endothelial cell injury, and inflammatory factor IL6, to clarify the correlation between lactic acid levels and vascular endothelial injury levels and body inflammation levels in patients with sepsis and sepsis ARDS;

[0024] Based on this, the present invention claims the following:

[0025] 1. Use of lactylated histone H3 in the preparation of a product for diagnosing and / or treating and / or preventing pulmonary microvascular endothelial cell dysfunction. Wherein, the lactylated histone H3 is a histone with the 14th amino acid being lactylated. Wherein, the pulmonary microvascular endothelial cell dysfunction is sepsis-mediated pulmonary microvascular endothelial cell dysfunction.

[0026] 2. Application of lactylated histone H3 as a target for treating pulmonary microvascular endothelial cell dysfunction, wherein the target is a drug target.

[0027] 3. Use of an inhibitor of histone H3 lactylation modification in the preparation of a product for diagnosing and / or treating and / or preventing pulmonary microvascular endothelial cell dysfunction, wherein the inhibitor comprises at least one of a glycolysis inhibitor and a LDHA inhibitor.

[0028] Figure 5The figure is a diagram of the mechanism of action of lactylated histone H3 in the preparation of treatment for pulmonary microvascular endothelial cell dysfunction in the present invention, and the corresponding Chinese and English abbreviations in the figure are as follows: Glycose: glucose; 2-DG: 2-deoxyglucose; Glycolysis: glycolysis; Pyruvate: pyruvate; LDH: lactate dehydrogenase; OX: sodium oxamate; Lactate: lactate; Lactyl-CoA: lactylated coenzyme A; MCTs: monocarboxylate transporters; p300 / HDAC: histone deacetylase; Nuclears: nuclei; H3K14la: lactylated lysine 14 of histone H3; Endothelial cell: endothelial cell; Endothelial dysfunction: endothelial dysfunction; Inflammation: inflammation; Permeability: permeability; Sepsis: sepsis.

[0029] The present invention is further described in detail below with reference to specific embodiments.

[0030] Sources of materials used in the examples

[0031] Cells: The present invention uses human umbilical vein endothelial cells (HUVEC) for in vitro experiments, and selects the umbilical cords of newborns delivered by healthy cesarean section mothers.

[0032] Reagents and antibodies: Endothelial cell culture medium, fetal bovine serum, endothelial cell growth factor, penicillin / streptomycin were from Sciencell, USA, 0.25% EDTA trypsin digestion solution was from Dalian Meilun Biotechnology, PBS buffer was from Wuhan Sewell Biotechnology, RIPA protein lysis, BCA protein detection kit, cell counting plate were from Thermo Fisher Scientific, USA, protease phosphatase inhibitors were from Roche, Switzerland, WB antibody rapid stripping buffer, PAGE gel rapid preparation kit, SDD-PAGE protein loading buffer (5×), Tris / glycine / SDS electrophoresis buffer (10×) were from Shanghai Yazyme Biotechnology, rapid transfer solution (20X), universal antibody diluent were from Suzhou Xinsaimei Biotechnology, HiPerFect transfection reagent was from QIAGEN, Germany, Feket ultrasensitive ECL luminescent solution was from Dalian Meilun Biotechnology, VCAM1 antibody, fibrin antibody, Ly6g antibody, CD31 antibody were from Abcam, UK, sodium oxalate reagent was from Selleck, USA, TF antibody was from Santa Clara, USA Cruz Company, 2-DG reagent from Selleck, USA, lipopolysaccharide (LPS) reagent, lactic acid reagent from Sigma, USA, sodium lactate reagent from Beijing Bioson Biotechnology, human MCP-1 / CCL ELISA kit, human IL-6 ultrasensitive ELISA kit, human E-selectin ELISA kit, human VCAM-1 / CD106 ELISA kit, human ICAM-1 / CD54 ELISA kit, mouse IL-6 ELISA kit, mouse TBF-α ELISA kit are all from Hangzhou Lianke Biotechnology, H3K14 lysine lactylation antibody, lactylysine pan-lactylation antibody are all from Hangzhou Jingjie Biotechnology, C646 reagent from Selleck, USA, HRP-labeled goat anti-rabbit secondary antibody, HRP-labeled goat anti-mouse secondary antibody, GAPDH antibody from Proteintech, USA, histone H3 antibody from Cell Signaling Technology, USA, DEPC water (DNase, RNase free) from Shanghai Bio-Tech, Trizol from Invitrogen, PAGERULER pre-stained protein from Thermometer Fisher Scientific, PVDF membrane from Bio-Rad, L-Lactate Assay Kit I-100Assays from EtonBioscience, isopropanol from Shanghai Jingzhouke Gene Technology, fluorescent secondary antibody ALEXA FLUOR488DONKEY, fluorescent secondary antibody ALEXA FLUOR 555GOAT from Thermometer Fisher Scientific.

[0033] Experimental animals: C57BL / 6J male mice, 6-8 weeks old, weighing 20-30 g, were used in the animal experiments, provided by Shanghai South Model Organism Research Center. The breeding conditions were temperature: 20-26°C; humidity: 40-70%; light: 12 hours on and 12 hours dark, and clean water and feed were provided. The animal experiment protocol was approved by the Animal Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine (No. 339092) and complies with the International Guide for the Care and Use of Laboratory Animals.

[0034] Statistical methods: The experimental data were analyzed using Prism 9. The data were expressed as mean ± standard deviation (Mean ± SD). All results represented at least 3 repeated experiments or were performed according to the instructions. The t-test was used for comparison between two groups, and one-way ANOVA analysis of variance was used for comparison between multiple groups. A two-sided P value < 0.05 was considered statistically significant.

[0035] The present invention is further described in detail below in conjunction with specific examples, including screening for histone H3K14la involved in endothelial dysfunction in sepsis-induced lung injury, inhibiting lactate production to significantly inhibit H3K14la levels and improving inflammatory responses in sepsis-induced lung injury in mice.

[0036] Example 1 Screening for histone H3K141a involved in endothelial dysfunction in sepsis-induced lung injury

[0037] In this example, a mouse sepsis model was established by intraperitoneal injection of LPS (5 mg / kg), and liquid chromatography tandem mass spectrometry (LC / MS / MS) non-targeted metabolomics was used to detect the lactate level in mouse lung tissue, Western blot was used to detect the pan-lactylation (Kla) modification level of lung tissue protein, LC-MS / MS whole protein lactylation modification omics of lung tissue, and lung tissue immunofluorescence staining were used to screen and identify histone H3K14la involved in endothelial dysfunction in sepsis-induced lung injury.

[0038] This embodiment is attached Figure 1 The corresponding Chinese and English abbreviations are as follows:

[0039] L-Lactic acid: lactic acid; Sham: sham operation group; LPS: endotoxin; CT: control group; Kla: lactylated; H3: histone 3; Tubulin: tubulin; VCAM1: vascular cell adhesion molecule 1; Ang2: angiopoietin 2; WTAP: WTAP (Wilms Tumor 1-Associated Protein) RNAN6-methyltransferase; H3K14la: lactylated lysine 14 of histone H3; CD31: platelet-endothelial cell adhesion molecule; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride, a fluorescent dye for cell nuclei.

[0040] The specific steps of this embodiment are as follows:

[0041] 1. Intraperitoneal injection of LPS (5 mg / kg) to establish a mouse sepsis model:

[0042] 1.1 Male C57BL / 6 mice (20-30 g) were used to prepare LPS solution. LPS was injected intraperitoneally at a dose of 5 mg / kg to establish a sepsis model. 30 minutes after the model was established, 0.5 ml of normal saline was injected intraperitoneally every 2 hours for a total of three times.

[0043] 1.2 Collect lung tissue 16 hours after modeling, cut it into small pieces, and immediately put it into liquid nitrogen with tweezers. Put the frozen tissue into a cryovial and store it at -80℃.

[0044] 2. Western blot was used to detect the level of pan-lactic acidification in lung tissue, the level of H3K14la, the level of WTAP, and the expression levels of VCAM1 and Ang2.

[0045] 1.1 Take out the tissue and wash it twice with an appropriate amount of cold PBS, centrifuge it at 4℃3000rpm for 3min, and discard the supernatant. Take 5mg of tissue on ice, add 1mL of lysis buffer to a 1.5mL EP tube and grind it. Use an ultrasonic cell disruptor to ultrasonically disrupt the lysis suspension in an ice bath, centrifuge it at 4℃12000rpm for 10min, discard the precipitated tissue and cell fragments, and take the supernatant as the protein suspension. Determine the protein concentration using the BCA method, add 250uL of 5*loading buffer to 1mL of protein suspension, denature the protein in a 100℃ metal bath for 10min, and store the protein sample at -20℃.

[0046] 1.2 Electrophoresis: Prepare 10% gel for SDS-PAGE gel electrophoresis, add 3uL marker on both sides of the lane, take out the sample, centrifuge and vortex, and load it according to the calculated protein concentration. Generally, add 20ug of sample. After all the samples are loaded, add electrophoresis solution to the electrophoresis tank and plug in the power supply. The upper gel is 80V and the lower gel is 120V for electrophoresis.

[0047] 1.3 Transfer: After electrophoresis, transfer the membrane. First, soak the PVDF membrane in methanol to activate it. Immerse the transfer clip, sponge and filter paper in the tray containing the transfer solution. From the negative electrode to the positive electrode, form a "transfer sandwich" in the order of black plate, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and white plate. Insert the transfer clip into the transfer rack and put it into the electrophoresis tank. Put an ice box in the electrophoresis tank, add the transfer solution and ice box, and transfer the membrane in a constant current 250mA ice water bath for 2 hours.

[0048] 1.4 Blocking: After the transfer is completed, put the membrane into 10% skimmed milk powder and block it at room temperature for 1 hour (slow speed).

[0049] 1.5 Primary antibody incubation: After blocking, wash the membrane three times with TBST on a shaker, 10 min each time (quickly), dilute Kla, H3, tubulin, VCAM1, Ang2, WTAP, H3K14la mouse antibodies with primary antibody diluent at a ratio of 1:1000, and incubate with the washed membrane at 4°C overnight.

[0050] 1.6 Secondary antibody incubation: After overnight, wash with TBST for 3 times, 10 min each time. Then add HRP-labeled mouse secondary antibody (1:5000 dilution) and incubate at room temperature for 1 h before washing with TBST.

[0051] 1.7 Development: Finally, use ECL chemiluminescent developer for development (1:1 mix, prepare and use immediately).

[0052] Western blot results Figure 1 As shown in B and 1D, compared with the control group, the level of lysine pan-lactation in the sepsis group was significantly increased, and histone H3K14la was significantly lactated. Figure 1 As shown in A, the lactate level in the lung tissue of septic mice was significantly increased.

[0053] 3. Immunofluorescence staining was used to detect H3K14la in microvascular endothelial cells of lung tissue of septic mice. Lung tissue immunofluorescence staining was performed at PTM Biolabs. The specific steps are as follows:

[0054] 3.1 Dewaxed and dehydrated lung tissue sections were boiled at 98°C for 10 minutes for antigen retrieval and then blocked with 10% normal goat serum for 1 hour at room temperature.

[0055] 3.2 Sections were incubated with designated antibodies overnight at 4°C.

[0056] 3.3 Incubate with Alexa Fluor-labeled specific isotype control secondary antibody (Service bio) for 1 hour at room temperature.

[0057] 3.4 Nuclei were stained with 4,6-diamino-2-phenylindole (Service bio) for 5 minutes at room temperature and then mounted with mounting medium (Sigma, MO, USA).

[0058] 3.5 Finally, the sections were stained with diaminobenzidine for microscopic examination (DS-U3; Nikon, Tokyo, Japan).

[0059] Immunofluorescence staining results Figure 1E shows that lactylated H3K14 co-localizes with the vascular endothelial cell marker CD31, suggesting the occurrence of lactylated modification of H3K14 in pulmonary microvascular endothelial cells (indicated by the white tip, Figure 1 E) Red: H3K14la (PTM1414, Hangzhou Jingjie Biotechnology, antibody specificity has been verified: http: / / www.ptm-biolab.com.cn / productDetail.html?id=5196); Green: CD31; Blue: DAPI (cell nucleus), white arrows indicate endothelial cells.

[0060] 4. LC-MS / MS non-targeted metabolomics and whole protein lactylation modification genomics of lung tissue were performed at PTM Biolabs, and LC-MS / MS detection was performed at PTM Biolabs. The specific implementation steps are as follows:

[0061] 4.1 The trypsin-digested peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile in water) and directly loaded onto a homemade reverse phase analytical column (25 cm length, 100 μm inner diameter).

[0062] 4.2 The peptides were separated using a gradient from 7% to 24% solvent B (0.1% formic acid in acetonitrile) in 42 min, then increased from 24% to 32% in 12 min, then increased to 80% in 3 min, and finally maintained at 80% for 3 min, all of which were performed on a Bruker Daltonics nanoElute UHPLC system at a constant flow rate of 450 nL / min.

[0063] 4.3 The peptide fragments then entered the timsTOF Pro (Bruker Daltonics) mass spectrometer through a capillary source, and the applied electrospray voltage was 1.75 kV.

[0064] 4.4 Precursors and fragments were analyzed on a TOF detector with an MS / MS scan range of 100 to 1700 m / z. The timsTOF Pro was operated in parallel accumulation tandem fragmentation (PASEF) mode.

[0065] 4.5 Precursors with charge states 0 to 5 were selected for fragmentation and 10 PASEF-MS / MS scans were acquired per cycle with dynamic exclusion set to 30 seconds.

[0066] The results of protein lactylation modification in lung tissue are as follows Figure 1 As shown in C, the 14th lysine of histone H3 is significantly lactylated (H3K14la).

[0067] Example 2 Inhibition of lactate production significantly inhibits H3K14la levels and improves inflammatory response in lung injury in septic mice

[0068] In this example, mice were treated with glycolysis inhibitor 2-DG and LDHA inhibitor Oxamate Sodium (OX), and a mouse sepsis model was induced by intraperitoneal injection of LPS (5 mg / kg). The lactate level in mouse plasma was detected by a lactate detection kit, the level of IL-6 in plasma was detected by an ELISA kit, the levels of pan-lactated Kla, H3K14la, Ang2 and VCAM1 in lung tissue were detected by Western blot, the degree of lung tissue damage was detected by H&E staining, and the expression level of VCAM1 in lung tissue and the adhesion level of CD45+ leukocytes in lung tissue were detected by IHC staining.

[0069] This embodiment is attached Figure 2 , 3 The corresponding Chinese and English abbreviations are as follows:

[0070] Plasma Lactate level: plasma lactate level; Sham: sham operation group; LPS: endotoxin; 2-DG: 2-deoxyglucose; OX: sodium oxamate; CT: control group; Kla: lactation; H3: histone 3; H3K14la: lactation of lysine 14 of histone H3; IL-6: cytokine interleukin 6; Ang2: angiopoietin 2; VCAM1: vascular cell adhesion molecule 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Lung leakage: Evans blue dye leaked from lung tissue; CD45: leukocyte common antigen.

[0071] The specific steps of this embodiment are as follows:

[0072] 1. Treat mice with glycolysis inhibitor 2-DG (250 mg / kg) and LDHA inhibitor Oxamate Sodium (750 mg / kg), and construct a mouse model of sepsis by intraperitoneal injection of LPS (5 mg / kg):

[0073] 1.1 Use male C57BL / 6 mice (20-30g), prepare glycolysis inhibitor 2-DG and LDHA inhibitor Oxamate Sodium (OX), and use dorsal restraint to grab the mouse, so that its head is slightly tilted back and its abdomen is facing up. The left hind limb of the mouse is fixed with the little finger. Then disinfect the injection site with a 75% alcohol cotton ball. Insert the syringe needle into the skin, and after entering the subcutaneous tissue, push forward 2-3mm along the subcutaneous tissue. The needle is inserted into the abdominal cavity of the mouse at a 45-degree angle to the skin. Draw back the needle plug. If there is no blood or liquid, the drug can be injected. After the injection is completed, gently rotate the needle and then slowly pull out the syringe to prevent liquid leakage.

[0074] 1.2. Prepare LPS solution and inject LPS intraperitoneally at a dose of 5 mg / kg to establish a sepsis model. 30 minutes after the model is established, inject 0.5 ml of normal saline intraperitoneally every 2 hours for a total of three times.

[0075] 1.3 Collect lung tissue 16 hours after modeling, cut it into small pieces, and immediately put it into liquid nitrogen with tweezers. Put the frozen tissue into a cryovial and store it at -80℃.

[0076] 2. L-Lactic Acid Detection Kit detects lactate level and LDH level in mouse plasma.

[0077] Human serum, mouse plasma, cell supernatant, and cell homogenate samples were collected to measure lactate levels according to the manufacturer's instructions (L-Lactic Acid Detection Kit, Eton Bioscience, 1200011002). The absorbance was measured at 490 nm. The LDH concentration in serum was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

[0078] 2.1 Direct determination of mouse plasma and cell supernatant.

[0079] 2.2 Intracellular lactate sample processing, all operations were carried out on ice.

[0080] 2.3 Remove the culture medium and wash with PBS three times to fully remove the residual liquid.

[0081] 2.4 Add 20 μl PBS to each well of the cell plate, scrape the adherent cells thoroughly with a cell brush, and collect the PBS into an EP tube.

[0082] 2.5 Use an ultrasonic device to fully lyse the cells, centrifuge at 4°C, 12,000×g for 10 minutes, and measure the lactate concentration in the supernatant, which is the intracellular lactate.

[0083] 2.6 Sample measurement and preparation of standards: Use a 96-well ELISA plate and add 50uL, 40uL, 30uL, 20uL, 10uL, 5uL, 1uL and 0uL of standard to each well in turn, and fill each well to 50uL with double distilled water.

[0084] 2.7 Add the sample to be tested: Continue to add 50 μl of the test sample and design 2 replicate wells for each sample.

[0085] 2.8 Add 50 μl of L-lactic acid content assay reagent to each well and incubate in a 37°C incubator for 30 minutes.

[0086] 2.9 Add 50 μl of 0.5 M acetic acid reagent to each well and stir gently to terminate the reaction. Use a needle tip to remove bubbles in the solution.

[0087] 2.10 Detection reading: Use an enzyme-labeled instrument to measure the absorbance at 490 nm. Draw a standard curve based on the standard 490 nm absorbance and concentration, and calculate the lactic acid concentration in the sample.

[0088] The results of lactate testing are as follows: Figure 2 As shown in A, 2-DG and OX pretreatment significantly suppressed the plasma lactate level.

[0089] 3. ELISA kit to detect IL-6 and Ang2 levels in plasma

[0090] The serum concentrations of IL6, sE-selectin, sICAM1, and sVCAM1 in healthy controls and sepsis patients, as well as the concentrations of IL6 and TNF∝ in mouse plasma and MCP1 in the supernatant of cultured HUVECs were measured using the corresponding ELISA kits (MultiSciences Biotechnology, Hangzhou, China) according to the manufacturer's instructions. The specific steps are as follows:

[0091] 3.1 Sample preparation: Mouse plasma: 1000 μl, centrifuge for 30 minutes, collect the supernatant for detection.

[0092] 3.2 Cell culture medium: 500 μl, centrifuge for 10 minutes, collect the supernatant for detection

[0093] 3.3 Sample measurement, preparation of standard: add 100 μl of standard with decreasing concentration gradient and 100 μl of standard dilution solution as blank control.

[0094] 3.4 Add the sample to be tested: Set up 2 replicate wells for each sample, calculate the required microplate, add the wash solution and soak for 30 seconds. Discard the wash solution and pat the microplate dry.

[0095] 3.5 Add sample: Add 100 μl of the sample to be tested to each sample well.

[0096] 3.6 Add detection antibody: Add 50 μl of diluted detection antibody to each well, seal the plate with sealing film, shake at 300 rpm, and incubate at room temperature for 1.5 hours.

[0097] 3.7 Washing: Remove the liquid, add 300 μl of washing solution, and wash 6 times. Remove the residual liquid thoroughly each time and pat dry.

[0098] 3.8 Enzyme incubation: Add 100 μl diluted horseradish peroxidase-labeled streptavidin, seal the plate with a sealing film, shake at 300 rpm, and incubate at room temperature for 30 minutes. Remove the liquid completely and wash thoroughly with washing solution 6 times.

[0099] 3.9 Add substrate for color development: Add 100 μl of color development substrate to each well and incubate at room temperature in the dark for 30 minutes.

[0100] 3.10 Add stop solution: Add 100 μl stop solution to each well.

[0101] 3.11 Detection readings: The microplate reader performs dual-wavelength detection. The OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm is measured by the microplate reader. The standard curve is drawn and the concentration of the sample to be tested is calculated.

[0102] ELISA results Figure 2 As shown in C, 2-DG and OX pretreatment significantly inhibited the level of cytokine IL-6 in plasma.

[0103] 4. Western blot was used to detect the levels of pan-lactated Kka, H13K14la, Ang2 and VCAM1 in lung tissue. The specific operation steps were consistent with those in Example 1.

[0104] Western blot results Figure 2 As shown in B, the levels of Kla and H3K14la in the lung tissues of mice treated with 2-DG and OX were significantly decreased.

[0105] 5. H&E staining to detect the degree of lung tissue damage:

[0106] 5.1 Dehydration: The tissue is dehydrated step by step using ethanol solutions of different concentrations from low to high.

[0107] 5.2 Transparency: After dehydration, place the tissue in a mixture of equal amounts of anhydrous ethanol and xylene for 15 minutes, and then place it in xylene until transparent.

[0108] 5.3 Wax penetration: After the tissue has been made transparent, place it in a mixture of equal amounts of xylene and paraffin for 15 minutes, and then place it in paraffin for 60 minutes. The wax penetration process is carried out in a constant temperature box at 55-60℃.

[0109] 5.4 Embedding and sectioning: Place the tissue into the wax mold and pour in the molten wax. Slice the tissue using a microtome, attach it to a glass slide, dry it in the oven, and then dry it.

[0110] 5.5 Hematoxylin-eosin (HE) staining a. Dewaxing and rehydrating paraffin sections: Keep the temperature at 60°C for 30 minutes until the wax melts. Place the paraffin sections in xylene I and xylene II for 10 minutes each, and then place them in different concentrations of ethanol from high to low for 5 minutes each to dewax.

[0111] 5.6 Hematoxylin staining: Soak the sections in PBS for 3 times, add hematoxylin to stain for 10 minutes, and rinse with double distilled water.

[0112] 5.7 Differentiation: The sections after hematoxylin staining were differentiated with 1% hydrochloric acid ethanol, and then rinsed with double distilled water. d. Blueing: 1% ammonia solution was used for blueing for 1 minute to stain the nucleus, and then rinsed with double distilled water.

[0113] 5.8 Eosin staining: Add eosin to the tissue sample for 3 minutes.

[0114] 5.9 Dehydration: After staining, the tissue sections are dehydrated again in a gradient manner.

[0115] 5.10 Air-drying and sealing: Soak the dehydrated tissue sample slices in xylene and finally use neutral gum to seal the slices.

[0116] HE staining results Figure 2 As shown in D, pretreatment with 2-DG and OX significantly alleviated the extent of lung injury in septic mice.

[0117] 6. Before killing the mice, inject Evans blue solution (Evans blue dye) into the tail vein of the mice. One hour later, the mice were killed and the content of Evans blue dye per unit weight of lung tissue was detected. The specific operation steps are as follows:

[0118] 6.1 Place mice under appropriate anesthesia (e.g., isoflurane).

[0119] 6.2 Inject Evans blue dye through the tail vein, the general dose is 10-30 mg / kg.

[0120] 6.3 The mice were killed, the chest cavity was opened quickly, the lung tissue was exposed, and the blood in the lung was removed by perfusion with physiological saline to reduce background staining.

[0121] 6.4 Tissue fixation: remove the lung tissue and fix it in 10% neutral formalin, usually for 24 hours.

[0122] 6.5 Tissue dehydration and embedding: The fixed lung tissue was dehydrated with gradient methanol and then transparentized with xylene. The tissue was embedded in paraffin and thin sections (4-5 μm) were prepared.

[0123] 6.6 Staining The paraffin sections were dewaxed in xylene and hydrated with gradient ethanol. The sections were stained in a solution containing Evans blue dye for 5-10 minutes.

[0124] 6.7 Dehydration and clearing Dehydrate with graded ethanol and then clear the sections with xylene.

[0125] 6.8 Mounting Use appropriate mounting medium to mount the slides.

[0126] 6.9 Microscope observation: Observe the stained sections under a microscope. Evans blue dye generally makes the tissue appear blue, and the vascular damaged areas will have more obvious staining. This process can help you detect and analyze changes in vascular permeability in lung tissue.

[0127] The results are as follows Figure 3 As shown in C, the level of Evans blue dye leakage into lung tissue was significantly reduced in 2DG- and OX-treated mice.

[0128] 7. Use IHC staining to detect the expression level of VCAM1 in lung tissue and the adhesion level of CD45+ leukocytes in lung tissue. The specific operation steps are as follows.

[0129] 7.1 Dewaxing and rehydrating: Keep the temperature at 60℃ for 30 minutes until the wax melts, and place the paraffin sections in xylene I and xylene II for 10 minutes each, and then place them in different concentrations of ethanol from high to low for 5 minutes each for dewaxing. Wash with double distilled water 3 times, 3 minutes each time.

[0130] 7.2 Antigen repair: Place in 10 nM citric acid antigen repair solution at pH 6.0, boil in a pressure cooker for 10 minutes, cool at room temperature for 30 minutes, and wash with PBS 3 times, 5 minutes each time.

[0131] 7.3 Inactivate enzyme: Soak the slices in 3% hydrogen peroxide-methanol at room temperature for 20 minutes, and wash with PBS three times, 5 minutes each time.

[0132] 7.4 Blocking: Add 5% BSA to the slices and block for 1 hour at room temperature.

[0133] 7.5 Incubate with primary antibody: Remove excess BSA, add primary antibody, and incubate overnight at 4°C. Wash with PBS 3 times, 5 minutes each time.

[0134] 7.6 Incubate with secondary antibody: Remove excess PBS, add secondary antibody, and incubate at room temperature for 1 hour. Wash with PBS 3 times, 5 minutes each time.

[0135] 7.7 Add DAB color developer: After washing with PBS, add DAB color developer and rinse with double distilled water after color development.

[0136] 7.8 Hematoxylin re-staining: Add hematoxylin for 2 minutes, rinse with double distilled water. Add 0.5% hydrochloric acid ethanol differentiation solution for 20 seconds, rinse with double distilled water. Soak in PBS for 20 seconds to turn blue, and continue to rinse with double distilled water.

[0137] 7.9 Dehydration and transparency: Dehydrate by soaking in ethanol with concentrations from low to high, 5 minutes each time. Soak in xylene I and xylene II for 5 minutes to make it transparent, 20 minutes each time.

[0138] 7.10 Sealing: Seal the slides with resin, let them dry, and then read the slides under a microscope.

[0139] IHC staining results Figure 3 As shown in D, the number of CD45-positive leukocytes adhering and infiltrating in the lung tissue was significantly reduced, and the black arrows indicate the pulmonary microvessels.

[0140] Example 3 H3K14 lactylation modification may regulate sepsis-related vascular endothelial function

[0141] This example uses LPS (1ug / mL) stimulated umbilical vein endothelial cells (HUVEC) as an in vitro endothelial injury model, collects supernatants at different times, and uses a lactate detection kit to detect lactate levels. After pre-treating the cells with 2-DG or Oxamate Sodium (OX), HUVECs were stimulated with LPS (1ug / mL), and Western blot was used to detect Kla and H3K14la protein levels in the cells. HUVECs were treated with different concentrations of lactic acid, and Western blot was used to detect Kla and H3K14la protein levels in the cells. Immunofluorescence staining was used to detect H3K14la levels in cells, and Western blot was used to detect H3K14la and Ang2 protein levels in cells.

[0142] This embodiment is attached Figure 4 The corresponding Chinese and English abbreviations are as follows:

[0143] Extracelluar Lactate: extracellular lactate level; CT: control group; LPS: endotoxin; Kla: lactation; H3: histone 3; H3K14la: lactation of lysine 14 of histone H3; Lactate: lactic acid; 2-DG: 2-deoxyglucose; OX: sodium oxamate; VCAM1: vascular cell adhesion molecule 1; Ang2: angiopoietin 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride, a fluorescent dye for cell nuclei.

[0144] The specific steps of this embodiment are as follows:

[0145] 1. Cell culture: Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord.

[0146] 1.1 Take the umbilical cord of a newborn baby delivered by healthy cesarean section, keep 15-20 cm of it, put it in sterile PBS and store it at 4℃ and transfer it to a sterile table.

[0147] 1.2 Wear sterile gloves during the whole process of cell extraction. Take out the umbilical cord and put it into a sterile dish. Wash the surface of the umbilical cord and both ends with PBS.

[0148] 1.3 Find the umbilical vein, squeeze out the remaining fluid in the umbilical vein, and repeatedly flush the umbilical vein with PBS through a syringe until no obvious blood remains.

[0149] 1.4 Use a vascular clamp to clamp one end of the umbilical vein, and inject trypsin into the umbilical vein from the other end until the vein is full and clamp it. Pay attention to clamping it fully to avoid trypsin leakage.

[0150] 1.5 Place the umbilical cord in sterile PBS in a 37°C water bath for 20 minutes for digestion.

[0151] 1.6 After sufficient digestion, squeeze the liquid in the umbilical vein into a 50ml centrifuge tube and add the prepared culture medium to terminate the digestion. Centrifuge at 800rpm for 10 minutes, discard the supernatant, suspend the precipitate with 1ml culture medium and transfer it to a culture bottle. Add 10ml culture medium and shake slightly, then place it in the incubator.

[0152] 1.7 After 24 hours, change the medium and discard the supernatant containing non-adherent cells and red blood cells. Observe the cell adhesion and growth under a microscope.

[0153] 1.8 After the cells have grown to full size in the culture flask, they are passaged or frozen. HUVECs were cultured in EGM-2MV medium (CC-3202, Lonza, Walkersville, MD USA) containing 10% fetal bovine serum (FBS, CC-4102B, Lonza), supplemented with streptomycin and penicillin, and then cultured in an incubator at 37°C in a humidified atmosphere containing 5% CO2. The HUVECs used in all experiments were from passage 1 to passage 4. The use of HUVECs complies with the guidelines for human research of Ruijin Hospital Affiliated to Shanghai Jiao Tong University.

[0154] 2. Drug treatment: HUVEC was inoculated in 24-well plates and 12-well plates, 500uL and 1mL of complete culture medium were added, the cell plate was gently shaken to allow the culture medium to fully cover the bottom of the culture well, and after the cell confluence reached more than 90%, LPS (1ug / mL) was used for stimulation to establish an in vitro endothelial injury model. For cells that need to be treated, the corresponding drugs are added according to the experimental requirements with a pipette after changing the liquid, and the cell plate is gently shaken to mix the liquid and placed in an incubator. After half an hour, the cell plate is taken out, and the culture medium does not need to be changed. LPS stimulation is directly given according to the above method. After drug treatment, the cells are placed in an incubator for culture, and the supernatant is collected at different times according to the experimental requirements. The lactic acid level is detected with a lactic acid detection kit. The specific operation of the lactic acid detection kit is consistent with Example 2.

[0155] Lactate test results Figure 4 As shown in A, umbilical vein endothelial cells (HUVEC) stimulated with LPS were used as an in vitro endothelial injury model. The lactate level in the cell supernatant increased significantly with the extension of LPS stimulation time.

[0156] 3. Stimulate HUVEC with LPS (1ug / mL), lyse the cells at different times, and then obtain the cells for protein extraction. Use Western blot to detect the protein levels of Kla and H3K14la in the cells. Before LPS stimulation, pretreat the cells with 2-DG or OX. The specific operation of Western blot is consistent with Example 1.

[0157] Western blot results Figure 4 As shown in B, LPS stimulation led to a significant increase in the levels of Kla and H3K14la in HUVEC. After HUVEC were pretreated with the glycolysis inhibitor 2-DG and the LDHA inhibitor Oxamate Sodium, the cells were stimulated with LPS. Figure 4 As shown in D and E, 2-DG and Oxamate Sodium significantly inhibited the level of H3K14la, and at the same time significantly downregulated the levels of inflammatory adhesion molecule VCAM1 and leakage marker Ang2 in endothelial cells.

[0158] 4. Treat HUVEC with different concentrations of lactic acid, and use Western blot to detect the protein levels of Kla and H3K14la in the cells. The specific operation of Western blot is consistent with Example 1.

[0159] The experimental results are as follows Figure 4 As shown in C, after HUVECs were stimulated with lactate, it was found that the levels of Kla and H3K14la in endothelial cells were significantly increased.

[0160] 5. Detect the H3K14la level in cells by immunofluorescence staining. The specific steps are as follows:

[0161] 5.1 Wash the cells with PBS and fix them with 4% paraformaldehyde for 15 minutes at room temperature.

[0162] 5.2 Permeabilize with 0.1% Triton X-100 for 20 minutes, and then block with 3% BSA (PBS) for 30 minutes.

[0163] 5.3 Primary antibodies (described in Methods) diluted in 3% BSA (PBS) were then incubated overnight at 4°C.

[0164] 5.4 After washing, add fluorescein-labeled secondary antibody diluted in PBS and incubate at room temperature for 1 hour.

[0165] 5.5 The cells were mounted with anti-fluorescence quenching mounting medium containing DAPI. Images were acquired using a microscope (DS-U3; Nikon, Tokyo, Japan).

[0166] Immunofluorescence staining results Figure 4 D, red: H3K14la; blue: DAPI (nucleus).

[0167] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. Application of lactylated histone H3 in the preparation of products for the treatment and / or prevention of pulmonary microvascular endothelial cell dysfunction.

2. The use according to claim 1, characterized in that: Lactated histone H3 is a histone protein that undergoes lactate modification at the 14th amino acid position.

3. The use according to claim 1, characterized in that: The pulmonary microvascular endothelial cell dysfunction is sepsis-mediated pulmonary microvascular endothelial cell dysfunction.

4. Application of lactylated histone H3 as a target for the treatment of pulmonary microvascular endothelial cell dysfunction.

5. The use according to claim 4, characterized in that: The target is a drug target.

6. Use of inhibitors of histone H3 lactylation modification in the preparation of products for the treatment and / or prevention of pulmonary microvascular endothelial cell dysfunction.

7. The use according to claim 6, characterized in that: The inhibitor is at least one of 2-deoxyglucose and sodium oxamate.

8. The use according to claim 6, characterized in that: The inhibitor includes at least one of a glycolysis inhibitor and a LDHA inhibitor.

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