Application of LECT2 / Tie1 / Src signal channel in preparation of medicine for preventing and treating endothelial cell injury

Abstract

The invention discloses an application of an LECT2 / Tie1 / Src signal channel in preparation of a medicine for preventing and treating endothelial cell injury. Research finds that LECT2 is combined with Tie1 to activate Src kinase to aggravate hypoxia and reoxygenation injury of vascular endothelial cells, so that liver ischemia reperfusion injury is aggravated. The Lect2 knocks down to relieve vascular endothelial cell injury, and the LECT2 recombinant protein aggravates vascular endothelial cell injury. Tie1 is knocked down, a TieIg3 segment recombinant protein inhibits LECT2 / Tie1 binding, and an Src inhibitor can relieve hypoxia injury of vascular endothelial cells. Liver ischemia reperfusion injury of the Lect2 knockout mouse is relieved, and vascular endothelial cell injury is relieved. The invention discloses a new mechanism of LECT2 / Tie1 / Src signal regulation and control of vascular endothelial cell hypoxia-reoxygenation injury, and provides a new target and strategy for treatment of liver ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of LECT2 / Tie1 / Src signaling pathway in the preparation of drugs for preventing and treating endothelial cell damage. Background Art

[0002] Ischemia-reperfusion (I / R)-induced liver injury is an important clinical problem after liver resection or transplantation. The important clinical features of liver I / R injury are failure of hepatic sinusoidal perfusion, leukocyte-endothelial cell interaction, loss of cell integrity and edema formation leading to microvascular dysfunction, including disturbance of vascular dilation and contraction balance, increased vascular permeability, inflammatory response of vascular endothelial cells, activation of coagulation and complement systems, etc.

[0003] At present, the regulatory mechanism of endothelial cell hypoxia-reoxygenation injury remains unclear. LECT2 plays a role in a variety of physiological activities and diseases, such as sepsis, diabetes, liver cancer, non-alcoholic fatty liver disease, and hematopoietic stem cell homeostasis. However, its functional regulation of endothelial cell function and whether it plays an important role in IRI are still unclear. Summary of the invention

[0004] The object of the present invention is to provide the use of a LECT2 / Tie1 / Src signaling pathway inhibitor in the preparation of a drug for preventing and / or treating endothelial cell damage.

[0005] The technical solution adopted by the present invention is:

[0006] A first aspect of the present invention provides a use of a LECT2 / Tie1 / Src signaling pathway inhibitor in the preparation of a medicament for preventing and / or treating endothelial cell damage.

[0007] Preferably, the factors causing endothelial cell damage include one or more of high glucose, hypoxia and / or reoxygenation, oxidative stress, ischemia, blood reperfusion, acidosis, severe infection, vascular disease, cardiovascular disease, and renin-angiotensin system.

[0008] Preferably, the vascular diseases include vasculitis, atherosclerosis, hypertension, lower limb artery occlusion, carotid artery stenosis, mesenteric artery stenosis, renal artery stenosis, polyarteritis, arterial dissection, arterial stenosis and occlusion, acute arterial thrombosis, aortic aneurysm, aortic dissection, visceral artery aneurysm, carotid artery aneurysm, lower limb artery aneurysm of various parts, venous reflux disorder, venous reflux disease, and vascular malformation.

[0009] Preferably, the cardiovascular diseases include valvular heart disease, arrhythmia, heart failure, congenital heart disease, ischemic heart disease, stroke, cardiomyopathy, rheumatic heart disease, hypertensive heart disease, endocarditis, arrhythmia, aortic disease, and peripheral arterial disease.

[0010] Preferably, the endothelial cells are vascular endothelial cells.

[0011] Preferably, the blood vessels include arterial vessels, venous vessels or capillaries.

[0012] Preferably, the LECT2 / Tie1 / Src signaling pathway inhibitor includes at least one of the following:

[0013] (1) an agent that inhibits gene expression or protein expression or protein activity of LECT2 and / or Tiel and / or Src;

[0014] (2) agents that inhibit the binding of LECT2 and Tiel;

[0015] (3) Agents that inhibit Src phosphorylation.

[0016] Preferably, the agent that inhibits gene expression or protein expression or protein activity of LECT2 and / or Tiel and / or Src includes a substance that inhibits LECT2 and / or Tiel and / or Src gene expression, silences or knocks out LECT2 and / or Tiel and / or Src genes, and / or a substance that inhibits the content and / or activity of LECT2 and / or Tiel and / or Src proteins.

[0017] Preferably, the agent for inhibiting gene expression or protein expression or protein activity of LECT2 and / or Tiel and / or Src comprises at least one of shRNA, siRNA, dsRNA, sgRNA, miRNA, cDNA, antisense RNA / DNA, small molecule compounds, peptides, and antibodies.

[0018] Preferably, the sequence of the siRNA that inhibits LECT2 is:

[0019] Lect2-siRNA: GGCAAGTCTTCCAATGAGA;

[0020] Preferably, the sequence of the siRNA that inhibits Tie1 is:

[0021] Tie1-siRNA:GCAACGGATCCTACTTCTA.

[0022] Preferably, the agent that inhibits the binding between LECT2 and Tiel includes at least one of Tie1 extracellular region fragment recombinant protein, Tie Ig3 segment recombinant protein, LECT2 fragment or full-length recombinant protein, and LECT2 blocking antibody.

[0023] Preferably, the agent that inhibits Src phosphorylation comprises Dasatinib.

[0024] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0025] Preferably, the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0026] Preferably, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

[0027] The beneficial effects of the present invention are:

[0028] The present study found that LECT2 regulates the oxidative stress response of vascular endothelial cells caused by hypoxia by binding to Tie1 and activating Src kinase, and plays an important role in liver ischemia-reperfusion injury; LECT2 aggravates the hypoxia-reoxygenation injury of vascular endothelial cells by binding to Tie1 and activating Src kinase, thereby aggravating liver ischemia-reperfusion injury. LECT2 knockdown reduces vascular endothelial cell injury, and LECT2 recombinant protein aggravates vascular endothelial cell injury. Tie1 knockdown, Tie Ig3 segment recombinant protein blocking LECT2 / Tie1 binding, and Src inhibitors can all reduce hypoxic injury to vascular endothelial cells. Lect2 gene knockout mice have reduced liver ischemia-reperfusion injury and reduced vascular endothelial cell injury. This study reveals a new mechanism by which LECT2 / Tie1 / Src signaling regulates hypoxia-reoxygenation injury of vascular endothelial cells, and provides new targets and strategies for the treatment of liver ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Knockout of LECT2 alleviates hypoxia-reoxygenation injury in EA.hy926 cells. Figure 1 Middle A: After Lect2 siRNA was transfected into EA.hy926 cells, the mRNA expression level of Lect2 gene was reduced. Figure 1 Middle B: CCK8 assay was used to indicate the cell proliferation capacity at different time points during reoxygenation after 8 h of hypoxia. Figure 1Middle C: After 8 h of hypoxia, SOD levels were measured at different time points during reoxygenation to indicate the level of cellular oxidative stress. Figure 1 Middle D: After 8 hours of hypoxia, LDH levels were measured at different time points during reoxygenation to indicate cytotoxicity. Data are representative of three independent experiments. All data are mean ± standard error. *p<0.05, **p<0.01, ****p<0.0001.

[0030] Figure 2 :rLECT2 aggravates hypoxia-reoxygenation injury in EA.hy926 cells. Figure 2 Middle A: CCK8 assay was used to indicate the cell proliferation capacity at different time points during reoxygenation after 8 h of hypoxia. Figure 2 Middle B: After 8 h of hypoxia, SOD levels were measured at different time points during reoxygenation to indicate the level of cellular oxidative stress. Figure 2 Middle C: After 8 hours of hypoxia, LDH levels were measured at different time points during reoxygenation to indicate cytotoxicity. Data are representative of three independent experiments. All data are mean ± standard error *p < 0.05.

[0031] Figure 3 :Tie1 knockout can reduce LECT2-induced hypoxia-reoxygenation injury in EA.hy926 cells. Figure 3 Middle A: After EA.hy926 cells were transfected with siTie1 RNA, the mRNA level of Tie1 gene was reduced. Figure 3 Middle B: CCK8 assay was used to indicate the cell proliferation ability of the siTie1 group and Tie1-siRNA+rLECT2 group at different time points during reoxygenation after 8 h of hypoxia. Figure 3 Middle C: During the reoxygenation process after 8 h of hypoxia, the SOD content of the siTie1 group and Tie1-siRNA+rLECT2 group was measured at different time points to indicate the level of cellular oxidative stress. Figure 3 Middle D: During reoxygenation after 8 hours of hypoxia, LDH release was measured at different time points in the siTie1 group and Tie1-siRNA+rLECT2 group to indicate cytotoxicity. Data are representative of three independent experiments. All data are mean ± standard error. *p<0.05, **p<0.01.

[0032] Figure 4 : Tie1-Ig3 blocks rLECT2-induced hypoxia-reoxygenation injury in EA.hy926 cells. Figure 4 Middle A: CCK8 assay was used to indicate the cell proliferation capacity at different time points during reoxygenation after 8 h of hypoxia; Figure 4Middle B: After 8 hours of hypoxia, SOD levels were measured at different time points during reoxygenation to indicate the level of cellular oxidative stress; Figure 4 Middle C: After 8 hours of hypoxia, LDH levels were measured at different time points during reoxygenation to indicate cytotoxicity. Data are representative of three independent experiments. All data are mean ± standard error. *p<0.05, **p<0.01.

[0033] Figure 5 :LECT2 / Tie1 signaling promotes Src phosphorylation. Figure 5 Middle A: After transfection with Lect2-siRNA, EA.hy926 cells were subjected to hypoxia-reoxygenation injury. Cell lysates were immunoblotted with the indicated antibodies. Figure 5 Middle B: rLECT2 was added to EA.hy926 cells, and cell lysates were immunoblotted with the indicated antibodies. Figure 5 Middle C: rLECT2 and rTie1-Ig3 were added simultaneously to EA.hy926 cells. Cell lysates were immunoblotted with the indicated antibodies. Data are representative of three independent experiments.

[0034] Figure 6 : Tie1-Ig3 blocks rLECT2-induced hypoxia-reoxygenation injury in EA.hy926 cells. Figure 6 Middle A: Inhibitory effect of Dasatinib on Src phosphorylation; Figure 6 Middle B: CCK8 assay was used to indicate the cell proliferation capacity at different time points during reoxygenation after 8 h of hypoxia. Figure 6 Middle C: After 8 h of hypoxia, SOD levels were measured at different time points during reoxygenation to indicate the level of cellular oxidative stress. Figure 6 Middle D: After 8 hours of hypoxia, LDH levels were measured at different time points during reoxygenation to indicate cytotoxicity. Data are representative of three independent experiments. All data are mean ± standard error. *p<0.05, **p<0.01.

[0035] Figure 7 For Lect2 - / - Mice showed reduced hepatic ischemia-reperfusion injury and sinusoidal endothelial cell damage. - / - Livers of mice and their littermate controls were clamped on the hepatic pedicle for 1 h and then reperfused. Serum and liver tissue samples were collected at different time points. Figure 7 Middle A: TUNEL staining of liver showed cell apoptosis; scale bar, 100 μm. Figure 7 Middle B: Cell apoptosis statistics. Figure 7 Middle C- Figure 7 Middle E: mRNA levels of IL-1β, IL-6, and TNF-β in the liver, respectively. Figure 7 Middle F: Liver LYVE1 staining shows apoptosis and staining statistics; scale bar, 100 μm. Data are representative of three independent experiments. n = 6 / group. Mean ± SEM. *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0037] Materials and Methods

[0038] EA.hy926 human umbilical vein cell fusion cells were maintained by our laboratory;

[0039] Small interfering RNA sequences:

[0040] Ctrl-siRNA:TTCTCGAACGTGTCACGT(SEQ ID NO.1);

[0041] Lect2-siRNA: GGCAAGTCTTCCAATGAGA (SEQ ID NO. 2);

[0042] Tiel-siRNA: GCAACGGATCCTACTTCTA (SEQ ID NO. 3).

[0043] Total SOD detection kit, TUNEL detection kit Bio-Tech Co., Ltd., total RNA extraction reagent AGRNAex Pro Aikerui Biotechnology Co., Ltd., reverse transcription kit Evo M-MLV, reverse transcription reagent premix, real-time quantitative PCR kit, Green Pro Taq HS Premixed qPCR Kit Acryl Biotech Co., Ltd.

[0044] Western Blot: β-actin antibody (1:10000, 20536) Wuhan Sanying Biotechnology Co., Ltd., secondary antibody goat anti-mouse IgG (1:10000, BA1050) goat anti-rabbit IgG (1:10000, BA1054) Boster Biotechnology Co., Ltd., anti-Src antibody (1:1000, 2109) anti-p-Src antibody (1:500, 2101), anti-mTOR antibody (1:1000, 2983), anti-p-mTOR antibody (1:1000, 5536), Cell Signaling Technology, Cell Counting Kit-8 (CCK-8) kit to detect cell viability GlpBio, USA.

[0045] Serum total SOD activity detection: CytoTox Non-Radioactive kit was used to detect SOD content, and Cytotoxicity Assay kit was used to detect LDH content, both from Promega;

[0046] TUNEL kit was used to detect cell apoptosis;

[0047] mouse:

[0048] Lect2 knockout mice (Lect2 - / - ) was generated by CRISPR / Cas9 strategy, and its genotype has been identified previously (see reference LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis). Briefly, the extracted genomic DNA was PCR amplified using specific primers (forward: 5'-GTGAGACTTAGATGTGGGAAGTTCCTG-3' (SEQ ID NO.4); reverse: 5'-CACCCTGAG GTATTCAGGCCATTAT-3' (SEQ ID NO.5)).

[0049] C57BL / 6 mice were obtained from Guangdong Medical Experimental Animal Center, Guangzhou, China.

[0050] Unless otherwise stated, all experiments were performed in male mice. The mice were housed under specific pathogen-free conditions with a standard 12-h light-dark cycle and had unlimited access to food and water. All mice used in the studies were healthy and immunocompetent. The animal study protocol complied with the U.S. Public Health Service policy on the use of laboratory animals and was approved by the Ethics Committee on the Care and Use of Animals of Southern Medical University.

[0051] To establish the liver ischemia-reperfusion model, 6-8 week old male mice were fasted for 12 h and anesthetized with isoflurane. A midline abdominal incision (3-5 cm) was made, and the intestines were gently moved aside to expose the portal vein. The liver hilum was separated from the surrounding tissue, and the portal triad (hepatic artery, portal vein, and bile duct) of the left and middle lobes was occluded using microvascular clamps. Successful clamping was confirmed by the whitening of the valve leaflets. After 60 min of ischemia, reperfusion was initiated by removing the clamps, resulting in an immediate color change in the middle and left lateral lobes. The incision was closed using a double-layer suture technique, and disinfectant was applied to the surgical area to reduce the risk of infection. The mice were then placed in heated, clean cages. Samples were collected at 0.5 h, 12 h, 24 h, and 48 h of reperfusion time. Recombinant human LECT2 (rLECT2) was prepared as described previously (for preparation methods, see reference LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis).

[0052] Histological analysis of liver necrosis by HE staining:

[0053] Liver tissues were collected at 0.5 h, 12 h, 24 h, and 48 h of reperfusion and then fixed in 10% neutral buffered formalin for 24 h. Tissues were processed, embedded in paraffin, and cut into 2.5 μm thick sections. Sections were stained with hematoxylin and eosin (HE) according to standard protocols. Stained sections were examined under a light microscope to assess liver morphology and determine necrotic areas. Necrosis was quantified using image analysis software, and the percentage of necrotic area relative to the total liver tissue area was calculated based on histological features such as loss of cell structure, eosinophilia, and cell debris.

[0054] Hypoxia-reoxygenation cell experiment:

[0055] When the cell density grows to 80%-90%, replace the full culture of the culture dish or well plate with an empty culture, and then put it into the anaerobic tank, tear open the anaerobic gas production bag packaging, take out the inner bag and put it into the card slot of the anaerobic tank, and put in the oxygen indicator, cover the lid of the anaerobic tank, fasten the buckle, and complete the airtightness. After 8 hours of hypoxia, open the lid of the anaerobic tank, take out the sample, replace the empty culture of the culture dish or well plate with the full culture, and continue to culture in the incubator, and then take 0h, 1h, 4h, and 8h cell samples in turn for the next test.

[0056] Cell Counting Kit-8 (CCK-8) cell viability assay:

[0057] After reoxygenation, discard the culture medium, add CCK8 detection reagent prepared in a ratio of 1:9 to cell culture medium in the dark, 100 μL per well, set up blank wells and control wells, place in a 37°C cell culture incubator and incubate for 1 hour. After the timing is up, use an enzyme reader to measure the absorbance at 450 nm.

[0058] CytoTox Non-Radioactive Cytotoxicity Assay (LDH) Cytotoxicity Assay:

[0059] Collect cells into 1.5mL EP tubes, centrifuge at 250g for 5 minutes at 4℃, take the supernatant as the sample to be tested, set up positive control and blank control, add 50μL of sample to each well and immediately add 50μL of reaction substrate solution, incubate for 25 minutes on a light-proof shaker, add 50μL of reaction stop solution to each well, incubate for 1 minute on a light-proof shaker, and measure the absorbance at 490nm using an enzyme reader.

[0060] Total SOD detection:

[0061] After washing the cells once with 4°C PBS buffer, add 200 μL of protein lysis buffer, thoroughly blow and lyse the cells and collect them in a 1.5 mL EP tube, add two steel beads, shake in a tissue grinder at 4°C for 10 seconds, centrifuge at 4°C 12000 rpm for 5 minutes, and take the supernatant as the sample to be tested.

[0062] Statistical analysis:

[0063] The data were analyzed using GraphPad Prism8 statistical software and appropriate statistical methods such as Student's t test or paired t test were used for statistical analysis. All data are expressed as mean ± sem, and p < 0.05 was considered statistically significant.

[0064] qPCR primers, the specific sequences are as follows:

[0065] GAPDH:

[0066] Forward:5'-AGAAGGTGGTGAAGCAGGCATC-3'(SEQ ID NO.6);

[0067] Reverse:5'-CGAAGGTGGAAGAGTGGGAGTTG-3'(SEQ ID NO.7);

[0068] Lect2:

[0069] Forward: 5'-GGACGTGTGACAGCTATGGC-3' (SEQ ID NO. 8);

[0070] Reverse:5'-TCCCAGTGAATGGTGCATACA-3'(SEQ ID NO.9);

[0071] Tie1:

[0072] Forward:5'-AATGCCGCGTATCGACTTCT-3'(SEQ ID NO.10);

[0073] Reverse: 5'-AATGCCGCGTATCGACTTCT-3' (SEQ ID NO. 11).

[0074] Example 1 LECT2 knockdown alleviates hypoxia-reoxygenation injury in EA.hy926 cells

[0075] Small interfering RNA (siRNA) was used to knock down the expression of Lect2 in EA.hy926 cells ( Figure 1 (A) After 8 h of hypoxic culture, the cells were sampled at multiple time points after reoxygenation to detect cell proliferation, oxidative stress, and cytotoxicity-related indicators.

[0076] The results showed that compared with the control group, Lect2 knockdown EA.hy926 cells showed stronger proliferation ability 1 h after reoxygenation ( Figure 1 Middle B);

[0077] Compared with the control group, the oxidative stress response of Lect2 knockdown EA.hy926 cells after reoxygenation was lower than that of the control group ( Figure 1 By detecting the release of cytotoxic LDH, it was found that the LDH release of EA.hy926 cells after Lect2 knockdown was significantly lower than that of the control group ( Figure 1 These results indicate that knockdown of Lect2 gene enhanced the ability of EA.hy926 cells to resist hypoxia-reoxygenation injury.

[0078] Example 2 LECT2 recombinant protein aggravates EA.hy926 cell hypoxia-reoxygenation injury

[0079] Using human LECT2 recombinant protein (rLECT2), three protein concentration gradients were set up and added to the cell culture medium immediately after hypoxia and reoxygenation to detect changes in various indicators at corresponding time points.

[0080] The results showed that after adding rLECT2 protein, the proliferation ability of EA.hy926 cells decreased ( Figure 2A), oxidative stress response is aggravated ( Figure 2 Middle B), cytotoxicity indexes were enhanced ( Figure 2 Middle C).

[0081] Example 3 Tie1 knockdown alleviates hypoxia-reoxygenation injury in EA.hy926 cells caused by LECT2 recombinant protein

[0082] The inventors' previous research results show that LECT2 is a functional ligand of receptor Tie1. In order to study whether LECT2 acts through Tie1, small interfering RNA (siRNA) was used to knock down the expression of Tie1 in EA.hy926 cells ( Figure 3 The results showed that Tie knockdown itself had no effect on EA.hy926 cell hypoxia-reoxygenation injury. However, rLECT2 significantly increased the hypoxia-reoxygenation injury of control EA.hy926 cells, but could not increase the hypoxia-reoxygenation injury of Tie knockdown EA.hy926 cells ( Figure 3 (in BD).

[0083] Example 4 Tie1 Ig3 segment recombinant protein blocks EA.hy926 cell hypoxia-reoxygenation injury caused by LECT2 recombinant protein

[0084] The inventors' previous studies found that LECT2 directly binds to the Tie1 Ig3 segment, and the Tie1 Ig3 segment recombinant protein (preparation method see reference LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis) can block the LECT2 / Tie1 binding and thus block the conduction of its downstream signaling pathway. The Tie1 Ig3 segment was used to detect changes in various indicators at corresponding time points.

[0085] The results showed that after adding Tie1 Ig3 segment recombinant protein, rLECT2 alleviated the hypoxia-reoxygenation injury of EA.hy926 cells ( Figure 4 (in AC).

[0086] Example 5 LECT2 / Tie1 promotes Src phosphorylation

[0087] In order to further study the mechanism by which LECT2 / Tie1 regulates hypoxia-reoxygenation injury in vascular endothelial cells, the activation of mTOR and Src kinases was detected.

[0088] The results showed that in the hypoxia-reoxygenation injury model of EA.hy926 cells, after Lect2 knockdown, the protein level and phosphorylation level of mTOR did not change; the protein level of Src did not change, but its phosphorylation level decreased ( Figure 5Middle A). After LECT2 recombinant protein treatment, the protein level and phosphorylation level of mTOR remained unchanged; the protein level of Src remained unchanged, but its phosphorylation level increased ( Figure 5 Middle B). Treatment with the recombinant protein of the Tie1 Ig3 segment reduced the phosphorylation level of Src ( Figure 5 Middle C).

[0089] Example 6 Src inhibitor alleviates EA.hy926 cell hypoxia-reoxygenation injury

[0090] To investigate the important role of Src kinase activation in LECT2 / Tie1 regulation of hypoxia-reoxygenation injury in EA.hy926 cells, the Src inhibitor Dasatinib was used.

[0091] The results showed that Dasatinib significantly inhibited Src phosphorylation ( Figure 6 The regulatory effects of LECT2 recombinant protein on the proliferation ability, oxidative stress level and cytotoxicity of EA.hy926 cells were blocked by Dasatinib ( Figure 6 This indicates that LECT2 regulates the hypoxia-reoxygenation injury of EA.hy926 cells by activating Src.

[0092] Example 7 LECT2 gene knockout mice have reduced liver ischemia-reperfusion injury and reduced liver sinusoidal endothelial cell damage

[0093] The liver ischemia-reperfusion injury model was established using LECT2 knockout mice. The results showed that compared with wild-type control mice in the same cage, the number of apoptotic liver cells in Lect2 knockout mice was reduced, and the expression of Il1b, Il6, and TNFa was reduced ( Figure 7 AE), showing milder levels of liver damage and inflammatory factors.

[0094] Normal hepatic sinusoidal endothelial cells highly express lymphatic endothelial cell hyaluronan receptor 1 (LYVE1), while injured hepatic sinusoidal endothelial cells lowly express or do not express LYVE1. - / - The expression of LYVE1 in endothelial cells of mice after ischemia-reperfusion was significantly higher than that in WT control mice ( Figure 7 F). Description Lect2 - / - The hepatic sinusoidal endothelial cells of mice were less damaged during ischemia-reperfusion.

[0095] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of LECT2 / Tie1 / Src signaling pathway inhibitors in the preparation of drugs for preventing and / or treating endothelial cell damage.

2. The use according to claim 1, characterized in that: The LECT2 / Tie1 / Src signaling pathway inhibitor includes at least one of the following; (1) an agent that inhibits gene expression or protein expression or protein activity of LECT2 and / or Tiel and / or Src; (2) agents that inhibit the binding of LECT2 and Tiel; (3) Agents that inhibit Src phosphorylation.

3. The use according to claim 2, characterized in that: The agent for inhibiting gene expression or protein expression or protein activity of LECT2 and / or Tiel and / or Src includes substances that inhibit LECT2 and / or Tiel and / or Src gene expression, silence or knock out LECT2 and / or Tiel and / or Src genes, and / or substances that inhibit LECT2 and / or Tiel and / or Src protein content and / or activity; Preferably, the agent for inhibiting gene expression or protein expression or protein activity of LECT2 and / or Tiel and / or Src comprises at least one of shRNA, siRNA, dsRNA, sgRNA, miRNA, cDNA, antisense RNA / DNA, small molecule compounds, peptides, and antibodies.

4. The use according to claim 3, characterized in that: The sequence of the siRNA that inhibits LECT2 is: Lect2-siRNA: GGCAAGTCTTCCAATGAGA.

5. The use according to claim 3, characterized in that: The sequence of the siRNA that inhibits Tie1 is: Tie1-siRNA: GCAACGGATCCTACTTCTA.

6. The use according to claim 2, characterized in that: The reagent for inhibiting the binding between LECT2 and Tiel comprises at least one of Tie1 extracellular region fragment recombinant protein, Tie Ig3 segment recombinant protein, LECT2 fragment or full-length recombinant protein, and LECT2 blocking antibody.

7. The use according to claim 2, characterized in that: The agents that inhibit Src phosphorylation include Dasatinib.

8. The use according to claim 1, characterized in that: The endothelial cells are vascular endothelial cells.

9. The use according to claim 1, characterized in that: The factors causing endothelial cell damage include one or more of high glucose, hypoxia and / or reoxygenation, oxidative stress, ischemia, blood reperfusion, acidosis, severe infection, vascular disease, cardiovascular disease, and renin-angiotensin system.

10. The use according to claim 1, characterized in that: The drug further comprises pharmaceutically acceptable excipients; preferably, the dosage form of the drug comprises at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, suppositories, enemas, aerosols, patches or drops; Preferably, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

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