Application of exosome in preparation of medicine for treating sepsis acute lung injury

Abstract

The invention belongs to the technical field of biological medicines, and provides application of an exosome in preparation of a medicine for treating sepsis acute lung injury aiming at the technical problem that a good medicine for treating sepsis acute lung injury cannot be obtained due to unclear pathogenesis of S-ALI at present. Researches show that endothelial progenitor cells transfer miR-218 to alveolar macrophages through exosomes, and the miR-218 is utilized to target HMGA1 genes, so that M1 polarization of the alveolar macrophages is inhibited, and then lung tissue injury, lung tissue leakage and lung tissue inflammatory response are inhibited to relieve sepsis acute lung injury. The invention proves that the functions of the miR-218 are not disclosed by the prior art in the development of the sepsis acute lung injury, reveals the pathological significance of the miR-218, provides a new thought and target for the prevention and treatment of the sepsis acute lung injury, and is beneficial to the development of a new medicine for treating the sepsis acute lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of exosomes in the preparation of a drug for treating sepsis-induced acute lung injury. Background Art

[0002] Sepsis-related acute lung injury (S-ALI) has become an important public health problem worldwide. However, the pathogenesis of S-ALI is not fully understood. S-ALI, known as acute respiratory distress syndrome (ARDS) caused by sepsis in humans, specifically refers to acute hypoxic respiratory failure caused by sepsis, secondary to alveolar damage caused by dysregulated inflammatory response. ARDS is characterized by diffuse interstitial and alveolar edema caused by damage to pulmonary capillary endothelial cells and alveolar epithelial cells, which can lead to acute hypoxic respiratory insufficiency or failure. In the decades since Ashbaugh first proposed ARDS in 1967, the diagnostic criteria for ARDS have been evolving and updating, and people's understanding of ARDS has been deepening. In 1994, the American-European Consensus Conference (AECC) proposed the diagnostic criteria for ARDS and they were widely accepted. In 2005, the Delphi criteria were proposed. In 2012, the Berlin Definition (diagnostic criteria) of ARDS modified and supplemented the previous diagnostic criteria for ARDS, and this version of the definition has the highest acceptance. Recently, the definition of ARDS has been updated again, and four major recommendations have been made: (1) including high-flow nasal oxygen with a minimum flow rate greater than or equal to 30 L / min; (2) using PaO2:FiO2≤300 mm Hg or blood oxygen saturation (blood oxygen saturation ≤97%) to detect hypoxemia; (3) retaining bilateral opacities as an imaging criterion, but adding ultrasound as an imaging modality, especially in resource-limited areas; (4) in resource-limited settings, positive end-expiratory pressure, oxygen flow, or specific respiratory support devices are not required. The incidence of ARDS cannot be ignored, and the mortality rate is high, especially in the ICU. An international, multicenter, prospective cohort study included 459 ICUs in 50 countries and collected data on 29,144 ICU patients, including 12,906 patients on mechanical ventilation, 3,022 of whom met the diagnostic criteria for ARDS. The incidence of ARDS in ICU patients exceeded 10%, and the incidence of ARDS in mechanically ventilated patients was 23.4%. The overall mortality rate of ARDS is 40%, and the mortality rate of severe ARDS is as high as 46.1%. Although the treatment options for ARDS are gradually improving, its overall mortality rate remains high, which may also be related to the pathogenesis of ARDS. Systemic inflammatory response syndrome (SIRS) caused by various injury factors (such as trauma and infection) is recognized as the core link and common pathway leading to ALI.The occurrence and development of ARDS are closely related to the body's over-activated inflammatory response, accompanied by the accumulation of a large number of pro-inflammatory cytokines and anti-inflammatory mediators. Pro-inflammatory cytokines can induce the activation of pulmonary vascular endothelial cells, and even lead to structural damage to the pulmonary vascular endothelium and biological metabolic dysfunction, thereby promoting the occurrence and development of ARDS. At present, the drugs that have been RCTed for ARDS include: (1) Interference with inflammation: corticosteroids, statins, vitamin D; (2) Antioxidant drugs: thiols, vitamin C, ulinastatin; (3) Drugs that stimulate fibrinolysis or block coagulation: aspirin, heparin, streptokinase; (4) Pulmonary vasodilators: inhaled NO, inhaled prostaglandins; (5) Prostaglandins (prostacyclin); (6) Prostaglandins (prostaglandin E1); (7) Fluid management: albumin replacement, β2-AR agonists; (8) Surfactant replacement therapy; (9) Neutrophil elastase inhibitors. Although multiple studies have identified various targets that, when pharmacologically or interventionally altered, can mitigate various pathological features of sepsis and ARDS experimentally (in vitro and in vivo) and in phase I and II clinical trials, no study has produced an effective treatment in a phase III randomized clinical trial. Therefore, the treatment of S-ALI still faces great challenges, and new treatments are urgently needed. Exosomes are tiny vesicles with a diameter of 30 to 200 nm secreted by cells. Exosomes promote intercellular communication by transferring functional proteins, lipids, and nucleic acid biomolecules between cells. Studies have found that almost all types of cells, including blood cells, immune cells, tumor cells, stem cells, etc., can produce exosomes for intercellular communication, thereby achieving a variety of biological functions such as cell proliferation, differentiation, immunoregulation, inflammation regulation, and cell apoptosis. As a potential drug delivery system, exosomes are similar to liposomes, with a unique bilipid membrane that wraps a hydrophilic core and can be loaded with hydrophilic and lipophilic materials. Because exosomes have different lipids, surface proteins, receptors, etc., they have better tissue and cell targeting. In addition, exosomes are produced by cells themselves, have good biocompatibility, low immunogenicity and low toxicity, and can prevent exogenous proteins, nucleic acids and other drugs from entering the body and being recognized and eliminated by the immune system. Although exosomes have the above advantages, there is no relevant research on whether exosomes can alleviate S-ALI and its corresponding relief mechanism. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art, provide an application of exosomes in the preparation of a drug for treating sepsis-induced acute lung injury, and explore the possibility that exosomes secreted by endothelial progenitor cells (EPCs) can alleviate S-ALI, and explore its possible mechanism.

[0004] The first object of the present invention is to provide an application of exosomes in the preparation of a drug for treating acute lung injury caused by sepsis, wherein the exosomes are obtained by secretion of endothelial progenitor cells.

[0005] Preferably, exosomes inhibit sepsis-induced acute lung injury by inhibiting lung tissue damage, lung tissue leakage, and inflammatory response of lung tissue.

[0006] Preferably, exosomes inhibit sepsis-induced acute lung injury by inhibiting lung tissue damage, lung tissue leakage and inflammatory response of lung tissue through miR-218.

[0007] Preferably, exosomes regulate the polarization of alveolar macrophages through miR-218, thereby inhibiting lung tissue damage, lung tissue leakage and inflammatory response of lung tissue.

[0008] Preferably, miR-218 inhibits lung tissue damage, lung tissue leakage and inflammatory response of lung tissue by promoting M2 macrophage polarization in alveolar macrophages and inhibiting M1 macrophage polarization in alveolar macrophages.

[0009] Preferably, miR-218 inhibits the polarization of M1 macrophages in alveolar macrophages and promotes the polarization of M2 macrophages in alveolar macrophages by inhibiting the protein expression level of the HMGA1 gene.

[0010] The second object of the present invention is to provide a drug for treating acute lung injury caused by sepsis, comprising exosomes obtained by secretion of endothelial progenitor cells.

[0011] Preferably, the exosomes are dissolved in physiological saline for injection to prepare an injection.

[0012] Preferably, the exosomes are administered by intravenous injection.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention shows that endothelial progenitor cells transfer miR-218 to alveolar macrophages through exosomes, and use miR-218 to target the HMGA1 gene, thereby inhibiting the M1 polarization of alveolar macrophages, thereby inhibiting lung tissue damage, lung tissue leakage and inflammatory response of lung tissue to alleviate sepsis acute lung injury. The present invention proves the function of miR-218 in the development of sepsis acute lung injury that has not been disclosed by the prior art, reveals its pathological significance, provides new ideas and targets for the prevention and treatment of sepsis acute lung injury, and helps to develop new drugs for the treatment of sepsis acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of in vitro uptake and distribution of exosomes secreted by EPCs are shown in Figure 2.

[0016] in, Figure 1 a is a graph showing the results of cell adhesion and differentiation; Figure 1 b is the result diagram of EPCs identification; Figure 1 c is a flow chart of ultracentrifugation method for extracting exosomes; Figure 1 d is the transmission electron microscopy image of exosomes; Figure 1 e is the particle size and concentration diagram of exosomes; Figure 1 f is the expression graph of exosome-specific positive and negative proteins; Figure 1 g is the expression level of miR-218 in exosomes secreted by EPCs;

[0017] Figure 2 This is the tissue distribution map of exosomes secreted by EPCs in vivo;

[0018] Figure 3 The expression diagram of exosomes secreted by EPCs in the lung;

[0019] Figure 4 The following are the results of survival rate, lung tissue HE staining and scoring, dry-wet ratio, Evans blue staining, BALF total protein and inflammatory factors in each group of mice;

[0020] in, Figure 4 a is the survival rate graph; Figure 4 b is the dry-wet ratio; Figure 4 c and Figure 4 d is the HE staining and scoring of lung tissue; Figure 4 e is Evans blue staining; Figure 4 f is BALF total protein; Figure 4 g is an inflammatory factor;

[0021] Figure 5 The figure shows the expression level of miR-218 in the lung tissues of mice in each group;

[0022] Figure 6 The following are the results of survival rate, lung tissue HE staining and scoring, dry-wet ratio, Evans blue staining, BALF total protein and inflammatory factors in each group of mice;

[0023] in, Figure 6 a is the survival rate graph; Figure 6 b is the dry-wet ratio; Figure 6 c and Figure 6 d is the HE staining and scoring of lung tissue; Figure 6 e is Evans blue staining; Figure 6 f is BALF total protein; Figure 6 g is an inflammatory factor;

[0024] Figure 7The results of the graph show the effect of miR-218 in exosomes secreted by EPCs on the polarization regulation of alveolar macrophages in the lung tissue of CLP-induced S-ALI mice;

[0025] in, Figure 7 a is the immunofluorescence image of M1 polarization of macrophages in lung tissues of each group; Figure 7 b is the percentage of M1 polarized macrophages in lung tissues of each group; Figure 7 c is the immunofluorescence image of M2 polarization of macrophages in lung tissues of each group; Figure 7 d is the percentage of M2 polarized macrophages in lung tissue of each group;

[0026] Figure 8 The results of the effect of exosomes secreted by EPCs on the M1 polarization of macrophages induced by LPS are shown in Figure 2.

[0027] in, Figure 8 a is a confocal microscopy image of AMs; Figure 8 b is the flow cytometric expression of AMs; Figure 8 c is the protein expression of AMs; Figure 8 d, RNA expression of AMs; Figure 8 e is the expression of inflammatory factors in AMs;

[0028] Fig. 9 The figure shows the effect of miR-218 in exosomes secreted by EPCs on the polarization regulation of macrophages induced by LPS;

[0029] in, Fig. 9 a is the protein expression of AMs; Fig. 9 b is the flow cytometric expression of AMs; Fig. 9 c is the RNA expression of AMs; Fig. 9 d is the expression of inflammatory factors in AMs;

[0030] Fig.10 This is a diagram of the validation results of the effects of EPCs exosomes in vitro;

[0031] in, Fig.10 a is a schematic diagram of the co-culture of EPCs and AMs; Fig.10 b shows the protein expression of AMs; Fig.10 c is the flow cytometric expression of AMs; Fig.10 d, RNA expression of AMs; Fig.10 e is the expression of inflammatory factors in AMs;

[0032] Fig.11 The polarization level detection results of each group of cells are shown in the figure;

[0033] in, Fig.11 a is the protein expression of AMs; Fig.11 b is the flow cytometric expression of AMs; Fig.11 c is the RNA expression of AMs; Fig.11 d is the expression of inflammatory factors in AMs;

[0034] Fig.12 This is a diagram showing the validation results of miR-218 targeting HMGA1;

[0035] in, Fig.12 a shows the bioinformatics analysis results of miR-218 and HMGA1; Fig.12 b is the relative activity result of dual luciferase of AMs; Fig.12 c is the relative activity result of dual luciferase of 293T; Fig.12 d is the result of the detection of HMGA1 protein level in AMs; Fig.12 e is a bar graph of HMGA1 protein levels in AMs;

[0036] Fig.13 The polarization level detection results of each group of cells are shown in the figure;

[0037] in, Fig.13 a is the protein expression of AMs; Fig.13 b is the flow cytometric expression of AMs; Fig.13 c is the RNA expression of AMs; Fig.13 d is the expression of inflammatory factors in AMs;

[0038] Fig.14 The polarization level detection results of each group of cells are shown in the figure;

[0039] in, Fig.14 a is the protein expression of AMs; Fig.14 b is the flow cytometric expression of AMs; Fig.14 c is the RNA expression of AMs; Fig.14 d Expression of inflammatory factors in AMs. DETAILED DESCRIPTION

[0040] The following is a description of the embodiments of the present invention. Figures 1 to 14 , the technical solutions in the embodiments of the present invention are clearly and completely described. 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, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] An embodiment of the present invention provides an application of exosomes in preparing a drug for treating acute lung injury caused by sepsis, wherein the exosomes are obtained by secretion of endothelial progenitor cells.

[0042] In the embodiments of the present invention, exosomes inhibit sepsis-induced acute lung injury by inhibiting lung tissue damage, lung tissue leakage, and inflammatory response of lung tissue.

[0043] In the embodiment of the present invention, exosomes inhibit sepsis-induced acute lung injury by inhibiting lung tissue damage, lung tissue leakage and inflammatory response of lung tissue through miR-218.

[0044] In the embodiments of the present invention, exosomes regulate the polarization of alveolar macrophages through miR-218, thereby inhibiting lung tissue damage, lung tissue leakage and inflammatory response of lung tissue.

[0045] In the embodiments of the present invention, miR-218 inhibits lung tissue damage, lung tissue leakage and inflammatory response of lung tissue by promoting polarization of M2 macrophages in alveolar macrophages and inhibiting polarization of M1 macrophages in alveolar macrophages.

[0046] In the embodiment of the present invention, miR-218 inhibits the polarization of M1 macrophages in alveolar macrophages and promotes the polarization of M2 macrophages in alveolar macrophages by inhibiting the protein expression level of HMGA1 gene.

[0047] An embodiment of the present invention also provides a drug for treating acute lung injury caused by sepsis, comprising exosomes obtained by secretion of endothelial progenitor cells.

[0048] Specifically, the exosomes are dissolved in physiological saline for injection to prepare an injection.

[0049] Specifically, the exosomes are administered by intravenous injection.

[0050] To facilitate understanding of the present invention, the present invention is further explained below with reference to specific embodiments in conjunction with the accompanying drawings, and the specific embodiments do not constitute a limitation on the embodiments of the present invention.

[0051] 1. Test methods

[0052] 1. Isolate exosomes from EPCs of C57BL / 6J mice by ultracentrifugation. Verify the uptake and distribution of exosomes in vitro and in vivo.

[0053] 2. Use miR-218inhibitor and miR-218mimic to study the function of exosomes in mice and their regulation on the polarization of alveolar macrophages (AMs). Use bioinformatics and dual luciferase reporter gene assays to explore the downstream targets of miR-218;

[0054] 3. Use si-HMGA1 and oe-HMGA1 to study the function of exosomes in regulating the polarization of AMs;

[0055] 4. In AMs, functional restoration experiments were performed to reveal the interaction between exosomal miRNA-218 and HMGA1.

[0056] 2. Test results

[0057] 1. Uptake and distribution of exosomes secreted by EPCs in vitro and in vivo

[0058] 1.1. Uptake and distribution of exosomes secreted by EPCs in vitro

[0059] In order to observe the difference between peripheral blood endothelial progenitor cells (EPCs) of the control group and patients with sepsis ARDS, the present invention firstly extracted peripheral blood EPCs of the above two groups, and the cells adhered and differentiated ( Figure 1 a) Double staining with Dil-Ac-LDL and FITC-UEA-I was used to identify EPCs. The results are shown in Figure 1 b. Figure 1 c Flowchart, exosomes were extracted by ultracentrifugation. The extracted exosomes were identified by transmission electron microscopy, and the results were shown in Figure 1 d. The extracted exosomes were analyzed for particle size and concentration using nanoparticles. The results are shown in Figure 1 e. The extracted exosomes were identified by WB for specific positive and negative proteins, and the results were shown in Figure 1 f. Figure 1 g It can be seen that compared with the control group, the expression level of miR-218 in the exosomes secreted by EPCs in the sepsis ARDS group was significantly reduced. The above results suggest that miR-218 in the exosomes secreted by EPCs may be involved in the occurrence and development of acute lung injury in sepsis.

[0060] 1.2 In vivo tracing of exosomes secreted by EPCs

[0061] In order to clarify the role of exosomes secreted by EPCs, it is crucial to first determine the distribution of EPCs exosomes in vivo. The present invention extracts EPCs from the bone marrow of C57BL / 6J mice, then cultures and identifies the extracted EPCs, and extracts exosomes by ultracentrifugation. The exosomes secreted by EPCs are labeled with DiR, and the labeled exosomes are injected into the tail vein of C57BL / 6J mice (6 weeks old, n=3), and the biodistribution of exosomes in vivo is evaluated at 0, 1, 6 and 24h after injection to determine the tissue distribution of exosomes. The results are shown in Figure 2. Figure 2 As shown. 24 hours later, the lung tissue of the mice was taken for imaging examination, and the results were as follows Figure 3 As shown. Figure 2The results showed that 1 hour after DiR-labeled exosomes were injected into the tail vein, high signals appeared immediately in the chest and abdomen of mice, and then gradually increased. Figure 3 showed that exosomes secreted from EPCs were highly expressed in the lung.

[0062] 2. Study on the mechanism of the effect of exosomes secreted by EPCs on acute lung injury induced by CLP in S-ALI mice

[0063] 2.1 Effects of exosomes secreted by EPCs on S-ALI

[0064] In order to verify the effect of EPCs exosomes on S-ALI, the mice in the sham operation group were used as the control, and a sepsis mouse model was established by CLP to obtain the CLP group, and then the CLP+PBS group and CLP+exo group were established, and the survival rate, lung tissue HE staining and score, dry-wet ratio, Evans blue staining, BALF total protein and inflammatory factors of the mice in the above groups were detected.

[0065] The CLP sepsis mouse model (CLP group) was established as follows:

[0066] (1) About 200 μL of 1% sodium pentobarbital was injected intraperitoneally into 6-week-old C57BL / 6J male mice to anesthetize the mice; (2) The skin of the middle abdomen was prepared; (3) About 1 cm of the abdominal cavity was exposed; (4) The cecum with thicker and thicker intestinal wall was found and exposed; (5) The feces in the mouse cecum was squeezed to the distal end of the cecum; (6) The base of the cecum and the distal end of the cecum were tied together at 1 / 2 of the length of the long axis with sutures, and then the lower cecum was pierced with a 22G needle to squeeze out feces about the size of a grain of rice; (7) The cecum was returned to the abdominal cavity; (8) The abdominal wall muscles and skin were sutured intermittently; (9) About 12 hours after the operation, the mice developed clinical symptoms related to sepsis, including fever, chills, hair erection, general weakness and decreased activity, thus obtaining the CLP sepsis mouse model.

[0067] Sham group: 6-week-old C57BL / 6J male mice were subjected to laparotomy in the same manner as the above-mentioned method for establishing the CLP sepsis mouse model, and then the laparotomy was immediately sutured without cecal ligation and puncture.

[0068] CLP+PBS group: PBS was intravenously injected into the mice of the CLP group, with 100 ul of PBS injected into each mouse.

[0069] CLP+exo group: Exosomes secreted by EPCs were intravenously injected into the mice of the CLP group, with 50ug (100ul) of exosomes injected into each mouse.

[0070] The survival rate, lung tissue HE staining and score, dry-wet ratio, Evans blue staining, BALF total protein and inflammatory factors of the mice in the Sham group, CLP group, CLP+PBS group and CLP+exo group were tested respectively. The test results are shown in Figure 4 As shown, through Figure 4 a It can be seen that compared with the CLP group, the mortality rate of mice in the CLP+exo group was significantly reduced, indicating that the exosomes secreted by EPCs have a protective effect on CLP-induced septic mice. Figure 4 b As can be seen, compared with the CLP group, the lung dry-wet ratio of mice in the CLP+exo group was significantly reduced, indicating that the exosomes secreted by EPCs alleviated the lung tissue leakage of CLP-induced S-ALI mice. Figure 4 As can be seen from the figure, compared with the CLP group, the infiltration of inflammatory cells in the lung tissue of mice in the CLP+exo group was significantly reduced, indicating that the exosomes secreted by EPCs alleviated the lung tissue damage induced by CLP in S-ALI mice. Figure 4 e It can be seen that compared with the CLP group, the Evans blue leakage of the lung tissue of mice in the CLP+exo group was significantly reduced, indicating that the exosomes secreted by EPCs alleviated the leakage of lung tissue in CLP-induced S-ALI mice. Figure 4 f As can be seen, compared with the CLP group, the total protein in the bronchoalveolar lavage fluid of the CLP+exo group was significantly reduced, indicating that the exosomes secreted by EPCs improved the lung tissue leakage of CLP-induced S-ALI mice. Figure 4 g It can be seen that compared with the CLP group, the levels of IL-6, TNF-α, and IL-1β in the bronchoalveolar lavage fluid of mice in the CLP+exo group were significantly decreased, indicating that EPCs exosomes alleviated the inflammatory response in the lung tissue of S-ALI mice induced by CLP.

[0071] 2.2 Effects of miR-218 on lung tissue of S-ALI mice

[0072] A control group (n=10), an LPS group (n=10) and a CLP group (n=10) were established, and the expression level of miR-218 in the lung tissues of each group of mice was detected.

[0073] Control group: 6-week-old C57BL / 6J male mice, without any treatment;

[0074] LPS group: LPS was directly injected into 6-week-old C57BL / 6J male mice via the peritoneal cavity, and the injection volume for each mouse was 10 mg / kg;

[0075] CLP group: The establishment method is the same as the above CLP group.

[0076] The results of the detection of miR-218 expression levels in the lung tissues of mice in the control group, LPS group, and CLP group are shown in Figure 5 As shown. Figure 5 The results showed that compared with the control group, the expression level of miR-218 in the lung tissue of mice in the LPS group and CLP group was significantly decreased, suggesting that miR-218 may be involved in the occurrence and development of S-ALI.

[0077] 2.3 Effects of miR-218 in exosomes secreted by EPCs on acute lung injury and pulmonary vascular permeability in CLP-induced S-ALI mice

[0078] The present invention verifies the effect of miR-218 in exosomes secreted by EPCs on S-ALI by using Sham group, CLP group, CLP+miR-218NC group, CLP+miR-218inhibitor group and CLP+miR-218mimic group of mice. The Sham group and CLP group in this part are the same as the Sham group and CLP group above.

[0079] CLP+miR-218NC group: exosomes secreted by EPCs miR-218 were intravenously injected into mice in the CLP group, with 50ug (100ul) of exosomes injected into each mouse;

[0080] CLP+miR-218inhibitor group: EPCs were transfected with miR-218inhibitor, and then the exosomes of miR-218inhibitor were collected and injected intravenously into the mice in the CLP group, with each mouse injected with 50ug (100ul) of exosomes;

[0081] CLP+miR-218mimic group: EPCs were transfected with miR-218mimic, and then the exosomes of miR-218mimic were collected and injected intravenously into the mice in the CLP group, with 50ug (100ul) of exosomes injected into each mouse;

[0082] The survival rate, lung tissue HE staining and score, dry-wet ratio, Evans blue staining, BALF total protein and inflammatory factors of the mice in the sham group, CLP group, CLP+miR-218NC group, CLP+miR-218inhibitor group and CLP+miR-218mimic group were detected respectively. The test results are as follows: Figure 6 As shown. Figure 6aThe results showed that compared with the CLP group, the mortality rates of mice in the CLP+miR-218NC group, CLP+miR-218inhibitor group, and CLP+miR-218mimic group were significantly reduced, suggesting that miR-218 in the exosomes secreted by EPCs has a protective effect on CLP-induced septic mice. Figure 6 b It can be seen that compared with the Sham group, the lung dry-wet ratio of mice in the CLP+miR-218NC group was significantly reduced; compared with the CLP+miR-218NC group, the lung dry-wet ratio of mice in the CLP+miR-218inhibitor group was significantly increased; compared with the CLP+miR-218NC group, the lung dry-wet ratio of mice in the CLP+miR-218mimic group was significantly reduced. Figure 6 c It can be seen that miR-218 in the exosomes secreted by EPCs alleviated lung tissue leakage in CLP-induced S-ALI mice. Figure 6 d It can be seen that compared with the Sham group, the inflammatory cell infiltration in the lung tissue of mice in the CLP+miR-218NC group was significantly reduced. Compared with the CLP+miR-218NC group, the inflammatory cell infiltration in the lung tissue of mice in the CLP+miR-218inhibitor group was significantly increased. Compared with the CLP+miR-218NC group, the inflammatory cell infiltration in the lung tissue of mice in the CLP+miR-218mimic group was significantly reduced. The above results indicate that miR-218 in the exosomes secreted by EPCs can alleviate CLP-induced lung tissue damage in S-ALI mice. Figure 6 e It can be seen that compared with the Sham group, the Evans blue leakage in the lung tissue of mice in the CLP+miR-218NC group was significantly reduced; compared with the CLP+miR-218NC group, the Evans blue leakage in the lung tissue of mice in the CLP+miR-218inhibitor group was significantly increased; compared with the CLP+miR-218NC group, the Evans blue leakage in the lung tissue of mice in the CLP+miR-218mimic group was significantly reduced. The above results indicate that miR-218 in the exosomes secreted by EPCs reduces the leakage of lung tissue in CLP-induced S-ALI mice. Figure 6 fIt can be seen that compared with the Sham group, the total protein content of bronchoalveolar lavage fluid of mice in the CLP+miR-218NC group was significantly decreased; compared with the CLP+miR-218NC group, the total protein content of bronchoalveolar lavage fluid of mice in the CLP+miR-218inhibitor group was significantly increased; compared with the CLP+miR-218NC group, the total protein content of bronchoalveolar lavage fluid of mice in the CLP+miR-218mimic group was significantly decreased. The above results indicate that miR-218 in the exosomes secreted by EPCs improves lung tissue leakage in CLP-induced S-ALI mice. Figure 6g It can be seen that compared with the Sham group, the levels of IL-6, TNF-α, and IL-1β in the bronchoalveolar lavage fluid of mice in the CLP+miR-218NC group were significantly decreased; compared with the CLP+miR-218NC group, the levels of IL-6, TNF-α, and IL-1β in the bronchoalveolar lavage fluid of mice in the CLP+miR-218inhibitor group were significantly increased; compared with the CLP+miR-218NC group, the levels of IL-6, TNF-α, and IL-1β in the bronchoalveolar lavage fluid of mice in the CLP+miR-218mimic group were significantly decreased. The above results indicate that miR-218 in the exosomes secreted by EPCs alleviates the inflammatory response of the lung tissue of CLP-induced S-ALI mice.

[0083] 2.4 Effect of miR-218 in exosomes secreted by EPCs on polarization regulation of alveolar macrophages in lung tissue of CLP-induced S-ALI mice

[0084] pass Figure 7 As shown in ad, in the CLP group, compared with the Sham group, the positive rate of F4 / 80+iNOS double staining in the lung tissue of the mice in the CLP group was significantly increased, but there was no difference in the positive rate of F4 / 80+Arg1 double staining; compared with the CLP group, the positive rate of F4 / 80+iNOS in the lung tissue of the mice in the CLP+miR-218NC group was significantly decreased, and the positive rate of F4 / 80+Arg1 double staining was significantly increased; compared with the CLP+miR-218NC group, the positive rate of F4 / 80+iNOS double staining in the lung tissue of the mice in the CLP+miR-218inhibitor group was significantly increased, and the positive rate of F4 / 80+Arg1 was significantly decreased; compared with the CLP+miR-218NC group, the positive rate of F4 / 80+iNOS double staining in the lung tissue of the mice in the CLP+miR-218mimic group was significantly decreased, and the positive rates of F4 / 80+Arg1 were significantly increased and decreased. These results suggest that miR-218 in exosomes secreted by EPCs reduces the M1 polarization of macrophages in the lung tissue of CLP-induced S-ALI mice and increases the M2 polarization of macrophages in the lung tissue of CLP-induced S-ALI mice. In summary, miR-218 in exosomes secreted by EPCs alleviates S-ALI, and its mechanism of action involves the polarization of AMs.

[0085] 3. Study on the mechanism of the effect of exosomes secreted by EPCs on LPS-induced acute lung injury in S-ALI mice

[0086] 3.1 Effects of exosomes secreted by EPCs on M1 polarization of LPS-induced macrophages

[0087] Exosomes secreted by EPCs were incubated with AMs for 24 h, labeled with Dil (red), cell nuclei with DAPI (blue), and cell membranes with phalloidin (green), and confocal microscopy images were taken as shown in Figure 2 Figure 8 As shown in a, through Figure 8 a clearly shows that AMs can engulf multiple EPCs exosomes.

[0088] The present invention verifies the effect of exosomes secreted by EPCs on M1 polarization of LPS-induced macrophages through a control group (control group), an LPS group, an LPS+PBS group, and an LPS+exo group.

[0089] Control group: AMs without any treatment;

[0090] LPS group: AMs were intervened with 100 ng / mL LPS for 24 hours.

[0091] LPS+PBS group: AMs were intervened with PBS (same volume as exosomes) 24 h in advance, and then 100 ng / mL LPS was used for intervention for 24 h.

[0092] LPS+exo group: EPCs exosomes (100 particles / cell) were used to intervene in AMs 24 hours in advance. After the intervention, 100 ng / mL LPS was used to intervene for 24 hours.

[0093] The polarization levels of cells in the control group, LPS group, LPS+PBS group and LPS+exo group were detected respectively. The test results are shown in Figure 8 b- Figure 8 As shown in e, through Figure 8 b It can be seen that compared with the control group, the iNOS expression level in the LPS group was significantly increased, and there was no significant difference in the CD206 expression level; compared with the LPS group, there was no significant difference in the iNOS and CD206 expression levels in the LPS+PBS group; compared with the LPS group, the iNOS expression level in the LPS+exo group was significantly decreased, and the CD206 expression level was significantly increased. Figure 8 c It can be seen that compared with the control group, the expression levels of iNOS and CD86 proteins in the LPS group were significantly increased, and there was no significant difference in the expression levels of CD206 and Arg1 proteins; compared with the LPS group, there was no significant difference in the expression levels of iNOS, CD86, CD206 and Arg1 proteins in the LPS+PBS group; compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+exo group were significantly decreased, and the expression levels of CD206 and Arg1 proteins in the LPS+exo group were significantly increased. Figure 8d It can be seen that compared with the control group, the expression levels of iNOS and CD86 mRNA in the LPS group were significantly increased, and there was no significant difference in the expression levels of CD206 and Arg1 mRNA; compared with the LPS group, there was no significant difference in the expression levels of iNOS, CD86, CD206 and Arg1 mRNA in the LPS+PBS group; compared with the LPS group, the expression levels of iNOS and CD86 mRNA in the LPS+exo group were significantly decreased, while the expression levels of CD206 and Arg1 mRNA were significantly increased. Compared with the control group, the expression levels of IL-1α, IL-1β, IL-6 and TNF-α in the cells of the LPS group were significantly increased. Figure 8 e It can be seen that compared with the LPS group, there was no significant difference in the expression levels of IL-1α, IL-1β, IL-6 and TNF-α in the LPS+PBS group; compared with the LPS group, the expression levels of IL-1α, IL-1β, IL-6 and TNF-α in the LPS+exo group were significantly reduced. The above results show that the exosomes secreted by EPCs can be phagocytosed by AMs, thereby reducing the M1 polarization of AMs under LPS stimulation and increasing the M2 polarization.

[0094] 3.2 Effect of miR-218 in exosomes secreted by EPCs on LPS-induced polarization of macrophages

[0095] The present invention verifies the role of miR-218 in the exosomes secreted by EPCs in vitro through a control group (control group), an LPS group, an LPS+miR-218NC group, an LPS+miR-218inhibitor group, and an LPS+miR-218mimic group.

[0096] Control group: AMs without any treatment;

[0097] LPS group: AMs were intervened with 100 ng / mL LPS for 24 h;

[0098] LPS+miR-218NC group: EPCs exosomes (100 particles / cell) were used to intervene AMs 24 h in advance, and after the intervention, 100 ng / mL LPS was used to intervene for 24 h;

[0099] LPS+miR-218inhibitor group: EPCs were transfected with miR-218inhibitor, and then the exosomes of miR-218inhibitor were collected and used to intervene in AMs 24 h in advance. After the intervention, 100 ng / mL LPS was used for intervention for 24 h;

[0100] LPS+miR-218mimic group: EPCs were transfected with miR-218mimic, and then the exosomes of miR-218mimic were collected. The collected exosomes were used to intervene in AMs 24 hours in advance. After the intervention, 100 ng / mL LPS was used to intervene for 24 hours.

[0101] The polarization levels of cells in the control group, LPS group, LPS+miR-218NC group, LPS+miR-218inhibitor group, and LPS+miR-218mimic group were detected. The test results are shown in Fig. 9 As shown, through Fig. 9 aIt can be seen that compared with the control group, the expression levels of iNOS and CD86 proteins in the LPS group were significantly increased, and there was no significant difference in the expression levels of CD206 and Arg1 proteins; compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218NC group were significantly decreased, and the expression levels of CD206 and Arg1 proteins were significantly increased; compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218inhibitor group were significantly increased, and the expression levels of CD206 and Arg1 proteins were significantly decreased; compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218mimic group were significantly decreased, and the expression levels of CD206 and Arg1 proteins were significantly increased. Fig. 9 b It can be seen that compared with the control group, the iNOS expression level in the LPS group was significantly increased, and there was no significant difference in the CD206 expression level. Compared with the LPS group, the iNOS expression level in the LPS+miR-218NC group was significantly decreased, and the CD206 expression level was significantly increased. Compared with the LPS+miR-218NC group, the iNOS expression level in the LPS+miR-218inhibitor group was significantly increased, and the CD206 expression level was significantly decreased. Compared with the LPS+miR-218NC group, the iNOS expression level in the LPS+miR-218mimic group was significantly decreased, and the CD206 expression level was significantly increased. Fig. 9c It can be seen that compared with the control group, the expression levels of iNOS and CD86 mRNA in the LPS group were significantly increased, and there was no significant difference in the expression levels of CD206 and Arg1 mRNA. Compared with the LPS group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218NC group were significantly decreased, and the expression levels of CD206 and Arg1 mRNA were significantly increased. Compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218inhibitor group were significantly increased, and the expression levels of CD206 and Arg1 mRNA were significantly decreased. Compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218mimic group were significantly decreased, and the expression levels of CD206 and Arg1 mRNA in the LPS+miR-218mimic group were significantly increased. Compared with the control group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the cells of the LPS group were significantly increased. Fig. 9 d It can be seen that compared with the LPS group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218NC group were significantly reduced; compared with the LPS+miR-218NC group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218inhibitor group were significantly increased; compared with the LPS+miR-218NC group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218mimic group were significantly reduced. The above experiments confirmed that exosomes secreted by EPCs can regulate LPS-induced AMs polarization through miR-218.

[0102] GW4869 inhibits EPCs exosomes from regulating AMs polarization via miR-218

[0103] GW4869 is a neutral sphingomyelinase inhibitor and the most widely used inhibitor in exosome production. The above experimental results show that EPCs exosomes can regulate the polarization of AMs through miR-218. In order to further verify the role of EPCs exosomes in vitro, the present invention established a transwell co-culture system of EPCs and AMs. EPCs intervened by miR-218NC, miR-218inhibitor and miR-218mimic were implanted in the lower chamber, and AMs were cultured in the upper chamber with or without GW4869. The schematic diagram of the co-culture of EPCs and AMs is shown in the figure. Fig.10 As shown in a. Fig.10b It can be seen that in the group without GW4869, compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218NC group were significantly decreased, and the expression levels of CD206 and Arg1 proteins were significantly increased; compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218inhibitor group were significantly increased, and the expression levels of CD206 and Arg1 proteins were significantly decreased; compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218mimic group were significantly decreased, and the expression levels of CD206 and Arg1 proteins were significantly increased; After GW4869 was added to the LPS+miR-218NC group, LPS+miR-218inhibitor group, and LPS+miR-218mimic group, the expression levels of iNOS and CD86 proteins in each group were significantly increased, and the expression levels of CD206 and Arg1 proteins were significantly decreased. Fig.10 c It can be seen that in the group without GW4869, compared with the LPS group, the iNOS expression level of the LPS+miR-218NC group was significantly decreased, and the CD206 expression level was significantly increased; compared with the LPS+miR-218NC group, the iNOS expression level of the LPS+miR-218inhibitor group was significantly increased, and the CD206 expression level was significantly decreased; compared with the LPS+miR-218NC group, the iNOS expression level of the LPS+miR-218mimic group was significantly decreased, and the CD206 expression level was significantly increased; after adding the exosome inhibitor GW4869 to the LPS+miR-218NC group, the LPS+miR-218inhibitor group, and the LPS+miR-218mimic group, the iNOS expression level of each group was significantly increased, and the CD206 expression level was significantly decreased. Fig.10dIt can be seen that in the group without GW4869, compared with the LPS group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218NC group were significantly decreased, and the expression levels of CD206 and Arg1 mRNA were significantly increased; compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218inhibitor group were significantly increased, and the expression levels of CD206 and Arg1 mRNA were significantly decreased; compared with the LPS+miR-218NC group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218mimic group were significantly decreased, and the expression levels of CD206 and Arg1 mRNA were significantly increased. After the exosome inhibitor GW4869 was added into the LPS+miR-218NC group, LPS+miR-218inhibitor group, and LPS+miR-218mimic group, the expression levels of iNOS and CD86 mRNA in each group were significantly increased, while the expression levels of CD206 and Arg1 mRNA were significantly decreased. Fig.10 e It can be seen that in the group without GW4869, compared with the LPS group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218NC group were significantly reduced; compared with the LPS+miR-218NC group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218inhibitor group were significantly increased; compared with the LPS+miR-218NC group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218mimic group were significantly reduced. After the exosome inhibitor GW4869 was added to the LPS+miR-218NC group, the LPS+miR-218inhibitor group, and the LPS+miR-218mimic group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in each group were significantly increased. These results indicate that the effect of EPCs on AMs polarization is mainly through miR-218 in exosomes.

[0104] 3.4. miR-218 in exosomes secreted by EPCs directly regulates the polarization level of AMs

[0105] The above experimental results show that miR-218 in the exosomes secreted by EPCs can regulate the polarization of AMs. So can the polarization of AMs be directly regulated by directly transfecting AMs with miR-218inhibitor and miR-218mimic? The present invention verifies the above hypothesis by using the control group, LPS group, LPS+miR-218inhibitor NC group, LPS+miR-218inhibitor group, LPS+miR-218mimic NC group and LPS+miR-218mimic group.

[0106] Control group: AMs without any treatment;

[0107] LPS group: AMs were intervened with 100 ng / mL LPS for 24 h;

[0108] LPS+miR-218inhibitor group: AMs were directly transfected with miR-218inhibitor 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0109] LPS+miR-218inhibitorNC group: AMs were directly transfected with miR-218inhibitorNC 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0110] LPS+miR-218mimic group: AMs were directly transfected with miR-218mimic 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0111] LPS+miR-218mimic NC group: AMs were directly transfected with miR-218mimic NC 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0112] The polarization levels of the cells in each group were tested. Fig.11 As shown. Fig.11a It can be seen that compared with the LPS group, there was no difference in the expression levels of iNOS, CD86, CD206, and Arg1 proteins in the LPS+miR-218inhibitorNC group and the LPS+miR-218mimicNC group; compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218inhibitor group were significantly increased, and the expression levels of CD206 and Arg1 proteins were significantly decreased; compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+miR-218mimic group were significantly decreased, and the expression levels of CD206 and Arg1 proteins were significantly increased. Fig.11 b It can be seen that compared with the LPS group, there was no difference in the expression levels of iNOS and CD206 in the LPS+miR-218inhibitorNC group and the LPS+miR-218mimicNC group; compared with the LPS group, the iNOS expression level in the LPS+miR-218inhibitor group was significantly increased, and the CD206 expression level was significantly decreased. Compared with the LPS group, the iNOS expression level in the LPS+miR-218mimic group was significantly decreased, and the CD206 expression level was significantly increased. Fig.11 c It can be seen that compared with the LPS group, there was no difference in the expression of iNOS, CD86, CD206, and Arg1 mRNA in the LPS+miR-218inhibitorNC group and the LPS+miR-218mimicNC group; compared with the LPS group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218inhibitor group were significantly increased, and the expression levels of CD206 and Arg1 mRNA were significantly decreased; compared with the LPS group, the expression levels of iNOS and CD86 mRNA in the LPS+miR-218mimic group were significantly decreased, and the expression levels of CD206 and Arg1 mRNA were significantly increased. Fig.11 d It can be seen that compared with the LPS group, there was no difference in the expression levels of IL-1α, IL-1β, IL-6, and TNF-α between the LPS+miR-218inhibitor NC group and the LPS+miR-218mimic NC group. Compared with the LPS group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218inhibitor group were significantly increased, while the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218mimic group were significantly decreased. The above results indicate that miR-218 can directly regulate the polarization level of AMs.

[0113] 4. Study on the role of HMGA1 in exosome regulation of AMs polarization

[0114] 4.1. miR-218 targets HMGA1

[0115] pass Fig.12 The bioinformatics analysis results of a showed that there was a target binding site between miR-218 and HMGA1. Fig.12 b and Fig.12 c It can be seen that after transfection of miR-218mimic into the HMGA1-WT group (wild-type HMGA1), the relative activity of dual luciferase was significantly reduced, but in the HMGA1-MUT (mutant HMGA1) group, after NC mimic was used as a control, the relative activity of dual luciferase was not significantly reduced. AMs were transfected with miR-218mimic and miR-218inhibitor, and then the HMGA1 protein level of transfected AMs was detected. The results are shown in Fig.12 de. Fig.12 It can be seen that after transfection with miR-218inhibitor, the HMGA1 protein level increased significantly, and after transfection with miR-218mimic, the HMGA1 protein level decreased significantly. These results indicate that miR-218 can target HMGA1 and regulate its protein level.

[0116] 4.2 HMGA1 directly regulates the polarization level of AMs

[0117] The control group, LPS group, LPS+si-HMGA1 NC group, LPS+si-HMGA1 group, LPS+oe-HMGA1 NC group and LPS+oe-HMGA1 group were used to verify whether HMGA1 directly regulates the polarization level of AMs. The control group and LPS group were the same as the control group and LPS group in Section 3.4, respectively.

[0118] LPS+si-HMGA1 NC group: AMs were directly transfected with si-HMGA1 NC 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0119] LPS+si-HMGA1 group: AMs were directly transfected with si-HMGA1 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0120] LPS+oe-HMGA1 NC group: AMs were directly transfected with oe-HMGA1 NC 24 h in advance, and after transfection, 100 ng / mL LPS was used for intervention for 24 h;

[0121] LPS+oe-HMGA1 group: AMs were directly transfected with oe-HMGA1 24h in advance. After transfection, 100ng / mL LPS was used to intervene for 24h. The cell polarization levels of the control group, LPS group, LPS+si-HMGA1 NC group, LPS+si-HMGA1 group, LPS+oe-HMGA1 NC group and LPS+oe-HMGA1 group were detected respectively. The test results are shown in Fig.13 As shown. Fig.13 a It can be seen that compared with the LPS group, there was no difference in the expression levels of iNOS, CD86, CD206 and Arg1 proteins in the LPS+si-HMGA1 NC group and the LPS+oe-HMGA1 NC group; compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+si-HMGA1 group were significantly decreased, and the expression levels of CD206 and Arg1 proteins were significantly increased; compared with the LPS group, the expression levels of iNOS and CD86 proteins in the LPS+oe-HMGA1 group were significantly increased, and the expression levels of CD206 and Arg1 proteins were significantly decreased. Fig.13 b It can be seen that compared with the LPS group, there was no difference in the expression levels of iNOS and CD206 between the LPS+si-HMGA1 NC group and the LPS+oe-HMGA1 NC group; compared with the LPS group, the iNOS expression level of the LPS+si-HMGA1 group was significantly decreased, and the CD206 expression level was significantly increased; compared with the LPS group, the iNOS expression level of the LPS+oe-HMGA1 group was significantly decreased, and the CD206 expression level was significantly decreased. Fig.13 c It can be seen that compared with the LPS group, there was no significant difference in the expression of iNOS, CD86, CD206 and Arg1 mRNA in the LPS+si-HMGA1 NC group and the LPS+oe-HMGA1 NC group; compared with the LPS group, the expression of iNOS and CD86 mRNA in the LPS+si-HMGA1 group was significantly decreased, and the expression of CD206 and Arg1 mRNA was significantly increased; compared with the LPS group, the expression of iNOS and CD86 mRNA in the LPS+oe-HMGA1 group was significantly increased, and the expression of CD206 and Arg1 mRNA was significantly decreased. Fig.13d It can be seen that compared with the LPS group, there was no significant difference in the expression levels of IL-1α, IL-1β, IL-6, and TNF-α between the LPS+si-HMGA1 NC group and the LPS+oe-HMGA1 NC group; compared with the LPS group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+si-HMGA1 group were significantly reduced, while the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+oe-HMGA1 group were significantly increased. The above results show that HMGA1 can directly regulate the polarization level of AMs.

[0122] 4.3. miR-218 regulates AMs polarization through HMGA1

[0123] The present invention knocks down miR-218 and HMGA1 in AMs cells (miR-218inhibitor+si-HMGA1), overexpresses miR-218 and HMGA1 in AMs cells (miR-218mimic+oe-HMGA1), and then interferes with 100 ng / mL of LPS to obtain LPS+miR-218inhibitor+si-HMGA1 group and LPS+miR-218mimic+oe-HMGA1 group, respectively, and then detects the polarization levels of cells in the above two groups, the control group, and the LPS group, respectively. The results are as follows: Fig.14 As shown. Fig.14 a It can be seen that compared with the LPS+miR-218inhibitor group, the iNOS and CD86 protein expression levels in the LPS+miR-218inhibitor+si-HMGA1 group were significantly decreased, and the CD206 and Arg1 protein expression levels were significantly increased; compared with the LPS+miR-218mimic group, the iNOS and CD86 protein expression levels in the LPS+miR-218mimic+oe-HMGA1 group were significantly increased, and the CD206 and Arg1 protein expression levels were significantly decreased. Fig.14 b It can be seen that compared with the LPS+miR-218inhibitor group, the iNOS expression level in the LPS+miR-218inhibitor+si-HMGA1 group was significantly decreased, and the CD206 expression level was significantly increased; compared with the LPS+miR-218mimic group, the iNOS expression level in the LPS+miR-218mimic+oe-HMGA1 group was significantly increased, and the CD206 expression level was significantly decreased. Fig.14c It can be seen that compared with the LPS+miR-218inhibitor group, the iNOS and CD86 mRNA expression levels in the LPS+miR-218inhibitor+si-HMGA1 group were significantly decreased, while the CD206 and Arg1 mRNA expression levels were significantly increased; compared with the LPS+miR-218mimic group, the iNOS and CD86 mRNA expression levels in the LPS+miR-218mimic+oe-HMGA1 group were significantly increased, while the CD206 and Arg1 mRNA expression levels were significantly decreased. Fig.14 d It can be seen that compared with the LPS+miR-218inhibitor group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218inhibitor+si-HMGA1 group were significantly increased; compared with the LPS+miR-218mimic group, the expression levels of IL-1α, IL-1β, IL-6, and TNF-α in the LPS+miR-218mimic+oe-HMGA1 group were significantly increased. The above results show that HMGA1 can directly regulate the polarization level of AMs. These results show that miR-218 regulates the polarization of AMs through HMGA1.

[0124] In summary, the results of the present study showed that the expression level of miR-218 was significantly reduced in S-ALI, EPCs delivered miR-218 to AMs via exosomes, and miR-218 targeted HMGA1, thereby inhibiting the M1 polarization of AMs and alleviating S-ALI. Therefore, the results of this study demonstrated a previously undisclosed function of miR-218 in the development of S-ALI and revealed its pathological significance. In the future, in-depth research on miR-218 or encapsulation of miRNA and siRNA into nanomaterials may be used for new therapeutic drugs. These new findings may help develop innovative S-ALI therapeutics.

[0125] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. An application of exosomes in the preparation of a drug for treating acute lung injury caused by sepsis, characterized in that: Exosomes are secreted by endothelial progenitor cells.

2. The use of the exosomes according to claim 1 in the preparation of a drug for treating acute lung injury caused by sepsis, characterized in that: Exosomes inhibit sepsis-induced acute lung injury by suppressing lung tissue damage, lung tissue leakage, and inflammatory response of lung tissue.

3. The use of the exosomes according to claim 2 in the preparation of a drug for treating acute lung injury caused by sepsis, characterized in that: Exosomes inhibit sepsis-induced acute lung injury by suppressing lung tissue damage, lung tissue leakage, and inflammatory response of lung tissue through miR-218.

4. The use of the exosomes according to claim 3 in the preparation of a drug for treating acute lung injury caused by sepsis, characterized in that: Exosomes regulate the polarization of alveolar macrophages through miR-218, thereby inhibiting lung tissue damage, lung tissue leakage, and inflammatory response of lung tissue.

5. The use of the exosomes according to claim 4 in preparing a drug for treating acute lung injury caused by sepsis, characterized in that: miR-218 inhibits lung tissue damage, lung tissue leakage and inflammatory response of lung tissue by promoting M2 macrophage polarization in alveolar macrophages and inhibiting M1 macrophage polarization in alveolar macrophages.

6. Use of the exosomes according to claim 5 in preparing a drug for treating acute lung injury caused by sepsis, characterized in that: miR-218 inhibits the polarization of M1 macrophages in alveolar macrophages and promotes the polarization of M2 macrophages in alveolar macrophages by suppressing the protein expression level of HMGA1 gene.

7. A drug for treating acute lung injury caused by sepsis, characterized in that: Includes exosomes secreted by endothelial progenitor cells.

8. The drug for treating acute lung injury caused by sepsis according to claim 7, characterized in that: Exosomes are dissolved in normal saline for injection and prepared into injections.

9. The drug for treating acute lung injury caused by sepsis according to claim 8, characterized in that: The exosomes were administered by intravenous injection.

Images