Application of exosome carrying miR-486 in pulmonary interstitial fibrosis

Abstract

The invention discloses an application of an exosome carrying miR-486 in pulmonary interstitial fibrosis, and belongs to the technical field of biological medicines, and the exosome carrying miR-486 is an exosome carrying miR-486 from umbilical cord stem cells; according to the application disclosed by the invention, the umbilical cord stem cell-derived exosome carrying the miR-486 is used for treating the pulmonary interstitial fibrosis for the first time, and the action mechanism of the umbilical cord stem cell-derived exosome carrying the miR-486 is that the umbilical cord stem cell-derived exosome carrying the miR-486 regulates fibroblast MRC-5 cell differentiation and collagen deposition through targeting FGF9; the development process of pulmonary interstitial fibrosis is improved and delayed by improving lung inflammation of mice with pulmonary interstitial fibrosis caused by bleomycin, reducing collagenous fiber deposition, inflammatory response, ECM process and pulmonary vascular structure reconstruction, and a new way is provided for treatment of the disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of exosomes carrying miR-486 in pulmonary interstitial fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is an inflammatory disease of the pulmonary interstitium caused by various reasons. The lesions mainly involve the pulmonary interstitium, but may also involve alveolar epithelial cells and pulmonary blood vessels.

[0003] Currently, apart from lung transplantation, the only treatment for this disease is pirfenidone, which is believed to slow the decline of patients' lung function. There are no other effective treatments. Repeated progression of the disease will progressively worsen and is irreversible. Oxygen inhalation can only help patients relieve pain and improve their quality of life.

[0004] In view of this, an application of exosomes carrying miR-486 in pulmonary interstitial fibrosis was designed to solve the above problems. Summary of the Invention

[0005] In order to solve the problems raised in the above background technology, the present invention provides an application of exosomes carrying miR-486 in pulmonary fibrosis, which has the characteristic of providing a new approach for the treatment of PF disease.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an application of exosomes carrying miR-486 in pulmonary interstitial fibrosis, wherein the exosomes carrying miR-486 are umbilical cord stem cell-derived exosomes carrying miR-486.

[0007] Furthermore, the steps for preparing the umbilical cord stem cell-derived exosomes carrying miR-486 are as follows:

[0008] Human umbilical cord-derived stem cells were cultured in serum-free stem cell culture medium;

[0009] Human umbilical cord-derived stem cells were transfected with miR-486-5p overexpressing lentivirus;

[0010] Collect the supernatant;

[0011] Remove large cell debris and cell membranes by centrifugation;

[0012] Exosomes from human umbilical cord-derived stem cells were collected by ultracentrifugation and resuspended in MEM medium.

[0013] Furthermore, the umbilical cord stem cell-derived exosomes carrying miR-486 regulate fibroblast differentiation and collagen deposition by targeting FGF9, thereby regulating the progression of pulmonary interstitial fibrosis.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. This invention proposes for the first time that umbilical cord stem cell-derived exosomes carrying miR-486 can be used to treat pulmonary interstitial fibrosis. Its mechanism of action is that umbilical cord stem cell-derived exosomes carrying miR-486 regulate the differentiation of fibroblasts MRC-5 cells and collagen deposition by targeting FGF9, participating in the regulation of pulmonary interstitial fibrosis disease progression, providing a new approach for the treatment of this disease.

[0016] 2. The umbilical cord stem cell-derived exosomes carrying miR-486 in the present invention can improve the progression of bleomycin-induced pulmonary fibrosis in mice by reconstructing pulmonary vascular structure, improving lung inflammation, and reducing collagen fiber deposition. This can provide new insights into the pathogenesis of pulmonary fibrosis and further improve research on the mechanisms related to the occurrence and development of pulmonary fibrosis.

[0017] 3. This invention can provide a theoretical basis and relevant experimental data support for early clinical diagnosis, early treatment, and future development of targeted miRNA drugs for the treatment of PF, and has innovative value and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 1 is a diagram showing the mechanism of action of miR-486-5p in TGF-β1-induced fibroblast differentiation according to the present invention, wherein A represents the expression of miR-486-5p after overexpression or silencing of miR-486-5p in MRC-5 cells; B represents the expression of Fn after TGF-β1 induction after overexpression or silencing of miR-486-5p in MRC-5 cells; C represents the expression of a-SMA after TGF-β1 induction after overexpression or silencing of miR-486-5p in MRC-5 cells; D represents the expression of Fn after TGF-β1 induction after overexpression or silencing of miR-486-5p in MRC-5 cells Or silencing miR-486-5p and inducing vimentin expression after TGF-β1; E is the expression of COL1A1 in MRC-5 cells after overexpression or silencing miR-486-5p and inducing TGF-β1; F is the expression of COL3A1 in MRC-5 cells after overexpression or silencing miR-486-5p and inducing TGF-β1; G is the expression of vimentin protein in MRC-5 cells after overexpression or silencing miR-486-5p and inducing TGF-β1 - western blot; H is the grayscale analysis of vimentin protein expression three times;

[0019] Figure 2Figure 1 is a diagram showing the mechanism of action of miR-486-5p targeting FGF9 to inhibit TGF-β1-induced fibroblast differentiation in the present invention, wherein A indicates that FGF9 is a target gene of miR-486-5p (dual luciferase reporter assay); B indicates that FGF9 is a target gene of miR-486-5p (qRT-PCR); C indicates that FGF9 is a target gene of miR-486-5p (Western blot); D indicates the expression of FGF9 after FGF9 silencing; E indicates the expression of Fn after FGF9 silencing; F indicates the expression of a-SMA e after FGF9 silencing; G indicates the expression of vimentin after FGF9 silencing; H indicates the expression of COL1A1 after FGF9 silencing; and I indicates the expression of COL3A1 after FGF9 silencing.

[0020] Figure 3 The following are the identification and concentration measurements of UC-MSCs-derived exosomes of the present invention, wherein A is exosome identification by electron microscopy; B is exosome identification by western blot; C is exosome size and concentration by NTA; D is exosome-transfected MRC-5 cells;

[0021] Figure 4 Figure 3 is a diagram showing the mechanism of action of UC-MSCs-derived exosomes carrying miR-486-5p in inhibiting TGF-β1-induced fibroblast differentiation by inhibiting FGF9, wherein A represents the expression of miR-486-5p after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes; B represents the expression of Fn after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; C represents the expression of a-SMA after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; D represents the expression of COL1A1 after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; E represents the expression of COL1A1 after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; F is the expression of COL3A1 after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; F is the expression of vimentin after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; G is the expression of FGF9 after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; H is the expression of vimentin protein after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1; I is the expression of FGF9 protein after treatment of MRC-5 cells with exosomes carrying miR-486-5p and control exosomes and induction of TGF-β1;

[0022] Figure 5Figure 1 is a graph showing the effects of UC-MSCs-derived exosomes carrying miR-486-5p on the proliferation and apoptosis of MRC-5 cells. Figure 1 shows the effects of UC-MSCs-derived exosomes carrying miR-486-5p on the proliferation and apoptosis of MRC-5 cells. Figure 1 shows the effects of UC-MSCs-derived exosomes carrying miR-486-5p on the apoptosis of MRC-5 cells.

[0023] Figure 6 These are the appearances of lung tissues of mice in different groups according to the present invention;

[0024] Figure 7 HE staining (100×) of lung tissues of mice in different groups of the present invention;

[0025] Figure 8 Masson staining (100×) of lung tissues of mice in different groups of the present invention;

[0026] Figure 9 This is a statistical diagram of collagen fibers in lung tissue of mice in different groups of the present invention;

[0027] Figure 10 Victoria blue staining (200×) of lung tissues of mice in different groups of the present invention;

[0028] Figure 11 This is a statistical diagram of elastic fibers in lung tissue of mice in different groups of the present invention;

[0029] Figure 12 The immunohistochemical staining (200×) of the lung tissues of mice in each group of the present invention is shown;

[0030] Figure 13 This figure shows the effect of exosomes carrying miR-486 on TGFβ and inflammatory factors. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] S1: In vitro experiments

[0033] S101: Select cells and culture them

[0034] The incubator conditions were set to a constant temperature of 37°C, 21% oxygen concentration, and 5% carbon dioxide concentration to simulate the physiological environment in the human body and ensure that the cells were in an optimal growth state. Human embryonic lung fibroblast MRC-5 cells were cultured in a complete medium containing 10% fetal bovine serum, 1% MEM non-essential amino acids, 1% sodium pyruvate, 1% glutamate-1, and 1% MEM basal medium in the incubator;

[0035] Human umbilical cord-derived stem cells (UC-MSCs) were cultured in serum-free stem cell culture medium.

[0036] S102: Cell transfection

[0037] Control lentivirus and miR-486-5p overexpressing lentivirus were purchased from Genema Gene and transfected into MRC-5 cells and UC-MSC cells at an MOI of 5;

[0038] S103: Exosome Preparation

[0039] The supernatant was collected, and large cell debris and cell membranes were removed by centrifugation. UC-MSC-derived exosomes were collected by ultracentrifugation and resuspended in MEM medium.

[0040] S104: RNA extraction

[0041] Total RNA was extracted from MRC-5 cells, UC-MSC cells, and exosomes using TRIzol reagent (Invitrogen) and miRNeasy mini kit (Qiagen);

[0042] S105: miR-486-5p blocks TGF-β1-induced lung fibroblast differentiation

[0043] Using U6 as an internal control, miR-486-5p mRNA in MRC-5 cells was detected according to the manufacturer's TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assay instructions (Life Technologies, Carlsbad, CA, USA);

[0044] Reverse transcriptase polymerase chain reaction results were analyzed using an ABI 7500 sequence analyzer (Applied Biosystems, Foster City, CA, USA). The expression of mesenchymal markers, including fibronectin (Fn), vimentin, α-smooth muscle actin (a-SMA), and collagen-related genes COL1A1 and COL3A1, was detected in MRC-5 cells after overexpression or silencing of miR-486-5p. The expression was normalized to β-actin using the following primers:

[0045]

[0046] The results are as attached Figure 1 As shown, it can be seen that:

[0047] Overexpression of miR-486-5p in MRC-5 cells can significantly upregulate the expression of miR-486-5p, while silencing miR-486-5p can downregulate the expression of miR-486-5p ( Figure 1 A);

[0048] Compared with the normal group, overexpression of miR-486-5p in TGF-β1-treated MRC-5 cells inhibited the expression of Fn, α-SMA, vimentin, COL1A1, and COL3A1 mRNAs; silencing of miR-486-5p in TGF-β1-treated MRC-5 cells downregulated the expression of Fn, α-SMA, vimentin, COL1A1, and COL3A1 mRNAs ( Figure 1 BF); In TGF-β1-treated MRC-5 cells, overexpression of miR-486-5p downregulated vimentin protein expression, while silencing of miR-486-5p upregulated vimentin protein expression ( Figure 1 GH);

[0049] S106: miR-486-5p blocks TGF-β1-induced lung fibroblast differentiation by targeting fibroblast growth factor 9 (FGF9)

[0050] Fibroblast growth factor 9 (FGF9) was predicted as a target gene of miR-486-5p by the target gene prediction website (www.targetscan.org);

[0051] The wild-type (WT) or mutant (MUT) sequence of FGF9 was inserted into the 3'UTR of the pmirGLO vector (wild-type, WT). 293T cells were cultured to approximately 70%-80% confluency in 6-well plates and co-transfected with mutant luciferase or wild-type reporter vectors, and mimicked miRNA or negative control (2 μg). After 48 h, luciferase activity was measured and normalized. The results are shown in the attached figure. Figure 2 As shown, it can be seen that:

[0052] When co-transfected with WT and NC or miR-486-5p mimics, the mimic miR-486-5p significantly reduced luciferase activity, however, no significant change in luciferase activity was observed after co-transfection with FGF9 MUT and miR-486-5pm mimics ( Figure 2 A);

[0053] Reverse transcriptase polymerase chain reaction results were analyzed using an ABI 7500 sequence detector (Applied Biosystems, Foster City, CA, USA) to detect the expression of mesenchymal-related markers, including FGF9 mRNA, after overexpression or silencing of miR-486-5p in MRC-5 cells and normalized to β-actin using the above primers;

[0054] MRC-5 cells were lysed in RIPA buffer containing 1% phenylmethylsulfonyl fluoride and 1% phosphatase (PMSF) inhibitor. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 20 μg of protein was loaded onto SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Primary antibodies, including anti-CD9, anti-CD63, anti-CD81, anti-HSP60, anti-HSP90, anti-GRP78, and anti-TSG101, were used for detection. Immunoreactive bands were detected with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies and visualized by chemiluminescence. Protein levels were analyzed using ImageJ software. The results are shown in the attached Figures. Figure 2 As shown, it can be seen that:

[0055] Overexpression of miR-486-5p inhibited the expression of FGF9 mRNA and protein; however, inhibition of miR-486-5p upregulated the expression of FGF9 mRNA but not FGF9 protein ( Figure 2 BC);

[0056] FGF9 silencing siRNA synthesized by Genetron Health was injected into the cells after 12 hours of culture. 3000 (Thermo Fisher Scientific) was transfected into cells. The cells were incubated at 37°C for 48 h before the experiment. The results are shown in the attached figure. Figure 2 As shown, it can be seen that:

[0057] FGF9 silencing can significantly downregulate the expression of FGF9 mRNA ( Figure 2 D);

[0058] Compared with the normal group, FGF9 silencing in TGF-β1-treated MRC-5 cells inhibited the expression of Fn, α-SMA, vimentin, COL1A1, and COL3A1 mRNA ( Figure 2 EI);

[0059] S107: Exosomal miR-486-5p inhibits fibroblast differentiation by targeting FGF9 exosomes

[0060] The resuspended exosomes (1 μg in 10 μl phosphate buffer solution (PBS)) were placed on a carbon-coated copper grid (200 mesh) and allowed to adsorb for 2 minutes. The grid was then washed twice with distilled water, incubated in 2% glutaraldehyde for 25 minutes, rinsed 8 times in PBS for 2 minutes each, and then stained with 8 μL of 2% uranyl acetate solution for 1 minute. After natural drying, the grid was observed using a transmission electron microscope. Figure 3 As shown, it can be seen that:

[0061] The size of exosomes is approximately 30-180 nm ( Figure 3 A);

[0062] Stem cell-derived exosomes were lysed using RIPA buffer, and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 20 μg of protein was loaded into each well and separated on a 10% SDS-polyacrylamide gel. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. Primary antibodies used were anti-CD9, anti-CD63, anti-CD81, anti-HSP60, anti-HSP90, anti-GRP78, and anti-TSG101. The membranes were incubated with appropriate dilutions of primary antibodies overnight at 4°C. Immunoreactive bands were detected with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies and visualized by chemiluminescence. Protein levels were analyzed using ImageJ software. The results are shown in the attached Figure 2. Figure 3 As shown, it can be seen that:

[0063] Expression of exosome-specific proteins CD9, CD63, CD81, HSP60, HSP70, HSP90, GRP78 and TSG101 ( Figure 3 B);

[0064] The exosome samples were diluted 10- to 100-fold in PBS so that the number of particles in the field of view was approximately 100 per frame. The NTA measurement conditions were set to 21-23.6°C temperature, 0.9-0.965 cP viscosity, 25 frames per second, and 60 seconds of measurement time. The detection thresholds were similar for all samples. The results show the average and standard deviation of at least three measurements. The results are shown in the attached figure. Figure 3 As shown, it can be seen that:

[0065] The exosome concentration was approximately 1.6×108±1.44×107 particles / ml ( Figure 3 C);

[0066] Compared with control exosomes, exosomes derived from overexpressed UC-MSCs had higher miR-486-5p expression; when MRC-5 cells were treated with exosomes, exosomes were found to aggregate around the cells ( Figure 3 D);

[0067] MRC-5 cells were treated with control exosomes and stem cell-derived exosomes carrying miR-486-5p. The results are shown in the attached figure. Figure 4 As shown, it can be seen that:

[0068] Treatment of MRC-5 cells with stem cell-derived exosomes carrying miR-486-5p also increased the expression of miR-486-5p in MRC-5 cells ( Figure 4 A), indicating that exogenous exosomes can mediate the transfer of miR-486-5p to MRC-5 cells;

[0069] Compared with control exosomes, treatment of MRC-5 cells with stem cell-derived exosomes carrying miR-486-5p also suppressed the mRNA expression of Fn, α-SMA, vimentin, COL1A1, and COL3A1 ( Figure 4 BF, 4H);

[0070] Treatment of MRC-5 cells with stem cell-derived exosomes carrying miR-486-5p inhibited the expression of miR-486-5p target gene FGF9 mRNA and protein ( Figure 4 G, 4I);

[0071] S108: Stem cell-derived exosomes carrying miR-486-5p inhibit TGF-β1-induced lung fibroblast differentiation by targeting FGF9 exosomes

[0072] MRC-5 cells were seeded in 96-well plates at a rate of 5000 cells per well in 100 μl of culture medium and then treated with NC-exosomes or miR-486-5p-overexpressing exosomes. After culturing for 24 h and 48 h, 10 μl of CCK-8 solution was added to each well, and the plates were returned to the incubator for 2 h. The reaction was measured using a microplate reader to detect the proliferation of MRC-5 cells;

[0073] 2 × 10 5 MRC-5 cells / well were seeded in 6-well plates and grown overnight. The culture supernatant was then replaced with fresh culture medium without fetal bovine serum and treated with control exosomes or miR-486-5p-overexpressing exosomes. The cells and cell culture supernatant were collected and washed twice with phosphate-buffered saline. The degree of cell apoptosis was analyzed using the Annexin-V / APC apoptosis detection kit according to the manufacturer's instructions. FACS Calibur (BD Biosciences company, NJ, USA) was used for apoptosis detection.

[0074] The results are as attached Figure 5 As shown, it can be seen that:

[0075] Compared with control exosomes, stem cell-derived exosomes carrying miR-486-4p slightly promoted the apoptosis of MRC-5 cells, but the effect was not statistically significant.

[0076] Compared with the control exosomes, exosomes carrying miR-486-4p had little effect on the proliferation of MRC-5 cells ( Figure 5 AB).

[0077] S2: In vivo experiments

[0078] S201: Establishing experimental animal models

[0079] Ten of 30 C57BL / 6 mice were randomly selected as the normal group, and the remaining 20 mice were used to establish the bleomycin PF model. Ten of the 20 mice were randomly selected from each group to be divided into the model group and the exosome group;

[0080] Ten rats in the model group were injected with phosphate-buffered saline (PBS) via the tail vein, and ten rats in the exosome group were injected with stem cell-derived exosomes carrying miR-486 via the tail vein;

[0081] S202: Effects of exosomes carrying miR-486 on vital signs in mice with pulmonary fibrosis

[0082] Observation of the survival status of mice showed that:

[0083] Normal group: The mice were in good general condition and had full morphology, with black and smooth hair;

[0084] Bleomycin was injected into the tail vein to establish the model: the mice showed increased respiratory rate, significantly reduced activity and food intake, became thin and shriveled, with hair loss and inverted hair. One mouse died on the third day after the start of bleomycin injection. On the tenth day, the survival condition stabilized and no further deaths occurred.

[0085] PF model group: PBS solution was injected into the tail vein starting from the second day after model establishment and was injected every other day until the end of the experiment on the 21st day;

[0086] Exosome group: Starting from the second day after model establishment, stem cell-derived exosomes carrying miR-486 were injected into the tail vein, and injected every other day until the end of the experiment on day 21. The mice's wheezing symptoms gradually alleviated, their activities improved, their individual morphology and hair were restored, and their overall condition was significantly better than that of the PF model group.

[0087] S203: Mouse lung tissue specimen results

[0088] Lung tissues of normal mice and mice with bleomycin-induced pulmonary fibrosis were collected. Figure 6 As shown, it can be seen that:

[0089] Normal group: lung tissue color and cord-like grooves were in good condition;

[0090] PF model group: lung tissue was darker in color, had a higher degree of edema, and had decreased elasticity, making it difficult to peel off;

[0091] Exosome group: lung tissue color and cord-like grooves, pulmonary edema and congestion showed varying degrees of improvement;

[0092] S204: Effects of exosomes carrying miR-486 on lung tissue structure

[0093] The sections of paraffin-embedded lung tissue were dewaxed in xylene I and II for 5 minutes each, then washed in 95% ethanol I and II for 1 minute each, then placed in 80% ethanol for 1 minute, then rinsed in tap water for 1 minute, then stained in Harris hematoxylin solution for 5 minutes, then rinsed in tap water for 2 minutes, then differentiated in 1% hydrochloric acid ethanol for 5 seconds, then rinsed in tap water to return to blue for 10 minutes, then stained in eosin solution for 1 minute, then rinsed in tap water, then placed in 80% and 95% ethanol for color adjustment and dehydration, then placed in anhydrous ethanol I and II for 2 minutes, then placed in carbolic acid xylene, xylene I and II for transparentization for 2 minutes each, sealed with neutral gum, and examined under a microscope to observe the pathological changes of lung tissue. The results are shown in the attached figure. Figure 7 As shown, it can be seen that:

[0094] Normal group: The surface membrane structure of the lung tissue is clear; the lung parenchyma is composed of the branches of the bronchus at all levels (black arrows) and the numerous alveoli at their terminals (purple arrows). There is no obvious abnormality in the structure of the bronchus at all levels. The alveolar wall is composed of a single layer of epithelium and has a clear structure. The interstitium, including the connective tissue and blood vessels in the lungs, has no obvious abnormalities. No obvious inflammatory cell infiltration is observed. The mouse alveoli have a fine and uniform structure, with no fibrin deposition or inflammatory cellulose exudate.

[0095] PF model group: The surface membrane structure of the lung tissue is clear; the lung parenchyma is composed of a large number of alveoli at all levels of bronchial branches and their terminals, and a small number of bronchiolar mucosal epithelial cells are irregularly arranged (gray arrows); a small amount of connective tissue hyperplasia (orange arrows) can be seen in a small area of ​​the alveolar wall, mild to moderate thickening of the alveolar wall, widening of the alveolar septum, a small number of alveoli are narrowed, the alveolar structure is unclear, accompanied by a small amount of granulocyte infiltration (blue arrows), and scattered necrotic cell fragments (purple arrows) can be seen; a small number of alveoli are compensatory dilated (black arrows), and the alveoli are of different sizes; a small amount of lymphocyte infiltration can be seen around a small number of blood vessels (red arrows); a small amount of interstitial vascular congestion (brown arrows), the mouse alveoli and terminal bronchioles are widely destroyed and fused, and a large amount of fibrin is deposited in the pulmonary interstitium;

[0096] In the exosome group, the surface membrane structure of the lung tissue was clear; the lung parenchyma consisted of numerous alveoli at the branches and terminals of the bronchial tubes at all levels within the lungs, with no obvious structural abnormalities of the bronchial tubes at all levels. A small amount of granulocyte infiltration (blue arrows) was observed in the alveolar walls, with mild thickening of the alveoli in the medium range, widening of the alveolar septa, and alveolar sizes varying. Treatment with exosomes carrying miR-486 significantly alleviated bleomycin-induced pulmonary fibrosis, with pathological damage, including thickening of the alveolar septa, interstitial edema, and inflammatory cell infiltration significantly reduced. The integrity of the alveoli was superior to that of the PF model group, and the structure of the alveoli and bronchioles of the mice was significantly improved. Large fibrin deposition and inflammatory exudates were rare in the pulmonary interstitium.

[0097] S205: Effects of exosomes carrying miR-486 on collagen fibers in lung tissue

[0098] Paraffin-embedded lung tissue sections were dewaxed in xylene I and II for 10 minutes each, then washed in 95% ethanol I and II for 1 minute each, then placed in 80% ethanol for 1 minute, then rinsed in tap water for 1 minute, then stained in Weigert iron hematoxylin for 5-10 minutes, then differentiated in acidic ethanol differentiation solution, then rinsed in tap water for 2 minutes, then placed in Masson blue solution to return to blue, then placed in distilled water for 1 minute, and then stained in Ponceau fuchsin staining solution for 5-10 minutes. During the operation, distilled water: weak acid solution was prepared in a 2:1 ratio to prepare a weak acid working solution, then placed in the weak acid working solution for washing for 1 minute, then placed in a phosphomolybdic acid solution for washing for 1-2 minutes, then placed in the weak acid working solution for washing for 1 minute, then placed in aniline blue staining solution for staining for 1-2 minutes, then placed in the weak acid working solution for washing for 1 minute, then placed in 95% ethanol for rapid dehydration, dehydrated with anhydrous ethanol 3 times, each time for 5-10 seconds, then placed in xylene for transparentization 3 times, each time for 1-2 minutes, sealed with neutral gum, and examined under a microscope to observe the collagen deposition in the lung tissue. The results are shown in the attached figure. Figure 8 and 9 As shown, it can be seen that:

[0099] Compared with the normal group, the PF model group showed a significant increase in collagen fibers, which were mainly located in the outer zone of the lung tissue, while the exosome group showed a significant decrease in lung tissue collagen fibers compared with the PF model group, indicating that exosomes carrying miR-486 reduced bleomycin-induced lung tissue collagen fibers;

[0100] This suggests that exosomes may reduce collagen expression in lung tissue, thereby reducing collagen I and collagen III deposition and delaying PF progression;

[0101] S206: Effects of exosomes carrying miR-486 on pulmonary angiogenesis

[0102] Place the paraffin-embedded lung tissue sections in xylene I and xylene II for 20 minutes each, then place them in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each, then place them in 75% ethanol for 5 minutes, rinse with running water for 5 minutes, draw circles around the tissue, and then add Victoria blue acidification solution (Victoria blue A and Victoria blue B are mixed in a ratio of 1:1 and taken out immediately after use) to oxidize the tissue for 5 minutes, then put the sections into two tanks of water for a total of 10 seconds, then gently shake the sections to remove excess water, use Victoria blue C to bleach the tissue for 2 minutes, soak the sections in 3 water washing tanks for 5 seconds each, gently shake to remove excess water on the sections, then place them in 70% ethanol for immersion, soak the sections in Victoria blue D for 24 hours (covered), and then Place in 70% ethanol soaking solution and wash twice, each time for about 10 seconds, until the surface of the glass slide is no longer stained, then rinse gently with running water, check the degree of staining of the elastic fibers under a microscope, dark stain, and then place in 75% ethanol to differentiate the slices for 5 seconds, rinse with tap water to complete the differentiation, and examine under a microscope. Repeat the differentiation, rinsing and microscopic examination until the elastic fibers are blue and the background is light blue or almost colorless. Use Victoria E blue dye to stain the slices for 1-5 minutes, then rinse with running water, and place the slices in anhydrous ethanol I, anhydrous ethanol II, and anhydrous ethanol III for 5 minutes each, and then place them in xylene I and xylene II for 5 minutes each to make them transparent, then seal the slices with neutral gum, and observe pulmonary angiogenesis under a microscope. The results are shown in the attached figure. Figure 10 and 11 As shown, it can be seen that:

[0103] Compared with the normal group, the lung tissues of the PF model mice showed damage, shedding, and necrosis of pulmonary vascular endothelial cells, proliferation of some endothelial cells and smooth muscle cells, thickening of the vascular wall, and even occlusion of the vascular lumen. The exosome group could alleviate bleomycin-induced alveolar epithelial and vascular damage in PF mice and inhibit the deposition of pulmonary vascular elastic fibers in the lung tissues.

[0104] This suggests that exosomes may delay PF progression by improving vascular remodeling;

[0105] S207: Effects of exosomes carrying miR-486 on extracellular deposition in lung tissue

[0106] In immunohistochemistry, the primary antibodies used were Collagen I, Collagen III, α-SMA, and FN;

[0107] The paraffin-embedded lung tissue sections were dewaxed and hydrated, antigen repaired, endogenous peroxidase blocked, serum blocked, primary antibody added, secondary antibody added, DAB color developed, cell nuclei stained again, dehydrated and mounted, and examined under a microscope. The results are shown in the attached Figure 12 As shown, it can be seen that:

[0108] Myofibroblasts are key cells in the formation of pulmonary fibrosis and are the main synthesizing cells of collagen I and collagen III. Excessive deposition of collagen I and collagen III can cause irreversible damage and fibrosis of lung tissue structure. α-SMA is its hallmark protein.

[0109] FN has a chemotactic effect on fibroblasts, which can promote the migration of fibroblasts to the site of injury, promote the division and proliferation of fibroblasts, and accelerate the synthesis and transcription of collagen cells;

[0110] The expression of α-SMA in the PF model group increased and was higher than that in the normal group, while the expression levels of α-SMA and Fn in the exosome group were lower than those in the PF model group;

[0111] Collagen I is the most common of collagen I and collagen III proteins. Fibroblasts and myofibroblasts are the main effector cells that synthesize and secrete collagen I in lung tissue.

[0112] Collagen III is closely related to inflammatory response and fibrosis. Fibroblasts can synthesize a large amount of collagen III. In PF, the accumulation of collagen III is a characteristic pathological change of PF, which is mainly manifested as excessive proliferation of ECM.

[0113] The lung tissues of mice in the normal group showed weak positive expression of CollagenⅠ and CollagenⅢ. The expression levels of CollagenⅠ and CollagenⅢ in the lung tissues of mice in the PF model group were significantly higher than those in the normal group. The expression levels of CollagenⅠ and CollagenⅢ in the exosome group were significantly lower than those in the PF model group.

[0114] This suggests that exosomes may reduce inflammatory responses, thereby reducing collagen I and collagen III deposition and delaying PF progression;

[0115] S208: Effects of exosomes carrying miR-486 on plasma TGFβ and inflammatory factors

[0116] In the enzyme-linked immunosorbent assay, an ELASA kit was used to dilute the antibody to 1-10 μg / ml with carbonate buffer, 100 ul of diluted antibody was added to a polystyrene fixed plate and kept in a 4°C refrigerator overnight. The next day, the well liquid was discarded and rinsed 3 times with a cleaning buffer solution, 3 minutes each time, 200 ul of blocking agent was added to each well, and incubated at 37°C for 1-2 hours. The sealing film was gently removed and placed in a plate washer for 3-5 times. 100 μl of appropriate mouse plasma from each group was added to the coated wells, the plate was sealed with a sealing film and incubated at 37°C for 1-2 hours. The washing method was the same as before. 100 μL of biotin-labeled antibody working solution was added to each well. The incubation and washing steps were the same as before. 100 μL of enzyme conjugate working solution was added to each well. The incubation and washing steps were the same as before. TMB matrix solution was added to each well and incubated at 37°C in the dark for 10-30 minutes until a clear color gradient appeared in the standard wells after serial dilution. 100 μL of sulfuric acid was added to each reaction well to change the color of the solution from blue to yellow. The blank control wells were adjusted to zero. The OD value of each well was measured at a wavelength of 450 nm to detect the expression of inflammatory factors TGF-β1, TNF-α, and IL-6 in the plasma of each group of mice.

[0117] GraphPad Prism (Version 9.0) software was used to analyze the data and plot them. Normally distributed measurement data were expressed as mean ± standard deviation, and one-way analysis of variance was used for inter-group comparisons. Skewedly distributed measurement data were expressed as M (P25, P75), and the rank sum test was used for inter-group comparisons.

[0118] The difference was considered statistically significant when p < 0.05;

[0119] The results are as attached Figure 13 As shown, it can be seen that:

[0120] TGF-β is a known growth factor with three subtypes identified: TGF-β1, TGF-β2, and TGF-β3. TGF-β1 regulates epithelial cell apoptosis, fibroblast proliferation, myofibroblast differentiation, collagen synthesis and deposition, and epithelial-mesenchymal transition, thereby promoting the massive release of other inflammatory factors (such as TNF-α and IL-6), further activating inflammatory cells to form a series of chain reactions, resulting in a cascade amplification effect and ultimately mediating PF. TGF-β1 is also upregulated in the lung tissue of PF patients, and overexpression of TGF-β1 can induce severe pulmonary interstitial fibrosis.

[0121] Macrophages can be divided into M1 macrophages and M2 macrophages after polarization. Th1 cytokines usually activate M1 macrophages, which then release a large number of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12, which increase inflammatory lung damage and promote fibrosis. TNF-α is an important indicator of inflammatory response because it links inflammatory response to M1 macrophage polarization.

[0122] IL-6 is a pleiotropic cytokine that acts as both a pro-inflammatory and a pro-fibrotic factor in PF. It sends inflammatory signals from local lesions to the whole body, stimulating the recruitment of immune cells, including monocytes / macrophages. In turn, these immune cells produce more inflammatory factors to recruit more macrophages, driving an inflammatory cascade, leading to persistent tissue damage and ultimately the development of pathological fibrosis. IL-6 promotes the phosphorylation and translocation of STAT3, which is associated with fibroblast activation, proliferation, and collagen I and collagen III protein deposition. M2 macrophages lead to the activation of the IL-6 / gp130 / Stat3 signaling pathway, which has also been shown to play a crucial role in the pathogenesis of PF.

[0123] Compared with the normal group, the plasma levels of TGFβ and inflammatory factors TNF-a and IL-6 in the PF model group were significantly increased, while the plasma levels of TGFβ, TNF-a and IL-6 in the exosome group were lower than those in the PF model group, and the differences were statistically significant.

[0124] These results indicate that exosomes can delay the progression of PF by regulating the inflammatory response of macrophages.

[0125] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An application of exosomes carrying miR-486 in pulmonary interstitial fibrosis, characterized in that: The exosomes carrying miR-486 are derived from umbilical cord stem cells.

2. The use of exosomes carrying miR-486 in pulmonary fibrosis according to claim 1, characterized in that: Preparation steps of the umbilical cord stem cell-derived exosomes carrying miR-486: Human umbilical cord-derived stem cells were cultured in serum-free stem cell culture medium; Human umbilical cord-derived stem cells were transfected with miR-486-5p overexpressing lentivirus; Collect the supernatant; Remove large cell debris and cell membranes by centrifugation; Exosomes from human umbilical cord-derived stem cells were collected by ultracentrifugation and resuspended in MEM medium.

3. The use of a stem cell-derived exosome carrying miR-486 in pulmonary fibrosis according to claim 2, characterized in that: The umbilical cord stem cell-derived exosomes carrying miR-486 regulate fibroblast differentiation and collagen deposition by targeting FGF9, thereby modulating the progression of pulmonary fibrosis. Furthermore, stem cell-derived exosomes carrying miR-486 can improve lung inflammation in mice with bleomycin-induced pulmonary fibrosis, reducing collagen deposition, inflammatory responses, ECM progression, and pulmonary vascular remodeling, thereby improving the progression of pulmonary fibrosis in mice.

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