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

Abstract

The invention relates to the field of acute lung injury caused by sepsis, in particular to application of lactobacillus rhamnosus exosome in preparation of medicine for treating and / or preventing acute lung injury caused by sepsis of subjects, and the acute lung injury caused by sepsis is acute complication of sepsis. The sepsis is caused by bacterial infection, and the particle size of the lactobacillus rhamnosus exosome ranges from 50 nm to 120 nm. The lactobacillus rhamnosus exosome provided by the invention can prevent and / or timely and effectively prevent the development of the disease course of a subject at the initial stage of sepsis acute lung injury, and finally, the treatment success rate of the subject is increased, and the prognosis of the subject is improved.

Description

Technical Field

[0001] The present invention relates to the field of acute lung injury caused by sepsis, and specifically to the use of Lactobacillus rhamnosus (LGG) exosomes in the preparation of drugs for preventing and treating acute lung injury caused by sepsis. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by an imbalance in the host's response to infection. It is one of the leading causes of death in critically ill patients worldwide and has become an important clinical problem facing the field of emergency and critical care medicine. The lungs are the most vulnerable and most susceptible target organs during sepsis. Sepsis-induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), with clinical manifestations of progressive dyspnea and refractory hypoxemia, has become a global public health issue that seriously endangers human health and needs to be urgently addressed. According to relevant epidemiological surveys, the incidence of acute lung injury in patients with sepsis is 68.2%, and the 90-day mortality rate of patients with acute lung injury is as high as 35.5%.

[0003] Acute lung injury (ALI) is caused by multiple pathogenic factors inside and outside the lungs, directly or indirectly (such as infection, trauma, burns, poisoning, pneumonia, blood transfusion, immune response, etc.), which damage the structure and function of pulmonary capillary endothelial cells and alveolar epithelial cells, and increase the permeability of pulmonary capillaries, resulting in non-cardiogenic diffuse pulmonary interstitial and alveolar edema. ALI is a common clinical complication of sepsis patients and an important cause of death in sepsis patients. In particular, when sepsis is caused by bacterial infection, toxic substances such as lipopolysaccharide (LPS) released by bacteria can cause multiple organ damage, further aggravating the symptoms of sepsis acute lung injury and the difficulty of treatment.

[0004] Sepsis-induced acute lung injury is a clinically critical disease with high morbidity and mortality, lack of effective prevention and treatment methods, and serious threat to human health. Therefore, early effective intervention in the early stage of the disease can improve the patient's prognosis and survival rate, which has important clinical significance. Chinese patent application CN115721653A discloses the use of corosolic acid in the preparation of a drug for treating sepsis-induced acute lung injury. Its therapeutic effect is relatively limited, and it needs to be continuously administered for at least one week to alleviate the symptoms of sepsis-induced acute lung injury to a certain extent, which is not conducive to practical clinical application. Summary of the invention

[0005] The present invention provides a use of Lactobacillus rhamnosus exosomes in the preparation of a drug for treating and / or preventing acute lung injury caused by sepsis in a subject, characterized in that the acute lung injury caused by sepsis is an acute complication of sepsis, the sepsis is caused by bacterial infection, and the particle size range of the Lactobacillus rhamnosus exosomes is 50-120 nm.

[0006] .In some embodiments, the drug is an oral preparation.

[0007] In some embodiments, a single dose of the drug comprises 1.5-2.5 mg exosomes / kg.

[0008] .In some embodiments, a single dose of the drug is 2 mg exosomes / kg (body weight).

[0009] .In some embodiments, the drug may also include a pharmaceutically acceptable carrier and / or adjuvant.

[0010] In some embodiments, the oral preparation includes tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups or lyophilized preparations for oral administration (or administration, dosing).

[0011] In some embodiments, the oral formulation is provided in individual dosage units so that a predetermined amount of active ingredient can be provided upon administration.

[0012] The term "pharmaceutically acceptable" as used in the present invention refers to compounds, raw materials, compositions and / or dosage forms that are, within the reasonable scope of medical judgment, suitable for contact with the tissues of the subject without excessive toxicity, irritation, allergic response or other problems and complications that exceed the reasonable benefit / risk ratio, and are effectively used for the intended purpose.

[0013] In some embodiments, the pharmaceutically acceptable carriers and / or adjuvants described in the present invention, such as solvents, diluents, excipients, are added at appropriate steps during the preparation process.

[0014] In some embodiments, the drug is prepared in the form of a dosage unit, in the form of an independent dosage form containing 90-150 mg of Lactobacillus rhamnosus exosomes for single use, and in the form of at least 3 independent dosage forms as one dosage unit.

[0015] In some embodiments, the drug is prepared in the form of a dosage unit, in the form of an independent dosage form containing 120 mg of Lactobacillus rhamnosus exosomes for one use, and in the form of at least 3 independent dosage forms as one dosage unit.

[0016] .In some embodiments, the drug is administered every day or every other day.

[0017] In some embodiments, the drug is administered once a day and at least three times a day.

[0018] .In some embodiments, the drug is used to directly target the lungs of the subject and be taken up by the lung tissue of the subject.

[0019] .In some embodiments, the subject has a lung injury score of 3 or 4. In some embodiments, the pathological manifestations of a lung injury score of 3 include: significant congestion and thickening of the alveolar wall, a decrease in the number of alveolar cavities, part of the cavity is occupied by serous fluid, and the nucleated cells on the alveolar wall are further increased. In some embodiments, the pathological manifestations of a lung injury score of 4 include: significant congestion and thickening of the alveolar wall, a decrease in the number of alveolar cavities, part of the cavity is occupied by serous fluid, the nucleated cells on the alveolar wall are further increased, local inflammatory cells are focally aggregated, and are accompanied by focal hemorrhage.

[0020] .In some embodiments, the drug is used to reduce the lung injury score of 3 or 4 points in a subject to 0 or 1 or 2 points. In some embodiments, the pathological manifestations of a lung injury score of 0 include: normal lung tissue structure, no obvious tissue pathological damage. In some embodiments, the pathological manifestations of a lung injury score of 1 include: mild lesions, mild thickening of the alveolar wall, slight increase in red blood cells and inflammatory cells on the wall, and a small amount of exuded serous fluid in the alveolar cavity. In some embodiments, the pathological manifestations of a lung injury score of 2 include: obvious thickening and congestion of the alveolar wall, obvious increase in nucleated cells on the wall, serous fluid and a small amount of leaked red blood cells in the alveolar cavity.

[0021] In some embodiments, the protein concentration of the exosomes is 0.9-1.1 μg / μL.

[0022] In some embodiments, the symptoms of acute lung injury caused by sepsis include one or more of localized hemorrhage in lung tissue, peri-alveolar inflammation, and alveolar inflammatory lesions.

[0023] .In some embodiments, the medicament is used to reduce the inflammatory response in the lungs of the subject.

[0024] As used herein, "subject" refers to a subject in need of disease treatment, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses and cows, but the present invention is not limited thereto.

[0025] In some embodiments, the method for preparing exosomes comprises:

[0026] .S101 When the strain density of Lactobacillus rhamnosus culture suspension reaches 2×10 9CFU / mL, the culture suspension was centrifuged at 2000 g for 10 to 15 minutes to obtain supernatant A;

[0027] .S102 centrifuging the supernatant A at 5000 g for 20 to 30 minutes to obtain supernatant B;

[0028] .S103 centrifuging the supernatant B at 10000 g for 30 to 40 minutes to obtain supernatant C;

[0029] .S104: filtering the supernatant C using a 0.45 μm filter, and ultracentrifuging the filtered supernatant C at 10,000 to 150,000 g for 70 minutes to obtain a precipitate D;

[0030] .S105 The precipitate D is resuspended and filtered using a 0.22 μm filter to obtain a filtrate E. The filtrate E is ultracentrifuged at 10,000 to 150,000 g for 70 to 120 minutes to obtain a precipitate F, i.e., the Lactobacillus rhamnosus exosomes.

[0031] Compared with the prior art, the beneficial technical effects of the present invention include at least the following aspects:

[0032] .1) Sepsis-induced acute lung injury is one of the thorny issues in the medical field. Compared with other types of acute lung injury (such as virus-induced lung injury and bleomycin-induced lung injury), bacteria-induced sepsis-induced acute lung injury is more severe and has a higher mortality rate. Therefore, the existing technology has poor clinical treatment effects on sepsis-induced acute lung injury, and usually requires continuous administration for at least one week to alleviate the symptoms of sepsis-induced acute lung injury to a certain extent. Unexpectedly, the Lactobacillus rhamnosus exosomes provided by the present invention only require a very small number of administrations (for example, 3 days) to better prevent sepsis-induced acute lung injury and significantly reduce the mortality rate of experimental animals (it should be emphasized that the 3-day mortality rate of mice with sepsis-induced acute lung injury that are not treated with the Lactobacillus rhamnosus exosomes provided by the present invention is as high as 100%). Experimental verification shows that, at a suitable dose (e.g., 1.5-2.5 mg Lactobacillus rhamnosus exosomes / kg), the Lactobacillus rhamnosus exosomes provided by the present invention can prevent and / or promptly stop the progression of the disease in subjects with septic acute lung injury in the early stages of septic acute lung injury, which is of great significance for improving the treatment success rate of the subjects and improving the prognosis of the subjects.

[0033] .2) Furthermore, the present invention found that Lactobacillus rhamnosus exosomes with a particle size range of 50-120 nm can be more absorbed by the lung tissue of the subject, thereby being able to better treat and / or prevent acute lung injury caused by sepsis in the subject.

[0034] .3) Experiments have shown that the Lactobacillus rhamnosus exosomes provided by the present invention can significantly improve pathological damage to lung tissue, downregulate the inflammatory response of lung tissue and exert a lung protective effect, and are particularly suitable for septic acute lung injury caused by bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0036] . Figure 1 is a representative transmission electron microscopy image of LGG exosomes 4 prepared in the present invention;

[0037] . Figure 2 Zeta potential diagram of LGG exosome 4 prepared in the present invention;

[0038] . Figure 3 This is a graph of the protein concentration of LGG exosomes 4 prepared in the present invention;

[0039] . Figure 4 HE staining diagram showing that the LGG exosomes prepared by the present invention can overall reduce lung tissue damage in mice with acute lung injury caused by sepsis;

[0040] . Figure 5 Representative HE staining images showing that the LGG exosomes prepared by the present invention reduce local bleeding, peri-alveolar inflammation and alveolar inflammation in the lung tissue of mice with acute lung injury caused by sepsis;

[0041] . Figure 6 This is a result diagram showing that the LGG exosomes prepared by the present invention reduce the mortality of mice with acute lung injury caused by sepsis;

[0042] . Figure 7 This is a graph showing the lung targeting effect detection of the LGG exosomes prepared in the present invention after 24 hours.

[0043] . Figure 8 This is a graph showing the lung targeting effect detection of the LGG exosomes prepared in the present invention after 48 hours.

[0044] . Fig. 9 The experimental results of alveolar epithelial cells MLE-12 taking up LGG exosomes with a particle size of 50-120 nm;

[0045] . Fig.10 The experimental results of alveolar epithelial cells MLE-12 taking up LGG exosomes with a particle size of 50-200 nm;

[0046] . Fig.11 This is a graph showing the experimental results of the LGG exosomes prepared in the present invention reducing the mRNA expression of inflammatory factors in lung tissue of mice with acute lung injury caused by sepsis. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 work are within the scope of protection of the present invention.

[0048] .Herein, "and / or" includes any and all combinations of one or more of the relevant listed items.

[0049] .In this article, "plurality" means two or more than two, that is, it includes two, three, four, five, etc.

[0050] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0051] .As used in this specification, the term "about" typically means + / -5% of the stated value, more typically + / -4% of the stated value, more typically + / -3% of the stated value, more typically + / -2% of the stated value, even more typically + / -1% of the stated value, even more typically + / -0.5% of the stated value.

[0052] .In this specification, certain embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0053] .Example 1

[0054] .The Lactobacillus rhamnosus (LGG) strain used in Preparation Example 1-Preparation Example 4 is the LGG strain with the species number BNCC136673 of Beina Biotechnology.

[0055] Preparation Example 1

[0056] A method for preparing Lactobacillus rhamnosus exosomes, comprising the following steps:

[0057] .Lactobacillus rhamnosus strains were cultured in MRS broth at 37°C for 40 hours;

[0058] When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 10 min to obtain supernatant A1;

[0059] The supernatant A1 was centrifuged at 5000 g for 20 minutes to obtain supernatant B1;

[0060] The supernatant B1 was centrifuged at 10000g for 30 minutes to obtain supernatant C1;

[0061] . The supernatant C1 was filtered using a 0.45 μm filter, and the filtered supernatant C1 was ultracentrifuged at 10000 g for 70 minutes to obtain a precipitate D1;

[0062] .The precipitate D1 was resuspended in phosphate buffered saline and filtered using a 0.22 μm filter to obtain filtrate E1; the filtrate E1 was ultracentrifuged at 10,000 g for 70 minutes to obtain precipitate F1, which was LGG exosome 1.

[0063] .Wherein, the concentration of the phosphate buffered saline is 0.01 mol / L and the pH is 7.30.

[0064] The obtained LGG exosomes 1 were stained in 1% phosphotungstic acid for 1-2 minutes at room temperature and observed under JEM-1400FLASH transmission electron microscope. The particle size and potential of LGG exosomes 1 were measured in a particle size analyzer. The average particle size of LGG exosomes 1 was 72.0±23.9nm, and the potential of LGG exosomes 1 was -17.65±3.52mV.

[0065] Preparation Example 2

[0066] A method for preparing Lactobacillus rhamnosus exosomes, comprising the following steps:

[0067] .Lactobacillus rhamnosus strains were cultured in MRS broth at 37°C for 41 hours;

[0068] When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / ml, the culture suspension was centrifuged at 2000 g for 12.5 min to obtain supernatant A2;

[0069] The supernatant A2 was centrifuged at 5000 g for 25 minutes to obtain supernatant B2;

[0070] The supernatant B2 was centrifuged at 10000g for 35 minutes to obtain supernatant C2;

[0071] . The supernatant C2 was filtered using a 0.45 μm filter, and the filtered supernatant C2 was ultracentrifuged at 80,000 g for 70 minutes to obtain a precipitate D2;

[0072] .The precipitate D2 was resuspended in phosphate buffered saline and filtered using a 0.22 μm filter to obtain filtrate E2; the filtrate E2 was ultracentrifuged at 80,000 g for 95 minutes to obtain precipitate F2, which was LGG exosome 2.

[0073] .Wherein, the concentration of the phosphate buffered saline is 0.01 mol / L and the pH is 7.35.

[0074] The obtained LGG exosomes 2 were stained in 1% phosphotungstic acid for 1-2 minutes at room temperature and observed under JEM-1400FLASH transmission electron microscope. The particle size and potential of LGG exosomes 2 were measured in a particle size analyzer. The average particle size of LGG exosomes 2 was 64.0±13.4nm, and the potential of LGG exosomes 2 was -16.25±4.18mV.

[0075] Preparation Example 3

[0076] A method for preparing Lactobacillus rhamnosus exosomes, comprising the following steps:

[0077] .Lactobacillus rhamnosus strains were cultured in MRS broth at 37°C for 42 hours;

[0078] When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 15 min to obtain supernatant A3;

[0079] The supernatant A3 was centrifuged at 5000g for 30 minutes to obtain supernatant B3;

[0080] The supernatant B3 was centrifuged at 10000g for 40 minutes to obtain supernatant C3;

[0081] . The supernatant C3 was filtered using a 0.45 μm filter, and the filtered supernatant C3 was ultracentrifuged at 150,000 g for 70 minutes to obtain a precipitate D3;

[0082] .The precipitate D3 was resuspended in phosphate buffered saline and filtered using a 0.22 μm filter to obtain filtrate E3; the filtrate E3 was ultracentrifuged at 150,000 g for 120 minutes to obtain precipitate F3, which was LGG exosome 3.

[0083] .Wherein, the concentration of the phosphate buffered saline is 0.01 mol / L and the pH is 7.40.

[0084] The obtained LGG exosomes 3 were stained in 1% phosphotungstic acid for 1-2 minutes at room temperature and observed under JEM-1400FLASH transmission electron microscope. The particle size and potential of LGG exosomes 3 were measured in a particle size analyzer. The average particle size of LGG exosomes 3 was 72.0±17.5nm, and the potential of LGG exosomes 3 was -14.84±4.89mV.

[0085] Preparation Example 4

[0086] A method for preparing Lactobacillus rhamnosus exosomes, comprising the following steps:

[0087] .Lactobacillus rhamnosus strains were cultured in MRS broth at 37°C for 40 hours;

[0088] When the bacterial density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / ml, the culture suspension was centrifuged at 2000 g for 10 min to obtain supernatant A4;

[0089] The supernatant A4 was centrifuged at 5000 g for 20 minutes to obtain supernatant B4;

[0090] The supernatant B4 was centrifuged at 10000g for 30 minutes to obtain supernatant C4;

[0091] . The supernatant C4 was filtered using a 0.45 μm filter, and the filtered supernatant C4 was ultracentrifuged at 150,000 g for 70 minutes to obtain a precipitate D4;

[0092] .The precipitate D4 was resuspended in phosphate buffered saline and filtered using a 0.22 μm filter to obtain filtrate E4; the filtrate E4 was ultracentrifuged at 150,000 g for 70 minutes to obtain precipitate F4, which was LGG exosome 4.

[0093] .Wherein, the concentration of the phosphate buffered saline is 0.01 mol / L and the pH is 7.30.

[0094] The obtained LGG exosomes 4 were stained in 1% phosphotungstic acid for 1-2 min at room temperature and observed using a JEM-1400FLASH transmission electron microscope. Figure 1 As shown. The particle size and potential of LGG exosome 4 were measured in a particle size analyzer. The average particle size of LGG exosome 4 was 77.0±24.4nm. The Zeta potential of LGG exosome 4 was -16.87±2.68mV, as shown Figure 2 shown.

[0095] The protein concentrations of LGG exosomes 1, LGG exosomes 2, LGG exosomes 3 and LGG exosomes 4 prepared in Preparation Examples 1 to 4 were measured.

[0096] An exemplary method for determining protein concentration comprises the following steps:

[0097] .The obtained LGG exosomes were quantified using the BCA protein assay kit. The specific steps are as follows:

[0098] (1) Dilute the protein standard with sterile PBS buffer to a final concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively; take 20 μL of each concentration of protein standard and add it to the standard wells of a 96-well plate to prepare a standard curve;

[0099] (2) Dissolving LGG exosomes in sterile PBS buffer to obtain LGG exosome solution; taking 20 μL of the kudzu root exosome solution and adding it to the sample wells of a 96-well plate;

[0100] (3) Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 min.

[0101] (4) Determine the A562 value or the absorbance at a wavelength of 540-595 nm using an enzyme-labeled instrument;

[0102] .(5) Calculate the protein concentration in the sample based on the standard curve.

[0103] The protein concentrations of LGG exosome 1, LGG exosome 2, LGG exosome 3 and LGG exosome 4 prepared in Preparation Examples 1 to 4 were 1.08 μg / μL, 1.10 μg / μL, 1.05 μg / μL and 0.95 μg / μL, respectively. The protein concentration of LGG exosome 4, as Figure 3 shown.

[0104] .Example 2

[0105] LGG exosomes alleviate lung tissue damage in mice with acute lung injury induced by sepsis

[0106] .Mice grouping and LGG exosome pretreatment: Adult healthy male C57 mice aged 8 weeks were randomly divided into 3 groups, with 8-10 mice in each group, namely sham operation group, cecal ligation and puncture (CLP) group (i.e., sepsis acute lung injury group), and CLP+LGG exosome group (i.e., LGG exosome treatment group). LGG exosome administration route and dosage: LGG exosomes were administered by oral gavage at a dose of 2μg / g (exosome protein content μg / mouse body weight g). Once a day for 3 consecutive days. The control group was pretreated with an equal amount of PBS by oral gavage at the same time. CLP or sham operation was performed 24 hours after the end of gavage.

[0107] The present invention adopts the cecal ligation and puncture (CLP) method to establish a mouse model of acute lung injury caused by sepsis, and the model can better simulate the pathogenesis of acute lung injury caused by sepsis in the human body. The specific construction steps are as follows: ① Weigh the mouse with an electronic scale, and continuously inhale isoflurane for anesthesia. After confirming that the mouse is completely anesthetized, fix the mouse on a sterile operating table; ② Shave the abdominal hair with a razor to expose the abdomen, and disinfect the abdominal skin with iodine cotton balls; ③ Use iodine-disinfected ophthalmic scissors to cut the abdominal skin along the mouse's abdominal linea alba. The surgical incision is 1-2 cm, and it is separated layer by layer to expose the mouse's abdominal cavity; ④ Use sterilized tweezers to pull the mouse's cecum out of the body and place it on an alcohol-disinfected sterile gauze, ligate it with surgical sutures at 50% of the end of the cecum, and use a sterilized No. 21 puncture needle to pierce twice between the distal end of the cecum and the ligation position to form two perforations, and squeeze out a little mouse feces by hand; ⑤ Return the cecum to the abdominal cavity, suture the surgical incision layer by layer, and disinfect the wound with iodine; ⑥ Sterile saline is injected subcutaneously in the neck at a dose of 1 mL / 20 g to resuscitate the mouse with fluid, and the mouse is placed on a warming pad for 30 minutes; ⑦ Give sufficient water. Sham operation group: After laparotomy, the cecum was exposed and turned over. After the cecum was found, it was not ligated or punctured, but sutured. The mice were killed 24 hours after surgery and samples were collected.

[0108] .The specific steps of paraffin embedding and sectioning of lung tissue are as follows: ① Fixation: immediately place the removed lung tissue in tissue fixative to completely immerse the lung tissue in it, and fix it in the fixative for 24 hours at room temperature; ② Dehydration: pour out the fixative and add ethanol solutions of different concentration gradients for dehydration. 70% ethanol for 30 min (tissue must be fully covered); 80% ethanol for 30 min; 90% ethanol for 30 min; 100% ethanol for 30 min (this step is repeated twice); ③ Transparency: immerse the tissue in a solution of xylene: anhydrous ethanol in a ratio of 1:1 for 30 min, then immerse it in xylene for 20 min, and repeat this step twice (depending on the transparency of the tissue. If the tissue is transparent, it can be directly immersed in wax after standing in the xylene solution for a few seconds); ④ Paraffin embedding: immerse the lung tissue in paraffin at 60°C for 3 hours, and then cool it at room temperature; ⑤ Sectioning: Fix the paraffin on the wax box, use a paraffin slicer to slice the lung tissue, set the section thickness to 5μm, float the slices in a pathology tissue bleaching and drying instrument, place the slices on a slide, and spread the slices in the machine.

[0109] HE staining of lung tissue: According to the instructions of the HE staining kit, the specific steps are as follows: ① Drying: Take the mouse lung slices prepared above and dry them for about 4 hours; ② Dewaxing and rehydrating: Dewaxing and rehydrating the paraffin slices at room temperature. The operation sequence is xylene 5 minutes twice, xylene: anhydrous ethanol 1:1 solution for 5 minutes. Then rehydration operation is carried out, which is 100% ethanol 2 minutes (2 times), 95% ethanol 2 minutes, 90% ethanol 2 minutes, 80% ethanol 2 minutes, 70% ethanol 2 minutes, double distilled water 2 minutes; ③ Hematoxylin staining: Put it in hematoxylin stain for 5 minutes (placed on a horizontal shaker); ④ Differentiation: Rinse with running water for 5 minutes, put it in differentiation solution and let it stand for 30 seconds, and soak it in distilled water (let it stand) for 15 minutes; ⑤ Eosin staining: Put it in eosin stain for 3 minutes (horizontal shaker), rinse with running water for 5 minutes, observe under a microscope, and restain it when the color is light. Soak in distilled water (let it stand) for 3 minutes; ⑥ Dehydration: 95% ethanol for 1 minute (if the eosin is darker, the time can be extended or dehydration can be started from 70% alcohol), 100% ethanol for 1 minute (2 times); ⑦ Transparent: xylene: anhydrous ethanol 1:1 solution for 1 minute, xylene for 5 minutes (2 times); ⑧ Sealing: Use neutral gum to seal (note that the cover glass cannot be moved and bubbles cannot be generated during the sealing process); ⑨ Observation: Observe the pathological changes of the lungs under an optical microscope on the above-stained sections.

[0110] .Pathological observation and scoring of lung tissue: Observe the histopathological changes of lung tissue under HE staining under a microscope to evaluate the severity of lung injury. The histopathological changes of acute lung injury in sepsis are mainly manifested as congestion and thickening of the alveolar wall, decreased air content in the alveolar cavity, leakage of red blood cells and infiltration of inflammatory cells. After HE staining, inflammatory cell infiltration, edema and damage of the entire left lung tissue can be observed under a low-power microscope to evaluate the distribution of lung damage; while under a high-power microscope, the alveolar structure can be identified, and the degree of lung injury can be evaluated and scored.

[0111] . Scoring method:

[0112] .0 points: normal lung tissue structure, no obvious pathological damage;

[0113] .1 point: The lesion is mild, with mild thickening of the alveolar wall, a slight increase in the number of red blood cells and inflammatory cells on the wall, and a small amount of exudate in the alveolar cavity.

[0114] .2 points: The alveolar wall is significantly thickened and congested, the number of nucleated cells on the wall is significantly increased, and there is serous fluid and a small amount of leaked red blood cells in the alveolar cavity.

[0115] .3 points: The alveolar wall is significantly congested and thickened, the number of alveolar cavities decreases, some cavities are occupied by serous fluid, and the number of nucleated cells on the alveolar wall increases further.

[0116] .4 points: On the basis of the above lesions, local inflammatory cells accumulate focally, accompanied by focal hemorrhage.

[0117] .This example verifies the effect of LGG exosomes prepared in Preparation Examples 1 to 4 on reducing (preventing) lung tissue damage in mice with acute lung injury caused by sepsis. The results are shown in Table 1.

[0118] Table 1 Lung tissue injury scores of mice with acute lung injury caused by sepsis in each group

[0119] . Figure 4 The overall lung injury of each experimental group is shown. Figure 5 The bleeding, peri-alveolar inflammation, and alveolar inflammatory lesions of each experimental group are shown. Figure 4 and Figure 5 As shown, the HE-stained pathological sections of the septic acute lung injury group showed that the mice in the septic acute lung injury group had severe local hemorrhage in the lung tissue, peri-alveolar inflammation, and alveolar inflammatory lesions; while the HE-stained pathological sections of the LGG exosome treatment group showed that the LGG exosomes prepared by the present invention can significantly reduce the lung tissue damage of mice with septic lung injury from multiple dimensions such as reducing local hemorrhage in the lung tissue, peri-alveolar inflammation, and alveolar inflammatory lesions.

[0120] .In order to study the effect of LGG exosomes on the survival rate of mice, LGG exosomes pretreatment was performed to observe the survival rate of mice.

[0121] .Mice grouping and LGG exosome treatment: Adult healthy male C57 mice aged 8 weeks were randomly divided into 3 groups, with 8 mice in each group, namely sham operation group, sepsis acute lung injury group, and LGG exosome treatment group. LGG exosome administration route and dosage: LGG exosomes were administered by oral gavage at a dose of 2μg / g (protein content μg / mouse body weight g). Once a day for 3 consecutive days. The control group was pretreated with an equal amount of PBS by oral gavage at the same time. CLP or sham operation was performed 24 hours after the end of gavage. Drinking water and diet were gradually restored 24 hours after the operation, and the mortality of mice was observed.

[0122] The results are as follows Figure 6 The results showed that the mortality rate of mice in the sham operation group was 0% 3 days after surgery, the survival rate of mice in the sepsis acute lung injury group was 100%, and the mortality rate of mice in the LGG exosome treatment group was 37.5%. The above results show that after pretreatment (treatment) with the LGG exosomes prepared by the present invention, the mortality rate of mice in the sepsis acute lung injury group was significantly reduced.

[0123] .Example 3

[0124] .To study the targeting effect of LGG exosomes at the whole animal level, the present invention administered LGG exosomes labeled with DIR fluorescent dye (red) to mice by gavage at a dose of 2 μg / g. After 24 or 48 hours, the fluorescence intensity of lung tissue was detected using a small animal in vivo imaging system (IVIS). Figure 7 As shown in the figure, the colored part is the LGG exosomes taken up by the lung tissue. After 48 hours, the fluorescence intensity of the lung tissue is as follows Figure 8 As shown, the colored marked part in the figure is the LGG exosomes taken up by the lung tissue. The results show that the LGG exosomes provided by the present invention can be directly taken up by the lung tissue.

[0125] .The steps of the experiment of LGG exosome uptake by alveolar epithelial cells MLE-12 are as follows: LGG exosomes were fluorescently labeled with the fluorescent dye PKH267 (green). Alveolar epithelial cells MLE-12 were incubated with PKH267-labeled LGG exosomes (0.2μg / mL) for 4 hours. After incubation, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. After staining with DAPI reagent for 3 minutes, the sections were sealed and observed under a fluorescence microscope. LGG exosomes were filtered twice using a 0.22μm filter before adding to the cells.

[0126] . Fig. 9 and Fig.10The results of the uptake of LGG exosomes with a particle size range of 50-120nm (determined by an exosome single particle size analyzer (chip)) and LGG exosomes with a particle size range of 50-200nm by alveolar epithelial cells MLE-12 are shown respectively. The above results show that alveolar epithelial cells can take up more LGG exosomes with a particle size range of 50-120nm.

[0127] .Example 4

[0128] LGG exosomes reduce the mRNA levels of inflammatory factors in lung tissue of mice with acute lung injury caused by sepsis.

[0129] The lung tissues of mice in each group were obtained as in Example 2.

[0130] . Extraction of total RNA from tissues: Extract total RNA from tissues according to the instructions of the RNAiso Plus kit. The specific steps are as follows: (1) Take out the frozen tissue from the liquid nitrogen tank, weigh 100 mg, put it into an RNase-free and autoclaved EP tube, add 1.0 mL of RNAisoPlus to each tube, and put it into a low-temperature fully automatic tissue grinding and homogenization instrument for homogenization. (2) Let the homogenized tissue stand on ice for 10 minutes. Centrifuge at 12000g at 4℃ for 5 minutes, and aspirate the supernatant into a new 1.5 mL enzyme-free EP tube. (3) Add 200 μl of chloroform to the tube. After confirming that the tube cap is tightly closed, shake the EP tube vigorously by hand for 15 seconds. After the solution is fully emulsified, let it stand on ice for 3 minutes. (4) Centrifuge at 12000g at 4℃ for 15 minutes. Carefully remove the EP tube from the low-temperature centrifuge. At this time, you can see that the liquid in the tube is divided into three layers: the top layer is the colorless supernatant, the middle layer is the white DNA-protein layer, and the bottom layer is the colored phenol-chloroform layer. Carefully aspirate the supernatant (RNA is present in this layer) and transfer it to another new RNase-free EP tube. (5) Add an equal volume of ice isopropanol solution to the supernatant, invert the tube to mix thoroughly, and then place it on ice for 10 minutes. (6) Centrifuge at 12000g in a low-temperature (4°C) centrifuge (set the time to 15 minutes). After centrifugation, a white precipitate appears at the bottom, which is RNA. (7) Carefully discard the supernatant, slowly add 75% ice ethanol along the tube wall (be careful not to touch the precipitate), gently invert it upside down for more than 10 times, thoroughly wash the tube wall, centrifuge it at 4°C, 12000g for 5 minutes, and then carefully discard the ethanol. (8) Dry the precipitate at room temperature for 2-5 minutes, observe the amount of precipitate with the naked eye, and add a certain amount of DEPC water according to the amount of RNA precipitate (you can use a pipette tip to gently blow) to completely dissolve the RNA precipitate. (9) Use a UV spectrophotometer to detect the concentration and purity of RNA. Record OD260 / 280 and OD260 / 230. RNA with a ratio between 1.8-2.0 and 2.0-2.4, respectively, is of good quality and can be used in subsequent experiments. All the above supplies were sterilized by high pressure first, and the operation was carried out on ice.

[0131] .RNA reverse transcription: Perform relevant operations according to the instructions of PrimeScript RT reagent Kit with gDNA Eraser.

[0132] .Reverse transcription reaction: Set the reaction program as follows: 37℃ for 15min; 85℃ for 5sec. The successfully reversed cDNA is stored at -20℃.

[0133] .PCR amplification: Prepare the reaction system and set the reaction program according to the TB Green Premix Ex Taq II (Takara) manual. The reaction was carried out on ice and 10 μl of the reaction system was used. After the reaction solution was prepared, gently mix it and centrifuge it for 10 seconds. Use the two-step PCR amplification standard program.

[0134] Primer design and sequence: Primer design was performed using Primer 6.0 software. The primer sequences for the following related genes were designed by comparing with the genes in the U.S. National Library gene bank:

[0135] The results are as follows Fig.11 As shown, the LGG exosomes prepared by the present invention can significantly reduce the mRNA expression of inflammatory factors in the lung tissue of mice with acute lung injury caused by sepsis, and can even reduce it to a level similar to that of the sham operation group.

[0136] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which are all protected by the present invention.

Claims

1. Use of Lactobacillus rhamnosus exosomes in the preparation of a medicament for treating and / or preventing acute lung injury caused by sepsis in a subject, characterized in that: The acute lung injury caused by sepsis is an acute complication of sepsis, the sepsis is caused by bacterial infection, and the particle size of the Lactobacillus rhamnosus exosomes ranges from 50 to 120 nm.

2. The use according to claim 1, characterized in that The medicine is an oral preparation.

3. The use according to claim 1, characterized in that A single dose of the drug includes 1.5-2.5 mg of Lactobacillus rhamnosus exosomes / kg.

4. The use according to claim 3, characterized in that The drug is prepared in the form of a dosage unit, and is formulated as an independent dosage form containing 90-150 mg of Lactobacillus rhamnosus exosomes for single use, and at least 3 independent dosage forms are formulated as one dosage unit.

5. The use according to claim 1, characterized in that The drug is intended to be directly targeted to the lungs of the subject and taken up by the lung tissue of the subject.

6. The use according to claim 1, characterized in that The subject's lung injury score was 3 or 4 points.

7. The use according to claim 1, characterized in that The protein concentration of the exosomes is 0.9-1.1 μg / μL.

8. The use according to claim 1, characterized in that The symptoms of acute lung injury caused by sepsis include one or more of local hemorrhage in lung tissue, peri-alveolar inflammation and alveolar inflammatory lesions.

9. The use according to claim 1, characterized in that The medicament is used to reduce the inflammatory response in the lungs of the subject.

10. The use according to claim 1, characterized in that The preparation method of the Lactobacillus rhamnosus exosomes comprises: S101 When the strain density of Lactobacillus rhamnosus culture suspension reaches 2×10 9 CFU / mL, the culture suspension was centrifuged at 2000 g for 10 to 15 minutes to obtain supernatant A; S102: centrifuging the supernatant A at 5000 g for 20 to 30 minutes to obtain supernatant B; S103 centrifuging the supernatant B at 10000 g for 30 to 40 minutes to obtain supernatant C; S104: filtering the supernatant C using a 0.45 μm filter, and ultracentrifuging the filtered supernatant C at 10,000 to 150,000 g for 70 minutes to obtain a precipitate D; S105: resuspend the precipitate D and filter it using a 0.22 μm filter to obtain a filtrate E. The filtrate E is ultracentrifuged at 10,000 to 150,000 g for 70 to 120 minutes to obtain a precipitate F, i.e., the Lactobacillus rhamnosus exosomes.

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