Use of lactobacillus-derived extracellular vesicles for prevention and / or treatment of lung injury and / or intestinal injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of lactobacillus-derived extracellular vesicles in prevention and / or treatment of lung injury and / or intestinal injury. The extracellular vesicles (LrEVs) derived from lactobacillus reuteri are extracted from a culture solution of lactobacillus reuteri, and have a bubble-shaped structure of a nano-sized double-layer membrane. The LrEVs synchronously reduce excessive secretion of proinflammatory factors in lung and small intestine tissues of a body, and pathological injury is relieved; meanwhile, imbalance and activation of innate lymphocyte subpopulations in the lung and the small intestine of the body are synchronously regulated, the immune homeostasis of the body is maintained, the weight loss of the body is delayed, and the survival rate of the body is improved. That is, the LrEVs has a one-drug double-target characteristic, shows that the LrEVs synchronously have a remarkable treatment effect on the lung injury and the intestine injury, and can be used for developing drugs or healthy functional foods to prevent and / or treat the lung injury and / or the intestine injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the use of extracellular vesicles derived from lactobacilli in preventing and / or treating lung injury and / or intestinal injury. Background Art

[0002] Influenza A virus (IAV) can cause zoonotic respiratory infections, posing a huge threat to human health. The main symptoms of infection include cough, fever and headache. In addition, both adult and pediatric patients experience gastrointestinal symptoms, including vomiting and diarrhea. Studies have reported that after influenza virus infection of mice, immune damage is not only manifested in the lungs, but also particularly in the small intestine. Currently, the most effective means of preventing and controlling influenza is vaccination, but influenza viruses continue to mutate through antigenic drift and antigenic shift, requiring continuous vaccine development and annual immunization. In addition, the immune response induced by influenza vaccines is not optimal in many subjects, especially in young children and the elderly who are at risk of severe influenza, further reducing the protective effect of influenza vaccines. Drugs are the second line of defense against influenza. Currently, clinical anti-influenza virus drugs all target important proteins of the virus. The high variability of influenza viruses makes it very easy to produce drug-resistant strains, making the drugs ineffective. In addition, taking antiviral drugs such as oseltamivir / mabaloxavir may cause gastrointestinal adverse reactions such as nausea, vomiting, diarrhea, and abdominal pain. Studies have shown that excessive natural immune responses are closely related to the severity and increased mortality of influenza virus infection. The body's excessive immune response, including immune cell infiltration and the production of cytokine storms, can lead to lung and intestinal immune pathological damage, causing acute respiratory distress syndrome and even multiple organ failure and death. Antiviral drug treatment cannot control the severe inflammatory response caused by influenza infection. Therefore, a therapeutic strategy that targets excessive inflammatory responses to reduce lung and intestinal pathological damage is the key to treating severe influenza. The development of new drugs for the simultaneous treatment of influenza lung and intestinal damage is an urgent problem to be solved in clinical practice.

[0003] Extracellular vesicles (EVs) are produced by species in all domains of life and play a key role in regulating physiological and pathological processes. Probiotic EVs are a type of tiny membrane vesicle structure secreted by probiotics that mediate the beneficial effects of probiotics while minimizing the potential risks of consuming live microorganisms. There is increasing evidence that probiotic EVs can regulate the host's innate and adaptive immune systems. Lactobacillus reuteri is an important probiotic, and its EVs have multiple biological activities and can play a key role in host health. There are significant functional differences in Lactobacillus reuteri extracellular vesicles from different sources, which are mainly reflected in the regulation of the host immune system, antibacterial activity, anti-inflammatory effects, etc. For example, the extracellular vesicles of Lactobacillus reuteri ATCC PTA 5289 (USPatent No.10,568,935B2 Probiotic compositions for oral health) have significant antibacterial activity and can inhibit the growth of pathogens in the oral cavity. The extracellular vesicles of Lactobacillus reuteri SD2112 have unique advantages in promoting skin health and reducing skin inflammation. However, EVs therapeutic drugs that can simultaneously treat influenza virus-mediated lung and intestinal damage are still very limited and are mostly in the research stage.

[0004] Therefore, the development of new drugs for the simultaneous treatment of influenza-induced lung and intestinal damage is an urgent clinical issue that needs to be addressed. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the problem that existing anti-influenza virus drugs can cause gastrointestinal adverse reactions such as nausea, vomiting, diarrhea, abdominal pain, etc., and have no therapeutic effect on intestinal symptoms. There are significant differences in the functions of extracellular vesicle-based drugs from different sources, and these defects and deficiencies are mainly reflected in the regulation of the host immune system, antibacterial activity, anti-inflammatory effects, etc., and to provide the use of extracellular vesicles derived from lactobacilli in the prevention and / or treatment of lung injury and / or intestinal injury.

[0006] The purpose of the present invention is to provide an extracellular vesicle.

[0007] Another object of the present invention is to provide a drug for preventing and / or treating lung injury and / or intestinal injury.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention protects the use of extracellular vesicles derived from lactobacillus in preventing and / or treating lung injury and / or intestinal injury. The lactobacillus is Lactobacillus reuteri (Lactobacillus reuteri) EHA2 strain, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2019, with a deposit number of GDMCC NO: 60758.

[0010] Preferably, the lung injury to be treated is lung injury mediated by influenza virus infection.

[0011] Preferably, the intestinal damage is intestinal damage mediated by influenza virus infection.

[0012] The above-mentioned lactobacillus-derived extracellular vesicles (LrEVs) are extracted from the culture fluid of Lactobacillus reuteri EHA2 and have a nano-sized double-layer membrane bubble structure. The LrEVs simultaneously reduce the excessive secretion of proinflammatory factors in the lungs and small intestinal tissues of the body infected with influenza virus, and reduce pathological damage; and simultaneously regulate the imbalance and activation of the inherent lymphocyte subpopulations in the lungs and small intestine of the body, maintain the body's immune homeostasis, delay the body's weight loss, and improve the body's survival rate. That is, the LrEVs have the characteristics of one drug with two targets, which is manifested as a significant therapeutic effect on influenza virus-mediated lung damage and intestinal damage at the same time, and can be used to develop drugs or health functional foods to reduce, prevent and / or treat lung damage and / or intestinal damage mediated by influenza virus infection.

[0013] Furthermore, the influenza virus is influenza A virus.

[0014] Furthermore, the influenza A virus includes H1N1 influenza virus.

[0015] Specifically, the H1N1 influenza virus is A / Puerto Rico / 8 / 34 H1N1 influenza virus, namely PR8 influenza virus.

[0016] The present invention protects an extracellular vesicle, which is prepared using lactobacillus. The lactobacillus is the EHA2 strain of Lactobacillus reuteri, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2019, with a deposit number of GDMCC NO: 60758.

[0017] Furthermore, the preparation method of the extracellular vesicles comprises the following steps: preparing an expanded culture medium of lactobacillus, collecting the obtained culture medium for a first centrifugation, collecting the supernatant for a first microfiltration, taking the microfiltrate for ultrafiltration, taking the ultrafiltrate for a second centrifugation, taking the supernatant for a second microfiltration, collecting the microfiltrate for ultracentrifugation, taking the precipitate for washing, and obtaining the extracellular vesicles.

[0018] Furthermore, the preparation of the expanded culture fluid of the lactobacillus comprises the following steps: using MRS solid culture medium to activate the bacteria, aseptically culturing at 37°C under anaerobic conditions for 36 to 48 hours, and then using MRS liquid culture medium to expand the culture for 36 to 48 hours.

[0019] Furthermore, the speed of the ultracentrifugation is 100000 g or above.

[0020] Furthermore, the conditions of the first centrifugation are (0-8)°C, (10,000-16000)g, (15-30)

[0021] min. In centrifugal conditions, g is used here as a unit of measurement for relative centrifugal force, indicating the magnitude of centrifugal force. During the centrifugation process, the centrifugal force exerted on the sample is determined by the rotation speed of the centrifuge and the centrifugal radius. In order to facilitate the comparison of the magnitude of centrifugal force under different centrifugal conditions, the concept of relative centrifugal force (RCF) was introduced and its unit was defined as g. The g here is actually a dimensionless unit, which represents the ratio of the centrifugal force exerted on the sample during the centrifugation process to the gravitational acceleration on the earth's surface.

[0022] Furthermore, the second centrifugation is performed twice, first centrifugation at a low speed, taking the supernatant and then performing high-speed centrifugation, the conditions of the low-speed centrifugation are (0-8)°C, (3,000-5000)g, (15-30)min, and the conditions of the high-speed centrifugation are (0-8)°C, (10,000-15000)g, (15-30)min.

[0023] Furthermore, the molecular weight cut-off of the ultrafiltration membrane is 80 to 120 kD, more preferably 100 kD.

[0024] Furthermore, the pore size of the filter membranes for the first microfiltration and the second microfiltration is 0.22 μm.

[0025] Furthermore, the washing is performed using a PBS (Phosphate Buffered Saline) solution.

[0026] Specifically, as an optional method, the method for preparing the extracellular vesicles comprises the following steps:

[0027] S1. The bacteria were activated using MRS solid medium and cultured in an anaerobic workstation at 37°C for 48 hours, and then expanded using MRS liquid medium for 36 hours to obtain a culture solution;

[0028] S2. Collect the culture fluid obtained in step S1, centrifuge at 15,000g for 25 minutes at 4°C, collect the supernatant and then filter it through a 0.22μm filter membrane; the filtrate is concentrated by an Amicon ultrafiltration system on a 100kD filter membrane, the concentrate is vortex-mixed and centrifuged at 4°C, 4,000g and 12,000g for 20 minutes, then filtered through a 0.22μm filter membrane, collect the filtrate, centrifuge it in an ultracentrifuge at 4°C and 160,000g for 100 minutes, remove the supernatant, retain the precipitate, and wash the precipitate three times with sterile PBS to obtain extracellular vesicles.

[0029] Furthermore, the extracellular vesicles are oval in shape and have a double-layer membrane bubble structure.

[0030] Furthermore, the particle size distribution of the extracellular vesicles is mainly between 50 and 200 nm, with a peak particle size of 99 nm.

[0031] The present invention protects a drug for preventing and / or treating lung injury and / or intestinal injury, wherein the drug comprises the extracellular vesicle and a pharmaceutically acceptable excipient.

[0032] Preferably, the lung injury and / or intestinal injury is lung injury and / or intestinal injury mediated by influenza virus infection.

[0033] Preferably, the influenza virus is influenza A virus.

[0034] Furthermore, the influenza A virus includes H1N1 influenza virus.

[0035] Specifically, the H1N1 influenza virus is A / Puerto Rico / 8 / 34 H1N1 influenza virus, namely PR8 influenza virus.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The lactobacillus-derived extracellular vesicles (LrEVs) of the present invention are extracted from the culture fluid of Lactobacillus reuteri and have a double-layer membrane bubble structure of nanometer size. The LrEVs diffuse to the lung tissue and intestinal tissue after oral absorption, synchronously reduce the excessive secretion of proinflammatory factors in the lung and small intestinal tissue of the body, and alleviate pathological damage; and synchronously regulate the imbalance and activation of the inherent lymphocyte subpopulations in the lung and small intestine of the body, maintain the body's immune homeostasis, delay the body's weight loss, and improve the body's survival rate. That is, the LrEVs have a dual-target characteristic of one drug, which is manifested as a significant therapeutic effect on lung damage and intestinal damage (especially lung damage and intestinal damage mediated by influenza virus infection). Therefore, the LrEVs of the present invention can be used to develop drugs or health functional foods to prevent and / or treat lung damage and / or intestinal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure a shows the appearance of exosomes under a transmission electron microscope, and Figure b shows the particle size distribution of exosome nanoparticle tracking analysis.

[0039] Figure 2 These are statistical graphs of fluorescence distribution data of internal organs of mice after continuous oral administration of fluorescently labeled LrEVs for 7 days, where Figure a is the fluorescence graph of each organ of the mice, and Figure b is the statistical graph of the fluorescence intensity of each organ of the mice.

[0040] Figure 3 These are statistical graphs showing the effects of LrEVs on the survival rate and weight changes of mice infected with the H1N1 influenza virus, wherein Figure a is a statistical graph showing the mortality rate of mice, and Figure b is a statistical graph showing the weight changes of mice.

[0041] Figure 4 Statistical graph of the effect of LrEVs on the virus titer in the lungs of mice infected with H1N1 influenza virus; data are expressed as mean ± SD and analyzed by t-test, asterisks indicate significant differences relative to the H1N1 group (H1N1 + PBS), * indicates P < 0.05; ** indicates P < 0.01.

[0042] Figure 5 Statistical graph showing the effect of LrEVs on the pathological changes in the lungs and small intestine of mice infected with H1N1 influenza virus.

[0043] Figure 6 Statistical graph of the effect of LrEVs on inflammatory factors in the lungs and small intestine of mice infected with H1N1 influenza virus; protein levels of inflammatory factors in the small intestine and BALF; data are expressed as mean ± SD and analyzed by t-test, asterisks indicate differences relative to the H1N1 group (H1N1 + PBS), * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; # indicates significant difference relative to the control group (PBS + PBS), # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001.

[0044] Figure 7 Figure 1 is a statistical diagram of the effect of LrEVs on ILCs in the lungs and small intestine of mice infected with H1N1 influenza virus; Figure a is a statistical diagram of the gating strategy of flow cytometry analysis of ILC1 in the small intestine of H1N1 infected mice, and Figures c and f are statistical diagrams of the gating strategy of flow cytometry analysis of ILC2 (c), NCR+ILC3 and NCR-ILC3 (f) in the small intestine of H1N1 infected mice; Figures b, d, e, and g are statistical diagrams of the gating strategy of flow cytometry analysis of ILC1, ILC2, NCR+ILC3 and NCR-ILC3 in the lungs and small intestine, respectively. + ILC3 and NCR -Statistical graph of the percentage of ILC3 cells (n=6); ILC1s were defined as: CD45+CD127+Lin-(CD3, CD45R, CD11b, Ly-G6)NKp46+RORγt-T-bet+Eomes-; ILC2s were defined as: CD45+CD127+Lin-RORγt-GATA3hi; NCR+ILC3s were defined as: NCR-ILC3s were defined as: CD45+CD127+Lin-RORγt+GATA3low NKp46-CCR6-; SI: small intestine; Lung: lung; Data are expressed as mean ± SD and analyzed by t-test, asterisks indicate differences relative to the H1N1 group (H1N1 + PBS), * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; # indicates significant difference relative to the control group (PBS + PBS), # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, ns indicates no significant difference.

[0045] Figure 8 Figure 1 shows the effect of LrEVs on the immune response of ILCs in the lungs and small intestine of mice infected with H1N1 influenza virus. Figure 1 shows the expression of IFN-γ in the lungs and small intestine. + Data statistics of the percentage of ILC1 (n=6); cd shows the percentage of IL-4 in the lung and small intestine. + Statistical diagram of the percentage of ILC2 (n=6); Figure ef shows IL-17 in the lung and small intestine + NCR - Statistical graph of the percentage of ILC3s (n=6); SI: small intestine; Lung: lung; Data are expressed as mean ± SD and analyzed by t-test, asterisks indicate differences relative to the H1N1 group (H1N1+PBS), * indicates P﹤0.05, ** indicates P﹤0.01, *** indicates P﹤0.001; **** indicates P﹤0.0001; # indicates significant difference relative to the control group (PBS+PBS), # indicates P﹤0.05, ## indicates P﹤0.01, ### indicates P﹤0.001; #### Indicates P﹤0.0001. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Lactobacillus reuteri EHA2 strain was deposited in Guangdong Microbiological Culture Collection Center on September 2, 2019, with the deposit number GDMCC NO: 60758

[0049] Figure 1 a means Figure 1 Figure a in Figure 2 a means Figure 2 Figure a in Figure 7 ab means Figure 7 The naming meanings of Figure a and Figure b in the figure are similar.

[0050] The H1N1 influenza virus used in the example is A / Puerto Rico / 8 / 34 H1N1 influenza virus, namely PR8 influenza virus.

[0051] Example 1 Cultivation of Lactobacillus reuteri EHA2 and Preparation of Extracellular Vesicles

[0052] (1) Cultivation of Lactobacillus reuteri A2:

[0053] The Lactobacillus reuteri EHA2 was activated by using MRS solid culture medium, and sterile static culture was carried out at 37° C. in an anaerobic workstation for 48 hours. Two loops of the activated strain were inoculated into MRS broth culture medium, and sterile static culture was carried out at 37° C. in an anaerobic workstation for 48 hours. Then, the culture was expanded, and 200 μL of the revived strain was aseptically inoculated into a 250 mL culture bottle containing 100 mL of MRS culture medium, and sterile culture was carried out at 37° C. in an anaerobic workstation for 36 hours to obtain a culture solution.

[0054] (2) Preparation of extracellular vesicles: The culture medium obtained in step (1) was centrifuged at 4°C and 12,000 g for 25 min. The supernatant was filtered through a 0.22 μm filter membrane to remove residual bacteria, cell debris, and large particles such as polymers. The filtrate was filtered through an Amicon ultrafiltration system (Merck Millipore) was concentrated on a 100kD filter membrane, the concentrate was retained, the filter membrane and filter were rinsed, and then transferred to a 50mL sterile centrifuge tube; the filter membrane and filter were rinsed twice with sterile PBS, and all the retained liquid was collected in a sterile centrifuge tube; the concentrate was vortexed and centrifuged at 4°C, 4,000g and 12,000g for 20min to remove the coagulant, and then the supernatant was filtered through a 0.22μm filter membrane and the filtrate was collected; the obtained filtrate was centrifuged at 4°C and 150,000g for 120min in an ultracentrifuge, the supernatant was removed, and the precipitate was retained, which was the extracellular vesicles containing a small amount of impurities; the extracellular vesicle precipitate was washed three times with 30mL sterile PBS, and then resuspended with 1mL PBS to obtain relatively pure extracellular vesicles (Lactobacillusreuteri EVs, referred to as LrEVs).

[0055] Example 2 Transmission electron microscopy detection of LrEVs

[0056] 1. Experimental methods

[0057] The transmission electron microscopy sample of LrEVs obtained in Example 1 was prepared by the hanging drop method. The main steps were as follows: the concentration of LrEVs was adjusted to about 1×10 11 particles / mL, and 10 μL of the sample suspension was carefully dropped onto a 200-mesh copper grid with a support membrane using a pipette; let it stand at room temperature for 1 min, and use filter paper to absorb the excess liquid. When it is almost completely dry, use a pipette to absorb a drop of 2% (w / v) sodium phosphotungstate dye (pH 7.0), dye for 1 to 2 min, and use filter paper to absorb the excess dye, let it stand at room temperature, and observe it after it is dried. The prepared LrEVs were observed and photographed using a HT7800 (JEOL, Tokyo, Japan) transmission electron microscope. The acceleration voltage of the transmission electron microscope was set to 80 kV, and other parameters were set to default parameters.

[0058] 2. Experimental results

[0059] The ultrastructure of LrEVs obtained in Example 1 under transmission electron microscopy is as follows Figure 1 As shown in a, the obtained extracellular vesicles LrEVs present an oval bubble structure containing a double membrane.

[0060] Example 3 Detection of LrEVs particle size distribution and concentration using nanoparticle tracking analysis (NTA)

[0061] 1. Experimental methods

[0062] The LrEVs obtained in Example 1 were adjusted to an appropriate concentration, and then 1000 μL was loaded into the NS300 nanoparticle tracking analyzer (Malvern Instruments, Malvern, UK) for detection. The parameters maintained during the experiment included a camera level of 7, a detection threshold of 10, a flow rate of 50, a capture number of 3, a capture duration of 40-60 s, and a temperature of 25°C. NanoSight NTA 3.3 software was used for data analysis. Other parameters used the default parameters.

[0063] 2. Experimental results

[0064] The particle size results of LrEVs are as follows Figure 1 As shown in b, the results show that the particle size distribution of LrEVs is mainly between 50-200nm, with a peak particle size of 99nm.

[0065] Example 4 Biodistribution of oral LrEVs in vivo

[0066] 1. Experimental methods

[0067] The LrEVs or PBS obtained in Example 1 were incubated with the near-infrared fluorescent dye DiR (5 μM, ThermoFisher) for 20 min at 37°C in the dark. The unbound dye was then removed by centrifugation at 150,000g and 4°C for 2 hours, and the LrEVs particles were resuspended in PBS. The mice were orally administered Dir-labeled LrEVs (10 mg / kg) once a day for 7 consecutive days. After 7 days, the mice were imaged using a small animal in vivo imager. In addition, organs were quickly removed from euthanized animals and imaged in vitro. The relative intensity was measured and compared with the vehicle (PBS) control and the free dye control.

[0068] 2. Experimental results

[0069] The results are as follows Figure 2 As shown, after oral administration for 7 consecutive days, strong fluorescent signals were detected in the liver (p<0.005), lung (p<0.005), and gastrointestinal tract (p<0.01) of mice, indicating that LrEVs had significant accumulation in these organs.

[0070] Example 5 Experiment on the life protection of mice infected with influenza virus by LrEVs

[0071] 1. Experimental methods

[0072] SPF BALB / c mice, female, 6-8 weeks old, were purchased from Hunan Slake Jingda Co., Ltd. The mice were randomly divided into a normal group (i.e., PBS), a virus infection group (i.e., H1N1), a LrEVs high-dose treatment group (i.e., LEVs-H, with a dosing concentration of 10 mg / kg / d), a LrEVs low-dose treatment group (i.e., LEVs-L, with a dosing concentration of 5 mg / kg / d), and an oseltamivir positive control drug treatment group (i.e., Oseltamivir or Ose, with a dosing concentration of 60 mg / kg / d), with 10 mice in each group. After the mice were anesthetized with isoflurane, the virus (2.5LD 50 / mouse, 50μL), and the control group mice were simulated infected with influenza virus by intranasal instillation of PBS (50μL). The mice in the LrEVs treatment group began to be administered by gavage 2 days before virus infection, once a day (0.2mL / time), for 7 consecutive days. The mice in the oseltamivir positive control drug treatment group began to be administered by gavage on day 0 after infection, once a day (0.2mL / time), for 5 consecutive days. The model group and the blank control group mice were given solvent PBS (Phosphate Buffered Saline) by gavage. The weight changes of mice were recorded every day, the incidence of mice was observed and the number of deaths was recorded. The observation was continued for 15 days, and the survival rate, average survival time and average weight change of each group of mice were calculated based on the observation results.

[0073] 2. Experimental results

[0074] The results showed that the mice in the normal group were in good spirits, responsive, and had shiny black fur. They also had normal breathing, good appetite, and normal food and water intake. During the entire observation period, no mice in the normal group died, and their health remained stable. In the model group, the mice gradually lost weight 3 days after being infected with the influenza virus. On the 5th day, the symptoms worsened further, with the mice huddled together, breathing rapidly, and having obvious abdominal breathing. Subsequently, the symptoms of the mice in the model group continued to worsen, with symptoms such as shivering and lethargy, and a sharp drop in weight. Starting on the 7th day, mice in the model group began to die. However, after the 12th day after being infected with the influenza virus, the mice that had not died began to gradually recover and their weight began to recover. Compared with the model group, the mice in the positive control drug oseltamivir group had the mildest symptoms, and the weight of the mice began to recover on the 10th day after being infected with the influenza virus ( Figure 3 b), no dead mice were found; the infection symptoms of mice in the high-dose LrEVs group were milder than those in the model group, and a few mice with severe symptoms died. The weight of mice began to recover on the 10th day after infection. Some mice in the low-dose LrEVs group died, and the weight of mice that did not die began to recover on the 11th day after infection. It can be seen from the survival curve ( Figure 3 a), the survival rate of mice in the normal group and the positive control drug group was 100%, and their survival curves overlapped; the survival rate of the model group was 10%, the survival rate of the high-dose LrEVs group was 70%, and the survival rate of the low-dose LrEVs group was 50%. Compared with the model group, the survival rate of the two dose groups of LrEVs was significantly different (p<0.01, p<0.05). The therapeutic effect of high-dose LrEVs (p<0.01) was better than that of low-dose (p<0.05). Therefore, high-dose LrEVs was used for further research in subsequent studies.

[0075] Example 6 Effect of LrEVs on Influenza Virus Titers in Mouse Lungs after H1N1 Influenza Virus Infection

[0076] 1. Experimental methods

[0077] (1) Infecting mice with H1N1 influenza virus

[0078] SPF BALB / c mice, female, 6-8 weeks old, were purchased from Hunan Slake Jingda Co., Ltd. The mice were randomly divided into a normal group, a virus infection group, a LrEVs treatment group (dosage concentration was 10 mg / kg / d), and a positive control drug oseltamivir treatment group (dosage concentration was 60 mg / kg / d). After isoflurane anesthesia, the mice were intranasally instilled with H1N1 influenza virus (1.5LD 50 / Mice in the model group and the blank control group were given solvent PBS (Phosphate Buffered Saline) by gavage. LrEVs treatment group mice were given drugs by gavage 2 days before infection, once a day (0.2 mL / time), for 7 consecutive days. Mice in the model group and the blank control group were given solvent PBS (Phosphate Buffered Saline) by gavage. Lung samples were taken on the 5th day after infection, with 8 mice in each group. Four mice were taken for alveolar lavage, and the alveolar lavage fluid (BALF) was collected. The supernatant was collected by centrifugation to measure the virus titer. The remaining 4 mice were fixed with 4% formaldehyde solution for tissue section observation.

[0079] (2) Collection of mouse bronchoalveolar lavage fluid

[0080] After the mice were anesthetized, the trachea was exposed, and the endotracheal cannula was placed. The bronchoalveoli were repeatedly flushed with PBS 3 times, 0.7 mL each time, and BALF was obtained by aspiration. The BALF volume of each mouse was recovered as close as possible. The obtained BALF liquid was centrifuged at 4°C (1500 rpm, 10 min) to collect the supernatant, which was frozen at -80°C for subsequent determination of inflammatory factors and viral titers.

[0081] (3) Effect of LrEVs on influenza virus content in bronchoalveolar lavage fluid (BALF) of mice infected with H1N1 influenza virus:

[0082] MDCK cells were inoculated in a 96-well plate overnight. After the cells were fully grown, mouse BALF supernatant diluted 10 times with DMEM / F12 basal culture medium was added, 100 μL / well, 6 replicate wells were set for each concentration, and a normal control group was set up, and an equal amount of DMEM / F12 basal culture medium was added as a blank control. After incubation for 2 hours in a 37°C, saturated humidity, 5% carbon dioxide incubator, the supernatant was discarded, the cells were washed twice with PBS, and the basal culture medium was added to continue culturing for 48 hours. The cell growth was observed every day. When the cells showed pathological phenomena such as atrophy, rounding, and shedding (i.e., cytopathic effect, CPE), the occurrence of CPE was recorded. The mouse BALF TCID was calculated using the Reed-Muench method. 50 value.

[0083] Among them, TCID 50 Tissue Culture Infective Dose 50 (TCID) is a measure of the ability of a virus to infect cells in culture, indicating the amount of virus that can cause 50% cell lesions (CPE). 50The calculation formula is as follows:

[0084]

[0085] TCID 50 = logarithm of virus dilution above 50% lesion rate + distance ratio

[0086] 2. Experimental results

[0087] Test results such as Figure 4 As shown, on the 5th day after H1N1 influenza virus infection, the virus content in the lungs of the oral positive drug (p<0.01) and LrEVs treatment groups was significantly reduced compared with the virus control group (p<0.05).

[0088] Example 7 Effects of LrEVs on Lung and Small Intestinal Pathology in Mice Infected with H1N1 Influenza Virus

[0089] 1. Experimental methods

[0090] SPF BALB / c mice, female, 6-8 weeks old, were purchased from Hunan Slake Jingda Co., Ltd. The mice were randomly divided into a normal group, a virus infection group, a LrEVs treatment group (dosage concentration was 10 mg / kg / d), and a positive control drug oseltamivir treatment group (dosage concentration was 60 mg / kg / d). After isoflurane anesthesia, the mice were intranasally instilled with H1N1 influenza virus (1.5LD 50 / Mice in the control group were injected with PBS (50 μL) intranasally to simulate viral pneumonia mice infection. Mice in the LrEVs treatment group were given drugs by gavage 2 days before virus infection, once a day (0.2 mL / time) for 7 consecutive days. Mice in the oseltamivir positive control drug treatment group were given drugs by gavage on day 0 after infection, once a day (0.2 mL / time) for 5 consecutive days. The model group and the blank control group were given solvent PBS (Phosphate Buffered Saline) by gavage. Lung samples were taken on the 5th day after infection, and the specific operations were as follows:

[0091] (1) Sampling: On the 5th day after virus infection, mice were anesthetized and killed, and lung and small intestinal tissues were obtained by dissection;

[0092] (2) Fixation: Fix the mouse lungs in 4% formaldehyde for 24 h (the amount of fixative is usually about 20 times the amount of the material block);

[0093] (3) Washing: After fixation, rinse in running water for several hours or overnight;

[0094] (4) Dehydration: Dehydrate in an automatic dehydrator overnight;

[0095] (5) paraffin wax permeabilization, embedding, and sectioning (4 μm);

[0096] (6) Bake at 42°C for 2 h;

[0097] (7) HE (Hematoxylin-eosin) staining: phenol TO (10 min) → TO (10 min) → anhydrous ethanol (2 min) → anhydrous ethanol (2 min) → 95% ethanol (2 min) → 95% ethanol (2 min) → 85% ethanol (2 min) → 75% ethanol (2 min) → water washing (1 min) → hematoxylin (5 min) → water washing (10 s) → 1% hydrochloric acid alcohol (3 s) → running water to blue (10 min) → eosin (20 s) → water washing (10 s) → 75% ethanol (10 s) → 85% ethanol (10 s) → 95% ethanol (30 s) → anhydrous ethanol (1 min) → anhydrous ethanol (1 min) → TO (1 min) → TO (1 min) → neutral gum sealing.

[0098] (8) Dehydration, transparency, and sealing.

[0099] 2. Experimental results

[0100] The results of pathological changes in lung tissue and small intestine of mice in each group are as follows Figure 5 As shown in the figure, the bronchi, blood vessels, lungs and alveolar walls of the normal control group mice were normal and there was no inflammatory reaction; more neutrophils and pyknotic epithelial cells were seen in the bronchial cavity of the model group mice, the bronchial mucosal epithelium was shed, and lymphocytes infiltrated around the wall; macrophages / tissue cells were seen in the alveolar cavity, and the interstitial was widened; more red blood cells and tissue cells were seen in the lung tissue cavity, and focal lymphocyte infiltration was seen; edema, congestion, hemorrhage, and scattered lymphocyte infiltration appeared around small blood vessels in the lung tissue sections. Compared with the virus group, mice orally administered LrEVs showed fewer neutrophils and pyknotic epithelial cells in the bronchial cavity, and the degree of lymphocyte infiltration in the alveolar cavity was not as good as that of the virus group mice. In addition, no obvious scattered lymphocyte infiltration was observed in the lung tissue cavity.

[0101] The normal group had normal intestinal villi, neatly arranged glands, and a small number of inflammatory cells in the mucosal lamina propria. After influenza virus infection, the small intestinal glands were disordered, and some of them fell off and disappeared. The above changes were alleviated after treatment with the positive drug oseltamivir and LrEVs.

[0102] Example 8 Effects of LrEVs on changes in immune cell subsets and inflammatory factor levels in the lungs and small intestine of mice infected with H1N1 influenza virus

[0103] 1. Experimental methods

[0104] SPF BALB / c mice, female, 6-8 weeks old, were purchased from Hunan Slake Jingda Co., Ltd. The mice were randomly divided into a normal group, a virus infection group, and a LrEVs treatment group (administration concentration was 10 mg / kg / d). After isoflurane anesthesia, the mice were intranasally instilled with H1N1 influenza virus (1.5LD 50 / Mice in the control group were injected with PBS (50 μL) intranasally to simulate viral pneumonia. Mice in the LrEVs treatment group were given oral administration (0.2 mL / time) 2 days before virus infection, once a day for 7 consecutive days, and mice in the model group and blank control group were given solvent PBS control by oral administration. The weight changes of mice were recorded every day, and samples were taken on the 5th day after infection.

[0105] (I) Effects of LrEVs on the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) and small intestine of mice infected with H1N1 influenza virus

[0106] The cytokines detected included TNF-α, IL-6, MIP-1α, RANTES, KC, and CCL-11, which were detected using MagneticAssay multiplex kits (BioRad). The cytokine content was detected using Bio-plex suspension chip technology in accordance with the technical instructions, as follows:

[0107] ① Preparation of standard products

[0108] Pipette 250 μL of standard diluent and add it to the freeze-dried powdered standard, vortex for 10 seconds, incubate on ice for 30 minutes, and after the standard powder is completely dissolved, dilute the standard in a 4-fold gradient (S1-S8).

[0109] ② Preparation of antibody pre-coated microspheres

[0110] Calculate the number of wells N to be tested. Each well requires 50 μL of 1X microspheres, and additionally calculate the 10% volume consumption. The total volume of 1X microspheres required is N×50×1.1 μL.

[0111] ③ Preparation of washing solution

[0112] After the 10X concentrated washing solution is restored to room temperature, it is diluted to 1X with detection buffer for later use.

[0113] ④ Preparation of detection antibodies

[0114] Calculate the number of wells to be tested, N. Each well requires 25 μL of detection antibody, and an additional 10% volume consumption is calculated. The total required detection volume is N×25×1.1 μL.

[0115] ⑤ Preparation of SA-PE solution

[0116] Calculate the number of wells to be tested, N. Each well requires 50 μL of SA-PE solution, and additionally calculate the 10% volume consumption. The total amount of SA-PE solution required is N×25×1.1 μL.

[0117] ⑥ Experimental steps

[0118] (1) Wash the thin-bottom 96-well plate with 1X washing buffer, 200 μL per well, at 300 rpm / min for 5 minutes at room temperature;

[0119] (2) Add 50 μL of standard into the standard well;

[0120] (3) Add 50 μL of detection buffer to the background wells;

[0121] (4) Add 50 μL of sample to the sample well;

[0122] (5) Add 50 μL of 1X microspheres to all wells;

[0123] (6) Incubate at room temperature in the dark for 30 min, setting the speed to 850 ± 50 rpm / min;

[0124] (7) Wash three times using an automatic plate washer;

[0125] (8) Add 25 μL of detection antibody to each well, incubate at room temperature in the dark for 30 min, and set the speed to 850±50 rpm / min;

[0126] (9) Wash three times using an automatic plate washer;

[0127] (10) Add 50 μL of SA-PE solution to each well and incubate at room temperature in the dark for 15 min. Set the speed to 850 ± 50 rpm / min.

[0128] (11) Wash three times using an automatic plate washer;

[0129] (12) Add 125 μL of detection buffer to each well, fully resuspend the microspheres, and then load them onto the Bio-Plex 200 for detection (50 microspheres / area).

[0130] (II) Flow cytometry detection of the effects of LrEVs on lung immune cells in mice infected with influenza virus

[0131] (1) Sampling: On the 5th day after mice were infected with influenza virus, the mice were anesthetized and killed, and their lungs were dissected and placed in a 12-well cell plate (containing 1 mL of 1640 medium (2% FBS)), and excess tissue was removed;

[0132] (2) Digestion: Cut the lung into small pieces and transfer them to a 15 mL centrifuge tube. Add 4 mL of cell digestion solution and place in a 37°C water bath for 30 min.

[0133] (3) Grinding: The digested lung tissue was ground on a 70 μm mesh (the mesh was placed in a six-well plate containing 1640 medium (2% FBS) and ground on ice);

[0134] (4) Filtration: After grinding, use a pipette to draw up the culture medium and rinse the mesh. Rinse each sample three times. Filter the cell suspension through a 70 μm mesh into a 50 mL centrifuge tube, centrifuge at 500 g for 7 min, and discard the supernatant.

[0135] (5) Red blood cell lysis: Add 6 mL of red blood cell lysis buffer and lyse on ice for 4-5 min. After lysis, add 20 mL of culture medium to terminate lysis and centrifuge at 400 g for 7 min.

[0136] (6) Washing: Add 10 mL of FACS buffer for washing, centrifuge and discard the supernatant, add 1.5 mL of FACS buffer to resuspend the cells, and transfer to a 2 mL EP tube;

[0137] (7) Blocking: Centrifuge at 400 g for 7 min, discard the supernatant, add 100 μL FACS buffer (containing 3 μL FC antibody / 100 μL FACS buffer) to resuspend, and incubate on ice for 10 min;

[0138] (8) Antibody incubation: Prepare antibody cocktail, add 1 μL of each antibody to each sample, calculate the volume of each antibody, mix the antibodies, add antibody diluent, and stain on ice in the dark for 30 minutes;

[0139] (9) Fixation: Add 1 mL of FACS Buffer to each tube for washing, centrifuge at 400 g for 7 minutes, and discard the supernatant.

[0140] For the clear solution, add 100 μL of Fix / Perm solution to each tube and fix on ice in the dark for 10 min;

[0141] (10) Washing: Add 1 mL of Wash buffer to each tube, centrifuge at 400 g for 7 minutes, discard the supernatant, add 1 mL of Wash buffer to each well, centrifuge at 400 g for 7 minutes, discard the supernatant, and add 1 mL of Wash buffer to each well.

[0142] Add 1 mL of FACS Buffer, transfer to a filter tube, and mark it;

[0143] (11) Detection: Add 200uL FACS Buffer to each tube and perform detection.

[0144] (III) Flow cytometry detection of changes in small intestinal immune cells of mice infected with influenza virus by LrEVs

[0145] (1) The intestines of 6-10 week old C57BL / 6 or Balb / c mice were collected and placed in a HBSS (w / o) culture dish.

[0146] (2) Clamp the small intestine with forceps and inject HBSS with a syringe to flush the intestine and remove feces.

[0147] (3) Remove residual fat tissue and Peyer's spots.

[0148] (4) Cut the intestines longitudinally first, then transversely, into pieces about 0.5 cm long.

[0149] (5) Transfer the tissue chunks into a 50 mL test tube containing 20 mL of pre-digestion solution.

[0150] (6) Incubate the 50 mL test tube containing the tissue fragments at 37°C with continuous rotation (220 rpm) for 20 minutes.

[0151] (7) After mixing for 10 seconds using a vortex mixer, the sample was applied onto a 100 μm mesh and placed in a 50 mL tube.

[0152] (8) Transfer the lamina propria tissue pieces (retaining the sieve pellet) to a new 50 mL tube containing 20 mL of fresh pre-digestion solution.

[0153] (9) Incubate the 50 mL tube containing the tissue fragments at 37°C with continuous rotation for 20 minutes.

[0154] (10) Mix using a vortex mixer for 10 seconds, apply the sample to a 100 μm mesh, and place in a 50 mL tube.

[0155] (11) Transfer the lamina propria tissue pieces (retaining the screen pellet) to a new 50 mL tube containing 20 mL HBSS (w / o).

[0156] (12) Incubate the 50 mL tube containing the tissue fragments at 37°C with continuous rotation for 20 minutes.

[0157] (13) Mix using a vortex mixer for 10 seconds, apply the sample to a 100 μm mesh, and place in a 50 mL tube.

[0158] (14) Add 100 μL of enzyme D, 50 μL of enzyme R, and 12.5 μL of enzyme A to a gentleMACSC tube containing 2.35 mL of preheated digestion solution and mix gently (using the Miltenyi lamina propria dissociation kit).

[0159] (15) Transfer the intestinal tissue into the gentleMACS C tube containing the enzyme mixture and close the C tube tightly.

[0160] (16) Using the heating function of the gentleMACS OctoDissociator with heater, run program 37C_m_LPDK_1

[0161] (17) After the program is terminated, tube C is separated from the gentlemacs dissociator and a short spin is performed at 300 × g to collect the sample at the bottom of the tube.

[0162] (18) Resuspend the sample, add 5 mL of FACS Buffer, filter the cell suspension through a 100 μm mesh, and add 10 mL of FACS Buffer to rinse the mesh.

[0163] (19) Discard the 100 μm sieve and centrifuge the cell suspension at 300 × g for 10 minutes. Aspirate the supernatant completely.

[0164] (20) Resuspend in 1640 (containing 10% FBS and 1% double antibody) to 0.6 ml

[0165] (21) Filter using a flow tube containing a 30 μm filter and rinse the filter with 0.5 mL of 1640 (containing 10% FBS and 1% double antibody) (a total of 1.1 mL)

[0166] (22) Count the cells and set aside.

[0167] (23) Blocking: Centrifuge at 400 g for 7 min, discard the supernatant, add 100 μL FACS buffer (containing 3 μL FC antibody / 100 μL FACS buffer) for resuspending, and incubate on ice for 10 min;

[0168] (24) Antibody incubation: Prepare antibody cocktail, add 1 μL of each antibody to each sample, calculate the volume of each antibody, mix the antibodies, add antibody diluent, and stain on ice in the dark for 30 minutes;

[0169] (25) Fixation: Add 1 mL of FACS Buffer to each tube for washing, centrifuge at 400 g for 7 min, discard the supernatant, add 100 μL of Fix / Perm solution to each tube, and fix on ice in the dark for 10 min.

[0170] (26) Washing: Add 1 mL of Wash buffer to each tube, centrifuge at 400 g for 7 minutes, discard the supernatant, add 1 mL of Wash buffer to each well, centrifuge at 400 g for 7 minutes, discard the supernatant, add 1 mL of FACS Buffer to each tube, transfer to a filter flow tube, mark it,

[0171] (27) Detection: Add 200uFACS Buffer to each tube and perform detection.

[0172] 2. Experimental results

[0173] (1) Effects of LrEVs on the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) and small intestine of mice infected with H1N1 influenza virus

[0174] The detected data were analyzed for data quality control using Bio-Plex Manager. Figure 6 As shown, on the 5th day after viral infection, the protein levels of inflammatory factors TNF-α, IL-6, MIP-1α, RANTES, CCL-11, and KC in the bronchoalveolar lavage fluid (BALF) and small intestine of mice infected with influenza virus were significantly increased. LrEVs treatment significantly reduced the protein expression levels of TNF-α (p<0.01), IL-6 (p<0.01), MIP-1α (p<0.01), RANTES (p<0.001), CCL-11 (p<0.01), and KC (p<0.001) in the BALF of mice, and significantly reduced the protein expression levels of TNF-α (p<0.001), IL-6 (p<0.05), MIP-1α (p<0.001), RANTES (p<0.001), CCL-11 (p<0.0001), and KC (p<0.01) in the small intestine of mice.

[0175] (2) Flow cytometry detection of the effects of LrEVs on immune cells in the lungs and small intestine of mice infected with influenza virus

[0176] The present invention first evaluated the changes in ILC subsets in the lungs and small intestine of mice after H1N1 infection. Figure 7 As shown, the frequency of ILC1s in the lungs of H1N1-infected mice was reduced, while the frequency of ILC1s in the small intestine was increased compared with control mice, while LrEVs treatment significantly inhibited the reduction of ILC1s frequency in the lungs and reduced the increase of ILC1s subsets in the intestine ( Figure 7 ab). Compared with normal control mice, the frequency of ILC2s in the lungs and intestines of mice infected with H1N1 increased, and LrEVs treatment significantly reduced the frequency of ILC2s in the lungs and intestines of mice ( Figure 7 cd). ILC3s can be further divided into NCR + ILC3s, NCR - ILC3s, NCRs in the lungs and intestines of H1N1-infected mice compared with controls + The frequency of ILC3s decreased, while NCR -The frequency of ILC3s increased; LrEVs treatment inhibited NCR in the lung + The frequency of ILC3s decreased, while NCRs in the intestine of H1N1-infected mice + The proportion of ILC3s did not change significantly ( Figure 7 ef). LrEVs treatment increased NCR in the lungs and intestines of H1N1-infected mice - The proportion of ILC3s decreased ( Figure 7 fg).

[0177] ILC1s are characterized by their ability to produce IFN-γ, while ILC2s can produce IL-4. Compared with H1N1 infection, LrEVs treatment significantly reduced IFN-γ in the lungs and small intestine of H1N1-infected mice. + The proportion of ILC1, increased IL-4 + The proportion of ILC2 Figure 8 ad). IL-17-producing NCR-ILC3s accumulated in the lung tissue and small intestine of H1N1-infected mice, and LrEVs treatment prevented IL-17A + NCR - The percentage of ILC3 Figure 8 ef). These data suggest that LrEVs attenuate the plasticity of ILCs that transdifferentiate into IL-17-producing ILCs in the small intestine and lung of H1N1-infected mice.

[0178] In summary, the LrEVs prepared by the present invention are absorbed and diffused to the lungs and intestines after oral administration, and simultaneously reduce the excessive secretion of proinflammatory factors in the lungs and small intestinal tissues of mice infected with H1N1, and alleviate pathological damage; and simultaneously regulate the imbalance and activation of innate lymphocyte subsets in the lungs and small intestines of mice, and maintain the immune homeostasis of the mouse body. In the lethal H1N1 infected mouse model, the weight loss of mice was delayed and the survival rate of mice was improved. That is, the LrEVs provided by the present invention have the characteristics of one drug and two targets, which is specifically manifested in the significant therapeutic effect on the lung damage and intestinal damage mediated by the influenza virus H1N1 at the same time. Therefore, the LrEVs of the present invention can be used to develop drugs or health functional foods, etc., to prevent or treat lung damage and / or intestinal damage mediated by influenza virus infection.

[0179] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of extracellular vesicles derived from lactobacilli in preventing and / or treating lung injury and / or intestinal injury, characterized in that: The lactobacillus is Lactobacillus reuteri EHA2, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 2, 2019, with the deposit number GDMCC NO: 60758.

2. The application according to claim 1, characterized in that: The lung injury is lung injury mediated by influenza virus infection.

3. The application according to claim 1, characterized in that: The intestinal damage is intestinal damage mediated by influenza virus infection.

4. The use according to claim 2 or 3, characterized in that: The influenza virus is influenza A virus.

5. The use according to claim 4, characterized in that: The influenza A virus includes the H1N1 influenza virus.

6. An extracellular vesicle, characterized in that The extracellular vesicles are prepared using lactobacillus, which is the Lactobacillus reuteri EHA2 strain, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2019, with a collection number of GDMCC NO: 60758.

7. The extracellular vesicle according to claim 6, characterized in that The preparation method of the extracellular vesicles comprises the following steps: preparing an expanded culture fluid of lactobacillus, collecting the obtained culture fluid for a first centrifugation, collecting the supernatant for a first microfiltration, taking the microfiltrate for ultrafiltration, taking the ultrafiltrate for a second centrifugation, taking the supernatant for a second microfiltration, collecting the microfiltrate for ultracentrifugation, taking the precipitate for washing, and obtaining the extracellular vesicles.

8. The extracellular vesicle according to claim 7, characterized in that The speed of the ultracentrifugation is 100000 g or above.

9. A drug for preventing and / or treating lung injury and / or intestinal injury, characterized in that: The drug comprises the extracellular vesicle according to any one of claims 6 to 8 and a pharmaceutically acceptable excipient.

10. The drug according to claim 9, characterized in that: The lung injury and / or intestinal injury is lung injury and / or intestinal injury mediated by influenza virus infection.

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