Lactobacillus reuteri and its application in preparing products for relieving lung diseases

Abstract

The invention discloses a Lactobacillus reuteri and application of the Lactobacillus reuteri in preparing a product for alleviating lung diseases. The Lactobacillus reuteri MLR1103 has a preservation number of CGMCC No.31162. Experiments show that the Lactobacillus reuteri or bacterial powder containing the Lactobacillus reuteri of the invention has the characteristics of tolerance to gastric acid, intestinal fluid and bile salts, can significantly alleviate acute lung injury caused by LPS in mice, lung injury caused by PM2.5, pulmonary fibrosis, asthma, pneumoconiosis and cough symptoms, alleviate lung pathological changes and restore lung function.

Description

Technical Field

[0001] The invention relates to Lactobacillus reuteri MLR1103, and belongs to the technical field of functional microorganisms. Background Art

[0002] Acute lung injury (ALI) refers to a serious disease in which the gas exchange function of the lungs decreases sharply due to infection, aspiration of harmful gases, trauma, shock and other non-cardiogenic factors. When the disease develops to the terminal stage, it is acute respiratory distress syndrome (ARDS), with specific clinical manifestations of respiratory distress, progressive hypoxemia, and imaging manifestations of exudative lesions or consolidation in both lungs. The common causes of ALI can be divided into direct injury and indirect injury. The causes of the former include pneumonia (caused by viruses, bacteria, fungi), aspiration lung injury (gastric contents, lung infection after drowning, toxic gases), pulmonary contusion, etc., and the causes of the latter include sepsis, multiple injuries, complications of massive blood transfusion, poisoning, burns, severe pancreatitis, pulmonary embolism, etc. At present, the only drug approved in my country for the treatment of ALI is sivelestat sodium, and its effect is relatively limited.

[0003] In addition to acute lung injury, chronic lung inflammation caused by PM2.5 is another common type of lung injury. PM2.5 refers to particles with an aerodynamic equivalent diameter of less than or equal to 2.5 microns in ambient air. PM2.5 has a small particle size, a large area, strong activity, and is easily accompanied by toxic and harmful substances (for example, heavy metals, microorganisms, etc.), and has a long residence time in the atmosphere and a long transportation distance, which has a great impact on air quality and human health. Its sources are very wide, including industrial emissions, motor vehicle exhaust, coal burning, biomass combustion, construction dust, road dust, and restaurant fumes. Because it can penetrate into the human respiratory tract and lungs, and even enter the blood circulation, it can cause a variety of health problems, such as respiratory diseases, cardiovascular diseases, and immune system damage. Therefore, the monitoring and control of PM2.5 is an important task in the field of environmental protection and public health. The harm of PM2.5 to the lungs mainly includes the following aspects: triggering an inflammatory response; PM2.5 can enter deep into the lungs, stimulate lung cells, and trigger an inflammatory response. Long-term inflammation can damage lung tissue and lead to decreased lung function. Damage to alveoli: Alveoli are important parts of the lungs for gas exchange. Harmful substances in PM2.5 can damage the structure and function of alveoli, affect the exchange of oxygen and carbon dioxide, and thus affect respiratory function. Increased risk of respiratory infections: PM2.5 weakens the defense mechanism of the respiratory tract, making it easier for bacteria, viruses and other pathogens to invade the respiratory tract and cause lung infections such as pneumonia. Lead to chronic diseases: Long-term exposure to high concentrations of PM2.5 may increase the risk of chronic obstructive pulmonary disease (COPD), asthma and other chronic lung diseases.

[0004] Pulmonary fibrosis (PF) is a very complex and difficult-to-treat disease among respiratory diseases. PF patients have a very short survival period after diagnosis and a high mortality rate, so it is called a "tumor-like disease" and seriously endangers human health. The commonly used anti-pulmonary fibrosis drugs in clinical practice mainly include glucocorticoids, pirfenidone, nintedanib, N-acetylcysteine, etc., but these drugs generally have many disadvantages such as many adverse reactions, high prices, and poor efficacy.

[0005] Asthma, also known as bronchial asthma in clinical practice, is an airway inflammation caused by various factors. Its pathogenic factors are complex, and genetics, allergic reactions, viral and bacterial infections, air pollution, and adverse reactions of some drugs may all induce asthma. The main symptoms of asthma are coughing, sputum, wheezing accompanied by wheezing. The pathogenesis of asthma involves multiple factors, including genetics, environment, immunity, and airway inflammation. The clinical manifestations of asthma include recurrent wheezing and shortness of breath, with or without symptoms such as chest tightness or coughing, accompanied by airway hyperresponsiveness and variable airflow limitation. At present, the efficacy of drugs for the treatment of asthma is limited and their applicability is poor.

[0006] Coughing is a common respiratory symptom and a protective reflex action of the human body. There are many reasons for coughing, the most common of which include respiratory infections: such as colds, influenza, pneumonia, bronchitis, etc. Pathogen infection leads to inflammation of the respiratory mucosa and increased secretions, which stimulates cough receptors and causes coughing. Allergies: Reactions to allergens such as pollen, dust mites, and animal dander can cause airway inflammation and coughing. Environmental factors: Air pollution, smoke, chemicals, dry air, etc. irritate the respiratory tract. Foreign bodies in the airway: Foreign bodies entering the airway can cause severe coughing. Side effects of drugs: Certain drugs may cause coughing. Respiratory diseases: such as asthma, chronic obstructive pulmonary disease, tuberculosis, etc. Gastroesophageal reflux disease: Gastric acid reflux irritates the esophagus and pharynx, causing coughing. The manifestations of coughing are also different. It may be a dry cough (without sputum) or accompanied by sputum (the nature, color and amount of sputum vary from disease to disease), and the frequency and severity of coughing are also different.

[0007] Pneumoconiosis is a systemic disease characterized by diffuse fibrosis of lung tissue caused by long-term inhalation of industrial dust during occupational activities and its retention in the lungs. The main cause of pneumoconiosis is long-term inhalation of inorganic mineral dust, such as silica dust, coal dust, asbestos dust, etc. Common types of pneumoconiosis include silicosis, coal worker's pneumoconiosis, asbestosis, etc. Symptoms of pneumoconiosis usually appear gradually after years of exposure to dust. In the early stages, they may manifest as coughing, sputum, chest pain, and dyspnea. As the disease progresses, dyspnea will gradually worsen, and may also be complicated by pulmonary tuberculosis, lung infection, chronic cor pulmonale, etc. At present, there is no cure or treatment for pneumoconiosis. Treatment measures are mainly to relieve symptoms, delay progression of the disease, and prevent complications.

[0008] Currently, there are reports on the use of microorganisms to alleviate the above diseases or symptoms, but there are generally problems with weak gastric acid resistance, intestinal fluid resistance, and bile salt resistance, and the effect is not ideal. Therefore, there is an urgent need for a probiotic that is resistant to gastric acid, intestinal fluid, and bile salts to treat and alleviate lung diseases or symptoms. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Lactobacillus reuteri with high yield of active metabolites.

[0010] The second object of the present invention is to provide a use of the above-mentioned Lactobacillus reuteri or bacterial powder containing the above-mentioned Lactobacillus reuteri in preparing a product for alleviating lung diseases.

[0011] The third object of the present invention is to provide a fermented food, probiotic preparation or pharmaceutical composition comprising the above-mentioned Lactobacillus reuteri.

[0012] The technical solution of the present invention is summarized as follows:

[0013] A Lactobacillus reuteri MLR1103, whose deposit number is CGMCC No.31162.

[0014] Application of the above-mentioned Lactobacillus reuteri or bacterial powder containing the above-mentioned Lactobacillus reuteri in preparing products for alleviating lung diseases.

[0015] The lung disease is preferably lung injury, pulmonary fibrosis, asthma, cough or pneumoconiosis.

[0016] The above products are medicines.

[0017] A fermented food, a probiotic preparation or a pharmaceutical composition comprising the above-mentioned Lactobacillus reuteri.

[0018] The fermented food is solid food, liquid food, semi-solid food, dairy product, vegetable and fruit food or freeze-dried food.

[0019] The Lactobacillus reuteri in the above-mentioned probiotic preparation is a fermentation product of live bacteria, dead bacteria or live bacteria.

[0020] Experiments have shown that the Lactobacillus reuteri MLR1103 (with a deposit number of CGMCC No. 31162, hereinafter referred to as MLR1103) of the present invention has the characteristics of tolerance to gastric acid, intestinal fluid, bile salts and high adhesion, and can significantly alleviate the acute lung injury caused by LPS, lung injury caused by PM2.5, pulmonary fibrosis, asthma, pneumoconiosis and cough symptoms in mice, alleviate lung pathological changes, and restore lung function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the HE staining image of the lungs in the evaluation of MLR1103 on the acute lung injury mouse model in Example 3;

[0022] Figure 2 The results of counting the number of white blood cells in the bronchial lavage fluid of the lungs of mice in the evaluation of MLR1103 on the acute lung injury mouse model in Example 3;

[0023] Figure 3 The protein content of bronchial lavage fluid in the lungs of mice in the evaluation of MLR1103 on the acute lung injury mouse model in Example 3 was determined;

[0024] Figure 4 The mRNA expression of inflammatory factors IL-1β, IL-6, and TNF-α in the lung tissue of mice in the evaluation of MLR1103 on the acute lung injury mouse model in Example 3;

[0025] Figure 5The protein expression of inflammatory factors IL-6 and TNF-α in the evaluation of MLR1103 on the acute lung injury mouse model in Example 3;

[0026] Figure 6 The expression of lactate dehydrogenase in the evaluation of MLR1103 on the acute lung injury mouse model in Example 3;

[0027] Figure 7 The CT scan results of the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0028] Figure 8 This is the HE staining image of the lungs in the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0029] Fig. 9 This is a MASSON staining image of the lungs in the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0030] Fig.10 The lung coefficient statistics in the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0031] Fig.11 The weight changes in the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0032] Fig.12 Determination of lung hydroxyproline content in the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0033] Fig.13 The mRNA expression of pulmonary fibrosis factors in the evaluation of MLR1103 on the pulmonary fibrosis mouse model in Example 4;

[0034] Fig.14 The CT scan results of the evaluation of MLR1103 on the asthma mouse model in Example 5;

[0035] Fig.15 This is the HE staining image of the lungs in the evaluation of MLR1103 on the asthma mouse model in Example 5;

[0036] Fig.16 This is the MASSON staining image of the lungs in the evaluation of MLR1103 on the asthma mouse model in Example 5;

[0037] Fig.17 This is the lung PAS staining image in the evaluation of MLR1103 on the asthma mouse model in Example 5;

[0038] Fig.18The statistics of the number of white blood cells in bronchial lavage fluid in the evaluation of MLR1103 on the asthma mouse model in Example 5;

[0039] Fig.19 The protein content of bronchial lavage fluid in the lungs in the evaluation of MLR1103 on the asthma mouse model in Example 5 is determined;

[0040] Fig. 20 Determination of serum IGE protein content in mice in the evaluation of MLR1103 on asthma mouse model in Example 5;

[0041] Fig.21 The expression of inflammatory factors IL-1β, IL-6, and TNF-α in lung tissue in the evaluation of MLR1103 on the asthma mouse model in Example 5;

[0042] Fig. 22 The lung CT scan results in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0043] Fig.23 This is the HE staining image of the lungs in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0044] Fig.24 Statistics of lung coefficient data in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0045] Fig.25 The weight changes in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0046] Fig.26 The expression of inflammatory factors IL-1β, IL-6, and TNF-α in lung tissue in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0047] Fig. 27 The expression of inflammatory factors IL-6 and TNF-α in lung tissue in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0048] Fig.28 The expression of lactate dehydrogenase in the evaluation of PM2.5-induced lung injury in mice by MLR1103 in Example 6;

[0049] Fig.29 The lung CT scan results in the evaluation of MLR1103 on the pneumoconiosis mouse model in Example 7;

[0050] Fig.30This is the change in the number of coughs in the evaluation of MLR1103 on the cough mouse model in Example 8. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with the embodiments and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Example 1

[0053] Strain isolation and identification

[0054] Fresh stool samples were collected from healthy adults who had lived in Dalian, Liaoning Province for a long time. The subjects had not taken antibiotics before the collection, had no history of taking probiotics, and had no history of gastrointestinal diseases. After the samples were diluted with sterile water, they were spread on MRS solid culture medium and cultured in a 37°C incubator for 3 days. When obvious colonies were observed, colonies with different morphologies were picked and cultured on new MRS solid culture medium for 2 days in a 37°C incubator. After monoclonal colonies were observed, monoclonal strains were continued to be cultured in MRS solid culture medium to obtain purified monoclonal intestinal bacteria. The strain was a white round colony with a smooth surface (numbered MLR1103). The bacterial solution was placed in a 20% glycerol aqueous solution and stored at -80°C. The collection center was entrusted for identification, RT-PCR was performed using 16S rRNA primers, and 16S rRNA gene sequencing analysis was performed on the isolated strains. Combined with the NCBI nucleic acid database BLAST comparison, its species was determined to be Lactobacillus reuteri (homology 100%). The strain of Lactobacillus reuteri MLR1103 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 3, 2024, with the deposit number CGMCC No.31162. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0055] The primers used in the identification process are as follows:

[0056] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1)

[0057] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0058] The identified gene sequence is shown in SEQ ID NO:3.

[0059] Example 2 Characteristic Detection of MLR1103

[0060] 1. Tolerance test

[0061] At China Industrial Microbiological Culture Collection Management Center ( https: / / www.china-cicc.org ) Lactobacillus reuteri Kandler et al. ATCC 23272 (hereinafter referred to as ATCC 23272, which is used as a reference strain) was purchased.

[0062] Wash the overnight cultured MLR1103 and ATCC 23272 with PBS three times, centrifuge and resuspend with PBS to obtain the resuspended bacterial solution. Use the resuspended bacterial solution to coat the MRS solid culture medium plate, and count as N0. Take 500μL of the resuspended bacterial solution and add 4.5mL of artificial gastric juice, artificial intestinal juice, pH=3 solution, and bile salt solution, respectively, coat the MRS solid culture medium plate, and culture at 37℃, 100r / min shaking incubator for 1h and 3h respectively, and the plate count is N1.

[0063] Survival rate (%) = N1 / N0×100%.

[0064] Preparation of artificial gastric juice: 0.2 g NaCl and 0.35 g pepsin were dissolved in 100 mL triple-distilled water, the pH was adjusted to 3, and filtered for sterilization.

[0065] Preparation of pH=3 solution: dissolve 0.2g citric acid in 100mL triple distilled water, adjust pH to 3, and filter to sterilize.

[0066] Preparation of bile salt solution: Dissolve 1.2 g of ox bile salt in 100 mL of triple-distilled water and filter to sterilize.

[0067] Preparation of artificial intestinal fluid: 0.2g NaCl and 0.1g trypsin were dissolved in 100mL triple distilled water, and the pH was adjusted to 8, which was liquid a. 1.2g ox bile salt was dissolved in 100mL triple distilled water, and the pH was adjusted to 8, which was liquid b; liquid a and liquid b were mixed in a volume ratio of 1:2 and filtered for sterilization.

[0068] Table 1 Survival rates of MLR1103 and ATCC 23272 in artificial gastric juice, pH 3 solution, bile salt solution, and artificial intestinal juice

[0069] strain Artificial gastric juice (pH=3) pH = 3 solution Bile salt solution Artificial intestinal fluid (pH = 8) MLR1103 50％ 47％ 822％ 110％ ATCC 23272 0％ 0％ 0％ 92％

[0070] 2. Comparison of bacterial metabolite content

[0071] The MLR1103 and ATCC 23272 reference strains were cultured in MRS medium at 37°C in a constant temperature incubator for 2 days; extracted with ethyl acetate, concentrated and dried, and methanol was added to prepare a 100 mg / ml test solution.

[0072] Succinic acid, 4-hydroxyproline-leucine, 4-hydroxyphenyl lactate, cyclo-4-hydroxyproline-phenylalanine, and salicylic acid were prepared with methanol to obtain 10 mg / ml, 5 mg / ml, 2.5 mg / ml, 1.25 mg / ml, and 0.625 mg / ml gradient solutions, respectively. The metabolite content of the reference strains MLR1103 and ATCC 23272 was analyzed by HPLC liquid phase. Under 10%-40% acetonitrile-water conditions, an ultraviolet detector was used with a wavelength of 210 nm and an analysis time of 40 min. The peak area with consistent retention time was used to calculate the content of each compound in the test solution (Table 2).

[0073] Table 2 Metabolite analysis of MLR1103 and ATCC 23272 reference strains (total metabolites are 100%)

[0074]

[0075] Example 3 Evaluation of the protective effect of MLR1103 on mice with acute lung injury induced by LPS

[0076] Male C57BL / 6J mice, aged 6-8 weeks, were randomly divided into four groups: blank group, model group (LPS modeling), MLR1103 group (LPS modeling + MLR1103 administration), and ATCC 23272 group (LPS modeling + ATCC 23272 administration), with 6 mice in each group.

[0077] After 3 days of adaptive feeding, the mice were gavaged with 2×10^7 cfu / kg MLR1103 in the MLR1103 group and 2×10^7 cfu / kg ATCC 23272 in the ATCC23272 group. The blank group and the model group were gavaged with 0.9% NaCl aqueous solution at 10 mL / kg body weight once a day for 7 days. All the above groups were intervened 7 days before modeling until the end of the experiment.

[0078] On the 7th day, the acute lung injury model of mice was established by intratracheal injection of LPS: tribromoethanol (300 mg / kg) was injected intraperitoneally for anesthesia. After anesthesia, the mice were fixed in a supine position. After detoxification, a 0.9% NaCl solution (10 mg / kg) of LPS was drawn by an intratracheal quantitative dosing device and inserted into the trachea for injection. The mice were then rotated upright so that the drug was evenly distributed in the lungs. After the animals woke up naturally, they were placed in cages. On the second day, the model was successfully established when the mice showed dull fur, erect hair, slow reaction, and curling. The blank group of animals were injected with 0.9% NaCl solution through the trachea using the same method as above.

[0079] The mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg), fixed in supine position, the abdominal cavity was opened, the trachea was exposed, and a 22G arteriovenous indwelling needle was inserted into the trachea 1 cm away, and 1 ml of 0.9% NaCl aqueous solution was injected into the lungs to recover the liquid. This was repeated twice, and the lung lavage fluid (BALF) was collected. The chest cavity was opened by extending the incision, the trachea was cut off near the thyroid cartilage, and the lungs were completely removed to observe the general morphology, elasticity, and presence of stasis of the lung tissue.

[0080] After the mice in each group were sacrificed, part of the lung tissue was removed from the right lobe of the lung, fixed with formaldehyde, embedded in paraffin, and sliced ​​at 4 μm. Conventional HE staining was performed and the degree of lung tissue inflammation was observed under a microscope. Figure 1 It can be seen that the lung tissue structure of the mice in the model group was destroyed, a large number of inflammatory cells infiltrated in the tissue, alveolar capillary congestion and dilation, and widened pulmonary interstitial and alveolar septa. Compared with the model group, the pathological changes in the MLR1103 group and the ATCC 23272 group were improved, the inflammatory cell infiltration of the lung tissue was reduced, the pulmonary interstitial and alveolar septa were narrowed, and the tissue structure was restored.

[0081] Take 1 ml of BALF fluid and use a cell counter to detect the number of white blood cells (WBC). Figure 2 It can be seen that the number of white blood cells in the lungs was significantly reduced, and the efficacy of the MLR1103 group was better than that of the ATCC 23272 group.

[0082] Take the remaining BALF solution, let it stand at room temperature for 5 minutes, centrifuge it at 3500rpm for 10 minutes, discard the supernatant, and resuspend it in 100μl PBS solution. Use the BCA protein quantification method to determine its protein content. Figure 3 It can be seen that the protein content was significantly reduced, and the efficacy of the MLR1103 group was better than that of the ATCC 23272 group.

[0083] Weigh 20 mg of mouse lung tissue, cut it into pieces, add zirconium oxide beads, add 1 ml of Trizol solution to lyse, put it in a cryogenic grinder and grind it for 10 minutes. After grinding, add 0.2 mL of chloroform, shake vigorously for 15 seconds, and leave it at room temperature for 5 minutes. After centrifugation at 12000 r / min for 10 minutes at 4°C, transfer the aqueous phase to a new centrifuge tube, gently mix the aqueous phase with an equal volume of pre-cooled isopropanol, incubate at 30°C for 10 minutes, centrifuge at 12 000 r / min for 15 minutes at 4°C, and discard the supernatant. Add 1 mL of pre-cooled 75% ethanol aqueous solution to wash the RNA precipitate. Centrifuge at 7500 r / min at 4°C for 5 minutes, remove the ethanol solution, and dry the RNA precipitate in air for 10 minutes. When dissolving RNA, add 20 μL of RNase-free water and blow it repeatedly 5 times, and store the RNA solution at -80°C. After detecting RNA concentration and purity, cDNA was synthesized as a fluorescent quantitative template using a reverse transcription kit and amplified on a PCR instrument. The reaction conditions for PCR amplification were: 95°C pre-denaturation for 30 seconds, 95°C denaturation for 5 seconds, 60°C annealing for 30 seconds, and 40 cycles of amplification. β-actin was used as an internal reference, and the 2-△△Ct method was used to calculate the mRNA expression of TNFα, IL-1β, and IL6 in mouse lung tissue. Figure 4 It can be seen that compared with the model group, the expression levels of inflammatory factor mRNA in the MLR1103 group and the ATCC 23272 group were significantly reduced, and the efficacy of the MLR1103 group was better than that of the ATCC23272 group.

[0084] Weigh 20 mg of mouse lung tissue, cut it into pieces, add steel beads, add 200 μL PBS solution, and grind it in a low-temperature grinder for 10 minutes. Collect the supernatant after centrifugation and store it at -80℃ for later use. ELISA was used to detect the content of IL-6 and TNF-α in mouse lung tissue. Figure 5 It can be seen that compared with the model group, the protein expression levels of inflammatory factors in the MLR1103 group and the ATCC 23272 group were significantly reduced, and the efficacy of the MLR1103 group was better than that of the ATCC 23272 group.

[0085] Weigh 20 mg of mouse lung tissue, cut it into pieces, add steel beads, add 200 μL PBS, and grind it in a cryogenic grinder for 10 minutes. Collect the supernatant after centrifugation and store it at -80°C for later use. Determine the lactate dehydrogenase content of the lung tissue of each group of mice according to the LDH lactate dehydrogenase kit method. Figure 6 It can be seen that compared with the model group, the lactate dehydrogenase content in the MLR1103 group and the ATCC 23272 group was significantly reduced, and the efficacy of the MLR1103 group was better than that of the ATCC 23272 group.

[0086] Example 4 Evaluation of the protective effect of Lactobacillus reuteri MLR1103 on bleomycin-induced mouse pulmonary fibrosis model

[0087] After 3 days of adaptive feeding, 6-8 week old male C57BL / 6J mice were randomly divided into four groups: blank group, model group (bleomycin modeling), perfenidone group (bleomycin modeling + perfenidone administration), and MLR1103 group (bleomycin modeling + MLR1103 administration), with 6 mice in each group.

[0088] On the first day, the mouse pulmonary fibrosis model was established by intratracheal injection of bleomycin: tribromoethanol (300 mg / kg) was injected intraperitoneally for anesthesia. After anesthesia, the mouse was fixed in a supine position. After sterilization, a 0.9% NaCl solution of bleomycin (3U / kg) was extracted by an intratracheal quantitative dosing device and inserted into the trachea. 0.05 mL of bleomycin solution was injected, and the mouse was immediately rotated upright so that the drug was evenly distributed in the lungs. After the animal naturally woke up, it was placed in a cage and raised. On the 7th day, the model was successfully established when the rats showed dull fur, erect hair, slow reaction, slow or decreased body weight growth, and curling. The modeling method of the blank group animals was the same as above, but only saline was used to replace bleomycin for intratracheal injection.

[0089] On the first day, the pifenidone group was gavaged at 200 mg / kg body weight daily, the MLR1103 group was gavaged at 2×10^7 cfu / kg daily, and the blank group and the model group were gavaged with 0.9% NaCl solution at 10 mL / kg body weight daily, once a day until the end of the experiment, for a total of 28 days.

[0090] On the 28th day, CT scans of the lungs of mice after fibrosis treatment were performed. The mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg) to ensure that the mice were completely anesthetized and had no pain response. The anesthetized mice were placed on the scanning table of the CT scanner. The position of the mice was adjusted to ensure that the lungs were within the scanning range. The scan was performed to obtain lung images. Figure 7 It can be seen that compared with the blank group, the lung CT of the model group showed reticular shadows and honeycomb changes. No obvious shadows were found in the lung CT of the pifenidone group and the MLR1103 group, and pulmonary fibrosis was improved.

[0091] Part of the lung tissue was removed from the right lobe of the lung, fixed with formaldehyde, embedded in paraffin, and sliced ​​at 4 μm. Conventional HE staining and Masson staining were performed, and the degree of inflammation and collagen fiber proliferation in the lung tissue was observed under a microscope. Figure 8It can be seen that the lung tissue structure of the blank group mice was normal, and no obvious inflammatory cell infiltration and bleeding were observed in HE staining. After bleomycin modeling, the lesions in the model group were unevenly distributed, the lung tissue structure was destroyed at the lesion site, the alveolar cavity was irregularly collapsed and fused, the alveolar septum was significantly widened, and inflammatory cell infiltration and fibroblast proliferation were observed. The changes in alveolar structure, inflammatory cell infiltration and collagen deposition in the lung tissue of the mice in the pifenidone group and MLR1103 group were improved compared with those in the model group, and the vascular dilation phenomenon was alleviated in the model group. Fig. 9 It can be seen that scattered fibrotic tissues were observed in the lungs of mice in the blank group by Masson staining, and obvious blue fibrotic areas were observed in the sections of the model group. The degree of fibrosis in the pifenidone and MLR1103 groups was improved compared with that in the model group.

[0092] The lungs were rinsed with 0.9% NaCl solution, dried with filter paper, weighed with an analytical balance, and the lung coefficient was calculated. The lung coefficient was calculated as follows: lung wet weight (mg) / body weight of the mouse at the time of death (g). Fig.10 It can be seen that compared with the blank group, the lung mass of the mice in the model group increased, the body weight decreased, and the lung coefficient increased. The lung coefficient of the mice in the phenanthate group and the MLR1103 group improved. Fig.11 It can be seen that compared with the model group, the body weight of the Pifenidone group and the MLR1103 group increased.

[0093] Hydroxyproline is one of the main amino acids constituting collagen, and collagen is a characteristic protein of fibrotic tissue. Therefore, measuring the hydroxyproline content in lung tissue can reflect the degree of fibrosis in lung tissue. Take 20 mg of lung tissue homogenate, and measure the hydroxyproline content in the lung tissue of each group of mice according to the hydroxyproline kit (alkaline hydrolysis method). Compared with the blank group, after bleomycin modeling, the hydroxyproline content in the lung tissue of mice in the model group was significantly increased, indicating an increase in fibrotic tissue; the hydroxyproline content in the lung tissue of mice in the pifenidone and MLR1103 groups was significantly lower than that in the model group, indicating a lower degree of fibrosis. Fig.12 .

[0094] Weigh 20 mg of mouse lung tissue, cut it into pieces, add zirconium oxide beads, add 1 ml of Trizol solution to lyse, put it in a cryogenic grinder and grind it for 10 minutes. After grinding, add 0.2 mL of chloroform, shake vigorously for 15 seconds, and leave it at room temperature for 5 minutes. After centrifugation at 12,000 r / min for 10 minutes at 4°C, transfer the aqueous phase to a new centrifuge tube, gently mix the aqueous phase with an equal volume of pre-cooled isopropanol, incubate at 30°C for 10 minutes, centrifuge at 12,000 r / min for 15 minutes at 4°C, and discard the supernatant. Add 1 mL of pre-cooled 75% ethanol aqueous solution to wash the RNA precipitate. Centrifuge at 7500 r / min at 4°C for 5 minutes, remove the ethanol solution, and dry the RNA precipitate in air for 10 minutes. When dissolving RNA, add 20 μL of RNase-free water and blow it repeatedly 5 times, and store the RNA solution at -80°C. After detecting RNA concentration and purity, cDNA was synthesized as a fluorescent quantitative template using a reverse transcription kit and amplified on a PCR instrument. The reaction conditions for PCR amplification were: 95°C pre-denaturation for 30s, 95°C denaturation for 5s, 60°C annealing for 30s, and 40 cycles of amplification. β-actin was used as an internal reference, and the 2-△△Ct method was used to calculate the expression of α-SMA, Fibronectin, E-cadherin, and Collagen I in mouse lung tissue. Fig.13 It can be seen that compared with the model group, the expression of fibrin-related genes in the pifenidone group and the MLR1103 group was significantly improved.

[0095] Example 5 Evaluation of the alleviating effect of Lactobacillus reuteri MLR1103 on asthma mouse model

[0096] After 3 days of adaptive feeding, 6-8 week old male C57BL / 6J mice were randomly divided into three groups: blank group, model group (OVA modeling), and MLR1103 group (OVA modeling + MLR1103 administration group), with 6 mice in each group.

[0097] Sensitization phase: 40 μg OVA ovalbumin and 4 mg 10% aluminum hydroxide (prepared in saline) were injected intraperitoneally on days 0 and 14.

[0098] Provocation phase: From the 21st day, 5% OVA was inhaled by nebulization for 20 minutes for 14 days to stimulate allergic reactions. 2×10^7 cfu / kg of bacteria was gavaged 1 hour before each provocation for a total of 42 days.

[0099] On the 41st day, CT scans of the mouse lungs were performed. Two weeks after fibrosis modeling, the mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg) to ensure that the mice were completely anesthetized and had no pain response. The anesthetized mice were placed on the scanning table of the CT scanner. The position of the mouse was adjusted to ensure that the lungs were within the scanning range. The scan was performed to obtain lung images. Fig.14 It can be seen that compared with the blank group, the model group showed ground-glass shadows, indicating inflammation or early fibrosis in the lungs, and the lungs of the MLR1103 group were significantly improved.

[0100] Part of the lung tissue was removed from the right lobe of the lung, fixed with formaldehyde, embedded in paraffin, and sliced ​​at 4 μm. HE, MASSON, and PAS staining were performed, and the degree of inflammation of the lung tissue was observed under a microscope. Fig.15 It can be seen that the lung tissue structure of the blank group mice was normal, and no obvious inflammatory cell infiltration and bleeding were observed in HE staining. The lesions in the model group were unevenly distributed, the lung tissue structure was destroyed at the lesion site, the alveolar cavity was irregularly collapsed and fused, the alveolar septum was significantly widened, and inflammatory cell infiltration and fibroblast proliferation were observed. The alveolar structure changes, inflammatory cell infiltration and collagen deposition in the lung tissue of the MLR1103 group mice were improved compared with those in the model group, and the vascular dilation phenomenon was alleviated in the model group. Masson staining of the lung fibrosis tissue of the blank group mice was scattered, and obvious blue fibrosis areas were observed in the model group sections. The degree of fibrosis in the MLR1103 group was improved compared with the model group, see the attached Fig.16 .Depend on Fig.17 It can be seen that PAS staining showed that the glycoprotein content in the model group increased, and the situation in the MLR1103 group was improved.

[0101] Depend on Fig.18 It can be seen that compared with the model group mice, the number of white blood cells in the lung lavage fluid (BALF) of the MLR1103-treated mice was significantly reduced. Fig.19 It can be seen that compared with the model group, the protein content of MLR1103 group was significantly reduced.

[0102] Blood was collected from mice in each group and centrifuged at 3500 r / min for 10 min. The supernatant was collected to obtain serum, and the IGE content in blood was determined by Elisa method. Fig. 20 It can be seen that compared with the blank group, the serum IGE content in the model group was significantly increased, and the IGE content in the MLR1103 group was significantly lower than that in the model group.

[0103] Total RNA was extracted from the lung tissues of mice in each group, and the levels of inflammatory factors TNF-α, IL-6 and IL-1β were tested. Fig.21 It can be seen that compared with the blank group, the level of inflammatory factors in the model group was significantly increased; compared with the model group, the expression of inflammatory factors in the MLR1103 group was reduced.

[0104] Example 6 Evaluation of the protective effect of Lactobacillus reuteri MLR1103 on a mouse model of lung injury induced by PM2.5

[0105] Male C57BL / 6J mice aged 6-8 weeks were randomly divided into three groups: blank group, model group (PM2.5 modeling), and MLR1103 group (PM2.5 modeling + MLR1103 administration), with 6 mice in each group.

[0106] After 3 days of adaptive feeding, the MLR1103 group was gavaged with 2×10^7 cfu / kg daily, and the blank group and model group were gavaged with 0.9% NaCl solution at 10 mL / kg body weight daily. Once a day, for a total of 7 days. All the above groups were intervened 7 days before modeling until the end of the experiment.

[0107] On the 7th day, the PM2.5 intratracheal injection method was used to replicate the mouse lung injury model: tribromoethanol (300 mg / kg) was injected intraperitoneally for anesthesia. After anesthesia, the mice were fixed in a supine position. After detoxification, the intratracheal quantitative dosing device was used to extract PM2.5 0.9% NaCl solution (10 mg / kg) and inserted into the trachea. 0.05 ml of PM2.5 suspension was injected. The model was maintained for 2 weeks (three times a week), and then the mice were rotated upright so that the drug was evenly distributed in the lungs. After the animals woke up naturally, they were kept in cages. The modeling method of the blank group animals was the same as above, except that PM2.5 was replaced with normal saline for intratracheal injection.

[0108] On the 21st day, CT scans of the mouse lungs were performed. Two weeks after PM2.5 modeling, the mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg) to ensure that the mice were completely anesthetized and had no pain response. The anesthetized mice were placed on the scanning table of the CT scanner. The position of the mouse was adjusted to ensure that the lungs were within the scanning range. The scan was performed to obtain lung images. Fig. 22 It can be seen that compared with the blank group, the model group showed ground-glass shadows, indicating inflammation or early fibrosis in the lungs, and the lungs of the MLR1103 group were significantly improved.

[0109] After the mice in each group were sacrificed, part of the lung tissue was removed from the right lobe of the lung, fixed with formaldehyde, embedded in paraffin, and sliced ​​at 4 μm. Conventional HE staining was performed and the degree of lung tissue inflammation was observed under a microscope. Fig.23 It can be seen that the lung tissue structure of mice in the model group was destroyed, a large number of inflammatory cells infiltrated in the tissue, alveolar capillary congestion and dilation, and widened pulmonary interstitial and alveolar septa. Compared with the model group, the pathological changes in the MLR1103 group were improved, inflammatory cell infiltration in the lung tissue was reduced, pulmonary interstitial and alveolar septa were narrowed, and the tissue structure was restored.

[0110] The removed lungs were rinsed with 0.9% NaCl solution, dried with filter paper, and weighed on an analytical balance. Calculation of lung coefficient = lung wet weight (mg) / body weight of the mouse at the time of death (g). Fig.24 It can be seen that compared with the blank group, the lung mass of the mice in the model group increased, the body weight decreased, the lung coefficient increased, and the lung coefficient of the mice in the MLR1103 group was significantly improved. Fig.25 It can be seen that during the experiment, the body weight of mice in the MLR1103 group was significantly improved compared with that in the model group.

[0111] Weigh 20 mg of mouse lung tissue, cut it into pieces, add zirconium oxide beads, add 1 ml of Trizol solution to lyse, put it in a cryogenic grinder and grind it for 10 minutes. After grinding, add 0.2 mL of chloroform, shake vigorously for 15 seconds, and leave it at room temperature for 5 minutes. After centrifugation at 12000 r / min for 10 minutes at 4°C, transfer the aqueous phase to a new centrifuge tube, gently mix the aqueous phase with an equal volume of pre-cooled isopropanol, incubate at 30°C for 10 minutes, centrifuge at 12 000 r / min at 4°C for 15 minutes, and discard the supernatant. Add 1 mL of pre-cooled 75% ethanol aqueous solution to wash the RNA precipitate. Centrifuge at 7500 r / min at 4°C for 5 minutes, remove the ethanol solution, and dry the RNA precipitate in air for 10 minutes. When dissolving RNA, add 20 μL of RNase-free water and blow it repeatedly 5 times, and store the RNA solution at -80°C. After detecting RNA concentration and purity, cDNA was synthesized as a fluorescent quantitative template using a reverse transcription kit and amplified on a PCR instrument. The reaction conditions for PCR amplification were: 95°C pre-denaturation for 30s, 95°C denaturation for 5s, 60°C annealing for 30s, and 40 cycles of amplification. β-actin was used as an internal reference, and the 2-△△Ct method was used to calculate the mRNA expression of TNFα, IL-1β, and IL6 in mouse lung tissue. Fig.26 It can be seen that compared with the model group, the expression level of related inflammatory factor mRNA in the MLR1103 group was significantly reduced.

[0112] Weigh 20 mg of mouse lung tissue, cut it into pieces, add steel beads, add 200 μL PBS solution, and grind it in a low-temperature grinder for 10 minutes. Collect the supernatant after centrifugation and store it at -80℃ for later use. ELISA was used to detect the content of IL-6 and TNF-α in mouse lung tissue. Fig. 27 It can be seen that compared with the model group, the levels of IL-6 and TNF-α in the MLR1103 group were significantly reduced.

[0113] Weigh 20 mg of mouse lung tissue, cut it into pieces, add steel beads, add 200 μL PBS solution, and grind it in a low-temperature grinder for 10 minutes. Collect the supernatant after centrifugation and store it at -80℃ for later use. Determine the lactate dehydrogenase content of the lung tissue of each group of mice according to the kit method. Fig.28It can be seen that compared with the model group, the LDH content in the MLR1103 group was significantly reduced.

[0114] Example 7 Evaluation of the protective effect of Lactobacillus reuteri MLR1103 on SiO2-induced pneumoconiosis model in mice

[0115] Male C57BL / 6J mice aged 6-8 weeks were randomly divided into three groups: blank group, model group (SiO2 modeling), and MLR1103 group (SiO2 modeling + MLR1103 administration), with 6 mice in each group.

[0116] After 3 days of adaptive feeding, the MLR1103 group was gavaged with 2×10^7 cfu / kg daily, and the blank group and model group were gavaged with 0.9% NaCl solution at 10 mL / kg body weight daily. Once a day, for a total of 7 days. All the above groups were intervened 7 days before modeling until the end of the experiment.

[0117] On the 8th day, the acute lung injury model of mice was established by intratracheal injection of SiO2: tribromoethanol (300 mg / kg) was injected intraperitoneally for anesthesia. After anesthesia, the mice were fixed in a supine position. After detoxification, the intratracheal quantitative dosing device was used to extract SiO2 0.9% NaCl solution (10 mg / kg) and inserted into the trachea, and 0.05 mL of SiO2 suspension was injected. The mice were then rotated upright to allow the drug to be evenly distributed in the lungs. After the animals woke up naturally, they were kept in cages. On the second day, the model was successfully established when the mice showed dull fur, erect hair, slow reaction, and curling. The modeling method for the blank group animals was the same as above, except that normal saline was used to replace SiO2 for intratracheal injection. On the 21st day, a CT scan of the mouse lungs was performed, and the mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg) to ensure that the mice were completely anesthetized and had no pain response. The anesthetized mice were placed on the scanning table of the CT scanner. The position of the mouse was adjusted to ensure that the lungs were within the scanning range. A scan was performed to obtain lung images. By Fig.29 It can be seen that compared with the blank group, the model group showed ground-glass shadows, indicating inflammation or early fibrosis in the lungs, and the lungs of the MLR1103 group were significantly improved.

[0118] Example 8 Evaluation of the antitussive effect of Lactobacillus reuteri MLR1103 on ammonia-induced cough model in mice

[0119] After 3 days of adaptive feeding, 6-8 week old male C57BL / 6J mice were randomly divided into three groups: model group (ammonia modeling), dextromethorphan group (ammonia modeling + dextromethorphan administration), and MLR1103 group (ammonia modeling + MLR1103 administration), with 6 mice in each group.

[0120] After 3 days of adaptive feeding, the MLR1103 group was gavaged with 2×10^7 cfu / kg daily, the model group was gavaged with 0.9% NaCl solution at 10 mL / kg body weight daily, and the dextromethorphan group was gavaged with 20 mg / kg body weight once a day for 7 days. All the above groups were intervened 7 days before modeling until the end of the experiment.

[0121] On the day of modeling, moisten a medical cotton ball with 0.5 mL of 10% ammonia water, place it in a 1000 mL beaker containing mice, cover it with a glass plate, and record the number of coughs of the mice within 3 minutes. Fig.30 It can be seen that the positive drugs dextromethorphan and MLR1103 can significantly inhibit the cough reaction of mice caused by ammonia.

[0122] Example 9 Preparation of bacterial powder containing Lactobacillus reuteri MLR1103:

[0123] (1) Inoculate MLR1103 into 50 mL of MRS liquid culture medium and culture it in an incubator at 37°C for 24 hours. Transfer 5% of the inoculum to 500 mL of sterilized MRS liquid culture medium and culture it at 37°C for 24 hours. Centrifuge the resulting fermentation broth, discard the supernatant and collect the bacterial precipitate, rinse it twice with PBS buffer, 10 minutes each time, and centrifuge it at a speed of 12000 rpm / min to obtain the MLR1103 bacterial precipitate.

[0124] (2) Take 4.1 g of trehalose, 2.8 g of sodium glutamate, and 8 g of sucrose, dissolve them in 10 mL of 40°C distilled water, filter through a 0.22 μm microporous filter membrane for sterilization, add sterile water to 100 mL, then add 15 g of skim milk powder to dissolve, and sterilize at 110°C to obtain a protective agent solution;

[0125] The MLR1103 bacterial precipitate obtained in step (1) is mixed with the protective agent solution at a mass ratio of 1:5 to obtain a bacterial suspension. 5 mL of the bacterial suspension is dispensed into 10 mL sterile stoppered glass bottles, which are pre-frozen at -80°C. After 2 hours, the suspension is taken out and freeze-dried in a vacuum freeze dryer. The freeze-drying conditions are a vacuum degree of 5 Pa, a partition heating temperature of 20°C, and a cold trap temperature of -55°C. After freeze-drying for 30 hours, the MLR1103 bacterial powder is obtained.

[0126] The MLR1103 bacteria powder was used to replace the MLR1103 bacteria in Examples 3 to 8 respectively. Other aspects were the same as those in the examples. The MLR1103 bacteria powder had similar effects to the MLR1103 bacteria in the examples.

[0127] The Lactobacillus reuteri MLR1103 or bacterial powder containing the Lactobacillus reuteri MLR1103 of the present invention is prepared into corresponding products by conventional techniques and with auxiliary materials acceptable to medicines.

[0128] A fermented food, a probiotic preparation or a pharmaceutical composition comprising the above-mentioned Lactobacillus reuteri.

[0129] The fermented food is solid food, liquid food, semi-solid food, dairy product, vegetable and fruit food or freeze-dried food.

[0130] The Lactobacillus reuteri in probiotic preparations is a fermentation product of live or dead bacteria or live bacteria.

Claims

1. A Lactobacillus reuteri MLR1103, characterized in that The deposit number is CGMCC No.31162.

2. Use of the Lactobacillus reuteri of claim 1 or bacterial powder containing the Lactobacillus reuteri of claim 1 in preparing a product for alleviating lung diseases; the product is a medicine; the lung disease is pulmonary fibrosis.

3. Use of the Lactobacillus reuteri of claim 1 or bacterial powder containing the Lactobacillus reuteri of claim 1 in preparing a product for alleviating lung diseases; the product is a medicine; and the lung disease is asthma.

4. Use of the Lactobacillus reuteri of claim 1 or bacterial powder containing the Lactobacillus reuteri of claim 1 in preparing a product for alleviating lung diseases; the product is a medicine; and the lung disease is pneumoconiosis.

5. Use of the Lactobacillus reuteri of claim 1 or bacterial powder containing the Lactobacillus reuteri of claim 1 in preparing a product for alleviating lung diseases; the product is a medicine; the lung disease is lung damage caused by PM2.

5.

6. Use of the Lactobacillus reuteri of claim 1 or bacterial powder containing the Lactobacillus reuteri of claim 1 in preparing a product for alleviating lung diseases; the product is a medicine; the lung disease is acute lung injury caused by LPS.

7. A fermented food, probiotic preparation or pharmaceutical composition comprising the Lactobacillus reuteri according to claim 1.

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