Leuconostoc mesenteroides and lactococcus lactis composition and uses

Abstract

The application discloses a Leuconostoc intestini and Lactococcus lactis composition and application, belongs to the field of microbial pharmacy, and relates to application of a Leuconostoc intestini and Lactococcus lactis composition to preparation of a drug for treating pulmonary fibrosis diseases; wherein the quantity of the Leuconostoc intestini and the Lactococcus lactis in the composition is equal in terms of colony forming units. Experimental results show that the Leuconostoc intestini and the Lactococcus lactis can effectively improve pulmonary fibrosis when being intervened alone or in combination. It is known to those skilled in the art that excessive deposition of collagen fibers is a core feature of pulmonary fibrosis diseases, and the Leuconostoc intestini and the Lactococcus lactis have a synergistic effect of inhibiting the content of lung tissue collagen in pulmonary fibrosis. Therefore, the Leuconostoc intestini and the Lactococcus lactis composition has a prospect of being developed into a drug for treating pulmonary fibrosis diseases (including idiopathic pulmonary fibrosis and secondary pulmonary fibrosis).

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceuticals, and relates to compound strains and their pharmaceutical uses, specifically to a composition of Leuconostoc mesenteroides and Lactococcus lactis and its uses. Background Technology

[0002] Pulmonary fibrosis (PF) is a lung disease characterized by chronic inflammation and excessive fibrosis of the lung interstitium and alveolar walls. Its main features are significant changes in lung tissue structure, composition, and stiffness, leading to deterioration of lung function. It begins with damage to alveolar epithelial cells, followed by the recruitment and activation of immune cells, ultimately resulting in abnormal post-traumatic repair. PF is divided into idiopathic pulmonary fibrosis (IPF) and secondary pulmonary fibrosis. IPF is a progressive lung disease caused by the combined effects of genetic and environmental factors, more common in middle-aged and elderly people, with a progressive worsening and poor prognosis. The main characteristic of IPF is damage to alveolar epithelial cells, leading to abnormal proliferation of fibroblasts and collagen deposition, ultimately resulting in interstitial fibrosis. Secondary pulmonary fibrosis is a disease state with a definite etiology, belonging to one of the important classifications of pulmonary fibrosis. Essentially, it is an independent clinical disease type caused by other known diseases or factors, with pulmonary tissue fibrosis as the core pathological change. The key to secondary pulmonary fibrosis lies in the "identifiable cause". Common causes can be divided into several categories: (1) underlying lung diseases: such as late-stage chronic obstructive pulmonary disease (COPD), lung tissue scarring after the cure of pulmonary tuberculosis, sequelae of lung abscess, etc., the underlying disease damages lung tissue for a long time and eventually develops into fibrosis; (2) systemic diseases affecting the lungs: such as systemic lupus erythematosus, scleroderma, systemic sclerosis, rheumatoid arthritis and other autoimmune diseases, which attack lung tissue through immune inflammatory response; (3) damage from external factors: such as long-term inhalation of dust (pneumoconiosis), harmful gases (such as formaldehyde, sulfur dioxide), biological particles (such as the cause of allergic pneumonia); or drug side effects (such as chemotherapy drug bleomycin, antiarrhythmic drug amiodarone), lung damage after chest radiotherapy; (4) infection or others: such as the recovery period of severe pneumonia (especially after viral pneumonia), abnormal fibrosis occurs during the lung tissue repair process.

[0003] Currently, there are two marketed anti-pulmonary fibrosis drugs: the multi-target intracellular tyrosine kinase receptor antagonist nintedanib and the novel oral pyridone derivative pirfenidone. Their efficacy and safety have been widely recognized. Pirfenidone was approved in my country for IPF in 2013. Nintedanib was approved in my country for IPF in 2017 and for systemic sclerosis-related interstitial lung disease and progressive fibrotic interstitial lung disease (PF-ILD) in 2020. However, existing anti-pulmonary fibrosis drugs can slow but not stop or reverse the fibrotic process, and these drugs also have certain adverse reactions, making them intolerable for some patients. Currently, the mortality rate for patients with IPF and PF-ILD remains high, and there is an urgent need for treatments with better efficacy and tolerability in treating pulmonary fibrosis.

[0004] Leuconostoc mesenteroides is an important species of the genus Leuconostoc in lactic acid bacteria. It is generally found on the surface of plants and is often used in fermented dairy products, silage, pickles and fruit wines.

[0005] Lactococcus lactis is a prokaryotic microorganism belonging to the phylum Firmicutes, class Bacillus, order Lactobacilli, family Streptococciaceae, and genus Lactococcus. It is also an important type bacterium in the genus Lactococcus, widely found in dairy and plant products, and widely used in the food industry. It is non-pathogenic to humans and animals and is recognized as a safe food-grade microorganism.

[0006] Currently, there are no reports on the combined use of Leuconostoc mesenteroides and Lactococcus lactis for the treatment of pulmonary fibrosis. This invention is proposed to enrich the clinical pool of anti-pulmonary fibrosis drugs. Summary of the Invention

[0007] The first objective of this invention is to provide a composition of Leuconostoc mesenteroides and Lactococcus lactis, and the second objective is to provide the use of this composition in the preparation of a medicament for treating pulmonary fibrosis.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] The use of a composition of Leuconostoc mesenteroides and Lactococcus lactis for the preparation of a medicament for treating pulmonary fibrosis; wherein, in the composition, the number of Leuconostoc mesenteroides and Lactococcus lactis are equal, based on colony-forming units.

[0010] Preferably, the Leuconostoc mesenteroides is Leuconostoc mesenteroides CICC21861.

[0011] Preferably, the lactococcus lactis is lactococcus lactis ATCC9936.

[0012] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

[0013] More preferably, the secondary pulmonary fibrosis is chronic obstructive pneumonia, Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, idiopathic dermatomyositis, scleroderma, or systemic sclerosis.

[0014] More preferably, the drug uses a combination of Leuconostoc mesenteroides and Lactococcus lactis as the active ingredient and is formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0015] More preferably, the excipient is a solid, liquid, or semi-solid.

[0016] More preferably, the dosage form is a powder, injection, tablet, pill, capsule, or oral liquid.

[0017] A bacterial composition comprising Leuconostoc mesenteroides and Lactococcus lactis, wherein the number of Leuconostoc mesenteroides and Lactococcus lactis in the composition is equal in terms of colony-forming units.

[0018] Beneficial effects:

[0019] The experimental results of this invention demonstrate that *Leuconostoc mesenteroides* and *Lactococcus lactis*, alone or in combination, can effectively improve pulmonary fibrosis. Those skilled in the art know that excessive collagen fiber deposition is a core characteristic of pulmonary fibrosis, and that combined administration of *Leuconostoc mesenteroides* and *Lactococcus lactis* has a synergistic effect in inhibiting collagen content in lung tissue during pulmonary fibrosis. Therefore, the combination of *Leuconostoc mesenteroides* and *Lactococcus lactis* shows promise for development into a medicament for treating pulmonary fibrosis diseases, including idiopathic and secondary pulmonary fibrosis. Attached Figure Description

[0020] Figure 1 The curves showing the changes in body weight of mice in each group over 28 days;

[0021] Figure 2 Micro-CT scans of the lungs of mice in each group were performed.

[0022] Figure 3 HE staining results of lung tissue from mice in each group;

[0023] Figure 4 The results of Masson's trichrome staining of lung tissue from each group of mice;

[0024] Figure 5 Collagen content in lung tissue of mice in each group;

[0025] Figure 6 IHC collagen staining was performed on the lung tissues of mice in each group. Detailed Implementation

[0026] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0027] I. Experimental Materials

[0028] 1. Laboratory animals

[0029] Thirty male C57BL / 6 mice, aged 7-8 weeks, were purchased from Jicui Pharmaceutical Co., Ltd. (Nanjing, China).

[0030] 2. Main reagents for the experiment

[0031] Leuconostoc mesenteroides CICC21861 (purchased from Unico (Shanghai) Life Science Co., Ltd.); Lactococcus lactis ATCC9936 (purchased from Unico (Shanghai) Life Science Co., Ltd.); Isoflurane (RWD); Xylene (Shanghai Nord); Ethanol (Shanghai Nord); Hematoxylin (Sigma-Aldrich); 1% Ethanol Hydrochloride (Shanghai Nord); 0.5-1% Ammonia (Shanghai Nord); Eosin (Sigma-Aldrich); Ponceau S-Acid Fuchsin (Sigma-Aldrich); Acetic Acid (Shanghai Nord); 1% Phosphomolybdic Acid (Sigma-Aldrich); Aniline Blue (Sigma-Aldrich); Neutral Gum (Beyotime).

[0032] 3. Experimental instruments

[0033] SkyScan 1276; Olympus VS200.

[0034] II. Experimental Methods

[0035] 1. Grouping, modeling, and drug administration

[0036] Male C57BL / 6 mice aged 7-8 weeks were housed in the SPF Animal Laboratory at the Frontier Medical Center of West China Hospital, Chengdu, Sichuan Province. Laboratory conditions were strictly controlled, with a temperature of 23°C, relative humidity of 46%, and a 12-hour light / dark cycle. This study was approved and supervised by the Laboratory Animal Ethics Committee of West China Hospital, Sichuan University.

[0037] Male C57BL / 6 mice (7-8 weeks old) were randomly assigned to five experimental groups after acclimatization in a specific pathogen-free (SPF) facility for 7 days: control group, model group, *Leuconostoc mesenteroides* group, *Lactococcus lactis* group, and combined group. Under isoflurane anesthesia (5% induction, 2% maintenance), pulmonary fibrosis was induced in the model group, *Leuconostoc mesenteroides* group, *Lactococcus lactis* group, and combined group via intratracheal infusion of bleomycin (3 mg / kg), while the control group received no treatment. Starting from day 7 after bleomycin intratracheal infusion, the *Leuconostoc mesenteroides* group was administered *Leuconostoc mesenteroides* via gavage daily (using physiological saline as the solvent, bacterial concentration 1×10⁻⁶). 8 CFU / mL, administration volume 200 μL), Lactococcus lactis group was administered Lactococcus lactis by gavage daily (using physiological saline as solvent, bacterial concentration 1×10⁻⁶). 8 The combined group was administered Leuconostoc mesentery and Lactococcus lactis via daily gavage (using physiological saline as the solvent, with a concentration of 1×10⁻⁶ CFU / mL, and a dosing volume of 200 μL). 8 Mice in both the control and model groups were administered the same volume of physiological saline by gavage (CFU / mL, administration volume 200 μL) until day 28. Lung tissue was collected on day 28 for histological analysis.

[0038] 2. Micro-CT for lung scanning imaging

[0039] On the day of the last administration, lung scans were performed on mice in each group. Mice were anesthetized (by isoflurane inhalation), securely fixed to the scanning table, and scanned using a SkyScan 1276. After setting key parameters, whole-lung scans were performed to acquire raw projection images. Tomographic reconstruction of the raw projection data was performed using the accompanying software NRecon.

[0040] 3. Lung histological analysis

[0041] Lung tissues from mice in each group were collected for histological analysis after lung scan imaging was performed on the day of the last administration.

[0042] (1) Hematoxylin-eosin (HE) staining

[0043] After dewaxing with xylene and dehydration with graded ethanol, lung tissue sections were stained with hematoxylin for 5-10 minutes, rinsed with tap water to remove excess stain, then differentiated with 1% hydrochloric acid ethanol for a few seconds until pale purple-red, rinsed with tap water, and then blued with 0.5% ammonia or saturated sodium bicarbonate for 3-5 minutes, and rinsed clean again. After dehydration with 80% and 95% ethanol for 1 minute each, they were stained with eosin for 3-5 minutes, then soaked in 95% ethanol for 2 minutes, followed by dehydration with anhydrous ethanol I and II for 2 minutes each, cleared with xylene I and II for 5 minutes each, and finally mounted with neutral resin.

[0044] (2) Masson trichrome staining

[0045] Lung tissue sections were washed with water and then stained sequentially: Wiegand's hematoxylin (5 min), rinsed with 0.2% acetic acid (2 min), Ponceau S-Acid Fuchsin (5 min), repeated acetic acid rinse, 1% phosphomolybdic acid differentiation (5 min), aniline blue reverse staining (5 min), and a final acetic acid rinse. Ethanol differentiation was performed before standard dehydration, xylene removal, and neutral resin mounting. Microscopic analysis was then performed on the stained sections.

[0046] (3) Immunohistochemical (IHC) staining

[0047] After dewaxing, antigen retrieval, and blocking, the sections were incubated overnight at 4 °C with primary antibody collagen I (rabbit polyclonal, MCE, HY-P81227). HRP-labeled secondary antibody was added, and the sections were incubated at 37 °C for 30 min. The sections were washed three times with PBS for 5 min each time. DAB chromogenic solution was added, and the chromogenic time was controlled under an optical microscope (approximately 3-5 min). Chlorination was stopped with distilled water. Hematoxylin was counterstained for 30 s, followed by differentiation with hydrochloric acid ethanol for a few seconds, and then rinsed with tap water for 10 min to achieve blue reversion. Using ImageJ v1.53k (NIH), DAB-positive cells (cytoplasmic / nuclear brown precipitate) in 10 randomly selected high-power fields (HPF; 20× objective, Leical DM IL LED) were counted for quantitative analysis.

[0048] 4. Statistical Analysis

[0049] All statistical analyses were performed using GraphPad Prism 7 (GraphPad software). All data are expressed as mean ± SEM. Normality was assessed using the Shapiro-Wilk test. One-way ANOVA and Tukey post-tests were used for comparisons among multiple groups. Pearson correlation coefficients quantified the linear relationship between variables. Statistical significance was defined as P < 0.05. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0050] III. Experimental Results

[0051] 1. Weight change trend

[0052] This study successfully established a C57BL / 6 mouse model of pulmonary fibrosis via bleomycin intratracheal instillation and evaluated the effects of Leuconostoc mesenteroides, Lactococcus lactis, and their combined gavage intervention on the pulmonary fibrosis model mice. Results are as follows: Figure 1As shown, the 28-day dynamic monitoring of body weight revealed that the control group mice showed a stable and normal growth trend in body weight; compared with the control group, the model group mice experienced severe inhibition of body weight growth after bleomycin induction; and compared with the model group, the inhibition of body weight growth in the Leuconostoc mesenteroides group, Lactococcus lactis group, and combined group mice was significantly alleviated.

[0053] 2. Micro-CT scan of the lungs to assess the severity of fibrotic lesions.

[0054] Lung scan imaging results as follows Figure 2 As shown, the imaging results clearly revealed the differences in lung structure among the groups of mice: In the control group, normal lung tissue structure and uniform lung field translucency were observed, with uniform lung parenchyma density, clear texture, and no abnormal high-density shadows or structural distortions, consistent with the characteristics of healthy lung tissue. In the model group, the lungs showed high-density consolidation in patches, mainly concentrated near the trachea. Irregular dilation of the bronchial lumen was also observed in some fibrotic lesion areas, and fine linear high-density shadows were occasionally seen in the peripheral lung fields, possibly due to thickening of the interlobular septa and formation of fibrotic strands. Compared with the model group, the lung field translucency was significantly improved and the number of fibrotic lesions was reduced in the *Leuconostoc mesenteroides* group, *Lactococcus lactis* group, and combined group. The *Lactococcus lactis* group and the combined group had the fewest areas of fibrotic consolidation. Furthermore, the lung structure of the mice in each treatment group was well preserved, with no obvious tractional bronchiectasis or severe lung tissue collapse, and the bronchial course was relatively natural.

[0055] Micro-CT imaging results showed that intervention with Leuconostoc mesenteroides and Lactococcus lactis, alone or in combination, could effectively improve the pathological imaging changes in mice with pulmonary fibrosis, significantly reduce the increase in lung tissue density, reduce the formation of fibrotic lesions, and better maintain the normal structure and translucency of lung tissue.

[0056] 3. Observe lung structure and inflammatory status by HE staining of lung tissue.

[0057] HE staining results are as follows Figure 3As shown, HE staining results clearly revealed the morphological differences in lung tissue among the different groups of mice: the lung tissue of the control group mice was intact and clear, with uniform alveolar cavities, thin and continuous alveolar walls, fine alveolar septa without significant thickening, and only a small number of scattered mononuclear cells in the lung interstitium, without inflammatory cell aggregation or exudation. The bronchial structure was normal, presenting typical morphological characteristics of healthy lung tissue. The lung tissue of the model group mice showed typical and severe pulmonary fibrosis and inflammatory pathological changes. A large number of normal alveolar structures disappeared, replaced by diffusely widened and fused alveolar septa, forming cystic cavities or areas of consolidation of varying sizes. The lung parenchymal structure was severely disordered, and the alveolar septa, alveolar cavities, and interpulmonary spaces were significantly affected. Numerous diffusely infiltrating inflammatory cells, mainly including neutrophils, lymphocytes, and macrophages, were observed in the lung tissue, forming obvious inflammatory exudative foci. Thickened septa and consolidation areas showed deposits of pinkish, homogeneous amorphous material, suggesting excessive accumulation of extracellular matrix such as collagen. Compared with the model group, the lung tissue structure of mice in the Leuconostoc mesenteroides group, Lactococcus lactis group, and combined group was significantly reduced, alveolar cavity structure was partially restored, alveolar septal widening was significantly reduced, alveolar collapse and fusion areas were reduced, the number of infiltrating inflammatory cells in the lung tissue was significantly reduced, and their distribution was more scattered. Inflammatory exudative foci shrank or disappeared, and the areas of pinkish amorphous matrix deposition were significantly reduced and thinned.

[0058] HE staining pathological results showed that Leuconostoc mesenteroides and Lactococcus lactis, alone or in combination, could effectively improve the inflammatory response in the lung tissue of mice with pulmonary fibrosis, reduce the destruction of alveolar structure, inhibit fibroblast proliferation and abnormal deposition of extracellular matrix, thereby alleviating the pathological process of pulmonary fibrosis.

[0059] 4. Masson's trichrome staining of lung tissue to assess collagen fiber deposition

[0060] Excessive collagen fiber deposition is a core characteristic of pulmonary fibrosis. To quantitatively evaluate the degree of pulmonary fibrosis and the effects of Leuconostoc mesenteriae and Lactococcus lactis, alone or in combination, on collagen metabolism, this study used Masson staining to specifically stain collagen fibers in the lung tissue of mice in each group. Figure 4 ), and perform collagen content analysis ( Figure 5Masson staining showed that the lung tissue structure of the control group mice was normal, with collagen fibers only appearing as thin, sparse linear distributions in the alveolar septa, blood vessels, and peribronchial basement membrane. Quantitative analysis also showed that the collagen content was extremely low (12.6% ± 0.01%). In the model group mice, a large number of dense, blue collagen fibers proliferated and filled the inflammatory infiltration areas and areas of alveolar structural damage and collapse, replacing the normal lung parenchyma structure. The collagen content was significantly higher than that of the control group (p < 0.001), reaching 62.5% ± The result was 0.06%, confirming the excessive synthesis and deposition of extracellular matrix, especially collagen, in the pulmonary fibrosis model. The degree of collagen deposition was reduced in mice in the Leuconostoc mesenteroides group, Lactococcus lactis group, and combined group. The area and density of the blue area stained by Masson staining were significantly lower than those in the model group. The widening of the blue area in the alveolar septa was reduced. The coarse collagen strands and diffuse blue deposition areas were reduced, thinned, and sparsed. The phenomenon of collagen replacing the structurally damaged areas was alleviated. The collagen content in the combined group was significantly reduced to the level of the control group.

[0061] Furthermore, the inhibition rates of *Leuconostoc mesenteroides* and *Lactococcus lactis* alone or in combination on lung collagen content were calculated based on the collagen content in the lung tissues of mice in the *Leuconostoc mesenteroides* group, *Lactococcus lactis* group, and combined group, as well as the collagen content in the lung tissue of mice in the model group. The Jin Zhengjun q-value method (Effects of Artesunate on Proliferation and Apoptosis of Human HepG2 Liver Cancer Cells and Anti-Hepatocellular Carcinoma Effect of Combined Chemotherapy Drugs, Chinese Journal of Pathophysiology, 2012) was used to determine whether there was a synergistic effect between *Leuconostoc mesenteroides* and *Lactococcus lactis* on lung collagen content (q < 0.85 indicates antagonistic effect, 0.85 ≤ q < 1.15 indicates additive effect, and q ≥ 1.15 indicates synergistic effect). The results are shown in Table 1. These results demonstrate that the combined administration of *Leuconostoc mesenteroides* and *Lactococcus lactis* has a synergistic inhibitory effect on lung collagen content in mice with pulmonary fibrosis.

[0062] Table 1

[0063] Inhibition rate of Lactococcus lactis group Inhibition rate of Leuconostoc mesenteroides group Combined group inhibition rate q value 45.62% 40.36% 81.18% 1.20

[0064] 5. Immunohistochemical staining of lung tissue to assess collagen content

[0065] IHC collagen staining of lung tissue showed ( Figure 6 Significant differences were found in the positive expression of collagen in the lung tissue of each group: compared with the control group, the positive expression of collagen in the lung tissue of the model group was significantly enhanced, and it was widely distributed in the thickened alveolar septa, lung interstitium and fibrotic lesion areas, with a large number of brown granules visible; after intervention with Leuconostoc mesenteroides and Lactococcus lactis alone or in combination, the positive expression of collagen in the lung tissue was significantly weakened compared with the model group, and brown granules were mainly visible in the local alveolar septa.

[0066] Jin Zhengjun's q-value method also showed that the combined administration of Leuconostoc mesenteroides and Lactococcus lactis had a synergistic effect in inhibiting the collagen content in the lung tissue of mice with pulmonary fibrosis (Table 2).

[0067] Table 2

[0068] Inhibition rate of Lactococcus lactis group Inhibition rate of Leuconostoc mesenteroides group Combined group inhibition rate q value 51.54% 40.90% 83.90% 1.18

[0069] The above experimental results indicate that *Leuconostoc mesenteroides* and *Lactococcus lactis*, alone or in combination, can effectively improve pulmonary fibrosis. Those skilled in the art know that excessive collagen fiber deposition is a core characteristic of pulmonary fibrosis, and that combined administration of *Leuconostoc mesenteroides* and *Lactococcus lactis* has a synergistic effect in inhibiting collagen content in lung tissue during pulmonary fibrosis. Therefore, the combination of *Leuconostoc mesenteroides* and *Lactococcus lactis* shows promise for development into a drug for treating pulmonary fibrosis diseases, including idiopathic and secondary pulmonary fibrosis.

[0070] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. The use of a composition of Leuconostoc mesenteroides and Lactococcus lactis in the preparation of a medicament for treating pulmonary fibrosis; wherein, In this composition, the number of Leuconostoc mesenteroides and Lactococcus lactis are equal, based on colony-forming units; the Leuconostoc mesenteroides is Leuconostoc mesenteroides CICC21861, and the Lactococcus lactis is Lactococcus lactis ATCC9936.

2. The use according to claim 1, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

3. The use according to claim 2, wherein the cause of the secondary pulmonary fibrosis is chronic obstructive pneumonia, Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, idiopathic dermatomyositis, scleroderma, or systemic sclerosis.

4. The use according to any one of claims 1 to 3, characterized in that: The drug uses a combination of Leuconostoc mesenteroides and Lactococcus lactis as its active ingredient, and is formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The auxiliary material is a solid, liquid, or semi-solid.

6. The use according to claim 4, characterized in that: The dosage form is powder, injection, tablet, pill, capsule or oral liquid.

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