Leuconostoc mesenteroides and lactococcus lactis composition and application

Abstract

The invention discloses a composition of leuconostoc mesenteroides and lactococcus lactis and application thereof, belongs to the field of microbial pharmacy, and requests to protect application of the composition of leuconostoc mesenteroides and lactococcus lactis in preparation of drugs for treating pulmonary fibrosis diseases. Wherein in the composition, the number of the leuconostoc mesenteroides is equal to that of the lactococcus lactis in a colony forming unit. Experimental results show that pulmonary fibrosis can be effectively improved through independent or combined intervention of leuconostoc mesenteroides and lactococcus lactis. Technicians in the field know that excessive deposition of collagenous fibers is the core characteristic of pulmonary fibrosis lesion, and combined administration of leuconostoc mesenteroides and lactococcus lactis has the effect of synergistically inhibiting the collagen content of lung tissues in pulmonary fibrosis. Therefore, the composition of the leuconostoc mesenteroides and the lactococcus lactis has the prospect of being developed into medicines for treating pulmonary fibrosis diseases (including idiopathic pulmonary fibrosis and secondary pulmonary fibrosis).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial pharmaceuticals, and relates to a composite bacterial strain and its pharmaceutical use, in particular to a Leuconostoc mesenteroides and Lactococcus lactis composition and use. BACKGROUND

[0002] Pulmonary fibrosis (PF) is a lung disease characterized by chronic inflammation and excessive fibrosis of the lung interstitium and alveolar wall. Its main feature is the significant change in lung tissue structure, composition and hardness, leading to deterioration of lung function. It begins with damage to alveolar epithelial cells, followed by recruitment and activation of immune cells, and eventually leads to abnormal repair after trauma. PF is divided into idiopathic pulmonary fibrosis (IPF) and secondary pulmonary fibrosis. IPF is a progressive lung disease caused by the combined action of genetic and environmental factors, mainly seen in the elderly, and is progressive and has a poor prognosis. The main feature of IPF is damage to alveolar epithelial cells, leading to abnormal proliferation of fibroblasts and collagen deposition, and eventually leading to pulmonary interstitial fibrosis. Secondary pulmonary fibrosis is a disease state with a clear cause, which is one of the important classifications of pulmonary fibrosis, and its essence is an independent clinical disease type with lung tissue fibrosis as the core pathological change triggered by other known diseases or factors. The key to secondary pulmonary fibrosis is that the cause can be found, and the common causes can be divided into several categories: (1) underlying lung disease: such as advanced chronic obstructive pulmonary disease (COPD), post-treatment sequelae of pulmonary tuberculosis, and sequelae of lung abscess, long-term damage to lung tissue by underlying diseases, and eventually developing into fibrosis; (2) systemic disease involving 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) external factors damage: such as long-term inhalation of dust (pneumoconiosis), harmful gases (such as formaldehyde and sulfur dioxide), biological particles (such as the cause of allergic pneumonitis); or drug side effects (such as the chemotherapeutic drug bleomycin and the antiarrhythmic drug amiodarone), lung damage after chest radiotherapy; (4) infection or others: such as severe pneumonia (especially after viral pneumonia) recovery period, abnormal fibrosis occurs during lung tissue repair.

[0003] Currently, there are two anti-pulmonary fibrosis drugs on the market, namely the multi-target intracellular tyrosine kinase receptor antagonist Nintedanib and the new oral pyridone derivative Pirfenidone, and their effectiveness and safety have been widely recognized. Pirfenidone was approved in China in 2013 for the IPF indication. Nintedanib was approved in China in 2017 for the IPF indication, and in 2020 for the systemic sclerosis-associated interstitial lung disease and progressive fibrotic interstitial lung disease (PF-ILD) indications. However, the existing anti-pulmonary fibrosis drugs can slow down but cannot stop or reverse the fibrosis process, and the above-mentioned drugs also have certain adverse reactions, which make patients unable to tolerate. At present, the mortality rate of IPF and PF-ILD patients is still very high, and there is an urgent need for treatment drugs with better anti-pulmonary fibrosis effect and tolerance.

[0004] Leuconostoc mesenteroides is an important strain of Leuconostoc in lactic acid bacteria, which generally exists on the surface of plants and is commonly used in fermented dairy products, silage, pickles and fruit wine.

[0005] Lactococcus lactis is a prokaryotic microorganism belonging to the Firmicutes phylum, Bacilli class, Lactobacillales order, Streptococcaceae family and Lactococcus genus, and is also an important model bacterium in the Lactococcus genus, widely exists in dairy products and plant products, is widely used in the food industry, and is non-pathogenic to humans and animals, is a recognized safe food-grade microorganism.

[0006] There is no related report on the combination of Leuconostoc mesenteroides and Lactococcus lactis for treating pulmonary fibrosis. In order to develop rich clinical anti-pulmonary fibrosis drugs, the present application is proposed. SUMMARY

[0007] The first object of the present application is to provide a Leuconostoc mesenteroides and Lactococcus lactis composition, and the second object is to provide the use of the composition for preparing a drug for treating pulmonary fibrosis disease.

[0008] The above objects of the present application are achieved by the following technical solutions:

[0009] The use of a Leuconostoc mesenteroides and Lactococcus lactis composition for preparing a drug for treating pulmonary fibrosis disease; wherein in the composition, the number of Leuconostoc mesenteroides and Lactococcus lactis is equal in terms of 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 disease is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

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

[0014] More preferably, the drug is a pharmaceutically acceptable dosage form prepared from a combination of M. filiformis and L. lactis as active ingredients and pharmaceutically acceptable excipients.

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

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

[0017] A bacterial composition consisting of M. filiformis and L. lactis, and in the composition, the number of M. filiformis and L. lactis is equal in terms of colony forming units.

[0018] Beneficial effects:

[0019] The experimental results of the present application show that M. filiformis and L. lactis alone or in combination can effectively improve pulmonary fibrosis. Those skilled in the art know that excessive deposition of collagen fibers is the core feature of pulmonary fibrosis lesions, and the combined administration of M. filiformis and L. lactis has a synergistic effect on inhibiting the collagen content in lung tissue in pulmonary fibrosis. Therefore, the combination of M. filiformis and L. lactis has the prospect of developing into a drug for treating pulmonary fibrosis diseases (including idiopathic pulmonary fibrosis and secondary pulmonary fibrosis). BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the weight change curve of each group of mice within 28 days;

[0021] Figure 2 is the Micro-CT scanning imaging of the lungs of each group of mice;

[0022] Figure 3 is the HE staining result of the lung tissue of each group of mice;

[0023] Figure 4 is the Masson's trichrome staining result of the lung tissue of each group of mice;

[0024] Figure 5 is the collagen content of the lung tissue of each group of mice;

[0025] Figure 6 is the IHC collagen staining of the lung tissue of each group of mice. DETAILED DESCRIPTION

[0026] The substantial content of the present application will be described in detail below in combination with examples, but the protection scope of the present application is not limited by this.

[0027] I. Experimental materials

[0028] 1. Experimental animals

[0029] C57BL / 6 mice, 7-8 weeks old, male, 30, purchased from Jiequ Pharmaceutical Company (Nanjing, China).

[0030] 2. Main reagents for experiment

[0031] Leuconostoc mesenteroides CICC21861 (purchased from Yuli Ke (Shanghai) Life Science Co., Ltd.); Lactococcus lactis ATCC9936 (purchased from Yuli Ke (Shanghai) Life Science Co., Ltd.); Isoflurane (RWD); Xylene (Shanghai Nuode); Ethanol (Shanghai Nuode); Hematoxylin (Sigma-Aldrich); 1% hydrochloric acid ethanol (Shanghai Nuode); 0.5-1% ammonia water (Shanghai Nuode); Eosin (Sigma-Aldrich); Ponceau acid fuchsin (Sigma-Aldrich); Acetic acid (Shanghai Nuode); 1% phosphomolybdic acid (Sigma-Aldrich); Aniline blue (Sigma-Aldrich); Neutral gum (Biyun Tian).

[0032] 3. Experimental instruments

[0033] SkyScan 1276; Olympus VS200.

[0034] II. Experimental methods

[0035] 1. Grouping, modeling and administration

[0036] 7-8-week-old male C57BL / 6 mice were raised in the SPF animal laboratory of the Frontier Medical Center of West China Hospital, Chengdu, Sichuan Province. The laboratory conditions were strictly controlled, with a temperature of 23°C, a relative humidity of 46%, and a light / dark cycle of 12 hours. This study was approved and supervised by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University.

[0037] Male C57BL / 6 mice (7-8 weeks old) were randomly divided into 5 groups after 7 days of adaptation in a specific pathogen-free (SPF) facility: control group, model group, Leuconostoc mesenteroides group, Lactococcus lactis group, and combined group. Under isoflurane anesthesia (5% induction, 2% maintenance), the model group, Leuconostoc mesenteroides group, Lactococcus lactis group, and combined group were induced by intratracheal instillation of bleomycin (3 mg / kg), and the control group was not treated. From the 7th day after intratracheal instillation of bleomycin, the Leuconostoc mesenteroides group was given Leuconostoc mesenteroides (1×10 8 CFU / mL, 200 μL of the drug was administered) every day, the Lactococcus lactis group was given Lactococcus lactis (1×10 8 CFU / mL, 200 μL of the drug was administered) every day, and the combined group was given Leuconostoc mesenteroides and Lactococcus lactis (both 1×10 8 CFU / mL, 200 μL of the drug was administered) every day. The control group and model group were given the same amount of normal saline by gavage, and the gavage was continued until the 28th day. The lung tissue was collected on the 28th day for histological analysis.

[0038] 2. Lung scanning imaging by Micro-CT

[0039] On the last day of administration, lung scanning imaging was performed on mice in each group. After anesthesia (isoflurane inhalation), the mice were fixed properly on the scanning bed, and SkyScan 1276 was used for scanning. After setting the key parameters, the whole lung of the mouse was scanned to obtain the original projection image. The original projection data was reconstructed into a tomographic image using the NRecon software provided with the device.

[0040] 3. Lung histological analysis

[0041] After lung scanning imaging on the last day of administration, the lung tissue of mice in each group was collected for histological analysis.

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

[0043] After xylene deparaffinization and gradient ethanol dehydration of the lung tissue sections, hematoxylin staining solution was used for 5-10 minutes, tap water was used to rinse off the color, then 1% hydrochloric acid ethanol was used for differentiation for a few seconds to a light purple red color, tap water was used for washing, then 0.5% ammonia water or saturated sodium bicarbonate was used for blueing for 3-5 minutes, and then washed clean; then after dehydration with 80% and 95% ethanol for 1 minute each, the tissue sections were immersed in eosin staining solution for 3-5 minutes, then immersed in 95% ethanol for 2 minutes, followed by dehydration with anhydrous ethanol I and II for 2 minutes each, transparency with xylene I and II for 5 minutes each, and finally dropped with neutral balsam for mounting.

[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 in the figure, the 28-day weight dynamic monitoring showed that the weight of the control group mice showed a stable and normal growth trend; compared with the control group, the weight growth of the model group mice was severely inhibited after bleomycin induction; compared with the model group, the weight growth inhibition of the Leuconostoc mesenteroides group, Lactococcus lactis group and combined group was obviously relieved.

[0053] 2. Lung Micro-CT evaluation of fibrosis lesion severity

[0054] The lung scan imaging results are shown in Figure 2 As shown in the figure, the imaging results clearly revealed the differences in lung structure of mice in each group: normal lung tissue structure and uniform lung field transparency were observed in the control group mice, the lung parenchyma density was uniform, the texture was clear, and no abnormal high-density shadow or structural distortion was observed, which was consistent with the characteristics of healthy lung tissue; the lung of the model group mice showed high-density consolidation, which was patchy, mainly concentrated near the bronchus, and part of the fibrosis lesion area showed irregular bronchial lumen dilation, and occasional fine linear high-density shadows were observed in the peripheral lung field, which may be due to interlobular septal thickening and fibrous cord formation; compared with the model group, the lung field transparency of the Leuconostoc mesenteroides group, Lactococcus lactis group and combined group was significantly improved, and the fibrosis lesions were reduced, among which the fibrosis consolidation area of the Lactococcus lactis group and the combined group was the least. In addition, the lung structure of mice in each administration group was well preserved, and no obvious traction bronchiectasis or severe lung tissue collapse was observed, and the bronchial course was relatively natural.

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

[0056] 3. Lung tissue HE staining to observe lung structure and inflammation

[0057] The HE staining results are shown in Figure 3As shown, HE staining results clearly revealed the morphological differences of lung tissues in each group of mice: the lung tissue structure of control mice was complete and clear, the alveolar cavity size was uniform, the alveolar wall was thin and continuous, the alveolar septum was fine, no obvious thickening was observed, only a small amount of single nucleus cells were observed in the lung interstitium, no inflammatory cell aggregation or exudation was observed, the bronchial structure was normal, and the typical morphological characteristics of healthy lung tissue were observed; the lung tissue of model mice showed typical and severe pulmonary fibrosis and inflammatory pathological changes, a large number of normal alveolar structures disappeared, and diffuse alveolar septum significantly widened and fused to form cystic cavities or consolidation areas of varying sizes, the lung parenchyma structure was severely disordered, a large number of diffuse inflammatory cells were observed in the lung alveolar septum, alveolar cavity and lung interstitium, mainly including neutrophils, lymphocytes and macrophages, forming obvious inflammatory exudation foci, the thickened septum and consolidation area showed powder-dyed and homogeneous amorphous substance deposition, suggesting excessive accumulation of extracellular matrix such as collagen; compared with the model group, the lung tissue structure of the Leuconostoc group, the Lactococcus group and the combined group was significantly reduced, the alveolar cavity structure was partially restored, the lung alveolar septum was significantly reduced, the lung alveolar collapse and fusion area was reduced, the number of inflammatory cells infiltrated in the lung tissue was significantly reduced, and the distribution tended to be scattered, the inflammatory exudation foci were reduced or disappeared, and the powder-dyed amorphous matrix deposition area was significantly reduced and thinned.

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

[0059] 4. Lung tissue Masson trichrome staining to evaluate collagen fiber deposition

[0060] Excessive deposition of collagen fibers is a core feature of pulmonary fibrosis. To quantitatively evaluate the degree of pulmonary fibrosis and the effect of Leuconostoc, Lactococcus alone or in combination on collagen metabolism, the lung tissue collagen fibers of each group of mice were specifically stained by Masson staining method ( Figure 4 ), and collagen content analysis was performed ( 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] Lactococcus group inhibition rate Leuconostoc group inhibition rate 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] The Q value method of Kim Jong-un also showed that Leuconostoc mesenteroides combined with Lactococcus lactis administration had a synergistic effect on inhibiting the lung tissue collagen content of the lung fibrosis model mice (Table 2).

[0067] Table 2

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

[0069] The above experimental results show that Leuconostoc mesenteroides, Lactococcus lactis alone or in combination can effectively improve pulmonary fibrosis. Those skilled in the art know that excessive deposition of collagen fibers is the core feature of pulmonary fibrosis lesions, and Leuconostoc mesenteroides combined with Lactococcus lactis administration has a synergistic effect on inhibiting the lung tissue collagen content in pulmonary fibrosis. Therefore, the composition of Leuconostoc mesenteroides and Lactococcus lactis has the prospect of developing into a drug for treating pulmonary fibrosis diseases (including idiopathic pulmonary fibrosis and secondary pulmonary fibrosis).

[0070] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

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.

2. The use according to claim 1, wherein the Leuconostoc mesenteroides is Leuconostoc mesenteroides CICC21861.

3. The use according to claim 1, wherein the lactococcus is lactococcus ATCC9936.

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

5. The use according to claim 4, wherein the secondary pulmonary fibrosis is chronic obstructive pulmonary disease, Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, idiopathic dermatomyositis, scleroderma, or systemic sclerosis.

6. The use according to any one of claims 1 to 5, 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.

7. The use according to claim 6, characterized in that: The auxiliary material is a solid, liquid, or semi-solid.

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

9. A bacterial composition, characterized in that: It is composed of Leuconostoc mesenteroides and Lactococcus lactis, and the number of Leuconostoc mesenteroides and Lactococcus lactis in the composition is equal in terms of colony-forming units.

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