Application of lactobacillus paracasei L9 in preparation of preparation for improving pulmonary fibrosis of aging mouse

Abstract

The invention belongs to the field of microorganisms, and particularly relates to application of lactobacillus paracasei L9 in preparation of a preparation for improving pulmonary fibrosis of an aged mouse. According to the lactobacillus paracasei L9 disclosed by the invention, the synthesis of I-type collagen is reduced by regulating and controlling gene expression, so that pulmonary fibrosis is relieved; meanwhile, the L9 improves the content of propionic acid and butyric acid in intestinal tracts and serum by regulating the abundance of SCFA-producing microbial flora in the intestinal tracts, so that pulmonary fibrosis is inhibited, and the L9 is a potential ideal preparation for improving or treating aging-related pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and relates to the application of probiotics in improving health conditions, specifically, the application of Lactobacillus paracasei L9 in the preparation of a preparation for improving pulmonary fibrosis in aging mice. Background Art

[0002] Pulmonary fibrosis is a complex, age-related, epithelium-driven interstitial lung disease, often found in the end stage of lung diseases. The cause of most pulmonary fibrosis is unknown, involving the coordinated action of multiple types of cells and the signal transduction mechanism across different organs and systems. The median survival time after diagnosis is less than 3 years. The lung function of most patients declines slowly and progressively, eventually leading to respiratory failure, while 10-15% of patients show an abnormally rapid decline within a few months. Recent studies have shown that the incidence of pulmonary fibrosis is closely related to aging, with an average onset age of 60-70 years, and aging male individuals have the highest susceptibility to pulmonary fibrosis. In recent years, with the aggravation of global aging, the incidence of pulmonary fibrosis has shown an upward trend worldwide, resulting in a rapid increase in the medical burden caused by pulmonary fibrosis.

[0003] The pathogenesis of pulmonary fibrosis involves impaired wound repair ability and dysregulated interaction between epithelium and mesenchyme, ultimately leading to pulmonary structure remodeling, mainly manifested by excessive deposition of extracellular matrix (ECM) such as collagen. The main components of ECM are composed of five substances: collagen, non-collagen, proteoglycan, elastin, and glycosaminoglycan. According to the distribution position in tissues, ECM can be divided into basement membrane and interstitial matrix. Collagen is the main structural protein constituting the ECM of lung tissue, and the excessive deposition of ECM during the occurrence of pulmonary fibrosis is mainly Type I collagen (Coll-Ⅰ) and Type III collagen (Coll-Ⅲ). In mammals, the components of ECM are mainly synthesized by fibroblasts and osteoblasts. The former is located in the skin, tendons, and other connective tissues, and the latter is located in the bones. After lung tissue is injured, fibroblasts transform from a resting state to an activated state, producing markers such as α-smooth muscle actin (α-SMA).

[0004] The intestinal flora is the largest and most diverse community in the human microbiome. They live in a mutually beneficial relationship with the host and perform a wide range of functions, which are the key to maintaining the homeostasis of the host's metabolism and immune system. [7]。The bidirectional interaction between the respiratory mucosa and the gut microbiota is called the gut-lung axis. It has been reported that implementing nutritional intervention strategies in elderly patients and affecting the diversity of gut microbiota and their metabolites is a potential adjuvant therapy for pulmonary fibrosis. Changes in the gut microbiota and their by-products (such as endotoxins, metabolites, cytokines, and hormones) may affect lung diseases. For example, microbial by-products can enter the lungs through the blood and have a more profound impact. Some studies have reported that manipulating specific gut bacteria that produce short-chain fatty acids (SCFAs) can effectively induce the production and enhanced function of regulatory T cells in the mouse colon, thereby affecting TGF-β signal transduction and enhancing the pulmonary immune response.

[0005] Lactobacillus paracasei L9 (L9, Yang J, Ren F, Zhang H, Jiang L, Hao Y, Luo X. Induction of Regulatory Dendritic Cells by Lactobacillus paracasei L9 Prevents Allergic Sensitization to Bovine β-Lactoglobulin in Mice[J]. 2015, 25(10): 1687-1696, CN 110339217 A, CGMCC NO.9800) is a probiotic isolated from the feces of healthy centenarians that is beneficial for regulating body homeostasis. Studies have shown that oral administration of L9 can reduce airway allergic reactions in an asthma mouse model by balancing the Th1 / Th2 immune response and regulating the IL-17 pro-inflammatory immune response. A large number of experiments have shown that oral administration of lactobacilli can improve respiratory diseases such as cystic fibrosis, asthma, cancer, and chronic obstructive pulmonary disease. However, there is currently no study on whether L9 can relieve pulmonary fibrosis. Summary of the Invention

[0006] The present invention aims to provide the application of L9 in alleviating aging-related pulmonary fibrosis and further clarify the important role of gut microbiota and their metabolites in diseases.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention is to provide the application of Lactobacillus paracasei L9 in the preparation of a preparation for treating pulmonary fibrosis.

[0009] Furthermore, the pulmonary fibrosis is aging-related fibrosis.

[0010] Furthermore, the preparation is an oral preparation.

[0011] Furthermore, the preparation also includes a pharmaceutically acceptable carrier.

[0012] Furthermore, the preparation reduces the synthesis of type I collagen by inhibiting the expression of HSP47 protein through the JNK-HSF1 pathway.

[0013] The second aspect of the present invention is to provide the use of Lactobacillus paracasei L9 in the preparation of a preparation for regulating the abundance of intestinal flora, characterized in that the regulation is to increase the abundance of short-chain fatty acid-producing flora and reduce the abundance of harmful flora.

[0014] Furthermore, the regulation is to increase the abundance of Eubacterium mucigenes, Clostridium leptum, Lachnospiraceae_UCG-006 and Rikenella.

[0015] Furthermore, the regulation is to reduce the abundance of intestinal harmful bacteria norank_f__norank_o__Clostridia_UCG-014 and Desulfovibrio.

[0016] Furthermore, the preparation is an oral preparation.

[0017] Furthermore, the preparation also includes a pharmaceutically acceptable carrier.

[0018] The third aspect of the present invention is to provide the use of Lactobacillus paracasei L9 in the preparation of a preparation for increasing the concentration of short-chain fatty acids in vivo.

[0019] Furthermore, the short-chain fatty acids in vivo are propionic acid and / or butyric acid.

[0020] Furthermore, the "in vivo" refers to in the intestine and / or in the serum.

[0021] Furthermore, the preparation is an oral preparation.

[0022] Furthermore, the preparation also includes a pharmaceutically acceptable carrier.

[0023] The fourth aspect of the present invention is to provide the use of Lactobacillus paracasei L9 in the preparation of a preparation for inhibiting the expression of HSP47, characterized in that the application is an in vitro cytological application.

[0024] Furthermore, the preparation inhibits the expression of the HSP47 transcription factor HSF1 by regulating the expression of the key targets HPK1, TAK1, MKK4, and MKK7 of the JNK pathway mediated by the PDGFRβ and TGFβR2 receptors.

[0025] The beneficial effects of the present invention include:

[0026] 1. Lactobacillus paracasei L9 can significantly reduce the expression of senescence marker proteins P16 and P21, and can significantly alleviate lung injury, pulmonary fibroblast foci and collagen deposition in senescent mice, and can significantly reduce the pulmonary fibrosis score and the expression of pulmonary fibrosis marker protein α-SMA in senescent mice.

[0027] 2. Lactobacillus paracasei L9 can significantly reduce collagen deposition, mainly reducing type I collagen, having no significant effect on type III collagen, and having no significant effect on the expression of degradation enzymes during the collagen degradation process and enzymes that crosslink with ECM.

[0028] 3. Lactobacillus paracasei L9 can significantly increase the content of uncut PINP during synthesis, indicating that L9 exerts its effect through the process of type I collagen synthesis.

[0029] 4. Lactobacillus paracasei L9 did not significantly inhibit the protein expression levels of key proteases P5CS, PSAT-1 and PHGDH for amino acid synthesis, nor did it significantly inhibit the assembly of procollagen molecules of C-Propeptide and the expression levels of Col1α1 chain and Col1α2 chain.

[0030] 5. Lactobacillus paracasei L9 limits the biosynthesis of type I collagen by reducing the expression of collagen synthesis molecular chaperone HSP47, thereby reducing the correct folding of type I collagen.

[0031] 6. Lactobacillus paracasei L9 inhibits the activation of MKK4 / 7 through the HPK1-TAK1 pathway by inhibiting the expression of receptors PDGFRβ and TGFβR2, and then inhibits the activation of the JNK pathway, thereby inhibiting the transcription factor HSF1, and ultimately affecting the expression of HSP47.

[0032] 7. Lactobacillus paracasei L9 can up-regulate the abundance of SCFA-producing bacteria in the intestines of mice, such as genera of bacteria including Eubacterium mucigenes, Clostridium leptum, Lachnospiraceae_UCG-006 and Rikenella, and reduce the abundance of genera of harmful intestinal bacteria such as norank_f__norank_o__Clostridia_UCG-014 and Desulfovibrio.

[0033] 8. Lactobacillus paracasei L9 can up-regulate the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid in the intestines of mice, with the most significant increase in propionic acid and butyric acid.

[0034] 9. Lactobacillus paracasei L9 can up-regulate the levels of propionic acid and butyric acid in the serum of mice, and the increase in the content of propionic acid and butyric acid is negatively correlated with the Ashcroft score of pulmonary fibrosis, the expression of α-SMA and type I collagen, that is, it is positively correlated with the improvement of pulmonary fibrosis symptoms, and the correlation of butyric acid is the strongest. Description of the Drawings

[0035] Figure 1 Collection and statistics of the body weights of mice in each group, where n = 8, Control: 24 months old, L9: 24 months old + L9.

[0036] Figure 2 HE staining and fibrosis scoring of the lungs of mice in each group. Among them, A is the optical microscope observation field of the HE-stained lung section of the mouse; B is the bar chart of the Ashcroft score of pulmonary fibrosis, n = 6, scale bar: 200 μm, Control: 24 months old, L9: 24 months old + L9.

[0037] Figure 3 Masson staining and collagen volume fraction of the lungs of mice in each group. Among them, A is the optical microscope observation field of the Masson-stained lung section of the mouse; B is the bar chart of the Masson collagen volume fraction. The average value is taken by randomly selecting five fields of view for each mouse, n = 6, scale bar: 200 μm, Control: 24 months old, L9: 24 months old + L9.

[0038] Figure 4 Sirius Red staining and analysis of collagen fiber composition of the lungs of mice in each group. Among them, A is the polarized light microscope observation field of the Sirius Red-stained lung section of the mouse; B is the Red / Green bar chart of the polarized light photograph of the Sirius Red staining. The average value is taken by randomly selecting five fields of view for each mouse, n = 6, scale bar: 200 μm, Control: 24 months old, L9: 24 months old + L9.

[0039] Figure 5 Effect of L9 on senescence markers in the lungs of mice and semi-quantitative analysis. Among them, A is the Western Blot detection of p21 and p16 proteins in the lung tissue of the mouse; B-C is the semi-quantitative analysis of the Western Blot detection of p21 and p16 proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0040] Figure 6Effect of L9 on pulmonary fibrosis markers in mice and semi-quantitative analysis, including A Western Blot detection of CollagenⅠ, CollagenⅢ, and α-SMA proteins in mouse lung tissue; B-D Semi-quantitative analysis of Western Blot detection of CollagenⅠ, CollagenⅢ, and α-SMA proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0041] Figure 7 Effect of L9 on the content of PINP in the lungs of mice, with 3 replicates and n = 6.

[0042] Figure 8 Effect of L9 on collagen assembly proteases in the lungs of mice and semi-quantitative analysis, including A Western Blot detection of LOXL2 and LOX proteins in mouse lung tissue; B-C Semi-quantitative analysis of Western Blot detection of LOXL2 and LOX proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0043] Figure 9 Immunofluorescence staining and fluorescence intensity analysis of LOXL2 and LOX in the lungs of mice in each group, including A-B Laser confocal microscopy photographs of immunofluorescence staining of LOXL2 and LOX proteins in mouse lung tissue; C-D Fluorescence intensity analysis of immunofluorescence staining of LOXL2 and LOX proteins. Five fields were randomly selected from each mouse and averaged, n = 6, scale bar: 50 μm, Control: 24 months old, L9: 24 months old + L9.

[0044] Figure 10 Effect of L9 on the expression of amino acid biosynthesis proteases in mouse lung tissue, including A Western Blot detection of P5CS, PSAT-1, and PHGDH proteins in mouse lung tissue; B-D Semi-quantitative analysis of Western Blot detection of P5CS, PSAT-1, and PHGDH proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0045] Figure 11 Effect of L9 on the expression of procollagen in mouse lung tissue, including A Western Blot detection of Col1α1, Col1α2, and cleaved type I collagen C-Propeptide proteins in mouse lung tissue; B-D Semi-quantitative analysis of Western Blot detection of Col1α1, Col1α2, and cleaved type I collagen C-Propeptide proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0046] Figure 12Effect of L9 on the expression of collagen synthesis molecular chaperones in mouse lung tissue. Among them, A: Western Blot detection of HSP47 protein in mouse lung tissue; B: Semi-quantitative analysis of Western Blot detection of HSP47 protein, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0047] Figure 13 Effect of L9 on the mRNA expression of HSP47 in mouse lung tissue, where n = 6, Control: 24 months old, L9: 24 months old + L9.

[0048] Figure 14 Effect of L9 on the expression of HSF1 in mouse lung tissue. Among them, A: Western Blot detection of HSF1 protein in mouse lung tissue; B: Semi-quantitative analysis of Western Blot detection of HSF1 protein, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0049] Figure 15 Effect of L9 on the activation of JNK pathway in mouse lung tissue. Among them, A: Western Blot detection of p-JNK, JNK, MKK4, and MKK7 proteins in mouse lung tissue; B-D: Semi-quantitative analysis of Western Blot detection of p-JNK, JNK, MKK4, and MKK7 proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0050] Figure 16 Effect of L9 on the expression of JNK pathway regulatory proteins in mouse lung tissue. Among them, A: Western Blot detection of ASK1, HPK1, and TAK1 proteins in mouse lung tissue; B-D: Semi-quantitative analysis of Western Blot detection of ASK1, HPK1, and TAK1 proteins, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0051] Figure 17 Effect of L9 on the expression of PDGFRβ and TGFβR2 receptors in mouse lung tissue. Among them, A: Western Blot detection of PDGFRβ and TGFβR2 receptors in mouse lung tissue; B-C: Semi-quantitative analysis of Western Blot detection of PDGFRβ and TGFβR2 receptors, n = 6, Control: 24 months old, L9: 24 months old + L9.

[0052] Figure 18Immunofluorescence staining and fluorescence intensity analysis of PDGFRβ in the lungs of each group of mice. Among them, A shows the field of view of immunofluorescence staining of PDGFRβ protein in mouse lung tissue photographed by a confocal laser scanning microscope; B shows the fluorescence intensity analysis of PDGFRβ protein immunofluorescence staining. Five fields of view were randomly selected from each mouse and averaged, n = 6, scale bar: 50 μm, Control: 24 months old, L9: 24 months old + L9.

[0053] Figure 19 Effect of L9 on the α-diversity of the intestinal flora in mice. Among them, A shows the inter-group difference in the α-diversity index of the intestinal flora at the genus level in mice, independent samples t-test, Shannon index; B shows the rarefaction curve of α-diversity, Shannon index, Con: 24 months old, L9: 24 months old + L9.

[0054] Figure 20 Effect of L9 on the PCoA analysis of the intestinal flora at the genus level in mice. Among them, A is the PCoA plot; B is the dysbiosis index plot. Con: 24 months old, L9: 24 months old + L9.

[0055] Figure 21 Effect of L9 on the inter-group differences in the intestinal flora of mice. Among them, A is a bar chart of the community composition at the phylum level, with the top 10 in abundance; B is a bar chart of the community composition at the genus level, with the top 30 in abundance, Con: 24 months old, L9: 24 months old + L9.

[0056] Figure 22 Analysis of the inter-group differences in the intestinal flora of mice by L9. Among them, A is a heatmap of the community composition at the genus level, with the top 30 in abundance, and the species-level clustering method is Average; B is a two-group difference test at the genus level, independent samples t-test, 95% confidence interval, Con: 24 months old, L9: 24 months old + L9.

[0057] Figure 23 Effect of L9 on short-chain fatty acids (SCFAs) in the intestines of mice. Among them, A is a clustering heatmap of the grouped expression patterns of intestinal SCFAs; B is a bar chart of the relative abundances of intestinal SCFAs; C is a box plot of the propionate content in the intestines; D is a box plot of the butyrate content in the intestines, n = 8, independent samples t-test, Control: 24 months old, L9: 24 months old + L9.

[0058] Figure 24 Effect of L9 on SCFAs in the serum of mice. Among them, A is a clustering heatmap of the grouped expression patterns of serum SCFAs; B is a box plot of the propionate content in the serum; C is a box plot of the butyrate content in the serum, n = 6, independent samples t-test, Control: 24 months old, L9: 24 months old + L9.

[0059] Figure 25 Heatmap of Pearson correlation analysis between serum SCFAs and lung fibrosis detection indicators.

[0060] Figure 26 Screening of the anti-fibrotic effects of four Lactobacillus paracasei strains: A is a Western Blot image, and B - D are grayscale analysis images of the expression of Coll-Ⅰ, Coll-Ⅲ, and α-SMA, respectively.

[0061] Note: In each figure, ns indicates no significant difference, * indicates *p < 0.05, ** indicates **p < 0.01, *** indicates ***p < 0.001, and **** indicates ****p < 0.0001 Detailed implementation manners

[0062] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0063] Example 1 Effect of Lactobacillus paracasei L9 on pulmonary fibrosis in senile mice

[0064] 1.1 Experimental animals

[0065] 1) C57BL / 6J mice at 15 months of age were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal experiments were approved by the Animal Welfare and Ethics Committee of China Agricultural University (Approval No.: AW32504202 - 5 - 8), and the recommendations of the World Health Organization's "Guide for the Care and Use of Laboratory Animals" were strictly followed. Before the formal experiment began, the mice were acclimated for 1 week. All mice had free access to food and water and were fed a standard diet;

[0066] 2) Grouping: The mice were randomly divided into two groups, namely the natural aging group (blank control group) and the Lactobacillus paracasei L9 intervention group (L9 group), with 8 mice in each group. The remaining 4 mice were euthanized after the acclimation feeding.

[0067] 3) Intervention: Starting from the formal experiment of the mice at 15 months of age, each mouse in the L9 group was intragastrically administered 100 μL of Lactobacillus paracasei L9 bacterial suspension at 3 pm every day, and the mice in the blank control group were intragastrically administered an equal volume of normal saline. The mice were euthanized nine months after the intervention.

[0068] 4) Sampling: Anesthetization and weighing: Mice were intraperitoneally injected with 1% sodium pentobarbital (75 mg / kg). Fixation and disinfection: The anesthetized mice were fixed and disinfected. An alcohol sprayer was used to spray 75% alcohol, and the mice were wiped with paper towels. Blood sampling from the apex of the heart: The thoracic cavity of the mice was opened, and a 1 mL sterile syringe was inserted into the right ventricle of the mice. After slowly collecting blood, the blood was quickly injected into a blood collection coagulation-promoting tube. Subsequently, the blood was placed at 4°C and allowed to stand for 30 min, and finally centrifuged (3500 g, 15 min) to obtain serum. Performing cardiac perfusion: The right auricle and liver were cut open, and the tip of a syringe filled with 4°C normal saline was slowly inserted into the right ventricle for perfusion, and the perfusion could be stopped when the lungs of the mice turned pale. Lung tissue processing: The upper lobe of the lung was immersed in 4% paraformaldehyde for 48 h, waiting for tissue sectioning and staining; the large lobe, middle lobe, lower lobe, and accessory lobe were placed in liquid nitrogen for temporary storage for subsequent detection.

[0069] 1.2 Post-sampling treatment experiments of animal models

[0070] 1) Hematoxylin-Eosin (HE) staining

[0071] Samples were processed according to the conventional HE staining method in this field, briefly summarized as follows: The lung tissue sections were dewaxed and hydrated, stained with hematoxylin staining solution, then immersed in distilled water until partially turned blue, the sections were immersed in eosin staining solution, and the excess staining solution was rinsed with distilled water. Dehydration was carried out in a system with increasing alcohol concentration, and after clearing with xylene, an appropriate amount of neutral gum was dropped on the glass slide, and after air-drying at room temperature, photographs were taken under an optical microscope.

[0072] 2) Masson staining

[0073] The lung samples were processed according to the conventional Masson staining method in this field, outlined as follows: First step, the lung tissue sections were immersed in iron hematoxylin staining solution, and the tissue sections were differentiated with acidic ethanol differentiation solution and rinsed with tap water. Second step, the lung tissue sections were blued in Masson bluing solution and rinsed with tap water. Third step, the lung tissue sections were stained with Biebrich scarlet fuchsin staining solution and the weak acid working solution was used. Fourth step, the lung tissue sections were immersed in phosphomolybdic acid solution and placed in the weak acid working solution. Fifth step, the lung tissue sections were immersed in aniline blue staining solution.

[0074] 3) Ashcroft scoring for pulmonary fibrosis

[0075] After photographing under a 10× magnification of an optical microscope, with each mouse as a unit, the Ashcroft score for pulmonary fibrosis of its histopathological sections was judged.

[0076] 4) Sirius Red staining

[0077] Sirius Red staining can detect abnormal proliferation of collagen fibers. Under ordinary optical microscopy, collagen fibers appear red and cell nuclei appear blue. When observed under polarized light microscopy, type I collagen fibers appear orange-yellow or red, and type III collagen fibers appear green.

[0078] Process the lung samples according to the conventional Sirius red staining method in this field: The steps of tissue embedding, sectioning, dewaxing and hydration are as described above. Immerse the tissue sections in the methyl blue staining solution for 10 min. Subsequently, wash the tissue sections with double-distilled water three times, and then stain the tissue sections with the Sirius red short-chain picric acid solution for 30 min. The steps of dehydration, clearing and mounting are as described above. Observe the tissue sections under a polarized light microscope and take pictures.

[0079] 5) Tissue protein extraction and Western Blot

[0080] Extract tissue proteins and perform Western blot detection according to the conventional method in this field: Add the tissue samples to the protease lysis solution, add grinding beads and grind in a liquid nitrogen environment. After cycling a certain number of times, centrifuge to obtain the supernatant. Determine the protein concentration using the BCA method and aliquot.

[0081] Prepare the stacking gel and separating gel. Add the protein samples to the loading buffer, boil them, load them onto the gel simultaneously with the Marker, perform electrophoresis, transfer the membrane, block with skim milk powder, incubate with antibodies, and develop the film.

[0082] 6) Tissue immunofluorescence staining

[0083] Perform immunofluorescence detection on tissue sections according to the conventional method in this field: After antigen repair, inactivation of endogenous enzymes, and serum blocking of the sections, add the primary antibody and incubate overnight. Add the secondary antibody, wash, and then mount and develop the color.

[0084] 7) Data processing

[0085] The experimental data are expressed in the form of Mean±SD. Use the software GraphPad Prism 9.0.0 for data analysis and drawing pictures. The independent samples t-test, two-tailed, was used to compare the quantitative data between the two groups. p < 0.05 was considered to have a significant difference, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0086] 2. Experimental results

[0087] 2.1 Effect of L9 on the body weight of mice

[0088] As the mice aged, the mental state of each group deteriorated, with symptoms such as reduced food intake, slowed movement, and hair loss. Compared with the blank control group, the mice in the L9 group had more positive manifestations in terms of mental state, food intake, hair, and movement. Among them, the breathing of the mice in the L9 group was smoother than that of the mice in the blank control group. As Figure 1 shown, at the beginning of the experiment, the initial weights of all mice showed a downward trend. As the mice aged, the average weight of the mice in the blank control group continued to decline, while the weight of the mice in the L9 group was relatively stable. In the later stage, the average weight gradually increased and was significantly different from that of the blank control group. Finally, there was no significant difference in the weight of the mice in the L9 group compared with the initial stage of intervention, indicating that L9 intervention could keep the weight of aging mice stable.

[0089] 2.2 Histopathological determination of lung tissue in mice intervened with L9 for pulmonary fibrosis

[0090] The histopathological determination of lung tissue was performed by HE staining, and the results were as Figure 2 shown in Figure A. The alveolar structure of 15-month-old mice was clear, and no collagen accumulation was observed. As the mice aged, the lung tissue pathology of 24-month-old mice in the blank control group deteriorated significantly, showing alveolar collapse, structural disorder, infiltration of a large number of inflammatory cells (such as lymphocytes, neutrophils, macrophages), and obvious large fibroblast foci, which were most obvious in the airway and around the damaged area. The lung tissue pathology of the mice administered Lactobacillus paracasei L9 since 15 months of age was significantly improved compared with the blank group. Specifically, the alveolar structure was not significantly changed, the infiltration of inflammatory cells in the lung tissue was significantly reduced, and the fibroblast foci were reduced. To further evaluate the pathological state of the mouse lung tissue, the Ashcroft score was used to indicate the degree of pulmonary fibrosis. The results were as Figure 2 shown in Figure B. L9 intervention could significantly reduce the pulmonary fibrosis score of lung tissue by 30% (p < 0.05).

[0091] 2.3 Effects of L9 on collagen fiber deposition and collagen volume fraction in mouse lung tissue

[0092] Collagen fiber staining was performed by Masson staining, and the results were as Figure 3 shown in Figure A. No disordered collagen deposition was observed in the lungs of 15-month-old mice. As the mice aged, a large amount of blue-stained collagen fiber deposition appeared in the lung tissue of 24-month-old mice in the blank control group, showing a trend of developing from the lung edge to the lung middle. At the same time, more blue-stained collagen fiber deposition was also observed in the ECM at the trachea and alveolar damage sites, which were obvious fibrosis symptoms. The collagen fiber deposition in the lung tissue of the mice administered Lactobacillus paracasei L9 since 15 months of age was significantly improved compared with the blank group. The blue-stained collagen fiber deposition in the field of view decreased, and the pulmonary fibrosis was significantly improved. To further evaluate the collagen fiber deposition in the mouse lung tissue, the collagen volume fraction was used to indicate the collagen fiber deposition in the lungs. The results were as Figure 3As shown in Figure B, L9 intervention significantly reduced the deposition of blue-stained collagen fibers in the lungs of mice by 40% (p < 0.05).

[0093] Mouse lung sections were stained using Sirius Red staining method and photographed using polarized light. The results are as Figure 4 shown. After Lactobacillus paracasei L9 intervention, the ratio of type I / III collagen in the lungs of mice decreased significantly (p < 0.05). Among them, Figure 4 A is the polarized light microscope observation field of Sirius Red staining of mouse lung sections; Figure 4 B is the Red / Green histogram of polarized light photography of Sirius Red staining.

[0094] 2.4 Effects of L9 on the expression of proteins related to lung aging and fibrosis in mice

[0095] The expression of senescence-related marker proteins in mouse lung tissues was detected by Western Blot. The experimental results ( Figure 5 ) showed that compared with the blank control group of mice with pulmonary fibrosis, the senescence marker proteins p16 and p21 in the lung tissues of the L9 group of mice decreased significantly (p < 0.05). Among them Figure 5 is the Western Blot detection of p21 and p16 proteins in mouse lung tissues; Figure 5 B - C Semi-quantitative analysis of Western Blot detection of p21 and p16 proteins

[0096] Figure 6 As shown by Western Blot in A, compared with the blank control group of mice, the pulmonary fibrosis marker proteins α-SMA ( Figure 6 D) and type I collagen in the L9 group of mice decreased significantly (p < 0.05). Among them, the content of type I collagen in the L9 group decreased significantly by 59% compared with the blank control group ( Figure 6 B), while the change in type III collagen was not significant ( Figure 6 C), indicating that the significant decrease in the ratio of type I / III collagen in the lungs of mice was caused by the decrease in type I collagen.

[0097] As Figure 7 shown: After Lactobacillus paracasei L9 intervention, PINP in the lungs of mice increased significantly by 61% (p < 0.05). Since the cleavage of PINP does not occur until the triple helix formation is completed, this result indicates that type I collagen may not be correctly folded in the endoplasmic reticulum after L9 intervention, which explains the decrease in the synthesis of type I collagen.

[0098] Figure 8As shown in A-C: After the intervention of Lactobacillus paracasei L9, there was no significant decrease in LOXL2, but there was a significant decrease in the expression of LOX (p < 0.05).

[0099] Figure 9 As shown in A-B, the results of immunofluorescence staining showed that LOX and LOXL2 were mainly expressed around the trachea, and Lactobacillus paracasei L9 could not significantly affect the expression of LOXL2 ( Figure 9 C), but could significantly reduce the expression of LOX by 27% (p < 0.05, Figure 9 D). In summary, the decrease in LOX enzyme may be due to a decrease in the content of synthesized collagen or an increase in the degradation of collagen, resulting in a decrease in the production of cross-linking enzymes.

[0100] Example 2 Mechanism of Lactobacillus paracasei L9 in regulating the synthesis of type I collagen in the lungs of aging mice

[0101] 1 Experimental methods

[0102] 1) Tissue immunofluorescence staining, tissue protein extraction and Western Blot were as described in Example 1;

[0103] 2) RNA extraction and real-time PCR

[0104] Sample RNA was extracted using conventional tissue RNA extraction methods in the art, primers and probes for related genes were designed, and the transcriptional expression levels of related genes were detected by fluorescence quantitative method;

[0105] 3) Data processing was as described in Example 1.

[0106] 2 Experimental results

[0107] 2.1 Effect of L9 on proteins related to the synthesis of type I collagen in mouse lung tissue

[0108] The results showed that ( Figure 10 A-D) Lactobacillus paracasei L9 did not significantly inhibit the protein expression levels of P5CS, PSAT-1 and PHGDH; Figure 11 A-D showed that although the protein expression levels showed a downward trend after the intervention of Lactobacillus paracasei L9, it did not significantly inhibit their protein expression levels.

[0109] In sharp contrast, Lactobacillus paracasei L9 significantly inhibited the expression of HSP47 by 61% (p < 0.05, Figure 12 A-B).

[0110] 2.2 Effect of L9 on the transcriptional level of HSP47 protein in mouse lung tissue

[0111] The expression level of the encoding gene Serpin H1 of HSP47 in the lungs of mice was detected by qPCR. The qPCR results showed ( Figure 13 ), that intragastric administration of Lactobacillus paracasei L9 could significantly inhibit the expression of Serpin H1 gene in the lungs of 61% of the mice (p < 0.05).

[0112] The expression level of the transcription factor HSF1 of Serpin H1 gene was detected by Western Blot. The results showed ( Figure 14 A - B): Compared with the mice in the blank control group, Lactobacillus paracasei L9 significantly reduced the content of HSF1 by 27% (p < 0.05).

[0113] The expression of several proteins in the JNK pathway was detected by Western Blot. The results showed ( Figure 15 A - D): Lactobacillus paracasei L9 significantly inhibited the phosphorylation activation of the JNK pathway (p < 0.05). Among them, L9 inhibited 85% of JNK phosphorylation, 43% of MKK4 and 22% of MKK7 protein expression respectively.

[0114] The upstream proteins of the JNK pathway were detected. The results were as Figure 16 shown in A: The expressions of ASK1 and TAK1 were significantly inhibited after the intervention of Lactobacillus paracasei L9 (p < 0.05), and the expression of HPK1 was also significantly inhibited (p < 0.05). To further clarify the most significant pathway by which L9 inhibits the JNK pathway, through Western blot gray scale analysis, the results were as Figure 16 shown in B - D. L9 inhibited 30% of TAK1 expression, and at the same time inhibited 31% of HPK1 expression, but only inhibited 18% of ASK1 expression, indicating that L9 intervention mainly inhibits the activation of the JNK pathway through the HPK1 - TAK1 pathway.

[0115] Two receptors were detected by Western blot experiment. The results were as Figure 17 shown in A - C. Lactobacillus paracasei L9 significantly inhibited the expressions of 30% of the PDGFRβ receptor and 29% of the TGFβR2 receptor (p < 0.05). Immunofluorescence staining ( Figure 18 A), it was found that the PDGFRβ receptor was significantly inhibited around the trachea and alveoli. Fluorescence intensity analysis ( Figure 18 B) showed that the expression of the PDGFRβ receptor was significantly inhibited by 28% (p < 0.05).

[0116] Example 3 Effects of Lactobacillus paracasei L9 on the intestinal flora and metabolites of aging mice

[0117] 1. Analysis of the effects of L9 on the intestinal flora of mice by fecal 16S rRNA sequencing

[0118] Extract fecal DNA, construct a library after PCR amplification, and use bioinformatics methods to analyze the differences in microbial community abundance by high-throughput sequencing.

[0119] α-diversity analysis can be used to estimate microbial species abundance and species diversity. The results show that there is no significant difference between the blank control group and the L9 group (Student’s t-test for Shannon index, p = 0.3756, Figure 19 as shown in A-B).

[0120] PCoA analysis shows that the plotted distance of L9 samples is greater than that of normal samples. The first (PC1) and second (PC2) principal coordinates account for 23.42% and 39.93% of the total variation, respectively. The dysbiosis index changes significantly after L9 intervention ( Figure 20 as shown in A-B).

[0121] Analysis of the identified intestinal bacteria at the phylum level of classification shows that significant differences between the L9 group and the blank control group are mainly observed in several bacterial phyla, including Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, the harmful bacterium Desulfobacterota, and Verrucomicrobia, etc. ( Figure 21 as shown in A). In this study, the community composition bar chart and pie chart were then used to analyze the top 50 microorganisms with different abundances at the genus level ( Figure 21 as shown in B). It is proved that the intestinal flora of mice changes at the genus level after L9 mouse intervention compared with that of naturally aging mice.

[0122] Using the Heatmap chart ( Figure 22 as shown in A), the corresponding microorganisms at the top 50 differential genus levels measured by relative abundance are highlighted. It is found that after L9 intervention, Lactobacillus paracasei significantly reduces the abundances of intestinal harmful bacteria such as norank_f__norank_o__Clostridia_UCG-014 and Desulfovibrio, etc., while increasing the abundance of lactic acid bacteria (p < 0.05). Comparing L9-intervened mice with naturally aging mice (Student’s t-test, 95% confidence interval), it is found that microorganisms such as Eubacterium mucigenum, Clostridium spiroforme, Lachnospiraceae_UCG-006, and Rikenella show a significant upward trend ( Figure 22 as shown in B)

[0123] 2. Fecal targeted metabolomics analysis

[0124] Use GS-MS to detect the content of non-short-chain fatty acid SCFAs in feces.

[0125] The results are as Figure 23As shown, it was found that in feces, L9 increased the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid, and the increases in propionic acid and butyric acid were the most significant (p < 0.05), with a 74% increase in propionic acid and a 64% increase in butyric acid.

[0126] 3. Blood targeted metabolomics analysis

[0127] The content of SCFA in serum was detected by LC-MS.

[0128] The results are as Figure 24 Shown in A - C, it was found that in serum, L9 significantly increased the levels of propionic acid by 97% and butyric acid by 193% (p < 0.05).

[0129] According to the Pearson correlation coefficient, correlation analysis was performed with Ashcroft pulmonary fibrosis score, α - SMA, type I collagen, collagen synthesis molecular chaperone HSP47 and pathway protein p - JNK. Figure 25 And the results in Table 1 showed that: First, in the mice intervened with Lactobacillus paracasei L9, the expression of butyric acid and propionic acid showed a significant positive correlation (p < 0.05); Second, the increased expression of propionic acid and butyric acid was also significantly negatively correlated with the development of pulmonary fibrosis (p < 0.05). At the same time, the expression of butyric acid was significantly negatively correlated with the expression of α - SMA and type I collagen (p < 0.05), the expression of propionic acid was negatively correlated with the expression of α - SMA and type I collagen, and its correlation with α - SMA was not significant (p = 0.076), only significantly negatively correlated with the expression of type I collagen (p < 0.05); Furthermore, it was found that the expression of butyric acid was significantly negatively correlated with the expression of p - JNK and HSP47 (p < 0.05), the expression of propionic acid was negatively correlated with the expression of p - JNK and HSP47, but its correlation with p - JNK was not significant (p = 0.063). In summary, the increase in the content of serum propionic acid and butyric acid was positively correlated with the improvement of pulmonary fibrosis, and the correlation of butyric acid was stronger. Finally, this study verified that the expression of p - JNK and HSP47, the expression of p - JNK and the development of pulmonary fibrosis, and HSP47 for the development of pulmonary fibrosis and the expression of type I collagen all showed significant positive correlations (p < 0.05).

[0130] In summary, the results of this study showed that the increase in the content of propionic acid and butyric acid inhibited the activation of the p - JNK pathway, further reduced the expression of HSP47, and ultimately improved pulmonary fibrosis, with butyric acid being more significant.

[0131] Table 1 Normality test table of detection indicators

[0132]

[0133] Example 4 Comparison of the inhibitory effects of different probiotics on senescence - related pulmonary fibrosis

[0134] The study used 10 ng / mL TGF-β1 to intervene in the mouse embryonic fibroblast cell line NIH / 3T3L9 to construct an in vitro simulated pulmonary fibrosis cell model, and co-cultured it with the supernatants of four strains of Lactobacillus paracasei (L9, LC01, 22.2, AM33). The results are as follows Figure 26 As shown in A-D, L9 significantly inhibited pulmonary fibrosis caused by TGF-β1 modeling. L9 significantly reduced the expression of Coll-Ⅰ, Coll-Ⅲ, and α-SMA (p < 0.05), and L9 showed the best anti-fibrotic effect among the four strains of Lactobacillus paracasei.

Claims

1. The application of Lactobacillus paracasei L9 in preparing a preparation for treating pulmonary fibrosis is characterized in that, The preparation inhibits the expression of HSP47 protein through the JNK-HSF1 pathway to reduce the synthesis of collagen I.

2. The use according to claim 1, characterized in that: The pulmonary fibrosis is aging-related fibrosis, and the preparation increases the content of short-chain fatty acids butyrate and / or propionate in the body.

3. The use according to claim 1, characterized in that: The preparation is an oral preparation.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation also includes a pharmaceutically acceptable carrier.

5. Application of Lactobacillus paracasei L9 in preparing a preparation for regulating the abundance of intestinal flora, characterized in that, The regulation is to increase the abundance of short-chain fatty acid producing bacteria and reduce the abundance of harmful bacteria.

6. The use according to claim 5, characterized in that: The regulation is to increase the abundance of Eubacterium mucosum, Clostridium, Lachnospiraceae_UCG-006 and Rikenella.

7. The use according to claim 5, characterized in that: The regulation is to reduce the abundance of intestinal harmful bacteria norank_f__norank_o__Clostridia_UCG-014 and Desulfovibrio.

8. Application of Lactobacillus paracasei L9 in preparing a preparation for improving the concentration of short-chain fatty acids in an organism, characterized in that: The short-chain fatty acids in the body are propionic acid and / or butyric acid.

9. The use according to claim 8, characterized in that: The in vivo refers to intestinal tract and / or serum.

10. The application of Lactobacillus paracasei L9 in preparing a preparation for inhibiting HSP47 expression, characterized in that: The application is an in vitro cytological application.

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