Application of Weissella fusion J1-1 and its exopolysaccharide in preventing and treating pulmonary fibrosis

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a fusion of Weissella J1-1 and its application. The fusion Weissella J1-1 is deposited with CCTCC NO: M 20232334 and is deposited in the China Center for Type Culture Collection. The fusion Weissella J1-1 can secrete exopolysaccharide, and the exopolysaccharide secreted can significantly improve the lung function of mice with pulmonary fibrosis. It was found by observing lung tissue sections that it can also significantly improve lung tissue fibrosis, has no effect on the weight and growth status of mice, and has no damage to important organs, with high safety. In addition, cell experiments have found that exopolysaccharide can also significantly improve the expression of fibrosis-related genes in lung fibroblasts, and has no effect on the vitality of fibroblasts. Therefore, the present invention fusion Weissella J1-1 and the exopolysaccharide secreted by it can be used to prepare a drug for alleviating or / and treating pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to application of Weissella fusion J1-1 and its exopolysaccharide in preventing and treating pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a common, chronic, progressive, and irreversible interstitial lung disease of unknown etiology. It is characterized by progressive dyspnea and worsening lung function. It progresses rapidly, is prone to recurrence, and is irreversible. Epidemiological surveys show that the incidence, prevalence, and mortality of IPF are increasing annually across regions, ethnicities, and even populations. The incidence rate in Europe and North America is approximately 2.8-9.3 per 100,000 people per year. The diagnosis of IPF requires high-resolution CT scans or lung biopsies, so the disease is often not discovered until it is in its later stages. The median survival after diagnosis is only 2-5 years, with a 5-year survival rate of 20-40%. The mortality rate is higher than that of cancer. The total medical cost of this disease is nearly US$2 billion, placing a heavy burden on patients and society. Current clinical treatments for IPF include anti-inflammatory, anticoagulant, immunosuppressive, antioxidant, and anti-fibrotic medications, as well as lung transplantation and pulmonary rehabilitation exercises. However, drug treatments are ineffective and have significant side effects. For example, the anti-fibrotic drugs pirfenidone and nintedanib, recommended in clinical guidelines, can slow the progression of IPF but are not effective in reversing the condition, and they can also cause numerous adverse reactions. Lung transplantation is currently the most effective treatment, but the shortage of lung donors, the high incidence of postoperative rejection, and the high cost of medical treatment make it difficult to widely promote and utilize. Therefore, there is an urgent need to strengthen research on the effective prevention and treatment of IPF in order to provide new strategies for its clinical treatment.

[0003] In Traditional Chinese Medicine (TCM), the lungs and large intestine are considered exterior and interior to each other. Both belong to the metal element in the Five Elements (wu xing). The lungs are yin and reside within the interior, while the large intestine is yang and resides on the exterior. Lung qi primarily functions by dispersing and descending, while the large intestine functions as a channel for the transport of waste. In other words, precisely because the intestines and lungs are exterior and interior to each other, their functions influence each other. In recent years, the role of the microbiome in the prevention and treatment of pulmonary fibrosis has garnered increasing attention. In TCM, the "lung-intestine" axis is closely linked, and microbiome modulation based on this axis has become a research hotspot as a potential therapeutic target for IPF. However, insufficient evidence exists regarding the safety of live microorganisms in specific preventive and treatment applications, including whether they can cause opportunistic infections in patients with low immunity and their inherent safety. Furthermore, the high environmental requirements for the storage of live microorganisms significantly limit their widespread clinical application.

[0004] Lactic acid bacteria exopolysaccharides are naturally occurring high-molecular-weight compounds that exhibit excellent biosafety and biocompatibility. They have the potential to modulate the immune system, acting as immunomodulators to regulate both innate and adaptive immunity. At the cellular level, they positively impact the activity and proliferation of various immune cell types, including macrophages, dendritic cells, and lymphocytes. Furthermore, they induce cytokine production, thereby enhancing the host immune system's defenses and reducing inflammatory responses. Inflammation is often a primary manifestation of early pulmonary fibrosis. Current research on exopolysaccharides has primarily focused on Lactobacilli, but Lactobacilli exopolysaccharide production is relatively low, and the specific mechanisms by which they contribute to the treatment of pulmonary fibrosis remain unclear. This has limited their widespread application in the treatment of pulmonary fibrosis. Therefore, identifying lactic acid bacteria with high exopolysaccharide production and further exploring the role and mechanisms of exopolysaccharides in the prevention and treatment of pulmonary fibrosis are of great significance.

[0005] Weissella confusa, a member of the lactic acid bacteria family, is widely distributed in the natural environment and is primarily found in saliva, breast milk, the human gastrointestinal tract, and traditional fermented foods, thus playing an important ecological role. Its exopolysaccharide production is high, and it is primarily used as a thickener, stabilizer, and gelling agent in areas such as food processing, cosmetics, and pharmaceuticals. Studies have shown that Weissella confusa exopolysaccharides have antioxidant, pathogenic bacteria inhibitory, lactic acid bacteria and bifidobacterium growth promoting, and immune regulation effects. However, there are currently no reports on the anti-fibrotic effects of Weissella confusa and its exopolysaccharides, particularly in the prevention and treatment of pulmonary fibrosis and its related mechanisms. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of fusion Weissella J1-1 and its exopolysaccharide in preventing and treating pulmonary fibrosis.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a fusion Weissella, wherein the nucleic acid sequence of the fusion Weissella contains the sequence shown in Sequence 1 (SEQ ID NO.1).

[0009] According to the above-mentioned fused Weissella, preferably, the fused Weissella is fused Weissella J1-1, fused Weissella J1-1 (Weissella confusa J1-1), the preservation number of the fused Weissella J1-1 is CCTCCNO: M 20232334, the preservation institution is China Center for Type Culture Collection (CCTCC), and the preservation institution address is China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is November 27, 2023.

[0010] The screening and identification process of the above-mentioned fusion Weissella J1-1 is as follows: sterile PBS and fermented wine lees are fully shaken and resuspended, the supernatant is taken, and gradient dilution is performed to 10 -5 , add the dilution to the MRS liquid culture medium for lactic acid bacteria growth, and incubate at 37℃ for 48h. -5 , then draw the dilution and spread it onto MRS solid medium, and incubate it at 37°C until a single colony grows. Pick the single colony into MRS liquid medium, incubate it at 37°C for 24 hours, take 0.5ml of the bacterial solution, centrifuge it at 4°C and 5000g for 5 minutes, remove the supernatant, and add sterile PBS to resuspend the bacterial pellet. The 16S rDNA sequence of the bacterial solution was amplified using primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'), and the amplified sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequencing results were entered into the NCBI BLAST database for identification. Ultimately, homologous sequence analysis confirmed that the isolated strain was Weissella fusion, named Weissella fusion J1-1, and the strain was deposited in the China Center for Type Culture Collection (CCTCC) with a strain accession number of CCTCC NO: M 20232334. The 16S rDNA sequence of Weissella fusion J1-1 is shown in Sequence 1 (SEQ ID NO. 1).

[0011] The second aspect of the present invention provides the use of the fused Weissella or a culture containing the fused Weissella described in the first aspect in the preparation of a product for preventing, alleviating or / and treating pulmonary fibrosis.

[0012] The third aspect of the present invention provides an exopolysaccharide secreted by the fused Weissella described in the first aspect.

[0013] According to the above-mentioned extracellular polysaccharide, preferably, the extracellular polysaccharide is obtained by fermenting Weissella fusogenum J1-1 and separating and purifying the fermentation culture liquid.

[0014] According to the above-mentioned extracellular polysaccharide, preferably, the preparation method of the extracellular polysaccharide is: inoculating the fused Weissella J1-1 described in the first aspect into a culture medium for fermentation culture to obtain a culture solution, removing the bacteria and removing the protein from the culture solution to obtain a supernatant; subjecting the supernatant to alcohol precipitation, collecting the precipitate, and subjecting the precipitate to dialysis to remove impurities and freeze-drying to obtain the extracellular polysaccharide.

[0015] According to the above-mentioned extracellular polysaccharide, preferably, the culture medium is MRS medium or mMRS medium (modified MRS medium).

[0016] According to the above-mentioned extracellular polysaccharide, preferably, the composition of the mMRS medium is: 5 g / L diammonium citrate, 100 g / L sucrose, 10 g / L peptone, 0.2 g / L magnesium sulfate, 1 ml / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L anhydrous sodium acetate, 0.05 g / L manganese sulfate, and the balance is water; the fermentation culture temperature is 30°C to 37°C. More preferably, the culture temperature is 37°C.

[0017] According to the above-mentioned extracellular polysaccharide, preferably, the dialysis is performed in distilled water, and the dialysis molecular weight cut-off is 8000-14000 Da.

[0018] According to the above-mentioned extracellular polysaccharide, preferably, the alcohol precipitation treatment is to add anhydrous ethanol to the supernatant, mix well and then let it stand at 4°C for 12 hours to 18 hours.

[0019] Based on the above-mentioned exopolysaccharide, preferably, the specific operation of protein removal is: adding trichloroacetic acid to the culture medium after the bacterial cells are removed, mixing uniformly to obtain a mixed solution, placing the mixed solution at 4°C for 12 to 18 hours, centrifuging, and removing the precipitate. More preferably, the amount of trichloroacetic acid used is such that the mass volume percentage concentration of trichloroacetic acid in the mixed solution is 4%.

[0020] According to the above-mentioned extracellular polysaccharide, preferably, the specific operation of removing the bacterial cells is: centrifuging the culture medium to remove the bacterial cells. More preferably, the centrifugation conditions are: 4°C 12000g centrifugation for 30 minutes.

[0021] The fourth aspect of the present invention provides the use of the extracellular polysaccharide described in the third aspect in the preparation of a medicine for preventing, alleviating or / and treating pulmonary fibrosis.

[0022] The fifth aspect of the present invention provides a drug for preventing, treating and / or alleviating pulmonary fibrosis, wherein the drug comprises an active ingredient, which is the fused Weissella or a culture containing fused Weissella described in the first aspect or the exopolysaccharide described in the second aspect.

[0023] According to the above-mentioned medicine, preferably, when the active ingredient is the fused Weissella or a culture containing the fused Weissella as described in the first aspect, the fused Weissella may be a living cell or a dead cell.

[0024] According to the above-mentioned medicine, preferably, the medicine is administered by oral administration, oral gavage or parenteral administration. More preferably, the method of parenteral administration is intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrapulmonary administration, intranasal administration or rectal administration.

[0025] According to the above-mentioned medicine, preferably, the medicine further comprises pharmaceutically acceptable excipients. More preferably, the excipients include excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants, lubricants or flavoring agents.

[0026] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0027] (1) The fused Weissella confusa J1-1 (W.confusa J1-1) screened by the present invention can secrete exopolysaccharides, which can significantly inhibit the proliferation and phenotypic transformation of pulmonary fibrosis cells and effectively play an anti-pulmonary fibrosis role; moreover, the exopolysaccharides have no effect on the weight and growth status of mice, do not damage the important organs of mice, and are highly safe.

[0028] (2) The present invention fuses Weissella J1-1 to screen the fermented lees, and its metabolite exopolysaccharide is also a natural component produced during the metabolism of lactic acid bacteria. From the perspective of clinical application, compared with other drugs for treating pulmonary fibrosis, the exopolysaccharide of Weissella J1-1 is safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Results of screening of fermentation medium and exopolysaccharide extraction of W.confusa J1-1; A is a photo of exopolysaccharide secretion by W.confusa J1-1 on MRS solid culture plates; B is a photo of exopolysaccharide secretion by W.confusa J1-1 on mMRS solid culture plates; C is a photo of freeze-dried exopolysaccharide samples;

[0030] Figure 2 This is the scanning electron microscopy result of the exopolysaccharide of Weissella J1-1;

[0031] Figure 3 This is the result of ultraviolet spectrum scanning analysis of the exopolysaccharide of Weissella J1-1;

[0032] Figure 4 This is the infrared spectrum scanning analysis result of the exopolysaccharide of Weissella J1-1;

[0033] Figure 5 This is the HPLC analysis result of the exopolysaccharide of Weissella J1-1;

[0034] Figure 6 The weight changes of mice in each experimental group at different time points are shown in the figure; Control represents the control group, Model represents the model group, J1-1 exopolysaccharide represents the exopolysaccharide treatment group, and PFD represents the PFD group; n=4, compared with Model: * P<0.05, *** P < 0.001;

[0035] Figure 7 Figure 2 is the lung function test results of mice in each experimental group; a is the EF50 test result; b is the TV test result; c is the MV test result; Control represents the control group, Model represents the model group, J1-1 exopolysaccharide represents the exopolysaccharide treatment group, and PFD represents the PFD group; n=4, compared with the control group: * P<0.05, ** P < 0.001; compared with Model: * P < 0.05;

[0036] Figure 8 HE staining and alveolitis score results of lung tissue of mice in each experimental group, among which A is a representative picture of HE staining of lung tissue of experimental mice in each group (scale bar = 20 μm); B is the alveolitis score of lung tissue of experimental mice in each group Results; Control represents the control group, Model represents the model group, J1-1 exopolysaccharide represents the exopolysaccharide treatment group, and PFD represents the PFD group; n=4, compared with Control: * P<0.05, ** P < 0.001;

[0037] Figure 9 Masson staining and pulmonary fibrosis score results of lung tissues of mice in each experimental group; A is a representative image of Masson staining of lung tissues of mice in each experimental group (scale bar = 20 μm); B is the Ashcroft score of pulmonary fibrosis of lung tissues of mice in each experimental group Results; Control represents the control group, Model represents the model group, J1-1 exopolysaccharide represents the exopolysaccharide treatment group, and PFD represents the PFD group; n=4, compared with Control: * P<0.05, ** P < 0.001;

[0038] Figure 10 The results of qRT-PCR detection of the expression of MRC-5 activation-related indicators (α-SMA, FN, COL I) in fibroblasts; n = 3;

[0039] Figure 11 The results show the effects of different concentrations of fused Weissella J1-1 exopolysaccharide on the activity of MRC-5 cells; n = 3, compared with the 0 mg / ml J1-1 exopolysaccharide group: *P < 0.05;

[0040] Figure 12 The results show the effects of different concentrations of Weissella J1-1 exopolysaccharide on α-SMA, FN and COLⅠ mRNA in MRC-5 cells n=3, compared with the Control group: * P<0.05. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0042] The following detailed descriptions are all exemplary and are intended to provide further explanation of the present invention. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, components and / or combinations thereof.

[0043] The experimental methods in the following examples without specifying specific conditions were all based on conventional techniques in the art or the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers were all conventional products that can be purchased commercially.

[0044] Example 1: Acquisition and identification of W. confusa J1-1

[0045] The fermented lees were chopped and ground, then added into sterile PBS and resuspended by shaking. The supernatant was taken and graded diluted to 10 -5, add the dilution to the MRS liquid culture medium for lactic acid bacteria growth, and incubate at 37℃ for 48h. -5 Then, aspirate the dilution and spread it onto MRS solid medium. Incubate at 37°C until a single colony grows. Pick the single colony into MRS liquid medium and incubate at 37°C for 24 hours. Then, take 0.5 ml of the bacterial solution and centrifuge at 4°C and 5000g for 5 minutes. After removing the supernatant, add sterile PBS to resuspend the bacterial pellet.

[0046] The 16S rDNA sequence of the bacterial culture was amplified using primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3') and the amplified sequence was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequenced 16S rDNA sequence of W. confusa J1-1 is shown in SEQ ID NO: 1.

[0047] The 16S rDNA sequencing results were entered into the NCBI BLAST database for identification. Finally, the isolated strain was confirmed to be Weissella confusa J1-1 based on homologous sequence analysis and named W. confusa J1-1. The strain was deposited in the China Center for Type Culture Collection (CCTCC) with the strain accession number CCTCC NO: M 20232334.

[0048] Example 2: Acquisition of exopolysaccharide from W. confusa J1-1

[0049] 1. Optimization of W.confusa J1-1 fermentation medium

[0050] By streaking the plates, W.confusa J1-1 was streaked onto MRS solid culture plates and mMRS solid culture plates, and then the solid culture plates were cultured at 37°C for 24-48 hours to observe the exopolysaccharide secretion of W.confusa J1-1 on the MRS solid culture plates and mMRS solid culture plates. Figure 1 As shown in A and B.

[0051] Depend on Figure 1 As shown in Figures A and B, the exopolysaccharide of W. confusa J1-1 grown in mMRS medium is more viscous than that grown in MRS medium, indicating that the exopolysaccharide production of W. confusa J1-1 is increased. Therefore, mMRS is preferred for fermentation of W. confusa J1-1.

[0052] 2. Extracellular polysaccharide acquisition

[0053] Thaw a cryovial of W. confusa J1-1 in a 37°C waterbath. Use a sterile inoculating loop to remove 1-2 drops of the bacterial solution and streak in three sections. Incubate in a 37°C incubator for 48 hours until a single colony is observed. Use a micropipette to pick the resulting colony and inoculate it into 5 ml of liquid MRS medium. Incubate at 37°C for 24 hours until the solution becomes turbid. Inoculate a 2% (v / v) inoculum into a conical flask of modified MRS liquid medium (mMRS medium). Incubate at 30°C for 48 hours until the medium becomes viscous, thereby obtaining the fermentation broth. Boil the broth in a 100°C waterbath for 15 minutes to inactivate bacteria and enzymes, then cool to room temperature. If the broth is viscous, dilute it with sterile deionized water. Centrifuge the boiled broth at 12,000g and 4°C for 30 minutes in a high-speed refrigerated centrifuge. Discard the bacterial cells and other precipitates to obtain the supernatant. To remove protein, add 80% (m / v) trichloroacetic acid (TCA) to a final concentration of 4% (m / v). After refrigerating at 4°C for 12 hours, centrifuge at 12,000 g for 30 minutes at 4°C in a high-speed refrigerated centrifuge. Collect the supernatant and repeat this step two to three times to remove as much protein as possible. Pre-chill the supernatant at 4°C with anhydrous ethanol. Add three volumes (v / v) of cold anhydrous ethanol to the supernatant after removing protein and other impurities. The supernatant is refrigerated at 4°C for 12 hours to precipitate the exopolysaccharides. Centrifuge at 12,000 g for 30 minutes at 4°C to obtain a precipitate. Dissolve the exopolysaccharide precipitate in pure water, and repeat the precipitation step two to three times. After alcohol precipitation, the exopolysaccharide is reconstituted and dialyzed in distilled water (molecular weight cutoff 8,000-14,000 Da) at 4°C for 48-72 hours to remove impurities. The water is changed every eight hours. The polysaccharide solution after dialysis was vacuum freeze-dried to obtain the extracellular polysaccharide freeze-dried powder (such as Figure 1 (as shown in C in the figure). Figure 1 As shown in Figure C, the extracellular polysaccharide freeze-dried powder is a fluffy, dry, white flocculent solid.

[0054] Example 3: Identification and Characterization of Weissella confusa J1-1 Exopolysaccharide

[0055] 1. Scanning electron microscope observation of morphology

[0056] Take an appropriate amount of the lyophilized powder of the extracellular polysaccharide of Weissella obtained in Example 2, dissolve it with ethanol, and undergo ultrasonic cleaning, sampling, infrared drying, gold spraying (10nm), and gluing, and then fix it under an electron microscope for observation. The surface micromorphology of the extracellular polysaccharide lyophilized powder was observed at 1000, 2000, 5000, and 10000 magnifications at an accelerating voltage of 5.0 kV. The scanning electron microscopy results are as follows: Figure 2 shown.

[0057] Depend on Figure 2 It can be seen that the exopolysaccharide of fusion Weissella J1-1 presents a regular porous structure.

[0058] 2. UV spectrum scanning

[0059] An appropriate amount of the lyophilized powder of the fused Weissella exopolysaccharide obtained in Example 2 was accurately weighed and dissolved in ultrapure water to prepare samples of different concentrations of 200 μg / ml, 400 μg / ml and 1000 μg / ml. The characteristic absorption peak in the wavelength range of 200 to 800 nm was then scanned using a spectrophotometer to observe the ultraviolet absorption of the exopolysaccharide derived from the fused Weissella exopolysaccharide at different wavelengths. The results are shown in FIG. Figure 3 shown.

[0060] Depend on Figure 3 It can be seen that the UV spectrum scanning results show that the UV absorption spectrum curve of the fusion Weissella J1-1 exopolysaccharide is smooth and flat after 280nm, and no obvious absorption peak is found, indicating that there is no protein and nucleic acid in the fusion Weissella J1-1 exopolysaccharide sample.

[0061] 3. Infrared spectrum scanning

[0062] Accurately weigh an appropriate amount of the lyophilized powder of the fusion Weissella exopolysaccharide obtained in the example, mix it with potassium bromide in a certain proportion, compress the mixed particles using the conventional potassium bromide (KBr) tableting method, and measure the sample at 500 cm using a Fourier transform infrared spectrometer. -1 -4500cm -1 The absorption spectrum within the range. The results are as follows Figure 4 shown.

[0063] Depend on Figure 4 It can be seen that at 500cm -1 -4500cm -1 Spectra at a wavelength range of 3420 cm reveal the vibrations of functional groups in the exopolysaccharide of Weissella fusilis J1-1. -1 The broad and strong peak at 2927cm is the characteristic peak of the stretching vibration of the hydroxyl group OH in the sugar component, which is a typical characteristic of sugar substances. -1 and 1645cm -1 The sharp peaks at the absorption peaks at 1423cm correspond to the stretching vibration of the CH single bond and the stretching vibration of the C=O double bond in the sugar ring. -1 and 1354cm -1 The absorption peak between 1000 cm and 1000 cm is attributed to the angular vibration of the C-H bond. -1 and 1200cm -1The absorption peaks between 1155 and 1017 cm are considered to be the fingerprint region of the polysaccharide. The broad absorption peaks of the polysaccharide at 1155 and 1017 cm are caused by the stretching vibration of the C-OH linking bond and the COC glycosidic bond stretching vibration of the sugar ring, which also indicates that the polysaccharide contains pyranose residues. -1 The peak at 1030-1150cm is the specific absorption peak of glycosidic bond, proving the existence of α-glycosidic bond. -1 There is no peak at 847 cm, which proves that there is no β-configuration of glycosidic bond. -1 The peak is caused by the stretching vibration of the α-anomeric carbon, indicating the α-configuration of the glycosidic bond in the polysaccharide. Therefore, the FT-IR spectrum shows that the exopolysaccharide of Weissella fusionensis J1-1 has pyranose residues and α-type glycosidic bonds.

[0064] 4. HPLC analysis of monosaccharide and uronic acid composition in the exopolysaccharide of Weissella fusion J1-1

[0065] Different monosaccharide components were separated using a chromatographic column, and the monosaccharide content of the monosaccharide components in different samples was quantitatively analyzed using the external standard method. Appropriate amounts of mannose, glucuronic acid, glucose, xylose, arabinose, and galactose reference substances were weighed and diluted to a concentration of 50 μg / ml in water to form a mixed reference solution. In a 5 ml EP tube, 250 μl of the mixed reference solution, 250 μl of 0.6 mol / l NaOH solution, and 500 μl of 0.4 mol / l PMP-methanol solution were added and incubated at 70°C for 1 hour. The mixture was then incubated in an ice-water bath for 10 minutes, neutralized with 500 μl (0.3 mol / l) HCl, and 1 ml of chloroform was added. The mixture was vortexed for 1 minute, centrifuged at 3000 r / min for 10 minutes, and the supernatant was extracted three times. The supernatant was used for HPLC analysis.

[0066] Accurately weigh approximately 5 mg of the lyophilized powder of the fused Weiss exopolysaccharide obtained in Example 2 into a 10 mL ampoule. Add 5.0 mL of 2 mol / L TFA, seal the tube, and acid-hydrolyze at 120°C for 4 h. Remove the sample and add methanol-nitrogen to evaporate the TFA. Reconstitute the sample with 5.0 mL of water. Transfer 250 μl of the sample solution to a 5 mL EP tube. Proceed as above.

[0067] The chromatographic conditions for HPLC analysis were as follows: instrument: Shimadzu LC-20AD, chromatographic column: Xtimate C18 4.6*200mm 5μm, column temperature: 30°C, flow rate: 1.0 ml / min, detection wavelength: 250 nm, injection volume: 20 μl, mobile phase: 0.05 M potassium dihydrogen phosphate solution (adjusted to pH 6.70 with sodium hydroxide solution)-acetonitrile.

[0068] Test results such as Figure 5 shown.

[0069] Depend on Figure 5It can be seen that the results of high performance liquid chromatography analysis showed that the exopolysaccharide of Weissella fusion J1-1 was glucan.

[0070] Example 4: Animal experiments verify the efficacy of W. confusa J1-1 exopolysaccharide in improving pulmonary fibrosis

[0071] 1. Experimental animals and groups

[0072] Sixty-four SPF C57BL / 6J mice (weight: 20 ± 2 g; Animal Quality Certificate Number: 110324221101674161) were purchased from Beijing Sibeifu Laboratory Animal Co., Ltd. (SCK [Beijing] 2019-0010). Mice were acclimated for 7 days to the laboratory room temperature (25 ± 1)°C, relative humidity (50 ± 10)%, ventilation rate 10–15 times / h, ammonia concentration ≤ 14 mg / m³, and noise level ≤ 60 dB. They were fed sterile feed and had free access to sterile water. The purification system was regularly inspected and maintained in a quiet environment.

[0073] 2. Experimental Grouping and Construction of Mouse Pulmonary Fibrosis Model:

[0074] Thirty-two C57BL / 6J mice were randomly divided into a control group, a model group, an exopolysaccharide treatment group, and a PFD (pirfenidone) group (i.e., a positive control group), with 8 mice in each group.

[0075] The mouse pulmonary fibrosis model was established using a single intratracheal instillation of bleomycin (BLM). Anesthetized mice were placed supine on an operating platform. A weight was hung to expose the mouse's mouth. The tongue was pulled out and oral mucus was wiped. A flashlight was shone into the mouse's throat to reveal a bright spot that opened and closed with breathing, representing the tracheal opening. A cannula was inserted into the trachea along the side of the mouse's mouth. The needle core was quickly removed. Successful intubation was verified with a filamentous cotton wool. BLM solution was then rapidly injected (control mice received the same dose of saline), followed by a 0.5 mL bolus of air. Finally, the mouse was gently rocked and rotated to evenly distribute the liquid throughout the lungs. The mouse's body temperature was maintained constant, and the mouse was returned to its cage after full recovery. The day of bleomycin instillation was designated day 0. After the mouse recovered and was returned to its cage, the mouse was housed normally for 28 days to establish the mouse pulmonary fibrosis model.

[0076] When constructing a mouse pulmonary fibrosis model, the day of tracheal instillation of BLM was set as day 0. After 28 days of normal feeding, drug intervention was carried out from day 29. Among them, the mice in the exopolysaccharide treatment group were given an exopolysaccharide solution at a dose of 100 mg / kg body weight per day (the exopolysaccharide solution was prepared by dissolving the exopolysaccharide obtained in Example 2 in a 0.5% CMC-Na solution. The concentration of the exopolysaccharide solution was specifically prepared at 100 mg / kg / d and 0.5 mL / 100 g mouse body weight. The concentration of the solution was determined based on the mouse The mice in the PFD group were gavaged with PFD solution (PFD solution was prepared by dissolving PDF in 0.5% CMC-Na solution. The concentration of PFD solution was 50 mg / kg / d, 0.5 mL / 100 g mouse body weight. The concentration of the solution varied according to the mouse body weight. The total daily intake was maintained at 100 mg / kg) at a dose of 50 mg / kg body weight per day. This lasted for 14 days. The experiment ended after gavage on the 42nd day. The drug dose was calculated using the equivalent dose coefficient conversion formula: D 小鼠 =D 人 ×(HI 小鼠 / HI 人 )×(W 小鼠 / W 人 ) 2 / 3 , where D represents dose, W represents body weight, and HI represents body shape index.

[0077] 3. Effects of W.confusa J1-1 exopolysaccharide on the growth of mice:

[0078] After the pulmonary fibrosis mouse model was successfully established, the feeding, mental state, and activity response of the mice in the control group, model group, exopolysaccharide treatment group, and PFD group were closely observed until the end of the experiment.

[0079] The results showed that the mice in the control group had smooth fur, bright eyes, and good spirits. Their diet, activity, and bowel movements were normal. Their weight maintained or increased slightly during the experiment, and no deaths occurred. However, the mice in the pulmonary fibrosis model group gradually lost their fur luster, showed signs of lethargy, decreased activity, and fatigue, and their food and water intake decreased significantly. After drug intervention, the mice in the exopolysaccharide treatment group and the PFD group showed significant improvement in the above symptoms compared to the model group. The mice had smooth and shiny fur, were more energetic, had increased activity, and significantly increased food and water intake.

[0080] 4. Effect of W.confusa J1-1 exopolysaccharide on mouse body weight

[0081] When constructing the mouse pulmonary fibrosis model, the day of tracheal instillation of BLM was set as day 0, and the weight changes of mice in the control group, model group, exopolysaccharide treatment group and PFD group were recorded on days 0, 7, 14, 21, 28 and 42. The results are shown in Figure 2. Figure 6 shown.

[0082] As shown in Table 6, weight gain slowed after endotracheal intubation in the model, exopolysaccharide, and PFD groups on day 0, and from day 7 onwards, weight was significantly lower than that in the control group (P < 0.01). After 14 days of drug intervention, the weight of mice in the exopolysaccharide and PFD groups was significantly higher than that in the model group (P < 0.01), and the rate of weight gain rebounded significantly. This suggests that the exopolysaccharide of Weissella J1-1 significantly improves the weight of mice with pulmonary fibrosis.

[0083] 5. Effect of W.confusa J1-1 exopolysaccharide on lung function in mice with pulmonary fibrosis

[0084] At the end of the experiment (the day of tracheal instillation of BLM in the establishment of the mouse pulmonary fibrosis model was set as day 0, and after 28 days of normal feeding, drug intervention was carried out from the 29th day, and the experiment was terminated after 14 days of drug intervention), the changes in lung function EF50 (Expiratory Flow, 50% tidal volume), TV (Tldal Volume, tidal volume) and MV (Maximum Ventilation Volume, minutely ventilated) of the mice in the control group, model group, exopolysaccharide treatment group and PFD group were measured. The results are shown in the figure. Figure 7 shown.

[0085] Depend on Figure 7 Compared with the control group, the model group showed a significant decrease in EF50, TV, and MV (P < 0.01). Compared with the model group, the exopolysaccharide group showed a significant increase in EF50 and MV (P < 0.05), and a significant increase in TV (P < 0.01). Compared with the model group, the PFD group showed an increase in EF50, MV, and TV, but without significant difference. This indicates that the exopolysaccharide of Weissella J1-1 significantly improves various lung function indicators in mice.

[0086] 6. Study on the effect of W.confusa J1-1 exopolysaccharide on lung tissue damage in mice with pulmonary fibrosis:

[0087] At the end of the experiment (the day of tracheal instillation of BLM in the establishment of mouse pulmonary fibrosis model was set as day 0, after 28 days of normal feeding, drug intervention was carried out from the 29th day, and the experiment was terminated after 14 days of drug intervention), the mice were killed and the lung tissues were collected for HE staining and Masson staining respectively. The staining results are shown in Figure 2. Figure 8 and Figure 9 shown.

[0088] Depend on Figure 8 It can be seen that compared with the control group, the alveolar wall of the lung tissue of mice in the model group was significantly thickened, and the rupture and fusion were severe. Obvious inflammatory exudate was visible in the alveolar cavity and the alveolitis score was significantly increased (P < 0.01). The extracellular polysaccharide group and the PFD group were able to effectively improve the above symptoms through drug treatment (P < 0.01).

[0089] Depend on Figure 9 It can be seen that compared with the control group, the alveolar walls of the lung tissue of mice in the model group were disordered, broken, and thickened, with large blue-stained areas, obvious interstitial collagen deposition, and increased pulmonary fibrosis scores (P < 0.01). The above symptoms were significantly improved after drug intervention in the extracellular polysaccharide group and the PFD group (P < 0.01).

[0090] HE and Masson staining results showed that fusion of Weissella J1-1 exopolysaccharide significantly improved various problems in the lung tissue of mice with pulmonary fibrosis, such as alveolar wall thickening, rupture, and fusion. It also improved interstitial collagen deposition and reduced alveolitis and pulmonary fibrosis scores. This suggests that fusion of Weissella J1-1 exopolysaccharide can be used to alleviate and treat pulmonary fibrosis.

[0091] Example 5: Cell experiment to verify the regulatory effect of W. confusa J1-1 exopolysaccharide on the phenotypic effect of fibroblasts

[0092] 1. Experimental cells, experimental groups and treatments

[0093] Cell line: Human embryonic lung fibroblast-like cell line (MRC-5) purchased from Wuhan Punosai Life Science Co., Ltd. Myofibroblasts are key effector cells in IPF. They are derived from multiple sources and undergo phenotypic transformation from fibroblasts to myofibroblasts. This process produces a large amount of ECM proteins, such as collagen and fibronectin, which in turn promotes tissue hardening. During this process, matrix hardness (such as a harder culture dish) can also play a role in promoting fibrosis.

[0094] The experimental groups included: experimental group (EPS J1-1 group) and control group (Control group). The experimental group (EPS J1-1 group) was added with fused Weissella J1-1 exopolysaccharide. The EPS J1-1 group was added with different concentrations of fused Weissella exopolysaccharide according to different functional tests. The control group was not added with exopolysaccharide.

[0095] 2. Identification of the degree of fibrosis in the MRC-5 cell line

[0096] MRC-5 cells were cultured statically in a dedicated complete culture medium in a 37°C, 5% CO2 incubator, and the medium was changed 2 to 3 times a week. The cell growth status was observed under a microscope, and the cells were subcultured when their density reached more than 70% of the bottom area of ​​the culture dish. During subculture, the original culture medium was aspirated, washed once with PBS, and 1.5 ml of 0.25% trypsin (containing EDTA) was added for digestion. When the cells were observed to shrink and become round under a microscope, an equal amount of complete culture medium was immediately added to terminate the digestion. The cells were gently blown down from the bottom of the dish, and the cell suspension was transferred to a centrifuge tube. Centrifuged at 1200 rpm / min for 5 minutes, the supernatant was discarded, 2 mL of complete culture medium was added for re-suspending, and subcultured to the culture dish at a ratio of 1:2. The day when the 0th generation cells recovered was recorded as Day 0. After 27 days of culture without any inducer, the expression of fibroblast activation-related indicators (α-SMA, FN, COL I) was detected by qRT-PCR. The results are as follows. Figure 10 shown.

[0097] Depend on Figure 10 It can be seen that after MRC-5 was revived and cultured in conventional culture dishes for 27 days, there was no significant difference in the expression of α-SMA, FN and COLⅠ mRNA in the cells, indicating that the purchased MRC-5 cells had obviously shown fibrosis under the stimulation of the hardness of the culture dish matrix, and can be directly used to verify the therapeutic efficacy of the fused Weissella J1-1 exopolysaccharide.

[0098] 3. Effect of W.confusa J1-1 exopolysaccharide on MRC-5 cell viability

[0099] MRC-5 cells in good growth condition were cultured at 8×10 3 The cells were seeded at a density of 100 cells / well in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for about 24 hours. When the cells grew to cover 70% to 80% of the bottom of the culture dish, 200 μL of different concentrations of Weissella fusion J1-1 exopolysaccharide solution (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) were added to induce for 48 hours; CCK8 (10 μL / well) was added for 1.5 to 2 hours, and the absorbance at a wavelength of 540 nm was detected using a microplate reader. The experimental results are shown in Figure 2. Figure 11 shown.

[0100] Depend on Figure 11 It can be seen that compared with the 0μg / ml concentration of Weissella J1-1 exopolysaccharide, the 1.0mg / ml concentration of J1-1 exopolysaccharide can significantly reduce the viability of MRC-5 cells (P<0.05), and the 0.2, 0.4, and 0.6mg / ml concentrations of J1-1 exopolysaccharide have no significant effect on the viability of MRC-5 cells. This shows that low and medium concentrations of Weissella J1-1 exopolysaccharide have no significant effect on the viability of fibroblasts MRC5.

[0101] 4. Improvement effect of W.confusaJ1-1 exopolysaccharide on fibrosis-related genes in MRC-5 cells

[0102] MRC-5 cells in good growth condition were cultured at a rate of 1.75×10 5 Cells were seeded into 6-well plates at 100 μg / well. When the cells grew to cover 70% to 80% of the bottom of the culture dish, the cells were divided into a control group (Control), different concentrations of J1-1 exopolysaccharide groups (0.2, 0.4, 0.6 mg / mL), and different concentrations of J1-1 exopolysaccharide groups (0.2, 0.4, 0.6 mg / mL) were added with corresponding concentrations of fusion Weissella J1-1 exopolysaccharide for intervention for 48 hours. Cells were collected using QIAzol and RIPA lysis buffer, and qRT-PCR technology was used to detect the gene expression of α-SMA, FN and COLⅠ. The experimental results are shown in the figure below. Figure 12 shown.

[0103] Depend on Figure 12 Compared with the control group, the expression of α-SMA, FN, and COLⅠ mRNA in the 0.2 mg / ml and 0.4 mg / ml J1-1 exopolysaccharide groups gradually decreased, and the 0.6 mg / ml J1-1 exopolysaccharide group significantly decreased α-SMA, FN, and COLⅠ mRNA (P < 0.05). This indicates that the fusion of Weissella J1-1 exopolysaccharide improves the fibrosis of MRC5 cells in a dose-dependent manner.

[0104] In summary, the fusion of Weissella J1-1 exopolysaccharide significantly improved various problems in the lung tissue of mice with pulmonary fibrosis, such as alveolar wall thickening, rupture, and fusion. It also improved interstitial collagen deposition and reduced alveolitis and pulmonary fibrosis scores. Furthermore, the fusion of Weissella J1-1 exopolysaccharide significantly improved the fibrosis of human embryonic lung fibroblast-like cells (MRC-5). Therefore, the fusion of Weissella J1-1 exopolysaccharide can be used to prevent, alleviate, and treat pulmonary fibrosis.

[0105] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the essence and scope of protection of the technical solutions of the present invention.

Claims

1. A fusion of Weissella ( Weissella confusa ), characterized in that, The fusion Weissella nucleic acid sequence contains the sequence shown in Sequence 1; the fusion Weissella is fusion Weissella J1-1, and the preservation number of fusion Weissella J1-1 is CCTCC NO: M 20232334.

2. An exopolysaccharide secreted by the fused Weissella according to claim 1, wherein the exopolysaccharide is prepared by inoculating the fused Weissella into a culture medium for fermentation and culture to obtain a culture solution, removing the bacteria and removing proteins from the culture solution to obtain a supernatant; subjecting the supernatant to alcohol precipitation, collecting the precipitate, dialyzing the precipitate to remove impurities, and freeze-drying to obtain the exopolysaccharide; the culture medium is MRS medium or mMRS medium; the fermentation and culture temperature is 30°C to 37°C; and the dialysis molecular weight cutoff is 8000 to 14000 Da.

3. The exopolysaccharide according to claim 2, characterized in that The alcohol precipitation treatment is to add anhydrous ethanol to the supernatant, mix evenly, and then stand at 4°C for 12 hours to 18 hours; the specific operation of the protein removal is to add trichloroacetic acid to the culture solution after removing the bacteria, mix evenly, stand at 4°C for 12 hours to 18 hours, centrifuge, and remove the precipitate.

4. Use of the exopolysaccharide according to any one of claims 2 to 3 in the preparation of a medicament for preventing, alleviating or / and treating pulmonary fibrosis.

5. A drug for preventing, alleviating or / and treating pulmonary fibrosis, characterized in that: The invention comprises an effective ingredient, wherein the effective ingredient is the extracellular polysaccharide according to any one of claims 2 to 3.

6. The drug according to claim 5, characterized in that The drug is administered orally, by gavage or parenterally.

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