Lipopolysaccharide capable of relieving chronic obstructive pulmonary disease as well as preparation method and application thereof

Abstract

The invention discloses lipopolysaccharide capable of relieving chronic obstructive pulmonary disease and a preparation method and application thereof, and belongs to the technical field of biological medicine. The lipopolysaccharide is a lipopolysaccharide capable of producing prevotella melaninogena, and the lipopolysaccharide is a lipopolysaccharide capable of producing prevotella melaninogena. The invention discovers and proves for the first time that the structure of the P.nigrum lipopolysaccharide is different from that of escherichia coli lipopolysaccharide, which is mainly reflected in that the acylation and phosphorylation levels of lipid A of the P.nigrum lipopolysaccharide are relatively low; the P.nigrum lipopolysaccharide can relieve the tobacco smoke induced mouse COPD (cyclooxygenase activated carbon); the P.nigra lipopolysaccharide can inhibit inflammatory reaction by inhibiting activation of a Tollis receptor mediated NF [kappa] B signal channel; the P.nigricans lipopolysaccharide can be used for inhibiting pyroptosis by inhibiting the activation of Caspase1. The invention can be applied to the fields of development of new drugs for preventing and treating chronic obstructive pulmonary diseases, research and development of new technologies and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a lipopolysaccharide that can alleviate chronic obstructive pulmonary disease, its preparation method, and its application. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung disease characterized by chronic respiratory symptoms caused by airway and / or alveolar abnormalities, typically manifesting as persistent, progressively worsening airflow obstruction. It is a common disease that seriously threatens human health, with a high global prevalence. Some large-scale epidemiological studies indicate a global prevalence of 10.3%, and the latest projections from the World Health Organization (WHO) show that by 2060, more than 5.4 million people will die annually from COPD and related conditions. The risk factors for COPD are complex and can be summarized as the combined effects of individual genetic and environmental factors.

[0003] Prevotella is a diverse genus of Gram-negative, obligate anaerobic bacteria. Prevotella melanogaster (… Prevotella melaninogenica Prevotella is a species in this genus, named for the black pigmentation of its colonies. Prevotella is relatively common in the human microbiota, being one of the most prevalent genera in the oral cavity, and also inhabits multiple parts of the body, including the respiratory tract, skin, vagina, and gastrointestinal tract. Studies have found a positive correlation between Prevotella in sputum and lung function, and a negative correlation with the severity of COPD. Other studies have found that Prevotella can inhibit the immune stimulation of dendritic cells (DCs) by COPD-associated pathogens (Haemophilus influenzae), inhibiting the production of IL-12p70 by DCs, which can promote the activation of inflammatory cells. This indicates that Prevotella has the function of regulating the immune response to specific pathogens. Simultaneously, studies have shown that dietary fiber in the intestine, fermented by Prevotella, can produce large amounts of succinic acid, a substrate for intestinal gluconeogenesis, which can significantly improve glucose tolerance and blood glucose levels in mice.

[0004] Lipopolysaccharide (LPS) in the cell wall plays a crucial role in the interaction between Gram-negative bacilli and their host. The mechanism by which pathogens induce inflammation after invasion involves a cascade reaction triggered by LPS acting on Toll-like receptor 4 (TLR4). Interspecies differences in LPS structure (such as structural variations in lipid A) affect TLR4 recognition, leading to variations in their ability to elicit an immune response. For example, *E. coli* LPS, with its lipid A structure comprising six acyl-containing fatty acid chains and two phosphate groups, produces a large amount of NF-κB-dependent inflammatory factors (TNF-α, IL-1β, and IL-6) upon stimulation of PBMCs. In contrast, LPS from *Bacteroides dorei* fails to elicit such a response regardless of dosage, likely due to the lower number of acyl and phosphate groups in its lipid A. However, research on this type of LPS from *Propionibacterium niger* is relatively limited. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention demonstrates for the first time that the structure of *Propionibacterium nervum* lipopolysaccharide differs from that of *Escherichia coli* lipopolysaccharide, and that it can alleviate tobacco smoke-induced COPD in mice. Furthermore, this function is related to its anti-inflammatory and anti-pyroptosis effects. This invention can be applied to the development of new drugs for the prevention and treatment of chronic obstructive pulmonary disease, as well as the research and development of new technologies.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide the application of Propionibacterium mellea lipopolysaccharide in the preparation of products for the prevention and treatment of chronic obstructive pulmonary disease.

[0007] Furthermore, the chronic obstructive pulmonary disease (COPD) prevention and treatment product inhibits the inflammatory response by suppressing the activation of the Tolls receptor-mediated NFκB signaling pathway.

[0008] Furthermore, the chronic obstructive pulmonary disease prevention and treatment product inhibits pyroptosis by suppressing the activation of Caspase1.

[0009] The second objective of this invention is to provide a lipopolysaccharide that can prevent and treat chronic obstructive pulmonary disease, wherein the lipopolysaccharide is the *Propionibacterium mellea* lipopolysaccharide mentioned above.

[0010] The third objective of this invention is to provide a method for preparing the lipopolysaccharide described above, characterized by comprising the following steps: (1) Anaerobic culture of the strain: Spread the bacterial suspension onto Buchner blood agar plates and incubate at 37°C in an anaerobic environment for 2 to 4 days until the plates are covered with black colonies. (2) Collection and disruption of bacterial cells: All bacterial cells were fully harvested, resuspended in deionized water to obtain a bacterial suspension, and then sonicated until the bacterial cell walls were fully disrupted. (3) Hot phenol-water extraction: Add an equal volume of phenol aqueous solution to the broken suspension to form a two-phase mixture, then stir and heat in a water bath at 60-70℃ for 20-40 minutes, then cool to 0-10℃ and stand for more than 6 hours; (4) Phase separation to obtain crude extract: After standing, the mixture is centrifuged at 0-10℃ and 3000-5000 × g for 20-40 minutes to separate and collect the upper aqueous phase, and obtain crude extract containing lipopolysaccharide. (5) Purification, concentration and freeze drying: The crude extract is concentrated by ultrafiltration, washed to remove small molecule impurities, and then purified and concentrated to obtain the lipopolysaccharide.

[0011] Furthermore, in step (2), the duration of the ultrasonic treatment is 8 to 12 minutes.

[0012] Furthermore, in step (3), the volume concentration of the phenol aqueous solution is 85% to 95%.

[0013] Furthermore, in step (5), the ultrafiltration concentration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to 10 kDa.

[0014] The fourth objective of this invention is to provide a product for the prevention and treatment of chronic obstructive pulmonary disease, which contains the lipopolysaccharide described above.

[0015] Furthermore, the product type includes drugs, and the dosage form of the drug is any one of capsules, tablets, microcapsules, injections, sprays, or ointments.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to discover that the structure of Przewalski's bacterium lipopolysaccharide is different from that of Escherichia coli lipopolysaccharide, mainly reflected in the lower acylation and phosphorylation levels of lipid A in the former.

[0017] 2. This invention is the first to discover that lipopolysaccharide from Propionibacterium mellea can alleviate tobacco smoke-induced COPD in mice.

[0018] 3. This invention is the first to discover that lipopolysaccharide from Propionibacterium mellea can inhibit inflammatory responses by suppressing the activation of the Tolls receptor-mediated NFκB signaling pathway.

[0019] 4. This invention is the first to discover that Przewalski's lipopolysaccharide can inhibit pyroptosis by inhibiting the activation of Caspase1.

[0020] 5. This invention can be applied to the development of new drugs for the prevention and treatment of chronic obstructive pulmonary disease and the research and development of new technologies. Attached Figure Description

[0021] Figure 1The results of the comparative analysis of the molecular structures of Pm-LPS and Ec-LPS in Example 1 of this invention are shown. (A) Fourier transform infrared spectrum: The spectra of the two samples are similar in general shape and characteristic peak type. The figure marks the CH3 bond (2933 cm⁻¹) in the molecular structure of lipopolysaccharide. -1 Nearby), CN bond (1243cm) -1 Nearby), C=O bond (1633cm) -1 Nearby), CO bond (1050cm) -1 Nearby), glycosidic bond (921cm) -1 Nearby) and OH bonds (1410cm) -1 (A) The position of major chemical bonds, including those near the target. (B) X-ray photoelectron spectroscopy (XPS), fine elemental spectrum and peak fitting sub-peaks of C1s, CC / CH bond (red sub-peak, binding energy around 284.8 eV), CO bond (blue sub-peak, binding energy around 286 eV), C=O bond (green sub-peak, binding energy around 288 eV). (C) X-ray photoelectron spectroscopy, fine elemental spectrum and peak fitting sub-peaks of O1s, PO4 group (red sub-peak, binding energy around 531 eV), CO bond (green sub-peak, binding energy around 532.3 eV), C=O bond (purple sub-peak, binding energy around 533 eV). The vertical axis of the XPS spectrum is the photoelectron signal intensity (or the number of points collected per second), and the horizontal axis is the binding energy. Pm-LPS, Proprynella niger lipopolysaccharide; Ec-LPS, Escherichia coli lipopolysaccharide.

[0022] Figure 2 This invention's Example 1 verifies that *Propionibacterium niger* lipopolysaccharide can significantly alleviate cigarette smoke-induced COPD in mice. (A) Mice were instilled with Pm-LPS or PBS intratracheally and collected after 6 months of exposure to tobacco smoke, with an air control set up. (B) Mouse weight gain trend and amount. (C) Representative images of nucleated cell counts and Giemsa staining in bronchoalveolar lavage fluid (400×), representative images of HE staining in lung tissue sections (200×) and mean alveolar intercepts, representative images of PAS staining in lung tissue sections (200×) and mean airway mucus thickness. (D) Statistics of mouse lung function indicators. Data are expressed as mean ± standard deviation or median ± interquartile range (n=6-8 per group). Statistical significance was determined using one-way ANOVA combined with Dunnett's multiple comparison test (ns P>0.05, P<0.01, P<0.001, ...). P<0.0001). CS: Cigarette smoke exposure group; Pm-LPS: *Prevotella melanogaster* lipopolysaccharide treatment group; CS+Pm-LPS: *Prevotella melanogaster* lipopolysaccharide treatment group after cigarette smoke exposure; PBS: Phosphate buffer; HE: Hematoxylin-eosin staining; PAS: Periodic acid-Schiff staining; BALF: Bronchoalveolar lavage fluid; Cchord: Static compliance; FRC: Functional residual capacity; FEV100 / FVC: Forced expiratory volume in 0.1 seconds to forced vital capacity.

[0023] Figure 3 This document describes the verification process of the significant inhibition of inflammatory response and pyroptosis in COPD mice by *Propionibacterium nervosa* lipopolysaccharide in Example 1 of this invention. (A) Immunoblotting showing the expression levels of p-NFKB, cleaved-caspase 1, and N-GSDMD in mouse lung tissue. (B) Immunofluorescence staining showing the expression levels of p-NFKB and N-GSDMD in mouse lung tissue (200×). (C) Serum levels of TNF-α, IL-8, IL-1α, and IL-1β in mice. Data are expressed as mean ± standard deviation or median ± interquartile range (n=6-8 per group). Statistical significance was determined using one-way ANOVA combined with Dunnett's multiple comparison test (nsP>0.05, P<0.01, P<0.001). P<0.0001). CS: Cigarette smoke exposure group; Pm-LPS: Prösprinae lipopolysaccharide-producing group; CS+Pm-LPS: Prösprinae lipopolysaccharide-producing group after cigarette smoke exposure.

[0024] Figure 4 This is a verification process for demonstrating that *Propionibacterium niger* lipopolysaccharide in Example 1 of this invention can inhibit the inflammatory response by suppressing the activation of the Tolls receptor-mediated NFκB signaling pathway. (A) Expression levels of p-NFκB in cells after 24 h of stimulation with different concentrations of CSE following 16HBE. (BC) Expression levels of p-NFκB in cells and the levels of TNF-α and IL-8 in the cell culture medium after 60 min of pretreatment with Pm-LPS or chloroquine (CQ) followed by 2% CSE stimulation for 24 h following 16HBE. Data are expressed as mean ± standard deviation or median ± interquartile range (n = 3 per group). Statistical significance was determined using one-way ANOVA combined with Dunnett's multiple comparison test (ns P > 0.05). P < 0.0001). CSE: Cigarette smoke extract; Pm-LPS: Prösprinae lipopolysaccharide.

[0025] Figure 5 This is a verification process of how lipopolysaccharide from *Propionibacterium niger* can inhibit pyroptosis by inhibiting the activation of Caspase 1, as described in Example 1 of this invention. (A) Expression level of cleaved-caspase 1 in cells after stimulating 16HBE with different concentrations of CSE for 48 h. (B) Expression levels of cleaved-caspase 1 and N-GSDMD in cells after 48 h of pretreatment with Pm-LPS or VX-765 followed by stimulation with 2% CSE, and the levels of lactate dehydrogenase (LDH), IL-1α, and IL-1β in the cell culture supernatant. (C) Representative image of live and dead cells stained with Calcein / PI (200×) and the proportion of PI-positive dead cells. Data are expressed as mean ± standard deviation or median ± interquartile range (n = 3 per group). Statistical significance was determined by one-way ANOVA combined with Dunnett's multiple comparison test (ns P > 0.05). P < 0.0001). CSE: Cigarette smoke extract; Pm-LPS: Prösprinae lipopolysaccharide. Detailed Implementation

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0027] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0028] Example 1 I. Sources, strain preservation, preparation and molecular structure determination of lipopolysaccharide from Propionibacterium mellea The standard strain of *Prevotella hepatica* produced in our laboratory was purchased from BNCC (product number BNCC371785). After obtaining the strain, gene sequencing was performed to identify the species, and the strain was simultaneously preserved using the following method: A well-grown *Prevotella hepatica* colony in the late logarithmic growth phase was scraped from a blood agar plate and added to 1 ml of sterile broth. The mixture was thoroughly shaken to prepare a bacterial suspension with three McFarland turbidities. 100 μl of this bacterial suspension was added to a bacterial preservation tube containing 1 ml of 20% sterile glycerol solution. The mixture was thoroughly mixed, and 2-3 drops of liquid paraffin were added to the surface of the mixture. The tube was then quickly and tightly capped to ensure a seal. The bacterial strain name, number, and preservation date were clearly marked on the tube. Finally, the preservation tube was placed directly in an ultra-low temperature freezer at -80°C for long-term storage.

[0029] The following method was used to prepare lipopolysaccharide from revived and preserved *Propionibacterium niger* strains: One strain preservation tube was taken from a -80°C ultra-low temperature freezer and rapidly thawed in 37°C warm water. 200 μl of the bacterial suspension was immediately spread onto an anaerobic-treated Brucella blood agar plate. The plate was placed in an anaerobic flask (containing an anaerobic gas-generating bag and anaerobic indicator) and incubated at 37°C for 2-4 days. Once black colonies had covered the plate, the bacterial lipopolysaccharide was extracted using the hot phenol-water method: Bacterial cells were fully harvested and resuspended in deionized water to prepare a homogeneous suspension. The suspension was sonicated for 10 minutes to fully disrupt the cell walls. An equal volume of 90% liquid phenol was added to form a two-phase mixture. This mixture was heated in a 65°C water bath with continuous stirring for 30 minutes to lyse the cells and release lipopolysaccharide. After heating, the mixture was immediately cooled to 4°C and allowed to stand overnight. Then, centrifuge (4000 × g for 30 minutes at 4°C) to clearly separate the solution into an upper aqueous phase (containing lipopolysaccharides, nucleic acids, etc.) and a lower phenolic phase (containing proteins), with an insoluble denatured protein layer in between. Carefully aspirate the upper aqueous phase. Transfer the collected aqueous phase to an ultrafiltration tube and centrifuge to discard the solvent and other components. Repeated washing with deionized water can be used to improve the recovery rate. Finally, freeze-dry the lipopolysaccharide-containing liquid obtained from ultrafiltration to obtain lipopolysaccharide powder, weigh it, and seal it for storage.

[0030] The differences in the types and elemental contents of surface functional groups of lipopolysaccharides from *Propionibacterium mellea* and *Escherichia coli* were analyzed using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy (Xps). The specific methods are as follows: 1. The chemical structure of the sample surface was characterized using a Fourier transform infrared spectroscopy (FT-IR) instrument (Brook, TENSOR27, USA), and the types of surface functional groups were analyzed by FT-IR. The samples were prepared by a pelleting method. First, 5 mg of sample and 250 mg of pure KBr were finely ground and uniformly placed in a mold. Then, the mold was gradually pressurized to a maximum of 20 MPa using a hydraulic press to press the sample into a transparent sheet. Finally, the infrared absorption spectrum was measured by FT-IR transmission mode.

[0031] 2. X-ray photoelectron spectroscopy (Xps) analysis was performed using a Thermo Scientific K-Alpha X-ray photoelectron spectrometer and its accompanying software (Avantage). The test parameters were as follows: radiation source: Al Kalph source; test tube voltage: 15 kV; tube current: 10 mA; X-ray energy: 1486.8 eV; background vacuum in the analysis chamber: 2 x 10⁻⁹ mbar; test spot size: 400 μm; test step size: generally 0.05 eV; number of scans: 5; analysis mode: CAE mode; pass energy: 30.0 eV; binding energy range: -5 to 1350 eV; instrument work function: 4.2 eV. The testing process is as follows: Blocks or films are directly cut to appropriate sizes and adhered to the sample stage with conductive adhesive to form conductive contact. The samples are then directly fed into the XPS instrument for testing. For powders, the powder is coated onto an Al foil with double-sided adhesive, pressed into a pellet using a pelleting device, and then the Al foil with the pellet sample attached is adhered to the sample stage with conductive adhesive. Finally, the sample is fed into the instrument for vacuum testing. The results yield the full spectrum and narrow spectrum of elements, including elemental valence states and their proportions. The detection limit is 0.5%-1%. The vertical axis of the XPS spectrum represents the photoelectron signal intensity (or the number of points collected per second), and the horizontal axis represents the binding energy.

[0032] II. Laboratory Animals Male C57BL / 6 mice (Guangdong Vital River Laboratory Animal Technology Co., Ltd.) were grouped according to the experimental design. Mice were exposed to tobacco smoke in a full-body fumigation chamber for 6 days / week for 24 weeks before data collection. *Prevotella mellea* lipopolysaccharide and PBS were administered via tracheal instillation to CS-exposed mice and control mice. The specific method was as follows: Mice were weighed and anesthetized by intraperitoneal injection of 2,2,2-Tribromoethanol (200 mg / kg). Under laryngoscope-assisted tracheal instillation of *Prevotella mellea* lipopolysaccharide (20 μg / 50 μl PBS) or 50 μl PBS (once weekly) was administered. After administration, mice were placed in a lateral decubitus position until recovery from anesthesia. Lung function tests, bronchoalveolar lavage, and lung tissue sections were performed on each mouse to confirm the COPD phenotype.

[0033] Mouse lung tissue proteins were used for Western blotting experiments, and lung tissue sections were used for Hematoxylin and Eosin staining, Periodic Acid-Schiff staining, and immunofluorescence staining. Mouse serum was used to detect various cytokines. The animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University, with ethics number 20250062.

[0034] III. Cell Experiments Bronchial epithelial cells (16HBE, Wuhan Pronosei Biotechnology Co., Ltd., catalog number CP-H009) were seeded into cell culture plates. After cell adhesion and growth, CSE stimulation was added, with appropriate pretreatment performed according to the experimental design before stimulation. After stimulation for a certain period of time, the activity of lactate dehydrogenase and the levels of various cytokines in the cell culture medium were measured, live and dead cells were counted, and cell proteins were extracted for Western blotting experiments.

[0035] IV. Statistical Analysis Continuous variables are expressed as mean (standard deviation) or median (interquartile range), while categorical variables are expressed as percentage (%). Comparisons between groups of continuous variables were performed using Student's t-test, Ordioay one-way ANOVA, Mann-Whitney U test, or Kruskal-Wallis test; analyses of categorical variables were performed using Pearson's chi-square test or Fisher's exact test. A two-sided p-value <0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 22.

[0036] V. Experimental Results, Technical Problems Solved, and Advantages Compared with Existing Technologies 1. The structure of Przewalski's lipopolysaccharide differs from that of Escherichia coli lipopolysaccharide. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy (Xps) were used to compare and analyze the differences in the types and elemental contents of surface functional groups of *Propionibacterium niger* lipopolysaccharide and *Escherichia coli* lipopolysaccharide. A comparison of their molecular structures revealed that, compared to *E. coli* lipopolysaccharide, *Propionibacterium niger* lipopolysaccharide showed weaker peaks for CH3, CN, and C=O bonds, while glycosidic and OH bonds showed stronger peaks. Figure 1 A); the proportion of C=O bonds in *Propionibacterium niger* lipopolysaccharide (C1s: 17.12%, O1s: 7.78%) is lower than that in *Escherichia coli* lipopolysaccharide (C1s: 21.05%, O1s: 11.68%), and the proportion of PO4 groups in *Propionibacterium niger* lipopolysaccharide (2.37%) is also lower than that in *Escherichia coli* lipopolysaccharide (2.60%). Figure 1 (BC). The above test results indicate that the structure of *Propionibacterium nervosa* lipopolysaccharide differs from that of *Escherichia coli* lipopolysaccharide, mainly reflected in the lower acylation and phosphorylation levels of lipid A in the former. This structural characteristic may determine that its biological function also differs from that of *Escherichia coli* lipopolysaccharide.

[0037] 2. Lipopolysaccharide from *Propionibacterium mellea* can alleviate tobacco smoke-induced COPD in mice. Mice exposed to tobacco smoke were administered *Prevotella mellea* lipopolysaccharide via intratracheal instillation, and samples were collected after 6 months. The specific method was as follows: Mice were weighed and anesthetized by intraperitoneal injection of 2,2,2-Tribromoethanol (200 mg / kg). Under laryngoscope-assisted instillation of *Prevotella mellea* lipopolysaccharide (20 μg / 50 μl PBS) or 50 μl PBS (once weekly) via intratracheal instillation. After administration, the mice were placed in a lateral decubitus position until they recovered from anesthesia. Results showed that *Prevotella mellea* lipopolysaccharide significantly alleviated COPD induced by tobacco smoke exposure in mice. Figure 2 ), inhibited the inflammatory response and pyroptosis in COPD mice ( Figure 3 ).

[0038] 3. Propionibacterium mellea lipopolysaccharide can inhibit the inflammatory response by suppressing the activation of the Tolls receptor-mediated NFκB signaling pathway. First, tobacco smoke extract (CSE) was prepared as follows: The smoke from two unfiltered cigarettes was inhaled into a glass syringe containing 5 ml of DMEM. The pH of the resulting suspension was adjusted to 7.4 using sodium hydroxide. After filtration and sterilization, the liquid was considered 100% CSE. It was then diluted with DMEM to obtain an appropriate concentration for subsequent experiments.

[0039] Then, human bronchial epithelial cells (16HBE) were stimulated with different concentrations of CSE (1%, 2%, 4%) for 24 hours. When the concentration reached 2%, the expression level of p-NFκB in the cells was significantly increased. Figure 4 A), therefore, this invention uses 2% CSE to re-stimulate 16HBE. After 24 hours, the expression level of p-NFκB in cells and the concentrations of TNF-α and IL-8 in cell culture medium are significantly increased. However, before stimulation, pretreatment of cells with Propionibacterium mellea lipopolysaccharide (Pm-LPS) (5 μg / ml) for 60 minutes can significantly reduce the levels of the above three indicators. Figure 4 (BC). Meanwhile, this invention uses chloroquine (CQ), an inhibitor of the Tolls receptor pathway (10 μg / ml), as a positive control, and the treatment method is the same as that for Pm-LPS.

[0040] The specific methods for cell culture and stimulation are as follows: 16HBE cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated in a 5% CO2, 37°C incubator. Cells were seeded into 6-well plates (1×10⁶ cells / wells). 6After the cells adhered and grew, CSE stimulation was added. Before stimulation, *Propionibacterium mellea* lipopolysaccharide (5 μg / ml) or chloroquine (10 μg / ml) could be added for pretreatment for 60 min (or no pretreatment). After 24 h of stimulation, the cell culture supernatant was collected, and cell proteins were extracted for subsequent experiments.

[0041] The above studies indicate that Przewalski's lipopolysaccharide can inhibit the inflammatory response by suppressing the activation of the Tolls receptor-mediated NFκB signaling pathway.

[0042] 4. Lipopolysaccharide from *Propionibacterium mellea* can inhibit pyroptosis by suppressing the activation of Caspase 1. Stimulation of 16HBE with different concentrations of CSE (1%, 2%, 4%) for 48 hours resulted in a significant increase in cleaved-caspase 1 expression in cells when the concentration reached 2%. Figure 5 A), therefore, this invention uses 2% CSE to re-stimulate 16HBE. After 48 hours, the expression levels of cleaved-caspase 1 and N-GSDMD in the cells, as well as the activity of lactate dehydrogenase (LDH) and the concentrations of IL-1α and IL-1β in the cell culture supernatant, were significantly increased. However, pretreatment of cells with Propionibacterium mellea lipopolysaccharide (Pm-LPS) (5ug / ml) for 120 minutes before stimulation could significantly reduce the levels of the above three substances. Figure 5 (BC). Meanwhile, this invention uses the Caspase1 inhibitor VX-765 (5uM) as a positive control, and the treatment method is the same as that for Pm-LPS.

[0043] The specific methods for cell culture and detection are as follows: Cells are seeded into 12-well plates (1×10⁻⁶ cells / well). 5 After cell / well culture, CSE stimulation was added after cell adhesion and growth. Pretreatment with *Propionibacterium mellea* lipopolysaccharide (5 μg / ml) or VX-765 (5 μM) for 120 min prior to stimulation (or no pretreatment) was performed. After 48 h of stimulation, lactate dehydrogenase activity in the cell culture was measured using an LDH activity assay kit. After aspirating the culture medium, cells were washed with PBS and then incubated in the dark with the Viability / Cytotoxicity Assay Kit for 30 min. Cell counts were performed under a fluorescence microscope.

[0044] The above studies indicate that Przewalski's lipopolysaccharide can inhibit pyroptosis by suppressing the activation of Caspase1.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Propionibacterium mellea ( Prevotella melaninogenica Application of lipopolysaccharide in the preparation of products for the prevention and treatment of chronic obstructive pulmonary disease.

2. The application according to claim 1, characterized in that, The chronic obstructive pulmonary disease (COPD) prevention and treatment product inhibits the inflammatory response by suppressing the activation of the Tolls receptor-mediated NFκB signaling pathway.

3. The application according to claim 1, characterized in that, The chronic obstructive pulmonary disease (COPD) prevention and treatment product inhibits pyroptosis by suppressing the activation of Caspase 1.

4. A lipopolysaccharide capable of preventing and treating chronic obstructive pulmonary disease, characterized in that, The lipopolysaccharide is the *Propionibacterium mellea* lipopolysaccharide used in the application described in claim 1.

5. A method for preparing the lipopolysaccharide as described in claim 4, characterized in that, Includes the following steps: (1) Anaerobic culture of the strain: Spread the bacterial suspension onto Buchner blood agar plates and incubate at 37°C in an anaerobic environment for 2 to 4 days until the plates are covered with black colonies. (2) Collection and disruption of bacterial cells: All bacterial cells were fully harvested, resuspended in deionized water to obtain a bacterial suspension, and then sonicated until the bacterial cell walls were fully disrupted. (3) Hot phenol-water extraction: Add an equal volume of phenol aqueous solution to the broken suspension to form a two-phase mixture, then stir and heat in a water bath at 60-70℃ for 20-40 minutes, then cool to 0-10℃ and stand for more than 6 hours; (4) Phase separation to obtain crude extract: After standing, the mixture is centrifuged at 0-10℃ and 3000-5000 × g for 20-40 minutes to separate and collect the upper aqueous phase, and obtain crude extract containing lipopolysaccharide. (5) Purification, concentration and freeze drying: The crude extract is concentrated by ultrafiltration, washed to remove small molecule impurities, and then purified and concentrated to obtain the lipopolysaccharide.

6. The preparation method according to claim 5, characterized in that, In step (2), the ultrasonic treatment time is 8 to 12 minutes.

7. The preparation method according to claim 5, characterized in that, In step (3), the volume concentration of the phenol aqueous solution is 85% to 95%.

8. The preparation method according to claim 5, characterized in that, In step (5), the ultrafiltration concentration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to 10 kDa.

9. A product for the prevention and treatment of chronic obstructive pulmonary disease, characterized in that, It includes the lipopolysaccharide as described in claim 2.

10. The chronic obstructive pulmonary disease prevention and treatment product according to claim 9, characterized in that, The product type includes drugs, and the dosage form of the drugs is any one of capsules, tablets, microcapsules, injections, sprays, or ointments.

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