Application of bear gall powder in preparation of medicine for treating acute lung injury

Abstract

The invention relates to the technical field of biology, and discloses an application of bear gall powder in preparation of a medicine for treating acute lung injury, the effective components of the medicine comprise Ursodeoxycholic acid, Tauroursodeoxycholic acid, Chenodeoxycholic acid, Deoxycholic acid, 3-Ketocholic acid and the like, and the effective components of the medicine comprise the Ursodeoxycholic acid, the Tauroursodeoxycholic acid, the Chenodeoxycholic acid, the Deoxycholic acid, the 3-Ketocholic acid and the like. During application, at the cellular level, the concentration of the bear gall powder is 6.25-12.5 mu g / mL; at the animal level, the concentration of the bear gall powder is 30-120mg / kg. According to the application of the bear gall powder in preparation of the medicine for treating the acute lung injury, the bear gall powder acts on Keap-1 / Nrf-2 / HO-1 and NF-kappa B signal channels to improve pathological changes of lung tissues and inhibit inflammatory response and oxidative injury, so that ALI is treated, and an earlier-stage experimental basis is provided for subsequent application of the bear gall powder in treatment of lung diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of bear bile powder in the preparation of drugs for treating acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a disease caused by multiple factors that damage alveolar epithelial cells and capillary endothelial cells. It is characterized by diffuse interstitial and alveolar edema, leading to acute hypoxic respiratory dysfunction. The main triggers for ALI include oxidative stress and inflammatory responses induced by endotoxins (lipopolysaccharide, LPS) and direct lung infection caused by sepsis. Its pathogenesis is complex, involving several aspects: inflammatory response, alveolar epithelial and endothelial cell damage, oxidative stress, immune response, and disruption of the alveolar-vascular barrier. Specifically, inflammatory mediators (such as TNF-α and IL-1) lead to damage to alveolar and small vessel endothelial cells, increasing alveolar permeability; excessive production of ROS (reactive oxygen species) further exacerbates cell membrane and DNA damage; and the aggregation of immune cells (such as neutrophils and macrophages) leads to the release of inflammatory factors and enzymes, creating a vicious cycle. Furthermore, multiple signaling pathways (such as the NF-κB pathway, the Nrf-2 signaling pathway, and autophagy) jointly regulate the occurrence and development of ALI. Currently, clinical treatments commonly employ methods such as anti-hypoxia, anti-inflammation, hemostasis, and thoracentesis. However, these methods have limitations, and long-term use of glucocorticoids may induce drug resistance. Therefore, exploring new targets and drugs for the treatment of ALI is particularly important.

[0003] Bear bile powder (BBP), a precious animal medicine in traditional Chinese medicine, contains bile acids, cholesterol, bile pigments, and amino acids, and possesses various pharmacological effects such as antioxidant, anti-inflammatory, and anti-cancer properties. Korean medical theory holds that bear bile has the effects of promoting blood circulation, removing blood stasis, clearing heat and detoxifying, inducing sweating, and expelling pathogenic factors from the lungs. The classic Korean medical text, *Dongui Saxiang Medical Treatise*, records that bear bile is called the "Lung Marshal" medicine, with the function of expelling pathogenic factors from the lungs, and has the effects of promoting blood circulation, removing blood stasis, clearing heat and detoxifying, and inducing sweating; *Dongui Saxiang New Compilation* records that bear bile powder is used to induce sweating and expel pathogenic factors from the lungs. Tanreqing Injection, composed of Scutellaria baicalensis, bear bile powder, goat horn, honeysuckle, and forsythia, is used to treat phlegm-heat obstructing the lungs syndrome in wind-heat lung disease. It is suitable for patients with symptoms such as fever, cough, difficulty expectorating phlegm, sore throat, thirst, red tongue, and yellow tongue coating, as well as those with early-stage pneumonia, acute bronchitis, acute exacerbations of chronic bronchitis, and upper respiratory tract infections exhibiting the above syndromes. Bear Bile and Fritillaria Oral Solution, composed of bear bile powder, Fritillaria cirrhosa, almond water, honey, and menthol, is suitable for symptoms such as phlegm-heat cough, persistent cough, and difficulty expectorating phlegm. The above studies indicate that bear bile powder may have certain preventive and therapeutic effects on lung diseases. Given the functions of bear bile powder, this study aims to investigate its anti-acute lung injury effects and possible mechanisms of action. Summary of the Invention

[0004] The purpose of this invention is to provide the application of bear bile powder in the preparation of drugs for treating acute lung injury, and to provide a preliminary experimental basis for the subsequent application of bear bile powder (BBP) in the treatment of lung diseases.

[0005] To achieve the above objectives, the present invention provides the application of bear bile powder in the preparation of drugs for treating acute lung injury, wherein the active ingredients of the bear bile powder include Ursodeoxycholic acid, Tauroursodeoxycholic acid, Chenodeoxycholic acid, Deoxycholic acid and 3-Ketocholanic acid, etc.

[0006] Furthermore, when applied, at the cellular level, the concentration of the bear bile powder is 6.25-12.5 μg / mL; at the animal level, the concentration of the bear bile powder is 30-120 mg / kg.

[0007] Furthermore, it is applied to: Cellular level: ① Reduce the level of cellular inflammatory factors; ② Reduce cellular oxidative stress, manifested as decreased cellular MDA and NO activity and increased SOD activity; ③ Reduce ROS levels in cells; Animal level: ④ Reduce pulmonary edema and lung injury levels; ⑤ It reduces the number of total cells and neutrophils, and decreases MPO activity.

[0008] Furthermore, bear bile powder improves lung tissue pathological changes and inhibits inflammatory response and oxidative damage by acting on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways.

[0009] To achieve the above objectives, the present invention also provides a drug for treating acute lung injury, comprising bear bile powder, wherein the active ingredients of the drug include Ursodeoxycholic acid, Tauroursodeoxycholic acid, Chenodeoxycholic acid, Deoxycholic acid, and 3-Ketocholanic acid, etc.

[0010] Furthermore, the drug is used for: ① Reduce the level of cellular inflammatory factors; ② Reduce cellular oxidative stress, manifested as decreased cellular MDA and NO activity and increased SOD activity; ③ Reduce ROS levels in cells; ④ Reduce pulmonary edema and lung injury levels; ⑤ It reduces the number of total cells and neutrophils, and decreases MPO activity.

[0011] Furthermore, the drug improves lung tissue pathological changes and inhibits inflammatory response and oxidative damage by acting on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways.

[0012] The advantages and positive effects of the bear bile powder described in this invention in the preparation of drugs for treating acute lung injury are as follows: 1. This invention employs network pharmacology and in vitro / in vivo experimental verification methods to investigate the in vivo and in vitro effects and mechanisms of bear bile powder on LPS-induced acute lung injury (ALI). Network pharmacology results show that the anti-ALI effect of bear bile powder mainly involves apoptosis, inflammatory response, and immune-related functions and pathways. In vitro / in vivo experimental results show that bear bile powder can alleviate LPS-induced inflammatory response and oxidative stress damage in RAW264.7 cells and ALI mice, and improve the pathological changes in lung tissue of ALI mice. Furthermore, BBP significantly upregulated the mRNA and protein levels of IκB-α, Nrf-2, and HO-1 in cells and tissues, and downregulated the mRNA and protein levels of Keap-1 and NF-κB (p65). In summary, BBP improves lung tissue pathological changes and inhibits inflammatory response and oxidative stress damage by acting on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways, thereby treating ALI, providing a preliminary experimental basis for the subsequent application of BBP in the treatment of lung diseases.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is a Venn diagram of the intersection target points of BBP and ALI in an embodiment of the present invention; Figure 2 This is a diagram of the common target PPI network structure in an embodiment of the present invention; Figure 3 This is a GO function enrichment analysis in an embodiment of the present invention; Figure 4 This is a KEGG functional enrichment analysis in an embodiment of the present invention; Figure 5 This is a network diagram of "disease-component-target" in an embodiment of the present invention; Figure 6This invention illustrates the effects of different concentrations of BBP on the viability of RAW 264.7 cells and LPS-induced viability of RAW 264.7 cells (x̄±s, n=3), where A represents RAW 264.7 cell viability, B represents LPS-induced RAW 264.7 cell viability, and compared with the control group, # indicates p<0.05, ##p<0.01, and ### indicates p<0.001; compared with the model group, This indicates that p < 0.01; Figure 7 In this embodiment of the invention, BBP was used to reduce the level of inflammatory factors in RAW264.7 cells induced by LPS (x̄±s, n=3), where A was IL-6, B was Cox-2, C was IL-1β, and D was TNF-α. Compared with the blank group, ## p<0.01, ### indicates p<0.001; compared with the model group, This indicates that p < 0.01; Figure 8 This invention illustrates the effect of BBP on LPS-induced oxidative factor levels in RAW264.7 cells (x̄±s, n=3), where A represents MDA, B represents NO, and C represents SOD. Compared to the control group, ##p<0.01, where ### indicates p<0.001; compared to the model group, This indicates that p < 0.05. This indicates that p < 0.01. p<0.001; Figure 9 The expression of ROS in each group of cells in the embodiments of the present invention (scale bar: 100 μm). Figure 10 In this embodiment of the invention, qPCR was used to detect the mRNA expression levels (x̄±s, n=3) of Keap-1, HO-1, Nrf-2, NF-κB (p65), TNF-α, IκB-α, and IL-1β in LPS-induced RAW264.7 cells, where A represents Nrf-2, B represents Keap-1, C represents HO-1, D represents NF-κB (p65), E represents IκB-α, F represents TNF-α, and G represents IL-1β. Compared with the control group, ### express p <0.001; compared with the model group, express p <0.01, p <0.001; Figure 11The figures show the H&E staining results of BBP on LPS-induced ALI mouse lung tissue in this embodiment of the invention. A represents HE staining of lung tissue from each group of mice, B represents lung injury score, and C represents the wet / dry weight ratio of mouse lung tissue. Compared with the blank group, ### indicates p<0.001; compared with the model group, This indicates that p < 0.01. *p<0.001; Figure 12 This invention illustrates the effects of BBP on the number and protein content of neutrophils in the BALF of LPS-induced ALI mice, where A represents the total number of cells in BALF; B represents the number of neutrophils in BALF; C represents the protein level in BALF; and D represents the MPO activity assay (x̄±s, n=6). Compared with the control group, ## indicates p<0.01; compared with the model group, This indicates that p < 0.05. This indicates that p < 0.01; Figure 13 In this embodiment of the invention, BBP was used to reduce LPS-induced lung inflammation and oxidative stress damage in ALI mice (x̄±s, n=6), where A represents NO level, B represents MDA level, C represents SOD level, D represents IL-1β, E represents Cox-2, F represents TNF-α, and G represents IL-6. Compared with the control group, ## indicates p<0.01; compared with the model group... This indicates that p < 0.05. This indicates that p < 0.01; Figure 14 The images show the immunohistochemical results (×400, scale bar: 50 μm) of Nrf-2, HO-1, IL-1β, and NF-κB (p65) in the lung tissue of mice in each group of this invention, where A represents HO-1, B represents NF-κB (p65), C represents IL-1β, and D represents Nrf-2. Compared with the blank group, ## indicates p<0.01, and ### indicates p<0.001; compared with the model group, This indicates that p < 0.05. This indicates that p < 0.01; Figure 15 This invention illustrates the effect of BBP on the protein levels of Keap-1, Nrf-2, HO-1, IκB-α, and NF-κB (p65) in the lung tissue of LPS-induced ALI mice. In this embodiment, A represents Keap-1, B represents Nrf-2, C represents HO-1, D represents NF-κB (p65), and E represents IκB-α. Compared to the control group, ## indicates p < 0.01, and ### indicates p < 0.001; compared to the model group, This indicates that p < 0.05. This indicates that p < 0.01. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0017] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments without specified sources are all commercially available raw materials. The bear bile powder used in this invention was purchased from the Yanbian Bear Farm (production batch number: 20220902).

[0018] Example 1. Experimental Methods: 1.1 Screening of active ingredients and target sites in bear bile powder: Rapid analysis of the chemical components of bear bile powder was performed using UHPLC-Q-Orbitrap-HRMS. 20 mg of bear bile powder was dissolved in 2 mL of 80% methanol-water solution, vortexed for 10 min, and centrifuged at 13000 rpm for 10 min. The supernatant was collected. A Thermo Scientific™ Hypersil Gold Vanquish column (2.1 × 100 mm, 1.9 μm) was used with acetonitrile (A) and 0.1% formic acid / water solution (B) as the mobile phase, and a flow rate of 0.3 mL / min for elution. Data were acquired in positive and negative ion modes using an electrospray ionization source, with a spray voltage of 3.5 kV (+) and 3.2 kV (-) and a capillary temperature of 320℃. Bile acid components were identified using reference standards, literature, and databases. Molecular structural formulas and CanonicalSMILES formats of the bile acid components from bear bile powder were searched using the PubChem database, and potential therapeutic targets were analyzed by integrating the HERB and STRING databases. The obtained targets are then standardized and named using the UniProt database to ensure data consistency.

[0019] 1.2 Prediction of targets in acute lung injury: Using "Acute lung injury" and "LPS" as keywords, we searched for targets related to acute lung injury in the GeneCards, OMIM, and Disease databases, removed duplicate values, and obtained potential targets for ALI. We then used the Venny 2.1.0 tool to screen for intersection targets between bear bile powder and ALI, and constructed a Venn diagram of drug-disease relationship.

[0020] 1.3 Construction of Protein-Protein Interaction (PPI) Network and Screening of Key Targets: Import the intersection target points into the STRING database and set parameters to generate a PPI network diagram. Import the selected target points into the Network Analyzer module of Cytoscape 3.10.0 software for topology analysis, and filter key and core target points based on network centrality parameters (degree, betweenness, closeness).

[0021] 1.4 Construction of the "Disease-Medicinal Material-Component-Target" Network: Cytoscape 3.9.0 software was used to construct an interaction network diagram of "disease-medicinal material-active ingredient-target" for the effect of bear bile powder on acute lung injury. The Network Analyzer module was used for topology analysis to screen core compounds.

[0022] 1.5 GO and KEGG enrichment analysis: GO enrichment analysis described the three dimensions of gene biological processes (BP), molecular functions (MF), and cellular components (CC); KEGG pathway enrichment analysis revealed the therapeutically relevant signaling pathways involved by the target. Key targets were imported into the DAVID bioinformatics platform for GO and KEGG enrichment analysis, and functional items and pathways were screened based on p-values. Bubble charts and bar charts were then generated using the MicroBioinformatics Cloud platform.

[0023] 1.6 Cell Culture: Mouse mononuclear macrophage leukemia cells (RAW 264.7) (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in complete culture medium (containing 10% newborn calf serum and 90% RPMI-1640 medium) and incubated at 37°C with 5% carbon dioxide.

[0024] 1.7 CCK-8 assay for cell viability: The cytotoxicity of BBP against macrophages was detected using the CCK-8 assay. RAW 264.7 cells were cultured at 5 × 10⁻⁶ cells / cells. 4Cells were seeded at a density of 10 cells / mL in 96-well plates and treated with different concentrations (0, 6.25, 12.5, 25, 50, 75, 100 μg / mL) of BBP for 24 h. After adding 10 μL of CCK-8 solution to each well, the OD value was measured at 450 nm using a microplate reader to calculate cell viability.

[0025] RAW264.7 cells were grown at a rate of 5 × 10⁻⁶. 4 Cells were seeded at 1 / mL in 96-well plates and incubated for 12 h. After pretreatment with different doses of BBP (6.25–50 μg / mL) or dexamethasone (Dex, 25 μg / mL) for 1 h, cells were stimulated with 0.5 μg / mL LPS for 24 h. 10 μL of CCK-8 solution was added to each well, and the OD value was measured at 450 nm using a microplate reader to calculate cell viability.

[0026] 1.8 Determination of the levels of inflammatory factors and oxidative products in cells: Cells were cultured and modeled according to the above method. After drug administration, the cell supernatant was aspirated, and the contents of TNF-α, COX-2, IL-1β, IL-6, MDA, SOD, and NO were detected according to the reagent instructions.

[0027] 1.9 ROS Detection: RAW264.7 cells were fed at a rate of 8 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and incubated for 12 h. They were then treated with bear bile powder (6.25 and 12.5 μg / mL) or dexamethasone (Dex, 25 μg / mL) for 1 h, followed by stimulation with 0.5 μg / mL LPS for 24 h. Cells were then incubated with 10 μM / L DCFH-DA at 37°C for 20 min. After incubation, the supernatant was discarded, and the cells were washed twice with PBS. Images were acquired and analyzed using a fluorescence microscope.

[0028] 1.10 qRT-PCR analysis of cell samples: Cells were cultured in 6-well plates. After culturing as described above, grouping, and administering drugs for 24 h, the supernatant was removed, and 1 mL of Trizol and 200 μL of chloroform were added to each well. The cells were incubated for 5 min, then centrifuged at 12000 r / min for 15 min at 4 °C. The supernatant was then aspirated, and an equal volume of isopropanol was added. The cells were incubated for 10 min, then centrifuged again at 12000 r / min for 10 min at 4 °C. The supernatant was removed, and 1 mL of 75% ethanol was added. The cells were centrifuged for 5 min, discarded, and dried at room temperature for 5 min. RNA was dissolved in 20 μL LEPC water and stored at -80 °C. Following the manufacturer's instructions, genomic cDNA was first removed, followed by reverse transcription, and finally amplification (p65, IκBα, IL-6, IL-1β, Nrf-2, HO-1, Keap-1, and GAPDH) was performed to obtain Ct values. The Ct values ​​were then used to calculate... Table 1 shows the required primer sequences.

[0029] Table 1 Primers used for RT-PCR

[0030] 1.11 Animal grouping: Thirty male C57BL / 6 mice were randomly divided into three groups: control group, model group, and dexamethasone group (5 mg / kg). -1 The mice were divided into three groups: a low-dose bear bile powder group (30 mg / kg) and a high-dose bear bile powder group (120 mg / kg), with six mice in each group. According to the experimental design, the mice were administered the medication continuously for 5 days. One hour after the last administration, all mice except the control group received an intratracheal infusion of LPS (4 mg / kg). -1 Mice were anesthetized 24 h later (10 μL / 20g) and lung tissue and bronchoalveolar lavage fluid (BALF) were collected.

[0031] 1.12 BALF Collection and Analysis: Bronchoalveolar lavage (BALF) was performed using 1 mL PBS in a syringe, repeated twice, and the BALF was recovered. The sample was centrifuged at 3000 rpm / min for 10 min at 4°C. The total cell and neutrophil counts in the BALF precipitate were determined using a Wright-Giemsa staining kit (Sangon Biotech, China). The protein concentration in the BALF was detected using a diquine formate assay kit (BCA) (Beyotime Biotechnology Co., Ltd., Shanghai, China).

[0032] 1.13 MPO Analysis: MPO activity, a sensitive and specific marker of ALI, can be used to assess the number of neutrophils aggregated in tissues and is an indicator of neutrophil infiltration in the lungs. 10 mg of left lower lung tissue was taken and homogenized in a tissue homogenizer at a mass / volume ratio of 1:9 with tissue homogenizing medium to prepare a 10% tissue homogenate. The MPO content in the lung tissue was determined strictly according to the kit instructions.

[0033] 1.14 Lung tissue wet-to-dry weight ratio: After the mice were sacrificed, lung tissue was collected, washed with PBS buffer, dried with filter paper, and weighed wet. The lung tissue was then incubated at 80°C for 48 hours and weighed again to obtain the dry weight of the lung tissue. The wet-to-dry weight ratio of the lung tissue was calculated to determine the degree of pulmonary edema. Lung tissue wet-to-dry weight ratio = lung tissue wet weight / lung tissue dry weight.

[0034] 1.15 HE staining for pathological changes in lung tissue: Histopathological examination was performed on fresh lung tissue from mice from which exudates were not collected. Lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 mm), and stained with hematoxylin and eosin for histological evaluation. Pathological changes in lung tissue were observed in each group of mice, and lung tissue damage was scored. The scoring criteria included four indicators: 1. alveolar congestion; 2. hemorrhage; 3. neutrophil infiltration or aggregation in the alveolar or vascular walls; 4. alveolar wall thickening or hyaline membrane formation. The severity of the lesions was scored from 0 to 4 points: 0 points for no lesions or very mild lesions; 1 point for mild lesions; 2 points for moderate lesions; 3 points for severe lesions; and 4 points for extremely severe lesions. The total score was 16 points, and the sum of the scores for the four evaluation criteria was the total score.

[0035] 1.16 Determination of inflammatory cytokines in lung tissue: Lung tissue was washed with cold PBS, weighed, minced, and homogenized in PBS at a ratio of 1:9. The homogenate was subjected to freeze-thaw cycles to further lyse the cells, and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was collected, and the levels of inflammatory factors TNF-α, COX-2, IL-1β, and IL-6 were determined according to the ELISA kit procedure.

[0036] 1.17 Determination of MDA, SOD, and NO content in lung tissue: Lung tissue was washed with cold PBS, weighed, minced, and homogenized in PBS at a ratio of 1:9. The homogenate was subjected to freeze-thaw cycles to further lyse the cells, and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was collected, and the levels of oxidative factors (MDA, SOD, NO) were determined according to the reagent instructions.

[0037] 1.18 Immunohistochemical analysis of lung tissue: Lung tissue paraffin sections were dewaxed. Treatment with 3% hydrogen peroxide for 10 minutes blocked endogenous peroxidase. Lung tissue was blocked with 5% BSA buffer for 1 h. Primary antibodies (p65, IL-1β, Nrf-2, and HO-1) were incubated overnight at 4°C, and secondary antibodies were incubated at 37°C in the dark for 1 hour. Samples were washed three times with PBS and stained with DAPI for 10 min. Fluorescence images were acquired using a fluorescence microscope.

[0038] 1.19 Western blot analysis of the expression of NF-κB and related proteins in the Keap1 / Nrf2 / HO-1 pathway in lung tissue: The collected lung tissue was placed in a culture dish and cut into small pieces of approximately 3 mm × 3 mm using surgical scissors. 1 mL of cold RIPA lysis buffer was added, and the tissue was homogenized using a glass homogenizer. The resulting tissue homogenate was centrifuged at 1000 r / min, 4 ℃, for 5 min, and the supernatant was collected. BCA working solution was prepared at a ratio of 50:1, and protein standard solutions of different concentrations were added. 200 μL of BCA working solution was added to each well, and the mixture was incubated at 37 ℃ for 30 min. The absorbance was measured at 562 nm. A standard curve was plotted. The sample was diluted to an appropriate concentration, BCA working solution was added, and the mixture was incubated at 37 ℃ for 30 min. The absorbance was measured, and the protein content of the sample was calculated.

[0039] Place a clean glass plate on the gel holder. Prepare a 10% SDS-PAGE lower separating gel and a 5% upper stacking gel. Pour the gels into the gels and allow them to solidify. Carefully remove the sample comb and add 1×Tris-Gly electrophoresis buffer. Aspirate an appropriate amount of sample and add it to the sample wells. Turn on the power at 60 V. Once the protein sample enters the separating gel, increase the voltage to 90 V. Stop electrophoresis when the target band has entered 2 / 3 of the gel. Lift the glass plate and place the membrane on filter paper soaked in transfer buffer. Then, place an NC / PVDF membrane on top, forming a transfer sandwich of "fiber pad—filter paper—gel—NC / PVDF membrane—filter paper—fiber pad". Place the transfer sandwich in the transfer tank and maintain a constant current of 200 mA for 60–120 min. Remove the label.

[0040] The NC / PVDF membrane was placed in 5% skim milk blocking buffer and shaken on a shaker for 1.5–2 h. After blocking, the membrane was washed with TBST for 5 min × 3 times. The membrane was then placed in primary antibody (IκBα, p65, Keap1, Nrf2, HO-1) culture medium and incubated overnight at 4 °C on a shaker. The next day, the membrane was removed and washed with TBST for 5 min × 3 times. The secondary antibody was diluted with 5% skim milk blocking buffer and reacted at room temperature with shaking for 1–2 h. After the secondary antibody reaction, the membrane was washed with TBST for 5 min × 3 times. Finally, the membrane was developed and fixed with ECL solution, and the band intensity was quantified using ImageJ gel analysis software.

[0041] 2. Results: 2.1 Analysis of bile acid components in bear bile powder: The components of bear bile powder were analyzed using UHPLC-Q-Orbitrap-HRMS. Twenty bile acid compounds were successfully isolated and identified (see Table 2).

[0042] Table 2. Information on bile acid compounds in bear bile powder characterized by UHPLC-Q-Orbitrap high-resolution mass spectrometry.

[0043] 2.2 Network pharmacology analysis: Using network pharmacology, 20 bile acid components were imported into the HERB and Swiss TargetPrediction databases, resulting in 71 targets. Combined with databases such as GeneCards, OMIM, and Disease, 5165 acute lung injury-related targets were obtained, ultimately identifying 64 overlapping targets. Figure 1 ). Constructing PPI network analysis Figure 2 As shown, functional enrichment analysis of GO and KEGG was performed. Figure 3 , Figure 4 (As shown). The results showed that the core targets mainly involve apoptosis, inflammatory response, and immune-related functions and pathways. Further, a "component-target-disease" network diagram was constructed ( Figure 5 As shown in Table 3, the key compounds screened out were Ursodeoxycholic acid, Tauroursodeoxycholic acid, Chenodeoxycholic acid, Deoxycholic acid, and 3-Ketocholanic acid.

[0044] Table 3 Information on Key Active Compounds

[0045] 2.3 BBP alleviates LPS-induced inflammatory response in RAW264.7 cells: CCK-8 results showed that BBP (6.25-50 μg / mL) had no significant effect on RAW 264.7 cell viability. However, BBP (75-200 μg / mL) significantly inhibited RAW 264.7 cell viability. p <0.05, p <0.01 or p <0.001)( Figure 6 (A). Subsequently, an ALI cell model was established by LPS induction. The viability of RAW264.7 cells was significantly reduced after LPS treatment. Treatment with different concentrations of BBP (12.5-50 μg / mL) significantly increased cell viability. Figure 6 (B). Therefore, 6.25 and 12.5 μg / mL were selected as low and high doses of BBP for subsequent cell experiments.

[0046] The levels of inflammatory factors in cells were detected using ELISA. The results are as follows: Figure 7 As shown, compared with the blank group, LPS significantly increased the levels of TNF-α, IL-6, Cox-2 and IL-1β in the model group cells. p <0.001). Compared with the model group, the BBP group (12.5 μg / mL) significantly reduced the levels of TNF-α, IL-6, Cox-2 and IL-1β. p <0.01). The results indicate that BBP has a certain anti-inflammatory effect on LPS-induced inflammation in RAW264.7 cells.

[0047] 2.4 Effects of BBP on LPS-induced oxidative stress in RAW264.7 cells: Oxidative stress plays a crucial role in various diseases. Therefore, a kit was used to detect the levels of SOD, MDA, and NO in cells. The results are as follows: Figure 8 As shown in the figure. Compared with the control group, the model group showed significantly increased MDA and NO activities and significantly decreased SOD activity. However, BBP (12.5 μg / mL) significantly reduced LPS-stimulated MDA and NO activities in RAW264.7 cells and increased SOD activity. p <0.01, p <0.001). The above results indicate that BBP has a certain antioxidant effect on LPS-induced RAW264.7 cells.

[0048] 2.5 Effect of BBP on LPS-induced ROS expression in RAW264.7 cells: The results are as follows Figure 9As shown, compared with the control group, the ROS content in the model group cells was significantly increased. However, after BBP treatment, the ROS content in the cells was significantly decreased.

[0049] 2.6 BBP protects LPS-induced inflammation and oxidative stress damage in RAW264.7 cells by activating the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways: The Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways have been reported to be involved in the regulation of oxidative stress and inflammatory responses. The mRNA expression levels of Keap-1, HO-1, Nrf-2, NF-κB, TNF-α, IκB-α, and IL-1β in cells were determined using qPCR. Results are as follows: Figure 10 As shown, compared with the control group, the mRNA expression levels of Keap-1, TNF-α, NF-κB (p65), and IL-1β were significantly increased in the model group, while the mRNA expression levels of IκB-α, Nrf-2, and HO-1 were significantly decreased. However, compared with the model group, BBP treatment significantly reduced the mRNA expression levels of Keap-1, TNF-α, NF-κB (p65), and IL-1β, and increased the mRNA expression levels of IκB-α, Nrf-2, and HO-1. This indicates that the protective effect of BBP against LPS-induced cell damage may be through its action on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways.

[0050] 2.7 Pathological changes in lung tissue of ALI mice due to BBP: To further evaluate the protective effect of BBP against ALI, an LPS-induced ALI animal model was established in this study. Pathological changes in lung tissue were observed using HE staining. Results are as follows: Figure 11 As shown in Figure A, compared with the control group, the model group exhibited significant lung histopathological changes, including neutrophil infiltration, alveolar damage, hemorrhage, and pulmonary edema. However, after treatment with BBP (120 mg / kg), the lung histopathological changes in LPS-induced ALI mice were significantly reduced. Furthermore, the lung injury scores showed a similar trend. Compared with the control group, the lung injury score in the model group was significantly increased, while the lung injury score in the BBP group (120 mg / kg) was significantly decreased. Figure 11 (B) The effect of BBP on the degree of lung edema in LPS-induced ALI mice was evaluated by measuring the lung wet / dry weight ratio in each group. The results are as follows: Figure 11 In mice with ALI, LPS stimulation significantly upregulated the lung wet-to-dry weight ratio compared to the control group, while treatment with BBP (120 mg / kg) significantly reduced this ratio. These results indicate that BBP has a protective effect against LPS-induced ALI in mice.

[0051] 2.8 Effects of BBP on neutrophil count and protein content in BALF of ALI mice: Neutrophil activation and infiltration play a crucial role in LPS-induced ALI. LPS can disrupt the alveolar-capillary barrier, leading to the leakage of plasma proteins and neutrophils into the alveolar spaces. Results are as follows... Figure 12 As shown in Figures A and B, LPS stimulation significantly increased the number of total cells and neutrophils compared to the control group. However, compared to the model group, the number of total cells and neutrophils in the BBP group (120 mg / kg) mice was significantly reduced. Furthermore, protein content in BALF directly reflects lung permeability. Compared to the control group, the protein concentration in the BALF of LPS-treated mice was significantly increased. However, this was significantly reduced in mice pretreated with BBP at a concentration of 120 mg / kg. Figure 12 (C). Myeloperoxidase (MPO) plays a crucial role in the occurrence and development of ALI. Results are as follows... Figure 12 As shown in Figure D, MPO activity was significantly increased in the model group mice compared to the control group. However, BBP (120 mg / kg) significantly reduced MPO activity compared to the model group.

[0052] 2.9 Effects of bear bile powder on LPS-induced inflammatory response and oxidative stress damage in ALI mice: Oxidative damage and inflammatory responses can lead to pathological changes in lung tissue with ALI. The levels of inflammatory and oxidative factors in lung tissue were detected using ELISA. Results are as follows: Figure 13 As shown, compared with the control group, the levels of Cox-2, IL-1β, IL-6, and TNF-α in the lung tissue of mice in the model group were significantly increased. However, BBP pretreatment significantly reduced the levels of Cox-2, IL-1β, IL-6, and TNF-α. Furthermore, compared with the blank group, the concentrations of MDA and NO in the lung tissue of mice in the model group were significantly increased, while the concentration of SOD was decreased. However, compared with the model group, BBP (120 mg / kg) treatment significantly reduced the concentrations of MDA and NO and increased the concentration of SOD.

[0053] 2.10 Effects of bear bile powder on the expression of Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathway-related proteins in the lung tissue of LPS-induced ALI mice: Immunohistochemical results showed that, compared with the control group, LPS stimulation significantly increased the expression of NF-κB (p65) and IL-1β in lung tissue, and decreased the expression of Nrf-2 and HO-1. In contrast, the expression levels of p65 and IL-1β in the BBP group were significantly lower than those in the model group, while the expression of Nrf-2 and HO-1 was significantly higher. Figure 14 ).

[0054] Western blot results are as follows Figure 15 As shown, compared with the control group, the levels of NF-κB (p65) and Keap-1 proteins in the lung tissue of the model group were significantly increased, while the levels of IκB-α, Nrf-2, and HO-1 proteins were significantly decreased. After BBP pretreatment, the levels of p65 and Keap-1 proteins in the lung tissue of ALI mice were significantly decreased, while the levels of IκB-α, Nrf-2, and HO-1 proteins were significantly increased.

[0055] This invention employs network pharmacology and in vitro / in vivo experimental validation methods to investigate the in vivo and in vitro effects and mechanisms of bear bile powder on LPS-induced acute lung injury. Cell viability was assessed using CCK-8 assays in LPS-mediated ALI cell and animal models. The levels of inflammatory factors (TNF-α, COX-2, IL-1β, IL-6) and oxidative factors (MDA, SOD, and NO) in cells and tissues were measured using ELISA and related kits. The expression of NF-κB and Keap-1 / Nrf-2 / HO-1 signaling pathway-related mRNAs in cells was determined by qPCR. The effects of bear bile powder on ALI were evaluated by detecting the number and protein count of immune cells in bronchoalveolar lavage fluid, lung pathological changes, lung wet-to-dryness ratio, and myeloperoxidase content. Furthermore, immunohistochemistry and Western blotting were used to assess the expression of NF-κB and Keap-1 / Nrf-2 / HO-1 signaling pathway-related proteins in lung tissue. Network pharmacology results indicate that bear bile powder exerts its anti-ALI effect primarily through apoptosis, inflammatory responses, and immune-related functions and pathways. In vitro and in vivo experiments showed that bear bile powder could alleviate LPS-induced inflammatory responses and oxidative stress damage in RAW264.7 cells and ALI mice, and improve pathological changes in lung tissue of ALI mice. Furthermore, compared to the model group, BBP significantly upregulated the mRNA and protein levels of IκB-α, Nrf-2, and HO-1 in cells and tissues, while downregulating the mRNA and protein levels of Keap-1 and NF-κB (p65).

[0056] Therefore, this invention utilizes the above-mentioned bear bile powder in the preparation of drugs for treating acute lung injury. BBP improves pathological changes in lung tissue and inhibits inflammatory response and oxidative damage by acting on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways, thereby treating ALI and providing a preliminary experimental basis for the subsequent application of BBP in the treatment of lung diseases.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The use of bear gall powder in the preparation of a drug for treating acute lung injury, characterized in that: The active ingredients of the bear bile powder include Ursodeoxycholic acid, Tauroursodeoxycholic acid, Chenodeoxycholic acid, Deoxycholic acid, and 3-Ketocholanic acid. ​ 2. The use of bear gall powder according to claim 1 in the preparation of a drug for treating acute lung injury, characterized in that: When applied, at the cellular level, the concentration of the bear bile powder is 6.25-12.5 μg / mL; at the animal level, the concentration of the bear bile powder is 30-120 mg / kg.

3. The application of the bear bile powder according to claim 1 in the preparation of a drug for treating acute lung injury, characterized in that, Applied to: Cellular level: ① Reduce the level of cellular inflammatory factors; ② Reduce cellular oxidative stress, manifested as decreased cellular MDA and NO activity and increased SOD activity; ③ Reduce ROS levels in cells; Animal level: ④ Reduce pulmonary edema and lung injury levels; ⑤ It reduces the number of total cells and neutrophils, and decreases MPO activity.

4. The application of the bear bile powder according to claim 1 in the preparation of a drug for treating acute lung injury, characterized in that: Bear bile powder improves lung tissue pathological changes and inhibits inflammatory response and oxidative damage by acting on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways.

5. A drug for treating acute lung injury, characterized in that: The drug includes bear bile powder, and its active ingredients include Ursodeoxycholic acid, Tauroursodeoxycholic acid, Chenodeoxycholic acid, Deoxycholic acid, and 3-Ketocholanic acid.

6. The medicament for treating acute lung injury according to claim 5, characterized in that: The drug is used for: ① Reduce the level of cellular inflammatory factors; ② Reduce cellular oxidative stress, manifested as decreased cellular MDA and NO activity and increased SOD activity; ③ Reduce ROS levels in cells; ④ Reduce pulmonary edema and lung injury levels; ⑤ It reduces the number of total cells and neutrophils, and decreases MPO activity.

7. The medicament for treating acute lung injury according to claim 5, characterized in that: The drug improves lung tissue pathological changes and inhibits inflammatory response and oxidative damage by acting on the Keap-1 / Nrf-2 / HO-1 and NF-κB signaling pathways.

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