Ganoderma lucidum spore powder extract for regulating intestinal flora to treat acute lung injury and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a ganoderma lucidum spore powder extract for treating acute lung injury by regulating intestinal flora and application thereof. The preparation method of the ganoderma lucidum spore powder extract (aqueous extract) comprises the following steps: selecting wall-broken ganoderma lucidum spore powder as a raw material, adding ultrapure water into the wall-broken ganoderma lucidum spore powder according to a solid-liquid ratio of 1g / (9-11) mL, firstly performing ultrasonic oscillation dissolution, then performing constant-temperature extraction, and then performing centrifugation to take supernate as a ganoderma lucidum spore powder extract solution. The invention also provides application of the ganoderma lucidum spore powder extract in preparation of medicines for regulating intestinal flora and treating acute lung injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a ganoderma lucidum spore powder extract for treating acute lung injury and application thereof. BACKGROUND

[0002] Ganoderma lucidum is a basidiomycete composed of multiple pore fungi, and is a medicinal and edible fungus that is tonic, invigorating, and beneficial to maintaining homeostasis of the body. Ganoderma lucidum spore powder is a small spore ejected from the cap of a mature ganoderma lucidum fruiting body, and is the reproductive cell of ganoderma lucidum. The double-layer cell wall of the ganoderma lucidum spore is composed of chitin and glucose, and is hard and acid-resistant, which hinders the dissolution of effective components, and thus seriously affects the digestion and absorption of the gastrointestinal tract. Crushing the outer wall of the ganoderma lucidum spore through a wall-breaking process can effectively improve the utilization of effective components by the organism and enhance the therapeutic effect.

[0003] Various viruses (such as influenza virus, novel coronavirus, respiratory syncytial virus, etc.) can cause viral pneumonia and other infectious lung diseases, which are generally characterized by high infectivity, extensive morbidity, and potential fatal risk. Inflammation is the main defense mechanism of the host against viral infection, and is characterized by the activation and mobilization of inflammatory cells such as macrophages, neutrophils, and lymphocytes. Excessive activation of inflammatory cells can lead to the release of cytokines and the production of harmful systemic immune responses, i.e., "cytokine storm". Acute lung injury (ALI) is an acute lung inflammation syndrome caused by various pathogenic factors (such as viral, bacterial infection, trauma, shock, aspiration, sepsis, etc.) in the lung or outside the lung. ALI is a dynamic process that can progress to acute respiratory distress syndrome (ARDS) in severe cases. Among the clinical diseases related to acute lung injury, viral pneumonia is the most common. At present, the main clinical treatment is supportive treatment, such as the use of ECMO, and there is no effective treatment to prevent and reverse serious consequences. Moreover, ALI develops rapidly and has a high mortality rate. Therefore, it is urgent to explore effective methods for treating ARDS / ALI.

[0004] The intestinal microbiota constitutes a complex and dynamic ecosystem, and plays a key role in regulating the immune system and overall physiological function. When the intestinal microecology is imbalanced, problems such as a decrease in the number of beneficial flora, excessive proliferation of harmful bacteria, damage to the intestinal barrier function, and disorders of the immune system will occur, ultimately leading to the occurrence of various diseases. Studies have shown that there is an interaction between the intestinal microbiome and the lungs, which is referred to as the "gut-lung axis". Changes in the composition, diversity, and function of the intestinal microbiota can affect lung immune function. More and more studies have shown that the type of intestinal microbiota and changes in its metabolic products are closely related to immune response, inflammatory response, and the progression of lung diseases. SUMMARY

[0005] The technical problem solved by the present application is to provide a broken wall ganoderma spore powder extract for regulating intestinal flora to treat acute lung injury and an application thereof.

[0006] To solve the above technical problem, the present application provides a preparation method of a broken wall ganoderma spore powder extract (water extract) for regulating intestinal flora to treat acute lung injury, comprising the following steps: selecting broken wall ganoderma spore powder as raw material, adding ultrapure water to the broken wall ganoderma spore powder at a material-liquid ratio of 1 g / 9-11 mL (preferably 1 / 10 mL), ultrasonic oscillation dissolution, constant temperature extraction, centrifugation to obtain supernatant as a ganoderma spore powder extract (water extract) solution.

[0007] Improvements of the preparation method of the ganoderma spore powder extract of the present application:

[0008] The constant temperature extraction is constant temperature extraction at 80±0.5℃ for 3±0.1h;

[0009] The ultrasonic oscillation dissolution is ultrasonic oscillation dissolution at an ultrasonic frequency of 40KHz, a power of 200W and a temperature of 4±0.5℃ for 30±1min; for example, 2g of broken wall ganoderma spore powder is used as raw material.

[0010] The centrifugation is centrifugation at 4000±200rpm for 10±0.5min.

[0011] The present application also simultaneously provides an application of the ganoderma spore powder extract (water extract) prepared by any of the above methods in the preparation of a drug for regulating intestinal flora to treat acute lung injury.

[0012] Improvements of the application of the present application: reducing lung inflammation (H&E staining shows that BGLSE reduces lung inflammatory cell infiltration), pulmonary edema and pulmonary tissue fibrosis (Masson staining shows that BGLSE reduces lung blue collagen fiber deposition) of an acute lung injury model mouse, and protecting the lung.

[0013] Further improvements of the application of the present application: playing an anti-inflammatory role by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway (immunohistochemistry shows that the expression levels of TLR4, MyD88, NF-κB p65 and phosphorylated NF-κB p65 are significantly reduced after BGLSE intervention).

[0014] Further improvements of the application of the present application: increasing the immune cell and immunoglobulin levels of a COVID-19 patient (the number of lymphocytes, the proportion of CD4+ T cells, the levels of immunoglobulin M (IgM) and IgG of the patient increase after BGLSE treatment), and reducing the levels of inflammatory cells and C-reactive protein (the white blood cell count and the proportion of neutrophils of the patient significantly decrease, and the C-reactive protein value decreases by an average of 68.4% after BGLSE treatment).

[0015] As a further improvement of the application: the intestinal flora and metabolites are changed.

[0016] Specifically: the intestinal flora of acute lung injury mice and COVID-19 patients is changed, the relative abundance of Proteobacteria (harmful bacteria) in acute lung injury mice decreases after taking Ganoderma lucidum spore powder, and the relative abundance of Actinobacteria (beneficial bacteria) increases, as shown in (C) of Figure 4 COVID-19 patients taking Ganoderma lucidum spore powder, the relative abundance of Bacteroidetes (beneficial bacteria) increases, as shown in (C)~(D) of Figure 8 (D)~(E) of Figure 5 (B) of Figure 9

[0017] As a further improvement of the application: the intestinal flora and metabolite joint analysis found that Bifidobacterium was significantly positively correlated with L-arginine, as shown in (A) of Figure 6 (A) of Figure 6 (C) of Figure 6 (D) of

[0018] That is, in acute lung injury mice, by adjusting the level of L-arginine, which is significantly positively correlated with Bifidobacterium, the mTOR signaling pathway is activated, and the excessive autophagy is inhibited to reduce the inflammatory response.

[0019] The Ganoderma lucidum spore powder extract (water extract) obtained by the present application has triterpenes, esters, nucleosides, sterols, organic compounds and other components, which endow the extract with the activity of regulating intestinal flora to treat acute lung injury.

[0020] The Ganoderma lucidum extract product for regulating intestinal flora to treat acute lung injury described in the present application includes but is not limited to the following types:

[0021] Medicine: the Ganoderma lucidum spore powder extract (water extract) is mixed with a pharmaceutically acceptable excipient to prepare tablets, capsules, oral liquids, injections and other dosage forms of medicines for regulating intestinal flora to treat acute lung injury;

[0022] Functional food: a proper amount of the Ganoderma lucidum spore powder extract (water extract) is added in the food processing process to develop functional foods with the function of assisting antiviral pneumonia, such as beverages, pastries, meal replacement powders, etc.

[0023] ​Health products: Ganoderma lucidum spore powder extract (water extract) is combined with other nutritional ingredients or functional substances to prepare health products in the form of capsules, soft capsules, powders, oral liquids, etc., which are used for enhancing human immunity and assisting in resisting viral pneumonia.

[0024] The Ganoderma lucidum spore powder extract (water extract) of the present application has a significant effect of treating acute lung injury. Meanwhile, the present application also provides that the extract can play a role by regulating intestinal flora, providing a new intervention option based on intestinal flora regulation for the treatment of acute lung injury, and highlighting the potential therapeutic value of the "gut-lung axis".

[0025] The present application selects broken Ganoderma lucidum spore powder as a raw material, carries out extraction and component analysis, establishes an acute lung injury mouse model, and carries out clinical experiments. The Ganoderma lucidum spore powder extract prepared by the present application has multiple active ingredients, reduces lung edema and pulmonary fibrosis in acute lung injury mice, has a protective effect on the lungs, plays an anti-inflammatory role by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway, increases the beneficial flora in the intestines of mice, reduces harmful flora, regulates the abundance of bifidobacteria, activates the mTOR signaling pathway by regulating the L-arginine level which is significantly positively correlated with bifidobacteria, and inhibits excessive autophagy to reduce inflammatory response; acute lung injury is most likely caused by viral infection, and clinical research on COVID-19 patients shows that broken Ganoderma lucidum spore powder can improve the immunity of patients, reduce inflammatory response, and regulate the intestinal flora and metabolites of patients. The present application provides a natural drug solution for the research and development of drugs for treating acute lung injury, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0027] Figure 1 ACQUITY UPLCTM I-Class / XevoG3XS positive ion mode analysis BGLSE triterpenoids chart;

[0028] Figure 1 In:

[0029] (A): Sanguisorba officinalis saponin II; (B): Arjunolic acid; (C): Ganoderma-8-enic acid LM2; (D): Ganoderma acid beta; (E): Ganoderma acid theta; (F): Ganoderma acid V; (G): Ganoderma acid X; (H): Ganoderma lactone; (I):

[0030] Ganoderma acid I methyl ester.

[0031] Figure 2 ALI mouse morphological and histological staining chart;

[0032] Figure 2 In:

[0033] (A): W / D ratio of lung tissue of ALI mice; (B): lung tissue morphology of mice; (C): H&E staining section image of lung tissue of ALI mice; (D): Masson staining section image of lung tissue of ALI mice.

[0034] Figure 3 Spatial proteomics analysis diagram of ALI mice;

[0035] Figure 3 In:

[0036] (A): Protein quantity statistical atlas; (B): Protein quantification difference result histogram; (C): KEGG pathway enrichment analysis bubble chart; (D): Immunohistochemical staining image of lung tissue of ALI mice

[0037] Figure 4 Fecal 16S rRNA analysis diagram of ALI mice;

[0038] Figure 4 In:

[0039] (A): Venn diagram of intestinal microbial flora based on amplicon sequence variation (ASV) distribution; (B): PCoA score table; (C): Column chart of relative abundance of intestinal flora of mice at the door level; (D)-(E): LefSe analysis of different species of intestinal microbial flora of mice.

[0040] Figure 5 Serum metabolomics analysis diagram of ALI mice;

[0041] Figure 5 In:

[0042] (A): Principal component analysis (PCA) score table of blank group and LPS group; (B): PCA score table of BGLSE-L group and LPS group; (C): PCA score table of BGLSE-H group and LPS group; (D): Volcano plot of differential metabolites in mouse serum under positive ion mode; (E): Volcano plot of differential metabolites in mouse serum under negative ion mode.

[0043] Figure 6 Combined analysis diagram of intestinal flora and metabolites of ALI mice;

[0044] Figure 6 In:

[0045] (A): Heat map of combined analysis of intestinal flora and metabolites; (B): KEGG enrichment pathway analysis of serum metabolites of mice;

[0046] (C): mTOR signaling pathway atlas; (D): Western blot analysis of mTOR, P-mTOR, P62 and LC3B expression.

[0047] Figure 7 Biochemical analysis chart for COVID-19 patients;

[0048] Figure 7 In:

[0049] (A): Lymphocyte count; (B): Total T lymphocyte CD3 percentage; (C): Serum immunoglobulin (Ig) level; (D): Serum IgG level; (E): CD4+ T cell percentage; (F): Number of white blood cells; (G): Percentage of neutrophils; (H): C-reactive protein content.

[0050] Figure 8 Fecal 16S rRNA analysis chart for COVID-19 patients;

[0051] Figure 8 In:

[0052] (A): Venn diagram of intestinal microflora based on amplicon sequence variant (ASV) distribution; (B): Bar chart of relative abundance of patient intestinal flora at the door level; (C)-(D): Linear discriminant analysis (LDA) analysis of different species of patient intestinal microbiota.

[0053] Figure 9 Urine metabolomics analysis chart for COVID-19 patients;

[0054] Figure 9 In:

[0055] (A): Plot of partial least squares discriminant analysis (PLS-DA) between EG, EG-2, CG and CG-2 groups; (B): Volcano plot of differential metabolites in positive and negative ion modes for patients in experimental groups; (C): Volcano plot of differential metabolites in positive and negative ion modes for patients in control group. DETAILED DESCRIPTION

[0056] The present application will be further described below in conjunction with specific examples, but the scope of protection of the present application is not limited to this:

[0057] Example 1, preparation of broken wall ganoderma spore powder extract and chemical component analysis

[0058] Raw material selection: broken wall ganoderma spore powder, the broken wall rate of ganoderma spore powder ≥98%.

[0059] Extraction method: 2 g of broken wall Ganoderma lucidum spore powder (BGLSE) was weighed by an analytical balance and placed in a 50 mL centrifuge tube, 20 mL of ultrapure water was added, and it was dissolved for 30 min in an ultrasonic oscillator (ultrasonic frequency was 40 KHz, power was 200 W, and temperature was 4 ℃), then it was immersed in an electric heating constant temperature water bath at 80 ℃ for 3 h (stir every 30 min), and then centrifuged at 4000 rpm for 10 min to obtain the supernatant. The concentration of the Ganoderma lucidum spore powder extract (water extract) solution was 100 mg / mL.

[0060] The broken wall Ganoderma lucidum spore powder extract solution was diluted to 10 mg / mL with ultrapure water, filtered through a 0.22 μm filter, and then loaded into a liquid phase bottle for machine preparation. The ultrapure water filtered through a 0.22 μm filter was used as a blank solution.

[0061] ACQUITY UPLCTMI-Class and Xevo G3XS liquid chromatography-mass spectrometry were used for analysis, and Waters BEH T3 1.8 m (2.1 mm x 150 mm) was used as the chromatographic column for chromatographic separation. The mobile phase was composed of 0.1% formic acid (A) and acetonitrile (B) aqueous solution, and the flow rate was 0.3 mL / min. The injection volume was 3 μL, and the column temperature was 40 ℃. The gradient elution conditions were as follows. 0-5 min, 5% B; 5-18 min, 5-55% B; 18-25 min, 55-80% B; 25-31 min, 80-90% B; 31-38 min, 90-95% B; 38-42 min, 95-5% B. Mass spectrometry was detected in positive ion mode, and the mass range collected was 50-1200 Da. The parameters were set as follows: capillary voltage 3 kV, ion source temperature 100 ℃, spray temperature 280 ℃, atomizer flow rate 800 L / h, cone hole voltage 40 V, and collision energy low / high energy (6 V / 20-30 V).

[0062] The established chromatographic conditions and mass spectrometry conditions were used for analysis, and the chemical components of the broken wall Ganoderma lucidum spore powder extract were analyzed by UNIFI 1.9.2 software compared with the Waters Traditional Medicine database, and the retention time and mass spectrometry data were comprehensively considered. Nine triterpenoids, two esters, two nucleosides, five sterols, and eight organic compounds were identified in the broken wall Ganoderma lucidum spore powder extract. Among them, the triterpenoids were sanguiluton Ⅱ, acerogenin acid, ganoderma-8-enic acid LM2, ganoderic acid β, ganoderic acid θ, ganoderic acid V, ganoderic acid X, ganoderma lactone, and ganoderic acid Ⅰ methyl ester. Figure 1 )

[0063] Example 2, morphological observation of lung tissue of acute lung injury mice

[0064] A solution of Ganoderma lucidum spore powder extract having a concentration of 100 mg / mL was obtained using the method of Example 1.

[0065] Model establishment: C57BL / 6 male and female mice (purchased from Shanghai Slek Experimental Animal Co., Ltd., experimental ethics approval number IACUC-20231204-03) were selected, with a body weight range of 18-22 g and a mouse age of 6-8 weeks. After the mice were acclimated to the environment for one week, 32 mice were randomly divided into four groups according to the number of male and female mice in each group, i.e., 4 mice each, namely, a blank group (Saline), a model group (LPS), a low-dose group (BGLSE-L), and a high-dose group (BGLSE-H). The blank group mice were administered normal saline (0.2 mL), the model group mice were administered normal saline (0.2 mL), the low-dose group mice were administered 0.8 g / kg of broken wall Ganoderma lucidum spore powder extract solution, and the high-dose group mice were administered 4 g / kg of broken wall Ganoderma lucidum spore powder extract solution.

[0066] Note: Taking the low-dose group as an example, 0.8 g represents 0.8 g of Ganoderma lucidum spore powder extract.

[0067] The mice were treated with normal saline or drugs for 7 days (i.e., daily gavage according to the above conditions), and on the eighth day, the blank group was injected intraperitoneally with normal saline 0.2 mL, while the model group, the low-dose group, and the high-dose group were injected intraperitoneally with LPS (20 mg / kg, 14 mg of LPS dissolved in 7 mL of normal saline) to construct an acute lung injury model in mice. Four hours after modeling, the mice in each group were euthanized, and the organs were removed for collection of lung tissue, periorbital blood samples, and intestinal contents for research.

[0068] Lung tissue morphology observation: The lung tissue of each group of mice was removed and photographed for record. The wet weight of the lung tissue of each group of mice was recorded, and then the lung tissue was dried in an 80°C oven for 48 h. The dry weight of the lung tissue was measured three times until the weight was stable. The lung edema of each mouse was evaluated by the W / D ratio. After paraffin embedding of the mouse lung tissue sections, H&E and Masson staining were performed, as follows:

[0069] The lung tissues of the blank group, model group, low-dose broken Ganoderma lucidum spore powder group and high-dose broken Ganoderma lucidum spore powder group were taken out and fixed with 4% paraformaldehyde, embedded with paraffin to prepare tissue sections with a thickness of 4 μm, dyed with hematoxylin and eosin staining solution after deparaffination, and the morphological changes of the lung tissues were recorded under a microscope after dehydration, xylene transparency and section sealing. The lung tissues of the blank group, model group, low-dose broken Ganoderma lucidum spore powder group and high-dose broken Ganoderma lucidum spore powder group were taken out and fixed with 4% paraformaldehyde, and the paraffin sections were deparaffinated and dyed with iron hematoxylin for 5 min, washed with distilled water, dyed with acidic magenta staining solution for 5 min, soaked in 2% glacial acetic acid for 1 min, phosphomolybdate for 3 min, aniline blue for 5 min, and 0.2% glacial acetic acid for 1 min, and the morphological changes were observed under a microscope after dehydration, transparency and section sealing.

[0070] The results showed that:

[0071] As shown in Figure 2 (A), compared with the blank group, the lung W / D ratio of the model group increased, and the lung edema was effectively alleviated after treatment with BGLSE-L and BGLSE-H.

[0072] The morphology of the lung tissues of the mice in each group was recorded, as shown in Figure 2 (B), no congestion was observed in the lungs of the blank control group; compared with the blank control group, the lungs of the LPS model group showed obvious lung congestion, and the shape of the lungs was in an expanded state; compared with the model group, the lung expansion of the mice in the BGLSE-L group was slowed down, and the lung surface showed a slight congestion state; compared with the model group, the lung expansion of the mice in the BGLSE-H group was slowed down, and the lung congestion was alleviated compared with the low-concentration group. The results showed that after treatment with broken Ganoderma lucidum spore powder, the lung edema of the mice was effectively improved. As shown in Figure 2 (C), hematoxylin and eosin staining showed that compared with the blank control group, the alveolar septum of the LPS-induced mice thickened, accompanied by inflammatory cell infiltration, and the lung inflammation of the mice pretreated with BGLSE was significantly reduced, indicating that BGLSE had a protective effect on the lung injury of the LPS-induced mice. As shown in Figure 2 (D), Masson showed that compared with the blank control group, the lung tissue fibrosis of the LPS-induced mice was significantly increased, with more blue collagen fiber deposition, obvious fibrous material filling in the bronchial cavity, and severe lung fibrosis, and the lung fibrosis of the mice pretreated with BGLSE was significantly improved, indicating that BGLSE had an improvement effect on the lung tissue fibrosis of the LPS-induced mice.

[0073] Example 3, Analysis of Spatial Proteomics of Lung Tissues of Acute Lung Injury Mice

[0074] Spatial proteomics analysis: The lung tissues of mice obtained in Example 2 were collected and stored at -80 °C after quick freezing in liquid nitrogen. For frozen samples, cryosection was performed using a Leica CM1950 cryostat with a section thickness of 10 pm. The tissue was transferred from the -80 °C freezer to the cryostat pre-cooled to -20 °C and left for 1 h. The sample was fixed for sectioning, and the cut sections were transferred to Leica-specific glass slides using a pre-cooled brush. The sections were allowed to warm up using a finger temperature, promoting adhesion of the sections to the glass slides, and then transferred to a vacuum desiccator for drying for 30 min. After vacuum packaging, the target sections were stored in a -80 °C freezer. HE staining was performed on the target sections, and the stained sections were imaged using a Leica Aperio CS2. Based on the HE staining images, LCM cutting was performed using a Leica laser microdissection instrument. The LCM-cut samples were subjected to in-solution enzymatic digestion. Reducing agent was added to a final concentration of 20 mM, and the reaction was performed at 30 °C for 2 h. After cooling to room temperature, an appropriate amount of alkylation reagent was added to a final concentration of 40 mM, and the mixture was shaken at 600 rpm for 1 min. The mixture was protected from light and incubated at room temperature for 30 min. 1.5 pL of reagent TCEP was added, and the mixture was mixed well. 2.5 pL of reagent IAA was added, and the mixture was incubated for 3 min. The reaction was terminated by heating at 95 °C. Trypsin was added, and the mixture was shaken at 600 rpm at 37 °C for 2 h. After desalting and lyophilization, the samples were reconstituted with 0.1% FA. The concentration of the peptide segments was determined by OD280. Then, 0.4 pg of the peptide segments was taken out and spiked with an appropriate amount of iRT standard peptide segments for DIA mass spectrometry detection. DIA analysis was performed using a nanoscale flow rate Nanoelute system for chromatographic separation. Buffer: A liquid was 0.1% formic acid aqueous solution, and B liquid was 0.1% formic acid acetonitrile aqueous solution, and a 90 min gradient was used. The samples separated by nanoscale high-performance liquid chromatography were subjected to DIA mass spectrometry analysis using a timsTOF mass spectrometer (Bruker). Detection mode: positive ion, MS and MSMS mass spectrometry scanning range: 100-1700 m / z. MS2 used DIA data acquisition mode, and 32 TIMS Scan acquisition windows were set, with a cumulative time of 100 ms for each window. In the PASEF mode, the collision energy size was linearly changed with the ion mobility 1 / K0 (the fragmentation energy from 20 to 59 eV corresponded to the ion mobility 1 / K0 = 0.6 to 1.6 Vs / cm2). The DIA data were processed using Spectronaut software, and the database was the same as that used for database construction. The software parameters were set as follows: retention time prediction type was set to dynamic iRT, interference on MS2 level correction was enabled, cross run normalization was enabled, and all results had to pass the filter parameter Q Value cutoff of 0.01 (equivalent to FDR < 1%).

[0075] Immunohistochemical staining: The lung tissues of the blank group, model group, low-dose broken Ganoderma lucidum spore powder group and high-dose broken Ganoderma lucidum spore powder group were taken out and fixed with 4% paraformaldehyde, and then paraffin-embedded to prepare tissue sections with a thickness of 4 pm. The sections were deparaffinized and the antigen was extracted with citric acid buffer. First, endogenous peroxidase was blocked with 3% hydrogen peroxide, and then blocked with 1% BSA. The primary antibody (1:100 dilution) was incubated overnight at 4°C. The next day, HRP-labeled secondary antibody was added, and finally the chemiluminescence signal was detected using ECL substrate and imaging system.

[0076] Results show:

[0077] As shown in Figure 3 (A): Protein analysis of lung tissue samples of mice in each group showed that the saline group, LPS group and BGLSE-L combined with BGLSE-H group had 7851 overlapping proteins. As shown in Figure 3 (B): The control group and the model group had 54 up-regulated proteins and 160 down-regulated proteins; the BGLSE-L group and the model group had 108 up-regulated proteins and 36 down-regulated proteins; the BGLSE-H group and the model group had 78 up-regulated proteins and 109 down-regulated proteins. As shown in Figure 3 (C): KEGG pathway enrichment analysis of differentially expressed proteins in each group showed that the main related pathway of the differentially expressed proteins in the LPS group and the BGLSE-L group was the NF-κB signaling pathway. As shown in Figure 3 (D): Compared with the control group, the expression of TLR4, MyD88, NF-κB p65 and phosphorylated NF-κB p65 in the lung tissue of LPS-treated mice increased. However, after BGLSE intervention, the expression of TLR4, MyD88, NF-κB p65 and phosphorylated NF-κB p65 decreased. The expression levels of inflammatory factors IL-6 and TNF-a were also detected. Compared with the control group, the expression of IL-6 and TNF-a in the LPS group increased significantly. In contrast, in the BGLSE group, a dose-dependent decrease in the expression of these cytokines was observed. These results indicate that BGLSE exerts its anti-inflammatory effect by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway.

[0078] Example 4, Study on Intestinal Flora and Metabolites of Acute Lung Injury Mice

[0079] 1) Analysis of intestinal flora by 16S rRNA: The intestinal contents of mice obtained in Example 2 were collected, rapidly frozen in liquid nitrogen and stored at -80°C until analysis. The genomic DNA of the samples was extracted and the purity and concentration of the DNA were detected by Nanodrop 2000. PCR was performed on the V3-V4 variable region of the 16S rRNA gene. The PCR product was detected by 2% agarose gel electrophoresis, and the target fragment was recovered by cutting the gel. The PCR amplification recovery product was detected and quantified by a fluorescence quantitative system. The TruSeq Nano DNA LT Library Prep Kit was used for library construction. The constructed library was detected by Agilent Bioanalyzer 2100 and Promega QuantiFluor before sequencing. Using Cutadapt software, the primer sequence was cut off, and DADA2 was used for quality filtering, noise reduction, splicing and de-embedding, etc. Quality control analysis to obtain representative sequence and ASV abundance table. After selecting the representative sequence of ASV using the QIIME 2 software package, all representative sequences were aligned and annotated with the database. 16S was aligned with the Silva (version 138) database to calculate the alpha and beta diversity indices.

[0080] The results showed that:

[0081] As shown in Figure 4 (A): compared with the control group, the number of species in the model group decreased, and the number of species in the BGLSE group increased compared with the model group, indicating that BGLSE treatment restored the number of intestinal flora species in ALI mice. As shown in Figure 4 (B): PcoA analysis showed that the species composition between the control group and the LPS model group was significantly different, and BGLSE changed the structure of the intestinal flora of ALI mice. As shown in Figure 4 (C): the species composition between groups was analyzed at the door level, and the biological abundance of each species was shown in a bar chart. The intestinal flora of the four groups of mice was mainly composed of Bacteroidetes, Firmicutes, Verrucomicrobia, Proteobacteria and Actinobacteria. Compared with the control group, the relative abundance of Proteobacteria increased and the relative abundance of Actinobacteria decreased in the LPS group, while the abundance of Proteobacteria decreased and the abundance of Actinobacteria increased in the BGLSE group compared with the LPS group. Linear discriminant analysis effect size (LefSe) method (LDA>2) was used for statistical analysis to compare the differences in bacterial abundance between groups. As shown in Figure 4 (D): compared with the LPS group, the BGLSE-L group mainly showed significant expression differences in Bacteroidetes-related species. These species are mainly involved in carbohydrate degradation, short-chain fatty acid synthesis and intestinal immune cell interaction, thereby helping to maintain intestinal immune homeostasis. As shown in Figure 4(E) shows: compared with the LPS group, the BGLSE-H group mainly showed expression differences of Lactobacillaceae_NK4A136 group and Ruminococcaceae. These flora are involved in the generation of short-chain fatty acids, which can be used as energy sources and can inhibit the growth of pathogenic bacteria.

[0082] 2) Non-targeted metabolic analysis: The mouse blood samples collected in Example 2 were centrifuged at 3000 rpm for 15 min to obtain serum, which was quickly frozen in liquid nitrogen and stored at -80°C until analysis. An appropriate amount of serum was added to a pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortexed, ultrasonicated for 30 min, and placed at -20°C for 10 min. The mixture was centrifuged at 14000 x g for 20 min, and the supernatant was vacuum dried. 100 μL of acetonitrile / water solution (acetonitrile:water = 1:1, v / v) was added, and the mixture was centrifuged at 14000 x g for 15 min. The supernatant was filtered through a 0.22 μm filter and transferred to a vial for UPLC-Q-TOF-MS analysis. Chromatographic conditions: The sample was separated by an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system and a HILIC chromatographic column, the column temperature was maintained at 25°C, the flow rate was 0.5 mL / min, and the injection volume was 2 μL. The mobile phase A was a mixture of water, 25 mM ammonium acetate and 25 mM ammonia water, and the mobile phase B was acetonitrile. Elution program: 0-0.5 min, 95% B; 0.5-7 min, 95-65% B; 7-8 min, 65-40% B; 8-9 min, 40% B; 9-9.1 min, B from 40% to 95% linearly; 9.1-12 min, 95% B. Mass spectrometry conditions: AB Triple TOF 6600 mass spectrometer was used to collect the spectrum of the serum sample. After separation by an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC), the sample was analyzed by a Triple TOF 6600 mass spectrometer (AB SCIEX) for mass spectrometry analysis. The sample was detected by electrospray ionization (ESI) positive ion and negative ion modes respectively. The ESI source parameters were as follows: auxiliary heating gas 1: 60, auxiliary heating gas 2: 60, curtain gas (CUR): 30 psi, ion source temperature: 600°C, spray voltage (ISVF): ±5500V; primary mass-to-charge ratio detection range: 60-1000 Da, secondary ion mass-to-charge ratio detection range: 25-1000 Da, primary mass spectrum scan accumulation time: 0.20 s / spectra, secondary mass spectrum scan accumulation time: 0.05 s / spectra; secondary mass spectrum was obtained by data-dependent acquisition mode (IDA), and peak intensity value screening mode was used, with a de-clustering voltage (DP) of ±60V (positive and negative modes), a collision energy of 35±15 eV, an IDA dynamic exclusion isotope ion range of 4 Da, and 10 fragment spectra were collected per scan.

[0083] Results show:

[0084] As Figure 5 (A)-(C) show: PCA cluster analysis shows that the LPS group is significantly separated from the other three groups. By differential analysis of all detected metabolites, the FC>1.5 or FC<0.67 metabolites detected by T test and p value<0.05 are defined as differential metabolites. As Figure 5 (D)-(E) show: In positive and negative ion modes, the drug group and the model group are up-regulated in organic acids and their derivatives, lipids and lipid molecules.

[0085] Example 5, combined analysis of intestinal flora and metabolites of ALI mice

[0086] The intestinal flora and differential flora and metabolites obtained in Example 4 are subjected to combined analysis, the correlation coefficient between the significant differential flora and significant differential metabolites screened from the experimental samples is analyzed by spearman statistical method, and matrix heat map, hierarchical clustering, correlation network and other analyses are performed by combining R language and Cytoscape software, so as to multi-angle mine the interaction relationship between flora and metabolites.

[0087] Results show:

[0088] As Figure 6 (A) shows: compared with the LPS group, the abundance of Bifidobacterium in the BGLSE-L group is significantly increased, and its abundance is significantly positively correlated with the level of l-arginine. As Figure 6 (B) shows: KEGG pathway enrichment analysis shows that the differential metabolites are enriched in a variety of biological pathways. As Figure 6 (C) shows: the mTOR signaling pathway closely related to L-arginine metabolism regulation is one of the top 10 enriched pathways. As Figure 6 (D) shows: WB analysis of mTOR signaling pathway. Compared with the control group, the phosphorylation level of mTOR protein in the LPS group is reduced. After drug treatment, the phosphorylation level of mTOR protein is increased. LC3B and P62 are proteins related to autophagy. After drug treatment, the expression level of P62 is up-regulated, and the expression level of LC3B II is down-regulated. These findings show that Ganoderma lucidum may regulate the mTOR signaling pathway by regulating the abundance of Bifidobacterium and the level of L-arginine, thereby inhibiting excessive autophagy, reducing inflammation, and having an important impact on the treatment of ALI.

[0089] Example 6, study on immunity and inflammatory response of COVID-19 patients

[0090] The clinical trial was conducted in Hangzhou Red Cross Hospital and approved by the Ethics Committee of Hangzhou Red Cross Hospital (approval number:

[2024] research and review No. (018)). All participants obtained written informed consent.

[0091] Inclusion criteria: (1) COVID-19 infection history within one year and persistent symptoms, including respiratory, cardiovascular, nervous, reproductive system and other clinical symptoms. The most common symptoms are fatigue, muscle weakness, cough and sleep difficulty; (2) age between 18 and 80 years old, not participating in other drug clinical research within one month. Exclusion criteria: patients with severe primary diseases of cardiovascular, liver, kidney and hematopoietic system, and mental illness; pregnant or lactating women; legally defined disabled persons; unwilling to accept research measures, or for other reasons cannot cooperate.

[0092] Clinical experimental scheme: A simple random number method was used to prepare a random card, which was sealed in an envelope for standby. Patients were screened according to the inclusion and exclusion criteria, and the envelope was opened according to the random card grouping. The control group was treated according to the specific performance of the new coronavirus sequelae, and the observation group was treated with oral ganoderma spore powder once a day, 2g each time, based on the treatment of the control group, for 1 month. The patient indicators were observed before and after 30 days of treatment.

[0093] A total of 100 participants were included in the cohort and randomly received conventional treatment (control group) or ganoderma spore powder treatment (observation group) on the basis of conventional treatment. 50 participants were included in the observation group and took 100 mg / mL of ganoderma spore powder extract solution (containing 2g of ganoderma spore powder extract) obtained by Example 1 daily for 30 days, and finally 15 participants completed all processes. 50 participants were included in the control group, and finally 20 participants completed all processes. The average age of the observation group was 54.6-74 years old, and the average age of the control group was 60.5-82.1 years old, with no statistically significant difference. Participants had no serious primary diseases and mental diseases. The survival quality score of the observation group before taking was 83.63±11.4, and the survival quality score after taking was 85.88±10.71.

[0094] The results showed that:

[0095] As shown in Figure 7 (A) and (B): Compared with the control group, the number of lymphocytes in the observation group treated with BGLSE increased, and the proportion of total T lymphocytes increased. As shown in Figure 7 (C): In the observation group, the proportion of CD4+ T cells in patients after taking BGLSE increased compared with that before taking the drug, while the proportion of CD4+ T cells in the control group decreased. As shown in Figure 7(D)~(E) show that the levels of immunoglobulin M (IgM) and IgG increase after taking Ganoderma lucidum spore powder. These results show that BGLSE can activate immune cells such as lymphocytes, promote the proliferation of T and B lymphocytes, and stimulate the production of immunoglobulins, thereby enhancing the immunity of COVID-19 infected patients. Patients infected with COVID-19 suffer lung damage due to excessive inflammatory response. Leukocytes play a key role in the inflammatory process, and we detected the number and proportion of leukocytes in the blood. Figure 7 (F)~(G) show that compared with the control group, the leukocyte count and the proportion of neutrophils in the observation group significantly decreased (27.8% and 18.8%, respectively) after taking BGLSE. As shown in Figure 7 (H) shows that the C-reactive protein (CRP) levels of most patients in the observation group were above the standard before treatment, and the CRP value decreased by an average of 68.4% after treatment, while the average decrease in the control group was 50.9%. These data show that BGLSE has a significant effect on relieving the inflammatory response in COVID-19 patients.

[0096] Example 7, Study of Gut Microbiota and Metabolites in COVID-19 Patients

[0097] 1. Analysis of gut microbiota by 16S rRNA: Collect fecal samples from patients in Example 6 before and after 30 days of treatment, and store them at -80°C until analysis. The subsequent steps refer to the steps for analyzing 16S rRNA in mice in Example 3.

[0098] The results show that:

[0099] As shown in Figure 8 (A), the control group before treatment (CG group), the control group after 30 days of treatment (CG-2 group), the observation group before treatment (EG group), and the observation group after 30 days of treatment (EG-2 group) have common gut microbiota. As shown in Figure 8 (B), the species composition of each group is analyzed at the door level, and the biological abundance of each species is shown in a bar chart. Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Clostridia, and Verrucomicrobia are found in each group, with Bacteroidetes and Firmicutes having the highest abundance. As shown in Figure 8 (C)~(D), compared with the EG group, the relative abundance of Bacteroidetes in the EG-2 group increases. Compared with the EG group, the differentially expressed microbial group in the EG-2 group is the Fusobacteriaceae family.

[0100] 2. Non-targeted metabolic analysis: Collect urine samples from patients in Example 6 before and after 30 days of treatment, and store them in a -80°C freezer until analysis. The subsequent steps refer to the non-targeted metabolic analysis of mice in Example 3.

[0101] The results show that:

[0102] As shown in Figure 9 (A): The score plot of partial least squares discriminant analysis (PLS-DA) showed that there was a clear separation between the control group before treatment (CG group), the control group after 30 days of treatment (CG-2 group), the observation group before treatment (EG group) and the observation group after 30 days of treatment (EG-2 group). Metabolites with t-test and p < 0.05, FC > 1.5 or FC < 0.67 were defined as differential metabolites by differential analysis of all metabolites detected. As shown in Figure 9 (B): Compared with the EG group, the up-regulated differential metabolites in the EG-2 group were mainly organic acids and their derivatives and benzene in the positive ion mode; and the up-regulated differential metabolites were lipids, lipid molecules and benzene in the negative ion mode. As shown in Figure 9 (C): In the control group, the down-regulated differential metabolites were mainly organic acids and their derivatives and lipids and lipid molecules in the positive ion mode; and the down-regulated differential metabolites were mainly organic heterocyclic compounds and organic acids and their derivatives in the negative ion mode. This shows that BGLSE can regulate the expression of metabolites in COVID-19 patients, mainly metabolites composed of organic acids and their derivatives, lipids and lipid molecules.

[0103] Comparative Example 1, replace the "water extract of broken wall ganoderma lucidum spore powder" of the present application with "water suspension of broken wall ganoderma lucidum spore powder" at the same concentration, and detect according to the above-mentioned example, it is found that:

[0104] In the mouse model, the broken wall ganoderma lucidum spore powder is weaker than the water extract in improving the lung dry / wet ratio, reducing pulmonary interstitial edema and fibrosis, and is more moderate in promoting Bacteroidetes and inhibiting Proteobacteria in the intestinal flora, and has a lower up-regulation amplitude of organic acids and their derivatives in serum. In COVID-19 patients, the broken wall ganoderma lucidum spore powder is less effective than the water extract in promoting the number of lymphocytes, the proportion of total T lymphocytes and the proportion of CD4+ T cells, and the growth of immunoglobulin IgM and IgG is slow, the reduction of white blood cell count, neutrophil proportion and CRP level is weaker, and the regulation of intestinal flora structure and the regulation effect of characteristic metabolites in urine are significantly lower than those of the water extract. This shows that the active ingredients of the broken wall ganoderma lucidum spore powder need to be extracted by water to more efficiently play a role, and direct use of its original powder cannot achieve the same therapeutic effect of acute lung injury.

[0105] Comparative Example 2, replace "ultrapure water" in Example 1 with "80% ethanol", and keep the volume unchanged, and the rest is the same as Example 1. The obtained is named ganoderma lucidum spore powder alcohol extract solution.

[0106] The "broken wall Ganoderma lucidum spore powder alcohol extract solution" was used to replace the "broken wall Ganoderma lucidum spore powder water extract solution" obtained in Example 1, and the above-mentioned examples were detected according to the above-mentioned examples. It was found that in the acute lung injury mouse model, the improvement of lung edema (W / D ratio) and lung fibrosis by alcohol extract was weaker than that by water extract, the up-regulation of actinobacteria and down-regulation of proteobacteria in intestinal flora was not significant, and the regulation effect of lipids and lipid molecules in serum was more moderate than that of water extract; in COVID-19 patients, its activation effect on lymphocytes, total T lymphocytes and CD4+ T cells was not as good as that of water extract, the growth amplitude of IgM and IgG was lower, the inhibition effect on white blood cells, neutrophils and CRP was weaker, and the regulation effect on Bacteroidetes in intestinal flora and organic acid derivatives in urine was inferior to that of water extract. This shows that the alcohol extraction process cannot efficiently retain or extract the key components for treating acute lung injury, and its activity is much lower than that of water extract.

[0107] Finally, it should also be noted that the above-mentioned is only a few specific embodiments of the present application. Obviously, the present application is not limited to the above-mentioned examples, and many variations can also be made. All variations that can be directly derived or inferred by those skilled in the art from the content disclosed in the present application should be considered as the protection scope of the present application.

Claims

1. A Ganoderma lucidum spore powder extract for regulating intestinal flora and treating acute lung injury, characterized in that... The preparation method includes the following steps: Select broken-cell wall Ganoderma lucidum spore powder as raw material, add ultrapure water to the broken-cell wall Ganoderma lucidum spore powder according to the material-liquid ratio of 1g / 9~11mL, first dissolve by ultrasonic vibration, then extract at constant temperature, and then centrifuge to take the supernatant as Ganoderma lucidum spore powder extract solution.

2. The Ganoderma lucidum spore powder extract according to claim 1, characterized in that: The constant temperature extraction was carried out at 80±0.5 ℃ for 3±0.1 h.

3. The Ganoderma lucidum spore powder extract according to claim 2, characterized in that: Ultrasonic dissolution was performed under the following conditions: ultrasonic frequency of 40 kHz, power of 200 W, and temperature of 4±0.5 ℃ for 30±1 min.

4. The Ganoderma lucidum spore powder extract according to claim 3, characterized in that: Centrifugation was performed at 4000±200 rpm for 10±0.5 min.

5. The use of the Ganoderma lucidum spore powder extract according to any one of claims 1 to 4 in the preparation of a drug for regulating intestinal flora to treat acute lung injury.

6. The application according to claim 5, characterized in that: It reduces lung inflammation, pulmonary edema, and pulmonary fibrosis in a mouse model of acute lung injury, thus protecting the lungs.

7. The application according to claim 5, characterized in that: It exerts its anti-inflammatory effect by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway.

8. The application according to claim 5, characterized in that: It increases the levels of immune cells and immunoglobulins in COVID-19 patients, and reduces the levels of inflammatory cells and C-reactive protein.

9. The application according to claim 5, characterized in that: This causes changes in the gut microbiota and metabolites.

10. The application according to claim 5, characterized in that: Joint analysis of gut microbiota and metabolites revealed that Bifidobacteria are significantly positively correlated with L-arginine; L-arginine regulates the mTOR signaling pathway; and it reduces inflammatory responses by inhibiting excessive autophagy.

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