CN117838741A

CN117838741A - Application of ganoderma lucidum alcohol extract in treating or relieving silicosis

Info

Publication number: CN117838741A
Application number: CN202311793597.5A
Authority: CN
Inventors: 谢林伸; 钱蕊; 周丁子; 姚于勤; 张本; 张凌; 张勤; 彭丽君; 杜文
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-04-09

Abstract

The invention discloses an application of an ganoderma lucidum alcohol extract in treating or relieving silicosis, belonging to the field of medicines. The invention is based on ultra-high liquid chromatography-quadrupole electrostatic field orbit trap mass spectrometry (UPLC-QE-MS) technology, network pharmacology, molecular docking and in vivo experiments, and explores the active ingredients and mechanism of the ganoderma lucidum alcohol extract in treating silicosis inflammation and fibrosis. The results show that the white ganoderma lucidum alcohol extract can effectively relieve productive dust-induced silicosis inflammatory reaction and pulmonary fibrosis, can obviously reduce inflammatory factors generated at a silicosis focus, inhibit TGF-beta/Smad pathway activation, reduce collagen deposition, block inflammatory reaction and fibrosis progress, and is an effective alternative scheme for treating or relieving productive dust-induced silicosis inflammatory reaction and pulmonary fibrosis by utilizing the modern traditional Chinese medicine technology.

Description

Application of ganoderma lucidum alcohol extract in treating or relieving silicosis

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of an ganoderma lucidum alcohol extract in treating or relieving silicosis.

Background

As one of the most widespread and oldest occupations worldwide, silicosis is most widespread in the industries of ore mining, metal processing, stone construction and the like, and the most significant cause of disease is the deposition of nondegradable free silica dust particles in the lungs. As the disease progresses, severe inflammatory reactions and fibrosis occur in the lungs of the afflicted population, which can be progressive and fatal, with irreversible damage to lung function. The pathogenesis of silicosis is not clear, and no effective treatment means exists at present, so that the disease deterioration can be relieved and the life quality can be improved only through various comprehensive management strategies such as dust contact prevention, alveolar lavage, inflammation progress inhibition, pulmonary fibrosis alleviation and the like.

The diagnosis of silicosis firstly carries out professional medical history investigation of contact with free silicon dioxide particles, further completes analysis and examination of chest films before and after high kilovolt X-ray, pulmonary function and arterial blood gas, and combines clinical manifestation and X-ray chest film for diagnosis. In the clinical treatment of Western medicine, silicosis has no cure method, and only aims at stage conditions, and the medicines are applied to the diseases. Lung nodules, which are often confused with silicosis, are solid or sub-solid shadows of the lung, which are represented by imaging as circles or quasi-circles with diameters less than or equal to 3cm and have increased density, and belong to a multisystem non-cheesed granulomatous disease with unknown etiology, which often invade the lung portals, mediastinal lymph nodes and lung tissues, and are a completely different diagnosis and treatment principle from silicosis.

Modern traditional Chinese medicine has a certain curative effect and advantages in silicosis treatment and effective delay of patient disease progress, and at present, research has shown that traditional Chinese medicine extracts are proved to be clinically effective, such as tetrandrine. Based on the dialectical theory of traditional Chinese medicine, the dialectical treatment of silicosis comprises 6 types of symptoms such as non-dispersing lung qi, lung-accumulated phlegm-heat, yin deficiency and lung heat, chest yang blockage, lung-spleen phlegm dampness, lung-kidney deficiency and the like, the patients are mainly concentrated on men, and the patients are regulated by activating blood circulation to remove blood stasis, dispersing lung qi and regulating qi so as to achieve the aim of improving the life quality of the patients, but the cure is difficult to achieve; the lung nodule is caused by qi and blood dampness and phlegm stagnation in the lung, and is mostly caused by qi deficiency and mass groups, wherein women are more, the treatment is carried out by taking strengthening body resistance to eliminate pathogenic factors as the core, and a doctor can regulate the lung from qi, blood and water to treat the nodule, so that the aim of complete cure is fulfilled.

The existing method for treating silicosis has the problems of poor treatment effect, high cost, large side effect of medicines and the like. And most silicosis patients belong to middle and low income groups, and high medical cost is difficult to support, so that the search for a high-efficiency and low-cost silicosis treatment scheme is urgent.

Although the white ganoderma lucidum and the alcohol extract thereof have the functions of regulating immune function, resisting aging and slowing down collagen deposition, no report on the alleviation or treatment of diseases such as lung injury or lung fibrosis caused by productive dust has been seen.

Disclosure of Invention

The technical problem solved by the invention is to provide the medical application of the ganoderma lucidum alcohol extract, and an effective alternative scheme is provided for treating or relieving silicosis, especially silicosis induced by production dust.

The technical scheme for solving the technical problems is as follows: provides the application of the ganoderma lucidum alcohol extract in preparing medicaments for treating or relieving silicosis.

In particular, the invention provides application of ganoderma lucidum alcohol extract in preparing medicines for treating or relieving silicosis injury or silicosis fibrosis of silicosis.

The silicosis injury or silicosis fibrosis is induced by productive dust.

Furthermore, the fibrosis process of silicosis is an early stage fibrosis process.

Optionally, the productive dust is silicon dust. Silicon dust is an inorganic dust with a crystalline free silica content of more than 10%.

The silicosis injury is silicosis inflammatory reaction.

The silicosis inflammatory response is an inflammatory response caused by an elevated level of inflammatory factors IL-6 and/or TGF-beta in the lung tissue.

The ganoderma lucidum alcohol extract is used for inhibiting abnormal expression of TGF-beta/Smad pathway related proteins in lung tissues.

The components in the white meat ganoderma lucidum alcohol extract comprise glycyrrhetinic acid, ganoderic acid DM, alisol C, ganoderic acid beta and red light sapogenin.

When the ganoderma lucidum alcohol extract is used for treating or relieving silicosis, pharmaceutically acceptable auxiliary materials or auxiliary components are added into the ganoderma lucidum alcohol extract to prepare an oral preparation.

The oral preparation comprises a solid preparation, a liquid preparation or a suspension preparation.

Further, the solid preparation comprises capsules, tablets, pills, powder or granules.

Further, the liquid preparation comprises emulsion, solution, suspension, syrup or tincture.

The invention provides a method for preparing an alcohol extract of ganoderma lucidum, which comprises the following steps:

grinding the dried white ganoderma lucidum into powder by a grinder, placing the powder in a Soxhlet extractor, and extracting by using ethanol solution under heating; and (5) drying the extracting solution, and freezing, storing and preserving for later use.

The heating temperature is recommended to be 60-80 ℃.

Ethanol solution is recommended to use ethanol with volume fraction of 60% -90%.

In the Soxhlet extractor, the feed liquid ratio comprises, but is not limited to, 0.5 to 5:50, the material is white ganoderma lucidum powder, and the liquid is ethanol solution.

The extraction time is at least 1h, and the proper extension of the extraction time is helpful for extracting more effective components.

Freezing may be employed including, but not limited to, temperatures below-80 ℃.

Under the support of network pharmacology and molecular docking technology, the key targets for finding the white ganoderma lucidum alcohol extract to relieve silicosis inflammation and fibrosis are 66 core targets including IL-6 and TGF-beta, and the white ganoderma lucidum alcohol extract is found to play a role by regulating and controlling interleukin signals, TGF-beta channels and other signal channels through KEGG enrichment channel analysis, and active ingredients such as glycyrrhetinic acid, ganoderic acid DM, alisol C, ganoderic acid beta, red light sapogenin and the like in the white ganoderma lucidum alcohol extract can be well combined with targets on disease signal channels through molecular docking. This is consistent with our in vivo experiments, verifying the effect of white ganoderma lucidum alcohol extract on antagonizing silicosis inflammatory reaction and fibrosis.

Compared with the prior art, the invention has at least the following beneficial effects:

the invention provides application of ganoderma lucidum alcohol extract in preparing a medicament for treating or relieving inflammatory reaction and fibrosis progress of silicosis. The invention discovers that the ganoderma lucidum alcohol extract can effectively relieve lung injury or diseases induced by productive dust, such as pneumonitis reaction and pulmonary fibrosis, inhibit immune reaction caused by silicon dioxide, block epithelial mesenchymal transition, and relieve the pulmonary fibrosis degree of a silicosis focus, has good application prospect in the aspects of relieving and recovering silicosis caused by the productive dust clinically, and provides a new effective choice for the treatment of silicosis.

Drawings

FIG. 1 is a schematic diagram of the network of the active ingredient-target-disease of the white ganoderma lucidum alcohol extract and a target Wen diagram.

FIG. 2 is a graph of PPI network interactions of the white ganoderma lucidum alcohol extract-silicosis common target.

FIG. 3 is a graph showing the GO function enrichment of white ganoderma lucidum alcohol extract against silicosis core targets.

FIG. 4 is a KEGG pathway enrichment analysis of white ganoderma lucidum alcohol extract against a core target of silicosis (front 15).

FIG. 5 is a graph of the docking binding energy of a key active ingredient to a core target.

FIG. 6 is a graph of a pattern of docking of key active ingredients to a core target.

FIG. 7 is a graph showing the weight gain of mice 14 days after the white ganoderma lucidum alcohol extract was used to intervene in silicosis model mice.

FIG. 8 is a graph of HE staining of lung tissue of mice (20X) 14 days after intervention of Ganoderma lucidum alcohol extract in silicosis model mice.

FIG. 9 is a map of the Masson staining of lung tissue of mice (20X) 14 days after intervention of white ganoderma lucidum alcohol extract in silicosis model mice.

FIG. 10 is a graph showing the weight gain of mice 35 days after the white ganoderma lucidum alcohol extract was used to intervene in silicosis model mice.

FIG. 11 is a graph of HE staining of lung tissue of mice (20X) 35 days after intervention of Ganoderma lucidum alcohol extract in silicosis model mice.

FIG. 12 is a map of the Masson staining of lung tissue of mice 35 days after intervention of white ganoderma lucidum alcohol extract in silicosis model mice (20X).

FIG. 13 is a graph showing the expression levels of N-cadherin, E-cadherin, vimentin, alpha-SMA, collagen I, collagen III proteins in lung tissue of mice 35 days after intervention of an alcoholic extract of Ganoderma lucidum in silicosis model mice.

FIG. 14 is a bar graph showing IL-6 inflammatory factor content in lung tissue of mice 14 days and 35 days after intervention of white ganoderma lucidum alcohol extract in silicosis model mice.

FIG. 15 is a bar graph of TGF-beta inflammatory factor content in lung tissue of mice 14 days and 35 days after intervention of white ganoderma lucidum alcohol extract in silicosis model mice.

FIG. 16 is a schematic representation of the mechanism of action of an alcoholic extract of Ganoderma lucidum in blocking the TGF-beta/Smad pathway and thus improving silicosis fibrosis.

Detailed Description

As used herein, "treatment" refers to therapeutic or prophylactic measures that can be achieved to alleviate or mitigate a disease, control or prevent an undesired physiological condition, disorder or disease, or to achieve a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; a reduction in the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; delay or slowing of the occurrence of a condition, disorder, or disease progression; improvement or alleviation (whether partial or complete) of a condition, disorder, or disease state, whether detectable or undetectable; or an improvement or amelioration of a condition, disorder, or disease. Treatment involves eliciting a clinically significant response without undue levels of side effects.

In the present invention, the "inflammatory reaction" refers to a reaction that induces immune cell aggregation and inflammatory factor release in the lung after inhalation and deposition from exogenous free silica particles. Early silica deposition in silicosis is recognized and engulfed by macrophages, but due to undigested macrophages autophagy or apoptosis, and release a large amount of inflammatory factors, attract other immune cells to aggregate, and induce inflammation. The inflammation in lung nodules is mostly caused by immunological disorder, which is different from silicosis.

Through a great deal of creative work, the invention provides the application of the ganoderma lucidum alcohol extract in preparing the medicines for treating or relieving silicosis. In the preparation of the medicine for treating or relieving silicosis, the weight ratio of the white ganoderma lucidum alcohol extract preparation material liquid medicine (white ganoderma lucidum powder and ethanol solution) is 1:50, the administration route is mainly oral.

When the white ganoderma lucidum alcohol extract is used for preparing the medicine for treating or relieving silicosis, the raw materials of the components with the weight proportion range or the optimal value of the white ganoderma lucidum alcohol extract can be taken, the white ganoderma lucidum alcohol extract is prepared into oral decoction according to a traditional Chinese medicine conventional preparation method, and pharmaceutically acceptable auxiliary materials or auxiliary components can be added to prepare other common preparations in the field, such as oral preparations including solid preparations, liquid preparations or suspension preparations; the solid preparation comprises capsules, tablets, pills, powder or granules; liquid formulations include emulsions, solutions, suspensions, syrups or tinctures.

The pharmaceutically acceptable auxiliary component has certain physiological activity, but the addition of the component does not change the dominant position of the pharmaceutical composition in the disease treatment process, but only plays auxiliary effects, and the auxiliary effects are only the utilization of the known activity of the component and are auxiliary treatment modes which are conventional in the medical field. If the auxiliary components are used together with the pharmaceutical composition of the present invention, the auxiliary components still fall within the scope of the present invention.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 detection of the alcohol extract component of Ganoderma lucidum

The ganoderma lucidum alcohol extract adopted by the invention is rich in active substances such as triterpenic acid, flavonoid, alkaloid and the like, and active ingredients of the ganoderma lucidum alcohol extract are predicted by ultra-high liquid chromatography-quadrupole electrostatic field orbit trap mass spectrometry (ultra performance liquid chromatography q exactive mass spectrometry, UPLC-QE-MS) detection and SWISS ADME website screening.

UPLC-QE-MS method for detecting chromatographic conditions of components of the white ganoderma lucidum alcohol extract: ACQUITY UPLC HSS T3 chromatography column (100 mm. Times.2.1 mm,1.8 um); column temperature: 45 ℃; mobile phase 0.1% (v/v) formic acid aqueous solution-acetonitrile (B); flow rate: 0.35ml/min; sample injection volume: 5 μl. Photodiode array scan range: 210-400 nm; gradient elution procedure: 0-2 min,5% B;4min,30% B;8min,50% B;10min,80% b;14-15min,100% B;15.1-16min,5% B. Mass spectrometry conditions: the ion source is a heating electrospray ionization source, and a positive and negative ion scanning mode is adopted for mass spectrum signal acquisition; positive ion spray voltage 3.8kV and negative ion spray voltage-3 kV; the ion source temperature is 320 ℃; sheath air flow rate 35Arb, auxiliary air flow rate 8Arb; detecting the ion range to 100-1200m/z; full scan resolution 70000; the second order fragmentation energy grading was 10eV, 20eV, 40eV in order.

The raw data obtained by the above method were subjected to baseline filtering, peak identification, integration, retention time correction, peak alignment and normalization by the processing software Progenesis QI v3.0 software. Identification of compounds is based on exact mass numbers, secondary fragmentation and isotope distribution, secondary fragmentation qualitative scores matched to database theoretical fragmentation, 100 full scores, and generally preference is given to > 80 score substances. The Swiss ADME database (http:// www.swissadme.ch /) database was used to collect small molecule ADME parameters and drug class data. The drug-like property is a common attribute of the existing clinical drugs, and the drug development can be accelerated by utilizing the drug-like property 5 principle, so that the drug success rate is higher, and the drug-like property is a screening index for preliminary analysis of the drug activity. The active pharmaceutical ingredients of ganoderma lucidum alcohol extract are screened according to the principle of drug-like property 5 (1. Molecular weight is less than 500;2. Number of hydrogen bond donors is less than 5;3. Number of hydrogen bond acceptors is less than 10;4. Lipid water distribution coefficient is less than 5;5. Number of rotatable bonds is not more than 10). The specific results of the screening are shown in Table 1.

TABLE 1 active ingredients of white glossy ganoderma alcohol extract

As can be seen from Table 1, the active ingredients of the white meat ganoderma lucidum alcohol extract comprise 36 kinds of glycyrrhetinic acid, ganoderic acid DM, alisol C, ganoderic acid beta, red light sapogenin and the like.

Example 2 validation of the mechanism of action of Ganoderma lucidum alcohol extract by means of network pharmacology and molecular docking

1. 36 drug-like components satisfying those shown in Table 1 were screened at the SWISS ADME site (http:// www.swissadme.ch /), and SWISS expression was obtained in the Pubchem database (https:// Pubchem. Ncbi. Lm. Nih. Gov). Target spots of active ingredients of the ganoderma lucidum alcohol extract are collected, 868 potential target spots of the active ingredients of the ganoderma lucidum alcohol extract are collected in a traditional Chinese medicine system pharmacology database and analysis platform (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, TCMSP, https:// old.tcmsp-e.com/tcmsp.php), SEA (Similarity ensemble approach) database (http:// sea. Bkslab. Org /), swiss Target Prediction database (http:// sea. Bkslab. Org /) and other databases, and target spot proteins are uniformly adjusted to gene names by means of a Uniprot website database (https:// www.uniprot.org /), so that the ganoderma lucidum alcohol extract drug target spot database is prepared, as shown in figure 1.

To ensure gene integrity as much as possible, the study collected silico disease target genes and excluded duplicate entries by integrating 3 disease gene databases, namely an online human Mendelian genetic database (Online Mendelian Inheritance in Man, OMIM, https:// www.omim.org /), a comprehensive database of human genes (the Human Genes Compendium, geneCards, https:// www.genecards.org /), and a DisGeNET database (https:// www.disgenet.org/home /), to obtain the silico disease targets 313. The potential target of the white ganoderma lucidum alcohol extract and the disease target genes of silicosis are compared and analyzed through an on-line tool of Venny 2.1.0 (https:// bioinonfogp. Cnb. Cis/tools/Venny /), a Wen graph (shown in figure 1) is drawn, and the intersection of the two is the potential target of the white ganoderma lucidum alcohol extract for interfering silicosis inflammation, and 67 intersection genes are obtained.

Inputting the confirmed targets into a STRING database (https:// cn. STRING-db. Org /) to construct a protein-protein interaction (protein-protein interaction, PPI) network between the targets, downloading a tav format file and importing a Cytoscape 3.9.1 for visual analysis, and performing a mutual mapping on the PPI network of the common target of the ganoderma leucovorin alcohol extract-silico. PPI network interaction is carried out on 67 intersection targets, colors and sizes of the targets are marked according to node Degree sequencing, target genes of the first 10 are TGFB1, IL6, TP53, BCL2, CASP3, IL1B, EGFR, JUN, MMP and PTGS2 respectively, the specific interaction relation is shown in FIG. 2, wherein the colors and the sizes of the nodes represent the sizes of node Degree values Degree, and the deeper the colors, the larger the node is.

The 67 intersection target genes are led into a DAVID database (https:// DAVID. Ncifcrf. Gov/, version 8.0) to carry out GO enrichment analysis and KEGG enrichment analysis, the results shown in the figures 3 and 4 are respectively obtained, the ordinate of the figure 4 shows the channel with the enrichment degree ranking at the front from top to bottom, the abscissa shows the proportion of the gene list enriched to the target channel factor accounting for the total genes contained in the gene list, and the larger the point shows the more target points enriched to the channel. GO enrichment analysis includes target gene annotation of 3 aspects of cellular components (cellular component, CC), molecular functions (molecular function, MF) and biological processes (biological process, BP), KEGG enrichment analysis is an annotation of signal pathways in which targets are involved. In GO enrichment analysis, BP results show that main biological processes comprise positive and negative feedback involved in an apoptosis process, positive regulation and control effects of proliferation and differentiation of smooth muscle/vascular smooth muscle cells and the like; CC results indicate that the cellular components involved mainly include cytoplasm, extracellular space, caspase complex, etc.; MF results indicate that the molecular function is mainly reflected in protein bond binding, enzyme activation, polypeptide enzyme activation, etc. (fig. 3). The results of the bubble figure enrichment analysis of the KEGG pathway revealed that the intersection gene was concentrated in inflammatory-related pathways such as interleukin signaling, cytokine signaling of immune cells, TNF signaling pathway, and the like, and was highly expressed in fibrosis-related pathways such as TGF- β signaling pathway, pulmonary fibrosis, and the like (fig. 4).

2. Molecular docking of active ingredients of ganoderma lucidum alcohol extract with potential targets for improving silicosis inflammation is used for evaluating the binding energy and binding sites of the active ingredients in ganoderma lucidum alcohol extract and potential intervention targets, and the molecular docking is simulated by using open source software Autodock Vina1.2.0 (molecular pattern laboratory of Stokes institute). Firstly, the crystal structure of TGF-beta 1 protein is obtained in an RCSB PDB database (https:// www.rcsb.org /), 3D structure files of active components in the table 1 are downloaded in a Pubchem website (https:// Pubchem. Ncbi. Lm. Nih. Gov /), SDF files are downloaded, and then the files are converted into PDB files through Open Babel 3.1.1 software. The prepared ligand (Table 1 active ingredient) and receptor (TGF-. Beta.1 protein) files were subjected to dehydration, hydrogenation and the like using PYMOL software, and stored as PDBQT format files. In the Grid Box mode setting receptor and ligand docking Box of AutoDock Tools software, molecular docking of small molecules and key proteins was performed using AutoDock Vina1.2.0, repeated 9 times, the conformation with the lowest binding energy was taken, and the results were visualized in PYMOL software. In general, a molecular binding energy of-5.0 kcal/mol is set as a threshold, and a smaller binding energy indicates a ligand and a receptorThe tighter the bulk binding, the darker the color, the lower the binding energy, and the shallower the color, the higher the binding energy, less than 0, indicating that the binding activity between molecules is less than-5.0 kcal. Mol, as shown in FIGS. 5 and 6 ^-1 Indicating that the molecules have stronger binding activity and the lower the energy is, the stronger the binding capacity is. From fig. 5, it is clear that 36 active ingredients may interfere with TGF- β signaling pathway, and that glycyrrhetinic acid, ganoderic acid DM, alisol C, ganoderic acid β, red sapogenin may be the most important ingredients for interfering with TGF- β signaling pathway.

Example 3 establishment of mouse lung injury model and in vivo animal experiment with Alternaria Ganoderma alcohol extract intervention

1. Silica-induced pulmonary disease modeling

1) Animal species

C57BL/6J, male mice, 8 weeks old, 23-25g.

2) Reagent(s)

Silica (brand: sigma), 200 ℃,2h dry heat sterilization, dissolved in sterile physiological saline for standby, and mixed into suspension by high-speed vortex before use.

White ganoderma lucidum alcohol extract: grinding the white ganoderma lucidum dry product into powder by a grinder, wherein the powder is 1: placing the feed liquid ratio of 50 in a Soxhlet extractor, and extracting with 80% ethanol at 70deg.C for 2 hr; oven drying the extractive solution, storing at-80deg.C, and performing ultrasonic reconstitution with purified water.

3) Method of

And injecting pentobarbital sodium (50-55 mg/kg) into the abdominal cavity according to the body weight to anesthetize the mice, gently pinching toe tips to observe whether obvious reflex reaction exists or not so as to judge whether the anesthesia depth is reached, and carrying out subsequent experiments.

Mice reaching the anesthesia depth were fixed on the console, and the incisors were fixed with surgical wires to fix the heads.

The tongue of the mouse is gently pulled out by forceps, the neck of the mouse is irradiated by a mercury lamp light source, and the mouth of the air tube is found by naked eye observation, so that the point which is suddenly and suddenly breathed by the mouse is found, namely the mouth of the air tube.

The hose of the indwelling needle is inserted into the trachea of a mouse, the mouse is lifted, 80 mu L of evenly mixed silicon dioxide suspension (50 mg/mL) is rapidly injected, the indwelling needle is pulled out, the forceps gently pull the tongue of the mouse, choking cough and choking are avoided, after the mouse breathes steadily, the mouse is put back into the cage, vital signs of the mouse are observed, and the mouse is recovered until the mouse wakes up.

2. Research method for alleviating silicon dioxide-induced lung diseases by ganoderma lucidum alcohol extract

1) Grouping and intervention

The control group (control group) and the white ganoderma lucidum alcohol extract administration control group (GLE-C group) were each air-instilled with physiological saline, the model group (model group) and the white ganoderma lucidum alcohol extract administration group (GLE group) were each air-instilled with a silica suspension, and the groups were each 8 animals after the molding was completed. The next day of gastric lavage, GLE group and GLE-C group were each filled with white ganoderma lucidum alcohol extract (200 mg/kg), and control group and model group were each filled with equal volumes of physiological saline.

2) Intervention method

Oral gastric lavage, dissolving white ganoderma lucidum alcohol extract with physiological saline to prepare working solution of 25mg/mL, and determining gastric lavage working solution according to body weight in GLE group and GLE-C group during intervention; the control group and the model group were perfused with an equal volume of physiological saline. Mice were sacrificed 14 days and 35 days after continuous dosing, respectively.

3) Evaluation index

The index comprises the weight of the mice, biochemical index of the blood of the mice, HE staining and scoring of lung tissues, masson staining and scoring of the lung tissues, inflammatory factors IL-6 of the lung tissues, TGF-beta content, N-cadherin of the lung tissues, E-cadherin, vimentin, alpha-SMA, collagen I and Collagen III protein expression quantity. The experimental results are shown in FIGS. 7-15, and tables 2-3, respectively.

3. In vivo animal experimental result of white ganoderma lucidum alcohol extract intervention

1) Weight change

Under normal physiological conditions, the weight of animals is increased until stable, and the weight is easy to change when the body is subjected to acute injury or gastrointestinal tract stimulation. As shown in fig. 7, after 14 days of silica induction, the weight of each group of mice showed a stable growth trend, which indicates that acute damage is not caused to the mice in the molding process, and stomach irritation is not caused to the mice by the stomach irrigation of the white ganoderma lucidum alcohol extract; as shown in fig. 10, after 35 days of silica induction, the body weight of each group of mice showed a stable growth trend and reached a stable state at a later stage, indicating that the long-term gastric lavage of the ganoderma lucidum alcohol extract did not produce significant toxic and side effects on the mice.

2) Biochemical index of blood

The blood biochemical index is a commonly used index for detecting animal metabolism, and can reflect the normal level of liver function and kidney function of animals. As shown in the results of table 2 and table 3, each group of indexes is at normal level, which indicates that the long-term intake of the ganoderma lucidum alcohol extract does not negatively affect the metabolic capability of the mice.

Table 2: biochemical index of blood of animals in 2 weeks group

Wherein: data represent mean ± standard deviation, 5 animals per group

ND represents no measurement; in the group, "2W means 2 weeks";

abbreviations: ALP2L represents alkaline phosphatase; ALTL represents alanine aminotransferase; ASTL stands for aspartate aminotransferase; HDLC4 represents high density lipoprotein; LDLC4 represents low density lipoproteins; TP2 represents total protein; UA2 represents uric acid; UREAL represents urea.

Table 3: biochemical index of 5-week group animal blood

Data represent mean ± standard deviation, 5 animals per group.

In the group, "5W means 5 weeks";

All values are expressed as mean ± SEM.

3) Lung tissue HE staining and scoring

Silicosis is a progressive disease and can be classified into an inflammatory phase, an inflammatory and fibrotic coexisting phase, and a fibrotic phase. The animal model adopted by the invention is a silicosis model of 2 weeks (14 days) and 5 weeks (35 days), and is in an early stage of silicosis, wherein 2 weeks are in an inflammatory stage, and 5 weeks are in an inflammation and fibrosis coexisting stage.

As shown in fig. 8 and 11, HE staining results showed that:

(a) The lung tissue structure of the control group and the white ganoderma lucidum administration control group (GLE-C) is complete, the alveolar wall is a monolayer cell, no obvious exudation, inflammatory cell infiltration and fibroblast proliferation exist in the alveolar cavity, and the lung tissue structure is a normal lung tissue structure.

(b) After induction with silica, the mouse alveolar structure was destroyed, the alveolar space was widened, massive lymphocyte infiltration was seen, and aggregation of macrophages, mast cells, fibroblasts occurred, accompanied by tissue vascular proliferation, evident alveolar lesions, alveolar wall thickening, fibroblast proliferation, and diffuse fibrosis of the lung tissue were seen in the lung tissue at day 35. The above manifestations suggest substantial lung injury and fibrosis formation.

(c) Compared with a model group, the interference of the white ganoderma lucidum alcohol extract (GLE group) can obviously improve the damage of the lung tissue structure, maintain the integrity of alveoli, relieve inflammatory infiltration and relieve the lung injury induced by silicon dioxide, and especially the lung tissue performance of the GLE group and a control group is close, so that the improvement of the white ganoderma lucidum alcohol extract on the lung tissue structure damage is obvious.

4) Lung tissue Masson staining and scoring

As shown in fig. 9 and 12, masson staining results showed that:

(a) After instillation through the silicon dioxide trachea, obvious collagen deposition exists in the lung clearance of the five-week silicosis model, dense fibrous scar balls appear in the existing partial areas, and immune cell aggregation is carried out on the periphery.

(b) The GLE group significantly inhibited this induction of silica compared to the model group, and the GLE group showed similar lung tissue performance as the control group.

5) Lung tissue inflammatory factor IL-6, TGF-beta content, lung tissue N-cadherin, E-cadherin, vimentin, alpha-SMA, collagen I, collagen III protein expression level

Under the stimulation of free silicon dioxide, immune cells can release pro-inflammatory and pro-fibrotic factors TGF-beta and IL-6, induce TGF-beta/Smad channel activation and various inflammatory reactions, promote collagen expression of fibroblasts and epithelial cells, and deepen the fibrosis progress of silicosis focus. As shown in figures 14-15, under the intervention of the white ganoderma lucidum alcohol extract, the TGF-beta and IL-6 inflammatory factor contents of lung tissues are reduced, and the fibrosis progress of silicosis is slowed down. Silicon dioxide induces lung injury, inflammatory reaction can occur at early stage of injury, and fibrosis reaction occurs, which is often accompanied by pathological phenomena such as TGF-beta/Smad pathway activation, collagen deposition and the like. As shown in FIG. 13, after 5 weeks of silica induction, obvious Collagen deposition phenomenon appears in lung tissues, and the expression level of Collagen I and Collagen III proteins is obviously increased; abnormal expression of the marker protein TGF-beta 1, the phosphorylated Smad2 and Smad3 proteins on the TGF-beta/Smad pathway also occurs. The interference of the white ganoderma lucidum alcohol extract can effectively reduce the expression quantity of type I (Collagen I) and type III (Collagen III) Collagen in lung tissues caused by silicon dioxide and correct the abnormal expression of TGF-beta/Smad pathway marker protein, which suggests that the white ganoderma lucidum alcohol extract can relieve the fibrosis reaction induced by the silicon dioxide.

The experimental results show that the ganoderma lucidum alcohol extract can inhibit the release of inflammatory factors, alleviate inflammatory reaction of lung tissues, slow down collagen deposition, relieve the process of silicosis fibrosis, and improve silica-induced pneumonia injury and pulmonary fibrosis, and a specific mechanism path diagram can be referred to as figure 16. The ganoderma lucidum alcohol extract is effective for inflammatory reaction (week 2 and week 5) and early fibrosis (week 5) in the progression of silicosis model. The ganoderma lucidum alcohol extract can block the further evolution to severe and irreversible fibrosis, and avoid the further worsening of the symptoms of patients suffering from silicosis.

Although the invention has been described herein with reference to illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the scope and spirit of the principles of this disclosure. More specifically, various variations and modifications may be made to the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure. In addition to variations and modifications in the component parts and/or arrangements, other uses will be apparent to those skilled in the art.

Claims

1. Application of Ganoderma lucidum alcohol extract in preparing medicament for treating or relieving silicosis is provided.

2. The use according to claim 1, wherein said white ganoderma lucidum alcohol extract is used for preparing a medicament for treating or relieving silicosis injury or silicosis fibrosis of silicosis.

3. The use according to claim 1 or 2, wherein the silicosis injury or silicosis fibrosis is induced by production dust.

4. The method according to claim 3, wherein the production dust is silicon dust.

5. The use according to claim 3, wherein the silicosis injury is a silicosis inflammatory reaction.

6. The use according to claim 5, wherein the silicosis inflammatory response is an inflammatory response caused by an increased content of inflammatory factors IL-6 and/or TGF- β in the lung tissue.

7. The use according to claim 3, wherein the white ganoderma lucidum alcohol extract is used for inhibiting abnormal expression of TGF- β/Smad pathway-related proteins in lung tissue.

8. The use according to any one of claims 1-2, 4-7, wherein the ingredients of the white ganoderma lucidum alcohol extract comprise glycyrrhetinic acid, ganoderic acid DM, alisol C, ganoderic acid β, red sapogenin.

9. The use according to any one of claims 1-2, 4-7, wherein the white ganoderma lucidum alcohol extract is obtained by extracting white ganoderma lucidum powder with an ethanol solution as a solvent under heating.

10. The use according to any one of claims 1-2, 4-7, wherein the medicament comprises an alcoholic extract of ganoderma lucidum and pharmaceutically acceptable auxiliary materials or auxiliary components, and is prepared into an oral preparation.