Manufacturing method of silica-induced lung injury rat model and application of Erchen decoction

Abstract

The invention provides a manufacturing method of a silica-induced lung injury rat model and application of Erchen decoction. A manufacturing method of a silica-induced lung injury rat model is used for researching medical application and pathological effects of Erchen decoction in treatment of silicosis, traditional Chinese medicines in the Erchen decoction comprise Pinellia ternate, dried orange peel, Poria cocos, honey-fried licorice root, dark plum and ginger, and the manufacturing method is characterized by comprising the following steps: S1, experimental animals and grouping; the invention provides a manufacturing method of a silica-induced lung injury rat model and an application of Erchen soup, and systematically verifies and deepens a microecological mechanism of silicosis on the basis of early discovery; a new accurate diagnosis and treatment target is provided; the modern breakthrough of pharmacological research of traditional Chinese medicines is realized; the dysbacteriosis type silicosis model established by the invention has the advantages of simple and convenient modeling method, good repeatability, clear pathological characteristics and clear key target spots.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of experimental animal medicine and pharmacology of traditional Chinese medicine, and particularly relates to a method for preparing a silica-induced lung injury rat model and application of Erchen Decoction. BACKGROUND

[0002] Silicosis is a chronic, progressive, diffuse pulmonary fibrosis disease caused by long-term inhalation of silica particles, which has high disability rate and mortality rate. So far, there is no specific treatment method in the world. The core pathological link is sustained and uncontrolled inflammation, but the fundamental cause of driving this chronic inflammation has not been fully elucidated, which leads to repeated setbacks in drug development.

[0003] In recent years, the development of microecology provides a new perspective for understanding chronic inflammatory diseases. The healthy lung, which was once considered "sterile", has now been proven to have a stable microbial community with low biomass but active function. The homeostasis of lung microecology is crucial for maintaining local immune balance and resisting pathogen invasion. More and more evidence shows that the occurrence and development of various chronic lung diseases, such as chronic obstructive pulmonary disease, asthma and idiopathic pulmonary fibrosis, are closely related to the structural disorder of lung flora. Lung flora imbalance can destroy airway immune homeostasis and trigger or exacerbate abnormal inflammation. However, as a non-infectious foreign body, how does silica directly affect the structure of the resident flora in the lung and mediate the subsequent immune imbalance and fibrosis process? This is a key scientific problem that needs to be answered in the field of silicosis research.

[0004] Existing silicosis animal models, whether by direct instillation of tracheal exposure or by complex dust inhalation chamber exposure, have their own limitations. The tracheal instillation method is traumatic and prone to secondary infection, which interferes with the study of lung flora. Although the dust exposure method is closer to the real occupational scenario, the equipment is expensive, the dose is difficult to control accurately, and few studies have focused on the direct impact of silica on lung microecology. Therefore, the existing model system largely ignores the possible key pathological chain of "silica exposure → lung flora disorder → chronic inflammation → fibrosis", which leads to the research always staying in the observation of the appearance of inflammatory damage itself.

[0005] Therefore, it is necessary to provide a method for preparing a silica-induced lung injury rat model and application of Erchen Decoction to solve the above technical problems. SUMMARY

[0006] The present application provides a method for preparing a silica-induced lung injury rat model, which solves the problems that the silicosis animal model in the prior art fails to focus on the changes in lung microecology, the pathogenesis is unclear, and the pharmacological mechanism of traditional Chinese medicine is unclear.

[0007] To solve the above technical problems, the application provides a method for preparing a silica-induced lung injury rat model to study the medical use and pathological effect of Erchen Decoction in treating silicosis, and the traditional Chinese medicines in the Erchen Decoction include pinellia, dried tangerine or orange peel, poria, fried licorice, dark plum and fresh ginger, and the method comprises the following steps:

[0008] S1: experimental animals and grouping; 36 SPF healthy male SD rats are selected, and after adaptive feeding for one week, the rats are randomly divided into 6 groups according to the weight, and each group contains 6 rats;

[0009] S2: setting a normal control group; no any treatment is performed, and only 1 mL of 0.9% normal saline is given for non-exposed tracheal instillation at the corresponding time point, and then 2 mL of normal saline is given for subsequent gavage;

[0010] S3: setting a model group; 1 mL of 50 mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and then 2 mL of normal saline is given for subsequent gavage;

[0011] S4: setting a positive control group; 1 mL of 50 mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and then 0.3 mg / kg dexamethasone is given for subsequent gavage;

[0012] S5: setting a low-dose Erchen Decoction group; 1 mL of 50 mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and 2 mL of 0.88 g of the water decoction of Erchen Decoction is given for daily gavage;

[0013] S6: setting a medium-dose Erchen Decoction group; 1 mL of 50 mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and 2 mL of 1.76 g of the water decoction of Erchen Decoction is given for daily gavage;

[0014] S7: setting a high-dose Erchen Decoction group; 1 mL of 50 mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and 2 mL of 3.52 g of the water decoction of Erchen Decoction is given for daily gavage.

[0015] Preferably, the non-exposed tracheal instillation modeling comprises the following steps: step one: after the rats are anesthetized by intraperitoneal injection of sodium pentobarbital, the rats are fixed on a specially designed adjustable angle inclined plate with an inclination angle of 45° with the back downward;

[0016] Step two: a small LED cold light source is used for transcutaneous illumination from the neck to clearly locate the trachea;

[0017] Step three: a front-end blunted 16G intravenous indwelling needle hose or a 16-gauge gavage needle is gently inserted into the trachea through the oral cavity and over the epiglottis, and the position of the catheter in the tracheal ring is confirmed by slightly touching the neck;

[0018] Step four: After confirming the tracheal entry, inject 50 mg / mL sterile suspension of silica dioxide prepared in advance and evenly dispersed by ultrasonic within 2-3 seconds through the connected microsyringe;

[0019] Step five: The normal control group is dripped with the same volume of sterile normal saline;

[0020] Step six: After the operation is completed, the rat is upright and rotated several times to promote the uniform distribution of the suspension in the lung, and is put back into the cage after its spontaneous breathing is stable, without the need for surgical incision of the neck skin and muscle, with minimal trauma, avoiding exogenous bacterial contamination.

[0021] Preferably, the next day after modeling, the Erchen Decoction low, medium and high dose groups are respectively given corresponding concentrations of Erchen Decoction decoction daily at regular intervals by gavage, and the dose is set to 0.5 times, 1 times and 2 times of the clinical adult equivalent dose; the model group and the normal control group are given the same volume of normal saline by gavage daily, and the positive control group is given the same amount of dexamethasone solution by gavage, for continuous intervention for 4 weeks.

[0022] Preferably, the selected rats are 6-8 weeks old and weigh 180-220g.

[0023] The application of Erchen Decoction in the silica dioxide-induced lung injury rat model systematically analyzes the intervention effect and dose-effect relationship of the "bacteria-inflammation" pathological axis of different doses of Erchen Decoction, including the following aspects:

[0024] (1) Identification of lung flora structure and key bacteria: the rats in each group are sacrificed at the 4th week, and lung tissue samples are collected aseptically to extract total DNA;

[0025] (2) Analysis of "bacteria-inflammation" correlation;

[0026] (3) Pathological damage verification;

[0027] (4) Evaluation of the "bacteria regulation-inflammation suppression" efficacy of Erchen Decoction.

[0028] Preferably, after collecting the lung tissue samples and extracting the total DNA, 16S rRNA gene high-throughput sequencing technology is used to analyze the α diversity and β diversity of the flora, and through bioinformatics algorithms, Lachnospiraceae-NK4A136-group is verified and locked again as the core differential bacteria genus of the model group.

[0029] Preferably, when analyzing the "bacteria-inflammation" correlation, ELISA technology is used to detect the content of representative inflammatory factors in the serum, and statistical methods are used to correlate the relative abundance of the key bacteria genus Lachnospiraceae-NK4A136-group with the content of inflammatory factors, so as to prove that the proliferation of the flora is significantly positively correlated with the severity of the pro-inflammatory response by data.

[0030] Preferably, when the pathological damage is verified, the left lung is taken for tissue fixation to make paraffin sections; hematoxylin-eosin staining is used to evaluate the degree of lung inflammation cell infiltration and alveolar structure destruction; the severity of damage in each group is quantified by semi-quantitative pathological scoring, and compared with the flora and inflammation data.

[0031] Preferably, when the "regulating flora-inhibiting inflammation" effect of Erchen Decoction is evaluated, all detection indexes of the model group and the low, medium and high dose groups of Erchen Decoction are comprehensively compared; it is proved that after intervention of Erchen Decoction, the abundance of Lachnospiraceae-NK4A136-group bacteria genus decreases, the content of inflammatory factors decreases, and the lung inflammation infiltration and fibrosis pathological score improves.

[0032] Compared with the related art, the method for preparing a silica-induced lung injury rat model provided by the application has the following beneficial effects:

[0033] The application provides a method for preparing a silica-induced lung injury rat model and application of Erchen Decoction, which further verifies and deepens the microecological mechanism of silicosis pathogenesis on the basis of the previous findings: not only to simply reproduce the phenomenon, but also to use the key bacteria genus Lachnospiraceae-NK4A136-group found in the previous stage as a clear target and biomarker, and to first prove the core driving role of the flora in the pathological process of silicosis from the causal relationship through drug intervention.

[0034] A new target for precise diagnosis and treatment is provided: Lachnospiraceae-NK4A136-group is successfully screened and verified as a core pathogenic bacteria genus in the process of silicosis through the model, which not only can be used as a potential biomarker for early diagnosis and prognosis evaluation, but also represents a new drug treatment target with great potential, and specific intervention on the bacteria genus is expected to become a precise strategy for future silicosis treatment;

[0035] A modern breakthrough in traditional Chinese medicine pharmacological research is achieved: the application first scientifically explains the mechanism of the classic prescription Erchen Decoction in treating lung diseases from the perspective of microecological regulation, and the research proves that the efficacy is closely related to the remodeling of the disordered lung flora, the inhibition of the key pathogenic bacteria and the alleviation of the inflammatory response, which provides modern scientific microscopic evidence for the "theory of principles, methods, prescriptions and drugs" of traditional Chinese medicine, and promotes the modernization and internationalization of traditional Chinese medicine;

[0036] Create an efficient drug screening and evaluation platform: The "dysbiosis" silicosis model established by the application has the advantages of simple modeling method, good repeatability, clear pathological characteristics and clear key target points. It is not only suitable for evaluating complex preparations such as Erchen Decoction, but also can be used as an efficient platform for screening any new candidate drugs intended to treat pulmonary fibrosis diseases by regulating lung microecology. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 A flowchart of the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure;

[0038] Figure 2 A schematic diagram of the body weight gain rate of rats in each group of the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure;

[0039] Figure 3 A graph showing the influence of the method for preparing a silica-induced lung injury rat model provided by the application on the morphological structure of lung tissue in each group is shown in the figure;

[0040] Figure 4 A schematic diagram showing the influence of Erchen Decoction on the PMLI rat lung Nlrp3, Gsdmd and Txnip mRNA according to the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure;

[0041] Figure 5 A comparison graph showing the influence of Erchen Decoction on the PMLI rat lung tissue NLRP3 and GSMDM protein expression according to the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure;

[0042] Figure 6 A schematic diagram showing the influence of Erchen Decoction on the PMLI rat serum inflammatory factor content according to the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure;

[0043] Figure 7 A schematic diagram showing the difference between groups of species according to the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure;

[0044] Figure 8 A heatmap showing the correlation analysis of lung flora and inflammatory factors, inflammatory mediators according to the method for preparing a silica-induced lung injury rat model provided by the application is shown in the figure. DETAILED DESCRIPTION

[0045] The application will be further described below in conjunction with the drawings and embodiments.

[0046] Please refer to Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 ,Figure 6 、 Figure 7 and Figure 8 wherein, Figure 1 is the flowchart of the method for preparing a silica-induced lung injury rat model provided by the application; Figure 2 is the schematic diagram of the body weight gain rate of rats in each group of the method for preparing a silica-induced lung injury rat model provided by the application; Figure 3 is the influence diagram of the morphological structure of lung tissue in each group of the method for preparing a silica-induced lung injury rat model provided by the application; Figure 4 is the influence diagram of the effect of Erchen Decoction on the lung Nlrp3, Gsdmd and Txnip mRNA of PMLI rats in the method for preparing a silica-induced lung injury rat model provided by the application; Figure 5 is the comparison diagram of the influence of Erchen Decoction on the lung tissue NLRP3 and GSMDM protein expression of PMLI rats in the method for preparing a silica-induced lung injury rat model provided by the application; Figure 6 is the influence diagram of the effect of Erchen Decoction on the serum inflammatory factor content of PMLI rats in the method for preparing a silica-induced lung injury rat model provided by the application; Figure 7 is the difference between the species in each group of the method for preparing a silica-induced lung injury rat model provided by the application; Figure 8 is the correlation analysis heat map of lung flora and inflammatory factors, inflammatory mediators in the method for preparing a silica-induced lung injury rat model provided by the application.

[0047] The method for preparing a silica-induced lung injury rat model is used to study the medical use and pathological effect of Erchen Decoction in the treatment of silicosis. The traditional Chinese medicines in the Erchen Decoction include Pinellia ternate, Citrus reticulata, Poria cocos, Radix Glycyrrhizae, Prunus mume and Zingiber officinale. The method comprises the following steps:

[0048] S1: Experimental animals and grouping; 36 SPF healthy male SD rats are selected, and after adaptive feeding for one week, they are randomly divided into 6 groups according to body weight, 6 rats in each group;

[0049] S2: Setting up a normal control group: without any treatment, only giving non-exposed tracheal instillation of 1mL of 0.9% normal saline at the corresponding time point, and subsequent gavage of 2mL of normal saline;

[0050] S3: Setting up a model group: 1mL of 50mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and 2mL of normal saline is used for subsequent gavage;

[0051] S4: Setting up a positive control group: 1mL of 50mg / mL silica suspension is used for non-exposed tracheal instillation modeling, and 0.3mg / kg dexamethasone is used for subsequent gavage;

[0052] S5: Set up low-dose group of Erchen decoction: 1 mL of 50 mg / mL silica suspension non-exposed tracheal instillation modeling, daily gavage 2 mL of 0.88 g of Erchen decoction water decoction;

[0053] S6: Set up medium-dose group of Erchen decoction: 1 mL of 50 mg / mL silica suspension non-exposed tracheal instillation modeling, daily gavage 2 mL of 1.76 g of Erchen decoction water decoction;

[0054] S7: Set up high-dose group of Erchen decoction: 1 mL of 50 mg / mL silica suspension non-exposed tracheal instillation modeling, daily gavage 2 mL of 3.52 g of Erchen decoction water decoction.

[0055] The non-exposed tracheal instillation modeling includes the following steps: step one: after the rats are anesthetized by intraperitoneal injection of sodium pentobarbital, the back is fixed on a specially designed adjustable angle inclined plate with an angle of 45°;

[0056] Step two: use a small LED cold light source to transdermally illuminate from the neck to clearly locate the trachea;

[0057] Step three: a blunt 16G intravenous catheter or a 16-gauge gavage needle is inserted into the trachea through the rat's mouth and over the epiglottis, and the position of the catheter in the tracheal ring is confirmed by gently touching the neck;

[0058] Step four: after confirming that it has entered the trachea, a pre-prepared and uniformly dispersed 50 mg / mL silica sterile suspension is quickly injected through the connected microsyringe within 2-3 seconds;

[0059] Step five: the normal control group is instilled with the same volume of sterile normal saline;

[0060] Step six: after the operation is completed, the rat is rotated several times to promote uniform distribution of the suspension in the lungs, and after the rat's spontaneous breathing is stable, it is returned to the cage without the need for surgical incision of the neck skin and muscle, with minimal trauma and avoiding external bacterial contamination.

[0061] The next day after modeling, the low, medium and high dose groups of Erchen decoction are given corresponding concentrations of Erchen decoction water decoction by gavage at regular intervals every day, with the dose set at 0.5 times, 1 times and 2 times the clinical adult equivalent dose; the model group and the normal control group are given the same volume of normal saline by gavage every day, and the positive control group is given the same amount of dexamethasone solution by gavage, for a continuous intervention of 4 weeks.

[0062] The rats selected are 6-8 weeks old and weigh 180-220 g.

[0063] In this embodiment, the application of SiO2-induced lung injury rat model of Erchen Decoction is provided. The intervention effect and dose-effect relationship of the "bacteria-inflammation" pathological axis are systematically analyzed by different doses of the Erchen Decoction, including the following aspects:

[0064] (1) Identification of lung flora structure and key bacteria: the rats in each group were sacrificed at the 4th week, and the lung tissue samples were collected aseptically, and total DNA was extracted;

[0065] (2) Analysis of "bacteria-inflammation" correlation;

[0066] (3) Pathological damage verification;

[0067] (4) Efficacy evaluation of Erchen Decoction "regulating bacteria and inhibiting inflammation".

[0068] After the lung tissue samples were collected and total DNA was extracted, 16S rRNA gene high-throughput sequencing technology was used to analyze the alpha diversity and beta diversity of the flora. Through bioinformatics algorithm, Lachnospiraceae-NK4A136-group was verified and re-locked as the core differential bacteria of the model group.

[0069] When analyzing the "bacteria-inflammation" correlation, ELISA technology was used to detect the content of representative inflammatory factors in the serum. Statistical methods were used to correlate the relative abundance of key bacteria Lachnospiraceae-NK4A136-group with the content of inflammatory factors, and data was used to prove that the proliferation of the flora was significantly positively correlated with the severity of the pro-inflammatory response.

[0070] When verifying the pathological damage, the left lung was taken for tissue fixation, and paraffin sections were prepared; hematoxylin-eosin staining was used to evaluate the degree of lung inflammatory cell infiltration and alveolar structure destruction; semi-quantitative pathological scoring was used to quantify the severity of damage in each group, and the data was compared with the flora and inflammation data.

[0071] When evaluating the "regulating bacteria and inhibiting inflammation" efficacy of Erchen Decoction, all detection indicators of the model group and the low, medium and high dose groups of Erchen Decoction were comprehensively compared; it was proved that after the intervention of Erchen Decoction, the abundance of Lachnospiraceae-NK4A136-group bacteria decreased; the content of inflammatory factors decreased; the lung inflammatory infiltration and fibrosis pathological score improved.

[0072] Example 1

[0073] 1. Experimental materials

[0074] Animals: 36 SPF male SD rats.

[0075] Reagents: Silica particles (80% of particle size 1-5 μm, Sigma-Aldrich); sodium pentobarbital; Rat TNF-α, IL-1β, IL-18 kits for ELISA detection; NLRP3, GSDMD polyclonal antibodies; DNeasy PowerSoil Pro Kit for DNA extraction.

[0076] Traditional Chinese medicine: Erchen Decoction includes Banxia (15g), Chenpi (15g), Fuling (9g), Zhigancao (6g), Wumei (5g), Shengjiang (12g); The preparation of Erchen Decoction water decoction liquid refers to the drug composition, dosage and preparation method of Erchen Decoction in "Prescription Science" and "Chinese Medicine Pharmacology": First decoction: after weighing the medicinal materials according to the prescription ratio, soak them in 10 times the volume of distilled water for 30 minutes, then boil and decoct for 30 minutes on low heat, and filter; Second decoction: add 7 times the volume of distilled water to the residue, boil and decoct for 20 minutes, and filter; Concentration: combine the two decoction liquids, concentrate under reduced pressure to the target concentration (0.44 g / mL, 0.88 g / mL and 1.76 g / mL), and store at 4℃ after dispensing.

[0077] 2. Detailed operation steps

[0078] Grouping and modeling: 36 rats were randomly divided into 6 groups; except for the normal control group, the remaining 5 groups of rats were given 50 mg / mL of silica suspension (1 mL / kg) by non-exposed tracheal instillation method after anesthesia.

[0079] Drug intervention: starting from the 2nd day after modeling, each group was given gavage at 10 am every day; the low, medium and high dose groups were given corresponding concentrations of Erchen Decoction solution (2 mL per rat); the model group and the normal control group were given the same volume of normal saline; the positive control group was given the same volume of dexamethasone solution. Continuous administration for 28 days.

[0080] Sample collection: on the 29th day, 2 hours after the last administration, the rats in each group were anesthetized, blood was collected from the abdominal aorta, and the blood was centrifuged at 3000 rpm for 15 minutes after standing at 4℃ for 2 hours; the supernatant serum was taken and stored at -80℃; then the chest was opened, the right lung was taken and stored at -80℃ for ELISA and 16S rRNA sequencing; the left lung was fixed with 4% paraformaldehyde for 24 hours for subsequent pathological section preparation; part of the right lung tissue was quickly placed in liquid nitrogen for quick freezing and stored at -80℃ for subsequent molecular biology detection.

[0081] 3. Detection analysis and expected results

[0082] 3.1 Effect of Erchen Decoction on the weight gain rate of PMLI rats

[0083] Compared with the normal control group, the body weight growth rate of the model group was significantly reduced; compared with the model group, the body weight growth rate of the Erchen Decoction group was significantly increased, and the body weight growth rate of the dexamethasone group was significantly decreased, which was much lower than that of each group, and the results are shown in Figure 2 .

[0084] Note: NC: normal control group; M: model group; DEX: dexamethasone group; ECL: low-dose Erchen Decoction group; ECM: medium-dose Erchen Decoction group; ECH: high-dose Erchen Decoction group; compared with the normal control group: *P<0.05 **P<0.01; compared with the model group: # P<0.05 ## P<0.01.

[0085] 3.2 Pathological examination

[0086] The H&E staining results are expected to show that, compared with the normal control group, the lung alveolar structure of the model group is severely damaged, the alveolar septum is thickened, and is accompanied by a large number of inflammatory cell infiltration, fibrous aggregation, multiple cellular nodules and fused nodules; compared with the model group, the lung tissues of the dexamethasone group and the low, medium and high dose Erchen Decoction groups are improved to different degrees, showing reduced inflammatory cell infiltration, reduced fibrous aggregation, reduced number and volume of cellular nodules and fused nodules; among them, the medium-dose Erchen Decoction group is the most improved, and the results are shown in Figure 3 .

[0087] Note: NC: normal control group; M: model group; DEX: dexamethasone group; ECL: low-dose Erchen Decoction group; ECM: medium-dose Erchen Decoction group; ECH: high-dose Erchen Decoction group.

[0088] 3.3 Effect of Erchen Decoction on the expression of inflammation-related genes in the lung tissue of PMLI rats

[0089] Compared with the normal control group, the relative expression levels of Nlrp3, Gsdmd and Txnip mRNA in the model group were increased (P<0.01), and compared with the model group, the relative expression levels of the above mRNA in the low, medium and high dose Erchen Decoction groups were significantly reduced (P<0.05 or P<0.01); among them, the low and high dose Erchen Decoction groups had no statistically significant down-regulation effect on the relative expression level of Txnip mRNA, and the results are shown in Figure 4 .

[0090] Note: NC: normal control group; M: model group; DEX: dexamethasone group; ECL: low-dose Erchen Decoction group; ECM: medium-dose Erchen Decoction group; ECH: high-dose Erchen Decoction group; compared with the normal control group: *P<0.05 **P<0.01; compared with the model group: # P<0.05 ## P<0.01 nsP>0.05.

[0091] 3.4 Effect of Erchen Decoction on the expression of NLRP3 and GSDMD proteins in lung tissues of PMLI rats

[0092] Compared with the normal control group, the expression of NLRP3 and GSDMD proteins in the lung tissues of the model group rats was significantly increased (P<0.01); compared with the model group, the expression of NLRP3 protein in the rats of the Erchen Decoction medium and high dose groups was significantly decreased (P<0.05), and the expression of GSDMD protein in the rats of the Erchen Decoction low, medium and high dose groups was significantly decreased (P<0.05), and the results are shown in Figure 5 .

[0093] Note: NC: normal control group; M: model group; DEX: dexamethasone group; ECL: low dose of Erchen Decoction group; ECM: medium dose of Erchen Decoction group; ECH: high dose of Erchen Decoction group; average optical density value (IHC, x400); compared with the normal control group: *P<0.05 **P<0.01; compared with the model group: # P<0.05 ## P<0.01 ns P>0.05.

[0094] 3.5 Inflammatory factor detection

[0095] The ELISA results are expected to show that compared with the normal control group, the levels of IL-1β, IL-18 and TNF-α in the serum of the model group rats were significantly increased (P<0.05 or P<0.01); compared with the model group, the levels of IL-1β, IL-18 and TNF-α in the rats of the Erchen Decoction low, medium and high dose groups were significantly decreased (P<0.05 or P<0.01), and the effect of the Erchen Decoction medium dose group was better, and the results are shown in Figure 6 .

[0096] Note: NC: normal control group; M: model group; DEX: dexamethasone group; ECL: low dose of Erchen Decoction group; ECM: medium dose of Erchen Decoction group; ECH: high dose of Erchen Decoction group; compared with the normal control group: *P<0.05 **P<0.01; compared with the model group: # P<0.05 ## P<0.01 ns P>0.05.

[0097] 3.6 16S rRNA sequencing analysis:

[0098] Screening of different species between groups

[0099] As Figure 7Compared with the normal control group, the relative abundance of Lachnospiraceae_NK4A136_group, Helicobacter, NK4A214_group and Eubacterium_siraeum_group in the model group was significantly increased, and after the intervention of Erchen Decoction, the relative abundance was significantly reduced. The relative abundance of Staphylococcus and Terrisporobacter in the model group was significantly reduced, and the relative abundance of the low, medium and high dose groups of Erchen Decoction was significantly increased, and the difference was statistically significant (P<0.05).

[0100] Table 1 Relative abundance of different bacterial genera in each group

[0101]

[0102] Note: NC: normal control group; M: model group; DEX: dexamethasone group; ECL: low dose of Erchen Decoction group; ECM: medium dose of Erchen Decoction group; ECH: high dose of Erchen Decoction group.

[0103] 3.7 Correlation analysis: Spearman correlation analysis is expected to reveal that the relative abundance of Lachnospiraceae-NK4A136-group is significantly correlated with inflammatory mediators.

[0104] (1) Correlation between dominant lung bacterial genera and three inflammatory factors (IL-1β, IL-18 and TNF-α)

[0105] The differential bacterial genus Lachnospiraceae_NK4A136_group was significantly positively correlated with IL-1β (r=0.525) and IL18 (r=0.383); Helicobacter was significantly positively correlated with IL-1β (r=0.37) (P<0.05);

[0106] The non-differential bacterial genus Streptococcus was significantly negatively correlated with IL-18 (r=-0.48, P<0.01); Dialister was significantly positively correlated with IL-18 (r=0.38, P<0.05); Escherichia-Shigella was significantly negatively correlated with TNF-α (r=0.42, P<0.05); Bergeyella was significantly positively correlated with TNF-α (r=0.47, P<0.05); Solibacillus was significantly negatively correlated with TNF-α (r=-0.40, P<0.05); Rikenellaceae_Rc9_gut_group was significantly positively correlated with IL-18 (r=0.45, P<0.05) (Table 2); Figure 8 ​

[0107] (2) Correlation between dominant lung bacteria and three inflammation-related genes (Nlrp3, Gsdmd and Txnip).

[0108] Differential bacteria Lachnospiraceae_NK4A136_group was significantly positively correlated with Nlrp3 (r = 0.48) and Txnip (r = 0.56) (P < 0.05); NK4A214_group was significantly positively correlated with Gsdmd (r = 0.57) and Txnip (r = 0.50) (P < 0.05), see Figure 8 and Table 3.

[0109] Note: A. Correlation analysis heat map between dominant lung bacteria and inflammatory factors (IL-1β, IL-18 and TNF-α); B. Correlation analysis heat map between dominant lung bacteria and lung tissue inflammatory mediators, *P < 0.05 **P < 0.01.

[0110] Table 2 Correlation analysis table of lung flora and inflammatory factors (IL-1β, IL-18 and TNF-α)

[0111]

[0112] Table 3 Correlation analysis table of lung flora and inflammatory mediators (Nlrp3, Gsdmd and Txnip)

[0113]

[0114] The working principle of the method for preparing the silica-induced lung injury rat model and the application of Erchen Decoction provided by the present application is as follows:

[0115] In work, first, the SD rats are divided into a normal control group, a model group, a positive control group and Erchen Decoction low-, medium- and high-dose intervention groups; a silica-induced lung injury model is constructed by a non-exposure tracheal instillation method; after 4 weeks of intervention, multi-dimensional and multi-level system detection is performed on each group, the core of which is 16S rRNA sequencing to analyze lung flora changes, combined with qPCR detection of inflammation-related genes, histological technology detection of inflammation-related proteins, ELISA detection of inflammatory factors and histopathological evaluation of lung injury; finally, correlation analysis is performed to correlate the flora, inflammation and pathological injury, and the dose-effect relationship of Erchen Decoction is systematically evaluated, and finally the core mechanism of "regulating bacteria, inhibiting inflammation and reducing damage" is clarified.

[0116] Compared with the related art, the method for preparing the silica-induced lung injury rat model provided by the present application has the following beneficial effects:

[0117] The application provides a method for preparing a silica-induced lung injury rat model and an application of Erchen Decoction, and based on the previous discovery, the microecological mechanism of the pathogenesis of silicosis is verified and deepened: not only the phenomenon is simply reproduced, but also the key genus Lachnospiraceae-NK4A136-group discovered in the previous stage is used as an explicit target and biomarker, and through the means of drug intervention, the core driving role of the bacterial group in the pathological process of silicosis is first confirmed from the causal relationship.

[0118] A new target for accurate diagnosis and treatment is provided: through the model, Lachnospiraceae-NK4A136-group is successfully screened and verified as a core pathogenic genus in the process of silicosis, the genus can not only be used as a potential biomarker for early diagnosis and prognosis evaluation, but also represents a highly potential new drug treatment target, and specific intervention on the genus is expected to become an accurate strategy for future silicosis treatment.

[0119] A modern breakthrough in traditional Chinese medicine pharmacological research is realized: the application first scientifically elucidates the mechanism of the classic prescription Erchen Decoction in treating lung diseases from the perspective of microecological regulation, and the research proves that the curative effect is closely related to the remodeling of the disordered lung flora, the inhibition of the key pathogenic bacteria and the relief of the inflammatory reaction, which provides modern scientific microscopic evidence for the theory of "principle, method, prescription and medicine" of traditional Chinese medicine, and promotes the modernization and internationalization of traditional Chinese medicine.

[0120] An efficient drug screening and evaluation platform is created: the "flora imbalance type" silicosis model established in the application has the advantages of simple modeling method, good repeatability, clear pathological characteristics and clear key target, and it is not only suitable for evaluating compound preparations like Erchen Decoction, but also can be used as an efficient platform for screening any new candidate drug aiming at treating pulmonary fibrosis diseases by regulating the lung microecology.

[0121] The above only describes the embodiments of the application, and does not limit the patent scope of the application, and any equivalent structure or equivalent flow transformation obtained by using the content of the specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the application.

Claims

1. A method for establishing a rat model of silica-induced lung injury, characterized in that, Includes the following steps: S1: Experimental animals and grouping; 36 healthy male SPF-grade SD rats were selected and, after one week of acclimatization, were randomly divided into 6 groups according to their body weight, with 6 rats in each group; S2: Set up a normal control group: No treatment was given, except that 1 mL of 0.9% saline was infused into the non-exposed trachea at the corresponding time point, followed by 2 mL of saline by gavage; S3: Set up the model group: Initiate the model by non-exposed tracheal infusion of 1 mL of 50 mg / mL silica suspension, followed by gavage administration of 2 mL of physiological saline; S4: Set up a positive control group: 1 mL of 50 mg / mL silica suspension was dripped into the non-exposed trachea to establish the model, followed by gavage administration of 0.3 mg / kg dexamethasone; S5: Set up a low-dose group of Er Chen Tang: 1 mL of 50 mg / mL silica suspension was dripped into the non-exposed trachea to establish the model, and 2 mL of 0.88 g of Er Chen Tang decoction was administered by gavage daily. S6: Set up the medium dose group of Er Chen Tang: 1 mL of 50 mg / mL silica suspension was dripped into the non-exposed trachea to establish the model, and 2 mL of 1.76 g of Er Chen Tang decoction was administered by gavage daily. S7: Set up a high-dose group of Er Chen Tang: 1 mL of 50 mg / mL silica suspension was administered via non-exposed tracheal infusion to establish the model, and 2 mL of 3.52 g of Er Chen Tang decoction was administered by gavage daily.

2. The method for creating a rat model of silica-induced lung injury according to claim 1, characterized in that, The non-exposed tracheal infusion modeling includes the following steps: Step 1: After anesthetizing the rats with sodium pentobarbital via intraperitoneal injection, fix them back-down on a specially designed adjustable inclined plate with a tilt angle of 45°. Step 2: Use a small LED cold light source to provide transdermal illumination from the neck to clearly locate the trachea; Step 3: Gently insert a blunt-tipped 16G intravenous catheter or a 16-gastric needle into the trachea through the rat's mouth and past the epiglottis. Confirm the position of the catheter within the tracheal ring by gently touching the neck. Step 4: After confirming entry into the trachea, inject a pre-prepared and ultrasonically dispersed 50mg / mL sterile silica suspension rapidly within 2-3 seconds using the connected micro-syringe. Step 5: The normal control group was infused with an equal volume of sterile saline. Step Six: After the operation is completed, rotate the rat upright several times to promote the even distribution of the suspension in the lungs. After its spontaneous breathing stabilizes, put it back in the cage. There is no need to surgically cut the neck skin and muscles, so the trauma is minimal and exogenous bacterial contamination is avoided.

3. The method for creating a rat model of silica-induced lung injury according to claim 1, characterized in that, Starting the day after modeling, the low, medium, and high dose groups of Er Chen Tang were given the corresponding concentration of Er Chen Tang decoction by gavage at regular intervals every day. The doses were set to 0.5 times, 1 times, and 2 times the equivalent dose for clinical adults, respectively. The model group and the normal control group were given an equal volume of physiological saline by gavage every day, while the positive control group was given an equal volume of dexamethasone solution by gavage. The intervention lasted for 4 consecutive weeks.

4. The method for creating a rat model of silica-induced lung injury according to claim 1, characterized in that, The selected rats were 6-8 weeks old and weighed 180-220g.

5. The application of Er Chen Tang in the rat model of silica-induced lung injury according to any one of claims 1-4, characterized in that, The intervention effect and dose-response relationship of the Er Chen Tang at different dosages on the "bacterium-inflammation" pathological axis were systematically analyzed. Including the following aspects: (1) Identification of lung microbiota structure and key genera: Rats in each group were sacrificed in week 4, lung tissue samples were aseptically collected, and total DNA was extracted. (2) Correlation analysis of "bacteria-inflammation"; (3) Pathological damage verification; (4) Evaluation of the "antibacterial and anti-inflammatory" effects of Er Chen Tang.

6. The application of Er Chen Tang in the rat model of silica-induced lung injury according to claim 5, characterized in that, After collecting lung tissue samples and extracting total DNA, 16S rRNA gene high-throughput sequencing technology was used to analyze the α-diversity and β-diversity of the bacterial community. Through bioinformatics algorithms, the Lachnospiraceae-NK4A136-group was verified and identified as the core differential bacterial genus of the model group.

7. The application of Er Chen Tang in the rat model of silica-induced lung injury according to claim 5, characterized in that, During the "bacteria-inflammation" correlation analysis, the levels of representative inflammatory factors in serum were simultaneously detected using ELISA technology. Statistical methods were used to correlate the relative abundance of the key bacterial genus Lachnospiraceae-NK4A136-group with the levels of inflammatory factors, demonstrating a significant positive correlation between the proliferation of this bacterial community and the severity of pro-inflammatory responses.

8. The application of Er Chen Tang in the rat model of silica-induced lung injury according to claim 5, characterized in that, During the pathological damage verification, tissue samples from the left lung were fixed and paraffin sections were prepared. Hematoxylin-eosin staining was used to assess the degree of inflammatory cell infiltration and alveolar structural damage in the lungs. The severity of damage in each group was quantified by semi-quantitative pathological scoring and compared with bacterial flora and inflammation data.

9. The application of Er Chen Tang in the rat model of silica-induced lung injury according to claim 5, characterized in that, When evaluating the "antibacterial regulation and anti-inflammatory" efficacy of Er Chen Tang, all detection indicators of the model group and the three dosage groups of Er Chen Tang (low, medium, and high doses) were comprehensively compared.

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