A traditional Chinese medicine composition for treating diffuse interstitial lung disease and its application

Abstract

The present invention belongs to the field of traditional Chinese medicine technology, and in particular relates to a traditional Chinese medicine composition for treating diffuse interstitial lung disease and its application. The traditional Chinese medicine composition of the present invention is made of the following raw materials in parts by weight: 10-12 parts of Curculigo, 10-20 parts of Epimedium, 30-45 parts of Astragalus, 20-30 parts of Ophiopogon, 15-30 parts of Clemati, 10-15 parts of Kadura Caulis, 10-15 parts of Trachelospermum officinale, 10-15 parts of Earthworm, 10-12 parts of Curcuma, 12-15 parts of Polygonum cuspidatum, 15-20 parts of Fritillaria thunbergii, and 6 parts of Licorice, which has better clinical treatment effect for diffuse interstitial lung disease. Further studies have shown that the above drugs have a synergistic effect after compatibility, and can inhibit alveolar epithelial cell-mesenchymal cell transformation and reduce excessive deposition of extracellular matrix by regulating the abnormal activation of TGF-β1 / Snail and Wnt3a / β-catenin signaling pathways, thereby treating IPF.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a traditional Chinese medicine composition for treating diffuse interstitial lung disease and application thereof. Background Art

[0002] Diffuse interstitial lung disease (ILD), also known as diffuse parenchymal lung disease (DPLD), is a general term for a group of diffuse lung diseases that primarily affect the pulmonary interstitium and alveolar space, leading to loss of alveolar capillary functional units. Clinical manifestations include gradually worsening dyspnea, progressive exertional shortness of breath, cough, or coughing up white sputum. Pulmonary function tests show restrictive ventilation dysfunction with decreased diffusion capacity, hypoxemia, and diffuse bilateral lung lesions on imaging.

[0003] Idiopathic pulmonary fibrosis (IPF), a type of interstitial lung disease (ILD), is a chronic, progressive, fibrotic interstitial lung disease with lesions localized to the lungs. Characteristically, lung histology and / or chest high-resolution CT (HRCT) findings suggest usual interstitial pneumonia (UIP). IPF lesions are often confined to the lungs, with minimal systemic symptoms. The primary manifestations are progressively worsening dyspnea, accompanied by restrictive ventilatory dysfunction and impaired gas exchange, hypoxemia, and even respiratory failure. Data from the UK and the US indicate an incidence of approximately 4.6-16.3 cases per 100,000 population, with a prevalence of 13-20 cases per 100,000 population. The incidence rate is higher in men than in women, at a ratio of approximately 1.5-1.7:1, and increases with age. The etiology of IPF remains unclear, but known risk factors include smoking and exposure to metal and wood dust. Approximately 0.5%-3.7% of patients have a family history of IPF. Once diagnosed with IPF, the average survival is only 3.2 years. Compared to other interstitial lung diseases, the 5-year survival rate for IPF patients is less than 20%.

[0004] Current studies have shown that immune-mediated inflammatory response is a key factor in the pathogenesis of IPF, and alveolar epithelial damage in the early stage of the disease is a key driving factor in the pathogenesis of IPF. Damage to type I alveolar epithelial cells and destruction of the alveolar epithelial cell layer lead to the release of fibrotic cytokines (such as chemokines, proteases, and transforming growth factor-β). After activation, fibroblasts destroy the repair mechanism of the lung matrix, resulting in alveolar epithelial to mesenchymal transition (EMT) and activation and accumulation of myofibroblasts, leading to the occurrence and development of pulmonary fibrosis.

[0005] Currently, pirfenidone and nintedanib are the two main medications recommended for the treatment of idiopathic pulmonary fibrosis in the 2015 "Recommended Guidelines for the Clinical Treatment of Idiopathic Pulmonary Fibrosis," jointly released by the American Thoracic Society, the European Respiratory Society, the Japanese Thoracic Society, and the Latin American Thoracic Society. Pirfenidone, when administered to IPF patients for approximately 50 weeks, can slow the decline in lung function indicators such as forced vital capacity (FVC) and DLCO, prolong progression-free survival (PFS), and reduce the risk of mortality. While this medication can slow the progression of pulmonary fibrosis to some extent, long-term use is prone to adverse reactions, such as liver dysfunction and gastrointestinal problems, which severely restricts its clinical application. Furthermore, its high price makes it prohibitive for many patients.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The first object of the present invention is to provide a Chinese medicine composition for treating diffuse interstitial lung disease in view of the deficiencies in the prior art.

[0008] The second purpose of the present invention is the use of the Chinese medicine composition.

[0009] To achieve the above first purpose, the technical solution adopted by the present invention is as follows:

[0010] A traditional Chinese medicine composition for treating diffuse interstitial lung disease is prepared from the following raw materials in parts by weight: 10-12 parts of curculigo, 10-20 parts of epimedium, 30-45 parts of astragalus, 20-30 parts of ophiopogon, 15-30 parts of clematis, 10-15 parts of kadura vine, 10-15 parts of trachelospermum serrata, 10-15 parts of earthworm, 10-12 parts of zedoaria, 12-15 parts of polygonum cuspidatum, 15-20 parts of thunbergia thunbergii, and 6 parts of liquorice.

[0011] Furthermore, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of Curculigo, 10 parts of Epimedium, 30 parts of Astragalus, 30 parts of Ophiopogon, 15 parts of Clematidis, 15 parts of Kadura Caulis, 15 parts of Trachelospermum jasminoides, 12 parts of earthworm, 10 parts of Curcuma, 12-15 parts of Polygonum cuspidatum, 20 parts of Fritillaria thunbergii, and 6 parts of Licorice.

[0012] Furthermore, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of Curculigo, 10 parts of Epimedium, 30 parts of Astragalus, 30 parts of Ophiopogon, 15 parts of Clematidis, 15 parts of Piper meridionalis, 15 parts of Trachelospermi Caulis, 12 parts of Earthworm, 10 parts of Curcuma, 12 parts of Polygonum cuspidatum, 20 parts of Fritillaria thunbergii, and 6 parts of Licorice.

[0013] Furthermore, the traditional Chinese medicine composition further comprises excipients, which include at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

[0014] Furthermore, the dosage form of the Chinese medicine composition is selected from any one of granules, ointments, pills, injections and oral liquids.

[0015] To achieve the above second purpose, the technical solution adopted by the present invention is as follows: use of a traditional Chinese medicine composition in the preparation of a drug for treating diffuse interstitial lung disease.

[0016] Furthermore, the diffuse interstitial lung disease includes idiopathic pulmonary fibrosis.

[0017] Furthermore, the drug takes effect in at least one of the following ways:

[0018] (1) Down-regulate the expression of at least one protein among TGF-β1, β-catenin, MMP-7 and MMP-9 in fibroblasts;

[0019] (2) Increased serum IFN-γ levels;

[0020] (3) Reduce serum IL-4 and Laminin levels;

[0021] (4) Reduce the relative expression level of at least one protein mRNA among β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 in fibroblasts.

[0022] The Chinese medicine composition of the present invention uses Curculigo and Epimedium as the main drugs, which play the role of warming and tonifying kidney yang, dispelling wind and removing dampness. The auxiliary drugs selected are Clematidis, Kadura Caulis, and Trachelospermum schrenkiana to dredge the lung meridian; Curcuma zedoaria breaks blood and eliminates symptoms, and the four drugs work together to eliminate numbness and dredge the meridians; Astragalus membranaceus nourishes lung qi, and Ophiopogon japonicus nourishes qi and yin, helping the main drugs to nourish kidney qi, and the mutual generation of metal and water. Earthworm dispels wind and dredges the meridians; Polygonum cuspidatum and Fritillaria thunbergii dredge the meridians, disperse stagnation, clear the lungs and resolve phlegm, and these three drugs are together as adjuvant drugs; Licorice moistens the lungs and relieves coughs and can harmonize the other drugs, serving as an adjuvant and guiding drug, ultimately forming a kidney-tonifying and meridian-dredgeing prescription. This prescription has excellent performance in clinical application, especially making the patient's signs of cyanosis of the lips and sublingual blood stasis more significantly improved, the symptoms of dry mouth, dry throat, coughing and spitting sticky phlegm are significantly alleviated, and the symptoms of wheezing and shortness of breath are improved. If any of the main, auxiliary, adjuvant and guiding drugs are replaced or omitted, the clinical effect is not ideal. For example, if the astragalus in the prescription is replaced with roasted astragalus, the Fritillaria thunbergii that clears the lungs, resolves phlegm and moistens the lungs and is compatible with Ophiopogon japonicus is replaced with Inula frutescens that eliminates phlegm and Schisandra chinensis that descends qi and astringes the lungs, and Pinellia ternata, dried ginger and Scutellaria baicalensis that have the effects of drying dampness, resolving phlegm and clearing lung heat are added, and at the same time the blood-breaking drug Trillium is added, the efficacy of the prescription is not as good as that of the present invention; for another example, if Curculigo orchioides, which is also the main drug, is omitted, Piper mestophylla is missing from the ministerial drug, Ophiopogon japonicus is missing from the auxiliary main drug, and Fritillaria thunbergii is missing from the adjuvant drug, although Luffa loofah and Millettia reticulata that can dredge the meridians and activate the collaterals, as well as raw Coix seed that can eliminate dampness and resolve phlegm are added, the efficacy of this prescription is not as good as that of the present invention. The Chinese medicine composition of the present invention has better clinical effects.

[0023] Further studies have shown that the above drugs have a synergistic effect when combined, and can inhibit alveolar epithelial cell-mesenchymal cell transformation and reduce excessive deposition of extracellular matrix by regulating the abnormal activation of TGF-β1 / Snail and Wnt3a / β-catenin signaling pathways, thereby treating IPF.

[0024] The present invention establishes a bleomycin-induced pulmonary fibrosis rat model, and finds that the preferred example (Example 1) can effectively increase the IFN-γ content in serum, and reduce the serum IL-4 and Laminin contents and the TGF-β1 content in the alveolar lavage fluid; increase the expression of E-cadherin and Cytokeratin 19 proteins in lung tissue, while reduce the expression of ColI, ColIII, α-SMA, Vimentin, Wnt3a, and β-catenin proteins, and also reduce the relative expression of Snail, Wnt3a, β-catenin, and TGF-β1 protein mRNA; have an inhibitory effect on inflammatory cell infiltration and fibrous tissue proliferation in the airway wall, alveolar cavity, and interstitial tissue, and significantly reduce the Masson percentage of fibrosis; significantly reduce collagen fiber deposition, the cell structure is relatively intact, and the basement membrane thickening is significantly alleviated. Therefore, animal experiments have shown that the Chinese medicine composition of the present invention (Bu Shen Tong Luo Fang) can reduce ECM deposition by inhibiting the EMT process of rat alveolar epithelial cells, regulate the IL-4 / IFN-γ balance, and thus improve bleomycin-induced pulmonary fibrosis in rats. Its mechanism may be related to its inhibition of the Wnt3a / β-catenin and TGF-β1 / Snail signaling pathways.

[0025] At the same time, the present invention establishes a cell model in which TGF-β1 intervenes in alveolar type II epithelial cells to cause excessive cell proliferation. The results of the cell experiments are as follows: (1) The serum containing the Chinese herbal composition of the present invention can more effectively inhibit the cell proliferation induced by 10ng / ml TGF-β1, and the serum containing the drug extracted from the blood taken 1 hour after 3 days of drug administration can inhibit the cell proliferation induced by TGF-β1 more than the serum containing the drug extracted from the blood taken 6 hours after 3 days of drug administration. (2) The wound healing percentages of cells containing the Chinese herbal composition of the present invention were all low at 4, 8, 12, and 24 hours after scratching, indicating that the Chinese herbal composition of the present invention can inhibit cell migration and repair; (3) The Chinese herbal composition of the present invention can promote cell apoptosis; (4) The Chinese herbal composition of the present invention can reduce the expression of TGF-β1, β-catenin, MMP-7 and MMP-9 proteins in fibroblasts; (5) The Chinese herbal composition of the present invention can reduce the relative expression of β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 protein mRNA in cells. Therefore, cell experiments have shown that the Chinese medicine composition of the present invention (Bu Shen Tong Luo Fang) can inhibit the proliferation and migration of rat alveolar epithelial cells and promote apoptosis, thereby improving TGF-β1-induced alveolar epithelial cell fibrosis. Its mechanism may be related to its inhibition of Wnt3a / β-catenin and TGF-β1 / Snail signaling pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Figure 2 is a graph showing the serum IFN-γ, IL-4, and BALFTGF-β1 levels in rats with pulmonary fibrosis;

[0028] Figure 2 This is a transmission electron microscopy image (×10 μm) of lung tissue in rats with pulmonary fibrosis;

[0029] Figure 3 Masson staining (Masson×200) and HE staining (HE×200) of lung tissue in rats with pulmonary fibrosis;

[0030] Figure 4 This is a graph of lung fibrosis area in rats with pulmonary fibrosis;

[0031] Figure 5 This is a graph of serum Laminin levels in rats with pulmonary fibrosis;

[0032] Figure 6 The staining images and average absorbance values ​​of ColI expression (×200) and ColIII expression (×200) in lung tissues of rats with pulmonary fibrosis;

[0033] Figure 7 This is the effect of Bushen Tongluo recipe on the localization and expression of E-cadherin and Cytokeratin 19 proteins in rat lung tissue (immunohistochemistry, ×200) and the average absorbance value;

[0034] Figure 8 This is the effect of Bushen Tongluo recipe on the localization and expression of α-SMA and Vimentin proteins in rat lung tissue (immunohistochemistry, ×200) and the average absorbance value;

[0035] Figure 9 The relative expression levels and band diagrams of Wnt3a and β-catenin proteins in rat lung tissues;

[0036] Figure 10 This is the scratch test picture;

[0037] Figure 11 is a picture of cell apoptosis;

[0038] Figure 12 Immunoblotting of MMP-7, MMP-9, TGF-β1, and β-catenin in fibroblasts. 1-1, 2-1, and 3-1 are blank groups, 1-2, 2-2, and 3-2 are model groups, 1-3, 2-3, and 3-3 are pirfenidone groups, 1-4, 2-4, and 3-4 are high-dose Chinese medicine groups, and 1-5, 2-5, and 3-5 are low-dose Chinese medicine groups.

[0039] Note: In the above figures, # compared with the blank control group, P < 0.05, ## compared with the blank control group, P < 0.01; * compared with the model group, P < 0.05, ** compared with the model group, P < 0.01, && compared with the pirfenidone group, P < 0.01, the differences are significant. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The process parameters for which specific conditions are not specified in the following examples are generally in accordance with conventional conditions.

[0041] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0042] The present invention will be further described in detail below with reference to specific examples and comparative examples.

[0043] All the Chinese medicinal raw materials used in the present invention comply with the description of the corresponding medicinal materials in the 2020 edition of the "Chinese Pharmacopoeia", especially the medicinal parts and the content of the effective ingredients. For example, the content of curculigoside (C22H26O11) in Curculigo must not be less than 0.080%. The raw materials used in the following examples are their corresponding medicinal material slices, which are all obtained by the processing method in the 2020 edition of the "Chinese Pharmacopoeia".

[0044] The Chinese medicine composition of the present invention can be prepared into the following dosage forms: granules, ointments, pills, injections, oral liquids, etc.

[0045] When preparing different dosage forms, corresponding excipients such as fillers, binders, disintegrants, lubricants, absorbents and diluents are added. The preparation of different dosage forms can be prepared by conventional methods in the art, which will not be described in detail here. The following test examples use decoctions or concentrates prepared using the raw materials (medicinal material slices) of the present invention, and the specific preparation methods are as follows:

[0046] Specific preparation methods for animal or cell experiments:

[0047] The raw material medicine (medicinal material slice) of embodiment 1 is sourced from Hebei Shennong Beijing Pharmaceutical Co., Ltd., and the above medicine is prepared into a concentrate by the preparation room of Xiyuan Hospital of China Academy of Chinese Medical Sciences. The Chinese medicine flavor of embodiment 1 is decocted twice. The first time, 8 times of water (8 times of the amount of crude drug) is added and decocted for 1.5 hours. The second time, 6 times of water is added and decocted for 1 hour. The mixture is filtered, the filtrate is combined, and concentrated into an extract (i.e., a concentrate, a fluid extract), and packaged. Each gram of concentrate contains 2.94g of crude drug, and the concentrate concentration is 1g / ml (i.e., 1g of crude drug is contained in each milliliter of concentrate).

[0048] Preparation method of clinical prescription:

[0049] In Example 1, Comparative Example 1, and Comparative Example 2, the raw materials (medicinal material slices) were all sourced from Hebei Shennong Beijing Pharmaceutical Co., Ltd. and prepared into water decoctions by the decoction room of Xiyuan Hospital, China Academy of Chinese Medical Sciences. Each dose of medicine was decocted twice with 500 ml of water, that is, 500 ml of water was added to each dose of medicine and decocted once, and then 500 ml of water was added to each dose of medicine and decocted once again. The two decoctions were decocted for a total of 1.5 hours, and finally 400 ml of water decoction was decocted per dose. The raw material formulas given in the following examples and comparative examples are all the dosages of one dose of medicine.

[0050] Example 1

[0051] Weigh the following raw materials according to the weight ratio: Curculigo 10g, Epimedium 10g, Astragalus 30g, Earthworm 12g, Clemati 15g, Trachelospermi Caulis 15g, Kadura Caulis 15g, Curcuma 10g, Polygonum cuspidatum 12g, Fritillaria thunbergii 20g, Ophiopogon japonicus 30g, and Licorice 6g.

[0052] Example 2

[0053] Weigh the following raw materials according to the weight ratio: Curculigo 10g, Epimedium 10g, Astragalus 30g, Earthworm 10g, Clemati 15g, Trachelospermi Caulis 15g, Kadura Caulis 15g, Curcuma 10g, Polygonum cuspidatum 13g, Fritillaria thunbergii 20g, Ophiopogon japonicus 30g, and Licorice 6g.

[0054] Example 3

[0055] Weigh the following raw materials according to the weight ratio: Curculigo 12g, Epimedium 20g, Astragalus 45g, Earthworm 15g, Clematidis 30g, Trachelospermi Caulis 10g, Kadura Caulis 10g, Curcuma 12g, Polygonum cuspidatum 14g, Fritillaria thunbergii 15g, Ophiopogon japonicus 20g, and Licorice 6g.

[0056] Example 4

[0057] Weigh the following raw materials according to the weight ratio: Curculigo 10g, Epimedium 10g, Astragalus 30g, Earthworm 12g, Clematidis 15g, Trachelospermi Caulis 15g, Kadura Caulis 15g, Curcuma 10g, Polygonum cuspidatum 15g, Fritillaria thunbergii 20g, Ophiopogon japonicus 30g, and Licorice 6g.

[0058] Comparative Example 1

[0059] Weigh the following raw materials according to the weight ratio: earthworm 15g, zedoary 10g, clematis 20g, epimedium 15g, curculigo 15g, inula flower 15g, roasted astragalus 60g, pinellia 10g, dried ginger 10g, scutellaria 10g, trillium 10g, and schisandra 10g.

[0060] Comparative Example 2

[0061] Weigh the following raw materials according to the weight ratio: earthworm 12g, zedoaria 10g, clematis 15g, epimedium 10g, polygonum cuspidatum 10g, trachelospermum 15g, astragalus 30g, raw coix seed 30g, luffa 15g, and millettia 15g.

[0062] Clinical trial examples

[0063] The Chinese medicine compositions of Example 1, Comparative Example 1 and Comparative Example 2 were prepared into water decoctions according to the aforementioned method and used for the treatment of cases diagnosed by clinical Western medicine as diffuse interstitial lung disease or idiopathic pulmonary fibrosis. It was found that: (1) the scutellaria baicalensis used for expectoration and the schisandra chinensis used for descending qi and consolidating the lungs in Comparative Example 1 were replaced by the thunbergia thunbergii which is an expectorant and dispersing stagnation drug; the three-edged sword was omitted; the pinellia ternata, dried ginger and scutellaria baicalensis which have the effects of drying dampness and resolving phlegm and clearing lung heat in Comparative Example 1 were replaced by the thunbergia thunbergii combined with ophiopogon japonicus which are an expectorant and moistening drug; the roasted astragalus root was replaced by astragalus root, and Piper mesenteriae and liquorice were added to obtain the formula of Example 1, which was more excellent in clinical performance, especially in improving the signs of cyanosis of the lips and sublingual blood stasis of the patients. The symptoms of dry mouth, dry throat and coughing up sticky phlegm of the patients were significantly relieved, and the symptoms of wheezing and shortness of breath were improved.

[0064] (2) The raw coix seed with the effect of removing dampness and resolving phlegm in comparative example 2 was replaced with thunbergia bulb combined with ophiopogon japonicus, which has the effect of clearing heat, resolving phlegm and moistening the lungs; the loofah and Spatholobi vine in comparative example 2 were replaced with Piper mesenteriae; and curculigo and licorice were added to obtain the formula of Example 1. The clinical performance was better, especially in improving the signs of cyanosis of the lips and sublingual hemostasis of the patients. The symptoms of dry mouth, dry throat, coughing up sticky phlegm of the patients were significantly relieved, and the symptoms of wheezing and shortness of breath were improved.

[0065] Here are a few specific cases:

[0066] Case 1, a 57-year-old male, visited our clinic on March 26, 2024; Chief complaint: wheezing and coughing for over 7 years. Current medical history: 7 years ago, wheezing and dry cough developed without obvious cause, but the patient ignored them. 1 month ago, wheezing and coughing worsened due to a cold, and he is now being treated in our outpatient clinic. Current symptoms: wheezing, cough with scant sputum, chest tightness and shortness of breath, sore waist and weak legs, dry mouth and throat, cyanotic lips, good appetite and sleep, 2 urinations at night, 1 soft bowel movement daily. Pale and dark tongue with a light yellow and slightly greasy coating, sublingual blood stasis, weak radial pulse, slippery cun pulse. Past medical history: denied. Auxiliary examinations: SpO2 (transcutaneous oxygen saturation) 90%; Lung CT scan on March 12, 2024: 1. Bilateral pulmonary interstitial fibrosis, 2. Bilateral emphysema, 3. Multiple small nodules in both lungs, 4. Bilateral pulmonary cords, 5. Calcification in the right lung, 6. Bilateral pleural thickening; Western medicine diagnosis: diffuse interstitial lung disease;

[0067] Take the water decoction prepared from the raw material medicine of Example 1, 1 dose per day, 2 times per day, 200 ml per time;

[0068] Effect after taking the medicine: The patient's wheezing was relieved half a month after taking the medicine. After taking the medicine for 3 months, the cough was relieved, occasional chest tightness and shortness of breath, dry mouth and throat disappeared, sublingual blood stasis was relieved, cyanosis of the lips was relieved, appetite and sleep were good, nocturia disappeared, bowel movements were once a day, the quality was moderate, SpO295%.

[0069] Case 2, male, 60 years old, visited the hospital on March 12, 2024; Chief complaint: intermittent cough for more than 2 months. Current medical history: Cough and sputum appeared without obvious cause in March 2024. The yellow sputum turned into white sticky sputum, which was easy to cough up. The throat itched and coughed. There was no obvious aggravation when inhaling cold air and irritating odors. Current symptoms: cough, coughing up white sticky sputum, occasional chest tightness and shortness of breath, dry mouth, fatigue, chills, good appetite and sleep, normal bowel movements, dark red tongue with thin and greasy coating and little fluid, stringy pulse, weak radial pulse. Past history: chronic obstructive pulmonary disease. Auxiliary examinations: lung CT: interstitial changes in both lungs; Western medicine diagnosis: diffuse interstitial lung disease;

[0070] Take the water decoction prepared from the raw material medicine of Example 1, 1 dose per day, 2 times per day, 200 ml per time;

[0071] Effect after taking the medicine: The patient felt that the cough and sputum were relieved after taking the medicine for 1 week, the symptoms of coughing up sticky sputum improved, the sputum could be coughed up, the chest tightness and shortness of breath were relieved, the dry mouth was improved, and the fear of cold was improved. After taking the medicine for 3 months, the patient basically stopped coughing, coughing up sticky sputum improved, sublingual blood stasis was relieved, dry mouth improved, chest tightness and shortness of breath improved, and the tongue coating changed from dark red to light red, indicating that the symptoms of blood stasis were improved.

[0072] Case 3, male, 73 years old, visited the hospital on April 14, 2024; Chief complaint: wheezing and shortness of breath for more than 3 years. Current medical history: The patient was diagnosed with interstitial lung disease 3 years ago. He is now wheezing and shortness of breath, which worsens after activity. His blood oxygen level is about 70% at rest and 91-92% at rest after oxygen inhalation. He also has cough, white sticky sputum, chills, cold hands and feet, loss of appetite, poor sleep, incontinence, pale tongue with thin and greasy coating, and weak radial pulse. Past medical history: postoperative rectal cancer, hypertension, coronary heart disease, cauda equina syndrome; Auxiliary examinations: 2024.5.12 lung CT: 1. Bilateral interstitial fibrosis, consistent with UIP manifestations, 2. Chronic bronchitis, emphysema, and bullae, 3. Patchy consolidation and calcification in the right upper lobe of the lung, which is considered to be chronic inflammation or old lesions, 4. Enlarged mediastinal lymph nodes; Western medicine diagnosis: idiopathic pulmonary fibrosis;

[0073] Take the water decoction prepared from the raw material medicine of Example 1, 1 dose per day, 2 times per day, 200 ml per time;

[0074] Effect after taking the medicine: After taking the medicine for 1 week, the patient felt that coughing and sputum were relieved, chest tightness and shortness of breath were relieved, dry mouth was improved, and chills were improved. The patient took the medicine for 3 months. After that, the patient occasionally coughed up sputum, occasionally had chest tightness and shortness of breath, no dry mouth, chills disappeared, and the pale and dark tongue turned into a light red tongue, indicating that the symptoms of blood stasis had improved.

[0075] Case 4, a 50-year-old female, presented on March 20, 2015. Chief complaint: dry cough for over six months. Presenting medical history: dry cough, sticky phlegm in the throat, chest tightness, shortness of breath, abdominal distension, epigastric pain, dark red tongue with a greasy white coating, slippery pulse, and weak radial pulse. Past medical history: denied. Auxiliary examination: Lung CT showed bilateral interstitial changes. Western medical diagnosis: diffuse interstitial lung disease.

[0076] Take the water decoction prepared from the raw material medicine of Comparative Example 1, 1 dose per day, 2 times per day, 200 ml per time;

[0077] Effect after taking the medicine: After taking the medicine for 1 month, the patient's dry cough, chest tightness and shortness of breath were alleviated, and abdominal distension and epigastric pain were relieved. After taking the medicine for 3 months, abdominal distension and epigastric pain were relieved. After that, the patient's dry cough, chest tightness and shortness of breath symptoms still existed, there was still sticky phlegm in the throat that was difficult to cough up, and the dark red tongue was not significantly relieved.

[0078] Case 5: Male, 68 years old, visited the hospital on September 18, 2023; Chief complaint: intermittent cough for more than 3 months. Current medical history: Cough and sputum appeared without obvious cause in June 2023, coughing up a small amount of white sticky sputum, which was difficult to cough up, severe cough at night, no obvious aggravation by inhaling cold air and irritating odors, chest tightness and shortness of breath. Current symptoms: cough, coughing up a small amount of white sticky sputum, which was difficult to cough up, occasional chest tightness and shortness of breath, dry mouth, abdominal distension, poor appetite, good sleep, regular urination, loose stools, dark red tongue with white greasy coating, slippery pulse, weak radial pulse. Past history: gastroesophageal reflux disease. Auxiliary examinations: Lung CT: interstitial changes in both lungs; Western medicine diagnosis: diffuse interstitial lung disease;

[0079] Take the water decoction prepared from the raw material medicine of Comparative Example 1, 1 dose per day, 2 times per day, 200 ml per time;

[0080] Effect after taking the medicine: After taking it for 3 weeks, the patient felt that his cough was relieved, sputum could be coughed up, and dry mouth was improved compared to before. He took the medicine for a total of 3 months. After that, the patient still had a cough, still coughed up sticky sputum, still had dry mouth, dark red tongue did not improve, abdominal distension improved, and appetite improved.

[0081] Case 6: Male, 68 years old, visited the hospital on September 25, 2023; Chief complaint: intermittent cough for more than 2 months. Current medical history: Cough and sputum appeared without obvious cause in July 2023, coughing up white sticky sputum, occasionally yellow sputum in the morning, easy to cough up with large amount, coughing with itchy throat, chest tightness. Current symptoms: cough, coughing up white sticky sputum, easy to cough up with large amount, occasionally yellow sputum in the morning, chest tightness, dry mouth, abdominal distension, poor appetite, good sleep, regular urination, loose stools, dark red tongue with white greasy and slightly yellow fur, slippery pulse, weak radial pulse. Past history: gastroesophageal reflux disease. Auxiliary examinations: Lung CT: grid shadows in both lower lungs, consistent with interstitial lung changes; Western medicine diagnosis: diffuse interstitial lung disease;

[0082] Take the water decoction prepared from the raw material medicine of Comparative Example 1, 1 dose per day, 2 times per day, 200 ml per time;

[0083] Effect after taking the medicine: After taking the medicine for 3 weeks, the patient felt that his cough and sputum were relieved, his dry mouth was improved, and his abdominal distension was relieved. After taking the medicine for 3 months, the patient still had a cough, the amount of sputum was reduced, but there was still white sticky sputum that was difficult to cough up, no abdominal distension, good appetite, no chest tightness, formed stool, and dark red tongue that had not been relieved.

[0084] Case 7, a 66-year-old female, presented on March 20, 2024. Chief complaints: hand joint pain for 5 years and shortness of breath for over 2 months. Presenting medical history: chest tightness, shortness of breath, sticky and difficult-to-expect phlegm in the throat, dry mouth, swelling, pain, and numbness in the joints of both hands, aggravated by activity, morning stiffness lasting 20 minutes, general fatigue, poor appetite and sleep, regular urination, unformed stools, red tip of tongue with white fur, sublingual blood stasis, slippery pulse, weak radial pulse. Past medical history: rheumatoid arthritis. Ancillary examination: Lung CT showed bilateral interstitial changes. Western medical diagnosis: diffuse interstitial lung disease.

[0085] Take the water decoction prepared from the raw material medicine of Comparative Example 2, 1 dose per day, 2 times per day, 200 ml per time;

[0086] Effect after taking the medicine: After taking it for 3 months, the patient's chest tightness and shortness of breath were relieved, but he still coughed up sticky phlegm. The sublingual blood stasis was not relieved significantly, the dry mouth was not relieved significantly, the swelling and numbness of the joints of both hands were relieved, and the fatigue was relieved.

[0087] Case 8, a 50-year-old female, presented on October 16, 2023; Chief complaint: Six years of hand joint pain and six months of shortness of breath. Present medical history: Chest tightness, shortness of breath, coughing after catching a cold, coughing up sticky yellowish-white sputum, frequent coughing, dry mouth, tenderness, numbness, and limited mobility in both metacarpophalangeal joints and both knees, stiffness lasting one hour daily in the morning, poor appetite and sleep, regular urination, unformed stools, dark red tongue with a thin, greasy, yellow coating, and a stringy, thready pulse. Past medical history: Rheumatoid arthritis. Auxiliary examinations: Lung CT showed reticular shadows in both lower lungs and minimal traction bronchiectasis, consistent with bilateral interstitial changes. Western medical diagnosis: Diffuse interstitial lung disease.

[0088] Take the water decoction prepared from the raw material medicine of Comparative Example 2, 1 dose per day, 2 times per day, 200 ml per time;

[0089] Effect after taking the medicine: After taking it for 3 months, the patient's chest tightness and shortness of breath were relieved, the amount of yellow and white sticky sputum coughed up was reduced, but it was still difficult to cough up, the dark red tongue was not significantly relieved, and there was still dry mouth. The swelling, numbness and limited movement of the joints of both hands were relieved, and the morning stiffness time was reduced.

[0090] Case 9, a 66-year-old female, presented on October 30, 2023; Chief complaint: Hand joint pain for three years, wheezing and shortness of breath for one month. Present medical history: Chest tightness, shortness of breath, worsened by activity, phlegm in the throat, coughing up small amounts of yellow, sticky phlegm, dry mouth, bitter taste, swelling and pain in both wrists and elbows, limited mobility, stiffness for half an hour daily in the morning, normal appetite and sleep, regular bowel movements, dark red tongue with a thin, greasy, yellow coating, and a thready pulse. Past medical history: Rheumatoid arthritis. Auxiliary examinations: Lung CT showed lattice shadows in the lower lungs, consistent with bilateral interstitial changes. Western medical diagnosis: Diffuse interstitial lung disease.

[0091] Take the water decoction prepared from the raw material medicine of Comparative Example 2, 1 dose per day, 2 times per day, 200 ml per time;

[0092] Effect after taking the medicine: After taking it for 3 months, the patient's chest tightness and shortness of breath were relieved, but coughing up yellow sticky sputum did not show obvious relief, and the patient still had dry mouth and bitter taste, and the tongue was still dark red. The swelling and pain in the wrist and elbow joints were relieved.

[0093] The above cases are only a subset of clinical observations, and the pharmaceutical composition used in Example 1 is also only a subset of the pharmaceutical composition of the present invention. In actual clinical use, the dosage of the specific drugs in the pharmaceutical composition can be arbitrarily selected within the scope of the present invention according to the different clinical symptoms of the patients. However, if one or more drugs are replaced, omitted, or added, the clinical efficacy is not as good as that of the present invention.

[0094] Testing and result analysis of animal and cell experiments

[0095] 1. Animal Experiments

[0096] 1.1 Materials

[0097] 1.1.1 Animals

[0098] Fifty healthy male Sprague-Dawley rats, weighing (200 ± 10) g, were purchased from Spayfor (Speifo, license number: SCXK2019-0010) and housed in the animal room of Xiyuan Hospital, China Academy of Chinese Medical Sciences (license number: SYXK2018-0018). They were provided with regular water, 12 h of light, a humidity of (55 ± 5)%, and a temperature of (22 ± 2)°C. This experiment was approved by the Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences (license number: 2018XLC011-1).

[0099] 1.1.2 Medicinal materials and their preparation

[0100] Embodiment 1 (kidney-tonifying and collateral-draining prescription): Curculigo 10g, Herba Epimedii 10g, Radix Astragali 30g, Pheretima 12g, Radix Clematidis 15g, Caulis Trachelospermi 15g, Caulis Kaduradi 15g, Rhizoma Curcumae 10g, Polygonum cuspidatum 12g, Bulbus Fritillariae Thunbergii 20g, Radix Ophiopogonis 30g, Radix Glycyrrhizae 6g.The crude drug (medicinal material slice) source is Hebei Shennong Beijing Pharmaceutical Co., Ltd., by the preparation room of Xiyuan Hospital, China Academy of Chinese Medical Sciences, above medicine is prepared into concentrated solution, embodiment 1 Chinese medicine taste, decoct altogether 2 times, add 8 times of water (8 times of crude drug amount) for the first time and decoct 1.5h, add 6 times of water and decoct 1h for the second time, filter, merging filtrate, be condensed into extractum (i.e. concentrated solution, fluid extract), package.Contain crude drug 2.94g in every g concentrated solution, concentrated solution concentration is 1g / ml (i.e. containing crude drug 1g in every milliliter of concentrated solution).

[0101] 1.1.3 Drugs, reagents, and instruments

[0102] Pirfenidone capsules (National Drug Approval No.: H20133376, Beijing Contini Pharmaceutical Co., Ltd.); Bleomycin (Cat. No.: r25001-8, Invitrogen, USA); Laminin kit (Cat. No.: Ab119573, abcam, USA), TGF-β1 kit (Cat. No.: ERC107b, Beijing Xinbosheng Biotechnology Co., Ltd.), IFN-γ kit (Cat. No.: ERC101G, Beijing Xinbosheng Biotechnology Co., Ltd.), IL-4 kit (Cat. No.: ERC002, Beijing Xinbosheng Biotechnology Co., Ltd.); E-cadherin antibody (Cat. No.: ab231303, abcam, USA); Cytokeratin 19 antibody (catalog number: 10712-1-AP, Proteintech, USA); α-SMA antibody (catalog number: ab7817, abcam, USA); Vimentin antibody (catalog number: ab92547, abcam, USA); ColI antibody (catalog number: ab254113, abcam, USA); ColIII antibody (catalog number: ab7778, abcam, USA); Wnt3a antibody (catalog number: ab219412, abcam, USA); β-catenin antibody (catalog number: ab16051, abcam, USA). JB-P7 embedding machine (Wuhan Junjie Electronics Co., Ltd.); RM2235 pathology slicer (Leica, Germany); MIchrome5 Pro imaging system CCD (Fuzhou Xintu Optoelectronics Co., Ltd.); electrophoresis apparatus (Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.); MULTISKANMK3 fully automatic multifunctional microplate reader (Thermo Fisher Scientific, USA); NikonCi-SJY300C inverted microscope (Nikon Corporation, Japan), etc.

[0103] 1.2 Methods

[0104] 1.2.1 Grouping, modeling, and drug administration

[0105] The experiment was started after 50 SD rats were adaptively fed for one week and then randomly divided into a blank control group (Control), a model group (Model), a high-dose Bushen Tongluo Recipe group (BTD-H, a high-dose Chinese medicine group), a low-dose Bushen Tongluo Recipe group (BTD-L, a low-dose Chinese medicine group), and a pirfenidone group (Pifenidone), with 10 rats in each group. The model group, the high-dose and low-dose Bushen Tongluo Recipe groups, and the pirfenidone group used the following method for establishing the pulmonary fibrosis rat model: the rats were anesthetized with 3% sodium pentobarbital solution (0.3 ml / 100 g) ip, the rats were fixed in a supine position, the trachea was opened in an inverted T-shape, and a bleomycin A5 (0.5 mg / 100 g) aqueous solution was injected into the trachea at one time. After the injection, the rats were rotated upright and sutured. After 28 days, two rats were taken from each group and anesthetized with 3% sodium pentobarbital solution. The left lung tissue was taken and stained with hematoxylin-eosin (HE) and Masson. The lung tissue was observed under an optical microscope to see whether collagen fiber deposition and fibrosis changes appeared to evaluate the success of the pulmonary fibrosis model. After the model was successfully established, the high-dose and low-dose groups of Bushen Tongluo Recipe were given 30.88 g·kg -1 ·d -1 , 15.44g·kg -1 ·d -1 Oral administration of pirfenidone group: 0.11 g·kg -1 ·d -1 Oral gavage: the model group and the blank control group were given an equal amount of normal saline for 90 consecutive days, after which the samples were collected. In addition, the high-dose and low-dose groups of the Bushen Tongluo Recipe were gavaged with the amount of raw medicine used per kg of rat per day. For example, the high-dose group of the Bushen Tongluo Recipe was gavaged with 30.88g of raw medicine per kg of rat per day, and the low-dose group was gavaged with 15.44g of raw medicine per kg of rat per day. In addition, the low-dose group of the Bushen Tongluo Recipe was a human equivalent dose, which was converted to a low dose for rat administration based on the conversion coefficient of 0.018 between human and rat body surface area. The drug dosage of the pirfenidone group was 0.11g of pirfenidone capsule contents per kg of rat per day, which was dissolved in normal saline and then gavaged.

[0106] Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution (0.2 ml / 100 g). Blood was collected from the abdominal aorta and then sacrificed. 6-8 mL of blood was collected. The arterial blood was centrifuged at 4°C, 3000 r / min for 10 min to separate the serum and stored in a -20°C refrigerator for later use. The thoracic cavity was cut open, the right main bronchus was clamped, and the left lung was bronchoalveolar lavage was performed 5 mL × 6 times with normal saline. The recovery rate was more than 80%. The recovered fluid was concentrated in a sterile centrifuge tube and centrifuged at 4°C, 1200 r / min for 15 min. The supernatant was collected and stored in a -80°C refrigerator for later use. The right middle lobe of the lung was fixed in 10% neutral formaldehyde solution. Three right lower lobes of each group were cut into 1*1*1 mm pieces, placed in 2.5% pentanediol, and stored at 4°C. The remaining right lower lobes were divided into 2 mL cryovials and stored in a -80°C refrigerator.

[0107] 1.2.2 Index detection

[0108] 1.2.2.1 Elisa test

[0109] Elisa kits were used to measure IFN-γ, IL-4, and laminin levels in rat abdominal aorta serum and TGF-β1 levels in bronchoalveolar lavage fluid (BALF). The procedures were performed according to the kit instructions. The absorbance was measured at 450 nm using a microplate reader. Standard curves were drawn and sample concentrations were calculated.

[0110] 1.2.2.2 Pathological observation of rat lung tissue

[0111] Lung tissue was fixed with 10% neutral formaldehyde solution, embedded in paraffin, and sectioned at a thickness of 4 μm using conventional methods. Sections were then stained with HE and Masson staining. Changes in lung tissue structure and the distribution of collagen fibers were observed under a light microscope. The percentage of collagen fiber area (Masson percentage) was calculated using ImagesProPlus. Lung tissue was fixed with 2.5% glutaraldehyde, fixed, dehydrated, sectioned, and stained, and cellular ultrastructure was observed using a transmission electron microscope.

[0112] 1.2.2.3 Immunohistochemistry

[0113] Paraffin-embedded lung tissue was used to prepare tissue microarrays. The preparation method was carried out according to conventional methods. Three fields of view were photographed under a high-power microscope, and the average absorbance A of E-cadherin, Cytokeratin19, α-SMA, Vimentin, ColI, and ColIII positive staining was calculated using Gel Image ststemver.4.00 software.

[0114] 1.2.2.4 Western blot detection

[0115] The right lower lobe of the lung was obtained from cryopreserved tubes and subjected to standard Western blotting. Quantity One software was used to analyze the grayscale values ​​of Wnt3a, β-catenin, and β-actin, and the ratio of the grayscale values ​​of Wnt3a, β-catenin, and β-actin was calculated as the relative expression levels.

[0116] 1.2.2.5 RT-PCR detection

[0117] Total RNA was extracted from 50 mg of frozen lung tissue and dissolved in nuclease-free high-purity water; 2 μg of total RNA was reverse transcribed in a 20 μL reaction system (incubated at 42°C for 50 min and heated at 85°C for 5 min to terminate the reaction) to synthesize cDNA; 2 μL of 10-fold dilution of cDNA was used in a 20 μL reaction system for real-time fluorescence quantitative PCR (pre-denaturation at 95°C for 10 min, denaturation at 95°C for 5 s, and 60°C for 30 s, for a total of 40 cycles); 2 －△△CT Process the experimental data as relative mRNA expression levels.

[0118] 1.2.3 Statistical methods

[0119] SPSS 25.0 statistical software was used for statistical analysis, and the measurement data were expressed as mean ± standard deviation. Indicates that the normal distribution test and variance homogeneity test were first performed. If they obeyed the normal distribution, one-way analysis of variance was used; if they did not conform to the normal distribution, a non-parametric test (Wilcoxon rank sum test) was used. If p>0.05, the difference was not statistically significant; if p<0.05, the difference was statistically significant; if p<0.01, it was statistically significant.

[0120] 1.3 Experimental Results

[0121] 1.3.1 Effects of Bushen Tongluo Recipe on Pulmonary Inflammatory Factors and Fibrotic Factors in Rats with Pulmonary Fibrosis

[0122] The levels of serum inflammatory factors IFN-γ and IL-4 and the level of fibrosis factor TGF-β1 in bronchoalveolar lavage fluid were measured. The results of Elisa test were as follows: Figure 1 As shown:

[0123] Serum IFN-γ: Compared with the blank control group, the IFN-γ level in the model group was significantly decreased (p<0.05); compared with the model group, the IFN-γ levels in the high-dose and low-dose Bushen Tongluo prescription groups were significantly increased (p<0.05).

[0124] Serum IL-4: Compared with the blank control group, the IL-4 level in the model group was significantly increased (p < 0.05); compared with the model group, the IL-4 levels in the pirfenidone group and the high- and low-dose Bushen Tongluo prescription groups were significantly decreased (p < 0.01).

[0125] TGF-β1 in bronchoalveolar lavage fluid: Compared with the blank control group, the TGF-β1 level in the model group was significantly increased (p < 0.01); compared with the model group, the TGF-β1 levels in the high- and low-dose Bushen Tongluo prescription groups were significantly decreased (p < 0.01), and the TGF-β1 level in the pirfenidone group was significantly decreased (p < 0.05).

[0126] From the above, it can be seen that compared with pirfenidone, the Bushen Tongluo prescription has a better inhibitory effect on TGF-β1; and for the inflammatory factors IFN-γ and IL-4, the increase of IFN-γ and the decrease of IL-4 after medication are more significant, indicating that the inflammation level is better reduced.

[0127] 1.3.2 Effects of Bushen Tongluo Recipe on Lung Pathology in Rats with Pulmonary Fibrosis

[0128] Transmission electron microscopy of lung tissue showed that: Figure 2 As shown in the results, the structure of the blank control group was relatively complete, the edges of the alveolar epithelial cells were clear, the cells were not swollen, there was no inflammatory infiltration, and there was no basement membrane thickening; a large amount of collagen fiber deposition was observed in the lung tissue of the rats in the model group, the basement membrane was significantly thickened, and the cell structure was incomplete; the cell structure in the pirfenidone group was less complete, and collagen fiber deposition was observed, which was less than that in the model group; the collagen fiber deposition in the high- and low-dose Bushen Tongluo Recipe groups was significantly less than that in the model group, the cell structure was relatively complete, and the basement membrane thickening was significantly alleviated compared with the model group.

[0129] HE and Masson staining showed: Figure 3 As shown, the morphology of the bronchial mucosal epithelium and mucosal folds in rats in the blank control group remained normal, without dilatation. The smooth muscle layer of the bronchial wall was not thickened, and no significant inflammatory cell infiltration was observed in the alveolar interstitium or alveolar cavity. In the model group, residual secretions were present in the bronchial cavity, the smooth muscle layer of the bronchial wall was thickened, the mucosal folds were flattened, and the ciliated columnar epithelial cells in the mucosal epithelium degenerated and sloughed. Numerous inflammatory cells infiltrated the airway walls, alveolar cavities, and interstitial tissue, and fibrous tissue proliferation was evident. The high- and low-dose Bushen Tongluo Recipe groups and the pirfenidone group exhibited the pathological morphology of the model group, but with significantly less severe symptoms than those in the model group. The low-dose Bushen Tongluo Recipe group exhibited the greatest inhibitory effect on inflammatory cell infiltration and fibrous tissue proliferation in the airway walls, alveolar cavities, and interstitial tissue. As shown above, the Bushen Tongluo Recipe can inhibit inflammatory cell infiltration and fibrous tissue proliferation in lung tissue.

[0130] The degree of fibrosis in rats (Masson staining): Figure 4As shown in the results, compared with the blank control group, the Masson percentage of fibrosis in the rats in the other groups was significantly increased (p < 0.01); compared with the model group, the Masson percentage of fibrosis in the rats in the pirfenidone group and the high-dose Bushen Tongluo recipe group was significantly decreased (p < 0.01), and the Masson percentage of fibrosis in the rats in the low-dose Bushen Tongluo recipe group was significantly decreased (p < 0.05); there was no difference in the Masson percentage of fibrosis in the rats in the other groups. This shows that Bushen Tongluo recipe can inhibit the proliferation of fibrous tissue.

[0131] 1.3.3 Effects of Bushen Tongluo Recipe on Extracellular Matrix and Lung Collagen Levels in Rats with Pulmonary Fibrosis

[0132] Laminin (LN) is an indicator of extracellular matrix deposition (ECM), reflecting the degree of excessive extracellular matrix deposition. The results of ELISA detection are as follows: Figure 5 As shown; the level of type I collagen and type I and II collagen, namely ColI expression and ColII expression, can also reflect the degree of fibrosis. The positive color development and the calculation results of the average absorbance A are shown in Figure 6 shown; specifically:

[0133] Laminin content in serum: Figure 5 As shown in the data, compared with the blank control group, the serum LN levels in the model group and pirfenidone group were significantly increased (p < 0.01), and the serum LN level in the high-dose Chinese medicine group was also significantly increased (p < 0.05); compared with the model group, the serum LN level in the low-dose Chinese medicine group was significantly decreased (p < 0.05).

[0134] ColI expression in lung tissue: Figure 6 The color was brown-yellow, and the coloring area was mainly concentrated around the bronchial wall and alveolar interstitium; compared with the blank control group, the type I collagen levels in the model group, pirfenidone group, and high- and low-dose Bushen Tongluo prescription groups were significantly increased (p < 0.01); compared with the model group, the type I collagen levels in the pirfenidone group, and high- and low-dose Bushen Tongluo prescription groups were significantly decreased (p < 0.01).

[0135] ColⅢ expression in lung tissue: Figure 6 The staining was brown-yellow, mainly concentrated around the bronchial wall and alveolar interstitium; compared with the blank control group, the level of type III collagen in the model group, pirfenidone group and high- and low-dose Bushen Tongluo prescription groups was significantly increased (p < 0.01); compared with the model group, the level of type III collagen in the high- and low-dose Bushen Tongluo prescription groups and pirfenidone group was significantly decreased (p < 0.05).

[0136] It can be seen from this that the Bushen Tongluo recipe can reduce serum LN, indicating that the Bushen Tongluo recipe has an inhibitory effect on LN and can avoid excessive deposition of extracellular matrix; at the same time, the Bushen Tongluo recipe can reduce the expression of ColI and ColIII in lung tissue, indicating that the Bushen Tongluo recipe has an inhibitory effect on ColI and ColIII and can reduce the degree of fibrosis.

[0137] 1.3.4 Effects of Bushen Tongluo Recipe on Epithelial and Interstitial Marker Proteins in Lung Tissue

[0138] The epithelial marker proteins E-cadherin and Cytokeratin 19 and the mesenchymal marker proteins α-SMA and Vimentin in lung tissue can illustrate epithelial-mesenchymal transition (EMT). The positive color development and the calculation results of the average absorbance A are shown in the following table. Figure 7 、 8 shown; specifically:

[0139] E-cadherin expression in lung tissue: Figure 7 The staining was brown-yellow and mainly concentrated in the cytoplasm of the bronchial mucosal epithelium; compared with the blank control group, the E-cadherin levels in the model group, pirfenidone group, and high- and low-dose Bushen Tongluo prescription groups were significantly decreased (p<0.01); compared with the model group, the E-cadherin levels in the pirfenidone group, and high- and low-dose Bushen Tongluo prescription groups were significantly increased (p<0.05).

[0140] Cytokeratin 19 expression in lung tissue: Figure 7 The staining was brown-yellow and mainly concentrated in the cytoplasm of the bronchial mucosal epithelium. Compared with the blank control group, the levels of Cytokeratin 19 in the model group, pirfenidone group, and high- and low-dose Bushen Tongluo prescription groups were significantly decreased (p<0.01). Compared with the model group, the levels of Cytokeratin 19 in the pirfenidone group and high- and low-dose Bushen Tongluo prescription groups were significantly increased (p<0.05).

[0141] α-SMA expression in lung tissue: Figure 8 The α-SMA was shown to be brownish yellow in the middle, and the colored areas were concentrated in the interstitial fibrous tissue and occasionally expressed in the epithelial cells. Compared with the blank control group, the α-SMA level in the model group was significantly increased (p<0.01); compared with the model group, the α-SMA level in the pirfenidone group and the high- and low-dose Bushen Tongluo prescription groups was significantly decreased (p<0.01).

[0142] Vimentin expression in lung tissue: Figure 8The staining was brown-yellow, and the colored area was concentrated in the interstitial fibrous tissue, and occasionally expressed in epithelial cells; compared with the blank control group, the Vimentin level in the model group was significantly increased (P < 0.01); compared with the model group, the Vimentin level in the pirfenidone group and the high- and low-dose Chinese medicine groups was significantly decreased (P < 0.01).

[0143] As shown above, the Bushen Tongluo formula can increase the expression of the epithelial marker proteins E-cadherin and Cytokeratin 19 in lung tissue, indicating that the Bushen Tongluo formula has a promoting effect on the epithelial marker proteins E-cadherin and Cytokeratin 19. Simultaneously, the Bushen Tongluo formula can also reduce the expression of the interstitial marker proteins α-SMA and Vimentin in lung tissue, indicating that the Bushen Tongluo formula has an inhibitory effect on the interstitial marker proteins α-SMA and Vimentin. The increase and decrease in the expression of these two proteins demonstrate that the Bushen Tongluo formula can inhibit the EMT process.

[0144] 1.3.5 Effects of Bushen Tongluo Recipe on Wnt3a and β-catenin Protein in Lung Tissue

[0145] like Figure 9 As shown in the results, compared with the blank control group, the Wnt3a level in the model group was significantly increased (p < 0.05); compared with the model group, the Wnt3a level in the low-dose Bushen Tongluo Recipe group was significantly decreased (p < 0.05). At the same time, compared with the blank control group, the β-catenin level in the model group was significantly increased (p < 0.01); compared with the model group, the β-catenin levels in the pirfenidone group and the high- and low-dose Bushen Tongluo Recipe groups were significantly decreased (p < 0.01). These results indicate that the Bushen Tongluo Recipe can reduce the expression of the pathway proteins Wnt3a and β-catenin in lung tissue, indicating that the Bushen Tongluo Recipe has an inhibitory effect on the pathway proteins Wnt3a and β-catenin and can inhibit pulmonary fibrosis.

[0146] 1.3.6 Analysis of relative mRNA expression of Snail, Wnt3a, β-catenin, and TGF-β1 proteins in rat lung tissue

[0147] The relative expression levels of Snail, Wnt3a, β-catenin and TGF-β1 protein mRNA in rat lung tissue are shown in Table 1:

[0148] Table 1: Analysis results of relative expression levels of Snail, Wnt3a, β-catenin and TGF-β1 protein mRNA

[0149]

[0150] Note: # compared with blank control group, P<0.05, ## compared with blank control group, P<0.01; * compared with model group, P<0.05, ** compared with model group, P<0.01, the differences are significant.

[0151] The above animal experiments proved that Bushen Tongluo prescription can reduce ECM deposition by inhibiting the EMT process of rat alveolar epithelial cells and regulating the IL-4 / IFN-γ balance, thereby improving bleomycin-induced pulmonary fibrosis in rats. Its mechanism may be related to its inhibition of Wnt3a / β-catenin and TGF-β1 / Snail signaling pathways.

[0152] 2. Cell experiments

[0153] 2.1 Preparation of drug-containing serum:

[0154] 30 SPF grade SD rats, male, were randomly divided into 5 groups, namely Chinese medicine group 1, Chinese medicine group 2, western medicine group, normal saline group 1, normal saline group 2, with 6 rats in each group; among them, Chinese medicine group 1 and Chinese medicine group 2 were gavaged with 30.88g / (kg·d) and 15.44g / (kg·d) of the Bushen Tongluo recipe of Example 1, respectively, that is, Chinese medicine group 1 was gavaged with 30.88g of crude drug per kg rat per day, and Chinese medicine group 2 was gavaged with 15.44g of crude drug per kg rat per day. The gavage medication of Chinese medicine group 1 and Chinese medicine group 2 was obtained according to the specific preparation method of the prescription in the aforementioned animal experiment or cell experiment or the steps in 1.1.2 medicinal materials and their preparation; the western medicine group was gavaged with 110mg / (kg·d), that is, per kg rat per day. Rats were orally gavaged with 110 mg of the contents of pirfenidone capsules, which were dissolved in normal saline and then gavaged. 1 ml of the solution was gavaged for every 100 g of rats, and the total amount of the contents of the pirfenidone capsules in each kg of rat gavage solution was 110 mg; normal saline group 1 and normal saline group 2 were gavaged with normal saline at 1 ml / 100 g; all five groups of rats were gavaged with the corresponding solution, once a day, for 3 consecutive days. They fasted for 12 hours before blood collection, and blood was collected from the abdominal aorta 1 hour and / or 6 hours after the last gavage. The blood was collected and centrifuged at 3000 r / min for 10 minutes. The top layer of serum was aspirated into a 2 ml EP tube, and the serum was inactivated in a 56°C constant temperature water bath for 30 minutes. The serum was filtered and sterilized with a 0.22 μm microporous filter to collect the serum of each group of rats. The collected rat drug-containing serum of the Western medicine group, the Chinese medicine group 1 and the Chinese medicine group 2 were respectively prepared on the basis of EMEM basal medium into medium containing 10% of rat drug-containing serum of pirfenidone, high-dose Bushen Tongluo Recipe and low-dose Bushen Tongluo Recipe respectively, i.e., EMEM basal medium with 10% volume concentration of rat drug-containing serum, referred to as 10% drug-containing serum medium, including 10% pirfenidone drug-containing rat serum medium (pirfenidone drug-containing serum medium), 10% high-dose Bushen Tongluo Recipe drug-containing rat serum medium (Chinese medicine high-dose drug-containing serum medium), and 10% low-dose Bushen Tongluo Recipe drug-containing rat serum medium (Chinese medicine low-dose drug-containing serum medium), for later use. At the same time, the collected rat blank serum of the normal saline group 1 and / or the normal saline group 2 were prepared on the basis of EMEM basal medium into medium with a volume concentration of 10% of rat blank serum, i.e., EMEM basal medium with 10% volume concentration of rat blank serum, referred to as 10% rat blank serum medium, for later use. The EMEM basal culture medium used in the present invention was purchased from Beina Chuanglian Biotechnology Co., Ltd. in the mall.

[0155] 2.2 Experimental cells:

[0156] RLE-6TN rat alveolar type II epithelial cells.

[0157] 2.3 Experimental steps and MTT assay (to detect cell proliferation):

[0158] 2.3.1 Cell culture:

[0159] RLE-6TN rat alveolar type II epithelial cells were digested with trypsin and resuspended in 0.5% fetal bovine serum medium. They were seeded in a 96-well culture plate with 100 μl of cell suspension per well (containing 300 RLE-6TN rat alveolar type II epithelial cells). The culture plate was cultured in a 37°C, 5% carbon dioxide incubator. When the cells filled the well plate, the old culture medium was gently aspirated and replaced with serum-free medium (EMEM basal medium) and cultured for 24 hours. There were approximately 500 RLE-6TN rat alveolar type II epithelial cells per well. The serum-free medium was aspirated and 100 μl of the corresponding spare culture medium in 2.1 above was added to each well for culture and related experiments.

[0160] 2.3.2 Experiment 1: Screening the modeling concentration of TGF-β1

[0161] The cells were cultured according to the experimental steps in 2.3.1. After aspirating the serum-free culture medium, 100 μl of 10% rat blank serum culture medium was added to each well (approximately 500 RLE-6TN rat alveolar type II epithelial cells). After 24 hours, the culture medium was replaced with 0.5% fetal bovine serum. The RLE-6TN rat alveolar type II epithelial cells on the 96-well culture plate were randomly divided into a blank control group and an experimental group. The experimental groups included a 5 ng / ml TGF-β1 group, a 10 ng / ml TGF-β1 group, a 15 ng / ml TGF-β1 group, a 20 ng / ml TGF-β1 group, a 25 ng / ml TGF-β1 group, a 30 ng / ml TGF-β1 group, and a 40 ng / ml TGF-β1 group.

[0162] Except for the blank control group, each experimental group was treated with the corresponding concentration of TGF-β1 and cultured for 24 hours. The cells were then tested using the MTT assay. MTT was added, the cells were cultured in the dark for another 4 hours, and the absorbance (A) was measured at 492 nm using a microplate reader. The experiment was repeated three times. The test results are shown in Table 2.

[0163] Table 2

[0164]

[0165] *Compared with the blank control group, P < 0.05; #Compared with the 20 ng / ml TGF-β1 group, P < 0.05.

[0166] As can be seen from Table 2, 5-40ng / ml TGF-β1 can induce cell proliferation, so 5ng / ml, 10ng / ml, and 15ng / ml TGF-β1 were used to induce cells, and then observe whether the drug-containing serum can inhibit TGF-β1-induced cell proliferation.

[0167] 2.3.3 Experiment 2: Observe whether drug-containing serum can inhibit TGF-β1-induced cell proliferation

[0168] The cells were cultured according to the experimental steps in 2.3.1. After aspirating the serum-free medium, the RLE-6TN rat alveolar type II epithelial cells on the 96-well culture plate were randomly divided into a blank control group, a model group, a high-dose Chinese medicine group, a low-dose Chinese medicine group, and a pirfenidone group, with approximately 500 RLE-6TN rat alveolar type II epithelial cells in each well.

[0169] The blank control group and the model group were cultured with 100 microliters of 10% rat blank serum culture medium, respectively. The high-dose Chinese medicine group was cultured with 100 microliters of 10% Chinese medicine high-dose serum culture medium 1 hour and 6 hours after three days of drug administration. The low-dose Chinese medicine group was cultured with 100 microliters of 10% Chinese medicine low-dose serum culture medium 1 hour and 6 hours after three days of drug administration. The pirfenidone group was cultured with 100 microliters of 10% pirfenidone serum culture medium 1 hour and 6 hours after three days of drug administration. After 24 hours of intervention in each group, except for the blank control group, the other groups were induced with 5ng / ml, 10ng / ml, and 15ng / ml of TGF-β1 for 24 hours, and then detected by MTT method, that is, MTT was added, and the cells were cultured in the dark for 4 hours. The absorbance (A) was measured at 492nm with a microplate reader. The experiment was repeated 3 times, and the results are as follows:

[0170] Results (1): After 6 hours of drug administration for three days, the drug-containing serum of each group could not inhibit the cell proliferation induced by 5ng / ml TGF-β1. See Table 3 for details:

[0171] Table 3

[0172]

[0173] *P<0.05 compared with the blank control group.

[0174] Results (2): After 6 hours of administration, high-dose serum containing Chinese medicine can inhibit cell proliferation induced by 10ng / ml TGF-β1. See Table 4 for details:

[0175] Table 4

[0176]

[0177] *P<0.05 compared with the blank control group, #P<0.05 compared with the model group.

[0178] Results (3): Three days after drug administration, the drug-containing serum of each group was unable to inhibit the cell proliferation induced by 5ng / ml TGF-β1. See Table 5 for details:

[0179] Table 5

[0180]

[0181] *P<0.05 compared with the blank control group.

[0182] Results (4): After 3 days of drug administration, high-dose serum containing Chinese medicine and serum containing pirfenidone can inhibit cell proliferation induced by 10ng / ml TGF-β1. See Table 6 for details:

[0183] Table 6

[0184]

[0185] *Compared with the blank control group, P<0.05; #Compared with the model group, P<0.05.

[0186] Results (5): After 6 hours of drug administration for three days, the drug-containing serum of each group could not inhibit the cell proliferation induced by 15ng / ml TGF-β1. See Table 7 for details:

[0187] Table 7

[0188]

[0189]

[0190] *P<0.05 compared with the blank control group.

[0191] Results (6): High-dose serum containing Chinese medicine can inhibit cell proliferation induced by 15ng / ml TGF-β1 after 1 hour of drug administration for 3 days. See Table 8 for details:

[0192] Table 8

[0193]

[0194] *Compared with the blank control group, P<0.05; #Compared with the model group, P<0.05.

[0195] Conclusion: Drug-containing serum is more effective in inhibiting cell proliferation induced by 10 ng / ml TGF-β1. Drug-containing serum extracted from blood drawn 1 hour after 3 days of drug administration is more effective than serum extracted 6 hours after administration. Therefore, all subsequent experiments will use cells induced with 10 ng / ml TGF-β1, and culture medium containing 10% drug-containing serum by volume will be used for drug administration.

[0196] 2.4 Scratch test:

[0197] 2.4.1 Experimental steps:

[0198] A blank area, called a "scratch / wound," is artificially created on a confluent cell monolayer. Cells at the edge gradually migrate into the blank area, healing the scratch / wound. Because this resembles the in vitro wound healing process, it is also known as the Wound-Healing Assay and is widely used to observe the effects of exogenous factors, such as drugs and genes, on cell migration and repair.

[0199] Cell scratch test process: On a monolayer of adherent cells cultured in vitro on a culture dish or plate, use a micropipette tip or other hard object to scratch the central area where the cells grow, remove the cells in the central part, and then continue to culture the cells for the set time of the experiment (different time points can be selected). Remove the cell culture plate and observe under a microscope to see whether the surrounding cells have migrated to the central scratch area, and take a picture.

[0200] The specific experimental steps include: marking the 6-well culture plate, inoculating about 200,000 cells in each well to plate the cells, streaking the cells with a 20uL pipette tip (sterilized), washing the cells with sterile PBS and removing the streaked cells, and cell culture and observation. These experimental steps can be carried out according to conventional methods. In the present invention, cells are removed after 0, 4, 8, 12, and 24 hours in the cell culture process, observed under a microscope, and photographed. Figure 10 shown.

[0201] 2.4.2 Data Analysis and Results

[0202] After opening the image using ImageJ software, 6 to 8 horizontal lines were randomly drawn and the mean of the intercellular distances was calculated. The results are shown in Table 9:

[0203] Table 9: Comparison of wound healing ability at different scratch times

[0204]

[0205] Note: # compared with the blank control group, P < 0.05; ## compared with the blank control group, P < 0.01; * compared with the model group, P < 0.05; ** compared with the model group, P < 0.01, the differences are significant. δ compared with the low-dose Chinese medicine group, P < 0.05; δδ compared with the low-dose Chinese medicine group, P < 0.01.

[0206] comprehensive Figure 10 As shown in Table 9, after the RLE-6TN rat alveolar type II epithelial cells were modeled with 10 ng / ml TGF-β1, cell proliferation increased and migration accelerated. After administration of the Bushen Tongluo prescription, migration was inhibited.

[0207] 2.5 Apoptosis assay (detected by flow cytometry)

[0208] 2.5.1 Experimental steps

[0209] Flow cytometry: Red blood cell fluorescence was detected by flow cytometry at 488 nm, and cell cycle distribution was analyzed using Modfit software.

[0210] The working principle of a flow cytometer is to place cells in a sample tube. Under gas pressure, the cells are forced into a single row and discharged from the nozzle flow chamber to form a cell column. By detecting a single row of cells in the liquid flow, cell light scattering and fluorescence indicators are obtained, and a series of physical and chemical properties such as cell volume, internal structure, DNA, RNA, protein, and antigens are analyzed.

[0211] In normal cells, phosphatidylserine (PS) is exclusively distributed within the lipid bilayer of the cell membrane. During the early stages of apoptosis, PS on the cell membrane is oriented in the opposite direction to the inner and outer membranes. Annexin V, a calcium-dependent phospholipid-binding protein, was fluorescently labeled with fluorescein (FITC) as a fluorescent probe. Propyl iodide (PI), a nucleic acid dye, cannot penetrate the entire cell membrane, necrotic cells, or cells in the late stages of apoptosis. Due to loss of cell membrane integrity, PI can enter the nucleus and stain it, producing red fluorescence. Annexin V, as a fluorescent probe, binds to PS extending to the cell surface, resulting in a dual-color fluorescence pattern in these cells. Using isothiocyanate (V-FUC) and PI staining, the following results were obtained: normal living cells: Annexin V-FITC (-), PI (-); early apoptotic cells: Annexin V-FITC (-), PI (+); necrotic and late apoptotic cells: Annexin V-FITC (+), PI (+); and cell debris: Annexin V-FITC (-), PI (+).

[0212] After drug treatment, the cells were processed according to the requirements of the flow cytometer and tested on the machine.

[0213] 2.5.2 Statistical analysis and results

[0214] All experiments were repeated more than 6 times. Data were expressed as mean ± standard deviation. The differences between normally distributed numerical variables were compared using t-test and analysis of variance. Graphpad Prism 6 software was used to create statistical graphs, and Howjo 7.0 software was used for flow cytometry data analysis. Modfit software was used to analyze cell cycle distribution and perform statistics. Figure 11 The statistical results are shown in Table 10:

[0215] Table 10: Comparison of cell apoptosis percentage in each group

[0216]

[0217] Note: # compared with blank control group, P<0.05, ## compared with blank control group, P<0.01; * compared with model group, P<0.05, ** compared with model group, P<0.01, the differences are significant.

[0218] pass Figure 11 As can be seen from Table 10, compared with the blank control group, the proportion of apoptotic cells in the model group under the action of 10 ng / ml TGF-β1 was significantly decreased (P < 0.05); compared with the model group, the proportion of apoptotic cells in the low-dose Chinese medicine group was significantly increased (P < 0.05); thus, it can be seen that the Chinese medicine composition of the present invention can promote cell apoptosis.

[0219] 2.6 Detection of EMT phenotype using immunofluorescence

[0220] 2.6.1 Immunofluorescence experiments and results

[0221] Cells were seeded onto a 24-well plate at a density of 4,000 cells / ml, with 500 μl per well. After 20 hours of culture, the culture medium was aspirated and 10% rat serum-free medium was added, with 500 μl per well incubated for another 24 hours. For the model group, 10 ng / ml TGF-β1 was added and cultured for another 24 hours. The culture medium was then discarded, and each well was washed three times with PBS. The PBS was discarded, and 1 ml of 4% paraformaldehyde was added to each well. The cells were sent to the company for testing. The results are shown in Table 11.

[0222] Table 11: Immunofluorescence detection of cell EMT phenotype

[0223]

[0224] Note: # compared with blank control group, P<0.05, ## compared with blank control group, P<0.01; * compared with model group, P<0.05, ** compared with model group, P<0.01, the differences are significant.

[0225] Table 11 shows that the E-cadherin level in the blank control group was significantly higher than that in the model group (P < 0.01). The CK19 level in the blank control group was significantly higher than that in the model group (P < 0.05). The α-SMA level in the model group was significantly higher than that in the blank control group (P < 0.05). The Vimentin level in the model group was significantly higher than that in the blank control group (P < 0.01). This indicates that 10 ng / ml TGF-β1 treatment of alveolar type II epithelial cells induces the transformation of epithelial cells into mesenchymal cells, resulting in the formation of fibroblasts.

[0226] Comparison of MMP-7, MMP-9, TGF-β1, Snail, Wnt3a, and β-catenin protein expression in fibroblasts (Western blotting)

[0227] 2.7.1 Experimental equipment and reagents

[0228]

[0229] 2.7.2 Experimental steps

[0230] 2.7.2.1 Total cell protein extraction

[0231] Suspension cells: 2000rpm, 4℃, 5min, collect cell pellets by centrifugation, every 10 6 Add about 250ul of RIPA lysis buffer to the cells and shake.

[0232] Adherent cells: Rinse cells 2-3 times with PBS and add an appropriate volume of RIPA lysis buffer (add protease inhibitors within a few minutes before use) to the culture plate / flask for 3-5 minutes. Scrape the cells and transfer them to a 1.5ml centrifuge tube. Lyse on ice for 30 minutes. Once cells are completely lysed, centrifuge at 12,000 rpm at 4°C for 10 minutes. Collect the supernatant, which is the total cell protein solution.

[0233] 2.7.2.2 Total protein extraction from tissues:

[0234] Wash the tissue block 2-3 times with pre-cooled PBS to remove blood stains, cut into small pieces and place in a homogenization tube. Add 1-2 3mm homogenization beads and 10 times the volume of tissue lysis buffer (add protease inhibitors within a few minutes before use). Set the homogenization program for homogenization. Remove the homogenized tube and place it in the lysis buffer on ice for 30 minutes to ensure that the tissue is completely lysed. Centrifuge at 12000 rpm, 4°C for 10 minutes and collect the supernatant, which is the total tissue protein solution.

[0235] 2.7.2.3 Nuclear and cytoplasmic protein extraction (nuclear and cytoplasmic protein extraction kit)

[0236] Use the nuclear protein and cytoplasmic protein extraction kit according to its instructions to extract nuclear protein, including: taking appropriate amounts of cytoplasmic protein extraction reagent A and nuclear protein extraction reagent for use, and adding PMSF within a few minutes before use to make the final concentration of PMSF 1mM;

[0237] Adherent cells: Wash once with PBS, scrape the cells or treat them with EDTA solution, then pipette the cells and collect them by centrifugation, leaving the cell pellet for later use;

[0238] For the reserved cell pellet, extract the cytoplasmic proteins: add 200 μl of cytoplasmic protein extraction reagent A supplemented with PMSF for every 20 μl of cell pellet; then completely suspend and disperse the cell pellet; incubate on ice for 10-15 minutes; add 10 μl of cytoplasmic protein extraction reagent B; vortex at the highest speed for 5 seconds, incubate on ice for 1 minute; vortex at the highest speed for 5 seconds, centrifuge at 12000-16000g at 4°C for 5 minutes; aspirate the supernatant into a pre-cooled EP tube, which is the extracted cytoplasmic protein.

[0239] Nuclear protein extraction: Add 50 μL of PMSF-added nuclear protein extraction reagent to the pellet. Completely suspend and disperse the cell pellet. Vortex vigorously for 15-30 seconds every 1-2 minutes for a total of 30 minutes in an ice bath. Centrifuge at 12,000-16,000 g for 10 minutes at 4°C. Pipette the supernatant into a pre-chilled EP tube to obtain the extracted nuclear protein.

[0240] 2.7.2.4 Protein concentration determination: Take the undenatured protein solution and measure the protein concentration using the BCA protein concentration assay kit. Refer to the kit instructions for the specific method.

[0241] 2.7.2.5 Protein denaturation: Add 5* reduced protein loading buffer to the protein solution at a ratio of 4:1, denature in boiling water for 15 min, and store in a -20°C refrigerator until use.

[0242] 2.7.2.6 Immune Response

[0243] Perform SDS-PAGE electrophoresis according to conventional methods, including cleaning the glass plate, preparing the gel, and loading the sample. Prepare separating gel and stacking gel of different concentrations according to experimental requirements as follows:

[0244]

[0245] When loading the sample for electrophoresis, use a voltage of 75V for the stacking gel and 120V for the separating gel. Run the electrophoresis gel approximately 1 cm below the bottom of the bromophenol blue solution. Transfer the membrane to the membrane according to conventional methods: peel off the separating gel and place it on filter paper. Place a PVDF membrane that has been pre-activated with methanol for 2 minutes on top of the gel. Cover the membrane with three pieces of filter paper and a sponge pad, taking care to avoid air bubbles. Wet transfer the membrane at a constant current of 300 mA for 30 minutes, cooling it with ice water during transfer.

[0246] Place the transferred membrane in an incubation tank filled with TBST, add primary and secondary antibodies according to the antibody instructions (diluted 1:5000 in TBST), and incubate and elute.

[0247] 2.7.2.7 Chemiluminescence and gel image analysis

[0248] The eluted PVDF membrane was pressed into film according to conventional methods, and the pressed film was developed and fixed with developer and fixer reagents. The film was scanned and archived, and the color was removed using Photoshop. The optical density of the target band was analyzed using the Alpha software processing system.

[0249] 2.7.3 Results of MMP-7, MMP-9, TGF-β1, Snail, Wnt3a, and β-catenin protein expression in fibroblasts (immunoblotting)

[0250] Immunoblotting of MMP-7, MMP-9, TGF-β1, and β-catenin in fibroblasts Figure 12 The specific expression results can be found in Table 12. In addition, the expression of Snail and Wnt3a in fibroblasts was found to be extremely low and difficult to detect.

[0251] Table 12: Expression of MMP-7, MMP-9, TGF-β1, and β-catenin proteins in fibroblasts

[0252]

[0253] Note: # compared with the blank control group, P < 0.05; ## compared with the blank control group, P < 0.01; * compared with the model group, P < 0.05; ** compared with the model group, P < 0.01, the differences are significant; δ compared with the low-dose Chinese medicine group, P < 0.05; δδ compared with the low-dose Chinese medicine group, P < 0.01; γ compared with the high-dose Chinese medicine group, P < 0.05; γγ compared with the high-dose Chinese medicine group, P < 0.01.

[0254] comprehensive Figure 12As shown in Table 12, Bushen Tongluo formula has a strong inhibitory effect on MMP7 and MMP9, which can mainly regulate the degradation and remodeling of the extracellular matrix. In addition, compared with pirfenidone, Bushen Tongluo formula has a better inhibitory effect on TGF-β1 (10 ng / ml).

[0255] 2.8 Detection of relative protein mRNA expression in fibroblasts (RT-PCR)

[0256] 2.8.1 Experimental equipment and reagents

[0257]

[0258]

[0259] 2.8.2 Fluorescence quantitative PCR experimental steps

[0260] Total RNA extraction included the following steps: 1) washing cells with 1 ml of 4°C pre-chilled PBS, removing the PBS, adding 1 ml of RNA extraction solution, disrupting the cells, transferring the cells to a centrifuge tube, adding 250 μl of chloroform, mixing, and centrifuging at 12,000 rpm for 10 min at 4°C; 2) transferring 400 μl of the supernatant and adding 0.8 times the volume of isopropanol, mixing, incubating at -20°C for 15 min, and centrifuging at 12,000 rpm for 10 min at 4°C. The white precipitate at the bottom of the tube was RNA.

[0261] 2) Wash the RNA pellet with 1.5 ml of 75% ethanol and centrifuge at 12,000 rpm for 10 min at 4°C. After removing the liquid, blow dry on a clean bench for 3 min. Then, add 15 μl of RNase-free water to dissolve the RNA and incubate at 55°C for 5 min.

[0262] 3) Use Nanodrop2000 to detect RNA concentration and purity, and make the final concentration 100-500 ng / μl.

[0263] Reverse transcription includes: preparation of the reverse transcription reaction system and program settings, as follows:

[0264]

[0265] Quantitative PCR includes: taking a 0.2 ml PCR tube, preparing the reaction system (2×qPCR Mix 7.5 μl; 2.5 μM gene primers (upstream + downstream) 1.5 μl; reverse transcription product (cDNA) 2.0 μl; ddH2O 4.0 μl), preparing 3 tubes for each reverse transcription product, and amplifying as follows:

[0266]

[0267] 2.8.3 Result processing and analysis

[0268] ΔΔCT method: A = CT (target gene, test sample) - CT (internal standard gene, test sample); B = CT (target gene, control sample) - CT (internal standard gene, control sample); K = AB; expression fold = 2-K;

[0269] The relative expression results of β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 protein mRNA in fibroblasts can be seen in Table 12. In addition, no valid data were detected for MMP-7.

[0270] Table 12 Relative mRNA expression levels of β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 proteins

[0271]

[0272] Note: # compared with the blank control group, P < 0.05; ## compared with the blank control group, P < 0.01; * compared with the model group, P < 0.05; ** compared with the model group, P < 0.01, the differences are significant; δ compared with the low-dose Chinese medicine group, P < 0.05; δδ compared with the low-dose Chinese medicine group, P < 0.01; γ compared with the high-dose Chinese medicine group, P < 0.05; γγ compared with the high-dose Chinese medicine group, P < 0.01.

[0273] As can be seen from Table 12, Bushen Tongluo recipe can reduce the relative expression levels of β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 protein mRNA, indicating that Bushen Tongluo recipe has an inhibitory effect on the relative expression levels of β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 protein mRNA, and can inhibit the metalloproteinase MMP-9, and inhibit the Wnt3a / β-catenin and TGF-β1 / Snail signaling pathways.

[0274] The above cell experiments proved that Bushen Tongluo prescription can inhibit the proliferation and migration of rat alveolar type II epithelial cells, promote apoptosis, and reduce excessive ECM deposition by inhibiting the EMT process of rat alveolar type II epithelial cells. Its mechanism may be related to the inhibition of Wnt3a / β-catenin and TGF-β1 / Snail signaling pathways.

[0275] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicine composition for treating diffuse interstitial lung disease, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-12 parts of Curculigo, 10-20 parts of Epimedium, 30-45 parts of Astragalus, 20-30 parts of Ophiopogon, 15-30 parts of Clemati, 10-15 parts of Kadura Caulis, 10-15 parts of Trachelospermum jasminoides, 10-15 parts of Pheretima, 10-12 parts of Curcuma, 12-15 parts of Polygonum cuspidatum, 15-20 parts of Fritillaria thunbergii, and 6 parts of Licorice.

2. The Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 parts of Curculigo, 10 parts of Epimedium, 30 parts of Astragalus, 30 parts of Ophiopogon, 15 parts of Clemati, 15 parts of Kadura Caulis, 15 parts of Trachelospermum jasminoides, 12 parts of Pheretima, 10 parts of Curcuma, 12 parts of Polygonum cuspidatum, 20 parts of Fritillaria thunbergii, and 6 parts of Licorice.

3. The Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition further comprises excipients, which include at least one of a filler, a binder, a disintegrant, and a lubricant.

4. The Chinese medicine composition according to claim 1, wherein: The dosage form of the Chinese medicine composition is selected from any one of granules, ointments, pills, injections and oral liquids.

5. Use of the traditional Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a medicament for treating diffuse interstitial lung disease.

6. The use according to claim 5, characterized in that: The diffuse interstitial lung disease is idiopathic pulmonary fibrosis.

7. The use according to claim 6, characterized in that: The drug works by at least one of the following ways: (1) Down-regulate the expression of at least one protein among TGF-β1, β-catenin, MMP-7 and MMP-9 in fibroblasts; (2) Increased serum IFN-γ levels; (3) Reduce serum IL-4 and Laminin levels; (4) Reduce the relative expression of at least one protein mRNA among β-catenin, Wnt3a, Snail, TGF-β1 and MMP-9 in fibroblasts.

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