A traditional Chinese medicine composition for treating lung cancer and its uses

Abstract

This invention discloses a traditional Chinese medicine composition for treating lung cancer, prepared from the following raw materials in parts by weight: 15-30 parts of raw Astragalus membranaceus, 10-15 parts of Adenophora stricta, 10-15 parts of Asparagus cochinchinensis, 10-15 parts of Ophiopogon japonicus, 10-15 parts of Poria cocos, 15-30 parts of Selaginella tamariscina, 15-30 parts of Hedyotis diffusa, 15-30 parts of Paris polyphylla, 15-30 parts of Cremastra appendiculata, 15-30 parts of Prunella vulgaris, 15-30 parts of Houttuynia cordata, and 10-15 parts of Ligustrum lucidum. This traditional Chinese medicine composition can inhibit the proliferation of lung cancer cells and promote their apoptosis. Simultaneously, it exerts an anti-invasion and metastatic effect on lung cancer by intervening in exosomes, improving clinical symptoms in cancer patients, regulating functional immune function, and improving quality of life.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine composition for treating lung cancer and its use. This traditional Chinese medicine composition is applied to the treatment of malignant tumors in clinical practice, mainly targeting lung malignant tumors, especially metastases. Background Art

[0002] The present invention mainly discovers and verifies a group of traditional Chinese medicine compositions that can be applied to the treatment of lung cancer, including the occurrence and metastasis of lung cancer. The main characteristics of the prior art related to the present invention are as follows: 1. Compound drugs: Under the combination of traditional Chinese medicine theory and modern pharmacology, a traditional Chinese medicine prescription for treating lung cancer is formulated, but its mechanism is difficult to clarify due to the complex composition; 2. Traditional Chinese medicine monomers: Taking the monomer of a certain traditional Chinese medicine as the research object to explain its anti-tumor effect and mechanism, but losing the "theory" essence of traditional Chinese medicine; 3. Regulating immunity: Immune function is one of the most important components of the healthy qi in traditional Chinese medicine. Reveal the essential role of traditional Chinese medicine in "strengthening healthy qi" from the perspective of regulating immunity; 4. Enhancing efficacy and reducing toxicity: Looking for evidence-based medical evidence of the effectiveness of traditional Chinese medicine from the perspective of enhancing the efficacy of modern medicine and reducing adverse reactions during the treatment process of modern medicine in clinical practice; 5. Quality of life: The existing traditional Chinese medicine compositions mainly focus on improving the quality of life and relieving clinical symptoms, lacking evidence-based medical evidence and research on the mechanism of preventing metastases and prolonging survival time.

[0003] The deficiencies of the technology most relevant to the present invention are mainly manifested as follows: First, the main action mechanism, active ingredients, and effective ingredients of traditional Chinese medicine compounds still need to be studied in depth; Second, the complex composition and numerous medicinal flavors of traditional Chinese medicine compounds lead to unclear action mechanisms. How to optimize traditional Chinese medicine compounds to form a contemporary prescription with the characteristics of "simple, convenient, inexpensive, and effective"; Third, the preparation process of traditional Chinese medicine compositions urgently needs to be improved, especially to improve their bioavailability and efficacy stability; Finally, the clinical safety evaluation and effectiveness assessment of traditional Chinese medicine compositions also need to be more scientific and strict, and more detailed evaluation indicators and systems need to be formulated. Summary of the Invention

[0004] In view of the above deficiencies of the prior art, according to the embodiments of the present invention, it is desired to provide and verify a traditional Chinese medicine composition for treating lung cancer, which is mainly applied to the treatment of lung cancer, including the occurrence and metastasis of lung cancer.

[0005] According to an embodiment, the present invention provides a traditional Chinese medicine composition for treating lung cancer, which is prepared from the following raw materials in parts by weight: 15-30 parts of raw Astragalus membranaceus, 10-15 parts of Adenophora stricta, 10-15 parts of Asparagus cochinchinensis, 10-15 parts of Ophiopogon japonicus, 10-15 parts of Poria cocos, 15-30 parts of Selaginella tamariscina, 15-30 parts of Lysimachia christinae, 15-30 parts of Paris polyphylla, 15-30 parts of Cremastra appendiculata, 15-30 parts of Prunella vulgaris, 15-30 parts of Houttuynia cordata, and 10-15 parts of Ligustrum lucidum. Preferably, the parts by weight of the raw materials are: 30 parts of raw Astragalus membranaceus, 15 parts of Adenophora stricta, 15 parts of Asparagus cochinchinensis, 15 parts of Ophiopogon japonicus, 15 parts of Poria cocos, 15 parts of Selaginella tamariscina, 15 parts of Lysimachia christinae, 30 parts of Paris polyphylla, 15 parts of Cremastra appendiculata, 15 parts of Prunella vulgaris, 15 parts of Houttuynia cordata, and 15 parts of Ligustrum lucidum.

[0006] Subsequent embodiments and experimental examples of this invention will demonstrate the effective components of the traditional Chinese medicine composition that enter the blood and lung tissue, the network pharmacological analysis of the traditional Chinese medicine composition and the disease-related targets of the traditional Chinese medicine composition, the cellular and animal experiments on the prevention and treatment of lung cancer occurrence and metastasis by the traditional Chinese medicine composition, and that the mechanism of action of the traditional Chinese medicine composition in preventing and treating lung cancer occurrence and metastasis may be related to the inhibition of exosome protein expression. That is, the traditional Chinese medicine composition of this invention can inhibit the proliferation of lung cancer cells and promote their apoptosis, while simultaneously exerting an anti-lung cancer invasion and metastasis effect through intervention in exosomes, improving the clinical symptoms of cancer patients, regulating functional immune function, and improving quality of life.

[0007] The preparation method of the traditional Chinese medicine composition of this invention is not particularly special. The raw material components that meet the requirements stipulated in the National Pharmacopoeia are mixed evenly according to the weight proportions described in claim 2; decocted into a soup, or processed into granules, pills (water pills or drop pills), or traditional Chinese medicine formula granules, or one or more excipients acceptable in pharmaceutical preparation are added, and the mixture is made into granules, water pills, tablets, or capsules according to conventional processes.

[0008] The advantages of the traditional Chinese medicine composition for treating lung cancer in this invention are mainly: 1. Significant efficacy: This traditional Chinese medicine composition selects medicinal materials with the effects of softening and dispersing masses, clearing heat and detoxifying, nourishing qi and yin, and strengthening the spleen and kidneys, based on the theory of traditional Chinese medicine. Through the scientific combination of drugs and dosages, they work together to inhibit cancer cells and improve the therapeutic effect; 2. Fewer side effects: Compared with some chemical drugs, the side effects of the traditional Chinese medicine composition are usually smaller, and the impact on the patient's quality of life is relatively lower. Moreover, the components of the traditional Chinese medicine composition of this invention are clearly defined and have high safety; 3. Holistic regulation: The traditional Chinese medicine composition of this invention emphasizes the holistic concept, starting from the three organs of the lungs, spleen, and kidneys. The composition not only focuses on inhibiting the tumor itself, but also on regulating the patient's overall condition, including enhancing immunity and improving physical constitution, thereby helping patients better cope with the disease; 4. Wide applicability: Guided by the theory of traditional Chinese medicine, the traditional Chinese medicine composition of this invention is formulated with personalized dosage and ingredients according to the clinical characteristics of lung cancer. Therefore, it can be applied to patients with different types and stages of lung cancer, especially postoperative lung cancer patients.

[0009] The social and economic value of the traditional Chinese medicine composition for treating lung cancer in this invention is mainly reflected in the following aspects: 1. Reducing the medical burden: Since traditional Chinese medicine compositions are generally relatively inexpensive and have few side effects, they can reduce the medical burden on patients to a certain extent, enabling more patients to enjoy effective treatment; 2. Promoting the industrialization of traditional Chinese medicine: As people's understanding and acceptance of the efficacy of traditional Chinese medicine increase, the development of the traditional Chinese medicine industry will be further promoted. This will help enhance the competitiveness of traditional Chinese medicine in the international market and promote the international development of the traditional Chinese medicine industry; 3. Improving social benefits: By promoting and applying effective traditional Chinese medicine compositions to treat lung cancer, the survival rate and quality of life of lung cancer patients can be improved, reducing the social burden caused by the disease, thereby improving social benefits. Attached Figure Description

[0010] Figure 1 This invention shows the main components and metabolites of the traditional Chinese medicine composition (hereinafter also referred to as Feiyanshu) in peripheral blood and lung tissue, as well as 8 prototype components.

[0011] Figure 2 The results showed that Lungyanshu had 182 overlapping targets with lung cancer metastasis.

[0012] Figure 3 The PPI network analysis of the intersection targets shows that TLR4 is located at position 20.

[0013] Figure 4 Displaying lung cancer targets.

[0014] Figure 5 This study demonstrates the KEGG pathway analysis of Lung Rock Soothing in the treatment of tumors.

[0015] Figure 6The David database analysis shows the fitting analysis between lung cancer targets and the KEGG signaling pathway.

[0016] Figure 7 GO analysis was performed on lung biopsy targets.

[0017] Figure 8 This demonstrates the drug-target analysis network of Lung Rock Soothing.

[0018] Figure 9 This study demonstrates the preventive and therapeutic effects of Feiyanshu on lung metastasis in a mouse tail vein lung metastasis model.

[0019] Figure 10 The results showed that Lung Rock Su could inhibit the expression of exosomal proteins in the lung tissue of tumor-bearing mice.

[0020] Figure 11 Studies demonstrating the cytotoxic effects of Lungyanshu on CTC-TJH-01 and H1299 cells.

[0021] Figure 12 This study demonstrates the effects of different concentrations of Lungyanshu on the invasion and migration of CTC-TJH-01 and H1299 cells.

[0022] Figure 13 This study demonstrates a co-culture tracing experiment of PKH26-labeled exosomes with tumor cells.

[0023] Figure 14 This study demonstrates the effect of Lungyanshu on the expression of exosomal proteins in the culture supernatant of CTC-TJH-01 and H1299 cells. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments should be understood as illustrative only and not as limiting the scope of protection of the present invention. After reading the description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0025] Example 1

[0026] Weigh out 30g of raw Astragalus membranaceus, 15g of Adenophora stricta, 15g of Asparagus cochinchinensis, 15g of Ophiopogon japonicus, 15g of Poria cocos, 15g of Selaginella tamariscina, 15g of Lysimachia christinae, 30g of Paris polyphylla, 15g of Cremastra appendiculata, 15g of Prunella vulgaris, 15g of Houttuynia cordata, and 15g of Ligustrum lucidum, all in accordance with the requirements of the Pharmacopoeia. Decoct into a soup, or process into granules, pills, or traditional Chinese medicine formula granules, or add one or more pharmaceutically acceptable excipients and prepare into granules, pills, freeze-dried powder, tablets, or capsules using conventional processes.

[0027] Test case

[0028] 1. Metabolites and distribution patterns of Lung-Rock-Soothing Agent in peripheral blood and lung tissue of mice.

[0029] 1.1 Instruments and Reagents

[0030] Waters H-Class Ultra-High Performance Liquid Chromatography System (Waters Technologies Inc.); AB Sciex Triple 4600 high-resolution mass spectrometer (SCIEX); KQ-300BD ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); SIGMA 3K15 high-speed centrifuge (SIGMA); LNG-T98 centrifugal concentrator and dryer (Taicang Huamei Biochemical Instrument Factory); methanol and acetonitrile, mass spectrometry grade, purchased from Merck; formic acid, mass spectrometry grade, purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0031] 1.2 Laboratory Animals

[0032] Twenty-four healthy male C57 mice, weighing 20-22g and 8 weeks old, were provided by Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., certificate number SCXK(Su)2020-0009. They were housed at Shanghai Xinying Biotechnology Co., Ltd., with an indoor temperature of 22-26℃, a relative humidity of 40%-70%, and a light / dark period of 12 hours each.

[0033] 1.3 Experimental Methods

[0034] A. Lung-Rock Relief Test Sample: The lyophilized powder (150.39g) prepared in Example 1 was stored at 4℃ for later use.

[0035] B. Weigh approximately 1.0g of the test sample accurately, place it in a 50ml stoppered conical flask, add 10ml of 50% methanol, sonicate (300W power, 40kHz frequency) for 30 minutes, take 2ml into a centrifuge tube, centrifuge at high speed (12000 rpm) for 5 minutes, and take the supernatant.

[0036] 1.4 Chromatographic and Mass Spectrometric Conditions

[0037] A. Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (2.1×150mm, 1.8μm); mobile phase A: acetonitrile; mobile phase B: 0.1% formic acid aqueous solution. Elution program: 0–4 min, 0% acetonitrile; 4–8 min, 0%–8% acetonitrile; 8–28 min, 8%–20% acetonitrile; 28–46 min, 20%–35% acetonitrile; 46–51 min, 35%–60% acetonitrile; 51–54 min, 60%–90% acetonitrile; 54–57 min, 90% acetonitrile; 57–57.1 min, 90%–0% acetonitrile; 57.1–60 min, 0% acetonitrile. Column temperature 30℃; flow rate 0.3ml / min; injection volume for detecting compound components 2μl, injection volume for detecting components entering blood and tissues 5μl; detection wavelength 254nm, 190~400nm.

[0038] B. Mass spectrometry conditions: Mass spectrometry detection mode: ESI-Negative / Positive ion mode; mass range: 50–1700; secondary mass range: 50–1250; spray gas pressure: 50 psi; declustering voltage: 100 V; auxiliary heating gas pressure: 50 psi; collision voltage: ±40 eV; curtain gas pressure: 35 psi; collision voltage swing: 20 eV; ionization voltage: -4500 / 5000 V; ion release delay: 30 ms; ion source temperature: 500 °C; ion beam width: 15 ms; declustering voltage: 100 V; collision voltage: 10 eV.

[0039] C. Collection of serum and tissue samples: Mice were randomly divided into 4 groups (blank, 1h, 2h, and 4h groups, 6 mice in each group). They were administered 12G via gavage. The blank group received ultrapure water, while the drug-treated groups received 130g / kg of serum. -1 The lyophilized liquid solution of Lung-Rock Soothing Powder was administered by gavage for 3 consecutive days (the dosage was calculated based on twice the equivalent dose for a 70kg adult). At the end of the third day, blood was collected from the orbital cavity into a 1.5ml EP tube without anticoagulation. The tube was centrifuged at 8000×g for 5 minutes to obtain serum. Lung tissue was dissected and the surface residual blood was washed with 0.9% physiological saline. The tissue was then blotted dry with filter paper, and all samples were stored at -80℃ for later use.

[0040] D. Serum Sample Processing: Combine blank serum (1.5 ml) and treated serum (3.5 ml) separately. Add 3 times the volume of methanol (mass spectrometry grade) to precipitate proteins, vortex for 5 minutes, let stand for 20 minutes (4℃), centrifuge for 15 minutes (12000 rpm), collect the supernatant, centrifuge to concentrate and dry, and store at -80℃. Before analysis, reconstitute the residue with 80 μl of 50% methanol, vortex for 3 minutes, centrifuge for 15 minutes (12000 rpm), and collect the supernatant.

[0041] E. Lung tissue sample processing: Weigh the lung tissues before drug administration (1 g) and the lung tissues after drug administration (3 g) respectively with precision, place them in several 2-ml centrifuge tubes, add 1.5 ml of ice-cold methanol, grind for 30 s, perform ultrasonic treatment in an ice bath (power 300 W, frequency 40 KHz) for 15 minutes, centrifuge for 15 minutes (12,000 rpm), combine the supernatant, concentrate and dry by centrifugation, and store at -80 °C. Before analysis, dissolve the residue with 200 μl of methanol, vortex for 3 minutes, centrifuge for 15 minutes (12,000 rpm), and take the supernatant to obtain the sample.

[0042] 1.5 Analysis software

[0043] The data acquisition software is Analyst TF 1.7.1, and the data processing software is Peakview 1.2.

[0044] 1.6 Results

[0045] The serum samples and lung tissue samples after administration of Feiyanshu were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS). According to the multi-stage mass spectrometry information of the samples, relevant literature and the identification results of the original formula, 56 components were identified from the serum samples after administration of Feiyanshu, including 9 prototype components and 47 metabolites; 40 components were identified from the lung tissue samples after administration of Feiyanshu, including 8 prototype components and 32 metabolites. Among them, 8 prototype components expressed in both serum and lung tissue, as Figure 1 shown, mainly derived from Astragalus membranaceus, Herba Selaginellae, and Ligustrum lucidum.

[0046] 2. Network pharmacology and action target analysis of Feiyanshu.

[0047] 2.1 Find and screen the drug components of Astragalus membranaceus, Glehnia littoralis, Asparagus cochinchinensis, Ophiopogon japonicus, Poria cocos, Herba Selaginellae, Salvia chinensis, Paris polyphylla, Cremastra appendiculata, Prunella vulgaris, Houttuynia cordata, and Ligustrum lucidum in the Symmap and Swiss ADME databases. After predicting the drug targets with SwissTargetPrediction, take the intersection with the Pulmonary metastases targets retrieved from the Genecard and OMIM databases to obtain 182 related targets, as Figure 2 shown.

[0048] 2.2 Organize the 182 related targets in the Cytoscape database, take the first half of the Degree values, that is, the first 91 targets to construct a PPI network diagram, as Figure 3 shown, and find that the primary target is Tp53, and the 20th target is TLR4.

[0049] 2.3 Organize the lung cancer Shuyu-disease targets in the Cytoscape database, as Figure 4 shown, to form a potential target interaction network.

[0050] 2.4 Through KEGG analysis of 91 targets, as Figure 5 shown, it was found that lung cancer Shuyu can treat tumors and may act through the NF-KappaB signaling pathway.

[0051] 2.5 Through the David database, analyze the lung cancer Shuyu-disease targets, and perform a fitting analysis on the obtained KEGG signaling pathways, as Figure 6 shown, it was found that lung cancer Shuyu affects signal transduction, cell growth and death in the process of lung metastasis disease, and plays a role in the immune system.

[0052] 2.6 Through the Metascape database, perform GO analysis on the lung cancer Shuyu-disease targets, as Figure 7 shown, it was found that in biological processes, lung cancer Shuyu mainly affects cell migration, phosphorylation regulation, transmembrane receptor protein tyrosine kinase signaling, and in cell components mainly affects cell membrane, focal adhesion, receptor complex, and in molecular functions mainly affects protein kinase activity, protein tyrosine kinase activity, phosphatase binding, etc.

[0053] 2.7 Analysis of the drug targets of lung cancer Shuyu and diseases, as Figure 8 shown.

[0054] 3. In vitro and in vivo experiments on the treatment of lung cancer occurrence and metastasis by lung cancer Shuyu.

[0055] 3.1 Materials and methods:

[0056] (1) Experimental animals

[0057] In this experiment, male C57BL / 6J mice, 5-6 weeks old, with a body weight of about 20±5 g, were provided by Jiangsu Jicui Biotechnology Co., Ltd. The experimental animals were housed in the Specific Pathogen Free (SPF) experimental animal center of the Shanghai Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine (certificate number: SYXK (Shanghai) 2020-0014). The experimental animal housing conditions were constant temperature and humidity (temperature about 24°C, humidity about 60%), with the lights alternating day and night every 12 h; food and water were supplied充足供给, the bedding was changed every 3 days, and the animals were adaptively housed in the SPF environment for 1 week. This experiment was reviewed and approved by the Experimental Animal Welfare and Ethics Committee of the Shanghai Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine (ethics number: 2023012). Animal experiment operations complied with the regulations of the national and the hospital's experimental animal center on animal research management.

[0058] (2) Experimental cells It should be noted that "充足供给" in the original text seems to be an incorrect expression. I translated it as "adequately supplied" according to the context. If there is a more accurate term, it can be adjusted accordingly.

[0059] The CTC-TJH-01 cell line was created by our research group and has been deposited in the Wuhan Cell Bank; Lewis lung cancer cells (2LL cell line) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0060] (3) Drug preparation

[0061] The lyophilized powder of lung cancer prepared in Example 1 was stored in a refrigerator at 4°C for later use.

[0062] (4) Reagents, consumables, and instruments

[0063] A. Main reagents: Hematoxylin-eosin staining solution (Zhuhai Beso Biotechnology Co., Ltd., C200401), BCA quantitative reagent kit (Shanghai Yamei Biotechnology Co., Ltd., ZJ101); antibody C9 (Affbiotech, DF3964), antibody CFHR5 (GeneTex, GTX109982), MBL2 (Affbiotech, DF4152), β-actin (Hangzhou Huaan Biotechnology Co., Ltd., ET1702), DAB colorimetric reagent kit (Beijing Solarbio Science & Technology Co., Ltd., DA1010), formic acid (Thermo Fisher Scientific, USA). Fisher Scientific (A117); SDS-PAGE protein loading buffer (Shanghai Yamei Biotechnology Co., Ltd., PS105); RIPA lysis buffer (Beyotime Biotechnology Co., Ltd., P0013c); dithiothreitol (Sigma-Aldrich, 43819-5G); BSA (Sangon Biotech Co., Ltd., A0332); Trypsin (Promega, V5117); DAPI (Affinity Biosciences, C0020); CCK8 kit (Dojin Chemical, CK04); ECL chemiluminescence kit (Shanghai Shenger Biotechnology Co., Ltd., SB-WB012); exosome tracing kit (Huaying Biotechnology, ESQ-G-001).

[0064] B. Main consumables: Cell counting chamber (IBIDI, Germany, 80816), trypsin (Shanghai Maclean Biotechnology Co., Ltd., T6325), fetal bovine serum (BI, Israel, 1809249), DMEM medium (CORNING, USA, 10-013-CVRC), penicillin and streptomycin (Hyclone, USA, SV30010), DMSO (Shanghai Maclean Biotechnology Co., Ltd., D806647), PBS buffer (Hyclone, USA, SH30256.01), 15 / 50ml centrifuge tubes (CORNING, USA, 430790, 430829), 1.5ml centrifuge tubes (AXYGEN, USA, MCT-150-C), cryovials (BD, USA, 430659), culture dishes / pipettes / pipettes (Eppendorf, Germany), invasion kit / Transwell chamber (CORNING, USA).

[0065] C. Major Instruments: Roche Cell Count Analyzer (Roche, Switzerland, Cedex XS), Cell Culture Incubator (SANYO, Japan, MCO-18AIC), Paraffin Microtome (Leica, Germany, RM2125RTS), Chemiluminescence Imaging System (Shanghai Tianneng Technology Co., Ltd., Tanon 5200), Microplate Reader (Biotek, USA, Synergy H4), Electrophoresis / Transfer System (Bio-Rad), Inverted Fluorescence Microscope (Leica, Germany, DMI3000B); High-Speed ​​Refrigerated Centrifuge (Eppendorf, Germany, 5804R / 5418R); Ultrasonic Disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., JY96-IIN); MP Fastprep-24 Homogenizer (MP, USA, MP Fastprep-24) 5G); Electronic balance (OHAUS Shanghai OHAUS International Trade Co., Ltd., AX324Z); Fully automated pathology slide scanner (Ningbo Jiangfeng Bioinformatics Technology Co., Ltd., KF-PRO-120).

[0066] (5) Animal experiments

[0067] A. Establishment of a mouse subcutaneous xenograft model: The modeling method followed previous literature from our research group. When the 2LL cell line was cultured to the logarithmic growth phase, the cells were counted at 1×10⁻⁶. 7 / ml, suspended in serum-free culture medium. 0.1ml of the above cell suspension was injected into the right axilla of mice using an insulin injection needle to establish a mouse subcutaneous xenograft lung metastasis model.

[0068] B. Model Grouping and Intervention Methods: On the 7th day after modeling, mice were randomly divided into a Lung Accumulation Formula group and a saline control group, with 8 mice in each group. The drug concentration for experimental animals was calculated to be 2 g / ml based on the human-animal drug conversion factor. The lyophilized powder was dissolved into a 2 g / ml solution, and 0.2 ml was administered each time for 21 consecutive days. After treatment, mice were anesthetized with 2% sodium pentobarbital. After anesthesia, the thoracic cavity was fully exposed, and both lungs were removed. The lung metastasis and the number of metastatic lesions were observed under a microscope, and a portion of the tissue was cut and fixed in 4% paraformaldehyde for later use.

[0069] 3.2 Experimental Index Detection Methods

[0070] A. CCK8 assay to detect the cytotoxicity of Lung-Rock Extract on CTC-TJH-01 / H1299 cell lines: CTC-TJH-01 and H1299 cells in logarithmic growth phase were collected, washed once with PBS, and the cell concentration was adjusted to 3×10⁻⁶ cells / cells using the prepared culture medium. 4 Cells were cultured at a concentration of 100 μl / ml, and 100 μl of cell suspension (3000 cells / well) was added to each well of a 96-well plate. After culturing for 24 h, drug concentrations of 0, 50, 100, 200, 400, and 800 μg / ml were added to the corresponding wells. After 48 h of drug treatment, 20 μl of CCK-8 solution was added to each well, and the plates were cultured for another 2 h. Finally, the absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated. [Cell viability = (Experimental group OD - Blank group OD) / (Control group OD - Blank group OD) × 100%]

[0071] B. Transwell assay of the effect of Lung-Yanshu on the invasion and migration of CTC-TJH-01 / H1299 cells: The invasion chamber kit was removed from a -20°C freezer, placed in a 24-well plate, and kept at room temperature. After the cells returned to room temperature, 500 μl of serum-free DMEM medium was added to both the upper and lower chambers, and the plates were incubated at 37°C for 2 h to allow the Matrigel matrix layer to rehydrate. CTC-TJH-01 / H1299 cells were cultured, digested, resuspended in serum-free medium, counted, and the cell concentration was adjusted to 1.6 × 10⁻⁶. 5 Cells were added at a density of 100 cells / ml to the upper and lower chambers, respectively. Then, 750 μl of drug-containing medium with 20% FBS was added to the lower chamber, and 500 μl of drug-containing cell suspension was added to the upper chamber. The cells were then incubated for 16 h. Afterward, the cells were removed and the medium was discarded. The cells were stained with Giemsa stain for 5 min, then gently rinsed with water to remove the Giemsa stain. The cells were then air-dried at room temperature. Five fields of view were randomly photographed under a microscope, and the cells were counted and statistically analyzed.

[0072] C. Cell cloning assay to detect the effect of Lung Accumulation Formula on the migration of CTC-TJH-01 / H1299 cells: Cells were seeded in 6-well plates (1×10⁶ cells / wells). 6 Cells were cultured in a 37°C, 5% CO2 cell culture incubator (cells / well). When the cells formed a confluent monolayer, the culture medium was removed and a "scratch" was gently made with the tip of a 200 μl pipette. The cells were then gently rinsed twice with cold PBS. Drug was added according to different drug concentrations. After adding culture medium, the cells were placed in an incubator. Cell migration was observed using an inverted microscope after 24 hours.

[0073] D. Western blot assay to detect the effect of Lung-Accumulating Formula on the expression of exosomal proteins CFHR5 / C9 / MBL2 in CTC-TJH-01 / H1299 cells: The expression level of exosomal proteins CFHR5 / C9 / MBL2 in the culture supernatant of CTC-TJH-01 / H1299 cells was detected by Western blot (WB). CTC-TJH-01 / H1299 cells treated with different gradients of Lung-Accumulating Formula were added to RIPA lysis buffer and lysed on ice for 30 min. After lysis, the cells were centrifuged at 4°C (12000 rpm, 20 min) and the supernatant was collected. The total protein concentration of lung tissue was determined by BCA method. 40 μL of protein was taken from each well for loading. According to the kit instructions, 10% separating gel and 5% stacking gel were prepared for protein separation (stacking gel voltage 80V, 30 min; separating gel voltage 120V, 45 min). The membrane was fixed in layers from bottom to top: filter paper-PVDF membrane-gel-filter paper. The target protein was separated by electrophoresis, and the separated protein was electrotransfer (360mA current for 90min) to the PVDF membrane. The PVDF membrane was placed in anhydrous methanol for a few seconds, then blocked with 5% BSA for 1h. After blocking, primary antibodies CFHR5 (1:1000), C9 (1:1000), and MBL2 (1:1000) were added, and the membrane was incubated overnight at 4°C with shaking. The membrane was washed with PBST, and secondary antibody IgG (1:1000) was added and incubated at room temperature with shaking for 90min. A chromogenic mixture was prepared according to the ECL chemiluminescence kit. The PVDF membrane was immersed in the mixture for a few seconds, then placed in a gel imaging system for development and band image acquisition. ImageJ software was used to analyze the target protein and internal control bands, and the band gray values ​​were calculated.

[0074] E. Exosome Tracing Assay: Exosomes were extracted, purified, and traced according to the instructions of the exosome tracing kit.

[0075] F. Immunohistochemistry (IHC) assay to detect the effect of Lung Biopsy on the expression of exosomal proteins CFHR5 / C9 / MBL2 in lung tissue of tumor-bearing mice: Fixed lung tissue was paraffin-embedded and sectioned, then placed in citrate antigen retrieval solution for antigen retrieval. After washing, it was incubated in 3% H2O2 at room temperature for 25 min, and then blocked in 3% BSA for 1 h. CFHR5 (1:500), MBL2 (1:500), and C9 (1:500) antibodies were added and incubated at 4°C overnight. After washing again, HRP-labeled secondary antibody IgG was added and incubated for 1 h. Then, color development was performed according to the DAB kit instructions, followed by washing and hematoxylin coverage. After washing again, it was immersed in 1% hydrochloric acid alcohol for differentiation, then returned to the basket with ammonia, rinsed with running water, dehydrated with ethanol, and blocked. The slides were scanned with a digital pathological scanner, and the optical density values ​​of positive cells were counted using Image-Pro Plus 6.0. The average optical density value was calculated and statistically analyzed.

[0076] G. Statistical Methods: GraphPad Prism 8.0 software was used for statistical analysis. t-tests were used for pairwise comparisons. When the data followed a normal distribution, one-way ANOVA was used to compare differences between groups. When the data did not follow a normal distribution, the rank-sum test was used. The least significant difference method was used for pairwise comparisons when variances were homogeneous; the Games-Howell method was used for pairwise comparisons when variances were unequal. The criterion for statistical significance was α = 0.05, and p ≤ 0.05 indicated a statistically significant difference (*p < 0.05, **p < 0.01, ***p < 0.001).

[0077] 3.3 Experimental Results:

[0078] (1) Inhibitory effect of Lung-Yanshu on lung metastasis in mice with subcutaneous xenograft tumors.

[0079] The presence of lung metastases is an important indicator for assessing clinical metastasis prevention and treatment. Microscopic observation of the number of lung metastases in the treatment and control groups showed a significant reduction in the number of metastases compared to the control group (p<0.05). Furthermore, there were no significant changes in body weight and tumor volume in the treatment group on day 21 compared to the control group (p>0.05). See below for detailed results. Figure 9 .

[0080] (2) Effect of Lung-Rock-Soothing on the expression of exosomal proteins CFHR5 / MBL2 / C9 in lung tissue.

[0081] The expression of CFHR5 / MBL2 / C9 in lung tissue was detected by IHC to clarify the regulatory role of pulmonary liposome extract on exosomal proteins. Compared with the control group, the expression of CFHR5 / MBL2 / C9 proteins in lung tissue was significantly reduced in the treatment group (p<0.05). Figure 10 .

[0082] (3) Effects of Feiyanshu on the proliferation of lung cancer cell lines CTC-TJH-01 and H1299.

[0083] In the above study, we confirmed that tumor cells can secrete exosomes, and exosomes can serve as important messengers for intercellular communication. To further verify the effect of Feiyanshu on intervening in lung cancer metastasis, we used CCK8 and cell cloning experiments to reveal the effect of Feiyanshu on the proliferation of lung cancer cells. The CCK8 results showed that Feiyanshu had no obvious cytotoxic effect on CTC-TJH-01 at different concentrations; while at high concentrations, it showed an obvious cytotoxic effect on H1299 cells ( Figure 11 ).

[0084] The above experiments confirmed that Feiyanshu can inhibit the proliferation of tumor cells and has no obvious cytotoxic effect; at the same time, it indicated that it is safe below 400 μg / ml, so the next experiment was carried out at a concentration below 400 μg / ml.

[0085] (4) Study on the invasion and migration of Feiyanshu on CTC-TJH-01 and H1299 cells.

[0086] Clinical studies have found that Feiyanshu has the effect of inhibiting lung cancer metastasis, but the specific mechanism needs to be further studied. In the above study, we found that Feiyanshu had no inhibitory effect on the proliferation of CTC-TJH-01 and H1299 cells. Therefore, we further carried out scratch and Transwell experiments to evaluate whether Feiyanshu has an inhibitory effect on the metastasis of lung cancer cells. The results of the scratch experiment showed that at a drug concentration of 50 μg / ml, Feiyanshu could inhibit the migration of CTC-TJH-01 and H1299 cells (p<0.05), and with the increase of the drug concentration, its inhibitory effect gradually increased (p<0.01). The results of the Transwell experiment also showed that Feiyanshu had an obvious inhibitory effect on the migration and invasion of CTC-TJH-01 and H1299 cells (p<0.05)( Figure 12 ).

[0087] The above research results suggest that Feiyanshu has an inhibitory effect on the metastasis of CTC-TJH-01 and H1299 cells in vitro.

[0088] (5) Exosome tracing experiment reveals the phagocytosis of CTC-TJH-01 and H1299 exosomes.

[0089] Exosomes are microvesicles encapsulated in a lipid bilayer. They can be clearly visualized using the lipophilic dye PKH26 (red fluorescence). Therefore, we conducted an exosome tracing experiment to observe whether exosomes secreted by tumor cells could be phagocytosed by other tumor cells. Immunofluorescence staining results showed that exosomes secreted by CTC-TJH-01 cells could be phagocytosed by H1299 cells, and similarly, exosomes secreted by H1299 cells could be phagocytosed by CTC-TJH-01 cells. The phagocytosed exosomes were mainly distributed in the cytoplasm. Figure 13 ).

[0090] The above results suggest that exosomes can be phagocytosed by tumor cells when co-cultured with them, and also suggest that exosomes act as "messengers" for communication between tumor cells and immune cells or distant target organs.

[0091] (6) Effect of Lung-Yan-Shu on exosome proteins in the culture supernatant of CTC-TJH-01 and H1299 cells.

[0092] Previous studies investigated differentially expressed exosomal proteins in metastatic and non-metastatic NSCLC, finding that Lung-Yan-Suppressant (LF-Suppressant) could inhibit the expression of identified exosomal proteins in lung tissue. Therefore, we further investigated the effect of LF-Suppressant on the expression of exosomal proteins in lung cancer cell lines using Western blotting. The results showed that LF-Suppressant had no significant regulatory effect on exosomal protein C9; however, at a concentration of 50 μg / ml, LF-Suppressant downregulated CFHR5 and MBL2 proteins in CTC-TJH-01 and H1299 cells (p<0.05), and the inhibitory effect became more significant with increasing LF-Suppressant concentration (p<0.01). Figure 14 ).

[0093] The above findings suggest that Lungyanshu has the effect of inhibiting the secretion of exosomal proteins by CTC-TJH-01 and H1299 cells.

Claims

1. A traditional Chinese medicine composition for treating lung cancer, characterized in that, The raw medicinal materials are prepared by weight parts: Radix Astragali 15-30 parts, North Adenophora 10-15 parts, Radix Asparagi 10-15 parts, Ophiopogon 10-15 parts, Poria cocos 10-15 parts, Berberis 15-30 parts, Radix Trifolii 15-30 parts, Yunnan rhizoma 15-30 parts, Cordyceps 15-30 parts, Prunella 15-30 parts, Houttuynia cordata 15-30 parts, Ligustrum lucidum 10-15 parts.

2. The traditional Chinese medicine composition for treating lung cancer according to claim 1, characterized in that, The raw medicinal materials are prepared by weight parts: Radix Astragali 15-30 parts, North Adenophora 10-15 parts, Radix Asparagi 10-15 parts, Ophiopogon 10-15 parts, Poria cocos 10-15 parts, Berberis 15-30 parts, Radix Trifolii 15-30 parts, Yunnan rhizoma 15-30 parts, Cordyceps 15-30 parts, Prunella 15-30 parts, Houttuynia cordata 15-30 parts, Ligustrum lucidum 10-15 parts.

3. Use of the traditional Chinese medicine composition of claim 1 or 2 in the preparation of a drug for treating lung cancer.

4. Use of the traditional Chinese medicine composition of claim 1 or 2 in the preparation of a drug for preventing and treating lung cancer metastasis.

5. Use according to 3 or 4, characterized in that, The traditional Chinese medicine composition can inhibit lung cancer cell proliferation, promote apoptosis, and play an anti-lung cancer invasion and metastasis role by interfering with exosomes, improve the clinical symptoms of tumor patients, regulate immune function, and improve the quality of life.

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