A traditional Chinese medicine compound for treating pneumoconiosis

Abstract

This invention provides a traditional Chinese medicine compound for treating pneumoconiosis, belonging to the field of biomedical technology. The compound is a modified version of Fangji Huangqi Decoction, Tingli Dazao Xiefei Decoction, Linggui Zhugan Decoction, and Jiegeng Decoction. The principal ingredient, Fangji Huangqi Decoction, tonifies Qi and strengthens the spleen, consolidating the foundation and preventing toxins, primarily addressing the underlying deficiency. The assistant ingredients, Linggui Zhugan Decoction + Tingli Dazao Xiefei Decoction + Ginseng, warm and resolve phlegm, purge the lungs and expel water, and greatly replenish vital energy, addressing both the root cause and symptoms. The adjuvant ingredients, Danggui, Chuanxiong, Ziwan, Xingren, Qianhu, and Jiegeng Decoction, invigorate blood circulation, clear the lungs and resolve phlegm, relieve stagnation and guide the flow of Qi, harmonize the other ingredients, and alleviate both symptoms and symptoms. This compound has a significant therapeutic effect on silicosis mice, significantly improving alveolar structural damage, inhibiting the formation of cellular nodules in lung tissue, reducing their number and size, and preventing their transformation into fibrous nodules, thus significantly delaying the fibrotic process of silicosis. This traditional Chinese medicine compound provides a new strategy and option for the treatment of pneumoconiosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a traditional Chinese medicine compound for the treatment of pneumoconiosis. Background Technology

[0002] Pneumoconiosis, also known as pulmonary dust deposition disease, refers to a group of diseases caused by the long-term inhalation of industrial dust during occupational activities, leading to dust retention in the lungs and diffuse fibrosis of the lung tissue. Silicosis and coal worker's pneumoconiosis are the most common types. Thanks to continuous improvements in the working environment and strict occupational protection measures, the incidence of pneumoconiosis in traditional industries such as mining and casting has been effectively controlled. However, with the development of modern industrial technology, such as sandblasting of denim fabrics and the manufacturing and processing of artificial quartz countertops, pneumoconiosis is showing a resurgence trend in emerging industries, forming a new epidemic and attracting widespread social attention. Its essence lies in the lung inflammation and irreversible progressive fibrosis induced by continuous dust stimulation.

[0003] Modern medicine's intervention in pneumoconiosis mainly focuses on symptomatic and supportive treatment, such as oxygen therapy and bronchodilators. For the core driver of disease progression—pulmonary fibrosis—there is currently a lack of specific drugs and therapies that can reverse or effectively halt its progression, leaving clinical needs far from being met. From the perspective of the evolution of the pathogenesis in Traditional Chinese Medicine (TCM), the early stage of pneumoconiosis is dominated by pathogenic factors, with dust toxins obstructing the lung channels, impairing the lung's function of dispersing and descending qi, and causing phlegm to stagnate, clinically manifested as cough, expectoration, and chest tightness. In the middle stage, deficiency and excess are mixed, with phlegm and blood stasis intertwined, and airway spasms, manifested as chest pain, severe shortness of breath, and cyanosis of the lips. In the late stage, the root cause is deficiency and the manifestation is excess, with the lung disease affecting the spleen and kidneys, leading to deficiency of qi, yin, and yang in the lungs, spleen, and kidneys, with phlegm, fluid retention, blood stasis, and internal accumulation of water and qi becoming the core pathogenesis of the middle and late stages. Faced with the predicament of modern medicine in anti-fibrotic treatment, TCM, with its unique advantages of multi-target and holistic regulation, provides a new approach to the treatment of silicosis by starting from the overall pathogenesis and conducting staged treatment, demonstrating unique potential and becoming an important clinical choice. Currently, the integrated approach of traditional Chinese and Western medicine for the treatment of pneumoconiosis has been widely used in clinical practice and has achieved good results, providing a new strategy for breaking through the bottleneck in the treatment of pneumoconiosis and improving the quality of life of patients. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the shortcomings of the existing technology, the present invention provides a traditional Chinese medicine compound for the treatment of pneumoconiosis.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a traditional Chinese medicine compound for treating pneumoconiosis. The compound is derived from the Fangji Huangqi Decoction and Tingli Dazao Xiefei Decoction from the *Synopsis of Prescriptions of the Golden Chamber*, and the Linggui Zhugan Decoction and Jiegeng Decoction from the *Treatise on Cold Damage*. The compound consists of the following medicinal materials by weight: Astragalus membranaceus 14-26g, Fangji 9-21g, Atractylodes macrocephala 9-21g, Tingli Zi 3-15g, Aster tataricus 3-15g, Cinnamomum cassia 3-15g, Poria cocos 9-21g, Jiegeng 3-15g, Ginseng 3-15g, Angelica sinensis 3-15g, Ligusticum chuanxiong 3-15g, Prunus armeniaca 3-15g, Peucedanum praeruptorum 3-15g, Jujube 9-21g, and Glycyrrhiza uralensis (processed) 3-15g.

[0009] Furthermore, the traditional Chinese medicine compound is composed of the following medicinal materials by weight: Astragalus membranaceus 17-23g, Stephania tetrandra 12-18g, Atractylodes macrocephala 12-18g, Lepidium apetalum 6-12g, Aster tataricus 6-12g, Cinnamomum cassia 6-12g, Poria cocos 12-18g, Platycodon grandiflorus 6-12g, Panax ginseng 6-12g, Angelica sinensis 6-12g, Ligusticum chuanxiong 6-12g, Prunus armeniaca 6-12g, Peucedanum praeruptorum 6-12g, Ziziphus jujuba 12-18g, and Glycyrrhiza uralensis (processed) 6-12g.

[0010] Furthermore, the traditional Chinese medicine compound is composed of the following Chinese medicinal materials by weight: Astragalus membranaceus 20g, Stephania tetrandra 15g, Atractylodes macrocephala 15g, Lepidium apetalum 10g, Aster tataricus 9g, Cinnamomum cassia 9g, Poria cocos 15g, Platycodon grandiflorus 9g, Panax ginseng 9g, Angelica sinensis 9g, Ligusticum chuanxiong 9g, Prunus armeniaca 9g, Peucedanum praeruptorum 9g, Ziziphus jujuba 15g, and Glycyrrhiza uralensis (processed) 9g.

[0011] This invention also provides the application of the above-mentioned traditional Chinese medicine compound in the preparation of a drug for treating pneumoconiosis. The drug is made from the above-mentioned weight of traditional Chinese medicinal materials as raw materials, and is prepared by conventional traditional Chinese medicine manufacturing methods, supplemented with pharmaceutically acceptable excipients, into any one of the following: decoction, oral liquid, granules, tablets, pills, and capsules.

[0012] The root cause of pneumoconiosis lies in the invasion of external pathogenic factors, particularly the dryness and toxicity of minerals, leading to deficiency of lung and spleen qi and weakened defensive qi. Lung qi deficiency results in an inability to defend against toxins and a deficiency of vital qi; spleen deficiency leads to impaired digestion and transformation, causing the accumulation of dampness and phlegm, which obstructs the lung channels. The core pathogenesis of mid-to-late stage pneumoconiosis is rooted in lung and spleen qi deficiency, with symptoms including internal retention of phlegm and fluid, water retention in the lungs, phlegm and blood stasis, and impaired lung qi circulation. Based on this pathogenesis, the herbal formula of this invention is derived from the Fangji Huangqi Decoction and Tingli Dazao Xiefei Decoction from the *Jinkui Yaolue*, and the Linggui Zhugan Decoction and Jiegeng Decoction from the *Shanghan Lun*, further modified with ginseng, angelica, chuanxiong, aster, apricot kernel, and angelica root.

[0013] In this invention's traditional Chinese medicine compound formula, Fangji Huangqi Decoction is the principal ingredient, primarily used to invigorate qi and strengthen the spleen, protect the exterior, nourish the earth element to generate metal, and promote diuresis and eliminate turbidity. Astragalus is used in large quantities to tonify lung qi, strengthen the exterior, and boost the ancestral qi, thus promoting blood circulation, water metabolism, and expelling toxins and pathogens. Fangji clears the lung channels, promotes diuresis and eliminates dampness, and expels stagnant toxins and dampness from the lung channels. Atractylodes macrocephala invigorates the spleen, dries dampness, nourishes the middle jiao, and eliminates the source of phlegm and fluid retention. Jujube and prepared licorice root tonify the middle jiao, relieve spasms, protect the spleen and stomach, and harmonize the properties of the other herbs. The principal ingredient specifically addresses the root cause, tonifying the lungs and spleen, strengthening the defensive qi, expelling toxins, and eliminating the root of phlegm and dampness, directly targeting the internal deficiency and pathogenesis of pneumoconiosis.

[0014] Then, the formula combines Linggui Zhugan Decoction, Tingli Dazao Xiefei Decoction, and ginseng as assistant herbs, integrating warming yang to resolve phlegm, purging the lungs and eliminating water, and greatly replenishing vital energy, addressing both the root cause and the symptoms, and simultaneously supporting the body's resistance and eliminating pathogenic factors. Linggui Zhugan Decoction (Poria, Cinnamon Twig, Atractylodes Macrocephala, and Prepared Licorice Root) warms yang to resolve phlegm, strengthens the spleen and promotes diuresis, and unblocks yang to descend rebellious qi. It is used for mid-to-late stage lung disease and spleen disorders, abnormal fluid metabolism, phlegm retention, and water retention affecting the heart and lungs. Poria is used to promote diuresis and guide phlegm downward; Cinnamon Twig warms yang to transform qi and promote lung function; Poria and Cinnamon Twig, combined with Atractylodes Macrocephala and Licorice Root, strengthen the spleen and control water, warm and transform cold phlegm, and resolve phlegm and water retention. Tingli and Jujube Lung-Clearing Decoction (Tingli and Jujube): Tingli is bitter and cold, with a strong downward effect, clearing the lungs, relieving asthma, expelling phlegm, promoting fluid metabolism, and opening the lungs to disperse stagnation. It directly purges the stagnant phlegm, fluid, and toxins in the lungs. Jujube is sweet and warm, moderately tonifying, and moderating the strong nature of Tingli, purging pathogens without harming the lung and spleen's vital energy. It is specifically used to treat lung distension, wheezing, and phlegm accumulation in the lungs. Ginseng greatly tonifies the lung and spleen's original qi, assisting the principal herb in strengthening the body's foundation and enhancing its ability to replenish qi and consolidate the body's vital energy. It improves symptoms of pneumoconiosis such as shortness of breath, lethargy, fatigue, spontaneous sweating, and weakness, supporting the body's resistance to expel pathogens. The assistant herbs work together to warm and transform internal phlegm and fluid retention, forcefully purge stagnant fluid in the lungs, and further tonify the lung and spleen's original qi. It combines tonification and purging, and purging while tonifying, to treat the key symptoms of deficiency in the root and excess in the branch.

[0015] The formula also uses Angelica sinensis, Ligusticum chuanxiong, Aster tataricus, Prunus armeniaca, and Peucedanum praeruptorum as adjuvants, combined with Platycodon grandiflorus, to invigorate blood circulation, clear phlegm, relieve stagnation, guide the medicine to the meridians, and harmonize the effects of other herbs. Angelica sinensis and Ligusticum chuanxiong nourish and invigorate blood, promote qi circulation, clear stagnation, and resolve stagnation. In cases of chronic pneumoconiosis, phlegm and blood stasis accumulate in the collaterals, obstructing the lung collaterals. These two herbs promote the flow of qi and blood in the lung collaterals, dissipate stagnation, and improve symptoms such as chest pain, cyanosis of the lips, and obstruction of the collaterals, clearing the collaterals without harming the body's vital energy. Aster tataricus warms and moistens, resolves phlegm, and stops coughing. Prunus armeniaca descends lung qi, relieves cough and asthma. Peucedanum praeruptorum disperses lung qi, eliminates pathogens, and resolves phlegm. The combination of these three herbs, one to disperse, one to descend, and one to resolve, restores the lung's function of dispersing and descending qi, clears the airways, resolves phlegm, and relieves chronic cough and asthma. Platycodon grandiflorum Decoction (Platycodon grandiflorum, prepared licorice root): Platycodon grandiflorum opens and disperses lung qi, relieves stagnation and dissipates nodules, and guides the medicine upwards, serving as a key guiding herb for the lung meridian, opening up the lung channels blocked by dust, toxins, and phlegm. Prepared licorice root relieves spasms, protects the lungs, clears heat and detoxifies, and harmonizes the cold, hot, tonifying, and purging properties of all the herbs in the formula. The overall effect of the adjuvant herbs is: to invigorate blood and unblock the channels to disperse stagnation in the lung channels, to promote the downward flow of qi and resolve phlegm to restore the lung qi mechanism, to open stagnation and guide the medicine into the lungs, to harmonize the other herbs so that tonification does not retain pathogens and purging does not harm the body's vital energy, and to assist the principal and assistant herbs in clearing away external pathogens and regulating qi, blood, and the lung channels.

[0016] In summary, the principal herbal formula of this invention uses Fangji Huangqi Decoction to invigorate qi and strengthen the spleen, consolidating the foundation and preventing toxins, primarily addressing the root cause of deficiency; the assistant formula uses Linggui Zhugan Decoction + Tingli Dazao Xiefei Decoction + Ginseng to warm and transform phlegm and fluid retention, drain the lungs and expel water, and greatly replenish vital energy, addressing both the root cause and the symptoms; the adjuvant formula uses Danggui, Chuanxiong, Ziwan, Xingren, Qianhu, and Jiegeng Decoction to invigorate blood circulation, clear the meridians, disperse lung qi and resolve phlegm, relieve stagnation and guide the flow of qi, harmonize the other herbs, and alleviate both the symptoms and the underlying condition. The entire formula is meticulously structured, with a strict hierarchy of principal, assistant, adjuvant, and adjuvant herbs, simultaneously tonifying the lungs and spleen, regulating qi, blood, and fluids, combining tonification and purgation, and treating both dispersing and descending effects, closely addressing the entire pathogenesis of late-stage pneumoconiosis characterized by lung and spleen qi deficiency, phlegm and fluid retention, phlegm and blood stasis, and lung qi stagnation.

[0017] Furthermore, this invention utilizes a silicosis mouse model to evaluate the application prospects of this traditional Chinese medicine compound in the preparation of drugs for treating pneumoconiosis. Experimental results show that the traditional Chinese medicine compound of this invention has a significant therapeutic effect on silicosis mice. The compound can significantly improve alveolar structural damage in silicosis mice, inhibit the formation of cellular nodules in lung tissue, reduce their number and size, and prevent their transformation into fibrotic nodules, thus significantly delaying the progression of silicosis fibrosis. Its mechanism of action may be through inhibiting lactate levels during glycolysis remodeling and downregulating the protein expression levels of key glycolytic enzymes LDHA, PFKFB3, and PKM2 to inhibit the progression of lung fibrosis. Attached Figure Description

[0018] Figure 1 The experimental procedure (A) and the HE and Mosson staining results of lung tissues of mice in each group at 14d and 28d after drug administration are shown.

[0019] Figure 2 This invention analyzes the energy metabolism of lung tissue in mice with silicosis after treatment with a traditional Chinese medicine compound. (A) Heatmap of differential expression profiles in lung tissue of each group of mice (D28-C: Saline group, D28-S: Silica group, D28-LS: JFHT+L+Silica group, D28-MS: JFHT+M+Silica group, D28-HS: JFHT+H+Silica group). (B) KEGG enrichment analysis results of differentially expressed genes. (C) Comparison of lactate content in lung tissue of mice in each group.

[0020] Figure 3 Expression results of key glycolytic enzymes in the lung tissue of mice in each group. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1

[0023] A traditional Chinese medicine compound for treating pneumoconiosis is derived from the Fangji Huangqi Decoction and Tingli Dazao Xiefei Decoction from the *Synopsis of Prescriptions of the Golden Chamber*, and the Linggui Zhugan Decoction and Jiegeng Decoction from the *Treatise on Cold Damage*. The compound consists of the following medicinal materials by weight: Astragalus membranaceus 14g, Fangji 9g, Atractylodes macrocephala 9g, Tingli Zi 3g, Aster tataricus 3g, Cinnamomum cassia 3g, Poria cocos 9g, Jiegeng 3g, Ginseng 3g, Angelica sinensis 3g, Ligusticum chuanxiong 3g, Apricot kernel 3g, Peucedanum praeruptorum 3g, Jujube 9g, and Prepared licorice root 3g.

[0024] The above-mentioned traditional Chinese medicine compound is used to prepare a drug for treating pneumoconiosis. The drug is made from the above-mentioned weight of traditional Chinese medicinal materials as raw materials, and is prepared according to conventional traditional Chinese medicine manufacturing methods, supplemented with pharmaceutically acceptable excipients, into any one of the following: decoction, oral liquid, granules, tablets, pills, and capsules.

[0025] Example 2

[0026] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following medicinal materials by weight: Astragalus membranaceus 26g, Stephania tetrandra 21g, Atractylodes macrocephala 21g, Lepidium apetalum 15g, Aster tataricus 15g, Cinnamomum cassia 15g, Poria cocos 21g, Platycodon grandiflorus 15g, Panax ginseng 15g, Angelica sinensis 15g, Ligusticum chuanxiong 15g, Prunus armeniaca 15g, Peucedanum praeruptorum 15g, Ziziphus jujuba 21g, and Glycyrrhiza uralensis (processed) 15g.

[0027] Example 3

[0028] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following Chinese medicinal materials by weight: Astragalus membranaceus 17g, Stephania tetrandra 12g, Atractylodes macrocephala 12g, Lepidium apetalum 6g, Aster tataricus 6g, Cinnamomum cassia 6g, Poria cocos 12g, Platycodon grandiflorus 6g, Panax ginseng 6g, Angelica sinensis 6g, Ligusticum chuanxiong 6g, Prunus armeniaca 6g, Peucedanum praeruptorum 6g, Ziziphus jujuba 12g, and Glycyrrhiza uralensis (processed) 6g.

[0029] Example 4

[0030] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following Chinese medicinal materials by weight: Astragalus membranaceus 23g, Stephania tetrandra 18g, Atractylodes macrocephala 18g, Lepidium apetalum 12g, Aster tataricus 12g, Cinnamomum cassia 12g, Poria cocos 18g, Platycodon grandiflorus 12g, Panax ginseng 12g, Angelica sinensis 12g, Ligusticum chuanxiong 12g, Prunus armeniaca 12g, Peucedanum praeruptorum 12g, Ziziphus jujuba 18g, and Glycyrrhiza uralensis (processed) 12g.

[0031] Example 5

[0032] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine compound is composed of the following Chinese medicinal materials by weight: Astragalus membranaceus 20g, Stephania tetrandra 15g, Atractylodes macrocephala 15g, Lepidium apetalum 10g, Aster tataricus 9g, Cinnamomum cassia 9g, Poria cocos 15g, Platycodon grandiflorus 9g, Panax ginseng 9g, Angelica sinensis 9g, Ligusticum chuanxiong 9g, Prunus armeniaca 9g, Peucedanum praeruptorum 9g, Ziziphus jujuba 15g, and Glycyrrhiza uralensis (processed) 9g.

[0033] Experimental Example 1

[0034] The inhibitory effect of the traditional Chinese medicine compound of this invention on the proliferation of TGF-β1-induced lung fibroblasts MRC-5

[0035] 1 Experimental Drugs

[0036] Test substance 1: Astragalus membranaceus 20g, Stephania tetrandra 15g, Atractylodes macrocephala 15g, Lepidium apetalum 10g, Aster tataricus 9g, Cinnamomum cassia 9g, Poria cocos 15g, Platycodon grandiflorus 9g, Panax ginseng 9g, Angelica sinensis 9g, Ligusticum chuanxiong 9g, Prunus armeniaca 9g, Peucedanum praeruptorum 9g, Ziziphus jujuba 15g, Glycyrrhiza uralensis (processed) 9g

[0037] Test substance 2: Astragalus membranaceus 20g, Stephania tetrandra 15g, Atractylodes macrocephala 15g, Lepidium apetalum 10g, Cinnamomum cassia 9g, Poria cocos 15g, Platycodon grandiflorus 9g, Ziziphus jujuba 15g, Glycyrrhiza uralensis (processed) 9g

[0038] Test substance 3: Lepidium seed 10g, Aster tataricus 9g, Cinnamon twig 9g, Poria cocos 15g, Platycodon grandiflorus 9g, Ginseng 9g, Angelica sinensis 9g, Ligusticum chuanxiong 9g, Apricot kernel 9g, Peucedanum praeruptorum 9g, Jujube 15g, Prepared licorice root 9g

[0039] Test substance 4: Astragalus membranaceus 20g, Stephania tetrandra 15g, Aster tataricus 9g, Platycodon grandiflorus 9g, Panax ginseng 9g, Angelica sinensis 9g, Ligusticum chuanxiong 9g, Prunus armeniaca 9g, Peucedanum praeruptorum 9g

[0040] The above-mentioned test substances were prepared into fluid extracts by the Drug Analysis Laboratory of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine according to the corresponding formulations. The extracts were dried under reduced pressure, ground into powder, and then prepared into a 10 ng / mL drug-containing culture medium before use.

[0041] 2 Experimental Methods

[0042] Human embryonic lung fibroblasts (MRC-5) from the logarithmic growth region were seeded into 96-well plates, with 10 cells per well. 4Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours to allow for full adhesion and growth. They were divided into 7 groups: control group (MRC-5 cells + fresh culture medium); TGF-β1 group (MRC-5 cells + 100 μL fresh culture medium + 10 ng / mL TGF-β1); test substance group (MRC-5 cells + 100 μL drug-containing culture medium (test substances 1-4, respectively) + 10 ng / mL TGF-β1); and blank group (100 μL fresh culture medium). All cell groups were cultured at 37°C in a 5% CO2 incubator for 24 hours. After culturing, 10 μL of CCK-8 solution was added to each well, mixed, and incubated for another 4 hours. The absorbance (OD value) of each well was then measured at 450 nm using a microplate reader, and cell viability was calculated. Each group had 6 replicates.

[0043] Cell viability = [OD] TGF-β1 / 受试物 -OD 空白 ] / [DO 对照 -OD 空白 ]}×100%

[0044] 3 Experimental Results

[0045] Compared with the control group, MRC-5 cells significantly proliferated under TGF-β1 induction (P<0.05). Compared with TGF-β1, the proliferation of MRC-5 cells was significantly inhibited after treatment with different drugs (P<0.05); among them, test substance 1 had a significantly stronger inhibitory effect on TGF-β1-induced MRC-5 cell proliferation than test substances 2-4 (P<0.05).

[0046] In vitro experimental results show that the traditional Chinese medicine compound of this invention can significantly inhibit TGF-β1-induced MRC-5 cell proliferation to achieve the purpose of preventing and treating fibrosis. Importantly, based on the compatibility of traditional Chinese medicine, the experimental results also verify that there is a synergistic effect among the components of the traditional Chinese medicine compound. In particular, based on the classic formulas Fangji Huangqi Decoction, Tingli Dazao Xiefei Decoction, Linggui Zhugan Decoction, and Jiegeng Decoction, the newly added Aster tataricus, ginseng, angelica sinensis, chuanxiong rhizome, apricot kernel, and angelica dahurica can significantly enhance the anti-fibrotic pharmacological effect of the entire formula.

[0047] Table 1. MRC-5 cell survival rates after different drug treatments.

[0048] Group Cell viability / % control group 101.03±3.21 TGF-β1 <![CDATA[198.90±26.01 ① <!-- 4 -->]]> Test substance 1 <![CDATA[116.76±10.04 ② ]]> Test substance 2 <![CDATA[147.03±9.52 ②③ ]]> Test substance 3 <![CDATA[155.59±9.31 ②③ ]]> Test substance 4 <![CDATA[162.18±6.41 ②③ ]]>

[0049] Note: Compared with the control group, ① P<0.05; compared with TGF-β1, ② P<0.05; compared with test substance 1, ③ P<0.05

[0050] Experimental Example 2

[0051] Evaluation of the Therapeutic Effect of the Traditional Chinese Medicine Compound of the Invention on Mice with Silicosis Model

[0052] 1 Materials

[0053] 1.1 Silicon Dioxide Dust

[0054] Crystalline silicon dioxide dust (SiO2, purity 99%; particle size 0.5 - 10 μm) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. The dust was fully ground, sterilized by high-pressure steam, and suspended in physiological saline containing penicillin (6000 U / mL) to prepare a suspension with a final concentration of 50 mg / mL.

[0055] 1.2 Experimental Animals

[0056] Male C57BL / 6N mice, 6 - 8 weeks old, weighing (18 - 22 g), were purchased from Beijing Speefoo Biotechnology Co., Ltd. and raised in the IVC animal room of the First Affiliated Hospital of Henan University of Chinese Medicine (SYXK(Yu)2022 - 0004) according to SPF-level feeding conditions. Approved by the Ethics Review Committee of the First Affiliated Hospital of Henan University of Chinese Medicine (YFYDW2025050). The mouse lung embryonic fibroblast cell line (NIH / 3T3) was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-515.

[0057] 1.3 Drugs and Reagents

[0058] The traditional Chinese medicine compound of the invention (abbreviated as JFHT), with the formula of 20 g of Astragalus membranaceus, 15 g of Stephania tetrandra, 15 g of Atractylodes macrocephala, 10 g of Lepidium apetalum, 9 g of Aster tataricus, 9 g of Cinnamomum cassia, 15 g of Poria cocos, 9 g of Platycodon grandiflorum, 9 g of Panax ginseng, 9 g of Angelica sinensis, 9 g of Ligusticum chuanxiong, 9 g of Armeniaca vulgaris, 9 g of Peucedanum praeruptorum, 15 g of Ziziphus jujuba, and 9 g of roasted Glycyrrhiza uralensis, was prepared into a liquid extract by the Drug Analysis Laboratory of the First Affiliated Hospital of Henan University of Chinese Medicine, dried under reduced pressure, and ground into powder.

[0059] Positive control drug: Tetrandrine Tablets (Jin Aikang, Zhejiang Jinhua Conba Biopharmaceutical Co., Ltd., batch number: PH2003006, specification: 20 mg / tablet × 36 tablets).

[0060] 2 Research Methods

[0061] 2.1 Preparation of Animal Model

[0062] Experimental mice were acclimatized in a barrier animal facility for one week. They were then randomly divided into two groups: a saline control group (Contorl group, n=10) and a silica treatment group (n=50). During treatment, mice in the silica group were anesthetized with isoflurane (RWD, China) by inhalation, and then 50 μL of silica suspension (concentration 50 mg / mL) was injected into their trachea via a non-exposed tracheal infusion method. The control group received an equal volume of saline via the same method.

[0063] 2.2 Animal grouping and drug intervention

[0064] Mice used to establish an experimental silicosis model were randomly divided into five groups: Saline group, Silica group, JFHT+L+Silica group (low dose), JFHT+M+Silica group (medium dose), JFHT+H+Silica group (high dose), and tetrandrine+Silica group (positive control). The JFHT+L+Silica group, JFHT+M+Silica group, and JFHT+H+Silica group were administered the corresponding doses of the drug via gavage on day 2 after silica exposure, at doses of 2.223 g / (kg·d), 4.446 g / (kg·d), and 8.892 g / (kg·d), respectively, once daily. The tetrandrine+Silica group (positive control) was administered tetrandrine via gavage at doses of 0.039 g / (kg·d), once daily, on day 2 after silica exposure. The Controll group and Silica group received saline via gavage (0.2 mL / (kg·time)) during the intervention period. Lung tissue was collected from three mice in each group on days 14 and 28 after silica exposure for subsequent experiments. The experimental flowchart is shown below. Figure 1 As shown in Figure A.

[0065] 2.3 Hematoxylin-eosin staining

[0066] Left lung tissue from mice was fixed in 4% paraformaldehyde (Servicebio, China) for 24 h, then embedded in paraffin to prepare 4 μm thick sections. After dewaxing to water, the sections were stained with hematoxylin and eosin (HE): hematoxylin (Servicebio, China) staining for 5 min, differentiation in 1% hydrochloric acid ethanol (Servicebio, China) for 30 s, blue reversion with ammonia (Servicebio, China), staining with 0.5% eosin (Servicebio, China) for 2 min, dehydrated, and mounted with neutral resin. Separate sections were stained with Masson's stain: Weigert hematoxylin (Servicebio, China) staining for 8 min, washing with water, staining with Ponceau S and fuchsin (Servicebio, China) for 5 min, differentiation in 1% phosphomolybdic acid (Servicebio, China) for 2 min, counterstaining with aniline blue (Servicebio, China) for 5 min, and finally mounted with neutral resin (Absin, China). After staining, the degree of fibrosis in the mouse lung tissue was observed and assessed under an optical microscope.

[0067] 2.4 Masson's Trichrome Staining

[0068] After fixation, mouse left lung tissue was embedded in paraffin and sections with a thickness of 4 μm were prepared using a paraffin microtome. After dewaxing to water, the sections were stained with Weigert hematoxylin (Servicebio, China) for 5–10 min, rinsed with running water, stained with Ponceau S and fuchsin (Servicebio, China) for 5 min, differentiated with 1% phosphomolybdic acid aqueous solution for 2 min, and counterstained with aniline blue (Servicebio, China) for 5 min. After staining, the sections were mounted with neutral resin (Absin, China), and finally, collagen deposition in the mouse lung tissue was observed under an optical microscope.

[0069] 2.5 Immunoblotting assay

[0070] Total protein was extracted from cells or tissues, quantified using the BCA method, and the loading volume was adjusted. Loading buffer was added, and the mixture was boiled for denaturation for 10 min. Proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane using wet transfer. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. Anti-LDHA antibody (1:1000, Proteintech, China), Anti-PKM2 antibody (1:1500, Proteintech, China), and Anti-HK2 (1:1000, Proteintech, China) were incubated overnight at 4°C. After washing the membrane three times with TBST, the corresponding HRP-labeled secondary antibody (1:10000, Proteintech, China) was added, and the membrane was incubated at room temperature for 1 h. The membrane was washed again. Development was performed using ECL chemiluminescence, and images were acquired using a gel imaging system. The expression level of the target protein was analyzed by the grayscale values ​​of the bands.

[0071] 3 Results

[0072] 3.1 The traditional Chinese medicine compound of this invention can effectively delay the fibrosis process in silicosis mice.

[0073] A mouse model of silicosis was established using a non-exposed tracheal infusion method. The traditional Chinese medicine compound of this invention was administered daily by gavage starting from the first day after silica exposure, and the fibrosis status of the mouse lung tissue was assessed on days 14 and 28 post-exposure (Figure 1A). HE staining showed that, compared with the Saline group, the Silica group mice exhibited obvious early silicosis pathological changes in lung tissue on day 14 post-silica exposure, with a large number of macrophages agglomerating to form typical granulomatous structures, accompanied by inflammatory cell infiltration and widening of alveolar septa; while the JFHT group showed significantly reduced granuloma formation, milder inflammatory response, and relatively intact alveolar structure. By day 28, the granulomas in the Silica group had further progressed, forming typical cellular nodules with fibroblast proliferation and early collagen deposition, and some nodules had fused together. In contrast, the JFHT group showed only a small amount of scattered inflammatory cell aggregation without forming obvious cellular nodules, and the lung tissue structure was significantly improved compared to the model group. Masson staining further confirmed that by day 14, the Silica group had a small amount of blue collagen deposition around the granulomas and in the alveolar septa, while the JFHT group had significantly less collagen deposition than the model group. By day 28, the Silica group showed extensive deposition of dense blue collagen within the cellular nodules, forming obvious fibrotic structures, while the JFHT group only showed mild collagen distribution in local areas (Figure 1B). JFHT showed a similar therapeutic effect to tetrandrine. The above results indicate that the traditional Chinese medicine compound of this invention can significantly delay the progression of silicosis fibrosis.

[0074] 3.2 Energy metabolism analysis of lung tissue in silicosis mice treated with the traditional Chinese medicine compound of this invention

[0075] Forty key metabolites involved in processes such as the tricarboxylic acid cycle, glycolytic pathway, oxidative phosphorylation, and pentose phosphate pathway were detected using targeted metabolomics. Hierarchical clustering heatmaps of the metabolites showed that the selected target metabolites were stably expressed in samples within the same group (Figure 2A). Metabolites with significant differences were screened using a Fold Change > 1.5 (or < 1 / 1.5) and p < 0.05 as the criterion. In a silicosis model, lung tissue underwent a significant glycolytic reprogramming process due to silica dust stimulation (Figure 2B), manifested as a significant increase in lactate levels; however, effective traditional Chinese medicine treatment could inhibit this process, causing lactate levels to decrease accordingly (Figure 2C).

[0076] 3.3 The traditional Chinese medicine compound of this invention can inhibit the expression of key glycolytic enzymes in the lung tissue of silicosis mice.

[0077] Compared with the control group, the protein expression levels of key glycolytic enzymes LDHA, PFKFB3, and PKM2 in the lung tissue of silicosis model mice showed a significant upregulation trend. These three enzymes play a synergistic role in promoting the process of pulmonary fibrosis: PFKFB3, as a key regulatory enzyme of glycolysis, is responsible for accelerating the production of fructose-2,6-bisphosphate, thereby activating the entire glycolytic pathway; PKM2 promotes the activation and proliferation of myofibroblasts by regulating aerobic glycolysis and mechanosensitive signaling; and LDHA, as a terminal enzyme, catalyzes lactate production. Lactic acid not only participates in energy metabolism but can also act as a signaling molecule to further induce the expression of pro-fibrotic factors such as transforming growth factor-β (TGF-β), forming a vicious positive feedback loop. Together, these three enzymes drive the metabolic reprogramming of fibroblasts in the lung tissue of silicosis mice, providing energy and biosynthetic precursors for excessive deposition of the extracellular matrix, thereby significantly aggravating pulmonary fibrosis. After intervention with the traditional Chinese medicine compound of this invention or the positive control drug tetrandrine, the expression levels of the above-mentioned enzymes were significantly reversed, showing a marked downregulation; among them, the intervention effect of the traditional Chinese medicine compound of this invention showed a clear dose-dependent effect, and its inhibitory effect was positively correlated with the administered dose (e.g., Figure 3 (As shown).

[0078] In summary, the traditional Chinese medicine compound of this invention has a significant therapeutic effect on silicosis mice. This compound can significantly improve alveolar structural damage in silicosis mice, inhibit the formation of cellular nodules in lung tissue, reduce their number and size, and prevent their transformation into fibrotic nodules, thus significantly delaying the progression of silicosis fibrosis. Its mechanism of action may be through inhibiting lactate levels during glycolytic remodeling and downregulating the protein expression levels of key glycolytic enzymes LDHA, PFKFB3, and PKM2, thereby inhibiting the progression of lung fibrosis.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine compound for treating pneumoconiosis, characterized in that, The herbal compound is composed of the following herbs by weight: Astragalus membranaceus 14-26g, Stephania tetrandra 9-21g, Atractylodes macrocephala 9-21g, Lepidium apetalum 3-15g, Aster tataricus 3-15g, Cinnamomum cassia 3-15g, Poria cocos 9-21g, Platycodon grandiflorus 3-15g, Panax ginseng 3-15g, Angelica sinensis 3-15g, Ligusticum chuanxiong 3-15g, Prunus armeniaca 3-15g, Peucedanum praeruptorum 3-15g, Ziziphus jujuba 9-21g, and Glycyrrhiza uralensis (processed) 3-15g.

2. A traditional Chinese medicine compound for treating pneumoconiosis according to claim 1, characterized in that, The traditional Chinese medicine compound consists of the following Chinese medicinal materials by weight Composition: Astragalus membranaceus 17-23g, Stephania tetrandra 12-18g, Atractylodes macrocephala 12-18g, Lepidium apetalum 6-12g, Aster tataricus 6-12g, Cinnamomum cassia 6-12g, Poria cocos 12-18g, Platycodon grandiflorus 6-12g, Panax ginseng 6-12g, Angelica sinensis 6-12g, Ligusticum chuanxiong 6-12g, Prunus armeniaca 6-12g, Peucedanum praeruptorum 6-12g, Ziziphus jujuba 12-18g, Glycyrrhiza uralensis (processed) 6-12g.

3. A traditional Chinese medicine compound for treating pneumoconiosis according to claim 1, characterized in that, The herbal compound is composed of the following herbs by weight: Astragalus membranaceus 20g, Stephania tetrandra 15g, Atractylodes macrocephala 15g, Lepidium apetalum 10g, Aster tataricus 9g, Cinnamomum cassia 9g, Poria cocos 15g, Platycodon grandiflorus 9g, Panax ginseng 9g, Angelica sinensis 9g, Ligusticum chuanxiong 9g, Prunus armeniaca 9g, Peucedanum praeruptorum 9g, Ziziphus jujuba 15g, and Glycyrrhiza uralensis (processed) 9g.

4. The application of the traditional Chinese medicine compound according to any one of claims 1 to 3 in the preparation of a drug for treating pneumoconiosis, characterized in that, The drug is made from the above-mentioned weight of Chinese medicinal herbs as raw materials, and prepared according to conventional Chinese medicine manufacturing methods, supplemented with pharmaceutically acceptable excipients, into any one of the following: decoction, oral liquid, granules, tablets, pills, and capsules.

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