A traditional Chinese medicine compound for treating diabetic pulmonary fibrosis and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine compound for treating pulmonary fibrosis of diabetes and application thereof. The traditional Chinese medicine compound comprises 70-90 parts of Huangqi, 20-40 parts of Danshen, 10-30 parts of Chuanxiong, 20-40 parts of Beishashen and 40-60 parts of Shanyao. The application also discloses application of the traditional Chinese medicine compound in preparation of a medicine for treating pulmonary fibrosis of diabetes of the type of deficiency of both qi and yin and blood stasis. The traditional Chinese medicine compound has reasonable formula, and uses the traditional Chinese medicine theory scientifically. The raw materials are matched according to the principle of monarch, minister, assistant and guide, and the traditional Chinese medicines are synergistically used, so that the traditional Chinese medicine compound can not only supplement deficiency of both qi and yin of diabetes, but also dredge stasis of collaterals of long-term disease, and achieve good clinical test effect by considering both the symptoms and the root cause.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine composition technology, and specifically relates to a traditional Chinese medicine compound for treating diabetic pulmonary fibrosis and its application. Background Technology

[0002] Diabetic-induced pulmonary fibrosis (DiPF) refers to pulmonary fibrosis (PF) caused by the hyperglycemic state of diabetes and is one of the major complications of diabetes. The pathogenesis of DiPF is multifactorial and highly complex, and the specific mechanism is currently unclear. It may be related to chronic low-grade inflammation, oxidative stress, advanced glycation end products (AGEs), and hyperinsulinemia. Studies show that the clinical characteristics of DiPF patients mainly include abnormalities in three aspects: pulmonary function, imaging, and pathology. Their pulmonary function is reduced, presenting as restrictive ventilatory dysfunction and diffusion impairment, manifested as lower forced vital capacity (FVC), forced expiratory volume in one second (FEV1), total lung capacity (TLC), peak expiratory flow (PEF), and carbon monoxide diffusing capacity (DLco). Imaging abnormalities are linear or reticular changes on high-resolution CT images, which are different from typical... The imaging changes in idiopathic pulmonary fibrosis (UIP pattern, including reticular and honeycomb patterns) are similar; the pathological abnormalities are characterized by significant fibrotic lesions, mainly manifested by a large number of fibroblast proliferation and extracellular matrix (ECM) deposition. The proliferation of fibroblasts and ECM deposition mainly originate from epithelial-mesenchymal transition (EMT). EMT refers to the process by which a fully differentiated epithelial cell transforms into a mesenchymal cell phenotype, forming fibroblasts and myofibroblasts. Under pathological conditions, unrestricted activation of myofibroblasts will lead to excessive ECM deposition. In patients with diabetes mellitus (DM), long-term hyperglycemia leads to the accumulation of AGEs, oxidative stress, and inflammatory responses in lung tissue, which further leads to the transformation of abnormal epithelial cells into fibroblasts and myofibroblasts, producing excessive ECM, destroying tissue structure, and ultimately inducing pulmonary fibrosis.

[0003] Existing studies have demonstrated that EMT participates in the pathological process of DiPF by activating the TGF-β1 signaling pathway. TGF-β1 can promote fibroblast proliferation and aggregation through the Smads-mediated signal transduction pathway, promote the transformation of fibroblast phenotype to myofibroblast phenotype, and participate in ECM deposition. Smad3 is a key mediator of TGF-β1 signaling and is closely related to the development of fibrosis in multiple organs. The most prominent feature of diabetic patients is glucose metabolism disorder. Studies have shown that abnormal glucose metabolism can affect TGF-β1 activity and TGF-β1 signaling, suggesting that glucose metabolism participates in and regulates the occurrence and development of pulmonary fibrosis.

[0004] In traditional Chinese medicine, pulmonary fibrosis is often classified under the categories of "pulmonary atrophy" and "pulmonary obstruction," while diabetes is classified under the category of "diabetes mellitus." However, some scholars believe that treating the two separately as "pulmonary collateral disease" and "diabetes collateral disease" is more in line with the clinical characteristics of the diseases in traditional Chinese medicine. Diabetic pulmonary fibrosis is a disease caused by persistently high blood sugar, which leads to fibrosis in the lungs. It meets the characteristics of both diabetes collateral disease and pulmonary collateral disease. Therefore, diabetic pulmonary fibrosis can be summarized as "pulmonary diabetes collateral disease." The etiology of "pulmonary fibrosis" is mostly due to insufficient endowment, overwork, improper diet, or emotional imbalance, leading to dysfunction of the internal organs and disordered metabolism of food essence, resulting in the endogenous generation of "sugar toxins." "Sugar toxins" act as both a pathological product and a pathogenic factor, further hindering the circulation of qi, blood, and body fluids, aggravating the dysfunction of the internal organs and meridians, manifesting as elevated blood sugar and damage to the collaterals. Over time, "pulmonary fibrosis" can lead to damage to the lung collaterals by "sugar toxins," resulting in lung deficiency and collateral stasis. When the lung collaterals are not nourished, symptoms such as intractable wheezing, shortness of breath, cough, and chest tightness appear, eventually forming "pulmonary fibrosis." A core pathogenesis of pulmonary fibrosis in traditional Chinese medicine is blood stasis. The causes of blood stasis in traditional Chinese medicine include qi stagnation, cold coagulation, blood heat, qi deficiency, yang deficiency, and phlegm dampness. The core pathogenesis of diabetic pulmonary fibrosis is qi deficiency + yin deficiency + blood stasis.

[0005] Diabetic pulmonary fibrosis (DiPF) is often subclinical because the lungs have a rich compensatory reserve capacity. Clinical symptoms only appear when lung function declines by more than 30%, leading to a delayed development of early DiPF and missing the optimal time for early intervention. Studies show that DiPF patients have higher hospitalization and mortality rates, posing significant potential harm to patients. Currently, pirfenidone and nintedanib are FDA-approved drugs for the treatment of idiopathic pulmonary fibrosis, but related clinical studies suggest that their effects on mortality, lung function, and respiratory symptoms are limited, and their high cost and adverse reactions restrict their widespread use. There are currently no research reports on pirfenidone and nintedanib in DiPF patients. Therefore, researching and developing an effective traditional Chinese medicine compound for the prevention and treatment of diabetic pulmonary fibrosis remains a key focus and challenge in current research. Summary of the Invention

[0006] The purpose of this invention is to provide a traditional Chinese medicine compound for treating diabetic pulmonary fibrosis and its application, thereby solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of this invention provides a traditional Chinese medicine compound for treating diabetic pulmonary fibrosis, comprising the following components in parts by weight: 70-90 parts of Astragalus membranaceus, 20-40 parts of Salvia miltiorrhiza, 10-30 parts of Ligusticum chuanxiong, 20-40 parts of Adenophora stricta, and 40-60 parts of Dioscorea opposita.

[0009] Preferably, the traditional Chinese medicine compound comprises the following components in parts by weight: 80-90 parts of Astragalus membranaceus, 30-40 parts of Salvia miltiorrhiza, 10-20 parts of Ligusticum chuanxiong, 20-30 parts of Adenophora stricta, and 50-60 parts of Dioscorea opposita.

[0010] More preferably, the traditional Chinese medicine compound comprises the following components in parts by weight: 80 parts Astragalus membranaceus, 30 parts Salvia miltiorrhiza, 15 parts Ligusticum chuanxiong, 20 parts Adenophora stricta, and 50 parts Dioscorea opposita.

[0011] Preferably, the traditional Chinese medicine compound comprises the following components in parts by weight: 70-90 parts of Astragalus membranaceus, 20-40 parts of Salvia miltiorrhiza, 10-30 parts of Ligusticum chuanxiong, 20-40 parts of Adenophora stricta, 40-60 parts of Dioscorea opposita, 20-40 parts of Codonopsis pilosula, and 20-40 parts of Atractylodes macrocephala.

[0012] Preferably, the traditional Chinese medicine compound comprises the following components in parts by weight: 70-90 parts of Astragalus membranaceus, 20-40 parts of Salvia miltiorrhiza, 10-30 parts of Ligusticum chuanxiong, 20-40 parts of Adenophora stricta, 40-60 parts of Dioscorea opposita, 20-40 parts of Ophiopogon japonicus, and 40-60 parts of Rehmannia glutinosa.

[0013] Preferably, the traditional Chinese medicine compound comprises the following components in parts by weight: 70-90 parts Astragalus membranaceus, 20-40 parts Salvia miltiorrhiza, 10-30 parts Ligusticum chuanxiong, 20-40 parts Adenophora stricta, 40-60 parts Dioscorea opposita, 20-40 parts Curcuma zedoaria, 20-40 parts Prunus persica, and 10-30 parts Eupolyphaga sinensis.

[0014] The second aspect of this invention provides the application of a traditional Chinese medicine compound for treating diabetic pulmonary fibrosis in the preparation of a drug for treating diabetic pulmonary fibrosis of the qi deficiency, yin deficiency and blood stasis type.

[0015] Preferably, the drug dosage form is any one of pills, granules, powders, capsules, tablets, mixtures, and decoctions.

[0016] Solution of the present invention:

[0017] Astragalus and Dioscorea are the principal herbs, both of which enter the lung and spleen meridians. In the Shennong's Classic of Materia Medica, both are considered superior herbs. Astragalus is sweet and warm, tonifying qi and raising yang, and is good at tonifying spleen yang. Dioscorea is sweet and neutral, tonifying the spleen and nourishing the lungs, nourishing yin and generating fluids, and is good at tonifying spleen yin. When used together, the yin and yang are in harmony, and together they have the effects of strengthening the spleen and stomach and lowering blood sugar. The combination of the two herbs also has the effects of tonifying the spleen and nourishing yin, generating fluids and benefiting the lungs. It is often used to treat chronic cough due to lung deficiency and diabetes due to deficiency of both qi and yin.

[0018] Danshen has a bitter taste and is slightly cold in nature. It is used as an assistant herb and there is a saying that "one dose of Danshen has the same effect as four other herbs". It can both invigorate blood and nourish blood, and remove blood stasis without harming the body's vital energy.

[0019] North American ginseng is used as an adjuvant medicine, entering the lung and stomach meridians. It nourishes lung yin and benefits the stomach by promoting the production of body fluids. It is a preferred medicine for treating dry cough, wheezing, and thirst. The imperatorin it contains has anti-inflammatory and anti-asthmatic effects. When combined with yam, it enhances the yin-nourishing effect.

[0020] Chuanxiong has a pungent and warm nature, and it can circulate through the twelve meridians. As an adjuvant, it has the function of promoting blood circulation and removing blood stasis. It is a "qi medicine in the blood". When combined with Danshen, it combines cold and heat, and combines tonification and purgation, promoting blood circulation without harming the body's vital energy.

[0021] The main prescription contains only five Chinese herbs, which are refined and highly effective. They address the root cause by invigorating qi and nourishing yin, and address the symptoms by promoting blood circulation and unblocking collaterals. In clinical application, depending on the specific symptoms and the emphasis of the diagnosis, Codonopsis pilosula and Atractylodes macrocephala are often added to enhance the qi-tonifying effect, Ophiopogon japonicus and Rehmannia glutinosa are added to enhance the yin-nourishing effect, and Curcuma zedoaria, Prunus persica, and Eupolyphaga sinensis are added to break up blood stasis and eliminate blood stasis.

[0022] 2. Regarding single-herb medicines

[0023] Astragalus, sweet in taste and slightly warm in nature, enters the spleen and lung meridians. It has the effects of invigorating qi and raising yang, promoting tissue regeneration and detoxification, consolidating the exterior and stopping sweating, and promoting diuresis and reducing swelling. It is often used to strengthen the body's resistance and consolidate its foundation. The earliest record of Astragalus is found in the "Mingyi Bielu" which points out that Astragalus has the function of "quenching thirst". The "Ben Cao Jing Shu" states that "when the qi of Astragalus is strong, body fluids are generated, thus quenching thirst". Astragalus contains a variety of effective active ingredients, mainly polysaccharides, flavonoids, saponins and folic acid, which have the effects of enhancing immunity, anti-tumor, anti-oxidation, regulating blood sugar and scavenging oxygen free radicals.

[0024] Yam, sweet in taste and warm in nature, enters the lung, spleen, and kidney meridians. It has the functions of tonifying the spleen and stomach, promoting body fluid and replenishing qi, and tonifying the kidney and astringing essence. The "Compendium of Materia Medica" points out that yam can be used to treat "cough due to lung deficiency". Zhang Xichun's "Explanation of Yam" mentions: "Yam is white and enters the lung, sweet in taste and enters the spleen, thick in liquid and benefits the kidney, can nourish blood vessels, consolidate qi transformation, calm cough and relieve asthma" and "As for treating cough due to lung deficiency and shortness of breath due to kidney deficiency, yam is the best". The main components of yam include starch, amino acids, polysaccharides, trace elements, saponins, etc., which have the effects of regulating gastrointestinal function, lowering blood sugar, enhancing immunity, and delaying aging.

[0025] Danshen (Salvia miltiorrhiza) is bitter and slightly cold in nature, and enters the heart and liver meridians. According to *Ben Cao Zheng Yi* (Correct Interpretation of Materia Medica), Danshen can "reach the internal organs and resolve stagnation, thus eliminating accumulations and breaking up masses." *Shen Long Ben Cao Jing Bai Zhong Lu* (Record of One Hundred Kinds of Herbal Medicines) states that it "treats all blood-related diseases with coagulation." Danshen has a mild medicinal property, removing blood stasis without harming the body's vital energy. When treating masses, it is often used in combination with Curcuma zedoaria and Sparganium. Danshen mainly contains tanshinone, cryptotanshinone, and isotanshinone, which have effects such as altering blood rheology, improving microcirculation, anti-oxidation, anti-inflammation, and anti-fibrosis.

[0026] Chuanxiong (Ligusticum striatum) is pungent and warm in nature. It has the functions of promoting blood circulation, removing blood stasis, and relieving pain. The Compendium of Materia Medica states that it is a "qi-regulating medicine in the blood," and the Correct Meaning of Materia Medica states that it can "travel along the limbs and joints and unblock the meridians." Modern pharmacology has found that ligustrazine and ferulic acid are the main active ingredients in Chuanxiong, which have the effects of altering blood rheology, anti-oxidation, anti-thrombosis, and improving myocardial ischemia.

[0027] North American ginseng, with a sweet and bitter taste and slightly cold nature, has the effects of nourishing yin and clearing the lungs, benefiting the stomach and promoting the production of body fluids. It is often used to treat dry cough due to lung heat, cough with phlegm and blood due to overwork, and thirst due to depletion of body fluids from febrile diseases. According to "Ben Cao Cong Xin", North American ginseng can "specifically nourish lung yin, clear lung fire, and treat chronic cough and lung weakness". North American ginseng contains polyacetylenes, phenylpropanoids, flavonoids, steroids, terpenes, polysaccharides and other components, and has the effects of immunomodulation, anti-tumor, anti-inflammatory, antioxidant and liver protection.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. The traditional Chinese medicine compound formula of this invention is reasonable and scientifically applies the theory of traditional Chinese medicine. The raw materials are matched according to the "principal, assistant, adjuvant and guide" principle. The various Chinese medicines work synergistically to not only replenish the deficiency of both qi and blood in diabetic patients, but also to clear the stagnation in the collaterals caused by long-term illness. It addresses both the root cause and the symptoms and can achieve good clinical trial results. The results of clinical trials show that the traditional Chinese medicine compound formula of this invention can significantly improve the comprehensive efficacy score of patients with diabetic pulmonary fibrosis (deficiency of both qi and yin with blood stasis). Specifically, it can improve patients' lung function, improve the HRCT pulmonary fibrosis score, reduce the TCM syndrome score, improve patients' exercise tolerance and quality of life, and reduce serum TGF-β, hsCRP, IL-10 and Hyp factors.

[0030] 2. The traditional Chinese medicine compound of this invention can improve the general condition, lung coefficient, and lung function of DiPF model rats, improve the alveolar structure and inflammatory infiltration of rats, inhibit alveolar hyperplasia, alveolar wall and pulmonary blood vessel thickening, and also inhibit the increase of hydroxyproline (Hyp) content in lung tissue caused by diabetic pulmonary fibrosis, inhibit the expression of TGF-β1 / Smad3 signaling pathway, reduce EMT changes in epithelial cells, and reduce the deposition of downstream product collagen, thereby playing a role in improving diabetic pulmonary fibrosis. Attached Figure Description

[0031] Figure 1 The main instruments and equipment involved in animal experiments;

[0032] Figure 2 The main experimental materials and reagents involved in animal experiments;

[0033] Figure 3 Changes in body weight of rats in each group;

[0034] Figure 4 HE staining and Masson staining results of lung tissue from rats in each group

[0035] Figure 5 Immunohistochemical staining images of α-SMA, Collagen I, E-cadherin, fibronectin, TGF-β1, and Smad3 in rat lung tissue;

[0036] Figure 6 Mean optical density values ​​of α-SMA, Collagen I, E-cadherin, fibronectin, TGF-β1, and Smad3 in rat lung tissue after immunohistochemical staining (# indicates comparison with group C, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; * indicates comparison with group M, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001);

[0037] Figure 7 These are Western blot images of the expression of α-SMA, Collagen I, E-cadherin, fibronectin, TGF-β1, and Smad3 proteins in rat lung tissue.

[0038] Figure 8 Statistical graph of the gray values ​​of the bands expressing α-SMA, CollagenⅠ, E-cadherin, fibronectin, TGF-β1, and Smad3 proteins in rat lung tissue and the ratio of gray values ​​of the internal reference band (# indicates comparison with group C, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; * indicates comparison with group M, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001);

[0039] Figure 9 The results show the mRNA expression of α-SMA, Collagen I, E-cadherin, fibronectin, TGF-β1, and Smad3 in rat lung tissue (# indicates comparison with group C, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; * indicates comparison with group M, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

[0040] Figure 10 These are Western blotting images of α-SMA, CollagenI, TGF-β1, and Smad3 protein expression in MRC-5 cells from each group.

[0041] Figure 11 The grayscale values ​​of α-SMA, CollagenI, TGF-β1, and Smad3 protein expression in MRC-5 cells of each group are statistically analyzed (*** indicates P < 0.001 between the two groups, ** indicates P < 0.01 between the two groups).

[0042] Figure 12 The expression results of α-SMA, Collagen I, TGF-β1, and Smad3 mRNA in MRC-5 cells of each group (*** indicates P < 0.001 between the two groups, ** indicates P < 0.01 between the two groups, and * indicates P < 0.05 between the two groups);

[0043] Figure 13 Statistical graphs of immunofluorescence intensity of α-SMA, Collagen I, TGF-β1, and Smad3 in MRC-5 cells of each group (*** indicates P < 0.001 between the two groups, ** indicates P < 0.01 between the two groups, and * indicates P < 0.05 between the two groups);

[0044] Figure 14 Immunofluorescence images of α-SMA, Collagen I, TGF-β1, and Smad3 in MRC-5 cells of each group (A, B, C, and D in the figure are labeled with red fluorescence to indicate α-SMA, Collagen I, TGF-β1, and Smad3, respectively; blue fluorescence indicates DAPI nuclear staining; the image showing the common localization of red fluorescent agent DAPI and blue fluorescence is Merge; scalebar = 100 μm). Detailed Implementation

[0045] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description. In the following embodiments, reagents and instruments not specifically mentioned are commercially available, and experimental operations not specifically mentioned are performed according to the manufacturer's instructions or conventional techniques in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention; 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 endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein.

[0046] This invention provides a traditional Chinese medicine compound for treating diabetic pulmonary fibrosis, the traditional Chinese medicine compound comprising the following components in parts by weight: Astragalus membranaceus 70-90 parts, Salvia miltiorrhiza 20-40 parts, Ligusticum chuanxiong 10-30 parts, Adenophora stricta 20-40 parts, and Dioscorea opposita 40-60 parts.

[0047] In some preferred embodiments of the present invention, the weight parts of Astragalus membranaceus can be: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90, or 60, or any range of two of the above values.

[0048] In some embodiments of the present invention, the weight fraction of the danshen may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or any range of two of the above values.

[0049] In some embodiments of the present invention, the weight fraction of the chuanxiong rhizome can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or a range of any two of the above values.

[0050] In some embodiments of the present invention, the weight fractions of the North American ginseng can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or any range of two of the above values.

[0051] In some embodiments of the present invention, the weight fraction of the yam may be 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60, or any range of two of the above values.

[0052] In some preferred embodiments of the present invention, the traditional Chinese medicine compound comprises the following components in parts by weight: Astragalus membranaceus 70-90 parts, Salvia miltiorrhiza 20-40 parts, Ligusticum chuanxiong 10-30 parts, Adenophora stricta 20-40 parts, Dioscorea opposita 40-60 parts, Codonopsis pilosula 20-40 parts, and Atractylodes macrocephala 20-40 parts; Codonopsis pilosula and Atractylodes macrocephala are added to enhance the qi-tonifying effect. In some preferred embodiments of the present invention, the traditional Chinese medicine compound comprises the following components in parts by weight: Astragalus membranaceus 70-90 parts, Salvia miltiorrhiza 20-40 parts, Ligusticum chuanxiong 10-30 parts, Adenophora stricta 20-40 parts, Dioscorea opposita 40-60 parts, Ophiopogon japonicus 20-40 parts, and Rehmannia glutinosa 40-60 parts; Ophiopogon japonicus and Rehmannia glutinosa are added to enhance the yin-nourishing effect.

[0053] In some preferred embodiments of the present invention, the traditional Chinese medicine compound comprises the following components in parts by weight: 70-90 parts of Astragalus membranaceus, 20-40 parts of Salvia miltiorrhiza, 10-30 parts of Ligusticum chuanxiong, 20-40 parts of Adenophora stricta, 40-60 parts of Dioscorea opposita, 20-40 parts of Curcuma zedoaria, 20-40 parts of Prunus persica, and 10-30 parts of Eupolyphaga sinensis; Curcuma zedoaria, Prunus persica, and Eupolyphaga sinensis are used to break up blood stasis and remove blood stasis.

[0054] This invention also provides the application of the above-mentioned traditional Chinese medicine compound for treating diabetic pulmonary fibrosis in the preparation of a drug for treating diabetic pulmonary fibrosis of the qi deficiency, yin deficiency and blood stasis type.

[0055] The drug dosage form is any one of pills, granules, powders, capsules, tablets, mixtures, and decoctions.

[0056] Example 1

[0057] A traditional Chinese medicine compound for treating diabetic pulmonary fibrosis includes the following components: Astragalus membranaceus 80g, Dioscorea opposita 50g, Salvia miltiorrhiza 30g, Ligusticum chuanxiong 15g, and Adenophora stricta 20g.

[0058] Example 2

[0059] A traditional Chinese medicine compound for treating diabetic pulmonary fibrosis includes the following components: Astragalus membranaceus 27g, Dioscorea opposita 17g, Salvia miltiorrhiza 10g, Ligusticum chuanxiong 5g, and Adenophora stricta 7g.

[0060] Example 3

[0061] A traditional Chinese medicine compound for treating diabetic pulmonary fibrosis includes the following components: Astragalus membranaceus 53g, Dioscorea opposita 33g, Salvia miltiorrhiza 20g, Ligusticum chuanxiong 10g, and Adenophora stricta 13g.

[0062] Example 4

[0063] A method for preparing a traditional Chinese medicine compound freeze-dried powder for treating diabetic pulmonary fibrosis includes the following steps:

[0064] (1) Take an appropriate amount of Astragalus membranaceus, Dioscorea opposita, Salvia miltiorrhiza, Ligusticum chuanxiong and Adenophora stricta, weigh them, put them in a round-bottom flask, add 10 times the amount of water of the medicine, soak for 30 minutes, heat to boiling for 60 minutes, filter, and keep the filtrate for later use.

[0065] (2) Add 8 times the amount of water to the dregs obtained by filtering in step (1) and boil for a second time. Heat to a boil for 40 minutes and then filter.

[0066] (3) Combine the filtrates obtained in steps (1) and (2), and concentrate the filtrates under reduced pressure at 65°C to about 4L.

[0067] (4) Divide the concentrate obtained in step (3) into 4 trays with a thickness of about 1 cm. Place the trays in a -40℃ cold trap for 4 hours to pre-freeze, then place the trays on a grid to freeze dry for about 72 hours. Take them out, crush them, and mix them evenly to obtain the final product.

[0068] Experimental Example 1

[0069] This experimental case is a clinical trial, the purpose of which is to study the safety and efficacy of the traditional Chinese medicine compound of the present invention in patients with diabetic pulmonary fibrosis.

[0070] 1. Case source: A total of 63 patients with diabetic pulmonary fibrosis (Qi and Yin deficiency with blood stasis) who met the inclusion criteria and were hospitalized or visited the First People's Hospital of Zigong City, the Traditional Chinese Medicine Hospital of Ziyang City, and the Traditional Chinese Medicine Hospital of Shuangliu District of Chengdu City between December 2022 and January 2024.

[0071] 2. Western medicine diagnostic criteria: There is currently no universally accepted diagnostic criteria for diabetes-induced pulmonary fibrosis. Therefore, referring to the epidemiological characteristics of this disease, the cutoff point is a diabetes course of more than 5 years. Western medicine diagnosis must first meet the characteristics of comorbidity of T2DM and pulmonary fibrosis, and the diagnosis of pulmonary fibrosis must be within 1 year.

[0072] (1) Diagnostic criteria for type 2 diabetes: Refer to the Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020 edition). Typical symptoms of diabetes (polydipsia, polyuria, polyphagia, unexplained weight loss) plus random blood glucose ≥11.1 mmol / L; or fasting blood glucose ≥7 mmol / L; or 2-hour blood glucose ≥11.1 mmol / L from an oral glucose tolerance test (OGTT). Those without typical symptoms of diabetes need to have their blood glucose retested on another day for confirmation. (Note: Blood glucose refers to venous plasma glucose; fasting state refers to at least 8 hours without calorie intake; random blood glucose refers to blood glucose at any time of day).

[0073] (2) Diagnostic criteria for pulmonary fibrosis: Patients newly diagnosed with PF, excluding those with incomplete medical records, a history of glucocorticoid use (>1 week), or a history of occupational dust exposure. The diagnostic criteria for PF refer to the 2011 American Thoracic Society (ATS) / European Respiratory Society (ERS) joint IPF diagnostic practice guidelines (① + ② or any one of ③):

[0074] ① Exclude other known causes of interstitial lung disease (e.g., family and occupational exposure, connective tissue diseases, drug toxicity-induced interstitial lung disease).

[0075] ② High-resolution CT (HRCT) of the lungs shows UIP type: the lesions are mainly in the subpleural and basal regions, and the distribution is usually heterogeneous, with or without cellular traction bronchiectasis or bronchiectasis.

[0076] ③ A combination of specific chest and lung HRCT findings and histopathological findings.

[0077] 3. Traditional Chinese Medicine Diagnostic Criteria: Based on the syndrome section of the National Standard of the People's Republic of China "Terminology of Clinical Diagnosis and Treatment in Traditional Chinese Medicine", the "Diagnostic Criteria for Syndromes of Diabetes Mellitus and Its Complications in Traditional Chinese Medicine" and the "Guidelines for the Prevention and Treatment of Diabetes Mellitus in Traditional Chinese Medicine", the diagnostic criteria for Qi and Yin Deficiency with Blood Stasis are proposed as follows.

[0078] Main symptoms: ① Fatigue and weakness; ② Thirst and excessive drinking; ③ Cough, wheezing and shortness of breath; ④ Stabbing pain in the chest and ribs; ⑤ Dark purple lips and nails.

[0079] Secondary symptoms: ① spontaneous sweating and susceptibility; ② dry eyes; ③ hot palms and soles; ④ numbness in the limbs; ⑤ rough and dry skin.

[0080] Tongue and pulse: ① Tongue appearance: Pale red or dark purple tongue body with petechiae and ecchymosis; thin white tongue coating, little moisture, thin coating or peeled coating; purplish and engorged sublingual veins. ② Pulse appearance: Thready, rapid and weak, thready and wiry, deep and hesitant.

[0081] A diagnosis can be made by presenting three primary symptoms plus one secondary symptom, or two primary symptoms plus two secondary symptoms, in conjunction with the tongue and pulse examination.

[0082] 4. Inclusion criteria: (1) Meeting the Western medical diagnostic criteria for both type 2 diabetes and pulmonary fibrosis and having a TCM syndrome of Qi and Yin deficiency with blood stasis;

[0083] (2) History of diabetes mellitus > 5 years and diagnosis of pulmonary fibrosis < 1 year;

[0084] (3) Blood glucose control is relatively stable, and there are no serious acute complications of diabetes;

[0085] (4) Age 18-80 years old, gender not limited, with independent behavioral capacity;

[0086] (5) Good compliance and ability to cooperate in completing the experiment;

[0087] (6) Voluntarily participate in this trial and sign an informed consent form.

[0088] Note: Only patients who meet all 6 criteria above are eligible for inclusion in this study.

[0089] 5. Exclusion criteria:

[0090] (1) Patients with advanced pulmonary fibrosis complicated by acute or chronic respiratory failure;

[0091] (2) Those who are currently participating in other clinical trials;

[0092] (3) Other causes of interstitial lung disease and idiopathic interstitial lung fibrosis;

[0093] (4) Those with serious diseases such as cardiovascular, digestive tract, liver and kidney dysfunction, malignant tumors and hematopoietic system diseases, as well as those with impaired consciousness;

[0094] (5) Individuals allergic to the drugs used in this study;

[0095] (6) Pregnant women or breastfeeding women.

[0096] 6. Treatment methods:

[0097] Patients were randomly divided into a control group and a treatment group, with 32 patients in the treatment group and 31 patients in the control group.

[0098] (1) Control group: received basic treatment for diabetes and pulmonary fibrosis;

[0099] (2) Treatment group: The basic treatment for diabetes and pulmonary fibrosis was adopted, and a traditional Chinese medicine compound was given at the same time. The traditional Chinese medicine compound consisted of Astragalus membranaceus 80g, Dioscorea opposita 50g, Salvia miltiorrhiza 30g, Ligusticum chuanxiong 15g, and Adenophora stricta 20g. Method: The traditional Chinese medicine was decocted with 5 times its weight of water, cooled and filtered to remove the residue. Each dose yielded 300ml of juice, 150ml / bag, and was divided into 2 doses of 150ml each in the morning and evening.

[0100] (3) The total treatment course is 8 weeks. The condition changes are observed and recorded weekly, and medication adjustments are made accordingly.

[0101] 7. Observation indicators:

[0102] (1) Changes in overall therapeutic efficacy before and after treatment in the two groups;

[0103] (2) Changes in TCM syndrome scores before and after treatment in the two groups;

[0104] (3) Using the same pulmonary function instrument and oxygen saturation instrument, the changes in pulmonary function and transcutaneous oxygen saturation (SpO2) before and after treatment were detected and observed in both groups:

[0105] (4) Changes in chest HRCT scores before and after treatment in the two groups;

[0106] (5) Changes in serum levels of Hyp, TGF-β, CRP, and IL-10;

[0107] 8. Statistical methods

[0108] All data were entered into Excel and, after verification, were statistically processed using SPSS 25.0 software. Appropriate statistical methods were selected based on the data type: count data were expressed as n (%) and analyzed using the chi-square test or Fisher's test; continuous data were expressed as x ± s. For within-group comparisons of differences before and after treatment, paired t-tests were used if the data conformed to a normal distribution and homogeneous variance; otherwise, the rank-sum test was used. For between-group comparisons, independent samples t-tests were used if the data conformed to a normal distribution and homogeneous variance; independent samples t' tests were used if the variances were unequal; and the rank-sum test was used if the data did not conform to a normal distribution. A p-value < 0.05 was considered statistically significant.

[0109] 9. Clinical trial results:

[0110] Table 1 Comparison of overall therapeutic effects between the two groups (cases / %)

[0111]

[0112] As shown in Table 1, the total effective rate of the treatment group was 93.7%, while that of the control group was 48.4%. The overall efficacy of the two groups was compared using the c-test. 2 The difference was statistically significant (P < 0.01), suggesting that the traditional Chinese medicine compound of the present invention can improve the overall efficacy in patients with diabetic pulmonary fibrosis.

[0113] Table 2 Comparison of TCM syndrome scores before and after treatment in the two groups of patients.

[0114]

[0115] Note: *P<0.01 for comparisons between the two groups before and after self-treatment; #P<0.01 for comparisons with the control group.

[0116] As shown in Table 2, after treatment with the traditional Chinese medicine compound of the present invention, the TCM syndrome score of the treatment group decreased significantly compared with that before treatment, and the difference was statistically significant (P<0.01); the TCM syndrome score of the control group did not change significantly compared with that before treatment (P>0.05); after treatment, there was a significant difference in the total TCM syndrome score between the two groups (P<0.01), with the treatment group being significantly lower than that of the control group, indicating that the traditional Chinese medicine compound of the present invention can reduce the TCM syndrome score of patients with diabetic pulmonary fibrosis.

[0117] Table 3 Comparison of pulmonary function and SpO2 before and after treatment in the two groups of patients.

[0118]

[0119]

[0120] Note: Normally distributed data are... The results indicate that the difference between the two groups before and after self-treatment is **P<0.01.

[0121] In Table 3, FVC%, TLC%, and DLCO% are the percentages of the patient's measured FVC, TLC, and DLCO values ​​relative to their predicted values. SpO2% refers to blood oxygen saturation, which is the percentage of the total hemoglobin volume in the human blood that is bound to oxygen.

[0122] As shown in Table 3, after treatment, the FVC%, TLC%, and DLCO% in the treatment group all increased compared with those before treatment, and the differences were statistically significant (P<0.01). In the control group, there were no significant changes in the three indicators of FVC%, TLC%, and DLCO% after treatment compared with those before treatment, and there was no statistically significant difference (P>0.05). There were no significant changes in transcutaneous oxygen saturation (SpO2) before and after treatment in both the treatment group and the control group (P>0.05). This suggests that the traditional Chinese medicine compound of the present invention has an improving effect on ventilation and diffusion function in patients with diabetic pulmonary fibrosis.

[0123] Table 4. Chest HRCT scores before and after treatment in the two groups.

[0124]

[0125] Note: Normally distributed data are... The skewed distribution data are represented by M(Q). For comparisons between the two groups before and after treatment, *P<0.05, **P<0.01.

[0126] As shown in Table 4, before treatment, there were no statistically significant differences between the two groups in terms of chest HRCT imaging characteristics (ground-glass opacity, reticular opacity, honeycomb opacity) and overall CT score (P>0.05). After treatment, the ground-glass opacity, reticular opacity, and overall CT score in the treatment group decreased compared to before treatment (P<0.05), while there were no significant changes in these characteristics in the control group (P>0.05). This indicates that the traditional Chinese medicine compound of this invention can improve the pulmonary fibrosis score on HRCT.

[0127] Table 5 Comparison of Hyp, TGF-β, CRP, and IL-10 levels before and after treatment in the two groups.

[0128]

[0129] Note: Normally distributed data are... It is shown that when comparing before and after the self-treatment in the two groups, *P<0.05, **P<0.01; when comparing between the two groups after treatment ##P<0.01.

[0130] As can be seen from Table 5: After treatment, the serum Hyp and TGF-β in the treatment group were significantly lower than those before treatment, and the difference was statistically significant (P<0.05). There was no significant difference in the change of Hyp in the control group before and after treatment (P>0.05). The inflammatory indexes such as hrCRP and IL-10 in the treatment group and the control group decreased after treatment compared with those before treatment, and the difference was statistically significant (P<0.05). After treatment, the treatment group was significantly lower than the control group when comparing between the two groups (P<0.01). There was no significant change in the indexes such as Hyp and TGF-β in the control group compared with those before treatment (P>0.05). It shows that the traditional Chinese medicine compound of the present invention has an improvement effect on collagen metabolism and inflammation in DiPF patients, and the treatment effect is better than simple basic treatment.

[0131] Experimental Example 2

[0132] This experimental example is an animal experiment, aiming to verify the prevention and treatment effect of the traditional Chinese medicine compound of the present invention on DiPF model mice. This animal experiment complies with the relevant regulations of the "Guiding Opinions on the Humane Treatment of Laboratory Animals" and has been approved by the Laboratory Animal Ethics Committee of the First People's Hospital of Zigong City and Southwest Medical University. Ethical number: 20221201-008.

[0133] 1. Experimental materials

[0134] 1.1 Experimental animals

[0135] 60 SPF-grade male rats (6-8 weeks old) with a body weight of 150±20 g were selected and provided by Chengdu Dashuo Experimental Animal Co., Ltd. The animal production license number: SCXK(Sichuan)2020-030.

[0136] 1.2 Drugs

[0137] Compound high-dose group: Astragalus membranaceus 80 g, Dioscorea opposita 50 g, Salvia miltiorrhiza 30 g, Ligusticum chuanxiong 15 g, Glehnia littoralis 20 g (Implementation 1);

[0138] Compound low-dose group: Astragalus membranaceus 27 g, Dioscorea opposita 17 g, Salvia miltiorrhiza 10 g, Ligusticum chuanxiong 5 g, Glehnia littoralis 7 g (Implementation 2);

[0139] Compound medium-dose group: Astragalus membranaceus 53 g, Dioscorea opposita 33 g, Salvia miltiorrhiza 20 g, Ligusticum chuanxiong 10 g, Glehnia littoralis 13 g (Implementation 3).

[0140] It is composed of Astragalus membranaceus (batch number 220201), Salvia miltiorrhiza (batch number 220207), Ligusticum chuanxiong (batch number 20220424), Glehnia littoralis (batch number 20120101), and Dioscorea opposita (batch number 21040201), and is purchased from Chengdu Xingfengrui Chinese Herbal Pieces Co., Ltd.

[0141] 1.3 Main reagents and instruments

[0142] The main instruments and equipment involved in this experiment are shown in Figure 1 , and the main experimental materials and reagents are shown in Figure 2 ;

[0143] In this experiment, the ordinary feed is provided by Chengdu Dashuo Animal Co., Ltd.; the high-fat feed is provided by Jiangsu Coordinated Pharmaceutical Biotechnology Co., Ltd., batch number: 20220601. The formula of the high-fat feed is: maintaining the basic feed 43.5%, lard 17.5%, sucrose 12%, whole milk powder 10%, casein 10%, experimental animal premix 2%, microcrystalline cellulose 1.9%, dihydrogen phosphate 2% and stone powder 1.1%;

[0144] 2. Experimental methods

[0145] 2.1 Grouping and treatment of rats

[0146] All animals are housed in the experimental animal house of Sichuan Sainst Biotechnology Co., Ltd., and the animal use license number is: SYSK (Sichuan) 2020-199; all rats are placed in the same feeding environment, freely eating and drinking water, at a room temperature of 22-25°C, a relative humidity of 55%-65%, and the day and night light and dark alternation time is 12 hours each. They are adaptively fed for 3 days, during which ordinary feed is given; after the adaptive feeding, 10 rats are randomly selected as the blank (C) group and 40 rats are selected as the model group. After grouping, the blank group is given ordinary normal feed, and the model group is given high-fat diet with high-fat feed for 4 weeks to establish a model. The feed, bedding, etc. are replaced in time by a special person.

[0147] 2.2 Rat model establishment: 12 hours before model establishment, the rats are fasted but not water-deprived. 50 rats in the model group fed with high-fat feed are intraperitoneally injected with STZ 35 mg / kg once to induce the establishment of a diabetes model, and the blank (C) group is intraperitoneally injected with an equal dose of 0.1% sodium citrate buffer solution; 72 hours after the injection, blood is collected from the tail vein for determination. If FPG ≥ 16.7 mmol / L, the model establishment is successful. Rats that do not meet the standard are intraperitoneally injected with STZ 35 mg / kg again, and the blood glucose is measured 72 hours later; the 40 rats with successful model establishment are randomly and equally divided into the model (M) group, the compound low-dose group (ZL), the compound medium-dose group (ZM), and the compound high-dose group (ZH).

[0148] 2.3 Drug administration to rats: The blank (C) group and the model group continued to be fed with normal diet and high-fat diet, respectively. The model group was also given distilled water and then different doses of compound (3.68 g / kg·d) for intervention. The blank (C) group was given the same distilled water. After 6 weeks of intervention, 3 rats in the model (M) group were randomly selected for dissection and lungs were taken. After HE staining and observation, and comparison with the lungs of the normal group, it was confirmed that the pulmonary fibrosis model induced by type 2 diabetes was successful. The remaining rats in each group continued to be fed and intervened until the 8th week.

[0149] 2.4 Measurement of lung function in rats of each group

[0150] After the last administration, the rats were fasted for 8 hours but allowed free access to water. The rats were weighed and anesthetized with 10% chloral hydrate solution via intraperitoneal injection at a rate of 0.3 ml / 100 g. After about 5 minutes, endotracheal intubation was performed, and the rats were placed in a supine position in a body scintigraphy chamber. The endotracheal tube was connected to the airway of the body scintigraphy chamber, and the forced vital capacity (FVC) pressure parameter was set to 30 cmH2O. Data collection began after the data curve stabilized. After processing by the software, the required values ​​of various pulmonary function indicators were calculated, including forced vital capacity (FVC), peak expiratory flow (PEF), total pulmonary resistance (RL), and dynamic compliance (Cdyn).

[0151] 2.5 Blood glucose levels were measured in each group of rats.

[0152] Fasting blood glucose (FBG) was measured in whole blood from the terminal capillaries of rats at weeks 0, 4, 6, 8 and 12 of feeding.

[0153] 2.6 General condition observation and weighing of rats in each group

[0154] During the experiment, the mental state, coat color, and excretion of each rat were closely observed, and the weight of each group of rats was measured every 2 weeks.

[0155] 2.6 Sacrifice of rats for sampling

[0156] After measuring the lung function of rats, the rats were fixed, the thoracic cavity was quickly opened, the lungs were cut off, rinsed with physiological saline, the lungs were separated, and the mass of the lung tissue was quickly measured as wet weight.

[0157] 2.7 Determination of hydroxyproline (Hyp) content in lung tissue of rats in each group

[0158] After preparing a 10% tissue homogenate from a portion of the lower lobe of the right lung, the hydroxyproline (HYP) content of the sample was determined using a hydroxyproline (HYP) kit.

[0159] 2.8 HE staining and Masson staining were used to observe pathological changes and collagen deposition in lung tissue.

[0160] The left lower lung was fixed in 4% paraformaldehyde for 24 hours, dehydrated, embedded in paraffin, and sectioned in paraffin (5 μm thick). After dewaxing to water, it was stained with hematoxylin and eosin (HE) and Masson's stain, dehydrated, and mounted. The pathological changes of the lung tissue after HE staining were observed under a light microscope at 200x and 400x magnification, and the images were acquired and analyzed. The pathological changes of the lung tissue after Masson's staining were observed under a light microscope at 200x magnification, and the images were acquired and analyzed.

[0161] 2.9 Immunohistochemical staining, protein expression, and mRNA expression of EMT markers (α-SMA, Collagen I, E-cadherin, fibronectin) and TGF-β1 and Smad3 were measured in each group of rats.

[0162] Immunohistochemical analysis was used to semi-quantitatively detect the protein expression of TGF-β1, Smad3, α-SMA, E-cadherin, fibronectin, and Collagen I in rat lung tissue. Western blotting was used to detect the protein expression of TGF-β1, Smad3, α-SMA, E-cadherin, fibronectin, and Collagen I in rat lung tissue of each group. RT-PCR was used to detect the mRNA expression of TGF-β1, Smad3, α-SMA, E-cadherin, fibronectin, and Collagen I in rat lung tissue of each group.

[0163] 3. Statistical methods

[0164] Experimental data were statistically analyzed using GraphPadPrism 8.0, and the results were displayed using graphing functions. Normality was tested for the continuous data; if they conformed to a normal distribution, they were expressed as mean ± standard error (Mean ± SEM). Next, one-way ANOVA was used for population comparisons. If the variances were homogeneous, one-way ANOVA was used for between-group comparisons; if multiple group comparisons were involved, one-way ANOVA was used for between-group comparisons; if only two groups were involved, the independent samples t-test was used for between-group comparisons. If the variances were unequal, the Games-Howell method was used for pairwise between-group comparisons. For data that did not conform to a normal distribution, the Kruskal-Wallis method was used for population comparisons between groups, and the Mann-Whitney U method was used for pairwise between-group comparisons.

[0165] 4. Experimental Results

[0166] 4.1 General condition observation and weighing of rats in each group

[0167] Throughout the experiment, rats in group C had good nutritional status, with clean, shiny, and soft fur, a gentle temperament, agile movements, moderate food and water intake, and normal bowel movements. The other four groups (groups M, ZL, ZM, and ZH) were emaciated, with dull, yellowish, and dry fur, lethargy, a preference for lying down, relatively slow movements, significantly increased urine output compared to group C, and inconsistent stool consistency. After intervention with different doses of Bufei Tongluo Decoction, compared to group M, groups ZL, ZM, and ZH showed no significant differences in fur color and bowel movements, but significant improvements were observed in lethargy and slow movement. A total of 8 rats died during the experiment: 1 from group C, 2 from group M, 1 from group ZL, 2 from group ZM, and 2 from group ZH. These results suggest that the herbal compound of this invention has the effect of improving the general condition of DiPF rats.

[0168] See weight results Figure 3 As shown, the body weight of rats in each group increased rapidly in the first 4 weeks. During weeks 4-12, the body weight of groups C and M continued to increase, but the body weight of group C was significantly higher than that of group M. The body weight of the other 4 groups (group M, group ZL, group ZM, and group ZH) showed a decreasing trend after 4 weeks. The body weight of group ZH rats decreased most significantly in week 12 (P < 0.01). This suggests that the traditional Chinese medicine compound of the present invention has the effect of reducing the body weight of DiPF rats, and the high-dose traditional Chinese medicine compound has the most obvious effect.

[0169] 4.2 Random blood glucose levels in each group of rats

[0170] The results are shown in Table 6 below: The random blood glucose level of rats in group C was significantly lower than that of the other four groups (group M, group ZL, group ZM, and group ZH). From week 6 onwards, the same dose of the traditional Chinese medicine compound (ZL, ZM, and ZH) had no statistically significant effect on the random blood glucose of DiPF rats (P>0.05). Therefore, it is suggested that this traditional Chinese medicine compound has no significant effect on improving the random blood glucose of DiPF rats.

[0171] Table 6. Random blood glucose changes in rats of each group.

[0172]

[0173] Note: * indicates comparison with group C, *** indicates P < 0.0001; # indicates comparison with group M, ## indicates P < 0.001, ### indicates P < 0.0001.

[0174] 4.3 Lung coefficient of rats in each group

[0175] The lung coefficient of rats was calculated as lung wet weight / body weight * 100. The results are shown in Table 7 below: Compared with group C, the lung coefficient of rats in group M was significantly increased (P < 0.0001); compared with group M, there was no statistically significant difference in the lung coefficient between groups ZL and ZM (P > 0.05), but the lung coefficient of rats in group ZH was significantly decreased (P < 0.001); at the same time, the body weight and lung weight of rats in group ZH were significantly lower than those in groups C, M, ZL, and ZM (P < 0.001); the above results indicate that the traditional Chinese medicine compound of the present invention can improve the lung index of rats, especially the high-dose traditional Chinese medicine compound has the most significant effect.

[0176] Table 7. Body weight, lung weight, and lung coefficient (Mean±SEM) of rats in each group.

[0177]

[0178] Note: * indicates comparison with group C, *** indicates P < 0.0001, ** indicates P < 0.001; # indicates comparison with group M, ## indicates P < 0.001, ### indicates P < 0.0001.

[0179] 4.4 Results of lung function tests in each group of rats

[0180] The results are shown in Table 8 below: Compared with group C, the FVC, PEF, and Cydn levels of rats in group M were significantly decreased (P < 0.0001), while the RL level was significantly increased (P < 0.0001); compared with group M, the FVC, PEF, and Cydn levels of rats in group ZH were significantly increased (P < 0.0001), while the RL level was significantly decreased (P < 0.0001); there was no significant change in FVC between groups ZL and ZM (P > 0.05); PEF and Cydn levels in group ZM were significantly increased compared with group M (P < 0.05), while the RL level was significantly decreased compared with group M (P < 0.05); there was no statistically significant difference in FVC, PEF, and Cydn levels between group ZL and group M, while the RL level was significantly decreased compared with group M (P < 0.001).

[0181] The above results indicate that the traditional Chinese medicine compound of the present invention has the effect of improving lung function in DiPF rats, especially the high-dose traditional Chinese medicine compound has the most significant effect.

[0182] Table 8. FVC, PEF, RL, and Cdyn (Mean ± SEM) in rats of each group.

[0183]

[0184] Note: * indicates comparison with group C, *** indicates P < 0.0001, ** indicates P < 0.001, * indicates P < 0.05; # indicates comparison with group M, # indicates P < 0.05, ## indicates P < 0.001, ### indicates P < 0.0001.

[0185] 4.5 Results of Hydroxyproline (Hyp) Content Determination in Lung Tissue of Rats in Each Group

[0186] The results are shown in Table 9 below: Compared with group C, 8 weeks after STZ modeling, the Hyp content in the lung tissue of group M was significantly increased (P<0.0001). Compared with group M, the Hyp content in groups ZL, ZM and ZH were all decreased to varying degrees, with the decrease being more significant in groups ZM and ZH (P<0.0001). The Hyp content in group ZH was significantly lower than that in group ZL, and there was no statistically significant difference between groups ZH and ZM (P<0.001).

[0187] The above results indicate that the traditional Chinese medicine compound of the present invention can inhibit the increase of hydroxyproline (Hyp) content in lung tissue caused by pulmonary fibrosis and reduce the content of collagen fibers, especially the high-dose traditional Chinese medicine compound, which has the most significant effect.

[0188] Table 9. Hyp content in lung tissue of rats in each group (Mean ± SEM)

[0189]

[0190] Note: * indicates comparison with group C, *** indicates P < 0.0001, ** indicates P < 0.001; # indicates comparison with group M, ## indicates P < 0.001, ### indicates P < 0.0001.

[0191] 4.6 Results of HE staining and Masson staining

[0192] Results of HE staining and Masson staining are as follows: Figure 4 As shown:

[0193] HE staining results showed that the lung tissue morphology and structure of group C were relatively normal, the bronchial epithelium was neatly arranged (black arrow), and the alveolar cavity and alveolar wall morphology and structure were relatively normal. The thickness of the pulmonary artery wall (blue arrow) in the right image did not show significant changes. In group M, interstitial lung hyperplasia, pulmonary hemorrhage (black arrow), perivascular inflammatory cell cuffs mainly composed of lymphocytes were observed (circled), the pulmonary artery wall was significantly thickened, and the arterial lumen was narrowed (blue arrow). In group ZL, interstitial lung hyperplasia, slight widening of the alveolar wall (black arrow), perivascular inflammatory cell cuffs mainly composed of lymphocytes were observed (circled), the pulmonary artery wall was slightly thickened (blue arrow), and the bronchial epithelial cells were neatly arranged (red arrow). In both ZM and ZH, slight widening of the alveolar wall was observed locally (black arrow), perivascular and peribronchial inflammatory cell infiltration mainly composed of lymphocytes was observed (yellow arrow), the pulmonary artery wall was slightly thickened, and bronchial epithelial cells were sloughed off (red arrow).

[0194] Masson staining results showed that the alveolar structure of rats in group C was intact, with a small amount of blue collagen deposits in the normal lung interstitium; the alveolar septa of rats in group M were thickened, the alveolar structure was destroyed, and a large amount of blue collagen deposits were visible in all regions of the lung tissue; the degree of blue collagen deposits in all regions of the lung tissue of rats in the three intervention groups ZL, ZM and ZH was reduced compared with that in group M, and the improvement was more obvious in group ZH.

[0195] The Masson staining results were analyzed using Image-ProPlus 6.0 software. The pixel area of ​​blue collagen fibers, the cumulative optical density (IOD) of positive areas, and the pixel area (AREA) of the tissue were measured. The pixel area of ​​the field of view was calculated, and the percentage of collagen fiber area (%) was calculated as follows: collagen fiber pixel area / pixel area of ​​field of view * 100. The areal density was calculated as follows: cumulative optical density (IOD) / pixel area (AREA) of the tissue. The results are shown in Table 10 below. Compared with group M, groups ZL, ZM, and ZH showed varying degrees of reduction in the percentage of collagen fiber area and areal density, with group ZH showing the most significant reduction.

[0196] The above comprehensive results indicate that the traditional Chinese medicine compound of the present invention can improve the alveolar structure and inflammatory infiltration of DiPF rats, and inhibit alveolar hyperplasia, alveolar wall and pulmonary blood vessel thickening. Among them, the high-dose traditional Chinese medicine compound has the best effect, which shows that the traditional Chinese medicine compound of the present invention has the effect of improving pulmonary fibrosis in DiPF rats.

[0197] Table 10. Masson staining results of rat lung tissue in each group.

[0198]

[0199]

[0200] Note: * indicates comparison with group C, *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05; # indicates comparison with group M, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001.

[0201] 4.7 Immunohistochemical staining results of EMT markers and TGF-β1 and Smad3 in lung tissue of rats in each group

[0202] Immunohistochemical staining results are shown in Figure 5As shown: After immunohistochemical staining, normal rat cells appeared blue, while positive expression of α-SMA, E-cadherin, fibronectin, Collagen I, TGF-β1, and Smad3 proteins appeared brownish-yellow. In group C, α-SMA, Collagen I, and fibronectin proteins showed lighter staining, while E-cadherin protein showed darker staining, and the opposite was true in group M. In the three intervention groups (ZH group, ZM group, and ZL group), the staining of each protein was visible. Compared with group M, the staining degree of α-SMA, fibronectin, Collagen I, TGF-β1, and Smad3 proteins was reduced, while the staining of E-cadherin protein was increased.

[0203] Immunohistochemical staining results were imaged under a light microscope at 200x magnification. Image-ProPlus 6.0 software was used to select the same brownish-yellow color as a unified standard for judging the positivity of all images. Each image was analyzed to obtain the cumulative optical density (IOD) and the area of ​​the tissue pixels (AREA). The average optical density (AO value) was calculated as AO = IOD / AREA. The results are shown below. Figure 7 ;

[0204] Depend on Figure 6 It was observed that the expression levels of α-SMA, Collagen I, fibronectin, TGF-β1, and Smad3 proteins in lung tissue of group M were increased (P < 0.001), while the expression of E-cadherin was decreased (P < 0.001). Among the three intervention groups (ZH, ZM, and ZL), the expression levels of α-SMA, Collagen I, and fibronectin in groups ZM and ZH were significantly lower than those in group M (see...). Figure 6 In the middle B, C, and F groups (P < 0.01), E-cadherin expression was increased in the ZH group compared to the M group (see [reference needed]). Figure 6 E (P < 0.05), but there was no statistically significant difference in E-cadherin between the ZM and M groups; compared with the M group, the expression of Smad3 in each of the ZH, ZM, and ZL groups was significantly reduced (see Figure 6 The expression of TGF-β1 in the ZH group was significantly lower than that in the M group (P < 0.05), while the changes in the ZL and ZM groups were not statistically significant compared with those in the M group (P > 0.05).

[0205] 4.8 Results of EMT markers and TGF-β1 and Smad3 protein expression in lung tissue of rats in each group;

[0206] The experimental results of protein expression are shown in the figure. Figure 7 and Figure 8As shown: Compared with group C, group M showed significantly increased α-SMA, Collagen I, and fibronectin protein levels (P < 0.001), and significantly decreased E-cadherin expression (P < 0.001), accompanied by increased TGF-β1 and Smad3 protein expression (P < 0.001). Compared with group M, the expression of α-SMA, Collagen I, fibronectin, TGF-β1, and Smad3 proteins in all intervention groups (ZH, ZM, and ZL groups) was lower than that in group M, with the most significant decrease in group ZH (P < 0.05). E-cadherin levels in all intervention groups (ZH, ZM, and ZL groups) were higher than those in group M, but only the difference in group ZH was statistically significant (P < 0.05).

[0207] The above results indicate that pulmonary fibrosis (EMT) may have occurred in the lung tissue of DiPF model rats through the TGF-β1 / Smad3 signaling pathway. High-dose traditional Chinese medicine compound and metformin sustained-release tablets both inhibited EMT, thereby improving pulmonary fibrosis. This process may be related to the inhibition of the TGF-β1 / Smad3 pathway.

[0208] 4.9 Results of EMT markers and mRNA expression of TGF-β1 and Smad3 in lung tissue of rats in each group;

[0209] The experimental results for mRNA expression are shown in the figure. Figure 9 As shown: Compared with group C, group M showed significantly increased mRNA expression of α-SMA, Collagen I, and fibronectin (P < 0.001), and significantly decreased expression of E-cadherin (P < 0.001), accompanied by increased expression of TGF-β1 and Smad3 proteins (P < 0.001). Compared with group M, the mRNA expression of α-SMA, Collagen I, fibronectin, TGF-β1, and Smad3 in each intervention group (ZH group, ZM group, ZL group) was lower than that in group M, with the most significant decrease in group ZH (P < 0.05). In terms of E-cadherin expression, the difference between group ZH and group ZM and group M was statistically significant (P < 0.05), while there was no statistically significant difference (P > 0.05) between group ZH and group ZM and group M.

[0210] The experimental results from 4.7 to 4.9 suggest that this traditional Chinese medicine compound reduces EMT changes in epithelial cells by decreasing the expression of the TGF-β1 / Smad3 signaling pathway, thereby reducing the deposition of downstream product collagen and alleviating pulmonary fibrosis in diabetic rats, especially with the high-dose traditional Chinese medicine compound showing the most significant effect.

[0211] Experimental Example 3

[0212] This experiment is a cell experiment, and its purpose is to investigate the effect of the traditional Chinese medicine compound of the present invention on the differentiation of MRC-5 cells into myofibroblasts under high glucose conditions through in vitro experiments.

[0213] 1. Experimental Materials

[0214] Experimental cells: Human embryonic lung fibroblasts MRC-5, purchased from Wuhan Procell Biotechnology Co., Ltd.;

[0215] Experimental drugs: Lyophilized compound traditional Chinese medicine powder: Each 1g lyophilized powder contains 6g of raw herbs; composed of Astragalus membranaceus (batch number 220201), Salvia miltiorrhiza (batch number 220207), Ligusticum chuanxiong (batch number 20220424), Adenophora stricta (batch number 20120101), and Dioscorea opposita (batch number 21040201), purchased from Chengdu Xingfengrui Traditional Chinese Medicine Pieces Co., Ltd., and lyophilized by Sichuan Yuqiang Herbal Biotechnology Co., Ltd., and certified as qualified; TGF-β1 agonist SRI-011381;

[0216] 2. Experimental Methods

[0217] 2.1 Cell resuscitation and inoculation

[0218] Human embryonic lung fibroblast MRC-5 cell line was placed in MEM medium containing 10% FBS and cultured in a constant temperature incubator at 37℃ and 5% CO2. MRC-5 cells in the logarithmic growth phase and in good cell condition were digested, terminated, and adjusted to a suitable cell concentration. They were then seeded into 96-well plates at 100 μL per well and cultured in a constant temperature incubator at 37℃ and 5% CO2 for 24 h.

[0219] 2.2 Determining the concentration of high glucose treatment and the concentration of traditional Chinese medicine compound treatment using CCK8.

[0220] MRC-5 cells from section 2.1 were taken. After cell adhesion, the culture medium was discarded, and the cells were washed twice. They were then cultured in complete culture medium containing different concentrations of glucose and mannitol for 48 hours. The supernatant was discarded, and serum-free culture medium was added. 10 μL of CCK8 solution was added to each well, and the cells were incubated at 37°C for 0.5–1 hour. The absorbance of each well was measured using an OD450 nm wavelength microplate reader. Cell viability was calculated, and cell activity was tested to determine the optimal concentration for high glucose modeling. The optimal high glucose concentration was found to be 50 mmol / L.

[0221] 2.3 CCK8 Determination of the Concentration of Traditional Chinese Medicine Compound Treatment

[0222] 100 mg of the lyophilized compound traditional Chinese medicine powder was dissolved in 10 ml of PBS solution. After thorough dissolution, centrifugation, and filtration, the solution was diluted with complete culture medium and finally added to 96-well plates containing cells from step 2.1 at concentrations of 100 ug / ml, 50 ug / ml, 20 ug / ml, 10 ug / ml, 5 ug / ml, and 2.5 ug / ml. After culturing the cells in an incubator for 24 h, the culture medium was discarded, serum-free culture medium was added, and 10 μL of LCK-8 solution was added to each well. The cells were incubated at 37°C for 0.5–1 h. The absorbance of each well was measured using an OD450 nm microplate reader to calculate cell viability and detect cell activity. The optimal concentration of the compound traditional Chinese medicine powder was determined to be 20 ug / mL, with no significant cytotoxicity.

[0223] 2.4 Cell grouping and treatment

[0224] Cells seeded in step 2.1 were randomly divided into 5 groups (control group, model group, traditional Chinese medicine compound group (BFTL), TGF-β1 agonist group (SRI), and TGF-β1 agonist + traditional Chinese medicine compound group (SRI+BFTL)). The cells were added to different culture media and incubated for 48 h. Cell samples were collected, and the relative expression levels of α-SMA and CollagenI were detected using Western blot (WB) and RT-PCR. GAPDH was used as an internal reference gene. The culture media for the 5 groups were as follows:

[0225] (1) Control group: 2 mL of complete culture medium containing mannitol (isotonic with the model group);

[0226] (2) Model group: 2 mL of complete culture medium with a glucose concentration of 50 mmol / L;

[0227] (3) Traditional Chinese medicine compound (BFTL) group: 20 μL of lyophilized powder solution of traditional Chinese medicine compound with a concentration of 5 mg / ml was taken into 1.98 mL of complete culture medium (the glucose concentration in the complete culture medium was 50 mmol / L);

[0228] (4) TGF-β1 agonist (SRI): Take 2 μL of SRI-011381 stock solution with a concentration of 10 μmol / L into 1.998 mL of complete culture medium (the glucose concentration in the complete culture medium is 50 mmol / L).

[0229] (5) TGF-β1 agonist + traditional Chinese medicine compound (SRI+BFTL): Take 20 μL of lyophilized traditional Chinese medicine compound solution with a concentration of 5 mg / ml and 2 μL of SRI-011381 stock solution into 1.78 mL of complete culture medium (the glucose concentration in the complete culture medium is 50 mmol / L).

[0230] 3. Observation indicators:

[0231] The relative expression levels of α-SMA, Collagen I, TGF-β1, and Smad3 in MRC-5 cells before and after model establishment, before and after intervention with traditional Chinese medicine compound, before and after administration of TGF-β agonist SRI-011381;

[0232] 4. Results

[0233] 4.1 Protein expression levels of α-SMA, Collagen I, TGF-β1, and Smad3 in MRC-5 cells of each group

[0234] This experiment used the Western blot (WB) method for detection, and the results are as follows: Figure 10 and Figure 11 As shown: The detection of α-SMA, Collagen I, TGF-β1, and Smad3 proteins in five groups of MRC-5 cells revealed that, compared with the control group, the Model group showed significantly increased levels of α-SMA, Collagen I, TGF-β1, and Smad3 proteins (P < 0.001), suggesting that high glucose can induce the transformation of MRC-5 cells into myofibroblasts, inducing a pulmonary fibrosis model in MRC-5 cells. The BFTL group showed significantly higher levels of α-SMA (P < 0.001), Collagen I (P < 0.001), TGF-β1 (P < 0.001), and Smad3 proteins compared to the Model group. The expression of all three proteins (P < 0.01) was significantly reduced, indicating that the traditional Chinese medicine compound had a protective effect on high glucose-induced MRC-5 cells. The SRI group had the highest expression levels of α-SMA, Collagen I, TGF-β1, and Smad3 proteins (P < 0.001). The SRI+BFTL group showed lower expression levels of α-SMA (P < 0.001), Collagen I (P < 0.001), TGF-β1 (P < 0.01), and Smad3 (P < 0.001) proteins compared to the SRI group, indicating that the transformation of MRC-5 cells into myofibroblasts is related to TGF-β1 and Smad3, and the traditional Chinese medicine compound can inhibit this process.

[0235] 4.2 mRNA expression levels of α-SMA, Collagen I, TGF-β1, and Smad3 in MRC-5 cells of each group

[0236] The results of this experiment, detected by rt-PCR, are as follows: Figure 12As shown: Analysis of mRNA expression levels of α-SMA, Collagen I, TGF-β1, and Smad3 in five groups of MRC-5 cells revealed that, compared to the control group, the Model group showed significantly increased mRNA expression of α-SMA (P < 0.001), Collagen I (P < 0.001), TGF-β1 (P < 0.05), and Smad3 (P < 0.001), suggesting that high glucose can induce the transformation of MRC-5 cells into myofibroblasts, inducing a pulmonary fibrosis model in MRC-5 cells. The BFTL group showed significantly higher levels of α-SMA (P < 0.001), Collagen I, TGF-β1, and Smad3 (P < 0.001) mRNA compared to the Model group. The expression levels of α-SMA (P<0.001), Collagen I (P<0.001), TGF-β1 (P<0.05), and Smad3 (P<0.001) mRNA were significantly reduced, indicating that the traditional Chinese medicine compound had a protective effect on high glucose-induced MRC-5 cells. The expression levels of various mRNAs were the highest in the SRI group. The expression levels of α-SMA (P<0.001), Collagen I (P<0.001), TGF-β1 (P<0.05), and Smad3 (P<0.001) mRNA in the SRI+BFTL group were lower than those in the SRI group, indicating that the transformation of MRC-5 cells into myofibroblasts is related to TGF-β1 and Smad3, and the traditional Chinese medicine compound can inhibit this process.

[0237] 4.3 Immunofluorescence expression of α-SMA, Collagen I, TGF-β1, and Smad3 in MRC-5 cells of each group

[0238] This experiment used the IF method to observe the fluorescence intensity of related proteins α-SMA, Collagen I, and key proteins of the TGF-β1 / Smad3 signaling axis in an early pulmonary fibrosis model of MRC-5 cells, and evaluated the effect of traditional Chinese medicine compound on this signaling axis. The results are as follows: Figure 13 and Figure 14As shown: In the Model group after high glucose intervention, the positive fluorescence signals of α-SMA and Collagen I were significantly increased, and the fluorescence intensity was enhanced, indicating that MRC-5 cells underwent transformation into myofibroblasts after high glucose induction, accompanied by an increase in extracellular matrix. Simultaneously, the expression of TGF-β1 and Smad3 was significantly increased, suggesting that this transformation may be related to the TGF-β1 / Smad3 signaling pathway. In the BFTL group, the fluorescence intensity of α-SMA, Collagen I, TGF-β1, and Smad3 (Merge red reagent) was lower than that in the Model group (P < 0.05), indicating that the traditional Chinese medicine compound of this invention... The formula can inhibit the transformation of MRC-5 cells into myofibroblasts. In the SRI group treated with TGF-β1 agonists, the fluorescence intensities of α-SMA, Collagen I, TGF-β1, and Smad3 were all increased compared with the Model group (P < 0.01). In the SRI+BFTL group treated with the traditional Chinese medicine compound of this invention, the fluorescence intensities of α-SMA, Collagen I, TGF-β1, and Smad3 were reduced (P < 0.05). This suggests that the traditional Chinese medicine compound of this invention can inhibit the transformation of MRC-5 cells into myofibroblasts induced by high glucose through the TGF-β1 / Smad3 signaling pathway.

[0239] The results in sections 4.1-4.3 above indicate that the traditional Chinese medicine compound of this invention can improve the differentiation of MRC-5 cells into myofibroblasts induced by high glucose; the mechanism of high glucose-induced differentiation of MRC-5 cells into myofibroblasts is related to the TGF-β1 / Smad3 pathway, and the regulation of the TGF-β1 / Smad3 pathway by the traditional Chinese medicine compound is one of its mechanisms of intervention in diabetic pulmonary fibrosis.

[0240] In summary, it should be noted that the above description is only a preferred embodiment of the present invention and should not be used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still make some simple deductions, substitutions, or equivalent substitutions of some technical features for the technical solutions described in the foregoing embodiments without departing from the concept of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine compound for treating diabetic pulmonary fibrosis, characterized in that: The traditional Chinese medicine compound is composed of the following components in parts by weight: Astragalus membranaceus 70-90 parts, Salvia miltiorrhiza 20-40 parts, Ligusticum chuanxiong 10-30 parts, Adenophora stricta 20-40 parts, and Dioscorea opposita 40-60 parts.

2. The traditional Chinese medicine compound for treating diabetic pulmonary fibrosis according to claim 1, characterized in that: The traditional Chinese medicine compound is composed of the following components in parts by weight: Astragalus membranaceus 80-90 parts, Salvia miltiorrhiza 30-40 parts, Ligusticum chuanxiong 10-20 parts, Adenophora stricta 20-30 parts, and Dioscorea opposita 50-60 parts.

3. A traditional Chinese medicine compound for treating diabetic pulmonary fibrosis according to claim 2, characterized in that: The traditional Chinese medicine compound consists of the following components in parts by weight: Astragalus membranaceus 80 parts, Salvia miltiorrhiza 30 parts, Ligusticum chuanxiong 15 parts, Adenophora stricta 20 parts, and Dioscorea opposita 50 parts.

4. The application of the traditional Chinese medicine compound for treating diabetic pulmonary fibrosis as described in any one of claims 1-3 in the preparation of a drug for treating diabetic pulmonary fibrosis of the qi deficiency, yin deficiency and blood stasis type.

5. The drug for treating pulmonary fibrosis of diabetic patients with qi deficiency, yin deficiency, and blood stasis as described in claim 4, characterized in that: The drug dosage form is any one of pills, granules, powders, capsules, tablets, mixtures, and decoctions.

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