Application of aleppo avens in preparation of medicine for treating pulmonary fibrosis diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of aleppo avens in preparation of medicines for treating pulmonary fibrosis diseases. The aleppo avens and the aleppo avens extract with the pulmonary fibrosis disease effect are specifically provided, the aleppo avens aqueous extract (LBZ) can remarkably improve the survival quality of mice, reduce the lung coefficient and the HYP level of pulmonary fibrosis disease individuals and remarkably relieve pathological injuries of the pulmonary fibrosis individuals; the TGF-beta 1 and alpha-SMA proteins are reduced to different degrees; the levels of MDA, serum IL-6 and TNF-alpha are reduced, and the SOD level is improved; the levels of IL-6 and TNF-alpha are reduced to different degrees, the lung tissue of an individual with pulmonary fibrosis is remarkably reduced, and the in-vivo inflammatory response is relieved; the SOD level in the lung tissue is increased, the MDA level is reduced, the oxidative stress injury of the lung tissue is relieved, and then the symptoms of an individual suffering from the pulmonary fibrosis disease are improved. An effective medicine and a new treatment thought are provided for treating the IPF.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of blue cloth in the preparation of medicines for pulmonary fibrosis diseases. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, cicatricial interstitial lung disease characterized by abnormal fibroblast proliferation, excessive deposition of extracellular matrix (ECM), inflammatory damage, and destruction of lung tissue function. Clinical manifestations include distorted lung structure and decreased lung function, which eventually lead to respiratory failure or even death. The average survival period is 3-5 years, and the incidence rate is increasing worldwide. Although a lot of research has been done on IPF, its pathogenesis is complex and the cause is still unclear. The main pathological changes are caused by inflammation, immune mechanism, epithelial-mesenchymal transition (EMT), and oxidative stress. At present, the treatment options for IPF are very limited. Lung transplantation is the only effective method for patients with advanced IPF and respiratory failure. The 2015 International Guidelines for Idiopathic Pulmonary Fibrosis recommend the use of pirfenidone and nintedanib as first-line clinical drugs, but they are expensive and have poor prognosis. Therefore, it is particularly urgent to find a safe and effective treatment for IPF.

[0003] In recent years, traditional Chinese medicine (TCM) has made significant progress in the prevention or treatment of pulmonary fibrosis through multi-target and systemic regulation. Through the synergistic regulation of multiple components, multiple targets and multiple pathways, it plays an important role in the prevention, treatment and rehabilitation of pulmonary fibrosis. Blue Buzheng is the dried whole herb of Geum japonicum Thunb. var. chinense Bolle and Geum aleppicum Jacq. of the Rosaceae family. It has the effects of invigorating qi and blood, nourishing yin, dispelling wind and relieving exterior symptoms, promoting blood circulation and detoxifying, moistening the lungs and resolving phlegm. It is used for symptoms such as consumptive tuberculosis and weak cough. It is recorded in classics such as "Guizhou Miao Medicine Research and Development", "Guizhou Chinese Medicine Resources", "Hunan Medicine Records", "Guizhou Herbs", and "Yunnan Chinese Herbs". It is a medicine used by ethnic minorities in our province. Modern pharmacological studies have shown that blue Buzheng has pharmacological activities such as anti-inflammatory and anti-tumor. In clinical practice, blue Buzheng is one of the main ingredients of the listed products "Fenghan Cold Capsules" and "Shengning Cold Liquid", and has a certain therapeutic effect on respiratory inflammatory diseases. However, there are no reports on Lanbu's research on improving and treating IPF. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of blue cloth in the preparation of pulmonary fibrosis disease drugs, which provides an effective drug and a new treatment idea for the treatment of idiopathic pulmonary fibrosis (IPF), as follows:

[0005] Lanbu is currently developing drugs for the treatment of pulmonary fibrosis.

[0006] Furthermore, the blue cloth is the dried whole herb of Geum japonicum Thunb. var. chinense Bolle and Geum aleppicum Jacq. of the Rosaceae family.

[0007] Furthermore, the indigofera is an indigofera extract.

[0008] Further, the indigofera extract is an indigofera water extract or an ethanol extract. Preferably, the indigofera water extract and the ethanol extract are obtained by crushing the whole indigofera plant into coarse powder, sieving, heating and refluxing with water or ethanol of different concentrations as solvent, combining the extracts, filtering, concentrating under reduced pressure, and freeze-drying.

[0009] Furthermore, the pulmonary fibrosis disease drug is a drug for treating, protecting or regulating pulmonary fibrosis disease.

[0010] Furthermore, the pulmonary fibrosis disease drug is a drug that reduces HYP levels.

[0011] Furthermore, the drug for treating pulmonary fibrosis has dosage forms including oral preparations, injection preparations and external preparations.

[0012] Furthermore, the pulmonary fibrosis disease drug also includes pharmaceutically acceptable excipients.

[0013] A pulmonary fibrosis drug contains a Lanbuzheng component, which may be Lanbuzheng or an extract thereof, and may also be added with pharmaceutically acceptable excipients to prepare a desired dosage form.

[0014] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0015] The present invention provides blue bud and its extracts with the effects of treating or protecting, regulating and preventing pulmonary fibrosis. Blue bud and its extracts can significantly improve the quality of life of mice, reduce the lung coefficient and HYP level of individuals with pulmonary fibrosis, and significantly alleviate the pathological damage of individuals with pulmonary fibrosis; reduce TGF-β1 and α-SMA protein to varying degrees; reduce MDA, serum IL-6, TNF-α levels, and increase SOD levels; reduce IL-6 and TNF-α levels to varying degrees, significantly reduce the lung tissue of individuals with pulmonary fibrosis and reduce the inflammatory response in the body; increase the SOD level in the lung tissue, reduce the MDA level, reduce the oxidative stress damage of the lung tissue, and then improve the symptoms of individuals with pulmonary fibrosis. It provides an effective drug and a new treatment idea for the treatment of idiopathic pulmonary fibrosis (IPF). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of LBZ on the survival rate, lung coefficient and hydroxyproline (HYP) of BLM-induced IPF mice. (A) Survival rate of mice in each group; (B) Lung coefficient of mice in each group; (C) HYP level of lung tissue of mice in each group; data are expressed as mean±SEM. Compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0017] Figure 2 The effects of LBZ on lung pathological damage and TGF-β1 and α-SMA protein levels in lung tissues of mice with pulmonary fibrosis model (staining ×400; immunohistochemistry ×400); (A) HE pathology of lung tissues of mice in each group; (B) Masson staining of lung tissues of mice in each group; (C, D) inflammation and fibrosis levels of mice in each group; (E, F, G and H) TGF-β1 and α-SMA protein expression levels in lung tissues of mice in each group, data are expressed as mean ± SEM. Compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0018] Figure 3 The effect of LBZ on the level of inflammation in the serum of BLM-induced IPF mice and the level of oxidative stress in the lung tissue of mice. (AB) The expression levels of IL-6 and TNF-α in the serum of each group of mice; (CD) The content of SOD and MDA in the lung tissue of each group of mice; Data are expressed as mean±SEM (n=8). Compared with the blank group, #P<0.05, ##P<0.01, ##P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0020] Example 1

[0021] Study on the intervention effect of blue bud extract on BLM-induced idiopathic pulmonary fibrosis model mice

[0022] 1. Materials and Methods

[0023] 1.1 Drugs and reagents

[0024] Lanbuzheng whole grass (batch number: 20220811, Guizhou Jinsha Chinese Medicine Market), bleomycin sulfate (BLM, product number B802467, Shanghai McLean Biochemical Technology Co., Ltd.); pirfenidone (PFD, product number S80784, Shanghai Yuanye Biotechnology Co., Ltd.); hydroxyproline (HYP, product number A030-2), malondialdehyde (MDA, product number A003-1-2), superoxide dismutase (SOD, product number A001-3-2) and glutathione peroxidase (GSH-Px, product number A006-1-1) kits were purchased from Nanjing Jiancheng Bioengineering Institute; mouse interleukin 6 (IL-6, lot.RX203049M) and mouse tumor necrosis factor (TNF-α, lot.RX202412M) kits were purchased from Ruixin Biotechnology Co., Ltd.

[0025] Weigh 500g of dried whole herb of Lanbuzheng, add 10 times the amount of distilled water, soak for 30min, boil, keep slightly boiling for 30min, filter with gauze while hot to obtain filtrate; decoct the residue once more in the same way, filter while hot, and combine the two filtrates. Concentrate under reduced pressure to 1g·mL -1 The clear paste was placed in a freeze dryer and freeze-dried to obtain 139.5 g of powdered solid, with an extraction rate of 27.9%. The powder was stored in a -20°C refrigerator and recorded as LBZ.

[0026] 1.2 Main instruments

[0027] ReadMax 1500 optical absorption full wavelength microplate reader (Shanghai Shanpu Biotechnology Co., Ltd.); DW-86L578J -80℃ refrigerator (Qingdao Haier Biomedical Co., Ltd.); DK-98-ⅡA electric water bath (Tianjin Test Instrument Co., Ltd.); MS105DC electronic balance (Switzerland Mettler); KQ-100E ultrasonic extractor (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); TGL16M high-speed centrifuge (Hunan Kaida Scientific Instrument Co., Ltd.); micro syringe (Shanghai Gaoge Industry and Trade Co., Ltd.)

[0028] 2. Experimental Methods

[0029] 2.1 Construction of pulmonary fibrosis mouse model and grouping and drug treatment

[0030] 90 SPF male C57BL / 6J mice (6-8 weeks old, weighing 18-20 g) were provided by Changsha Biotechnology Co., Ltd. (License No. SCXK (Xiang 2022-0011)). The animal experiment protocol was approved by the "Animal Experiment Ethics Review Form of Guizhou Medical University" in accordance with the guidelines specified in the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health. The animals were kept in a controlled environment with a temperature of 25±5°C, a relative humidity of 60±5%, and a light / dark cycle of 12h, and they were free to eat and drink. After 7 days of adaptive feeding, the mice were divided into a normal group (Control, n=15), a model group (BLM, n=15), and a pirfenidone group (PFD, 273 mg·kg -1 , n = 15), LBZ low-dose group (LBZL, 1.67 g kg -1 , n = 15), LBZ medium dose group (LBZM, 3.33 g kg -1 , n = 15), LBZ high-dose group (LBZH, 6.67 g kg -1 , n=15); except for the normal group, the other 5 groups were injected with bleomycin (BLM, 3.5 mg / kg) into the tracheal ring to establish the IPF model. On the first day after modeling, each group was orally gavaged with the corresponding drug, and the normal group and the model group were given an equal volume of distilled water for 21 days. One hour after the last administration, the mouse serum was collected and centrifuged at 3000 rpm for 10 min at 4°C to separate the serum, and then stored at -80°C. The right lobe lung tissue sections were taken from each mouse, fixed with 10% paraformaldehyde, and embedded in paraffin. The remaining lung tissues were stored at -80°C for testing.

[0031] 2.2 Calculation of mouse lung coefficient

[0032] After the last intragastric administration, the lung tissue of the mice was peeled off, washed with physiological saline, and the surface moisture of the lung tissue was wiped dry with filter paper and weighed. The lung coefficient was calculated according to the following formula.

[0033] Lung coefficient = [(lung weight (g) / body weight (g)] × 100%

[0034] 2.3 Alkaline water method to detect HYP level in lung tissue

[0035] Accurately weigh the lung tissue, add 9 times the volume of normal saline, mechanically homogenize in an ice-water bath, centrifuge at 2500r / min for 10min, take the supernatant and dilute it with normal saline at a ratio of 1:9 to make 1% tissue homogenate, and determine the HYP content using a kit.

[0036] 2.4 Pathological examination of lung tissue

[0037] The right upper lobe of the mouse lung was selected, 3 mice in each group, and fixed in 4% paraformaldehyde for 24 hours. After dehydration, the sections were embedded in paraffin and sliced. Then the sections were fixed on slides and stained with hematoxylin-eosin (HE) and Masson, and sealed. Finally, the prepared lung sections were observed pathologically under a light microscope.

[0038] 2.5 Immunohistochemical analysis

[0039] Paraffin sections of lung tissue were obtained and cut into 5 μm sections using a microtome, and the operation was performed according to the instructions. The protein expressions of TGF-β1 and α-SMA in lung tissue of each group were detected.

[0040] 2.6 Detection of serum inflammation levels in mice by ELISA

[0041] The mouse serum was collected after the last administration, and the levels of IL-6 and TNF-α in the serum of each group of mice were detected according to the instructions.

[0042] 2.7 Enzyme activity detection of oxidative stress level in mouse lung tissue

[0043] Accurately weigh the lung tissue mass, add 9 times the volume of normal saline, mechanically homogenize in an ice-water bath, centrifuge at 2500r / min for 10min, take the supernatant and dilute it with normal saline at a ratio of 1:9 to make 1% tissue homogenate, detect the content of MDA and T-SOD according to the instructions of the kit, and perform the operation according to the instructions of the kit.

[0044] 2.8 Statistical analysis

[0045] All data were analyzed by analysis of variance (ANOVA). The results were expressed as mean ± standard deviation (mean ± SEM). SPSS 22.0 software (IBM, Armonk, USA) was used. P values ​​less than 0.05 (P < 0.05) were considered statistically significant.

[0046] 3. Results

[0047] Effects of LBZ on survival rate, lung coefficient and hydroxyproline (HYP) in BLM-induced IPF mice

[0048] Compared with the normal group, the survival rate of the model group was significantly reduced (p<0.01), and the HYP level and lung coefficient were significantly increased (p<0.01); compared with the model group, the positive group and the drug-treated group could increase the survival rate of mice, the low-dose and medium-dose LBZ groups could reduce the lung coefficient and HYP level of mice (p<0.05), and the positive group and the high-dose LBZ group could significantly reduce the lung coefficient and HYP level of mice (p<0.01). Figure 1 .

[0049] Effects of LBZ on pathological damage in BLM-induced IPF mice

[0050] HE staining results showed that the pathological scores of lung tissue sections of mice in the model group were higher than those in the normal group, showing thickening of the alveolar wall, disappearance of some alveolar cavity structures, massive proliferation of pulmonary interstitial tissue, infiltration of a large number of inflammatory cells in the interstitial tissue, and degeneration of bronchial epithelial cells. Compared with the model group, the damage to the lung tissue structure of mice in each drug-treated group was alleviated to varying degrees. Masson staining results showed that the blue part of the model group had significantly more collagen fibers than the normal group, and the collagen fiber deposition was significantly reduced after LBZ administration, indicating that LBZ can improve the pathological damage of mice with pulmonary fibrosis. Figure 2 .

[0051] Effects of LBZ on TGF-β1 and α-SMA protein levels in lung tissues of BLM-induced IPF mice

[0052] Compared with the normal group, the levels of TGF-β1 and α-SMA proteins in the lung tissue of the model group were significantly increased (p<0.05 or p<0.01); compared with the model group, the levels of TGF-β1 and α-SMA proteins in the lung tissue of the positive group) and the low, medium and high doses of LBZ were decreased to varying degrees (p<0.05 or p<0.01). Figure 2 3.4 Effect of LBZ on the level of inflammation in the serum of BLM-induced IPF mice

[0053] Proinflammatory cytokines such as IL-6 and TNF-α play an important role in the inflammatory process that leads to the progression of idiopathic pulmonary fibrosis (IPF). In order to evaluate the effect of LBZ on BLM-induced IPF in mice, the levels of IL-6 and TNF-α in mouse serum were detected by ELISA. The results showed that compared with the normal group (Controll), the levels of IL-6 and TNF-α in the serum of the model group (BLM) were significantly increased (p<0.05 or p<0.01); compared with the model group (BLM), the positive group (PFD) was able to reduce the levels of IL-6 and TNF-α, but there was no significant difference. Low, medium and high doses of LBZ can reduce the levels of IL-6 and TNF-α to varying degrees (p<0.05 or p<0.01). ( Figure 3 )

[0054] Effects of LBZ on oxidative stress in lung tissue of BLM-induced IPF mice

[0055] Oxidative stress response can lead to the progression of pulmonary fibrosis. SOD is an antioxidant with oxidase function in the body, which can reduce oxidative stress. MDA can interfere with biological macromolecules and initiate the release of inflammatory cytokines. Therefore, we used a biochemical kit to evaluate the levels of SOD and MDA in lung tissue. The results showed that compared with the normal group, the SOD level in the lung tissue of the model group was significantly decreased (p<0.01), and the MDA level was significantly increased (p<0.01); compared with the model group, the positive group and low, medium, and high doses of LBZ can increase the SOD level in the lung tissue to varying degrees and reduce the MDA level (p<0.05 or p<0.01). ( Figure 3 4. Conclusion

[0056] After intervention with bluebutensis water extract (LBZ), the survival rate of mice with pulmonary fibrosis was significantly improved, the quality of life of mice was significantly improved, the lung coefficient and HYP level of mice were reduced, and the pathological damage of mice with pulmonary fibrosis was significantly alleviated; TGF-β1 and α-SMA proteins were reduced to varying degrees; MDA, serum IL-6, and TNF-α levels were reduced, and SOD levels were increased; IL-6 and TNF-α levels were reduced to varying degrees, and the lung tissue of mice with pulmonary fibrosis was significantly reduced, and the inflammatory response in the body was reduced; SOD levels in lung tissue were increased, and MDA levels were reduced, which alleviated oxidative stress damage in lung tissue and thereby improved the symptoms of mice with pulmonary fibrosis model.

[0057] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. Lanbu is being used in the preparation of drugs for pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The blue cloth is the whole herb of the Rosaceae plant, the pubescent green grass or the green grass.

3. The use according to claim 1, characterized in that: The indigofera is an indigofera extract.

4. The use according to claim 3, characterized in that: The indigofera extract is a water extract or an ethanol extract of indigofera.

5. The use according to claim 4, characterized in that: The water extract and ethanol extract of Lanbuzheng are prepared by crushing the whole Lanbuzheng herb into coarse powder, sieving it, and then heating and refluxing it with water or ethanol as solvent. The extracts are combined, filtered, concentrated under reduced pressure, and freeze-dried.

6. The use according to claim 1, characterized in that: The pulmonary fibrosis disease medicine is a medicine for treating, protecting or regulating pulmonary fibrosis disease.

7. The use according to claim 1, characterized in that: The pulmonary fibrosis disease drug is a drug for reducing the HYP level.

8. The use according to claim 1, characterized in that The pulmonary fibrosis disease medicine has dosage forms including oral preparations, injection preparations and external preparations.

9. The use according to claim 1, characterized in that The pulmonary fibrosis disease medicine also includes pharmaceutically acceptable excipients.

10. A drug for pulmonary fibrosis, characterized in that: Contains blueberry ingredient.

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