Application of Nintedanib and plant-derived cyclic peptide combined medicine in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention belongs to the technical field of medicines, and provides application of Nintedanib and a plant-derived cyclic peptide and radix pseudostellariae cyclic peptide B combined medicine in preparation of a medicine for treating pulmonary fibrosis, the Nintedanib and the plant-derived cyclic peptide and radix pseudostellariae cyclic peptide B combined medicine can play a synergistic effect, and the inhibition effect on the pulmonary fibrosis process is remarkably improved. The toxic and side effects of diarrhea and intestinal mucosa injury caused by nintedanib are reduced, and a good choice is provided for clinical treatment of pulmonary fibrosis.

Description

Technical Field

[0001] The invention provides application of nintedanib and a plant-derived cyclic peptide in combination in preparing a medicine for treating pulmonary fibrosis, belonging to the technical field of medicines. Background Art

[0002] Pulmonary fibrosis (PF) is a chronic, progressive and difficult-to-cure lung disease characterized by excessive deposition of extracellular matrix (ECM) and lung structural remodeling[1]. Common clinical symptoms include dry cough, fatigue, chest discomfort and weight loss. In acute episodes of PF, patients may also experience dyspnea, respiratory failure and even death[2]. The etiology of PF in most patients is unknown and is known as idiopathic interstitial pneumonia, a major type of interstitial lung disease[3]. Currently, there is still a lack of effective treatments to curb the progression of PF. In terms of non-drug treatments, lung transplantation cannot be used as a routine treatment due to its high surgical cost, limited lung sources and high risks[4]. Stem cell therapy has certain potential in the treatment of PF, but its safety and effectiveness are not yet fully understood and currently cannot meet clinical needs[5]. Currently, the most commonly used drugs for the treatment of pulmonary fibrosis are nintedanib and pirfenidone approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)[6]. Several clinical trials have shown that nintedanib can effectively improve the lung function of patients with pulmonary fibrosis[7]. However, patients with pulmonary fibrosis often experience severe diarrhea and other adverse intestinal reactions after taking nintedanib, which makes them almost afraid to go out and seriously affects their quality of life[8].

[0003] The traditional Chinese medicine Pseudostellaria heterophylla is the dried root of Pseudostellaria heterophylla (Miq.) Pax exPax et Hoffm. of the Caryophyllaceae family. It is used to treat symptoms such as poor appetite, fatigue, cough with little sputum, and dry cough caused by yin deficiency and fluid damage. It is more suitable for patients with both qi and fluid damage, as well as those with chronic illness and weakness [9]. Pseudostellaria heterophylla cyclic peptide extracts can be used to improve COPD (patent: CN105566441A). Heterophyllin B (HB) is a quality indicator component of the traditional Chinese medicine Pseudostellaria heterophylla specified in the Chinese Pharmacopoeia. It is a cyclic octapeptide composed of L-type amino acids from the medicinal and edible plant Pseudostellaria heterophylla. Its safety has been clearly reported. HB can improve memory and / or prevent and treat Alzheimer's disease (patent: WO2021017925A1), improve diabetic retinopathy (patent: CN116603051A), and inhibit bacteria (patent: CN201810536760.2). Previous in vivo and in vitro studies by this research team have confirmed that HB can also be used to treat pulmonary fibrosis (patent: CN202111220534.1A) and ulcerative colitis (patent: CN202111234058.9A), and has great development potential and application prospects.

[0004] Faced with the dilemma of nintedanib, a drug for the clinical treatment of pulmonary fibrosis, there is an urgent need to determine a more effective treatment plan. On the basis of ensuring the efficacy, how to reduce the clinical dose of nintedanib and its intestinal adverse reactions such as diarrhea and abdominal pain is a technical problem that needs to be solved in this field. Summary of the invention

[0005] Purpose of the Invention

[0006] The present application provides an application of a pharmaceutical composition in the preparation of a drug for preventing or treating pulmonary fibrosis. The nintedanib described in the present invention combined with a plant-derived cyclic peptide, Pseudostellariae cyclotide B, has the effect of alleviating pulmonary fibrosis, and the nintedanib drug dosage used is lower than the original dosage. The source of Pseudostellariae cyclotide B is stable and safe, and the effect after combined use is significant, etc., and it is worthy of wide promotion.

[0007] The present application relates to the field of medical application technology, and discloses the use of a pharmaceutical composition in the preparation of a drug for preventing or treating pulmonary fibrosis. The raw materials of the pharmaceutical composition include nintedanib and Pseudostellariae cyclotide B, a plant-derived cyclotide, to provide new therapeutic drugs and means for the prevention and treatment of patients with pulmonary fibrosis, thereby providing technical support for the rational use of drugs.

[0008] Technical Solution

[0009] The use of nintedanib and a plant-derived cyclic peptide in combination for preparing a drug for treating pulmonary fibrosis is characterized in that:

[0010]

[0011] The nintedanib is any one or more of itself, its stereoisomers, its tautomers, its polymorphs, its solvates and its pharmaceutically acceptable salts;

[0012] The plant-derived cyclic peptide Pseudostigma cyclic peptide B is any one or more of itself, its stereoisomers, its tautomers, its polymorphs, its solvates and its pharmaceutically acceptable salts.

[0013] The application is characterized in that 10 mg / kg nintedanib and 10 mg / kg pseudostellaria cyclopeptide B are used in combination.

[0014] The use of a pharmaceutical composition in the preparation of a drug for treating pulmonary fibrosis is characterized in that the pharmaceutical composition contains nintedanib and Pseudomonas aeruginosa cyclotide B, and at least one pharmaceutically acceptable carrier or excipient.

[0015] The application is characterized in that the dosage form of the pharmaceutical composition is solution, granules, tablets, pills, drops, injection, capsules, powders or decoctions.

[0016] Specifically:

[0017] This application is proved by in vivo pharmacological experiments that the combination of nintedanib and Pseudostellariae cyclotide B can improve the degree of bleomycin-induced pulmonary fibrosis in mice: bleomycin is instilled into the trachea once to induce pulmonary fibrosis in mice. On the 7th day after modeling, nintedanib and / or Pseudostellariae cyclotide B are gavaged for 14 consecutive days, once a day. During this period, the weight changes and survival rates of mice were observed and recorded, and the experiment was terminated on the 21st day. The changes in the lung tissue pathological sections of each group of mice were observed by HE and Masson staining, and the infiltration of inflammatory cells, alveolar atrophy and collapse, alveolar wall thickening and collagen deposition were observed, and the synergistic pharmacological effects of Pseudostellariae cyclotide B on nintedanib were comprehensively evaluated.

[0018] This application is to prove that Pseudostellariae Cyclic Peptide B can improve the intestinal damage of mice with pulmonary fibrosis caused by Nintedanib through in vivo pharmacological experiments: bleomycin was instilled into the trachea once to induce pulmonary fibrosis in mice. On the 7th day after modeling, Nintedanib and / or Pseudostellariae Cyclic Peptide B were gavaged for 14 consecutive days, once a day. During this period, the weight changes and survival rates of mice were observed and recorded, and the experiment was terminated on the 21st day. The LPS level in the serum of each group of mice was detected by ELISA kit. The colon length of each group of mice was measured, the changes in the pathological sections of colon tissue HE staining were observed, and the integrity of the colon tissue structure, the arrangement of colon glands, the crypt structure, the integrity of the mucosal epithelial structure, and the infiltration of neutrophils in each group of mice were observed. Comprehensive evaluation of the improvement effect of Pseudostellariae Cyclic Peptide B on intestinal damage in mice with pulmonary fibrosis caused by Nintedanib.

[0019] The use of nintedanib combined with a plant-derived cyclic peptide Pseudostellariae cyclotide B in the preparation of a drug or functional product for the treatment of pulmonary fibrosis. Here: the administration route of the drug or functional product includes oral drugs, injection drugs, implant drugs, external drugs, sprays, inhaled drugs or a combination of the above drugs; the dosage form of the drug or functional product can be any of tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powders, injections, syrups, wine preparations, tinctures, dews or films, etc.; the treatment includes relief.

[0020] Beneficial Effects

[0021] Common drugs taken by patients with clinical pulmonary fibrosis include nintedanib, etc. Considering that the therapeutic drug nintedanib often causes adverse reactions such as diarrhea and abdominal pain in patients with pulmonary fibrosis. The previous in vitro and in vivo studies of this research team have confirmed that HB can also be used to treat pulmonary fibrosis (patent: CN202111220534.1A) and ulcerative colitis (patent: CN202111234058.9A), and it has great development potential and application prospects. The combination of Pseudostellariae Cyclic Peptide B and nintedanib may provide a feasible clinical treatment for pulmonary fibrosis, that is, a drug combination that improves the efficacy of nintedanib and reduces its gastrointestinal adverse reactions. Therefore, exploring the pharmacological effects and mechanisms of nintedanib combined with Pseudostellariae Cyclic Peptide B in the treatment of pulmonary fibrosis may provide a new strategy for the clinical treatment of pulmonary fibrosis.

[0022] Prior art shows that patients taking nintedanib at a commonly used clinical dose (300 mg / day) experience severe diarrhea and other adverse reactions. This therapeutic dose corresponds to a therapeutic dose of about 40 mg / kg for mice with pulmonary fibrosis used in the present invention. The effective dose of Pseudostellariae Cyclotide B in CN202111220534.1A is 20 mg / kg, and its effect in treating pulmonary fibrosis is limited.

[0023] The innovation of the present invention is that the combined use of 10 mg / kg of the tyrosinase inhibitor nintedanib and 10 mg / kg of Pseudostellariae Cyclotide B can significantly improve bleomycin-induced pulmonary fibrosis in mice, and its improvement effect is equivalent to the therapeutic effect of nintedanib alone at a dose of 40 mg / kg.

[0024] The present invention found that: when 10 mg / kg of nintedanib or 10 mg / kg of Pseudostellariae Cyclic Peptide B was administered alone, there was no significant difference between the group and the model group, indicating that the administration of 10 mg / kg of nintedanib or 10 mg / kg of Pseudostellariae Cyclic Peptide B alone was ineffective. However, if 10 mg / kg of nintedanib and 10 mg / kg of Pseudostellariae Cyclic Peptide B were used in combination to treat pulmonary fibrosis, they could play a synergistic role, significantly improve the inhibitory effect of nintedanib on the degree of pulmonary fibrosis, reduce its side effects such as diarrhea and intestinal mucosal damage, and provide a good treatment plan for the clinical treatment of pulmonary fibrosis.

[0025] The details are as follows:

[0026] In the bleomycin-induced mouse pulmonary fibrosis model: as the alveolar function is impaired, the animals develop respiratory disorders, significantly reduce their activity, curl up, reduce the glossiness of their fur, and eat less and lose weight. Pathological examination showed that the inflammatory infiltration and fibrosis of the lung tissue of mice with pulmonary fibrosis were severe. Compared with the fibrosis model, the combination of 10 mg / kg nintedanib and 10 mg / kg Pseudostellariae Cyclic Peptide B can significantly reduce the abnormal increase in lung coefficient caused by bleomycin (P<0.05) and the lung function and survival rate of mice after treatment on the 7th day of onset; HE and Masson stained lung tissue pathological section analysis showed that the treatment group of nintedanib combined with Pseudostellariae Cyclic Peptide B can significantly inhibit alveolar inflammatory infiltration and fibrillar collagen deposition in lung tissue. In summary, nintedanib combined with Pseudostellariae Cyclic Peptide B can significantly reduce weight loss, lung inflammation and fibrosis process, improve mortality, and has the pharmacological effect of anti-pulmonary fibrosis.

[0027] In addition, the present invention uses bleomycin-induced fibrosis mice as a model, and administers nintedanib alone and / or in combination with pyogenes cyclotide B (HB) to observe the signs, diarrhea and intestinal-pulmonary tissue damage of pulmonary fibrosis model mice before and after the combined medication. The experimental results show that compared with the nintedanib alone medication group, the combined medication with HB can significantly and effectively alleviate the diarrhea and intestinal mucosal barrier damage of nintedanib-induced fibrosis mice. Compared with the effective dose (40 mg / kg) of nintedanib used alone to treat pulmonary fibrosis mice, the nintedanib anti-pulmonary fibrosis medication dose (10 mg / kg) can be significantly reduced after combined gavage with HB. Nintedanib is a small molecule tyrosine kinase inhibitor, which is mainly used in the treatment of interstitial lung diseases such as pulmonary fibrosis in clinical practice. Patients with pulmonary fibrosis often have adverse symptoms such as diarrhea and abdominal pain after taking this drug. In summary, pyogenes cyclotide B has a good application prospect in the preparation of medicines or health foods for improving nintedanib-induced acute diarrhea, and can provide a new and effective treatment for patients with pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. The drawings in the following description are only some embodiments of the present invention.

[0029] Figure 1 Survival rate curves of mice in each group in the example.

[0030] Figure 2 Body weight changes of mice in each group in the examples.

[0031] Figure 3 The terminal weight of each group of mice in the embodiment. CON blank group; BLM model group; BLM+NDN nintedanib administration group; BLM+HB pyralidin B administration group; BLM+NDN+HB nintedanib combined with pyralidin B administration group. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0032] Figure 4 The lung coefficients of mice in each group in the embodiment. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0033] Figure 5 The degree of inflammation in the lung tissue of each group of mice in the example (H&E staining) (×200, scale bar = 50 μm).

[0034] Figure 6 The lung tissue pathological inflammation scores of each group of mice in the embodiment. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0035] Figure 7 The degree of fibrosis in the lung tissue of each group of mice in the example (Masson staining) (×200, scale bar=50 μm).

[0036] Figure 8 The pathological fibrosis scores of the lung tissues of each group of mice in the embodiment. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0037] Fig. 9 Expression of E-cadherin, α-SMA, Vimentin and GAPDH proteins in lung tissues of mice in each group in the example. A is WB result, B is statistical result; 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0038] Fig.10 The LPS content in the serum of each group of mice in the embodiment. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0039] Fig.11 Schematic diagram of the colon length of each group of mice in the Examples.

[0040] Fig.12 Statistical graph of colon length of each group of mice in the embodiment. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0041] Fig.13 HE staining of colon pathological sections of mice in each group in the example (×200).

[0042] Fig.14 Inflammation degree scores of colon tissue pathological sections of mice in each group in the embodiment. 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. Compared with the nintedanib group, @ P<0.05, @@ P<0.01, @@@ P<0.001.

[0043] Fig.15 Expression of Occludin, Claudin1 and GAPDH proteins in lung tissues of mice in each group in the example, A, WB results, B, statistical results. 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

[0044] The experimental methods in the following examples of the present invention without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0045] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The following is an explanation in conjunction with specific embodiments.

[0046] Example 1: Observation of the anti-pulmonary fibrosis effect of nintedanib combined with Pseudostigmine B, comprising the following steps:

[0047] 1) Establishment of pulmonary fibrosis model:

[0048] One hundred 8-week-old adult male C57BL / 6 mice (23-25 ​​g) (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., Jiangsu, China, animal license SYXK (#) 2023-0019) were randomly divided into 5 groups. The mice were housed in clean cages under stable room temperature (22 ° C) and lighting conditions (12-h light / dark cycle), and free access to food and water was allowed. After one week of adaptive feeding, the mice were randomly divided into five groups: blank control group (Control, C), bleomycin model group (Bleomycin, BLM), Taizishen cyclic peptide B administration group (BLM + HB, 10 mg / kg), nintedanib administration group (BLM + NDN, 10 mg / kg), nintedanib combined with Taizishen cyclic peptide B administration group (BLM + HB + NDN, 10 mg / kg). After 12 hours of fasting treatment before modeling, mice were injected intratracheally with bleomycin (3.5 mg / kg, 0.9% sterile saline) or 0.9% sterile saline (control). Seven days after bleomycin modeling, the group of Pseudospermum officinale B (BLM+HB, 10 mg / kg), the group of Nintedanib low-dose group (BLM+NDN, 10 mg / kg) and the group of Nintedanib combined with Pseudospermum officinale B (BLM+HB+NDN, 10 mg / kg) were gavaged once a day for 14 days, while the control group and the model group received the same amount of solvent. The weight and survival status of the mice were recorded every day.

[0049] 2) Experimental results analysis:

[0050] like Figure 1As shown in the figure, compared with the blank control group, the mice in the bleomycin model group began to die from the 8th day, and the survival rate of the mice in the bleomycin model group was only 65% ​​by the 21st day after modeling. The mortality rate of mice treated with nintedanib combined with Pseudostellariae Cyclic Peptide B was significantly lower than that of the bleomycin model group. In addition, the nintedanib group began to die from the 9th day, and the mortality rate of mice was no different from that of the bleomycin model group; the Pseudostellariae Cyclic Peptide B group began to die from the 9th day, and the mortality rate of mice was no different from that of the bleomycin model group.

[0051] like Figure 2 As shown in the figure, the weight gain of mice in the bleomycin model group showed a significantly slower trend than that of the blank control group from the 5th day. The weight loss of mice in the nintedanib combined with Pseudostellariae Cyclic Peptide B treatment group was improved compared with the bleomycin model group. From the second week, the weight gain of nintedanib combined with Pseudostellariae Cyclic Peptide B was significantly higher than that of the bleomycin model group (P<0.001); the improvement effect of nintedanib or Pseudostellariae Cyclic Peptide B alone on the weight of mice was not much different (P>0.05).

[0052] like Figure 3 As shown in the figure, the final body weight of mice in the pulmonary fibrosis model group was significantly lower than that in the group treated with nintedanib combined with Pseudostigmine B (P<0.001), and nintedanib combined with Pseudostigmine B treatment could significantly improve the weight loss rate of mice (P<0.001). In addition, the final body weight of mice treated with nintedanib or Pseudostigmine B alone was similar to that of the bleomycin model group, with no statistical difference (P>0.05). In summary, it can be seen that nintedanib combined with Pseudostigmine B has a significant improvement in the body weight and survival rate of mice with bleomycin-induced pulmonary fibrosis.

[0053] Example 2: Collection and analysis of lung tissue samples from each group before and after treatment with nintedanib combined with Pseudostellariae Cyclotide B

[0054] On the 21st day after the administration of bleomycin, the mice were anesthetized with 0.3% sodium pentobarbital, and the blood and lung tissue of the mice were collected. Lung tissue samples were fixed in 4% paraformaldehyde solution for at least 24 hours, then dehydrated, transparent, and paraffin-embedded. After cutting into 4-7 μm thick sections, hematoxylin-eosin (H&E) and Masson trichrome staining were performed. Representative images were obtained using a digital pathology section scanning system (purchased from GPBio, Shanghai, China). All data are expressed as mean ± SD GraphPad Prism 8 statistical software was used for data analysis, and one-way ANOVA was used for statistics. P<0.05 indicated statistically significant differences.

[0055] like Figure 4As shown in the figure, compared with the blank control group, after 21 days of bleomycin intervention, the lung coefficient of mice was significantly higher than that of the blank control group (P<0.001), indicating that the pulmonary fibrosis and collagen fiber deposition of mice in the bleomycin model group were more serious. Compared with the bleomycin model group, the lung coefficient of mice in the nintedanib combined with Pseudostigmine Cyclic Peptide B group was reduced (P<0.001), indicating that the pulmonary fibrosis and collagen fiber deposition of mice in the nintedanib combined with Pseudostigmine Cyclic Peptide B group were improved.

[0056] like Figure 5 and Figure 6 As shown in the figure, compared with the blank control group, after 21 days of bleomycin intervention, the smooth muscle of the mice lungs was significantly damaged, the inflammatory cell infiltration broke through the basement membrane, a large number of inflammatory cell infiltrations were seen in the lung interstitium, and the alveolar wall was hyperplastic and destroyed. Compared with the bleomycin model group, the lung conditions of mice in the group treated with nintedanib combined with Pseudostellariae Cyclic Peptide B were improved. Inflammatory cell infiltration was still visible in some lung interstitium of mice in the group treated with nintedanib or Pseudostellariae Cyclic Peptide B alone, and the alveolar septa were thickened, accompanied by alveolar wall destruction. In the group treated with nintedanib combined with Pseudostellariae Cyclic Peptide B, no inflammatory cell infiltration in the lung interstitium and thickening of the alveolar septa were observed, some alveolar septa were thinned, alveolar atrophy and collapse were rare, the marginal alveoli were intact, and the alveolar structure was restored.

[0057] like Figure 7 and Figure 8 As shown in the figure, compared with the blank control group, after 21 days of bleomycin intervention, accompanied by destruction of alveolar structure, obvious collagen fiber infiltration was observed in the lung interstitium. Compared with the bleomycin model group, the lung conditions of mice in the group treated with nintedanib combined with Pseudospermum cyclotide B were improved. Some collagen fiber infiltration was still observed in the group treated with nintedanib or Pseudospermum cyclotide B alone. After 14 days of intervention with nintedanib combined with Pseudospermum cyclotide B, the collagen fibers in the lung interstitium of mice were significantly reduced.

[0058] Example 3: Changes in E-cadherin, α-SMA and Vimentin protein levels in lung tissues of each group before and after treatment with nintedanib combined with Pseudomonas aeruginosa cyclotide B

[0059] The lung tissue stored in the ultra-low temperature refrigerator was taken out, and about 50 mg of lung tissue samples were cut with scissors and placed in an EP tube containing two magnetic beads and placed on ice. The pre-configured lysis mixture (RIPA strong lysis solution: PMSF: phosphatase inhibitor = 100:1:1) was added, and the sample was ground in a tissue grinder. The supernatant was aspirated to obtain the lung tissue protein extract. After the tissue protein extract was quantified by the BCA protein quantification kit, 5× loading buffer was added for denaturation. The relative expression levels of E-cadherin, α-SMA and Vimentin proteins in each group of samples were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

[0060] like Fig. 9 As shown in the figure, compared with the blank control group, the expression of E-cadherin in the lung tissue of mice in the bleomycin model group was significantly reduced, and α-SMA and Vimentin were significantly increased (P<0.001), indicating that bleomycin induced the occurrence of pulmonary fibrosis in mice. Nintedanib combined with Pseudostigmine B can significantly increase the expression of E-cadherin and reduce the protein expression of α-SMA and Vimentin, indicating that Nintedanib combined with Pseudostigmine B significantly alleviated pulmonary fibrosis in mice. Abnormal expression of E-cadherin, α-SMA and Vimentin proteins was still observed in the groups treated with Nintedanib or Pseudostigmine B alone.

[0061] Example 4: Collection and analysis of serum and colon samples from each group before and after treatment with nintedanib combined with Pseudostellariae Cyclotide B

[0062] On the 21st day after bleomycin administration, the mice were anesthetized with 0.3% sodium pentobarbital, and blood and colon tissue were collected. The abdominal cavity of the mice was cut open with sterilized forceps and scissors, and the colon was completely removed. The length of the colon of the mice was measured and photographed. The colon was washed with saline, and 1 cm of the lower part of the colon near the anus was cut off for pathological section preparation and histopathological scoring. The rest was divided into two equal parts by volume for subsequent experiments.

[0063] like Fig.10 As shown, the serum LPS content was detected using an ELISA kit. Compared with the normal control group, the LPS content in the serum of mice in the bleomycin model group increased significantly (P<0.001); in addition, the LPS content in the serum of mice in the nintedanib single-administration group also increased significantly; nintedanib combined with Pseudostigmine Cyclotide B can significantly reduce the LPS content in the serum of mice. Fig.11 and Fig.12 As shown in the figure, compared with the normal control group, the colon length of mice in the bleomycin model group was significantly shortened and the difference was statistically significant (P<0.001); compared with the colon length of mice in the model group, the shortening of the colon was alleviated after the administration of nintedanib combined with Pseudospermum officinale B, with a significant difference (P<0.001). This suggests that the administration of nintedanib combined with Pseudospermum officinale B can improve the colon damage of mice with pulmonary fibrosis and reduce the colon damage caused by nintedanib.

[0064] like Fig.13 and Fig.14 Compared with the blank control group, the results of HE staining and pathological histology showed that

[0065] Compared with the normal control group, the crypts and epithelium of the colon mucosal layer of mice in the bleomycin model group were mostly lost, accompanied by inflammatory cell infiltration, and inflammation invaded the submucosal layer and mucosal layer; the degree of colon mucosal lesions in mice in the nintedanib combined with scutellaria baicalensis cyclic peptide B group was alleviated, inflammatory cell infiltration was reduced, epithelial and crypt integrity was improved, and colon mucosal damage was significantly improved. The results of pathological histology showed that compared with the control group, the lesion score in the bleomycin model group was significantly increased (P < 0.001); compared with the model group, the pathological score in the nintedanib combined with scutellaria baicalensis cyclic peptide B group was significantly reduced (P < 0.001).

[0066] like Fig.15 As shown in the figure, compared with the blank control group, the expression of Occludin and Claudin 1 in the colon tissue of mice in the bleomycin model group was significantly reduced (P<0.001), indicating that the expression of intestinal tight junction proteins in mice with pulmonary fibrosis was reduced. Nintedanib combined with Pseudostigmine B can significantly increase the protein expression of Occludin and Claudin 1, indicating that Nintedanib combined with Pseudostigmine B significantly restored the expression of intestinal tight junction proteins in mice. The Occludin and Claudin1 proteins were still significantly reduced in the groups treated with Nintedanib or Pseudostigmine B alone.

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

[0068] References:

[0069] 1. Noble, PW, CE Barkauskas, and D. Jiang, Pulmonary fibrosis: patterns and perpetrators. The Journal of clinical investigation, 2012.122(8): p.2756-2762. 2.Glass, DS, et al., Idiopathic pulmonary fibrosis: Current and future treatment. 2022.16(2):p.84-96.

[0071] 3.Podolanczuk,A.J.,et al.,Idiopathic pulmonary fibrosis:state of theart for 2023.2023.61(4).

[0073] 4.Wynn,T.A.and T.R.J.N.m.Ramalingam,Mechanisms of fibrosis:therapeutictranslation for fibrotic disease.2012.18(7):p.1028-1040.

[0074] 5.Li,Z.,et al.,Stem cell therapy for COVID-19,ARDS and pulmonaryfibrosis.2020.53(12):p.e12939.

[0076] 6.Flaherty,K.R.,et al.,Safety of nintedanib added to pirfenidonetreatment foridiopathic pulmonary fibrosis.2018.52(2).

[0077] 7.Maher,T.M.,et al.,Biomarkers of extracellular matrix turnover inpatients withidiopathic pulmonaryfibrosis given nintedanib(INMARK study):arandomised,placebo-controlled study.2019.7(9):p.771-779.

[0079] 8.Lederer,D.J.and F.J.Martinez,Idiopathic pulmonary fibrosis.NewEnglandJournal of Medicine,2018.378(19):p.1811-1823.

[0080] 9.Hu,D.-j.,et al.,Chemistry,pharmacology and analysis ofPseudostellariaheterophylla:a mini-review.2019.14(1):p.21。

Claims

1. The use of nintedanib and a plant-derived cyclic peptide Pseudostellariae cyclic peptide B in combination for the preparation of a drug for the treatment of pulmonary fibrosis, characterized in that: The nintedanib is any one or more of itself, its stereoisomers, its tautomers, its polymorphs, its solvates and its pharmaceutically acceptable salts; The Pseudostellariae cyclotide B is any one or more of itself, its stereoisomers, its tautomers, its polymorphs, its solvates and its pharmaceutically acceptable salts.

2. The use according to claim 1, characterized in that: 10mg / kg nintedanib and 10mg / kg pseudoginseng cyclopeptide B were used in combination.

3. Use of a pharmaceutical composition in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The pharmaceutical composition contains nintedanib and Pseudostellariae Cyclotide B as described in claim 1 or 2, and at least one pharmaceutically acceptable carrier or excipient.

4. The use according to claim 3, characterized in that: The dosage form of the pharmaceutical composition is solution, granules, tablets, pills, drops, injection, capsules, powders or decoctions.

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