Application of Liancheng Liancheng Herb oral liquid in COPD disease

Abstract

The invention relates to the technical field of medicine, in particular to application of Liancheng and Radix Astragali oral liquid in COPD (chronic obstructive pulmonary disease). The research result shows that when the Liancheng and Radix Astragali oral liquid is given to a model animal, the levels of WBC, Mon, TNF-alpha and IL-6 of alveolar lavage fluid can be reduced, the percentage of CD4 +, CD4 + / CD8 + can be increased, airway resistance can be reduced, lung compliance can be enhanced, and pathological changes of animal lung tissues can be relieved; the Liancheng Ligusticum oral liquid is prompted to have the effects of resisting inflammation, improving immunity, improving lung functions and repairing lung tissue injury. Therefore, when the Liancheng oral liquid is applied to treatment and prevention of COPD, the Liancheng oral liquid can obviously relieve the pulmonary ventilation dysfunction of COPD animals, reduce the level of inflammatory factors of alveolar lavage fluid, improve immune cell expression and obviously relieve the lesion degree of lung tissues, and the Liancheng oral liquid can be known to have obvious prevention and treatment effects on COPD.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the use of Wuqi Oral Liquid in the treatment of COPD disease. Background Art

[0002] Chronic obstructive pulmonary disease, abbreviated as COPD, is a heterogeneous disease affected by genetic and environmental factors, characterized by persistent airflow limitation and corresponding respiratory symptoms. The course of COPD is long, the condition is protracted and not easily cured, and it is prone to repeated attacks, and various complications may occur. Its serious complications may affect the development and fatality rate of the disease. The high incidence and large social burden of COPD make it a key focus of public health concern worldwide. Acute exacerbation of COPD refers to the aggravation of the original symptoms of the patient, and even the manifestation of respiratory failure, often requiring a change in the original treatment plan. Acute exacerbation can lead to a continuous decline in lung function and gradually develop into cor pulmonale, ultimately threatening the life and health of the patient. Therefore, preventing and reducing the deterioration of the condition is a key goal in the management of COPD. Summary of the Invention

[0003] In patients with chronic obstructive pulmonary disease, exposure to harmful factors such as cigarette smoke and dust directly stimulates macrophages to release inflammatory factors such as IL-6 and TNF-α, resulting in damage to lung tissue. At the same time, it activates neutrophils to produce IL-6, exacerbating the inflammatory response, which is an important indicator reflecting the severity of COPD. CD4+ cells are an important part of the immune system, and the change in their percentage directly reflects the state of the body's immune function. In patients with COPD, a decrease in the CD4+ percentage indicates that the patient's immune function is inhibited, the body's ability to resist pathogens declines, and various infections are more likely to occur, leading to recurrence or exacerbation of the condition. The change in the CD4+ / CD8+ ratio reflects the disruption of the balance between helper T cells and cytotoxic T cells in the immune system. When this ratio decreases, it indicates that the immune regulation function is disordered, which may lead to the persistence and amplification of the inflammatory response, and is not conducive to the control and recovery of the condition. The degree of change in CD4+ and CD4+ / CD8+ is related to the degree of lung function damage in patients with COPD. The lower the CD4+ cell percentage and the CD4+ / CD8+ ratio, the more severe the lung function damage of the patient may be, and the faster the disease may progress. Pulmonary function examination is an objective indicator for judging airflow limitation with good repeatability and is the gold standard for diagnosing COPD. Through the data of various indicators obtained from pulmonary function tests, doctors can judge the degree of lung function damage of the patient and thus evaluate the severity of the COPD condition.

[0004] The results of this study showed that the levels of WBC, Neu, Lym, Mon, TNF-α, and IL-6 in the BALF of model rats were significantly increased, the airway resistance of lung function increased, and the lung compliance decreased. Moreover, there were pathological changes such as interstitial thickening and inflammatory cell infiltration in both the lungs and bronchi, indicating the successful establishment of the rat COPD model. Administration of Wuqi Oral Liquid to the model animals could reduce the levels of WBC, Mon, TNF-α, and IL-6 in the alveolar lavage fluid, increase the percentages of CD4+ and CD4+ / CD8+, decrease the airway resistance, enhance the lung compliance, and simultaneously alleviate the histopathological changes in the lung tissue of the animals; suggesting that Wuqi Oral Liquid has the effects of anti-inflammation, improving immunity, improving lung function, and repairing lung tissue damage.

[0005] Beneficial effects:

[0006] When Wuqi Oral Liquid is applied to the treatment and prevention of COPD, it can significantly relieve the pulmonary ventilation dysfunction in COPD animals, reduce the levels of inflammatory factors in the alveolar lavage fluid, increase the expression of immune cells, and significantly alleviate the degree of lung tissue lesions. It can be known that Wuqi Oral Liquid has obvious preventive and therapeutic effects on COPD. Description of the drawings

[0007] Figure 1 This is the HE staining diagram of the lung tissue pathological section during the experiment of the present invention;

[0008] Figure 2 This is the HE staining diagram of the bronchial tissue pathological section during the experiment of the present invention. Detailed implementation manners

[0009] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0010] Before explaining the specific scheme in this embodiment, the following explanations are made for the symbol abbreviations mentioned in this embodiment:

[0011] BALF: Bronchoalveolar Lavage Fluid, a sample used to detect lung inflammation and immune responses.

[0012] WBC: White Blood Cells count, an important indicator reflecting systemic or local inflammatory responses.

[0013] Neu: Neutrophils, a type of white blood cell involved in acute inflammatory responses.

[0014] Lym: Lymphocytes, a type of white blood cell that mainly participates in the adaptive immune response.

[0015] Mon: Monocytes, a type of white blood cell that can further differentiate into macrophages and dendritic cells.

[0016] TNF-α: Tumor Necrosis Factor alpha, an important pro-inflammatory cytokine.

[0017] IL-6: Interleukin-6, a multifunctional cytokine that plays an important role in the inflammatory response.

[0018] CD3+: CD3-positive T cells, a subset of T lymphocytes, representing the total number of T cells.

[0019] CD4+: CD4-positive T cells, a subset of helper T cells, whose main function is to assist other immune cells.

[0020] CD8+: CD8-positive T cells, a subset of cytotoxic T cells, whose main function is to kill infected cells.

[0021] CD4+ / CD8+ percentage: The ratio of CD4-positive and CD8-positive T cells, used to evaluate the balance of the immune system.

[0022] FVC: Forced vital capacity, which refers to the maximum volume of air that can be exhaled as quickly as possible after a maximum inhalation.

[0023] Quarantine-qualified SPF-grade SD rats: SPF-grade refers to rats without specific pathogens. Laboratory animals of this grade live in a barrier system and do not carry pathogens of zoonotic diseases and highly contagious animal diseases. SD rats are Sprague-Dawley rats, a commonly used laboratory rat strain. Such rats are usually used in experiments such as drug testing and toxicity research.

[0024] To verify the preventive and therapeutic applications of the proposed Wushi oral liquid in COPD, the following experimental content is proposed:

[0025] 1. Selection of test materials

[0026] 1.1 Test substances

[0027] Wuqi Oral Liquid, batch number: 2407002, specification: 10 mL / ampoule, content: 10.65 g (raw herbs) / 10 mL, properties: red-brown to dark-brown liquid, sweet in taste, slightly bitter, expiration date: June 2026, dosage and administration: 20 mL / day, 21.3 g (raw herbs) / day, functions and indications: used for dizziness, blurred vision, fatigue, palpitations, insomnia, loss of appetite, dry cough in the mouth and throat, hoarse cough, etc. caused by physical weakness, old age or qi-yin deficiency after chronic diseases, manufacturer: Hunan Tianjin Pharmaceutical Co., Ltd.

[0028] 1.2 Reference Substances

[0029] Glycine Theophylline Sodium Tablets, batch number: 2309151, expiration date: August 2025. Dosage and administration: 0.165 g / tablet, 1 tablet once, 3 times a day. Functions and indications: applicable to relieve wheezing symptoms in bronchial asthma, asthmatic bronchitis, obstructive pulmonary emphysema, etc.; can also be used for asthma caused by cardiogenic pulmonary edema. Provider: Hunan Tianjin Pharmaceutical Co., Ltd.

[0030] 1.3 Experimental Animals

[0031] 60 SPF-grade male SD rats, weighing 390.7 - 640.2 g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (SCXK(Xiang)2021 - 0002), raised in Area C of the barrier environment animal laboratory, experimental animal use license number: SYXK(Xiang)2020 - 0015.

[0032] 1.4 Main Reagents

[0033] 0.9% Sodium Chloride Injection, batch number: 24053101C, manufacturer: Hunan Kangyuan Pharmaceutical Co., Ltd.; Zoletil 50, batch number: 91CRA, manufacturer: Virbac S.A.; Lipopolysaccharide (LPS), batch number: 24157403, specification: 10 mg / ampoule, manufacturer: biosharp; Furong brand cigarettes (tar content: 11 mg, nicotine content in mainstream smoke: 0.9 mg, carbon monoxide in mainstream smoke: 13 mg), manufacturer: Hunan Changde Cigarette Factory.

[0034] 1.5 Main Instruments

[0035] PFT Pulmonary Function Detection System (Shanghai Tawang Intelligent Technology Co., Ltd.), Spectra Max i3x Multifunctional Microplate Reader (MD, USA), DFC 420C Pathology Imaging System (Leica, Germany), etc.

[0036] 2. Test Methods

[0037] 2.1 Grouping, Modeling and Drug Administration

[0038] Sixty SPF male SD rats qualified for quarantine were divided into a normal control group (10 rats) and a model group (40 rats). The rats in the model group were used to establish an obstructive pulmonary disease model by repeated smoking combined with intratracheal instillation of lipopolysaccharide. The model rats were placed in a smoking chamber, and cigarettes were burned at a rate of 1 cigarette every 5 minutes using an automatic cigarette smoker, and the burned smoke was introduced into the smoking chamber. Each smoking session lasted for 1 hour, once a day for 4 consecutive weeks. At the same time, 0.2 mL of 1 mg / mL lipopolysaccharide (LPS) solution was injected into the trachea of each rat, once every 1 week. The rats in the normal control group were injected with an equal volume of 0.9% sodium chloride injection into the airway using the same method. Two rats were randomly selected from the normal control group and the model group before drug administration for pulmonary function and lung tissue pathological examinations to confirm the successful establishment of the model. After modeling, 48 model rats were randomly divided into a model control group, a sodium glycinate theophylline tablet group (45 mg / kg), a sodium glycinate theophylline tablet combined with Wuqi Oral Liquid group (45 mg / kg + 2 g crude drug / kg), and low, medium, and high dose groups of Wuqi Oral Liquid (2, 4, 8 g crude drug / kg), with 8 animals in each group. The animals in each test substance group were given the corresponding concentration of the drug solution by gavage at a dose of 10 mL / kg, once a day for 28 consecutive days; the normal control group and the model control group were given an equal volume of pure water by gavage.

[0039] 2.2 Dose Design

[0040] The clinical intended dose of Wuqi Oral Liquid is 21.3 g crude drug per day. Calculated based on an average adult body weight of 70 kg and an average rat body weight of 0.2 kg, the equivalent dose ratio calculated by body surface area conversion is 0.018. According to the body surface area method, the equivalent dose for rats is 21.3 g × 0.018 / 0.2 kg ≈ 2 g crude drug / kg. In this experiment, the low, medium, and high doses of Wuqi Oral Liquid were set at 1, 2, and 4 times the clinical equivalent dose, namely 2, 4, and 8 g crude drug / kg.

[0041] The clinical intended dose of sodium glycinate theophylline tablets is 495 mg per day. Calculated based on the body surface area method, the equivalent dose for rats is 495 mg × 0.018 / 0.2 kg ≈ 45 mg / kg. Therefore, 45 mg / kg was designed as the dosing dose for the sodium glycinate theophylline tablet group in this experiment. The specific grouping and doses are shown in Table 1.

[0042] Table 1 Grouping and Dose Design

[0043]

[0044] Note: The adult body weight is calculated as 70 kg.

[0045] 2.3 Detection Indexes

[0046] 2.3.1 General Physiological Observation

[0047] Observe the physiological status and mortality of the animals before and after drug administration.

[0048] 2.3.2 Pulmonary function test

[0049] On the day after the last drug administration, the rats in each group were anesthetized with 60 mg / kg Zoletil 50. The trachea was exposed surgically and the rats were fixed supine in a plethysmograph chamber. A ventilation hose was inserted into the trachea, and a small animal pulmonary function analyzer was used to measure the airway resistance (Penh), pulmonary compliance (Cpyn), tidal volume (VT), forced vital capacity (FVC), and forced expiratory volume in 0.2 seconds (FEV 200 ).

[0050] 2.3.3 Detection of the content of white blood cells and inflammatory factors in bronchoalveolar lavage fluid

[0051] On the day after the last drug administration, the rats in each group were anesthetized with 60 mg / kg Zoletil 50. The lungs were removed. After ligating the left lung, the right lung was repeatedly lavaged with 0.9% sodium chloride injection, and the lavage fluid was collected. A five-class hematology analyzer was used to detect the total number of white blood cells, neutrophils, monocytes, and lymphocytes in the bronchoalveolar lavage fluid, and an ELISA method was used to detect the contents of IL-6 and TNF-α in the bronchoalveolar lavage fluid.

[0052] 2.3.4 Detection of immune cells in bronchoalveolar lavage fluid

[0053] On the day after the last drug administration, the rats in each group were anesthetized with 60 mg / kg Zoletil 50. Blood was collected from the abdominal aorta, and flow cytometry was used to classify lymphocytes (CD3 + , CD4 + , CD8 + ) in the whole blood.

[0054] 2.3.5 Histopathological examination of lung tissue

[0055] On the day after the last drug administration, the rats in each group were anesthetized with 60 mg / kg Zoletil 50. The bronchi and the left lung were removed, fixed in 10% neutral formalin, embedded in paraffin, sectioned, stained with HE, and placed under a light microscope to observe the histopathological changes of the bronchial and lung tissues.

[0056] 2.4 Data statistics

[0057] The significant figures of the data in this experiment were rounded according to the rule of rounding up or down, and statistical analysis was performed according to the SOP regulations. The software used for statistics was SPSS 23.0. The mean ± standard deviation was used Represent and conduct normality and homogeneity of variance tests. If normality is satisfied (P>0.05), one-way analysis of variance (ANOVA) is used for statistical analysis, and LSD+Dunnet (for homogeneous variance) or Tamhane’s T2 (for heterogeneous variance) is selected for comparison analysis according to the homogeneity of variance situation. If normality is not satisfied (P≤0.05), then Nonparametric Tests are used. The statistical results take α = 0.05 as the test limit, where P≤0.05 indicates statistical significance, and P≤0.01 indicates very significant difference in the test.

[0058] 3. Experimental results

[0059] 3.1 General physiological observations

[0060] No animal deaths occurred during the animal model establishment period and the administration period; during the animal model establishment period in the model group, symptoms such as piloerection, rapid breathing, and reduced activity were observed, and the symptoms of the animals were alleviated to varying degrees after administration.

[0061] 3.2 Effects on rat lung function indexes

[0062] As shown in Table 1, compared with the normal control group, Vt, Cpyn, FVC, and FEV 200 in the animal model control group were all significantly decreased (P≤0.01 or P≤0.05), and the Penh level was significantly increased (P≤0.05); compared with the animal model control group, the Penh levels in the medium- and high-dose groups of Wuqi Oral Liquid and the Theophylline Sodium Glycinate Tablets group were significantly decreased (P≤0.01 or P≤0.05), the Cpyn level in the high-dose group of Wuqi Oral Liquid was significantly increased (P≤0.05), and the FEV 200 levels in the low-, medium-, and high-dose groups of Wuqi Oral Liquid and the Theophylline Sodium Glycinate Tablets group were significantly increased (P≤0.05).

[0063] Table 1 Effects of Wuqi Oral Liquid on rat lung function ( n = 8)

[0064]

[0065] Note: Compared with the normal control group + P≤0.05, ++ P≤0.01; compared with the animal model control group * P≤0.05, ** P≤0.01. 3.3 Effects on the white blood cell content in rat bronchoalveolar lavage fluid

[0066] As shown in Table 2, compared with the normal control group, the contents of WBC, Neu, Lym, and Mon in the alveolar lavage fluid of the animals in the model control group were all significantly increased (P≤0.01 or P≤0.05); compared with the model control group, the content of WBC in the alveolar lavage fluid of the animals in the high-dose group of Wuqi Oral Liquid and the group of Sodium Glycinate Theophyllinate combined with Wuqi Oral Liquid was significantly decreased (P≤0.05); the content of Mon in the alveolar lavage fluid of the animals in the high-dose group of Wuqi Oral Liquid was significantly decreased (P≤0.05).

[0067] Table 2 Effects of Wuqi Oral Liquid on the content of white blood cells in the alveolar lavage fluid of rats ( n = 8)

[0068]

[0069] Note: Compared with the normal control group ++ P≤0.01, + P≤0.05, compared with the model control group * P≤0.05.

[0070] 3.4 Effects on the content of inflammatory factors in the alveolar lavage fluid of rats

[0071] As shown in Table 3, compared with the normal control group, the contents of IL-6 and TNF-α in the alveolar lavage fluid of the animals in the model control group were both significantly increased (P≤0.01); compared with the model control group, the contents of IL-6 and TNF-α in the alveolar lavage fluid of the low-, medium-, and high-dose groups of Wuqi Oral Liquid and the Sodium Glycinate Theophyllinate group were all significantly decreased (P≤0.01 or P≤0.05).

[0072] Table 3 Effects of Wuqi Oral Liquid on the content of inflammatory factors in the alveolar lavage fluid of rats ( n = 8)

[0073]

[0074] Note: Compared with the normal control group ++ P≤0.01, compared with the model control group ** P≤0.01, * P≤0.05.

[0075] 3.5 Effects on immune cells in the alveolar lavage fluid of rats

[0076] As shown in Table 4, compared with the normal control group, the percentages of CD4 + , CD4 + / CD8 + in the alveolar lavage fluid of the animals in the model control group were both significantly decreased (P≤0.01); compared with the model control group, the percentage of CD4 +The percentage increased significantly (P≤0.05). In the low-, medium-, and high-dose groups of Wuqi Oral Liquid and the group of Sodium Glycinate Theophyllinate Tablets combined with Wuqi Oral Liquid, the CD4 + / CD8 + percentage increased significantly (P≤0.01 or P≤0.05).

[0077] Table 4 Effects of Wuqi Oral Liquid on immune cells in bronchoalveolar lavage fluid of rats ( n = 8)

[0078]

[0079] Note: Compared with the normal control group ++ P≤0.01, compared with the model control group ** P≤0.01, * P≤0.05.

[0080] 3.6 Effects on rat lung histopathology

[0081] As Figure 1 shown, it is the pathological section of lung tissue. Among them, A is the normal control group; B is the model control group; C is the Sodium Glycinate Theophyllinate Tablets group; D is the group of Sodium Glycinate Theophyllinate Tablets combined with Wuqi Oral Liquid; E is the low-dose group of Wuqi Oral Liquid; F is the medium-dose group of Wuqi Oral Liquid; G is the high-dose group of Wuqi Oral Liquid. On the day after the last administration, no obvious abnormality was observed in the lung tissue of animals in the normal control group under the microscope; in the lung tissue of animals in the model control group, there were inflammatory cell infiltration in the fine alveolar cavity and thickening of the lung interstitium with inflammatory cell infiltration and other lesions; the lung tissue lesions in the low-, medium-, and high-dose groups of Wuqi Oral Liquid, the Sodium Glycinate Theophyllinate Tablets group, and the group of Sodium Glycinate Theophyllinate Tablets combined with Wuqi Oral Liquid were alleviated to varying degrees compared with the model control group.

[0082] As Figure 2 shown, it is the pathological section of bronchial tissue. Among them, A is the normal control group; B is the model control group; C is the Sodium Glycinate Theophyllinate Tablets group; D is the group of Sodium Glycinate Theophyllinate Tablets combined with Wuqi Oral Liquid; E is the low-dose group of Wuqi Oral Liquid; F is the medium-dose group of Wuqi Oral Liquid; G is the high-dose group of Wuqi Oral Liquid. On the day after the last administration, no obvious abnormality was observed in the bronchial tissue of animals in the normal control group under the microscope; in the bronchial tissue of animals in the model control group, there was hyperplasia of lymphoid tissue in the mucosa and submucosa; no obvious improvement was observed in each administration group.

[0083] As shown in Table 5, compared with the normal control group, the pathological scores of the lung and bronchial tissues of animals in the model control group increased significantly (P≤0.01); compared with the model control group, the pathological scores of the lung tissues of animals in the low-, medium-, and high-dose groups of Wuqi Oral Liquid decreased significantly (P≤0.05).

[0084] Table 5 Effects of Wuqi Oral Liquid on histopathology of rat lung and bronchus ( n = 8)

[0085]

[0086] Note: Compared with the normal control group ++ P ≤ 0.01, compared with the model control group * P ≤ 0.05.

[0087] From the above experiments, it can be seen that the levels of WBC, Neu, Lym, Mon, TNF-α, and IL-6 in the BALF of the model rats were significantly increased, the airway resistance of lung function increased, the lung compliance decreased, and there were pathological changes such as interstitial thickening and inflammatory cell infiltration in the lungs and bronchi, indicating that the construction of the rat COPD model was successful. Administering Wuqi Oral Liquid to the model animals could reduce the levels of WBC, Mon, TNF-α, and IL-6 in the alveolar lavage fluid, increase the CD4 + 、CD4 + / CD8 + percentage, reduce airway resistance, enhance lung compliance, and at the same time alleviate the histopathological changes of the animal lung tissue; suggesting that Wuqi Oral Liquid has the effects of anti-inflammation, improving immunity, improving lung function, and repairing lung tissue damage.

[0088] In summary, Wuqi Oral Liquid can significantly relieve the pulmonary ventilation dysfunction in COPD animals, reduce the levels of inflammatory factors in the alveolar lavage fluid, increase the expression of immune cells, and significantly alleviate the degree of lung tissue lesions, indicating that Wuqi Oral Liquid has obvious preventive and therapeutic effects on COPD, and its effective dose is the low dose (2 g crude drug / kg).

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. Use of Wuqi Oral Liquid produced by Hunan Tianjin Pharmaceutical Co., Ltd. in preventing and / or treating COPD diseases.

2. The application according to claim 1, characterized in that, The minimum effective dose is 2 g of crude drug / kg.

3. Use of Wuqi Oral Liquid produced by Hunan Tianjin Pharmaceutical Co., Ltd. in anti-inflammatory.

4. Use of Wuqi Oral Liquid produced by Hunan Tianjin Pharmaceutical Co., Ltd. in enhancing immunity.

5. Use of Wuqi Oral Liquid produced by Hunan Tianjin Pharmaceutical Co., Ltd. in improving lung function.

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