CN111135179B - Application of iron wintergreen acid in preparation of medicine for treating pneumonia - Google Patents

Abstract

The invention discloses an application of iron wintergreen acid as a unique active ingredient in preparing a medicament for treating pneumonia. The invention has a certain protection effect on pneumonia model mice, and the mechanism of the invention is probably related to the inhibition of proinflammatory cytokine release in lung.

Description

Application of iron wintergreen acid in preparation of medicine for treating pneumonia

Technical Field

The present invention relates to the field of medicine. More specifically, the invention relates to an application of iron wintergreen acid as a unique active ingredient in preparing a medicament for treating pneumonia.

Background

Community Acquired Pneumonia (CAP) refers to inflammation of the terminal airways, alveoli and lung interstitium, and is caused by pathogenic microorganisms, physicochemical factors, immune injury, allergy and drugs outside the hospital. Pathogens causing pneumonia, such as bacteria, fungi, chlamydia, viruses, etc., of which bacteria are the most common. In recent years, the incidence of pneumonia has been increasing. The pneumonia fatality rate is counted by scholars, wherein the number of outpatients is less than 1% -5%, the number of inpatients is about 12%, and the number of patients who enter an intensive care unit is as high as 40%. Pneumonia accounts for about half of the causes of severe sepsis and septic shock. Respiratory diseases are one of the main causes of death of the population in China, account for the first cause of death of the population in rural areas and the fourth cause of death of the population in cities, and the main causes of death are chronic obstructive pulmonary disease and pneumonia. Viral pneumonia (Viral pneumonia) is a lung inflammation caused by upper respiratory tract virus infection spreading downward, and the pathological changes occur in the lung interstitium, and the Viral pneumonia is characterized in that interstitial pneumonia, particularly connective tissue edema, congestion and inflammatory cell infiltration between alveoli and bronchioles cause thickening of alveolar walls, but the alveolar cavities are only slightly inflammatory, and less inflammatory cells and serous fluid seep out. Bacterial pneumonia is a series of inflammatory reactions caused by bacteria invading lower respiratory tract, activates inflammatory cells, releases various inflammatory factors and bioactive substances, damages lung, has pathological changes in lung parenchyma, mainly shows bronchopneumonia or lobar pneumonia, and has more bronchiolitis and alveolitis-induced secretion. The main treatment method of bacterial pneumonia is antibiotics at present, but with the application of antibiotics, the problem of drug resistance of bacteria is increasingly highlighted. The abuse of antibiotics makes bacteria complex, makes clinical infection difficult to control, even endangers the life of patients, prolongs the hospitalization time of the patients and obviously increases the economic burden of the patients. Therefore, the search for new effective treatment methods is urgent. The traditional Chinese medicine and the western medicine are combined to treat the bacterial pneumonia, so that the advantages are complemented, the clinical curative effect is improved, and a new way is brought to clinically treating the bacterial pneumonia. The invention patent with application number 2006100742986 discloses the use of ilex plant extract saponin and flavone in preparing medicine for treating diseases caused by virus. Iron ilexonic acid (RA) is one of the main chemical components of holly bark of Aquifoliaceae, and has been found to have pharmacological actions such as antibiosis, anti-tumor, antioxidation, immunoregulation and the like, but no report on the treatment of bacterial pneumonia is found. The research finds that RA has a good protective effect on bacterial pneumonia.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide the application of the iron rhein serving as the only active ingredient in preparing the medicament for treating the pneumonia, the iron rhein has a certain protective effect on a pneumonia model mouse, and the mechanism of the iron rhein is probably related to the inhibition of the release of proinflammatory cytokines in the lung.

To achieve these objects and other advantages in accordance with the present invention, there is provided a use of iron ilexolic acid as a sole active ingredient in the manufacture of a medicament for treating pneumonia.

Preferably, the use of iron wintergreen acid as the sole active ingredient in the manufacture of a medicament for inhibiting the release of proinflammatory cytokines in the lung.

Preferably, the application of the iron wintergreen acid as the only active ingredient in preparing the medicine for treating pneumonia septicemia is provided.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

Preferably, the medicine is one of tablets, capsules and nasal sprays.

Preferably, the minimum effective amount of the iron wintergreen in the medicine is 1 mg/kg.

The invention at least comprises the following beneficial effects:

the invention utilizes Lipopolysaccharide (LPS) to establish a mouse Acute Lung Injury (ALI) model, discusses the protection effect and possible mechanism of the iron ilexonic acid (RA) in the ovate leaf holly bark on the acute lung injury, provides basis for preventing and treating ALI caused by endotoxin, compared with a control group, the contents of Lymphocyte, Neutrophil, WBC, TNF-alpha, IL-6, IL-1 beta and MPO in an LPS group are obviously increased, and the pathological changes of lung tissues mainly show that epithelial cells of an airway are obviously proliferated, the lumen of the airway is reduced, inflammatory cell infiltration is caused, and smooth muscles are obvious; compared with the LPS group, the levels of TNF-alpha, IL-6, IL-1 beta and MPO and the levels of Lymphocyte, Neutrophil and WBC in the RA group are obviously reduced, and the pathological damage degree of lung tissues is obviously reduced compared with the LPS group;

the lung function condition of LPS (lipopolysaccharide) tracheal instillation molding mice is observed by using an AniRes2005 animal lung function analysis system, compared with a control group, the lung inspiration Resistance (RL) and expiration resistance (Re) of the LPS group mice are obviously increased, and the dynamic lung compliance (Cdyn), the expiration flow peak value (PEF) and the ventilation volume per Minute (MVV) are obviously reduced; compared with LPS group, RL and Re of RA group are obviously reduced, and Cdyn, PEF and MVV are obviously increased;

the mouse ear swelling model caused by xylene is utilized to discuss the influence of the iron wintergreen on the mouse ear tissue inflammation, and compared with a control group, the xylene group mouse ear swelling is obviously increased; ear swelling degree of the RA group mice is obviously reduced compared with that of the xylene group;

the influence of the iron rhein on the death of the mouse septicemia is verified by using an LPS (lipopolysaccharide) induced mouse septicemia death model, and compared with a control group, mice in an LPS group die completely within 84h after molding; survival rate of RA group mice was significantly improved (about 30%).

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows the effect of RA on the number of lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBCs) in the blood of mice;

figure 2 HE staining for lung histomorphology (x 200);

FIG. 3 is a graph showing the changes in TNF- α, IL-6 and IL-1 β after ALI induction in mice;

FIG. 4 is a graph of the change in lung homogenate MPO levels following ALI in mice;

FIG. 5 is a graph of the effect of RA on lung function in mice;

FIG. 6 is a graph of the effect of RA on xylene-induced ear swelling in mice;

FIG. 7 is a graph showing the effect of RA on LPS-induced sepsis death in mice.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

1 Material

1.1 Experimental animals

SPF-grade healthy BALB/C mice, with a weight of 20-22 g and a week of 7-8, are male and purchased from Schlekschada laboratory animals Co., Ltd, Hunan, all the laboratory animals are raised in a controlled environment at a room temperature of 18-24 ℃ and a humidity of 40-50%, the animals eat and drink water freely during the experiment, and the circadian rhythm is normal.

1.2 major drugs and reagents

Iron aspartic acid (ddu philid biotechnology limited); dexamethasone sodium phosphate injection (Henan hong pharmaceutical Co., Ltd., Chinese medicine standard H41020330); lipopolysaccharide (Sigma, usa); paraformaldehyde, xylene (Chinese medicine analytically pure); 0.9% sodium chloride injection (Jiangxi east Asia pharmaceutical Co., Ltd., Tubifui group); tumor necrosis factor (TNF-alpha) kit, interleukin (IL-6, IL-1 beta) kit (Invitrogen, USA). Myeloperoxidase (MPO) kit (Nanjing institute of bioengineering).

A quantitative nebulizer for pulmonary administration (Shanghai Yuyan scientific instruments, Inc.); AniRes2005 animal lung function analysis system (bevaciz technologies ltd.); electronic balances (mettler toledo instruments shanghai ltd); digital pipette gun (Eppendorf, germany); high speed centrifuges (Eppendorf, Germany); inverted microscope (Leica, Germany) 4 degree refrigerator, -20 degree refrigerator, -80 degree refrigerator (product of Auckima); microplate reader (Bio-Tek, USA); a full-automatic blood cell analyzer for animals (Shenzhen Merrill bio-medical electronics, Inc.); constant temperature cultivation shaker (Shanghai-Hengchun scientific instruments Co., Ltd.); digital display constant temperature water bath (Changzhou Guohua electric appliance Co., Ltd.).

2 method

2.1 creation and grouping of LPS-induced Acute Lung Injury (ALI) models

60 SPF BALB/C mice are male, have the physical quality of 20-22 g, are suitable for being raised in the environment for 3 days, and have the room temperature of 25 +/-5 ℃ and the relative humidity of 40-70 percent in a laboratory. The feed is fed by conventional feed, and the water is freely drunk. Mice were randomly divided into 6 groups: control group, model group (LPS group), RA (10 mg/kg, 20 mg/kg, 40 mg/kg) group, positive drug group (dexamethasone, DEX group, 5 mg/kg), 10 per group. LPS dose was 4 mg/kg, and the intracerobrospinal route of administration (intrathecal injection) replicated LPS acute lung injury mouse model. After each mouse is anesthetized by injecting 0.4% sodium pentobarbital into the abdominal cavity of the mouse at a ratio of 0.12 mL/10 g, the mouse is placed on the back on an operating table, the mouth of the mouse is opened by a mouse mouth gag, LPS (LPS is injected into the same amount of sterile physiological saline into a control group) is injected into the lung from the opening of epiglottis by a micro atomizer, the establishment of the model is completed, and water and feed are fed after the model is revived. The RA group is molded according to the dose, the celiac injection is performed 2 h before molding and 4h after molding to give the iron aspartic acid, the control group is given with the same amount of sterile normal saline, the DEX group is molded according to the dose, and the celiac injection is performed 1 time after 0.5 h after molding to give the dexamethasone injection, and the remaining administration time point is performed to give the same amount of normal saline. The animals were fasted for 12 h before molding and were sacrificed 12 h after molding, with free access to water. The hematology analyzer measures the quantity of blood lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBCs) of each group of mice; cutting left lung tissue, fixing with 4% paraformaldehyde solution, HE staining, and performing pathological observation; alveolar Lavage Fluid (BALF), serum and remaining lung were collected for TNF-a, IL-6, IL-1 β, MPO detection.

2.2 measurement of the number of lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBCs) in blood

In each group, whole mouse eyeball blood was collected 12 hours after molding, 40. mu.L of blood was collected, and the numbers of lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBCs) in the blood were measured by a Meyer's hematology analyzer.

2.3 Lung histopathological examination

Fixing lung tissue sample with 4% paraformaldehyde solution, dehydrating with gradient alcohol, decalcifying, processing with paraffin embedding, slicing, HE staining, and observing the shape change of the left lung tissue of the mouse under microscope.

2.4 detection of TNF-alpha, IL-6, IL-1 beta and Myeloperoxidase (MPO)

The levels of TNF-alpha, IL-6, IL-1 beta in alveolar lavage fluid, serum and lung homogenate were measured by ELISA following the kit protocol. The lung homogenate was assayed for MPO activity according to MPO kit instructions.

2.5 Effect of Ferulic acid on Lung function

60 SPF BALB/C mice are male, have the physical quality of 20-22 g, are suitable for being raised in the environment for 3 days, and have the room temperature of 25 +/-5 ℃ and the relative humidity of 40-70 percent in a laboratory. The feed is fed by conventional feed, and the water is freely drunk. Mice were randomly divided into 6 groups: control group, model group (LPS group), RA (10 mg/kg, 20 mg/kg and 40 mg/kg) group, positive drug group (dexamethasone, DEX group, 5 mg/kg), 10 per group. LPS was replicated in a mouse model of LPS acute lung injury by the intracerebrospinal administration route (intrathecal injection) at a dose of 4 mg/kg. After each mouse is anesthetized by injecting 0.4% sodium pentobarbital into the abdominal cavity of the mouse at a ratio of 0.12 mL/10 g, the mouse is placed on the back on an operating table, the mouth of the mouse is opened by a mouse mouth gag, LPS (LPS is injected into the same amount of sterile physiological saline into a control group) is injected into the lung from the opening of epiglottis by a micro atomizer, the establishment of the model is completed, and water and feed are fed after the model is revived. The RA group is molded according to the dose, the celiac injection is performed 2 h before molding and 4h after molding to give the iron aspartic acid, the control group is given with the same amount of sterile normal saline, the DEX group is molded according to the dose, and the celiac injection is performed 1 time after 0.5 h after molding to give the dexamethasone injection, and the remaining administration time point is performed to give the same amount of normal saline. Fasting is carried out 12 h before each model is built, water is freely drunk, each group is anesthetized by injecting 0.4% sodium pentobarbital 0.12 mL/10 g into the abdominal cavity 12 h after the model is built, a trachea cannula is arranged after about 5 min, a mouse is placed into a body tracing box in a supine position, the trachea cannula and a gas circuit of the body tracing box are connected, and after a data curve is stable, lung function indexes of the mouse are collected by an AniRes2005 animal lung function analysis system: pulmonary inspiratory Resistance (RL), expiratory resistance (Re), dynamic lung compliance (Cdyn), Peak Expiratory Flow (PEF), and minute ventilation (MVV). The same procedure was performed for the remaining mice 12 h after molding.

2.6 Effect of Ferulic acid on ear swelling of mice by Paralylene

50 SPF-grade BALB/C mice are male, have the physical mass of 20-22 g, are suitable for being raised in the environment for 3 days, and have the room temperature of 25 +/-5 ℃ and the relative humidity of 40-70 percent in a laboratory. The feed is fed by conventional feed, and the water is freely drunk. Mice were randomly divided into 5 groups: control group, model group (Xylene group), RA (20 mg/kg and 40 mg/kg) group, group of positive drugs (dexamethasone, DEX group, 5 mg/kg), 10 of each group. Xylene (40 mu L) is smeared on the right ear of a mouse for molding, the left ear of the mouse is used as a control, RA (20 mg/kg and 40 mg/kg) groups are molded according to the dose, and are injected intraperitoneally for 2 h before molding to give the iron ilexonic acid, DEX groups are molded according to the dose, and are injected intraperitoneally for 1 time after 0.5 h after molding to give the dexamethasone injection, and the remaining administration time points are injected intraperitoneally for giving the same amount of physiological saline. Fasting is carried out 12 h before each group is molded, water is freely drunk, mice are dislocated and killed 2 h after each group is molded, ear tissues of the mice are taken by a punching instrument with the diameter of 7 mm, then the weight is precisely weighed, the remaining ear tissues of the mice are soaked in 10% formalin for HE staining, and the influence of the iron rhein on the ear tissue inflammation of the mice is observed.

2.7 Effect of Ferro-wintergreen on LPS-induced death from mouse septicemia

50 SPF-grade BALB/C mice are male, have the physical mass of 20-22 g, are suitable for being raised in the environment for 3 days, and have the room temperature of 25 +/-5 ℃ and the relative humidity of 40-70 percent in a laboratory. The feed is fed by conventional feed, and the water is freely drunk. Mice were randomly divided into 5 groups: control group, model group (LPS group), RA (20 mg/kg and 40 mg/kg) group, positive drug group (dexamethasone, DEX group, 5 mg/kg), 10 of each group. LPS replicates the LPS acute lung injury mouse model at a dose of 15 mg/kg, intracerebrospinal administration route (intrathecal injection). After each mouse is anesthetized by injecting 0.4% sodium pentobarbital into the abdominal cavity of the mouse at a ratio of 0.12 mL/10 g, the mouse is placed on the back on an operating table, the mouth of the mouse is opened by a mouse mouth gag, LPS (LPS is injected into the same amount of sterile physiological saline into a control group) is injected into the lung from the opening of epiglottis by a micro atomizer, the establishment of the model is completed, and water and feed are fed after the model is revived. The RA group is modeled according to the dose, before modeling, 2 h of intraperitoneal injection is carried out on the iron ilexonic acid, the control group is given with the same amount of sterile normal saline, the DEX group is modeled according to the dose, after the DEX group is injected into the abdominal cavity for 1 time after the modeling, dexamethasone injection is given into the abdominal cavity for injection after 0.5 h, and the remaining administration time point is given with the same amount of normal saline through intraperitoneal injection. Fasting is carried out 12 h before each model is built, water is freely drunk, the survival condition of the mice of 144 h is continuously observed after each model is built, detailed records are made, and the influence of the iron rhein on the septicemia and death of the mice is observed.

2.8 statistical treatment

Data were statistically analyzed using GraphPad Prism 6.0 software and compared between groups by one-way analysis of variance. P < 0.05 is a significant event.

3 results

3.1 Effect on the number of lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBCs) in the blood of groups of mice

As shown in fig. 1, (a) the content of Lymphocyte in blood of mouse; (B) the content of neutrophile in the blood of the mice; (C) the WBC content in the blood of the mice, after the LPS treatment for 12 hours, the number of lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBC) in the blood of the mice in the LPS group is obviously higher than that of the mice in the control group; in contrast, the numbers of lymphocytes (lymphocytes), neutrophils (neutrophiles) and White Blood Cells (WBCs) in the blood of the mice in the RA and DEX groups were significantly lower than those in the LPS group (. P < 0.05,. P < 0.01,. P < 0.001).

3.2 Effect on pathological changes in mouse Lung tissue

As shown in fig. 2, (a) control group; (B) model group (LPS 4 mg/kg); (C) RA (10 mg/kg); (D) RA (20 mg/kg); (E) RA (40 mg/kg); (F) DEX (5 mg/kg), Scale bar: 300 mu m, and the observation through a microscope shows that the airway epithelial cells of the LPS group are obviously proliferated, the airway lumen is reduced, inflammatory cells infiltrate, and the smooth muscle is obviously thickened; the RA group and the DEX group all showed a significant reduction in the expression compared with the LPS group.

3.3 Effect of RA on mouse TNF-a, IL-6, IL-1 β and MPO levels

As shown in FIG. 3, (A) TNF-. alpha.content in mouse alveolar lavage fluid; (B) the IL-6 content in mouse alveolar lavage fluid; (C) the content of IL-1 beta in mouse alveolar lavage fluid; (D) the content of TNF-alpha in mouse serum; (E) the content of IL-6 in mouse serum; (F) the content of IL-1 beta in mouse serum; (G) the TNF-alpha content of the mouse lung homogenate; (H) the IL-6 content of the homogenate of mouse lung tissues; (I) the results of the mice lung homogenate containing IL-1 β, as shown in FIG. 4, after 12 h LPS treatment, showed that the levels of TNF- α, IL-6 and IL-1 β in the lung homogenate, and MPO activity in the lung homogenate were significantly increased in the mice alveolar lavage fluid, serum and lung homogenate compared to the control group, TNF- α, IL-6 and IL-1 β in the RA group, and MPO activity in the lung homogenate compared to the LPS group (P < 0.05, P < 0.01, P < 0.001), and TNF- α, IL-6, IL-1 β and MPO in the DEX group were significantly decreased in the LPS group (P < 0.05, P < 0.01, P < 0.001). The results suggest that RA can reduce TNF-alpha, IL-6, IL-1 beta and MPO levels, and may be one of its mechanisms of action in the treatment of LPS-induced ALI.

3.4 Effect of RA on pulmonary function indices in mice

As shown in fig. 5, (a) mouse pulmonary inspiratory Resistance (RL); (B) mouse exhalation resistance (Re); (C) dynamic lung compliance (Cdyn) in mice; (D) peak Expiratory Flow (PEF) in mice; (E) ventilation per Minute (MVV), after 12 h LPS treatment, lung function indicators were measured in mice using the AniRes2005 animal lung function analysis system and the results showed a significant increase in lung inspiratory Resistance (RL), expiratory resistance (Re), dynamic lung compliance (Cdyn), Peak Expiratory Flow (PEF) and ventilation per Minute (MVV) in LPS group compared to control group (P < 0.05,. P < 0.01,. P < 0.001). Compared with the LPS group, RL and Re were significantly decreased in the RA group, and Cdyn, PEF and MVV were significantly increased (P < 0.05, P < 0.01, P < 0.001).

3.5 Effect of RA on xylene-induced ear swelling in mice

As shown in fig. 6, ear swelling was significantly increased in the model group mice compared to the control group, while ear swelling was significantly reduced in the RA group mice compared to the xylene group (. P < 0.05,. P < 0.01,. P < 0.001).

3.6 Effect of RA on LPS-induced death of mouse septicemia

As shown in fig. 7, the LPS group mice all died within 84h after molding, while the RA group mice showed a significant increase in survival rate (about 30%).

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims (4)

1. Application of iron wintergreen acid as a unique active ingredient in preparing a medicament for treating septicemia caused by pneumonia.

2. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

3. The use of claim 1, wherein the medicament is one of a tablet, a capsule, and a nasal spray.

4. The use according to claim 1, wherein the minimum effective amount of iron wintergreen in the medicament is 1 mg/kg.

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