CN115227772B

CN115227772B - Traditional Chinese medicine formula for improving fine particulate matter-induced lung injury

Info

Publication number: CN115227772B
Application number: CN202210886459.0A
Authority: CN
Inventors: 张媛媛; 潘子倩; 寇俊萍; 张力元; 阎琨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2023-07-11
Anticipated expiration: 2042-07-26
Also published as: CN115227772A

Abstract

The invention discloses a traditional Chinese medicine formula for improving lung injury induced by fine particles, belonging to the field of natural medicines. The traditional Chinese medicine formula consists of honey mulberry leaf, dwarf lilyturf tuber, common anemarrhena rhizome, glehnia root and ginseng. According to the invention, a fine particle-induced mouse subacute lung injury model is adopted, and indexes such as lung wet-dry ratio, pathological morphology observation, inflammatory factor expression, oxidative stress level and the like prove that the formula has the effect of obviously improving the fine particle-induced subacute lung injury, and the formula is indicated to be used for preventing and treating the fine particle-induced subacute lung injury.

Description

Traditional Chinese medicine formula for improving fine particulate matter-induced lung injury

Technical Field

The invention belongs to the field of natural medicines, and particularly relates to a traditional Chinese medicine formula for improving lung injury induced by fine particles.

Background

Air pollution is listed as the fifth greatest risk of death worldwide. In 2019, the worldwide number of deaths from air pollution reached 667 tens of thousands of people ((1) Munzel T, gori T, al-Kindi S, et Al effects of gaseous and solid constituents of air pollution on endothelial function. European heart journ [ J ].2018;39 (38): 3543-3550. (2) GBD 2019 Risk Factors Collaborators.Global burden of 87 risk factors in 204 countries and territories,1990-2019:a systematic analysis for the Global Burden of Disease Study 2019[J ]. Lancet.2020;396 (10258): 1223-1249.). Fine particulate matter, also referred to as PM2.5, refers to particulate matter having an aerodynamic diameter of 2.5 μm or less in ambient air. The PM2.5 after adsorbing harmful substances has strong adsorptivity due to the characteristics of small particle size and relatively large surface area, and the main chemical components of the PM2.5 are elemental carbon, organic carbon compounds, sulfates, nitrates, ammonium salts and the like ((3) Xu Jing, ding Guoan, yan Peng, etc.,. Component characteristics and source analysis [ J ]. Applied to Beijing PM_ (2.5), the weather report, 2007, (05): 645-654. (4) Zhang Y, lang J, cheng S, et al, chemical composition and sources of PM1 and PM2.5 in Beijing in autumn [ J ]. The Science of the total environmental.2018; 630:72-82. (5) Niu Y, chen R, xia Y, et al fine particulate matter constituents and stress hormones in the hypothalamus-piping-additional axes [ J ]. Environmental inter-5.2018; 119:186-192.). When the fine particles reach the lungs, most of them will be deposited in the alveoli ((6) Kulkarni N, piese N, rushton L, et al carbon in airway macrophages and lung function in children J The New England and Journal of Medicine,2006,355 (1): 21-30.), and the fine particles deposited in the alveoli can partially penetrate the blood gas barrier and reach the whole body with the action of blood circulation, so that besides the respiratory system, the fine particles have obvious relevance to the lesions of organs outside the lungs such as the heart, liver, spleen and kidney, and the like, and in recent years, more and more researches have found that air pollution has relevance to the occurrence of various diseases such as cardiovascular diseases, diabetes, tumors and strokes ((7) Li D, li Y, li G, et al fluorescint reconstitution on deposition of PM 2.5.5 in lung and extrapulmonary organs J, PNAS,2019,116 (7): 2488-2493 (8) schraufmagel DE, balms JR, cowl, et al CT Pollution and Noncommunicable Diseases: A Review by the Forum of International Respiratory Societies', environmental Committee, t 2:Air Pollution and Organ Systems[J, and the like.

Aiming at respiratory system injury caused by environmental pollution, the prior effective personal protective measures mainly adopt filter devices such as an air purifier, a mask and the like, no targeted marketed medicines or health products ((9) Rajagopalan S, brauer M, bhatnagar A, et al personal-level protective actions against particulate matter air pollution exposure: a scientific statement from the American Heart Association [ J ]. Circulation,2020,142 (23): e411-e 431.) are available, and the clinically commonly used therapeutic medicines are glucocorticoid medicines such as dexamethasone, but the long-term use of the glucocorticoid medicines can bring more adverse reactions ((10) Zhaotao, huang Cibo. Rational use of the glucocorticoid [ J ]. Journal of clinical medicine treatment, 2010,8 (01): 23-28.). The Chinese herbal compound is prepared from two or more medicinal materials, and is a prescription designed for relatively determined symptoms, and has the characteristics of multiple components and multiple targets, so that the Chinese herbal compound has unique advantages in the aspect of preventing and treating lung injury caused by air pollution. In addition, because the toxic and side effects are small, the advantages of improving the compliance of patients and the like can be realized, so that the medicine has a considerable market, and the medicine becomes a research hot spot gradually.

Disclosure of Invention

The invention aims at providing a traditional Chinese medicine formula and a preparation method thereof.

The traditional Chinese medicine formula comprises honey mulberry leaf, dwarf lilyturf tuber, common anemarrhena rhizome, glehnia root and ginseng.

Further, the traditional Chinese medicine comprises the following components in parts by mass: 12-14 parts of honey mulberry leaf, 1-4 parts of dwarf lilyturf tuber, 7-11 parts of common anemarrhena rhizome, 7-11 parts of glehnia root and 2-6 parts of ginseng.

The preparation method of the traditional Chinese medicine composition comprises the steps of mixing traditional Chinese medicine decoction pieces according to a ratio of 1: adding 8 volumes of pure water, decocting twice each for one hour, mixing filtrates, and concentrating under reduced pressure. The concentrated solution is added with pharmaceutically acceptable carriers and/or excipients, and can be prepared into any clinically acceptable dosage forms such as tablets, pills, capsules, granules, medicinal granules, oral liquid and the like by adopting the traditional preparation process.

The second purpose of the invention is to provide the application of the traditional Chinese medicine composition in preparing the medicine for treating the subacute lung injury induced by the fine particles.

The subacute lung injury is caused by various internal and external pathogenic factors such as harmful gas inhalation, serious infection, wound, poisoning, shock and the like, and is caused by progressive and anoxic respiratory insufficiency or decay, and acute respiratory distress syndrome is caused by serious patients.

In the embodiment 1-3, a fine particle induced mouse subacute lung injury model is adopted, and indexes such as lung wet-dry ratio, pathological morphology observation, inflammatory factor expression, oxidative stress level and the like prove that the compound MAG has the effect of obviously improving the fine particle induced subacute lung injury, and the compound MAG is suggested to be used for preventing and treating the fine particle induced subacute lung injury.

The invention also aims to provide an application of the traditional Chinese medicine composition in preparing medicines for preventing and treating pulmonary diseases, wherein the pulmonary diseases comprise emphysema, chronic obstructive pulmonary disease, pulmonary arterial hypertension and pulmonary fibrosis.

Emphysema refers to a pathological condition of hypoairway elasticity at the distal end of the terminal bronchiole, excessive inflation, and an increase in lung volume or concomitant destruction of the airway wall; chronic obstructive pulmonary disease refers to chronic bronchitis and/or emphysema characterized by airflow obstruction associated with abnormal inflammatory responses of noxious gases and noxious particles; pulmonary hypertension refers to a hemodynamic and pathophysiological state in which pulmonary arterial pressure rises above a threshold; pulmonary fibrosis is an end-stage change in pulmonary disease characterized by proliferation of fibroblasts and massive extracellular matrix aggregation with concomitant inflammatory injury and destruction of tissue architecture.

According to the embodiment 4 of the invention, through the weight and the diet water intake of the mice, the measurement of the index of each viscera, the observation of the tissue morphology of each viscera and the measurement of the glutamic oxaloacetic transaminase and creatinine content, the high-dose compound MAG (20 g/kg) has no obvious side effect on male and female mice, has no hepatorenal toxicity, and has a wide application prospect.

The traditional Chinese medicine has reasonable formula components, interaction, lung injury improving and anti-inflammatory effects, does not generate any toxic or side effect, is easy to popularize, simple to prepare and convenient to operate, and can be used for industrial production.

Drawings

FIG. 1 shows the experimental results of improving lung tissue morphology and edema in mice with subacute lung injury induced by fine particles with Compound MAG of example 1

n=6). Wherein: ^### P<blank group 0.001 vs; ^** P<0.01, ^*** P<model group 0.001vs.

FIG. 2 shows experimental results of the compound MAG of example 1 for improving lung tissue pathological structural damage of fine particulate matter-induced sub-acute lung injury mice

FIG. 3 is a graph showing the results of the compound MAG of example 2 in improving inflammatory factor expression in serum of mice with subacute lung injury induced by fine particlesResult of [ (]

n=6). Wherein: ^## P<0.01, ^### P<blank group 0.001 vs; ^* P<0.05， ^** P<0.01 ^*** P<model group 0.001vs.

FIG. 4 shows experimental results of improving the content of malondialdehyde in lung tissue of mice with subacute lung injury induced by fine particles with Compound MAG of example 3

n=6). Wherein: ^### P<blank group 0.001 vs; ^* P<0.05， ^** P<model group 0.01vs.

FIG. 5 shows the effect of high-dose MAG on body weight and food intake of male and female mice in example 4

n＝10，ns:no significance)。

FIG. 6 shows the effect of the high dose of MAG on the morphology of organs and tissues of male and female mice in example 4.

FIG. 7 shows the effect of high-dose MAG on organ index of male and female mice in example 4

n＝10,ns:no significance)。

FIG. 8 shows the effect of high-dose MAG on glutamic-oxaloacetic transaminase and creatinine in male and female serum in example 4

n＝10，ns:no significance)。

Detailed Description

The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.

The novel traditional Chinese medicine composition MAG of the embodiment of the invention comprises 10 parts of honey mulberry leaves, 1.3 parts of dwarf lilyturf tuber, 9.3 parts of rhizoma anemarrhenae, 9.3 parts of radix glehniae and 4 parts of ginseng, wherein decoction pieces are purchased from Nanjing Shangyuan Tang medicine Co., ltd according to the weight ratio of 1:8 by volume, 2 times each for 1 hour, combining the two filtrates, and concentrating under reduced pressure.

Example 1

Improvement effect of compound MAG on fine particle-induced subacute lung injury mice lung pathological injury

1. Experimental method

a) Determination of the wet/dry weight ratio of lung tissue

Male ICR mice were randomized into 6 groups after 3 days of adaptive rearing: blank, model, model+low dose compound MAG (1.25 g/kg), model+medium dose compound MAG (5 g/kg), model+high dose compound MAG (20 g/kg), model+dexamethasone (Dex, 1 mg/kg). Model mice were molded by inhaling 40 μl (10 mg/kg) of PM suspension from the oropharynx on

days

1,3,5,7,9, 11, 13 and the same volume of PBS from the oropharynx of the blank group. The specific operation method of oropharyngeal inhalation comprises the following steps: the mice are placed in an isoflurane anesthesia box, and when the mice lose the positive reflection and cough vigorously, the mice are taken out, and the nose of the mice is pinched by the left hand, so that the bodies of the mice naturally droop. The right hand is used for holding the pipette gun, the gun head is used for pressing down and fixing the tongue, PM suspension is injected from the tongue root of the mouse immediately, the tongue is kept fixed, and after the mouse breathes violently for 5 times, the mouse is put back into the cage in the supine position, so that the mouse wakes up naturally. Mice in each dosing group were given the corresponding drug by gavage daily on days 1-14, and mice in the blank group were gavaged with the same volume of physiological saline. The dosing time for the odd days was 1h before PM inhalation. Mice were sacrificed on day 15, the left lung was removed, the left lung weight was accurately determined, recorded as the lung tissue wet weight, and the lung tissue was placed in an oven at 60 ℃ and dried for 48 hours until the weight was constant, recorded as the lung tissue dry weight. The lung tissue wet weight/lung tissue dry weight ratio was calculated and reported as lung wet weight/dry weight.

b) Pathological observation of lung tissue

Taking the upper leaf of the right lung of the mouse,the sections were fixed in 10% formalin, embedded in normal paraffin, approximately 4 μm thick, HE stained, read and scored by pathology professionals. Scoring criteria: the lesions were scored according to their severity score of 0.5 score (mild), 1 score (mild), 2 score (moderate), 3 score (severe), 4 score (severe), and no apparent lesions were 0 score. Adding all the scores to calculate the average score of each animal in each group

2. Experimental results

a) Compound MAG (MAG) improves lung tissue morphology and edema of mice with subacute lung injury induced by fine particles

As shown in fig. 1 a, the lung of the blank group had a smooth color, smooth surface and good elasticity, and no obvious abnormal damage in appearance, while the lung tissue of the model group had a large amount of black fine particles, and the color was dark, and diffused outward from bronchi. After the compound MAG (1.25,5, 20 g/kg) and dexamethasone (1 mg/kg) are dosed at different doses, the lung tissue injury is relieved to different degrees, the compound MAG has dose dependency, and the high-dose group has the most remarkable improvement effect. As shown in fig. 1B, the medium-high dose of the compound MAG has a significant improvement effect on pulmonary edema of mice with subacute lung injury induced by fine particles.

b) Compound MAG (MAG) for improving pathological structural damage of lung tissue of mice with acute lung injury induced by fine particles

As shown in fig. 2 a, the blank group had a clear structure, no congestion of the alveolar walls, no infiltration of inflammatory cells, no increase in goblet cells in epithelial cells, and no inflammatory exudates in the lumen. The lung tissue structure of the model group disappears and becomes solid, and a large amount of exudates mainly containing neutrophils are filled in the alveolus cavity. The lung tissue bronchi and perivascular may be seen as mild or mildly fibrous tissue hyperplasia with fine particulate matter deposited in the lung interstitium. After different doses of compound MAG (1.25,5, 20 g/kg) and dexamethasone (1 mg/kg) were administered, the lesions were significantly reduced. As shown in fig. 2B, the lung tissue pathology scoring result shows that the compound MAG (1.25,5, 20 g/kg) with different doses has a significant inhibitory effect on pathological lesions under the model, and the high-dose improvement effect is optimal.

Example 2

Improvement effect of compound MAG on fine particle-induced subacute lung injury mouse lung inflammation

1. Experimental method

Detection of inflammatory factors in serum

The mouse orbit of example 1 was bled in a 1.5mL EP tube, allowed to stand at room temperature for 30min, centrifuged at 3500rpm for 10min, and the supernatant was taken as serum and stored at-70 ℃. And detecting the expression of the serum inflammatory factors according to ELISA kits.

2. Experimental results

Compound MAG (MAG) for reducing inflammatory factor expression in serum of mice with subacute lung injury induced by fine particles

As shown in FIG. 3, compared with the blank group, the level of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the alveolar lavage fluid of the model group is increased, the compound MAG (1.25,5, 20 g/kg) and dexamethasone (1 mg/kg) with different doses have inhibition effect on the expression of the inflammatory factors, and the high dose group (20 g/kg) has the most remarkable effect.

Example 3

Improvement effect of compound MAG on fine particle-induced subacute lung injury mice oxidative stress level

1. Experimental method

Detection of malondialdehyde content in mouse lung tissue

The lung tissue of the mice of example 1 was accurately weighed in weight (g): volume (mL) =1:9 ratio, adding 9 times volume of physiological saline, mechanically homogenizing, taking the homogenate to be measured, and measuring the content of malondialdehyde in lung tissues according to malondialdehyde kit instruction.

2. Experimental results

Compound MAG reduces malondialdehyde content in lung tissue of mice with subacute lung injury induced by fine particles

As shown in FIG. 4, the malondialdehyde content in lung tissue was significantly increased in the model group compared to the blank group, and the malondialdehyde content in lung tissue was decreased by administration of high-dose compound MAG (20 g/kg) and dexamethasone (1 mg/kg).

Example 4

Compound MAG safety evaluation

1. Experimental method

a) Body weight and drinking water and food intake change of mice

SPF-class ICR mice were divided into four groups at random, a male blank group, a male administration group, a female blank group, and a female administration group, each group being 10, each half of the male and female animals after 3 days of adaptive breeding. The administration group is continuously administered with high-dose compound MAG (20 g/kg) for 14 days, the blank group is administered with physiological saline with corresponding volume, mice are normally fed after the stomach irrigation is finished, death and growth activity of the mice within 14 days are observed and recorded, and the weight, ingestion and drinking conditions of the mice are recorded.

b) Organ index determination

Each group of mice was sacrificed and dissected after blood was collected from the eyeballs, their hearts, livers, spleens, lungs, kidneys, brains were taken, weighed and recorded using an electronic balance, and the relevant organ indexes were calculated.

c) Organ tissue morphology changes

After blood is taken from eyeballs, the mice in each group are sacrificed and dissected, and the heart, liver, spleen, lung, kidney and brain of the mice are taken for photographing.

d) Liver and kidney injury assay

The serum obtained by separation is used for detecting indexes such as glutamic oxaloacetic transaminase, creatinine and the like by using a kit, and specific operation steps are carried out by referring to the specification of the related kit.

2. Experimental results

a) The high-dose compound MAG has no obvious influence on the weight of mice and drinking water and food intake

Weight gain is one of the important indicators for evaluating the growth of mice. As shown in fig. 5 a, mice in the compound MAG group had the same trend of weight gain within 14 days as the blank group, and both male and female mice grew normally. Statistical results showed that there was no significant difference in weight gain between the dosing group and the blank group mice. As shown in fig. 5, B, C, the drinking water and food consumption of mice in the administration group are not significantly changed in comparison with the administration group within 14 days, and the result shows that the high-dose compound MAG has no obvious influence on the weight increase and drinking water and food consumption of the mice.

b) The high-dose compound MAG has no obvious influence on the morphology of organs and tissues of mice

After the experiment, animals were sacrificed, and as shown in fig. 6, no obvious morphological differences were observed in the heart, liver, spleen, lung, kidney, brain and other major organs.

c) The high-dose compound MAG has no obvious influence on the organ indexes of various organs of mice

The main viscera index of the high-dose compound MAG administration group was not significantly changed from that of the blank group as shown in FIG. 7, which was obtained by weighing the main viscera such as heart, liver, spleen, lung, kidney, brain, etc. Experimental results show that the high-dose compound MAG has no obvious influence on the index of the main organs of the mice, and the compound MAG can be primarily considered to have no obvious targeting toxicity on the main organs of the mice.

d) The high-dose compound MAG has no obvious influence on liver and kidney injury of mice

As shown in figure 8, the results show that the serum glutamic-oxaloacetic transaminase and creatinine content in the serum of the administration group and the serum of the blank group of the male and female mice are not obviously changed, and the high-dose compound MAG has no obvious hepatorenal toxicity to the male and female mice.

Claims

1. Application of a Chinese medicinal composition in preparing medicine for preventing and treating subacute lung injury induced by fine particles,

the traditional Chinese medicine composition is prepared from 10 parts of mulberry leaves, 1.3 parts of dwarf lilyturf tuber, 9.3 parts of rhizoma anemarrhenae, 9.3 parts of radix glehniae and 4 parts of ginseng by mass, and is prepared by mixing the mulberry leaves, the dwarf lilyturf tuber, the rhizoma anemarrhenae, the radix glehniae and the ginseng according to a volume ratio of 1:8 adding pure water, decocting twice, each for one hour, mixing filtrates, and concentrating under reduced pressure.

2. A subacute lung injury prevention and treatment drug, which is characterized in that: the active ingredients of the traditional Chinese medicine composition are traditional Chinese medicine composition, the traditional Chinese medicine composition is prepared from 10 parts of mulberry leaves, 1.3 parts of dwarf lilyturf tuber, 9.3 parts of common anemarrhena rhizome, 9.3 parts of glehnia root and 4 parts of ginseng by mass, and the preparation method comprises the steps of mixing the mulberry leaves, the dwarf lilyturf tuber, the common anemarrhena rhizome, the glehnia root and the ginseng according to the volume ratio of 1:8 adding pure water, decocting twice, each for one hour, mixing filtrates, and concentrating under reduced pressure to obtain the final product;

the subacute lung injury is induced by fine particulate matter.