CN116059293B - Application of traditional Chinese medicine composition in preparation of medicine for treating acute exacerbation stage of chronic obstructive pulmonary disease - Google Patents

Abstract

The invention relates to the technical field of traditional Chinese medicines, and particularly discloses application of a traditional Chinese medicine composition in preparing a medicine for treating acute exacerbation phase of chronic obstructive pulmonary disease, and researches prove that the medicine can inhibit application of Clara cells to goblet cell differentiation medicines, increase application of CCSP protein expression medicines, promote AQP5 protein or AQP5 mRNA expression and reduce MMP-9 or IL-13 factor expression; it has also been demonstrated that the medicament of the invention can protect the cilia structure of airway epithelium, in particular in increasing cilia length, increasing cilia number, up-regulating beta-Tubulin IV protein expression, improving cilia direction, protecting cilia from ultrastructural damage; protecting airway cilia function and increasing cilia pulsation frequency.

Description

Application of traditional Chinese medicine composition in preparation of medicine for treating acute exacerbation stage of chronic obstructive pulmonary disease

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to application of a traditional Chinese medicine composition in preparation of a medicine for treating acute exacerbation of chronic obstructive pulmonary disease.

Background

Acute exacerbation of chronic obstructive pulmonary disease (Acute exacerbations of chronic obstructive pulmonary disease, AECOPD) refers to acute exacerbation of respiratory symptoms in a patient's COPD progression, resulting in the need for additional treatment. AECOPD can be caused by a number of factors, commonly upper respiratory tract and tracheal, bronchial infections. Acute exacerbations can be caused by physical and chemical factors such as smoking, air pollution, inhaled allergens, air temperature changes, etc., and irregular or interrupted treatment in the stationary phase. The main symptoms of AECOPD are exacerbation of shortness of breath, frequent wheezing, chest distress, cough aggravation, increased sputum volume, changes in sputum color and/or viscosity, fever, etc., and symptoms of general discomfort, insomnia, somnolence, fatigue, depression, mental disturbance, etc. can also appear. Patient exercise endurance decline, fever and/or chest X-ray imaging abnormalities may be signs of COPD exacerbation. Increased sputum and appearance of purulent sputum often suggest bacterial infection.

Airway mucus hypersecretion is an important pathological feature of AECOPD, an independent risk factor for AECOPD exacerbation and death. Airway mucus is usually the first line of defense of the respiratory tract, and can remove harmful substances, keep the airway moist, and protect the surface of the respiratory tract of a human body from being damaged by inhalation of the harmful substances. AECOPD interacts with airway mucus hypersecretion, which alters the airway microenvironment, providing favorable conditions for infection by microorganisms such as viruses, bacteria, etc. in the airways, making AECOPD infection difficult to control. While pathogenic microbial infection and its subsequent inflammatory products further cause mucus hypersecretion, forming a vicious circle, leading to AECOPD airway obstruction, airflow limitation, and reduced lung function. Airway hypersecreting patients the risk of AECOPD death is 3.5 times that of non-airway hypersecreting patients. Improving airway mucus hypersecretion can effectively improve the curative effect of AECOPD. Thus, inhibition of airway mucus hypersecretion is of clinical importance in the treatment of AECOPD.

The mucociliary clearance defense mechanism of the respiratory tract consists of mucus and cilia. Cilia are widely present in the human respiratory tract and are a major component of the mucociliary clearance system. Cilia regularly continue to oscillate in the mucus-fiber blanket, pushing harmful particles and pathogens out of the airways. Physiological change characteristics of AECOPD include mucus hypersecretion and cilia dysfunction. Inflammation caused by an acute episode can alter Ciliated Beat Frequency (CBF) by inflammatory and/or epithelial cells releasing mediators or causing cilia injury, epithelial cell loss and basement membrane injury. CBF and wave patterns are important biological indicators of epithelial cell function and may be altered during an acute episode, resulting in secretion accumulation and impaired beating. Structure-function studies help to determine the ciliary target sites involved in regulating cilia beat, and cilia characteristics that may lead to pulmonary disease include cilia function (cilia beat frequency-CBF), cilia length, cilia density, and cilia structure, which can alter CBF and motor quality. Random cilia orientation has also been reported as a possible cause of respiratory disease. Clinically, an expectorant is commonly used to treat airway mucus hypersecretion in combination with glucocorticoids and antibiotics. Although expectorants and glucocorticoids temporarily alleviate the symptoms of airway mucus hypersecretion, they do not fundamentally reduce the production of sputum. At the same time, bacterial resistance and double infection by antibiotics prevent disease prognosis. Due to excessive injury to airway mucus secretion, it is necessary to administer targeted therapies. However, the pathophysiological mechanisms of mucus-scavenging obstruction may be different for each disease, and therefore there may be no single treatment available in the area of respiratory obstruction, and there is a need to find drugs that are effective in improving airway mucus hypersecretion.

Disclosure of Invention

Aiming at the technical problems existing in the acute exacerbation stage of the existing chronic obstructive pulmonary disease, the invention provides application of a traditional Chinese medicine composition in preparing a medicine for treating the acute exacerbation stage of the chronic obstructive pulmonary disease.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

the application of a traditional Chinese medicine composition in preparing a medicine for treating acute exacerbation of chronic obstructive pulmonary disease is provided, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 52-86 of ephedra; gypsum 194-324; fructus forsythiae 194-324; 78-130 parts of radix scutellariae; white mulberry root bark 194-324; 78-130 parts of fried bitter apricot kernel; 78-130 parts of radix peucedani; 78-130 parts of pinellia ternate; 78-130 parts of dried orange peel; 78-130 parts of fritillary bulb; 78-130 parts of burdock; 78-130 parts of lonicera japonica; 39-65 parts of rheum officinale; radix Platycodi 46-76; 39-65 parts of liquorice.

Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: herba Ephedrae 52; gypsum 324; fructus forsythiae 194; radix Scutellariae 78; cortex Mori 194; parching semen Armeniacae amarum 130; radix Peucedani 78; pinellia ternate 130; dried orange peel 78; bulbus Fritillariae Thunbergii 78; fructus Arctii 130; flos Lonicerae 130; rhubarb 39; radix Platycodi 76; licorice 65.

Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: herba Ephedrae 86; gypsum 194; fructus forsythiae 324; radix Scutellariae 130; cortex Mori 324; parching semen Armeniacae amarum 78; radix Peucedani 130; pinellia tuber 78; dried orange peel 130; bulbus Fritillariae Thunbergii 130; burdock 78; flos Lonicerae 78; rhubarb 65; radix Platycodi 46; licorice 39.

Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: ephedra 69; gypsum 259; fructus forsythiae 259; radix Scutellariae 104; cortex Mori 259; parching semen Armeniacae amarum 104; radix Peucedani 104; pinellia ternate 104; dried orange peel 104; bulbus Fritillariae Thunbergii 104; fructus Arctii 104; flos Lonicerae 104; rhubarb 52; radix Platycodi 61; licorice 52.

Further, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: herba Ephedrae 55; gypsum 254; fructus forsythiae 318; radix Scutellariae 107; white mulberry root bark 203; a fried bitter almond 107; radix Peucedani 82; pinellia ternate 105; dried orange peel 84; bulbus Fritillariae Thunbergii 125; burdock 122; flos Lonicerae 113; rhubarb 42; radix Platycodi 60; licorice root 50.

The traditional Chinese medicine composition mainly comprises ephedra, gypsum, fructus forsythiae, radix scutellariae, cortex mori radicis and the like, plays the integral regulation advantages of the compound traditional Chinese medicine, and has obvious curative effects on acute exacerbation stage of chronic obstructive pulmonary disease through animal verification by organically combining the functions of eliminating pathogenic factors, relieving symptoms and regulating immunity.

The traditional Chinese medicine can be replaced by traditional Chinese medicines with the same or similar effects, and the traditional Chinese medicines can be processed according to national traditional Chinese medicine processing standards or Chinese medicine dictionary.

The active ingredients of the traditional Chinese medicine composition are prepared by the following steps:

A. Weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 40-70% ethanol for 2 times, each for 1-4 hr, adding 8-10 times of the extractive solution for the first time and 6-9 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.14-1.16 at 60deg.C;

C. weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for two times, 1-4 hours each time, adding 9-11 times of the weight of the first time and 7-9 times of the weight of the second time, merging decoction, filtering, decompressing filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.14-1.16 measured by heat at 60 ℃, merging with the clear paste obtained in the step B for standby;

the fine powder obtained in the step A and the combined fluid extract obtained in the step C jointly form the active ingredient of the pharmaceutical composition. The medicament of the invention is in the dosage forms of capsules, tablets, powder, granules, oral liquid, soft capsules, pills, tinctures, syrups, suppositories, gels, sprays or injections.

To enable the above dosage forms, pharmaceutically acceptable excipients are added in the preparation of these dosage forms, for example: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, matrices, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, and the like; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, crosslinked sodium carboxymethyl cellulose, and the like; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the binder includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; the sweetener comprises: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; the flavoring agent comprises: sweetener and various flavors; the preservative comprises: nipagin, benzoic acid, sodium benzoate, sorbic acid and salts thereof, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil and the like; the matrix comprises: PEG6000, PEG4000, insect wax, and the like.

Wherein the tablet is prepared by the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 40-70% ethanol for 2 times, each for 1-4 hr, adding 8-10 times of the extractive solution for the first time and 6-9 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.14-1.16 at 60deg.C;

C. weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for two times, 1-4 hours each time, adding 9-11 times of the weight of the first time and 7-9 times of the weight of the second time, merging decoction, filtering, decompressing filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.14-1.16 measured by heat at 60 ℃, merging with the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

200-355 of the spray powder obtained in the step D; the fine powder obtained in the step A is 54-145; 10-15.5 parts of sodium carboxymethyl starch; microcrystalline cellulose 7-12; 1.5 to 3.5 percent of magnesium stearate, adding starch, and preparing tablets according to a conventional preparation method.

The preparation method of the preferred tablet comprises the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 50% ethanol for 2 times and 3 hr each time, adding 10 times of the extractive solution for the first time and 6 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.15 at 60deg.C;

C. weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for 2 hours each time, adding 10 times of the weight for the first time and 7 times of the weight for the second time, merging the decoctions, filtering, decompressing the filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.15 measured by heat at 60 ℃, merging the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

step D, spraying powder 282.6; the fine powder 101 obtained in the step A; 12.5 parts of sodium carboxymethyl starch; microcrystalline cellulose 9.0; 2.25 parts of magnesium stearate, adding starch, and making into tablet by conventional preparation method.

The preparation method of other dosage forms of the medicine comprises the following steps: the raw materials are weighed according to the proportion and prepared by adopting a conventional preparation method, for example, a preparation process recorded in Fan Biting traditional Chinese medicine pharmacy (1 st edition of 1997 of Shanghai science publishing Co., ltd.) is adopted to prepare a pharmaceutically acceptable conventional dosage form.

The traditional Chinese medicine composition is used for treating acute exacerbation of chronic obstructive pulmonary disease, and is preferably used for inhibiting Clara cells from differentiating into goblet cells, increasing CCSP protein expression, promoting AQP5 protein or AQP5 mRNA expression, reducing MMP-9 or IL-13 factor expression and treating lung injury.

The traditional Chinese medicine composition is applied to treating acute exacerbation of chronic obstructive pulmonary disease, preferably to drugs for protecting cilia structures of airway epithelium, and particularly to drugs for increasing cilia length, increasing cilia quantity, up-regulating beta-Tubulin IV protein expression, improving cilia direction and protecting cilia ultrastructural damage.

The traditional Chinese medicine composition is applied to the treatment of acute exacerbation stage of chronic obstructive pulmonary disease, preferably to the drug for protecting the cilia function of the airway, in particular to the drug for increasing the cilia beating frequency.

The traditional Chinese medicine composition is applied to treating acute exacerbation of chronic obstructive pulmonary disease, preferably to improving airway mucus hypersecretion of patients.

Drawings

Fig. 1: lung tissue morphology (HE staining, ×200) of rats of each group;

fig. 2: comparison of average alveolar intercept (MLI) and average alveolar number (MAN) of groups of ratsN=5), a. Normal group; B. a model group; lhqk low dose group; dose group in lhqk; lhqk high dose group; F. ambroxol group. P < 0.01 compared to normal group; compared with the model group, the # P is less than 0.05, and the # P is less than 0.01; comparison with the Positive group of drugs DeltaDeltaP<0.01；

Fig. 3: positive expression of rat airway goblet cells (AB-PAS staining, ×200) in each group;

fig. 4: comparison of positive relative coloring area of AB-PAS of airway of rats in each groupN=5), a. Normal group; B. a model group; lhqk low dose group; dose group in lhqk; lhqk high dose group; F. ambroxol group. P < 0.01 compared to normal group; compared with the model group, the # P is less than 0.01; delta.P compared to ambroxol group<0.01；

Fig. 5: positive expression of CCSP in the airways of rats of each group (immunofluorescent staining, ×200);

fig. 6: lung tissue MUC5AC positive expression (immunohistochemical staining, ×200) in each group of rats;

Fig. 7: the lung tissue of each group of rats expressed AQP5 positively (immunohistochemical staining, ×200);

fig. 8: positive expression comparison of MUC5AC and AQP5 in lung tissue of ratsN=5), a. Normal group; B. a model group; lhqk low dose group; dose group in lhqk; lhqk high dose group; F. ambroxol group. P < 0.05 compared to the normal group; comparing to the model group, #P < 0.05; compared with ambroxol group, deltaP<0.05；

Fig. 9: comparison of MUC5AC and AQP5 mRNA expression in Lung tissue of rats of each groupN=4), a. Normal group; B. a model group; lhqk low dose group; dose group in lhqk; lhqk high dose group; F. ambroxol group. P < 0.05 compared to the normal group; comparing to the model group, #P < 0.05;

fig. 10: TNF-alpha, IL-1 beta and MMP-9 content in BALF of each group of rats is comparedpg/mL, n=8), a. Normal group; B. a model group; lhqk low dose group; dose group in lhqk; lhqk high dose group; F. ambroxol group. P < 0.01 compared to normal group; compared with the model group, the # P is less than 0.01; delta.P compared to ambroxol group<0.01；

Fig. 11: IL-13 content comparison in serum of rats of each grouppg/mL, n=8), a. Normal group; B. a model group; lhqk low dose group; dose group in lhqk; lhqk high dose group; F. ambroxol group. P < 0.01 compared to normal group; compared with the model group, the # P is less than 0.01; compared with ambroxol group, deltaP <0.05，△△ P<0.01；

Fig. 12: a is LHQK, which improves the degree of lung parenchymal injury in AECOPD rats, HE stains lung tissue (x 40). Red arrows show alveolar fusion and inflammatory cell infiltration. B Mean Lung Intercept (MLI), data shown as mean ± standard deviation (n=5), P <0.01 compared to control group, #p <0.01 compared to cs+lps group;

fig. 13: a airway H & E staining to assess airway epithelial cilia morphology change map, (x 40); b scanning electron microscope (X200, X1500, X6000);

fig. 14: a is SEM longitudinal section (x 12000); b cilia length (μm), data expressed as mean ± standard deviation, # P <0.01 compared to control group, # P <0.01 compared to cs+lps group;

fig. 15: detecting the expression of cilia related protein beta-Tubulin IV by adopting an immunofluorescence method; B-C Western Blot detects the expression of cilia-associated protein beta-tubulin IV (cilia-specific marker) in the trachea. Data are expressed as mean ± standard deviation, # P <0.01 compared to control group, # P <0.05 compared to cs+lps group;

fig. 16: a, analyzing the tracheal ring CBF by Image J software, wherein an arrow indicates one cilia swing; b tracheal rings CBF (n=3). P <0.01 compared to control group, #p <0.01 compared to cs+lps group; c in vitro ciliated cells CBF (n=3)..p <0.01 compared to control group, #p <0.01 compared to cs+lps group;

Fig. 17: a is TEM (x 2500). DM is peripheral microtubule, CM is central microtubule, green arrow indicates ciliated membrane rupture and disintegration; the black arrows B indicate microtubule displacement, the red arrows indicate ciliated membrane vesicles, and the blue arrows indicate large cilia; c cilia ultrastructural anomaly rate;

fig. 18: the TEM (x 15000) line is the line through the central microtubule, called the cilia axis, a line perpendicular to the cilia axis also pointing in the direction of cilia beat.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

Prescription:

52 g of ephedra; 324 grams of gypsum; 194 g of weeping forsythia; 78 g of baikal skullcap root; 194 g of white mulberry root-bark; 130 g of fried bitter almond; radix peucedani 78 g; 130 g of pinellia ternate; 78 g of dried orange peel; 78 g of fritillary bulb; 130 g of burdock; 130 g of lonicera japonica; 39 g of rheum officinale; 76 g of platycodon grandiflorum; 65 g of liquorice.

The preparation method comprises the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 50% ethanol for 2 times and 3 hr each time, adding 10 times of the extractive solution for the first time and 6 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.15 at 60deg.C;

C. Weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for 2 hours each time, adding 10 times of the weight for the first time and 7 times of the weight for the second time, merging the decoctions, filtering, decompressing the filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.15 measured by heat at 60 ℃, merging the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

step D, spraying powder 282.6; the fine powder 101 obtained in the step A; 12.5 parts of sodium carboxymethyl starch; microcrystalline cellulose 9.0; 2.25 parts of magnesium stearate, adding starch, and making into tablet by conventional preparation method.

Example 2

Prescription:

herba Ephedrae 86; gypsum 194; fructus forsythiae 324; radix Scutellariae 130; cortex Mori 324; parching semen Armeniacae amarum 78; radix Peucedani 130; pinellia tuber 78; dried orange peel 130; bulbus Fritillariae Thunbergii 130; burdock 78; flos Lonicerae 78; rhubarb 65; radix Platycodi 46; licorice 39.

The preparation method comprises the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 40% ethanol for 2 times and 4 hr each time, adding 8 times of the extractive solution for the first time and 9 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.14 at 60deg.C;

C. Weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for two times, wherein the amount of the water is 9 times for each time for 4 hours, the amount of the water is 7 times for the first time, the decoctions are combined, filtered, the filtrate is decompressed and ethanol is recovered, and the filtrate is concentrated to the clear paste with the relative density of 1.16 measured by heat at 60 ℃ and is combined with the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

step D, spraying powder 200; the fine powder 145 obtained in step a; sodium carboxymethyl starch 10; microcrystalline cellulose 12; 1.5, adding starch, and making into tablet by conventional method.

Example 3

Prescription:

ephedra 69; gypsum 259; fructus forsythiae 259; radix Scutellariae 104; cortex Mori 259; parching semen Armeniacae amarum 104; radix Peucedani 104; pinellia ternate 104; dried orange peel 104; bulbus Fritillariae Thunbergii 104; fructus Arctii 104; flos Lonicerae 104; rhubarb 52; radix Platycodi 61; licorice 52.

The preparation method comprises the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 70% ethanol for 2 times (1 hr each time), adding 10 times of the extractive solution for the first time, adding 6 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, and concentrating to obtain fluid extract with relative density of 1.16 at 60deg.C;

C. Weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for two times, 1 hour each time, adding 11 times of the weight for the first time, adding 7 times of the weight for the second time, merging decoction, filtering, decompressing filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.14 measured by heat at 60 ℃, merging with the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

step D, spraying powder 355; the fine powder 54 obtained in the step A; 15.5 parts of sodium carboxymethyl starch; microcrystalline cellulose 7; 3.5, adding starch, and making into tablet by conventional method.

Example 4:

the formula of the raw materials is as follows:

herba Ephedrae 55; gypsum 254; fructus forsythiae 318; radix Scutellariae 107; white mulberry root bark 203; a fried bitter almond 107; radix Peucedani 82; pinellia ternate 105; dried orange peel 84; bulbus Fritillariae Thunbergii 125; burdock 122; flos Lonicerae 113; rhubarb 42; radix Platycodi 60; licorice root 50.

The preparation method comprises the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 60% ethanol for 2 times and 2 hr each time, adding 9 times of the extractive solution for the first time and 7 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.15 at 60deg.C;

C. Weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the formula, adding water and decocting for 2.5 hours each time, adding 10 times of the weight of the mixture for the first time, adding 7 times of the weight of the mixture for the second time, merging the decoctions, filtering, decompressing the filtrate, recovering ethanol, concentrating the filtrate to obtain clear paste with the relative density of 1.14 measured by heat at 60 ℃, merging the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

the spray powder 300 obtained in the step D; the fine powder 132 obtained in step a; sodium carboxymethyl starch 14; microcrystalline cellulose 10.2; magnesium stearate 1.75, adding starch, and making into tablet by conventional method.

Experimental example 1:

to elucidate the effect of the drug of the present invention on improving the acute exacerbation phase of chronic obstructive pulmonary disease, the following experimental study was conducted using a tablet (hereinafter referred to as the drug of the present invention or LHQK) prepared in the same manner as in example 1.

1 Material

1.1 animals

150 SPF-class male Wistar rats, 5-6 weeks old, have a mass (200+ -20) g, and are purchased from Beijing Veitz laboratory animal technologies Co., ltd, animal quality certification No. 110011210114505818, certification No. SCXK [ Beijing ]2021-0011, approved by the national institutes of medicine, hebei, and No. N2021162.

1.2 drugs and Agents

LHQK (shijia, division of the kaolin pharmaceutical industry); ambroxol hydrochloride tablet (Shandong Yuxin pharmaceutical Co., ltd., batch number H20163194); lipopolysaccharide (LPS, sigma Co., USA); AB-PAS staining kit (Beijing Solaro Co., ltd., product number G1285); MUC5AC antibody (Abcam, UK, cat# ab 3649); AQP5 antibody (Abcam, uk, cat No. ab 78486); CCSP antibody (Abcam, uk, cat No. ab 213203); fluorescent secondary antibodies (Abcam, UK, cat# ab 150064); immunohistochemical staining kit (Beijing Zhonghua Jinqiao Co., ltd., product number PV-9001); rat tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), matrix metalloproteinase-9 (MMP-9) enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, USA, cat# KE20001, KE20005, KE20006, respectively); rat interleukin-13 (IL-13) enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Yillere Corp., cat# E-EL-R0563 c); reverse transcription kit, fluorescent quantitative kit (TaKaRa Co., japan); rat GAPDH, MUC5AC, AQP5 primer (Beijing Ding Guo Changsheng biotechnology Co., ltd.)

1.3 instruments

ME104E analytical balance (meltrefoil torrado, usa); TEC5 type Tissue embedding machine (sakura Tissue-Tek Co., japan); RM2235 rotary paraffin microtome, DM6000b optical microscope, LSM710 laser confocal fluorescence microscope (Leica company, germany); synergy 4-type multifunctional microplate reader (Bio Tek company, usa); HZ-2011KC type constant temperature shaking table (Tai cang Hua Li to experimental facilities Co., ltd.); 7900HT type real-time fluorescence quantitative PCR instrument (ABI company of America)

2. Method of

2.1 AECOPD rat model preparation

A method of smoke combined airway instillation Lipopolysaccharide (LPS) is adopted to prepare an AECOPD airway mucus high secretion rat model. LPS (1 mg/kg body weight) was instilled into the trachea of rats 24h before the experiment was carried out on days 1 and 14 and the materials were obtained, the same amount of physiological saline was instilled into the blank group (no smoking was carried out on the instillation day), rats were placed in a self-made smoking box (100 cm. Times.70 cm. Times.40 cm) for smoking on day 2, the smoke concentration was 180mg/m3, and smoking was carried out once every day and afternoon for 1h (15-20 min ventilation every 1 h), 40 cigarettes/h and 5 d/week. After smoking and administration for 12 weeks, the model was evaluated for pathology and the materials were anesthetized.

2.2 grouping and administration

150 rats were randomly assigned to a blank (Control), model (Model), low-dose (LHQK-L), medium-dose (LHQK-M), high-dose (LHQK-H) and Ambroxol (Ambroxol) group after one week of adaptive rearing. The low, medium and high doses of the medicine of the invention are respectively given with 1.47, 2.93 and 5.86 g crude drug/(kg.d) suspension for gastric lavage, wherein the medium dose is equivalent to 8 times of clinical dosage of people. Ambroxol group was given 30 mg/(kg.d) of ambroxol suspension and the model group was given an equivalent dose of 0.5% sodium carboxymethyl cellulose (CMC) solution for 12 weeks. The blank group was fed with water freely. The equivalent dose coefficient conversion formula was used to calculate the lavage dose [ [ i ] ] as D rat = D human x (HI rat/HI human) x (W rat/W human) 2/3, D as dose, HI as body size coefficient, W as body mass.

2.3 detection index

2.3.1 general observations of rats

The growth condition and activity of rats in each group are observed, and the skin and hair has smooth color, mental state, cough, asthma, etc.

2.3.2 HE staining for observing lung tissue pathology

Rat lung tissue was fixed with 4% paraformaldehyde, paraffin embedded, tissue sections were then HE stained and examined under a microscope for pathological changes such as alveolar size, structure and inflammatory cell infiltration. 3 alveolar visual field pictures are randomly cut from each lung tissue slice, a cross is drawn in the center of the picture, the alveolar number (Na) and the alveolar septum number (Ns) of each visual field are calculated, the length (L) and the area (S) are measured, and the calculation formula is as follows: average alveolar intercept (mean linear intercept, MLI) (μm) =l/Ns, average alveolar number (mean alveolar number, MAN) (/ mm) ² )=Na/S。

2.3.3 AB-PAS staining for observing rat airway goblet cell metaplasia and mucus secretion

Dewaxing rat lung tissue slices to water, adding aliskirin blue dye solution, dyeing for 15 min, washing with distilled water for 3 times each for 2 min, oxidizing with 1% periodate aqueous solution for 5 min, washing with distilled water for 3 times, and dyeing with Schiff solution for 15 min. Flushing with running water for 10 min; hematoxylin staining for 1 min, and washing; differentiating the acidic differentiation liquid for 2-5s, and washing with water; returning the Scott blue-dissolving liquid to blue for 3min, and washing with water for 3min; dehydrated in absolute ethyl alcohol, transparent xylene and sealing with neutral gum. After staining, the proliferation of the mucus of the lung tissue is observed under an optical lens, the acidic mucus material or cells are blue, the neutral mucus material or cells are red, and the mixed mucus material or cells are purple. The positive expression area ratio was calculated using Image-Pro Plus 6.0 analysis software.

2.3.4 Immunofluorescence observation of lung tissue Clara Cell Secretion Protein (CCSP) expression

Dewaxing rat lung tissue paraffin sections, repairing antigens, sealing 3% hydrogen peroxide at 37 ℃ for 10 min in a dark place, sealing 5% BSA at room temperature for 30 min, adding CCSP primary antibody (1:100), incubating overnight at 4 ℃, adding fluorescent secondary antibody (1:500), incubating for 50 min in a dark place, counterstaining with DAPI, sealing with anti-fluorescence quenching sealing tablets, and observing under a confocal fluorescence microscope.

2.3.5 Immunohistochemical observation of lung tissue mucin 5AC (MUC 5 AC) and aquaporin 5 (AQP 5) expression

Rat lung tissue paraffin section, antigen restoration after dewaxing, then dripping peroxidase blocking agent, dripping MUC5AC primary antibody (1:2000), AQP5 primary antibody (1:2000), incubating overnight at 4 ℃, adding secondary antibody, incubating at 37 ℃ for 30 min, DAB color development, hematoxylin counterstain, alcohol dehydration, xylene transparency, neutral resin sealing, image analysis by Image-Pro Plus 6.0 software, and calculating positive expression area ratio.

2.3.6 Real-time PCR detection of MUC5AC and AQP5 mRNA expression in lung tissue

Taking rat lung tissue about 0.05 g, adding 1 mL Trizol, fully grinding and homogenizing in a sterilization homogenizer, extracting total RNA by using a chloroform extraction method, measuring the concentration of RNA and the absorbance at 260 and 280 nm by using an ultraviolet spectrophotometer, and calculating the purity. cDNA synthesis was performed in a 20. Mu.L system according to the reagent instructions, and the first strand of cDNA was synthesized by reacting at 42℃for 2 min, followed by heating at 37℃for 15 min and 85℃for 5 s, and then cooling to terminate the reaction. RT-PCR amplification was performed using 1. Mu.L of primer, using the GAPDH gene as an internal reference, and the primer sequences are shown in Table 1. Reaction conditions: 95. 40 cycles of 30 s,95 ℃, 5 s, 60 ℃, 34 s. PCR reactions and data collection were recorded on quantsudio real-time quantitative PCR systems, and relative mRNA expression of the target gene in each sample was calculated using the relative quantitative formula 2- Δct, where Δct value = target gene Ct value-GAPDH gene Ct value.

2.3.7 ELISA detection of TNF-alpha, IL-1 beta, MMP-9 content in alveolar lavage fluid (BALF) and IL-13 content in serum

Centrifuging an alveolar lavage fluid, taking a supernatant, adding 100 mu L of a sample or a standard substance into a detection hole, sequentially adding a biotinylated antibody working fluid, an enzyme conjugate working fluid, a substrate solution and a stop solution according to the incubation time and conditions of an ELISA kit instruction, detecting an Optical Density (OD) value at the wavelength of a nm enzyme labeling instrument, and calculating the contents of TNF-alpha, IL-1 beta, MMP-9 and IL-13 of the sample according to a standard curve.

2.4 statistical treatment

Analysis was performed using SPSS22.0 statistical software. Experimental dataThe representation shows that the method obeys normal distribution and has uniform variance, the comparison among multiple groups uses single factor variance analysis, and the comparison between every two groups uses an LSD method; non-parametric test for comparison between groups without normal distribution, pairwise comparison with pairwise multiple comparison, P<A difference of 0.05 is statistically significant.

3 results

3.1 general conditions

Compared with the normal group, the model group rats have audible and airway phlegm sound and cough, dry hair and reduced activity; compared with the model group, the drug group and ambroxol group of the invention have better glossiness of the rat hair and increased activity.

3.2 Effect of the drug of the present invention on model rat pulmonary tissue pathological changes

The lung tissue structure under the normal group rat lens is normal, and no pathological change is seen; alveolar space is expanded under a model group lens, alveolar walls are broken, bronchiole basement membrane is thickened, and more inflammatory cells infiltrate around the bronchiole basement membrane; the low, medium and high dose groups and ambroxol groups of the medicine have the alveolus structures tending to be regular, and the pathological changes are improved, so that the medium and high dose groups of the medicine are obvious. Model group alveolar mean intercept (MLI) increased significantly (p < 0.01) and Mean Alveolar Number (MAN) decreased significantly (p < 0.01) compared to normal group; compared with the model group, the medicine has obviously reduced MLI (p <0.05, p < 0.01) of low, medium and high doses and ambroxol group, and obviously increased MAN (p < 0.01); compared with ambroxol group, the MLI of the low, medium and high dose groups of the medicine has no significant difference (p > 0.05), the MAN of the low dose group of the medicine has significantly reduced (p < 0.01), and the MAN of the medium and high dose groups has no significant difference (p > 0.05). See fig. 1 and 2.

3.3 Effect of the drug of the present invention on model rat airway goblet cell metaplasia and mucus secretion

The research result shows that the airway and bronchi of the normal group rats have few cup-shaped cells, and no obvious mucus secretion exists in the airway. Compared with the normal group, the model group has the advantages that the airway of the rat has a large number of cup cells with blue dye particles to generate, the cup cells are prompted to secrete a large number of acid mucins, and the mucous secretion of the airway is obviously increased (P < 0.01); the results of the medium and high dose groups of the medicine have no significant difference (p > 0.05). See fig. 3, 4.

3.4 Effect of the drug of the invention on model rat lung tissue CCSP

The rat airway epithelium is damaged by combination of fumigation and LPS airway instillation, so that Clara cells are differentiated into goblet cells, and the medicine can relieve airway mucus hypersecretion by inhibiting the differentiation of Clara cells into goblet cells. The results show that the CCSP expression of the epithelial cells on the surface of the lung airway has positive expression, compared with a normal group, the CCSP expression of a model group is obviously reduced, and compared with the model group, the CCSP expression level of the medicine provided by the invention, a high-dose group and an ambroxol group is obviously increased. See fig. 5.

3.5 Effect of the drug of the invention on model rat pulmonary tissue MUC5AC, AQP5

The normal group showed a small amount of MUC5AC positive expression, and the model group showed significantly increased MUC5AC positive expression (P < 0.05) and significantly decreased AQP5 positive expression (P < 0.05) in airway epithelial cells as compared with the normal group. Compared with the model group, the MUC5AC positive expression of the low, medium and high dose groups and the ambroxol group of the medicine is obviously reduced (P < 0.05), the AQP5 positive expression is obviously increased (P < 0.05), and the high dose and the ambroxol group of the medicine are obvious. Compared with ambroxol group, the MUC5AC positive expression of the low-dose group of the medicine is obviously increased (p < 0.05), the medium-dose group and the high-dose group have no significant difference (p > 0.05), and the AQP5 positive expression of the low-dose group of the medicine is obviously reduced (p < 0.05); positive expression was significantly increased in medium and high dose groups (p < 0.05), see fig. 6, 7, 8.

3.6 The effect of the medicine on the expression of MUC5AC and AQP5 genes in lung tissue of model rat

Compared with the normal group, the lung tissue MUC5AC mRNA expression of the rat in the model group is obviously increased (P < 0.05), and the AQP5 mRNA expression is obviously reduced (P < 0.05); compared with the model group, the MUC5AC mRNA expression of the medium-dose group, the high-dose group and the ambroxol group of the medicine is obviously reduced (P < 0.05), and the low-dose group of the medicine has no statistical difference (P > 0.05); the expression of the AQP5 mRNA in the high-dose group of the medicine is obviously increased (P is less than 0.05), and the low-dose group, the medium-dose group and the ambroxol group of the medicine have no statistical difference (P is more than 0.05); compared with ambroxol group, the drug of the invention has no statistical difference (P > 0.05) between MUC5AC and AQP5 mRNA in low, medium and high dose groups, as shown in figure 9.

3.7 Effect of the drug of the invention on TNF- α, IL-1β, MMP-9 and IL-13 content in serum in model rat BALF

TNF- α, IL-1β and MMP-9 levels were significantly elevated in BALF in model group rats compared to normal group (P < 0.01); compared with the model group, the low-dose group of the medicine has no statistical difference (P > 0.05), and the levels of TNF-alpha, IL-1 beta and MMP-9 in the middle-dose group, the high-dose group and the ambroxol group of the medicine are obviously reduced (P < 0.01); compared with ambroxol group, the low-dose group of the medicine has obviously increased TNF-alpha and MMP-9 levels (P < 0.01), the dose group of the medicine has obviously reduced IL-1β levels (P < 0.01), the MMP-9 levels are obviously increased (P < 0.01), the high-dose group of the medicine has obviously reduced TNF-alpha and IL-1β levels (P < 0.01), and the MMP-9 levels are obviously increased (P < 0.01), as shown in figure 10.

The serum IL-13 levels were significantly elevated in rats in the model group compared to the normal group (P < 0.01); compared with the model group, the low-dose group of the medicine has no statistical difference (P > 0.05), and the IL-13 level of the middle-dose group, the high-dose group and the ambroxol group of the medicine is obviously reduced (P < 0.01); compared with ambroxol group, the IL-13 level of the low dose group of the medicine is obviously increased (P < 0.05); the IL-13 level in the high dose group of the present invention was significantly reduced (P < 0.01), see FIG. 11.

The medicine can maintain the airway mucus state balance by inhibiting MUC5AC expression and promoting AQP5 expression, and simultaneously reduces the lung inflammation by reducing the levels of inflammatory factors such as TNF-alpha, IL-1 beta, MMP-9, IL-13 in serum and the like in BLAF, thereby effectively improving the airway mucus high secretion state in the AECOPD acute exacerbation stage, relieving the illness state and providing basis for treating the AECOPD acute exacerbation stage by clinical traditional Chinese medicine.

Experimental example 2:

to further elucidate the effect of the drug of the present invention on improving the acute exacerbation phase of chronic obstructive pulmonary disease, the following experimental study was conducted using the tablet prepared in the method of example 1 (hereinafter referred to as the drug of the present invention or LHQK), which interferes with AECOPD airway mucus hypersecretion by modulating ciliated structure and function.

2. Materials and methods

2.1 Animals

Animal experiments were conducted according to the ethical guidelines of the ethical committee of the medical research institute (N2021162) in the northwest of the river. In this study, male Wistar rats (200-250 g, about 5-6 weeks old) were purchased from beijing velariwa laboratory animal technologies limited (beijing, china). Water is fed daily. Coat color, food intake and behavior were recorded.

2.2 grouping and administration

After one week of feeding, rats were randomly divided into 6 groups and placed in the laboratory at the animal center of the medical institute in Hebei, where the ambient temperature was 22.+ -. 2 ℃, the light/dark period was 12, and the humidity was 55.+ -. 5%.

The mice were divided into 6 groups, and 150 rats were treated with 3 doses according to the random digital schedule, including the normal control group, the CS+LPS control group, the LHQK-L, LHQK-M, LHQK-H group, and the ELP group. Rats of the control group and CS+LPS group were perfused with 0.5% sodium carboxymethyl cellulose (CMC) solution, and LHQK-L, LHQK-M, LHQK-H group rats were perfused with suspensions of 1.47g crude drug/kg/d, 2.93g crude drug/kg/d, and 5.86g crude drug/kg/d, respectively. Positive control rats were perfused with ELP (E.Eucalyptus capsule) (0.3 g/kg/d) suspension.

2.3 modeling

After 1 week of adaptive feeding, cigarette smoke in combination with lipopolysaccharide induced a model of chronic obstructive pulmonary disease in rats. Except for Normal Control (NC), rats were anesthetized with 2% sodium pentobarbital at day 1, day 14, 24 before drawing, and 200. Mu.L of 1 g/L LPS (sigma L2880) was administered, and no smoke was generated on the day of instillation. Rats were placed in a 100 cm x 70 cm x 40 cm stainless steel smoke box and exposed to smoke from 40 cigarettes of fire smoke from day 2 to day 90. Rats were exposed to smoke twice daily (1 h each at noon and afternoon), and allowed to rest for 2 days per week, and experimental models were established. CS exposure and dosing follow-up for 12 weeks.

2.4 hematoxylin and eosin (H & E)

Lung tissue was perfused with 4% paraformaldehyde solution to prevent alveolar collapse and ligate the trachea. Lung tissue was then fixed by embedding in paraformaldehyde solution, gradient solution dehydration, paraffin embedding, sectioning, hematoxylin eosin staining kit (H & E) staining (Beyotime, shanghai, china). Finally, the sections were photographed under a fully automated biological microscope (Leica DM6000B, wetzlar, germany). Average linear intercept (MLI) was measured to assess the extent of emphysema.

2.5 Scanning Electron Microscope (SEM)

Specimens (17-18 week old rats) were fixed with 2.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1M sodium phosphate buffer, pH 7.3, at room temperature for 3 hours, and then 2 hours in 2% osmium tetroxide 0.1 sodium phosphate buffer. After dehydration in gradient ethanol, the samples for Scanning Electron Microscopy (SEM) were dried in a critical point dryer (poled, woad, uk), mounted on sample roots, and coated with gold-palladium in a cold sputter coater (Fisons Instruments Uckfield, uk). The specimens were examined using a scanning electron microscope DSM 960 (Zeiss Oberkochen, germany). Finally, the morphology and length of the tracheal epithelial cilia were observed by scanning electron microscopy. At least 3 cilia on each ciliated cell in each image (n=3-5 images per sample) were measured by ImageJ software tool, and the average length of cilia in SEM images of the culture (x 1000 magnification) was calculated. The cilia length distribution was determined using a frequency histogram distribution tool in GraphPad Prism.

2.6 immunofluorescence analysis

Rat trachea was fixed with 4% paraformaldehyde and 5 μm paraffin sections. The trachea is dewaxed for antigen retrieval, PBS is cleaned, hydrogen peroxide is incubated for 10 min at 37 ℃, PBS is cleaned for 3 times, and 5% BSA is blocked for 30min at room temperature. The trachea was incubated with beta-Tubulin IV (abcam, 1:500) overnight at 4 ℃. The trachea was washed with PBS and incubated with secondary antibody (gold Anti-Rabbit IgG H & L (DYlight 488, proteintech, 1:200) for 50min at room temperature after three more washes with PBS, the nuclei were stained with DAPI (Solarbio, beijin, china) for 10 min.

2.7Western blot analysis

Protein was extracted from rat lung tissue using RIPA lysis buffer containing the protease inhibitor PMSF. Protein concentration was then determined using BCA protein assay kit (Biyuntian, china). An appropriate amount of 5 Xprotein loading buffer, 100 XC, was added to the sample and boiled for 10 minutes. After cooling, the proteins were separated on a 4-20% SDS-PAGE gel (GenScript Biotech Corporation, china) and transferred to PVDF (Life Sciences, mexico). The membrane was blocked with an odxe blocking buffer (LI-COR, lincoln USA) for 1 hour at 37% C and incubated overnight with primary antibody (beta-Tubulin IV, abcam, 1:500) at 4% C. After 3 washes with TBST (10 min each), the membranes were further incubated with secondary antibodies (goat anti-rabbit IgG H & L) for 1 hour at 37C. After 3 more washes with TBST, the membrane was scanned with an Ordersai imager (LI-COR, lincoln USA). GAPDH is used as an internal reference.

2.8 analysis of the frequency of cilia swing of the tracheal annulus

Rats were anesthetized with pentobamidine, the trachea was removed and placed in DMEM medium. The tracheal ring (main trachea) was cut to about 0.5mm thickness with a scalpel and equilibrated in DMEM for 20-30 minutes. Then, 3-5 different fields of view were randomly selected under a high-speed camera, an inverted microscope and a 40-fold phase difference objective to record cilia motion. The time is 2.0-3.0 seconds, and the rate is 200-250 frames/second. The resulting image was imported into ImageJ, 3-6 regions of interest (ROIs) were selected, and the average gray values were measured within the ROI range of each frame. The variation of the gray intensity of the individual regions of interest is caused by repeated movements of the cilia, as shown. The beat period is calculated by dividing the number of complete beats by the time spent, with the mean ± standard deviation expressed in Hz.

2.9 in vitro ciliated cell cilia swing frequency analysis

Euthanasia was performed on 5 Wistar rats, 70% ethanol was sprayed on the neck portion, and the surface of the abdomen was cut laterally in the neck. Subcutaneous fat is removed, exposing the trachea. The adherent adipose tissue was harvested and washed and spread. The treated tracheal tissue was placed in Pronase E (100 mg/ml; from Streptomyces griseus, MCE, hy114158 a), DNase I (Solarbio, D8071) and DMEM/F12 (gibco, C11330500 BT) and digested overnight at 4℃for 18h. Then, the airway epithelial cells were gently dispersed with 10% Fetal Bovine Serum (FBS) upside down 15 times, the mixture was collected into a new tube, the collection medium containing 10% fetal bovine serum was added, and the mixture was gently inverted upside down 20-30 times, so as to obtain more epithelial cells, the above procedure was repeated twice, and all the mixture was collected into the same tube. The trachea is discarded. The mixture was centrifuged at 500g at 4℃for 10min. The supernatant was carefully discarded and the bottom cell pellet was resuspended in 2ml of culture medium. The suspension was placed in a 12-well plate and 20-30 ciliated cells were randomly selected under a high-speed camera, an inverted microscope and a phase contrast objective (40 x). The shooting criteria and statistical analysis are the same as for the tracheal rings CBF.

2.10 Transmission Electron Microscope (TEM)

Samples (17-18 week old rats) were sectioned into small sections (1 x 1 mm) using Transmission Electron Microscopy (TEM) and fixed in 2.5% glutaraldehyde (ph=7). 4) Standing at 4deg.C for 6-8 hr. Washed and fixed in 2% OsO4 for 1 hour at 4 ℃. Tissues were embedded in alalite CY212 after increasing ethanol concentration. Cut half sheet (1 μm) and stained with toluidine blue. And cutting ultrathin sections (60-80 nm), and dyeing with uranyl acetate and alkaline lead citrate.

2.11 cilia direction analysis

At 15000 magnification, at least 10 cilia cross-sections were captured per field of view, with the center pair clearly visible to measure cilia direction within the cilia shaft. On each image, a line was drawn on the center pair of each cross section, and the angle of each line was measured with a computer (vertical upward=0°; horizontal rightward=90°; vertical downward=180°). Thus, in each image, the directional angle of each cilia is obtained, and the standard deviation of the axial angle of each cilia is calculated. The mean standard deviation of the cilia axis represents an overall measure of cilia disorientation in the subject. Each animal was given 5 pictures and each group was given 3 animals at random.

2.12 cilia ultrastructural analysis

3 animals were randomly selected for each group, and 3-5 images were randomly selected for each animal. The total number of cilia and the number of abnormal cilia were counted in a TEM image at 15000x, and the percentage of abnormal cilia was calculated. Cilia defects include ciliated membrane vesicles, loss of power arms, microtubule defects, composite cilia, and giant cilia.

2.13 statistical analysis

All results are expressed as mean ± standard deviation and analyzed using SPSS 26.0 software. The data sets were compared using one-way analysis of variance (ANOVA). In all analyses, P < 0.05 was statistically significant and P < 0.01 was highly statistically significant. In addition, all graphics were created using GraphPad Prism (8.0) software.

3. Results

3.1 LHQK can alleviate pathological damage of pulmonary tissue of AECOPD rat

Lung tissue pathology was assessed using H & E staining (fig. 12A). The results show that the difference is statistically significant in the AECOPD model group under the microscope compared to the control group. The alveolar septum is thickened, inflammatory cells are increased, the increased alveoli are fused to form lung large bubbles, and the bronchial ciliated columnar epithelial cells are partially shed, denatured and necrotized, and goblet cells are increased, which indicates that the model is successfully prepared. The control group has complete and uniform alveolar structure, clear alveolar space and fewer inflammatory cells in the alveolar space. Compared with the model group, the LHQK group and the positive drug group have reduced alveolus quantity, partial alveolus is combined into large alveolus, small amount of inflammatory change occurs in the alveolus, and the shedding, the denaturation and the necrosis of bronchial epithelial cells are reduced.

Lung tissue pathologic injury was assessed by measuring average alveolar intercept (fig. 12B). The results showed that the average alveolar intercept was significantly higher than the control (P < 0.01). The average alveolar intercept of LHQK and ELP groups was significantly smaller than that of cs+lps group (P < 0.01). The above results indicate that cs+lps exacerbates pathological lesions in lung tissue in AECOPD rats, whereas the medium, high and ELP groups of LHQK reverse this change, with the dose effect of LHQK-M being most pronounced.

3.2 LHQK can improve CS+LPS-induced AECOPD rat airway epithelial cilia morphology changes

Airway epithelial cilia morphology changes were assessed by tracheal H & E staining (fig. 13A). The results show that the airway epithelium of the cs+lps group is shortened and deleted compared to the control group, whereas the airway epithelium of the LHQK, high dose group and ELP group is slightly damaged, the cilia still being aligned and a small number of deletions.

To further observe changes in morphology of airway epithelial cilia, airway epithelial cilia morphology was assessed by Scanning Electron Microscopy (SEM) cross-section (fig. 13B). The results show that the tracheal epithelium of the CS+LPS group has sparse cilia, shortened cilia length, disordered cilia arrangement, falling cilia, inconsistent swing, partial cilia fracture and more reticulate secretion attached to the cilia surface. The degree of airway epithelialization of LHQK, medium and high dose groups and ELP groups is increased, the cilia length is obviously increased, cilia are orderly arranged, the swinging directions of cilia are consistent, and no obvious reticular secretion is seen on the cilia surface.

3.3 LHQK reduces CS+LPS induced AECOPD rat cilia shortening

Cs+lps may shorten AECOPD rat ciliary length and LHQK may increase AECOPD rat ciliary length. To further investigate the change in cilia length, we used scanning electron microscopy longitudinal sections (fig. 14) to evaluate cilia length. The results show that the cilia length of the cs+lps group is significantly shortened compared to the control group. The cilia length was significantly increased in LHQK, both in the medium, high dose and ELP groups.

3.4 LHQK increases reduction of airway epithelial cilia under CS+LPS action

Cs+lps may decrease airway epithelial cilia, while LHQK may increase airway epithelial cilia number. To further investigate changes in cilia number, immunofluorescence and Western Blot were used to detect expression of the cilia marker β -Tubulin IV protein. Immunofluorescence (FIG. 15A) and Western Blot (FIGS. 15B-C) results showed that the CS+LPS group beta-Tubulin IV expression was significantly down-regulated (P < 0.01) compared to the control group. In LHQK, high dose and ELP groups beta-Tubulin IV expression was up-regulated (P < 0.05) compared to CS+LPS group.

3.5 LHQK significantly improves the reduced cilia beat frequency of cs+lps

Cs+lps reduced CBF in AECOPD rats, while LHQK ameliorated this injury. The high speed camera captures video images, which are analyzed with Image J software (fig. 16A-B), and the tracheal annulus cilia CBF is evaluated. CBF was significantly reduced in cs+lps group compared to control group (p < 0.01). The CBF was significantly improved in LHQK, in the high dose group and in the ELP group compared to the cs+lps group (P < 0.01).

To observe the CBF of cilia in vitro, we assessed CBF of individual cilia cells of each group using low temperature enzymatic digestion to acutely isolate cilia cells of AECOPD rats. The cs+lps group single ciliated cells CBF was significantly reduced compared to the control group (P < 0.01). The medium, high dose and ELP groups of LHQK significantly improved single ciliated CBF (P < 0.01) compared to the cs+lps group. The results of in vitro and in vivo studies were consistent. The above results indicate that cs+lps group can reduce CBF in and out of AECOPD rats, while LHQK can significantly improve CBF in and out of AECOPD rats.

3.6 LHQK can improve cilia ultrastructural damage caused by CS+LPS

CS+LPS causes cilia ultrastructural damage, and LHQK can improve cilia ultrastructural damage, and cilia ultrastructural is observed by transmission electron microscopy. The cilia ultrastructure is shown in fig. 17. The results showed that the cs+lps group had a smaller number and uneven distribution of cilia structures compared to the control group, most of the structures were significantly damaged, the cilia membrane damage disintegrated, and cilia membrane blebs, central microtubules or outer Zhou Weiguan shift significantly increased. While the number of cilia in the medium, high dose and positive drug groups of LHQK is significantly increased. The cilia structure was essentially intact, with no apparent membrane damage or disintegration. Ciliated membrane vesicles, central microtubules or outer Zhou Weiguan displacement are significantly reduced. The above results indicate that the cilia ultrastructural lesions of the CS+LPS group are significantly aggravated, while LHQK can reverse the lesions.

3.7 CS+LPS may cause the cilia to be non-uniform in direction and LHQK may be improved.

The cilia direction was assessed from the TEM photograph by measuring the angle between the plane defined by the central tubule and the reference lead. The cilia direction is shown in fig. 18. The results showed that the ciliated direction of the cs+lps group was disordered compared to the blank group. The cilia direction of the medium, high dose and positive drug groups in LHQK are relatively consistent. These results indicate that cs+lps has a damaging effect on cilia direction, while LHQK has an improving effect on cilia direction.

By studying the structure and function of cilia in AECOPD rats, it was found that the protective effect of the drug of the present invention on AECOPD rats may be related to the structural and functional changes of cilia, which play an important role in clearing airway mucus.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims (22)

1. The application of the traditional Chinese medicine composition in preparing the medicine for treating the acute exacerbation stage of the chronic obstructive pulmonary disease is characterized in that the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 52-86 of ephedra; gypsum 194-324; fructus forsythiae 194-324; 78-130 parts of radix scutellariae; white mulberry root bark 194-324; 78-130 parts of fried bitter apricot kernel; 78-130 parts of radix peucedani; 78-130 parts of pinellia ternate; 78-130 parts of dried orange peel; 78-130 parts of fritillary bulb; 78-130 parts of burdock; 78-130 parts of lonicera japonica; 39-65 parts of rheum officinale; radix Platycodi 46-76; 39-65 parts of liquorice.

2. The use of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: herba Ephedrae 52; gypsum 324; fructus forsythiae 194; radix Scutellariae 78; cortex Mori 194; parching semen Armeniacae amarum 130; radix Peucedani 78; pinellia ternate 130; dried orange peel 78; bulbus Fritillariae Thunbergii 78; fructus Arctii 130; flos Lonicerae 130; rhubarb 39; radix Platycodi 76; licorice 65.

3. The application of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: herba Ephedrae 86; gypsum 194; fructus forsythiae 324; radix Scutellariae 130; cortex Mori 324; parching semen Armeniacae amarum 78; radix Peucedani 130; pinellia tuber 78; dried orange peel 130; bulbus Fritillariae Thunbergii 130; burdock 78; flos Lonicerae 78; rhubarb 65; radix Platycodi 46; licorice 39.

4. The application of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: ephedra 69; gypsum 259; fructus forsythiae 259; radix Scutellariae 104; cortex Mori 259; parching semen Armeniacae amarum 104; radix Peucedani 104; pinellia ternate 104; dried orange peel 104; bulbus Fritillariae Thunbergii 104; fructus Arctii 104; flos Lonicerae 104; rhubarb 52; radix Platycodi 61; licorice 52.

5. The application of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: herba Ephedrae 55; gypsum 254; fructus forsythiae 318; radix Scutellariae 107; white mulberry root bark 203; a fried bitter almond 107; radix Peucedani 82; pinellia ternate 105; dried orange peel 84; bulbus Fritillariae Thunbergii 125; burdock 122; flos Lonicerae 113; rhubarb 42; radix Platycodi 60; licorice root 50.

6. Use according to any one of claims 1-5, characterized in that the active ingredient of the medicament is made by the steps of:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 40-70% ethanol for 2 times, each for 1-4 hr, adding 8-10 times of the extractive solution for the first time and 6-9 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.14-1.16 at 60deg.C;

C. weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for two times, 1-4 hours each time, adding 9-11 times of the weight of the first time and 7-9 times of the weight of the second time, merging decoction, filtering, decompressing filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.14-1.16 measured by heat at 60 ℃, merging with the clear paste obtained in the step B for standby;

the fine powder obtained in the step A and the combined fluid extract obtained in the step C jointly form the active ingredient of the pharmaceutical composition.

7. The use according to any one of claims 1 to 5, characterized in that the pharmaceutical dosage form is a tablet, capsule, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.

8. Use according to claim 7, characterized in that the tablet is made up of the following steps:

A. weighing Bulbus Fritillariae Thunbergii according to the proportion of the prescription, and pulverizing into fine powder for use;

B. weighing herba Ephedrae, fructus forsythiae, parched semen Armeniacae amarum, rhizoma Pinelliae, fructus Arctii, and radix et rhizoma Rhei, reflux extracting with 50% ethanol for 2 times and 3 hr each time, adding 10 times of the extractive solution for the first time and 6 times of the extractive solution for the second time, mixing the extractive solutions, filtering, recovering ethanol from the filtrate under reduced pressure, concentrating to obtain fluid extract with relative density of 1.15 at 60deg.C;

C. weighing gypsum, white mulberry root-bark, peucedanum root, dried orange peel, lonicera japonica, platycodon grandiflorum and liquorice according to the proportion of the prescription, adding water and decocting for 2 hours each time, adding 10 times of the weight for the first time and 7 times of the weight for the second time, merging the decoctions, filtering, decompressing the filtrate, recovering ethanol, concentrating to obtain clear paste with the relative density of 1.15 measured by heat at 60 ℃, merging the clear paste obtained in the step B for standby;

D. c, spraying and drying the combined fluid extract obtained in the step C, and collecting spraying powder for later use;

E. the weight proportion of the raw materials required by tabletting is as follows:

step D, spraying powder 282.6; the fine powder 101 obtained in the step A; 12.5 parts of sodium carboxymethyl starch; microcrystalline cellulose 9.0; 2.25 parts of magnesium stearate, adding starch, and making into tablet by conventional preparation method.

9. The use according to any one of claims 1 to 5, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for inhibiting the differentiation of Clara cells into goblet cells.

10. The use according to any one of claims 1 to 5, characterized in that the use of the Chinese medicinal composition for the preparation of a medicament for increasing CCSP protein expression is described.

11. The use according to any one of claims 1 to 5, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for promoting the expression of AQP5 protein or AQP5 mRNA.

12. The use according to any one of claims 1 to 5, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for reducing the expression of MMP-9 or IL-13 factor.

13. The use according to any one of claims 1-5, characterized in that the use of the traditional Chinese medicine composition for the preparation of a medicament for treating lung injury.

14. The use according to any one of claims 1-5, characterized in that the use of the traditional Chinese medicine composition for the preparation of a medicament for protecting the cilia structure of airway epithelium.

15. The use according to claim 14, characterized by the use of said Chinese medicinal composition for the preparation of a medicament for increasing cilia length.

16. The use according to claim 14, characterized by the use of said Chinese medicinal composition for the preparation of a medicament for increasing the quantity of cilia.

17. The use according to claim 16, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for up-regulating the expression of β -Tubulin IV protein.

18. The use according to claim 14, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for improving cilia direction.

19. The use according to claim 14, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for protecting cilia from ultrastructural damage.

20. The use according to any one of claims 1-5, characterized in that the use of the traditional Chinese medicine composition for the preparation of a medicament for protecting airway cilia function.

21. The use according to claim 20, characterized by the use of said Chinese medicinal composition for the preparation of a medicament for increasing cilia beat frequency.

22. The use according to any one of claims 1-5, characterized in that the use of said Chinese medicinal composition for the preparation of a medicament for improving airway hypersecretion in patients suffering from acute exacerbation of chronic obstructive pulmonary disease.