CN116236543A

CN116236543A - Application of Sang Juqing decoction in preparation of medicines for preventing or treating pulmonary fibrosis

Info

Publication number: CN116236543A
Application number: CN202310089133.XA
Authority: CN
Inventors: 戴卫波; 王珠强; 彭伟文; 曾聪彦; 罗佳; 黄梨婷
Original assignee: Zhongshan Hospital of TCM
Current assignee: Zhongshan Hospital of TCM
Priority date: 2023-02-09
Filing date: 2023-02-09
Publication date: 2023-06-09

Abstract

The invention discloses a new application of Sang Juqing decoction in preparing medicines for preventing or treating pulmonary fibrosis, and results show that compared with a model group, each dosing group of Sang Juqing decoction reduces pulmonary fibrosis of mice to different degrees, has similar treatment effect to positive medicine pirfenidone, and further proves that histopathological changes observed through HE and MASSON staining, szapiel scoring and Ashcroft scoring results: sang Juqing and Shang Neng delay the development of pulmonary fibrosis of mice, improve survival rate to a certain extent, have certain protection and alleviation effects on pulmonary fibrosis, and Sang Juqing decoction can reduce TNF-alpha and TGF-beta in serum of mice ₁ The expression also has the regulation and control effects on SOD, IL-6, MDA and GSH in lung tissues, and provides a new idea for developing high-quality anti-pulmonary fibrosis new drugs.

Description

Application of Sang Juqing decoction in preparation of medicines for preventing or treating pulmonary fibrosis

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to an application of Sang Juqing decoction in preparing a medicine for preventing or treating pulmonary fibrosis.

Background

Pulmonary fibrosis (pulmonary fibrosis, PF) is a chronic disease in which the process is that pulmonary fibroblasts proliferate, differentiate and accumulate in the extracellular matrix, causing irreversible damage to the lungs, eventually progressing to fibrotic pulmonary interstitial inflammatory disease. The median survival time of the patient with pulmonary fibrosis is about 2-3 years, the survival rate of 5 years is basically less than 30%, and the probability of the patient with pulmonary fibrosis suffering from lung cancer is 5 times that of the common people. Pulmonary fibrosis occurs in patients recovering from acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) at different levels.

The Western medicine has few medicines which can be selected in the treatment of pulmonary fibrosis, the currently known recommended medicines are Nidamib and pirfenidone, but both medicines cannot reverse the pulmonary tissue which is already fibrosed, and also cannot obviously prolong the life cycle of a patient, and besides, the oral administration of pirfenidone can cause central reactions such as nausea, vomiting, dizziness and the like and gastrointestinal reactions such as diarrhea and the like. The traditional Chinese medicine treatment mainly adopts syndrome differentiation treatment, has the advantages of less adverse reaction, multiple targets and the like, so that a traditional Chinese medicine prescription effective for relieving and treating pulmonary fibrosis is developed, and has important clinical value and significance.

Sang Juqing decoction has effects of clearing heat and eliminating phlegm, relieving cough, and removing toxic substances, and can be used for treating cough due to lung heat, dry throat, excessive phlegm, etc. Early-stage researches show that Sang Juqing decoction has a certain protection effect on the lung injury of rats caused by passive smoking and chronic smoking, has definite curative effect on treating chronic obstructive phlegm-heat-accumulated lung diseases, can effectively improve the traditional Chinese medicine symptoms and arterial blood qi indexes of patients, regulate the immune indexes of the patients and improve the exercise capacity and the lung functions of the patients. However, the document report of Sang Juqing decoction in preparing medicines for preventing or treating pulmonary fibrosis is not yet known.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of Sang Juqing decoction in preparing a medicament for preventing or treating pulmonary fibrosis.

The Sang Juqing soup is prepared from the following components in parts by weight:

10-20 parts of white mulberry root-bark, 5-15 parts of wild chrysanthemum flower, 10-40 parts of semen benincasae, 5-15 parts of snakegourd peel, 5-15 parts of plantain seed, 5-15 parts of fritillaria cirrhosa, 2-10 parts of liquorice, 6-12 parts of tabasheer, 10-35 parts of reed rhizome and 5-15 parts of earthworm.

As a further preferable aspect, the Sang Juqing soup is prepared from the following components in parts by weight:

15 parts of white mulberry root-bark, 10 parts of wild chrysanthemum, 20 parts of white gourd seed, 10 parts of snakegourd peel, 10 parts of plantain seed, 10 parts of fritillaria cirrhosa, 5 parts of liquorice, 10 parts of tabasheer, 25 parts of reed rhizome and 10 parts of earthworm.

The invention induces the pulmonary fibrosis of mice by bleomycin, and divides 63 male C57BL/6 mice into a blank group, a model control group and a pirfenidone group (0.2 g.kg of pirfenidone is given) ^-1 (10mL·kg ^-1 ) 1 time per day), sang Juqing decoction high and low dose group (low dose administration amount is 16.2 g.kg) ^-1 (5mL·kg ^-1 ) The dosage of the high dose group was 32.4 g.kg ^-1 (10mL·kg ^-1 ) 1 time per day) for 14d, obtaining lung tissues, performing HE and Masson staining, detecting the contents of TNF-alpha (tumor necrosis factor-alpha) and TGF-beta (transforming growth factor-beta) by serum, homogenizing the rest lung tissues, and detecting the contents of SOD (superoxide dismutase), IL-6 (interleukin-6), MDA (malondialdehyde) and GSH (glutathione). The histopathological results indicate that the Sang Juqing decoction can improve the pulmonary fibrosis state after administration, and the effect is similar to that of pirfenidone; sang Juqing decoction can reduce TNF-alpha, TGF-beta in mouse serum ₁ The expression also has regulating and controlling effects on SOD, IL-6, MDA and GSH in lung tissues.

Preferably, the pharmaceutical dosage form is a tablet, powder, mixture, pill, capsule or granule.

Compared with the prior art, the invention has the following excellent effects:

the invention provides a new application of Sang Juqing decoction in preparing a medicament for preventing or treating pulmonary fibrosis, and results show that each dosage administration group and model of Sang Juqing decoctionGroup comparison, the lung tissue fibrosis of mice is reduced to different degrees, and the effect is similar to that of positive drug pirfenidone treatment, and the histopathological changes observed through HE and MASSON staining, and the Szapiel scoring and Ashcroft scoring results further prove that: sang Juqing and Shang Neng delay the development of pulmonary fibrosis of mice, improve survival rate to a certain extent, have certain protection and alleviation effects on pulmonary fibrosis, and Sang Juqing decoction can reduce TNF-alpha and TGF-beta in serum of mice ₁ The expression also has the regulation and control effects on SOD, IL-6, MDA and GSH in lung tissues, and provides a new idea for developing high-quality anti-pulmonary fibrosis new drugs.

Drawings

FIG. 1 is a graph of mice survival (blank: normal; model: model; SJ-L: sang Juqing solution low dose; SJ-H: sang Juqing solution Shang Gao dose; PFD: pirfenidone group);

FIG. 2 shows the tissue appearance of mice in each group (A. Normal group; B. Model group; C. Sang Juqing solution low dose group; D. Sang Juqing solution Shang Gao dose group; E. Pirfenidone group);

FIG. 3 shows the HE staining (x 200) of mice in each group (A. Normal group; B. Model group; C. Sang Juqing solution low dose group; D. Sang Juqing solution Shang Gao dose group; E. Pirfenidone group);

FIG. 4 shows Masson staining (x 200) of mice in each group (note: A. Normal group; B. Model group; C. Sang Juqing solution low dose group; D. Sang Juqing solution Shang Gao dose group; E. Pirfenidone group).

Detailed Description

The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention.

1 Material

1.1 animals

63 male C57BL/6 mice, grade SPF, weight 18+ -2 g, purchased from medical laboratory animal center, guangdong province, license number SCXK (Guangdong) 2022-0002; use license number: SYXK 2020-0109, the raising condition is that the temperature is 22-26 ℃ and the humidity is 35-50%. The experiment passed ethical committee review approval (No. 2022038).

1.2 preparation of drugs and reagents

Sang Juqing decoction preparation: taking Sang Juqing decoction (comprising cortex Mori 15g, flos Chrysanthemi Indici 10g, semen Benincasae 20g, pericarpium Trichosanthis 10g, semen plantaginis 10g, bulbus Fritillariae Cirrhosae 10g, glycyrrhrizae radix 5g, concretio silicea Bambusae 10g, rhizoma Phragmitis 25g, lumbricus 10 g) 250g crude drug, bagging semen plantaginis with gauze, soaking distilled water 2.5L for 30min, decocting for 1.5h, filtering the liquid medicine with 8 layers of gauze, taking distilled water 2L, decocting for 1.5h, mixing the two filtrates, concentrating to 77ml (3.24 g.ml) ^-1 ) And supplementing 154mL of sterile water, sub-packaging into three branch pipes, and storing in a refrigerator at the temperature of minus 37 ℃ for standby.

Preparation of molding agent bleomycin hydrochloride: taking bleomycin hydrochloride (specification: 15mg, batch number: 610740, japanese Kagaku Co., ltd.) powder, dissolving in injectable physiological saline, and making into 0.3 mg.ml ^-1 A solution.

Positive control drug pirfenidone solution preparation: pirfenidone capsules (Ai Sairui) (specification: 100mg, lot number: 20211105, beijing Kandin pharmaceutical Co., ltd.) were taken as one pellet and dissolved in 5ml of 0.5% CMC-Na solution, and prepared for immediate use.

2 method

2.1 grouping and model establishment of pulmonary fibrosis mice

After adaptively feeding 63C 57BL/6 mice for 1 week, they were randomly divided into 5 groups according to body weight: blank group 12, model group 13, sang Juqing solution low dose group 13, sang Juqing solution Shang Gao dose group 13, positive control pirfenidone group 12.

The pulmonary fibrosis mouse model is prepared by a method of intratracheal instillation of bleomycin sulfate solution (3mg.kg-1): mice were intraperitoneally injected with 1% sodium pentobarbital (anesthetic dose: 50mg kg) ^-1 ) The mouse is fixed on the mouse board in a supine mode, the board head side is lifted by 45-60 degrees, the tongue of the mouse is pulled by the left hand, the right hand tube is held, and the right hand tube is tightly attached to the upper jaw along the radian of the right hand tube to drive the tracheal cannula through the tracheal cartilage annular gap. After successful cannula, the catheter was connected to a syringe, and 0.2ml (3 mg. Kg) of bleomycin sulfate solution was injected ^-1 ) Normal mice were intubated with saline as a blank. After each administration, the mice were rapidly treatedThe upright lifting is rotated anticlockwise for several times, so that the medicines are uniformly distributed in the lung as much as possible. And then closely monitoring the life state of the mice, and after the mice are awake, carrying out conventional feeding.

Model group and blank group after 24h model formation according to weight of 10 mL.kg of mice ^-1 The stomach was irrigated with 0.5% CMC-Na solution. Sang Juqing decoction low-dose group dosage is 5mL kg ^-1 (equivalent to 16.2 g.kg of crude drug) ^-1 ) The dosage of the high-dose group is 2 times of the clinical equivalent dosage, namely 10 mL.kg ^-1 (equivalent to 32.4 g.kg of crude drug) ^-1 ) And (5) pouring corresponding doses of medicines into the stomach. The positive control drug pirfenidone group is 0.2 g.kg of the weight of the mice ^-1 (10mL·kg ^-1 ) And (5) stomach irrigation. All once daily for 14 consecutive days. During the period, each group of mice can eat and drink water freely, and the activity of the mice, the death phenomenon and the like are observed.

2.2 sample collection

After 24 hours from the last dose on day 14 of the experiment, the body weight was weighed and serum was taken for measuring various indexes of serum. The whole double lung and bronchus are picked up, rinsed in physiological saline to remove blood stains, and the excessive water on the surface of the lung is sucked by gauze and then weighed. The lung coefficients are calculated.

2.3 pulmonary histopathological observations

A part of the lung tissue of the mouse is fixed by 4% paraformaldehyde, and after coronal surface sampling, block repairing, dehydration and paraffin embedding, continuous coronal sections are carried out, and HE staining and Masson staining are carried out on the sections. The stained sections were observed under a microscope and alveolar inflammation and collagen deposition were assessed using the Szapiel scoring system and the Ashcroft scoring system, respectively. Lung tissue sections from each mouse were scored for 5 independent areas and averaged. The scoring tables are shown in tables 1 and 2.

TABLE 1Szapiel scoring System

TABLE 2Ashcroft scoring System

2.4 enzyme-linked immunosorbent assay (ELISA) for detecting the content of each index of mice

2.4.1 content of TNF-alpha, TGF-beta in serum

Serum samples of each group of mice were taken and thawed on ice. According to the instructions, the standard is diluted, loaded, incubated for 30min at 37 ℃, washed 5 times, enzyme is added, again incubated for 30min at 37 ℃ and washed 5 times, color development is performed, the reaction is terminated, and finally the absorbance (OD value) is measured. The actual concentration of the sample is calculated.

2.4.2 content of SOD, IL-6, MDA, GSH in pulmonary tissue

Taking the residual lung tissues of each group of mice, weighing, shearing, homogenizing, respectively carrying out operations such as sample adding, enzyme adding, incubation, liquid preparation, washing, color development, termination, measurement and the like according to the specifications of the SOD, IL-6, MDA and GSH kits, detecting absorbance values (OD values) of each group by an enzyme-labeling instrument, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample.

2.5 statistical methods

The data were counted using SPSS 23.0 software to measure the data results

And (3) representing. If the data accords with normal distribution and the variances are uniform, adopting single-factor analysis of variance (One-way ANOVA), and adopting a Least Significant Difference (LSD) method for pairwise comparison; when the variance is uneven, the Duunett's T method is used, rank sum test is adopted for the hierarchical data, and P<0.05 indicates that the difference is statistically significant.

3 results

3.1 Sang Juqing Effect of soup on bleomycin-induced pulmonary fibrosis mice

3.1.1 Effect on survival of mice in groups

Animal death during the experiment is shown in figure 1.

Model group: 1 die after molding, 2 die after molding in 3-7 days, the survival rate is 77%, 3 die after molding in 8-14 days, and the survival rate is 54%.

The survival rate of the administration group is obviously improved: no death is seen in 3-7 days after molding, 2 of the Sang Juqing soup-releasing low-dose groups die in 8-14 days after molding, 1 of the Sang Juqing soup-releasing Shang Gao dose groups die, and 3 of the pirfenidone groups die, with survival rates of 85%, 92% and 77% respectively.

3.1.2 body weight changes

The body weight of the mice was recorded during the experiment and the results are shown in Table 3. At 7d, mice from each model group lost weight with significant differences (P < 0.01) compared to the blank group; on days 7d-14d, mice in the model group continued to lose weight with significant differences (P < 0.01) compared to the blank group, and each drug group had increased body weight with significant differences (P < 0.05) compared to the model group in the Sang Juqing solution Shang Gao dose group and the positive control drug pirfenidone group.

Table 3 variation of body weight of mice in each group

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Note that: comparison with blank group, P<0.05，**P<0.01; in comparison with the set of models, ^# P<0.05， ^## P<0.01

3.1.3 visual inspection of Lung tissue appearance

The double lungs of the mice in the blank group have fresh pink appearance, the lung lobes are shiny and non-tarnish, the elasticity is good, and no obvious bleeding point exists; the model group rats have dark red and dark and matt lungs, harder textures, poor elasticity and locally visible bleeding points; compared with the lung tissue of a model group of mice, the lung tissue of a Sang Juqing decoction-releasing low-dose group, a Sang Juqing decoction Shang Gao-dose group, a Pirfenidone (PFD) group of mice is improved in terms of color, elasticity, texture and the like to different degrees, the lung tissue of the mice is slightly better than the lung tissue of the mice of the model group, and the lung tissue of the mice of the model group is obviously improved by the high-dose group and the pirfenidone group. See fig. 2 for a set of diagrams.

3.1.4 cases of pulmonary coefficients of mice of each group

The lung coefficient of mice in the model group was increased, with very significant differences (P < 0.01) compared to the blank group; the lung coefficients of the Sang Juqing solution low-dose group, the high-dose group and the positive drug pirfenidone group are reduced, wherein the Sang Juqing solution Shang Gao dose group and the positive drug pirfenidone group have significant differences (P < 0.05) compared with the model group. See table 4.

Table 4 pulmonary coefficients of mice of each group

3.2 Effect on Lung histomorphology in mice of groups

3.2.1HE dyeing

The observation under the HE staining mirror shows that: the lung tissue of the mice in the blank group has clear internal structure, and the alveolar space is not thickened; the pulmonary tissue alveolus interval of the mice in the model group is obviously thickened, the lumen is narrow, and a fibrotic focus is formed; sang Juqing the lung fibrosis degree of the low and high dose groups and the pirfenidone group showed clear alveolar structure, and was significantly reduced compared with the model group (see fig. 3).

3.2.2Masson staining

Blank groups shown by Masson staining are normal lung tissue structures, and less blue collagen is deposited; the model group lung tissue can be seen to have a large number of blue collagen hyperplasia in the bronchus and the tissues around the vascular branches in the lung; sang Juqing the low and high dose groups and the pirfenidone group showed some destruction of alveolar structure and a different degree of reduction of blue collagen fibers (see figure 4).

3.2.3 Szapixel score and Ashcroft score

To further objectively evaluate the degree of fibrosis in the lung tissue of each group of mice, the lung tissue of the mice was scored using the Szapiel scoring and Ashcroft scoring system, and both groups of scoring results showed: the lung tissue score of the mice in the model group is obviously higher than that of the mice in the normal group; the scores of the administration groups are reduced, and compared with the scores of the mice in the model group, the scores of the administration groups have statistical significance (P is less than 0.01). The results are shown in Table 5.

Table 5 Sang Juqing effect of untangling on lung fibrosis mice lung tissue Szapiel score and Ashcroft score

3.3 influence of Sang Juqing decoction on the content of various indices of mice

3.3.1 TNF-alpha, TGF-beta in serum ₁ Horizontal comparison

As shown in Table 6, the mice in the model group had TNF-. Alpha.and TGF-. Beta.in comparison with the blank group ₁ The level was elevated and the difference was statistically significant (P < 0.05). Compared with the model group, the Sang Juqing decoction low dose group, sang Juqing decoction Shang Gao dose group and pirfenidone group serum TNF-alpha, TGF-beta ₁ The level was reduced and the difference was statistically significant (P < 0.05). Suggesting Sang Juqing decoction for reducing TNF-alpha, TGF-beta in serum of mice with pulmonary fibrosis ₁ Is contained in the composition.

TABLE 6 Sang Juqing decoction for the treatment of pulmonary fibrosis in mice serum TNF-alpha, TGF-beta ₁ Influence of level

3.3.2 comparison of SOD, IL-6, MDA and GSH levels in pulmonary tissue

As shown in Table 7, the effect of each group on the SOD level, IL-6 level and MDA level of pulmonary fibrosis mice is compared with that of a blank group, the SOD level of pulmonary tissue of a model group is reduced, the IL-6 level and MDA level are increased, the difference is statistically significant (P is less than 0.05), and the GSH level difference is not statistically significant; compared with the model group, the Sang Juqing decoction low dose group, the Sang Juqing decoction Shang Gao dose group and the pirfenidone group all have the advantages that the SOD and GSH levels of lung tissues are increased, the MDA level is reduced, and the difference is statistically significant (P is less than 0.05).

Although there was no significant difference in changes in lung tissue IL-6 levels in pirfenidone groups (p= 0.0893) compared to model groups, sang Juqing solution low dose group, sang Juqing solution Shang Gao dose group both reduced lung tissue IL-6 levels and the difference was statistically significant (P < 0.05). It is suggested that Sang Juqing untangling can improve inflammatory and oxidative/antioxidant imbalance states in lung tissue.

TABLE 7 influence of Sang Juqing De-soup on pulmonary fibrosis mouse pulmonary tissue SOD, IL-6, MDA, GSH levels

in conclusion, the pharmacodynamic experiments show that compared with the model group, the Sang Juqing decoction-released dosing groups of mice treated by bleomycin can reduce the pulmonary fibrosis of the mice to different degrees, and the treatment effect is similar to that of positive drug pirfenidone, and the histopathological changes, szapiel scoring and Ashcroft scoring results observed by HE and MASSON staining further prove that: sang Juqing and Shang Neng delay the development of pulmonary fibrosis of mice, improve survival rate to a certain extent, have certain protection and alleviation effects on pulmonary fibrosis, and Sang Juqing decoction can reduce TNF-alpha and TGF-beta in serum of mice ₁ The expression also has the regulation and control effects on SOD, IL-6, MDA and GSH in lung tissues, and provides a new idea for developing high-quality anti-pulmonary fibrosis new drugs.

Claims

1. Application of Sang Juqing decoction in preparing medicine for preventing or treating pulmonary fibrosis is provided.

2. The use according to claim 1, wherein the Sang Juqing soup is prepared from the following components in parts by weight:

10-20 parts of white mulberry root-bark, 5-15 parts of wild chrysanthemum, 10-40 parts of semen benincasae, 5-15 parts of snakegourd peel, 5-15 parts of plantain seed and Sichuan style

5-15 parts of fritillaria, 2-10 parts of liquorice, 6-12 parts of tabasheer, 10-35 parts of reed rhizome and 5-15 parts of earthworm.

3. The use according to claim 2, wherein the Sang Juqing soup is prepared from the following components in parts by weight:

15 parts of white mulberry root-bark, 10 parts of wild chrysanthemum, 20 parts of white gourd seed, 10 parts of snakegourd peel, 10 parts of plantain seed, 10 parts of bulbus fritillariae cirrhosae,

5 parts of liquorice, 10 parts of tabasheer, 25 parts of reed rhizome and 10 parts of earthworm.

4. The use according to claim 1, wherein the pharmaceutical dosage form is a tablet, powder, mixture, pill, capsule or granule.