CN111956655B - Application of pulsatilla saponin B4 in preparation of medicine for treating chronic obstructive pulmonary disease - Google Patents
Application of pulsatilla saponin B4 in preparation of medicine for treating chronic obstructive pulmonary disease Download PDFInfo
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- CN111956655B CN111956655B CN202010840016.9A CN202010840016A CN111956655B CN 111956655 B CN111956655 B CN 111956655B CN 202010840016 A CN202010840016 A CN 202010840016A CN 111956655 B CN111956655 B CN 111956655B
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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Abstract
The invention discloses application of pulsatilla saponin B4 in preparation of a medicine for treating chronic obstructive pulmonary disease. The cigarette filter tip has a protective effect on COPD rats caused by the dropping of LPS into the trachea of cigarette combination, the mechanism of the cigarette filter tip is probably related to the regulation of IL-12/STAT4 and IL-4/STAT6 signal channels, so that the regulation of Th1/Th2 cell differentiation is realized, a good treatment effect is realized on COPD chronic inflammation and airway remodeling induced by imbalance of Th1/Th2 cell differentiation, and a theoretical basis is provided for preventing and treating chronic obstructive pulmonary diseases.
Description
Technical Field
The invention relates to the technical field of medicines. More specifically, the invention relates to an application of pulsatilla saponin B4 in preparing a medicine for treating chronic obstructive pulmonary disease.
Background
Chronic Obstructive Pulmonary Disease (COPD) is a lung disease characterized by airflow limitation that is not completely reversible, progresses progressively, and is associated with abnormal inflammatory responses of the lung to harmful gases or particles such as cigarette smoke. The disease has high morbidity, mortality and disability rate, serious harm, difficult treatment and obviously shortened life expectancy, and is one of the main diseases harming the physical and mental health of the public. Smoking-stimulated T cell-mediated autoimmune imbalance is one of possible pathogenesis of chronic obstructive pulmonary diseases, and imbalance of Th1/Th2 cell differentiation can induce COPD chronic inflammation and airway remodeling.
Pulsatillae radix is dried root of Pulsatilla chinensis (Bunge) Regel of Pulsatilla of Ranunculaceae, and has effects of clearing heat and detoxicating, cooling blood and relieving dysentery. Modern pharmacological research shows that the saponins in the pulsatilla chinensis is the main active component of the pulsatilla chinensis, and has the effects of enhancing the immune function, resisting inflammation and the like. The pulsatilla saponin B4 is pentacyclic triterpenoid saponin extracted from pulsatilla and having pharmacological activity, and has pharmacological effects of resisting inflammation, resisting bacteria, regulating immunity, resisting tumors, resisting oxidation, resisting viruses and the like, but the effect of the pulsatilla saponin B4 on treating chronic obstructive pulmonary disease is not reported.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide the application of the pulsatilla saponin B4 in preparing the medicine for treating the chronic obstructive pulmonary disease, the pulsatilla saponin B4 has a protective effect on COPD rats caused by the dropping of LPS into the trachea in combination with cigarettes, the mechanism of the pulsatilla saponin B4 can be related to the regulation of IL-12/STAT4 and IL-4/STAT6 signal pathways, so that the regulation and the regulation of Th1/Th2 cell differentiation are realized, a good treatment effect is realized on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and a theoretical basis is provided for preventing and treating the chronic obstructive pulmonary disease.
To achieve these objects and other advantages in accordance with the present invention, there is provided a use of pulsatillae radix saponin B4 for the manufacture of a medicament for the treatment of chronic obstructive pulmonary disease.
Preferably, the medicament contains the pulsatilla saponin B4 with a therapeutically effective amount and a pharmaceutically acceptable carrier.
Preferably, the medicament is formulated into a pharmaceutically acceptable dosage form.
Preferably, the medicament is formulated as an injection.
Preferably, the drug inhibits the IL-12/STAT4 and IL-4/STAT6 signaling pathways.
Preferably, the dosage of the pulsatilla saponin B4 is not less than 2.5 mg/kg-d.
The invention at least comprises the following beneficial effects:
the invention uses ELISA kit to detect the content of relevant inflammatory cytokines in rat alveolar lavage fluid, real-time fluorescence quantitative method (RT-PCR) to detect the mRNA expression level of relevant genes in rat lung tissue, and Western blot to detect the expression level of relevant proteins in rat lung tissue. The results show that compared with a model control group, the Chinese pulsatilla saponin B4 low-dose group, the Chinese pulsatilla saponin B4 medium-dose group and the Chinese pulsatilla saponin B4 high-dose group have different-degree increasing effects on the body weight of rats; the content of inflammatory cytokines in rat alveolar lavage fluid is improved to different degrees; the IL-12/STAT4 signal pathway key protein T-beta in rat lung tissue and the protein expression levels of mRNA of IL-12 and STAT4 have reducing effects in different degrees; the mRNA and protein expression levels of IL-4/STAT6 signal pathway key proteins GATA3, IL-4 and STAT6 are increased to different degrees. The cigarette composition has a protective effect on COPD rats caused by the dropping of LPS into the trachea in combination with cigarettes, the mechanism of the cigarette composition is probably related to the regulation of IL-12/STAT4 and IL-4/STAT6 signal pathways, so that the regulation and the regulation of Th1/Th2 cell differentiation are realized, a good treatment effect is realized on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and a theoretical basis is provided for preventing and treating chronic obstructive pulmonary diseases.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a graph showing the effect of pasqueoside B4 of the present invention on IL-12 in alveolar lavage fluid;
FIG. 2 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on IFN- γ in alveolar lavage fluid;
FIG. 3 is a graph showing the effect of pasqueoside B4 of the present invention on IL-4 in alveolar lavage fluid;
FIG. 4 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on T-beta/IL-12/STAT 4mRNA levels in lung tissue of COPD rats;
FIG. 5 is a graph showing the effect of pasqueflower saponin B4 of the present invention on GATA3/IL-4/STAT6 mRNA levels in lung tissue of COPD rats;
FIG. 6 is a graph showing the effect of pasqueoside B4 of the present invention on levels of T-beta/IL-12/STAT 4 protein in lung tissue of COPD rats;
FIG. 7 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on GATA3/IL-4/STAT6 mRNA levels in lung tissues of COPD rats.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Application of pulsatilla saponin B4 in preparing medicine for treating chronic obstructive pulmonary disease is provided.
The pharmaceutical preparation contains the pulsatilla saponin B4 with effective treatment amount, and the balance of pharmaceutically acceptable carriers and excipients which are nontoxic and inert to human and animals.
The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical formulation auxiliaries. The pharmaceutical preparation of the present invention is used in the form of a dose per unit body weight. The extract of the present invention can be administered to a patient in need of treatment by oral administration or injection. For oral administration, it can be made into tablet, sustained release tablet, controlled release tablet, capsule, dripping pill, pellet, suspension, emulsion, powder or granule (nanometer preparation), oral liquid, etc.; for injection, the composition can be made into sterilized aqueous or oily solution, sterile powder for injection, liposome or emulsion.
In the figures 1-7, the blank control group is abbreviated as model group, the negative control group is abbreviated as DEX 5mg/kg, the low-dose group of pulsatilla saponin B4 is abbreviated as B4.5 mg/kg, the medium-dose group of pulsatilla saponin B4 is abbreviated as B4 mg/kg, and the high-dose group of pulsatilla saponin B4 is abbreviated as 10mg/kg.
1. Material
1.1 Experimental animals
Healthy male SD rats, 60, SPF grade, body weight (180 ± 20) g, provided by the experimental animals center of the university of medical, guangxi, animal certification number: SCXK Gui 2009-0002. All experimental animals are raised in a controllable environment at the room temperature of 18-24 ℃ and the humidity of 40-50%, the animals eat and drink water freely during the experiment, and the circadian rhythm is normal.
1.2 major drugs and reagents
Pulsatillae radix saponin B4 (Jiangxi Bencao Tiangong science and technology, LLC, batch number: 2018042205); lipopolysaccharide/LPS (Sigma Co.); physiological saline (Sichuan Koran pharmaceutical Co., ltd., batch No. L219012211); ELISA assay kit (synbose); dexamethasone (Guangdong south China pharmaceutical industry group Co., ltd., code: A14202002484).
1/1000 precision balance (Metler-Tollido instruments Shanghai, inc., model: ME 204E); low temperature high speed centrifuge (Eppendorf Co., model 5425R); 722sp visible spectrophotometer (shanghai prism technologies ltd); an enzyme-linked immunoassay instrument (BioTek company, USA, model: SYNERGYH 1).
2. Method of producing a composite material
2.1 establishment and grouping of animal models
60 healthy male SD rats are adaptively fed for 1 week and are randomly divided into 6 groups of 10 rats each, namely a blank control group, a model control group, a positive control group (DEX, 5 mg/kg), pulsatilla saponin B4 (2.5 mg/kg), pulsatilla saponin B4 (5 mg/kg) and pulsatilla saponin B4 (10 mg/kg). After grouping, marking and numbering the rats in each group, and feeding the rats in groups with feed and normal drinking water.
A rat model of chronic obstructive pulmonary disease is replicated by adopting a method of smoking cigarettes and instilling LPS in trachea. Molding a model control group, a positive control group (DEX, 5 mg/kg), pulsatilla saponin B4 (2.5 mg/kg), pulsatilla saponin B4 (5 mg/kg) and pulsatilla saponin B4 (10 mg/kg). In a normal room temperature environment, model rats are anesthetized on days 1 and 14 in an experiment, LPS (1 mg/mL) is instilled through a trachea after the model rats are anesthetized, and on days 2-28 (no smoking is performed on the day of dropping LPS), the model control group, the negative control group (dexamethasone), the anemone saponin B4 (2.5 mg/kg), the anemone saponin B4 (5 mg/kg) and the anemone saponin B4 (10 mg/kg) are given to the rats to smoke one cigarette for one time per day, and each rat smokes for 1 hour. The passive smoking method comprises the following steps: the rat is put into an autonomous smoking box, and the cigarette is ignited from the smoke inlet hole at one side, so that the rat is passively smoked and infected. A proper amount of silica gel desiccant is placed at the bottom of the box to reduce the influence of water vapor generated by cigarette combustion on rats. The drug treatment is carried out on the groups from the 4 th day of the experiment to the 3 rd day after the molding is finished, namely 4 to 31 days, and the total time is 28 days. The blank control group and the model control group are given physiological saline with equal volume in the tail vein every day; 2.5mg/kg,5mg/kg and 10mg/kg of pulsatilla chinensis saponin B4 are respectively injected into each group of pulsatilla chinensis saponin B4 through tail vein; the positive control group was administered with Dexamethasone (DEX) at 5mg/kg intraperitoneally once daily for 28 consecutive days.
2.2 taking materials
(1) Alveolar lavage fluid (BLAF) collection: after blood sampling of abdominal aorta, cutting off neck skin of rat, exposing organs, continuously opening thoracic cavity, stripping thymus gland and pericardium covering lung tissue, finding out trachea branch, ligating right main bronchus, injecting into left main bronchus and left lung through trachea with 6mL of 0.9% sterile normal saline, slowly and repeatedly pumping back for 4 times, centrifuging at4 ℃,3000r/min for 10min, sucking supernatant, placing in 2mL cryostraw, and storing at-80 ℃. After cell pellets obtained after centrifugation of alveolar lavage fluid were resuspended in 1mL sterile PBS, they were stored in 2mL cryovials and stored in a refrigerator at4 ℃ for white blood cell counting and differential counting.
(2) Lung tissue fixation method: ligating the left lung, loosening the right lung, injecting 5mL of 4% paraformaldehyde solution through the trachea, fixing for 30s, taking out the right lung middle lobe, and storing in 4% paraformaldehyde. For HE staining.
(3) Collecting lung tissue, removing alveolar lavage fluid and fixing lung tissue, taking out the rest lung tissue, placing in a cryopreservation tube, storing at-80 ℃, and using for Western blotting and mRNA detection.
2.3 detection of IL-12, IFN-. Gamma.and IL-4 in alveolar lavage fluid
Diluting alveolar lavage fluid supernatant by 10 times, measuring the contents of IL-12, IL-12R, IFN-gamma and IL-4 in BALF by enzyme linked immunosorbent assay (ELISA), and carrying out the experimental steps according to the operation of an ELISA kit specification.
2.4 detection of mRNA levels of T-beta/IL-12/STAT 4 and GATA3/IL-4/STAT6 in Lung tissue
Taking out the lung tissue stored in an ultra-low temperature refrigerator at minus 80 ℃, adding 1mL Trizol reagent into every 50-100 mg of the tissue, and cracking the tissue in a homogenizer; centrifuging, removing the precipitate, taking the supernatant into a new EP tube, adding 0.2mL of chloroform ice, standing for 1-5 min, centrifuging at 12000g and 4 ℃ for 10min, taking the upper aqueous phase into the new EP tube, adding isopropanol with the same volume, fully mixing uniformly, placing on ice for 10min, centrifuging at 12000g and 4 ℃ for 10min, removing the supernatant, and adding 1mL of 75% ethanol for washing; 12000g, centrifuging at4 deg.C for 5min, discarding supernatant, sucking liquid on the tube wall as much as possible, drying at room temperature for 5-10min, adding DEPC water to dissolve RNA, and storing in refrigerator at-80 deg.C. The expression level of each mRNA was detected by a fluorescent real-time quantitative RT-PCR instrument.
2.5 detection of protein levels of T-beta/IL-12/STAT 4 and GATA3/IL-4/STAT6 in Lung tissue
Lung tissue was lysed with RIPA lysate and 1mL RIPA and 10. Mu.L PMSF and 10. Mu.L phosphatase inhibitor were added per 100mg of tissue. The homogenizing machine is used for homogenizing, and supernatant is taken after centrifugation. Quantifying by using a BCA kit, diluting a sample, adding the sample, performing electrophoresis, 75V,30min,110V, and 1h; transferring the membrane, keeping the pressure at 70V for 2h, sealing, incubating the primary antibody overnight, washing the membrane for 10 min/time by TBST (tert-butyl-tert-butyl ether) for 3 times; after incubation at room temperature with secondary antibody, 1h, TBST was washed, and developed.
2.6 statistical treatment
The single-factor analysis of variance and t-test data were performed using STATA8.0 statistical software, with P <0.05 as the difference, which was statistically significant.
3. As a result, the
3.1 Effect of Pulsatillae saponin B4 on body weight of COPD rats
Compared with the blank group, the 14d and 28d of the model control group are obviously reduced after the model is subjected to body weight modeling, and the anemonin B4 and the dexamethasone positive drug group can obviously improve the weight of the rat, so that the anemonin B4 has an improvement effect on the weight reduction of the COPD rat, and the details are shown in Table 1.
TABLE 1
P <0.05 compared to model control
3.2 Effect of Pulsatillae saponin B4 on detection of IL-12, IFN-gamma and IL-4 in COPD rat alveolar lavage fluid
After modeling, the IL-2 and IFN-gamma levels in the alveolar lavage fluid of the model control group are obviously increased, which indicates that LPS (lipopolysaccharide) can promote the secretion of Th1 type cytokines of a COPD rat model through smoking cigarette and instilling air pipe; pulsatillae radix saponin B4 (5 mg/kg), pulsatillae radix saponin B4 (10 mg/kg), positive control group (DEX, 5 mg/kg) significantly reduced IL-1 and IFN- γ levels in alveolar lavage fluid, and pulsatillae radix saponin B4 (10 mg/kg) was more significant than positive control group (DEX, 5 mg/kg), indicating that pulsatillae radix saponin B4 has a down-regulating effect on Th1 type cytokines, see fig. 1, fig. 2, P <0.05, <0.01, > P <0.001, in fig. 2, compared to the model control group, > P <0.05, > P <0.01. The IL-4 level in the alveolar lavage fluid of the model control group is obviously reduced, the IL-4 level in the alveolar lavage fluid is obviously increased by the pulsatilla saponin B4 (5 mg/kg), the pulsatilla saponin B4 (10 mg/kg) and the positive control group (DEX, 5 mg/kg), and the IL-4 level is shown in figure 3, wherein P is less than 0.05 and P is less than 0.01 in the figure 3 compared with the model control group. The results show that the anemoside B4 has a regulating effect on the expression of cytokines in alveolar lavage of rats with chronic obstructive pulmonary diseases, and the high-dose effect is obvious.
3.3 Effect of Pulsatillae saponin B4 on T-beta/IL-12/STAT 4mRNA levels in COPD rat Lung tissues
Compared with a blank control group, the T-beta/IL-12/STAT 4mRNA level in the lung tissue homogenate of the model control group is obviously increased, the T-beta, IL-12 and STAT4mRNA levels in the lung tissue of COPD rats can be obviously reduced by the pulsatilla saponin B4 (2.5 mg/kg), the pulsatilla saponin B4 (5 mg/kg), the pulsatilla saponin B4 (10 mg/kg) and the positive control group (DEX, 5 mg/kg), and detailed results are shown in figure 4, and compared with the model control group, P is less than 0.05, and P is less than 0.01 in figure 4.
3.4 Effect of Pulsatillae saponin B4 on mRNA levels of GATA3/IL-4/STAT6 in COPD rat Lung tissues
Compared with a blank control group, the lung tissue homogenate of the model control group has obviously increased GATA3/IL-4/STAT6 mRNA level, and the pulsatilla saponin B4 (2.5 mg/kg), pulsatilla saponin B4 (5 mg/kg), pulsatilla saponin B4 (10 mg/kg) and positive control group (DEX, 5 mg/kg) can obviously reduce the GATA3/IL-4/STAT6 mRNA level in the lung tissue of COPD rats, and detailed in figure 5, compared with the model control group, P is less than 0.05, P is less than 0.01, and P is less than 0.001.
3.5 Effect of Pulsatillae saponin B4 on levels of T-beta/IL-12/STAT 4 protein in COPD rat Lung tissue
Compared with a blank control group, the levels of T-beta/IL-12/STAT 4 in the lung tissue homogenate of the model group are obviously increased, and the levels of T-beta, IL-12 and STAT4 in the lung tissue of COPD rats can be obviously reduced by pulsatilla saponin B4 (2.5 mg/kg), pulsatilla saponin B4 (5 mg/kg), pulsatilla saponin B4 (10 mg/kg) and a positive control group (DEX, 5 mg/kg), as shown in figure 6.
3.6 Effect of Pulsatillae saponin B4 on GATA3/IL-4/STAT6 protein levels in COPD rat Lung tissues
Compared with a blank control group, the levels of GATA3/IL-4/STAT6 in lung tissue homogenate of the model group are obviously increased, and the levels of GATA3/IL-4/STAT6 in lung tissue of COPD rats can be obviously reduced by pulsatilla saponin B4 (2.5 mg/kg), pulsatilla saponin B4 (5 mg/kg), pulsatilla saponin B4 (10 mg/kg) and a positive control group (DEX, 5 mg/kg), as shown in detail in figure 7.
4. General theory of the invention
Experimental results show that the pulsatilla saponin B4 has a protective effect on COPD rats caused by LPS (lipopolysaccharide) instillation in cigarette combined trachea, the mechanism of the pulsatilla saponin B4 can regulate IL-12/STAT4 and IL-4/STAT6 signal pathways, so that Th1/Th2 cell differentiation is regulated and regulated, a good treatment effect is achieved on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and a theoretical basis is provided for preventing and treating chronic obstructive pulmonary diseases.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (4)
1. Application of pulsatilla saponin B4 in preparing medicine for treating chronic obstructive pulmonary disease is provided.
2. The use according to claim 1, wherein the medicament comprises a therapeutically effective amount of pasqueflower saponin B4 and a pharmaceutically acceptable carrier.
3. The use of claim 2, wherein the medicament is formulated into a pharmaceutically acceptable dosage form.
4. The use according to claim 3, wherein the medicament is formulated as an injection.
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