CN117017960A - Application of Caryopterin A in preparation of medicines for treating pulmonary inflammation - Google Patents
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- CN117017960A CN117017960A CN202311183381.7A CN202311183381A CN117017960A CN 117017960 A CN117017960 A CN 117017960A CN 202311183381 A CN202311183381 A CN 202311183381A CN 117017960 A CN117017960 A CN 117017960A
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24D—CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES FOR CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
- A24D3/00—Tobacco smoke filters, e.g. filter-tips, filtering inserts; Filters specially adapted for simulated smoking devices; Mouthpieces for cigars or cigarettes
- A24D3/06—Use of materials for tobacco smoke filters
- A24D3/061—Use of materials for tobacco smoke filters containing additives entrapped within capsules, sponge-like material or the like, for further release upon smoking
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Pulmonology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Toxicology (AREA)
- Materials Engineering (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to the technical field of biological medicines, in particular to application of Caryopterin A in preparation of a medicine for treating pulmonary inflammation. The invention provides an application of Caryopterin A in preparing a medicament for treating pulmonary inflammation. The experiment provided by the invention shows that the Caryopterin A has good anti-inflammatory effect, is in a dose-dependent relationship, and has excellent technical effect on treating lung inflammation caused by cigarette smoke.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of Caryopterin A in preparation of a medicine for treating pulmonary inflammation.
Background
Long-term inhalation of cigarette smoke or other toxic particulate matter can cause changes in lung structure and weakness of lung function, and clinically presents chronic bronchitis, emphysema, pulmonary fibrosis and the like. Smoking is an important factor causing chronic obstructive pulmonary disease, and can cause degeneration of lung cells and damage of airway mucosa, which can cause poor sputum excretion of patients, bacterial colonization, high responsiveness, leading to airway epithelial barrier function, and increase oxidative stress, promoting airway epithelial cell aging and activation of pro-inflammatory signaling pathway.
Studies have shown that when a smoker's lungs are infected, the immune function of the body is significantly reduced, bacterial pneumonia and severe pneumonia occur in the lungs with a high probability, and the mortality rate of the illness is increased. When a smoker inhales the irritant gas for a long time, the cigarette smoke can destroy the sensory nerve endings of the lungs, so that the discharge of the secretion of the respiratory tract is blocked, the secretion mucus of glands is increased, and the purifying function of bronchus mucous membrane is destroyed; in addition, smoking can activate lung inflammatory cell activity, trigger respiratory tract inflammatory reaction, cause chronic lung inflammation, and increase lung infection incidence.
The chronic obstructive pulmonary disease is a heterogeneous pulmonary state, is characterized by chronic respiratory tract symptoms such as dyspnea, cough, expectoration and the like, is characterized by persistent airflow obstruction caused by airway abnormality such as bronchitis and bronchiolitis and pulmonary alveolus abnormality such as emphysema and the like, and has the characteristics of high morbidity, high disability rate and high mortality rate. Because of complex pathogenesis, specific therapeutic drugs are still lacking in clinic at present, so that the discovery of novel chronic obstructive pulmonary disease resistant drugs with novel structures and unique action mechanisms is one of important tasks of current pharmaceutical research.
Disclosure of Invention
The invention aims to provide an application of Caryopterin A in preparing a medicine for treating pulmonary inflammation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of Caryopterin A in preparing a medicament for treating pulmonary inflammation.
Preferably, the chemical formula of the Caryopterin is shown in formula 1.
Preferably, the lung inflammation is a lung inflammation caused by cigarette smoke.
The invention also provides an application of the Caryopterin A in preparing cigarettes or cigarette products.
Preferably, the smoking article is a cigarette additive, a cigarette filter or a holder.
The mongolian Caryopter factor of the invention is derived from an article published in the Natural product research journal book entitled "Anew abietane diterpene tuurgan a from caryopteris mongholica".
Compared with the prior art, the invention has the following beneficial effects:
the results of the invention show that under the stimulation of CSE, the expression of inflammatory factors such as IL-6, IL-8, TNF-alpha, MCP-1, CXCL10, MIP-1 alpha, MIP-1 beta mRNA and the like in the supernatant of A549 cells is obviously increased, which indicates that the A549 cells are in an inflammatory injury state and similar to inflammatory reaction of patients with COPD; the expression of inflammatory factors such as IL-6, IL-8, TNF-alpha, MCP-1, CXCL10, MIP-1 alpha, MIP-1 beta mRNA and the like in the supernatant of the A549 cells can be inhibited after the intervention of the Mongolian Caryopterin A, which shows that the Mongolian Caryopterin A has good anti-inflammatory effect and is in a dose-dependent relationship. Experiments prove that the Caryopterin A has excellent technical effect of treating lung inflammation caused by cigarette smoke.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing IL-6 content of different experimental groups of example 1;
FIG. 2 is a graph showing TNF- α levels of the various experimental groups of example 1;
FIG. 3 is a graph showing IL-8 content of different experimental groups of example 1;
FIG. 4 is a graph showing the expression level of IL-6mRNA in different experimental groups of example 2;
FIG. 5 is a graph showing the expression level of IL-8mRNA in different experimental groups of example 2;
FIG. 6 is a graph showing the expression level of TNF-. Alpha.mRNA in different experimental groups of example 2;
FIG. 7 is a graph showing the expression level of MCP-1mRNA in different experimental groups of example 2;
FIG. 8 is a graph showing MIP-1β mRNA expression levels of different experimental groups of example 2;
FIG. 9 is a graph showing the MIP-1α mRNA expression level of the different experimental groups of example 2;
FIG. 10 is a graph showing the expression level of CXCL10mRNA in different experimental groups according to example 2;
FIG. 11 is a graph showing the results of immunofluorescence experiments performed in different experimental groups of example 3;
FIG. 12 is a graph showing the effect of cytokine release from different experimental groups of example 3;
FIG. 13 is a bar chart showing the results of protein expression associated with the intervention of Caryopterin A on CSE-induced A549 cells in example 4;
FIG. 14 is an electrophoretogram of the related protein expression results of the Caryopterin A for CSE induced A549 cell intervention of example 4;
FIG. 15 shows the effect of varying concentrations of CSE on cell viability in example 1.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1.1 preparation of sample solution and administration concentration
Precisely weighing Caryopterin 0.1mol, adding 0.1L dimethyl sulfoxide (DMSO) for dissolving to obtain Caryopterin solution with Caryopterin concentration of 1mol/L, filtering with sterile filter head, adding cell culture medium (90% DMEM culture medium+10% HAKATA foetal calf serum+1% streptomycin) to obtain Caryopterin solution of 0.0 μm, 10 μm, 100 μm and 1000 μm, and preserving at 4deg.C for use.
1.2 preparation and screening of Cigarette Smoke Extract (CSE)
A cigarette (tar content: 10mg, smoke nicotine content: 1.0mg, smoke carbon monoxide content: 11 mg) is connected to a silica gel tube with similar diameter, the other end of the silica gel tube is connected with a 50mL syringe, the cigarette is lighted, the cigarette smoke extraction speed is similar to that of a normal person, 25mL of the cigarette smoke is injected into a 50mL centrifuge tube containing 10mL of culture medium (DMEM serum-free culture medium) each time, the mixture is fully and uniformly mixed, the cigarette smoke is completely dissolved in the culture medium, the culture medium containing the cigarette smoke is obtained by 8 times according to the method, and the culture medium containing the cigarette smoke is defined as CSE stock solution after filtration and sterilization by a 0.22 mu m sterile filter membrane, namely 100% CSE stock solution.
A549 cells were prepared according to 5×10 5 Density of wells/wells were incubated in 96-well plates at 37℃for 18h with 5%, 10%, 20%, 30% and 40% final concentrations of CSE stock (each diluted with DMEM medium) as a blank with an equal amount of DMEM medium added. The effect of blank and varying concentrations of CSE on cell viability was determined by the CCK-8 method. Results: the levels of inflammatory factors IL-8, IL-6 and TNF- α were significantly increased (P < 0.01) in the supernatant of model A549 cells after 10% stimulation with CSE compared to the placebo group, confirming an optimal CSE concentration of 10% (FIG. 15, statistically significant from 10%).
1.3 Effect of Caryopterin A on inflammatory injury of CSE-induced A549 cells
At optimal CSE concentration (10%), model groups were incubated with 0.0, 10.0, 100.0, 1000.0. Mu.M of a sample solution of Caryopterin for 24h, and the amounts of the pro-inflammatory factors IL-6, IL-8 and TNF-. Alpha.in the supernatants of the control group (control) and the cell culture of each of the model groups were determined (following the protocol).
Results: as shown in fig. 1 to 3, compared with the model group (10% cse, 0.0 group of mongolian Caryopterin), the administration of the mongolian Caryopterin A at three concentrations of 10 mu m, 100 mu m and 1000 mu m can obviously reduce the content of IL-6, TNF-alpha and IL-8 to obviously raise the level (P is less than 0.01), and the mongolian Caryopterin A is in a dose-dependent relationship, so that the mongolian Caryopterin A has a good anti-inflammatory effect.
Example 2
RT-PCR experiments
Example 1 after 24h of administration, a portion of the supernatant from each cell group was carefully aspirated, total RNA from each cell group was extracted by Trizol procedure, the concentration and purity of RNA was measured using Nanodrop 2000, and RNA at an excessive concentration was diluted in an appropriate ratio to a final concentration of 1000 ng/. Mu.L and reverse transcribed (10. Mu.L system) to cDNA at 25℃300s,42℃180 s,85℃5s. The primer sequences and reaction system solutions are shown in tables 1 and 2, and the change of the gene expression level is calculated by using GAPDH as an internal reference and a ΔΔct method (wherein a=ct (target gene, sample to be measured) -CT (internal reference gene, sample to be measured), b=ct (target gene, control sample) -CT (internal reference gene, control sample), k=a-B, and expression multiple=2-K).
TABLE 1 primer sequences for related genes
| Cytokines and methods of use | Primer sequences |
| IL-6-F | CGGGAACGAAAGAGAAGCTCTA |
| IL-6-R | CGCTTGTGGAGAAGGAGTTCA |
| IL-8-F | TTGGCAGCCTTCCTGATTTC |
| IL-8-R | TATGCACTGACATCTAAGTTCTTTAGCA |
| TNF-α-F | AACATCCAACCTTCCCAAACG |
| TNF-α-R | GACCCTAAGCCCCCAATTCTC |
| MCP-1-F | CAAGCAGAAGTGGGTTCAGGAT |
| MCP-1-R | AGTGAGTGTTCAAGTCTTCGGAGTT |
| CXCL10-F | CCACGTGTTGAGATCATTGCT |
| CXCL10-R | TGCATCGATTTTGCTCCCCT |
| MIP-1α-F | CTGCATCACTTGCTGCTGACA |
| MIP-1α-R | CACTGGCTGCTCGTCTCAAAG |
| MIP-1β-F | AAAACCTCTTTGCCACCAATACC |
| MIP-1β-R | GAGAGCAGAAGGCAGCTACTG |
| GAPDH-F | CAAGCAGAAGTGGGTTCAGGAT |
| GAPDH-R | AGTGAGTGTTCAAGTCTTCGGAGTT |
As shown in SEQ NO:1 to 16.
Table 2 reaction system liquid preparation table
| Component (A) | Dosage of |
| 2SYBR Gree qPCR Master Mix | 5.0μl |
| Forward primer | 0.2μl |
| Reverse primer | 0.2μl |
| Nuclease-Free Water | 2.6μl |
| cDNA | 2.0μl |
| Totals to | 10.0μl |
The results are shown in FIGS. 4 to 10: after stimulation of A549 cells by CSE, the inflammatory factors IL-6, IL-8, TNF- α, MCP-1, MIP-1β, MIP-1α and CXCL10 of the cells were increased in mRNA expression (control and CSE groups); after being treated by Caryopterin A, each administration group obviously inhibits the mRNA expression of inflammatory factors IL-6, IL-8, TNF-alpha, MCP-1, MIP-1 beta, MIP-1 alpha and CXCL 10; and is dose dependent. The Mongolian Caryopteris can inhibit the expression of inflammatory factors, thereby achieving good anti-inflammatory effect.
Example 3
Immunofluorescence experiment of Paraffin section
Example 1 after 24 hours of dosing, a portion of the supernatant from each cell group was carefully aspirated, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 15min; washing with PBS, and treating with 0.5% Triton (X-100) at room temperature for 10min to rupture cell membranes, rinsing with PBS 3 times, 5 min/time. After the sections were slightly dried, a circle was drawn around the tissue with a histochemical pen (to prevent antibody from running away), 3% BSA was added dropwise into the circle to cover the tissue uniformly, and the tissue was blocked at room temperature for 30min. The blocking solution is gently thrown away, PBS is dripped on the slice, the slice is horizontally placed in a wet box for incubation at 4 ℃ for overnight. The slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping secondary antibody covering tissues (primary antibody: p-NF-kB (Affinity, rabbit antibody)) corresponding to the primary antibody into the circle after the sections are slightly dried, and incubating for 50min at room temperature in a dark place. The slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping DAPI dye solution into the ring after the slices are slightly dried, and incubating for 10min at room temperature in a dark place. The slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (5) after the slices are slightly dried, sealing the slices by using an anti-fluorescence quenching sealing tablet. The sections were observed under a nikon inverted fluorescence microscope and images were acquired as shown in fig. 11. (ultraviolet excitation wavelength 330-380 nm, emission wavelength 420nm, FITC green excitation wavelength 465-495 nm, emission wavelength 515-555 nm, CY3 red excitation wavelength 510-560, emission wavelength 590 nm).
Results: FIGS. 11-12 show immunofluorescence experiments that, compared with the normal group, after stimulation of A549 cells by CSE (model group), NF- κB enters the nucleus and up-regulates the expression of phosphorylated NF- κ B p 65; compared with the model group, after the intervention of the Caryopterin A (the final concentration is 1000 mu M), the NF- κB can be obviously inhibited from entering the cell nucleus.
Example 4
Westernblot experiment
Example 1 after 24h of administration, a portion of the supernatant from each cell group was carefully aspirated, well lysed (on ice) and the total protein was extracted by high-speed freeze centrifugation (13000 g centrifugation at 4 ℃ C. For 5 min). The total protein concentration was determined by BCA method, 4:1 adding loadingbuff-er (5×), mixing thoroughly, boiling in water for 10min, cooling in ice bath, centrifuging (13000 g centrifuging at 4deg.C for 5 min), running electrophoresis (sodium dodecyl sulfate-polyacrylamide gel), wet rotary molding, and sealing at room temperature.
Taking out the PVD film, washing the sealing liquid, and placing 1: incubating for one pot at 4 ℃ in the primary antibody solution prepared in proportion of 1000; washing sealing liquid, and placing 1:5000 proportion of secondary antibodies (four primary antibodies are respectively NF-kB P65 (P65/GAPDH group) (Servicebio, rabbit antibody), P-NF-kB P65 (P-P65/GAPDH group) (CST, rabbit antibody), ERK (Servicebio, rabbit antibody), P-ERK (ABclonal, rabbit antibody) and secondary antibodies (goat anti-rabbit) are prepared, incubated for 1h at room temperature, and then the sealing solution is washed, exposed, developed and fixed. The X-ray film is washed by tap water, naturally aired, scanned and the optical density value of the target strip is analyzed by image processing software.
Statistical analysis
Statistical analysis of experimental data between the two groups was performed using IBM SPSS Statistics software, with P < 0.05 considered statistically significant differences.
Results: the expression level of the related protein in a549 cells was detected by western blotting. The results are shown in FIGS. 13 and 14 (FIG. 13 groups: control, CSE, 10. Mu.M, 100. Mu.M, 1000. Mu.M Caryopterin A; FIG. 14 lighter the band color, lower the protein expression level), compared with the normal group, ERK1/2MAPK signal pathway is activated after CSE induction of A549 cells, and pERK1/2 content is significantly increased; compared with the model group, the content of pERK1/2 is obviously reduced after the intervention of the Caryopterin A.
In order to determine whether the abietane diterpenoid components in the mongolian Caryopteris have the activity of resisting chronic obstructive pulmonary disease, the invention adopts a cigarette smoke extract to induce A549 cells to establish an in vitro inflammatory injury model, takes the content of inflammatory factors, mRNA level and protein level of cell supernatant as evaluation indexes, and preliminarily examines the inflammatory injury protection effect of the mongolian Caryopteris A.
NF-. Kappa.B is a multifunctional nuclear transcription factor that exists predominantly in the form of the P65 subunit in lung tissue. When lung tissue is stimulated by various cytokines, viruses, harmful particles and the like, the lung tissue enters the cell nucleus and is combined with a kappa B site on DNA to start transcription of various inflammatory response genes to participate in various biological responses. MAPK comprises 3 main families such as ERK1/2, JNK, P38MAPK and the like, and a signal pathway mediated by MAPK is involved in various physiological and pathological processes such as inflammatory response, cell proliferation, apoptosis and the like. After stimulating A549 cells by CSE, NF- κB moves into the nucleus and combines with target genes, activates MAPK signal pathway, activates transcription of various inflammatory genes, causes inflammatory reaction, and causes inflammatory reaction of lung tissue and airway. When the Caryopteris clathrata A interferes with the A549 cells induced by CSE, the P-P65 can be obviously inhibited from entering the nucleus, and the phosphorylation degree of NF-kappa B P65 and ERK1/2MAPK can be reduced.
Taken together, the mongolian Caryopterin can inhibit the inflammatory response of A549 cells induced by CSE, and is dose-dependent, and the action mechanism of the mongolian Caryopterin is related to the inhibition of NF- κB and ERK1/2MAPK phosphorylation.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. Application of Caryopterin A in preparing medicine for treating pulmonary inflammation is provided.
2. The use according to claim 1, wherein the lung inflammation is a lung inflammation caused by cigarette smoke.
3. The application of Caryopterin A in preparing cigarette or cigarette product is provided.
4. Use according to claim 3, wherein the smoking article is a cigarette additive, a cigarette filter or a mouthpiece.
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