CN110664797A

CN110664797A - Application of isorhamnetin in preparation of medicine for inhibiting pulmonary artery smooth muscle cell proliferation

Info

Publication number: CN110664797A
Application number: CN201910871896.3A
Authority: CN
Inventors: 周茹; 常智; 郝银菊; 周玮; 徐清斌; 王佳玲; 裴延敏; 余洋
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2020-01-10

Abstract

The invention discloses an application of Isorhamnetin (Isorhamnetin) in preparing a medicament for inhibiting pulmonary artery smooth muscle cell proliferation. The experimental result shows that when the aloperine injection is used in a safe dosage range, the aloperine with the dosage of 100 mu M can obviously inhibit the proliferation of human pulmonary artery smooth muscle cells, obviously regulate the expression of BMP signal channel related protein, prove that the isorhamnetin can inhibit the pulmonary vascular remodeling and further inhibit the pulmonary hypertension, the cardiovascular malformation or the chronic obstructive pulmonary disease.

Description

Application of isorhamnetin in preparation of medicine for inhibiting pulmonary artery smooth muscle cell proliferation

Technical field

The invention belongs to the field of cardiovascular diseases, and particularly provides application of isorhamnetin in preparation of a medicament for inhibiting pulmonary artery smooth muscle cell proliferation.

Two background art

Pulmonary vascular remodeling refers to a pathological change process of a vascular wall tissue structure and function after a pulmonary artery is subjected to various injuries and stimulation, and the pathological change process comprises smooth muscle cell proliferation, collagen deposition, small vessel lumen closure and the like. Pulmonary vascular remodeling may be caused by immune diseases such as kawasaki disease, hypoxic-ischemic state, exogenous toxic substances such as smoking, etc., which are clinical syndromes characterized by pulmonary hypertension (PAH), a group of which are progressively increased in pulmonary artery pressure and pulmonary vascular resistance, eventually leading to right heart failure, and the pathogenesis of which is complex, the prognosis is poor, and no effective delay or treatment drugs are available, cardiovascular malformation, Chronic Obstructive Pulmonary Disease (COPD), etc., which are physiological bases of various cardiopulmonary diseases.

Isorhamnetin (ISO) is a known flavonoid compound and widely exists in flowers, fruits and leaves of various plants such as ginkgo biloba, sea buckthorn and the like. In recent years, researches show that isorhamnetin has various cardiovascular and cerebrovascular protecting effects of protecting endothelium, resisting atherosclerosis, resisting myocardial ischemia, protecting cardiac muscle cells, inhibiting cardiac muscle fibrosis, lowering blood pressure, resisting thrombus, platelet aggregation, resisting oxidation and the like, and also has various effects of inhibiting adipocyte differentiation, resisting anoxia, resisting tumors, lowering blood sugar, resisting inflammation, resisting viruses and the like. Studies have shown that isorhamnetin dose-dependently inhibits noradrenaline-promoted vascular smooth muscle cell proliferation and DNA synthesis. The pharmacological action of isorhamnetin on the cardiovascular system is a scientific problem which has been concerned by the subject group for a long time, and the previous researches show that: isorhamnetin has protective effect on pulmonary hypertension induced by monocrotaline. However, the mechanism of action of isorhamnetin on the proliferation of pulmonary artery smooth muscle cells and pulmonary vascular remodeling is not reported at present. The medicine has extremely high potential value and social significance when being developed into the medicine for treating diseases such as pulmonary hypertension and the like.

Disclosure of the invention

The research of the applicant finds that isorhamnetin can inhibit the proliferation of pulmonary artery smooth muscle cells, and the mechanism of isorhamnetin is probably to inhibit the expression of BMP signal channel related protein. On the basis, the isorhamnetin can be used for treating pulmonary vascular reconstruction and further inhibiting pulmonary hypertension, heart blood malformation or COPD.

In one aspect, the application provides a use of isorhamnetin in preparing a medicament for inhibiting lung smooth muscle cell proliferation, wherein the structural formula of the isorhamnetin is shown as a formula (1):

further, the single application dose of isorhamnetin is a dose which does not cause central inhibition.

Further, isorhamnetin is applied at a concentration of 25-100 μ M to human pulmonary artery smooth muscle cells.

Further, isorhamnetin was used at a concentration of 25. mu.M.

Further, isorhamnetin was used at a concentration of 50. mu.M.

Further, isorhamnetin was used at a concentration of 100. mu.M.

Further, inhibition of lung smooth muscle cell proliferation can inhibit pulmonary vascular remodeling.

Further, inhibition of pulmonary vascular remodeling can inhibit pulmonary hypertension, cardiac malformation, or COPD.

In another aspect, the present invention provides a medicament for inhibiting lung smooth muscle cell proliferation, which comprises isorhamnetin as the only active ingredient and pharmaceutically acceptable excipients.

Further, the drug can inhibit pulmonary vascular remodeling and further inhibit pulmonary hypertension, cardiovascularisation or COPD.

The application of isorhamnetin as a medicament for treating pulmonary hypertension provided by the invention has the following beneficial effects:

(1) isorhamnetin can obviously inhibit the proliferation of human pulmonary artery smooth muscle cells;

(2) isorhamnetin can inhibit the expression of BMP signal channel related protein.

Description of the four figures

FIG. 1: figure showing the effect of isorhamnetin on the proliferation rate of human pulmonary artery smooth muscle cells;

FIG. 2: effect of isorhamnetin on TNF- α -induced levels of BMPR2 protein expression in pulmonary artery smooth muscle cells (compared to normal group:^##P<0.01, comparison with model group:^＊P<0.05，^＊＊P<0.01(

n＝6))；

FIG. 3: effect of isorhamnetin on TNF- α -induced expression levels of p-smad1/5 protein in pulmonary artery smooth muscle cells (compared to normal group:^##P<0.01, comparison with model group:^＊P<0.05，^＊＊P<0.01(

n＝6))；

FIG. 4: effect of isorhamnetin on the TNF- α induced expression level of Id1 mRNA in pulmonary artery smooth muscle cells (compared to normal group:^##P<0.01, comparison with model group:^＊P<0.05，^＊＊P<0.01(

n＝6))；

FIG. 5: effect of isorhamnetin on the TNF- α induced expression level of Id3 mRNA in pulmonary artery smooth muscle cells (compared to normal group:^##P<0.01, comparison with model group:^＊P<0.05，^＊＊P<0.01(

n＝6))。

detailed description of the preferred embodiments

The present invention will be described in further detail with reference to examples, in which the aloperine used is a compound represented by the above formula (1).

Example 1

The isorhamnetin is used as a medicine for treating pulmonary hypertension, wherein the single application dose of the isorhamnetin is 100 mu M of human pulmonary smooth muscle cells, and the dosage form of the medicine is a solution dosage form.

Example 2

Example 3

The effects of examples 1 to 3 can be confirmed by the following animal experiments and the results thereof:

first, experimental material

1.1 cells

Human pulmonary artery smooth muscle cells (purchased from Shanghai Qiao Xinzhou Biotech Co., Ltd.)

1.2 major drugs and reagents

Isorhamnetin (from Beijing Zhongke biologics), DMEM (from Gibco, 11965092, USA), fetal bovine serum (from Gibco, C2027010, USA), trypsin (from Gibco, 15050065, USA), CCK-8 (M8180-250, Beijing Solebao Tech technologies, Inc.), dimethyl sulfoxide (DMSO) (from Sigma, D8418, USA), whole protein extraction kit, protein content determination kit (from Kyoto Kagyo Biotech GmbH, KGP2100, KGPBCA), TEMED (from Sigma, T8090, Ammonium Persulfate (AP) (from Sigma, A8090, 10% SDS (Shanghai Tongshan, P61014), 30% acrylamide (from Double-Helix, P31031), protein loading buffer 5X (from Beijing Biotech, P114, Kyowa Bioluminescence substrates, USA 6283, Adnve-10), antibody BMPR2(Cat NO: ab-6671) was purchased from AbcamBiotechnology, Inc. (CA, USA), and p-smad1/5(Cat NO:41D10) was purchased from Cell Signaling technology (MA, USA). RNA reverse transcription kit and TransStart Tip Green qPCR SuperMix from TransGen Biotech, Beijing.

1.3 Main instruments

Ultra clean bench (SW-CJ-2FD, Antai technologies, Suzhou), carbon dioxide incubator (Thermoscinentific, USA), vertical pressure steam sterilizer (Shanghai Shenan medical treatment facility), centrifuge (Hypothermia, Z323K, Siemens Co.), water bath cabinet (constant temperature, DK-420S, Shanghai Jinghong laboratory facilities Co.), microplate reader (Model 550, Thermo scientific, USA), BH-N I C-B type inverted microscope (Japan Olympus optical Co.), laser confocal microscope (TCS-SP, Germany LEICA), electronic analytical balance (AL104 Switzerland Mettler-Toleco Group Co.), cryogenic refrigerator (BCD-215KCM, China sea), refrigerator (994-80. C, Germany' S ice maker (SIM-F124, Japan Sanyo corporation), Miliic-10A, millipore, USA), a dry box (9140, Shanghai Jinghong laboratories), a vortexer (8K-II, Shanghai surgical instruments), a fluorescence quantitative gradient PCR instrument (Jena, Germany).

1.4 cell Resuscitation

Taking out the cell freezing tube (with protective glasses and gloves) from the liquid nitrogen tank, quickly placing the tube into a water bath pot with the temperature of 37 ℃, shaking continuously, thawing as soon as possible to cut off a gauze pocket, taking out the freezing tube, wiping the tube with alcohol for disinfection, opening a cover on a purification bench, moving the tube into a centrifuge tube, adding culture solution, blowing cells, sucking cell suspension out by a suction tube, adding the cell suspension into a culture bottle, and placing the bottle in an incubator with the temperature of 37 ℃ for resting culture. Culturing with complete culture solution, and replacing the culture solution the next day

1.7 subculture

After cell fusion, the old culture medium was removed, a suitable amount of pancreatin was added for observation, 1ml of medium was added after cell contraction to stop digestion and remove the digestive juice, and the cells were centrifuged in a10 ml centrifuge tube at 1000rpm for 10 min. Taking a new culture bottle, discarding the supernatant containing pancreatin from the centrifuged cells, blowing the suspension by using a culture medium, transferring the suspension, supplementing the culture solution, observing and recording under a microscope, and putting the suspension into a culture incubator for culture. The complete culture solution of the subculture cells can be changed into the complete culture solution containing calf serum from the third generation for culture, and the culture solution is changed every other day. The human pulmonary artery smooth muscle cells can be transmitted for at least six generations, and the cells can be frozen from the second generation. The first generation of well-grown cells was taken for subsequent experiments.

1.8 cell observations

The growth of the cells including the morphology, size, fusion status, presence or absence of contamination, etc. was observed under an inverted microscope every day after cell inoculation, and the photographs were retained as reference.

1.9 culture of test cells

Cells in logarithmic growth phase were taken, trypsinized, counted on a hemocytometer, and plated at the desired density in cell culture plates/flasks. The medium was changed every other day until confluency was reached. Starvation was replaced with serum-free medium to synchronize the cells. Cells are then harvested for relevant assays. Liquid culture, and replacing the culture solution every other day.

Second, the experimental procedure

(I) CCK-8 method for detecting influence of isorhamnetin on proliferation of TNF-alpha induced HPASMCs

1.1 Experimental procedures

(1) Digestion and plating: pancreatin digestion of logarithmic phase pulmonary artery smooth muscle cells, centrifugal collection after termination, adjusting cell suspension concentration, and adjusting cell count to 5-10 × 10⁴And/ml. The volume of each well is 100ul, the density of the cells to be tested is adjusted to 5000-10000/well during plating, and the marginal wells are filled with sterile PBS.

(2) Administration: 5% CO₂Oven incubation at 37 ℃ followed by next day monolayer confluency of cells to the bottom of wells (96 well flat bottom plate) and addition of isorhamnetin at 20. mu.M, 40. mu.M, 80. mu.M, 160. mu.M concentration gradient, 100ul per well.

(3)5％CO₂Incubating at 37 ℃ for 12h, and observing the action effect of the medicament under an inverted microscope.

(4) Add 10. mu.L of CCK solution to each well (care was taken not to generate bubbles in the wells which would affect the OD readings).

(5) The plates were incubated in an incubator for 4 h.

(6) Absorbance at 450nm was measured with a microplate reader.

(7) The zero setting wells (medium, CCK-8) and the control wells (cells, culture medium, CCK-8) were set simultaneously.

And (3) activity calculation: cell viability [ (%) [ a (medicated) -a (blank) ]/[ a (0 medicated) -a (blank) ] × 100 wherein: a (dosing): absorbance of wells with cells, CCK solution and drug solution; a (blank): absorbance of wells with medium and CCK solution without cells; a (0 dosing): absorbance of wells with cells, CCK solution, but no drug solution. Cell viability: cell proliferation activity or cytotoxic activity.

1.2 Experimental results: different concentrations of isorhamnetin have an effect on the cell proliferation activity of Human Pulmonary Artery Smooth Muscle Cells (HPASMCs). The cell proliferation rate was significantly increased 24 hours after TNF- α -stimulated HPASMCs compared to the normal control group (P < 0.05); after isorhamnetin (25. mu.M, 50. mu.M, 100. mu.M) intervention, CCK-8 results showed that the proliferation rate of cells in the isorhamnetin-intervened group was reduced (P < 0.05, P < 0.01) compared to the TNF-alpha model group without isorhamnetin, indicating that isorhamnetin can inhibit TNF-alpha induced proliferation of HPASMCs (FIG. 1).

(II) detecting the influence of aloperine on the expression of p-smad1/5 and BMPR2 proteins in HPASMCs induced by TNF-alpha by Western blot

2.1 Experimental procedures

(1) Protein extraction (Whole process low temperature operation)

Cell preparation: HPASMCs were seeded into 60mm dishes and starved for 12h to synchronize when 80% of cells were confluent. 5ml of fresh culture medium containing 20ng/ml of TNF-alpha is added into each culture dish, fresh culture medium is added into a normal control group, the culture medium is replaced by a normal group and a model group after the culture is placed in an incubator for 24 hours, and the administration group is added with 0.5mM aloperine for further culture for 24 hours. Each group of cells was taken out of the incubator, washed 3 times with precooled PBS, and the cells were digested with pancreatin, transferred to a 1.5ml EP tube, and centrifuged. (1000rpm for 10min, 4 ℃) the supernatant was discarded, and the pellet was suspended in 1mL of precooled PBS and centrifuged again, as above. The above procedure was repeated 2 times (pellet i.e.cells).

Cell lysates were prepared as described: preparing according to requirements: to 1ml of cold Lysis Buffer, 10. mu.l of phosphatase inhibitor, 1. mu.l of protease inhibitor and 5. mu.l of 100mM PMSF were added, and stored on ice until use. Note that the preparation is now used.

Cell lysis: after 2 times of centrifugation, the supernatant was discarded, 60. mu.l of lysate was added to the pure cells, mixed well and placed on a shaker and shaken at 4 ℃ for 30 minutes (shaker rotation speed was adjusted to maximum).

Centrifugal separation: each set of EP tubes was trimmed and centrifuged in a 4 ℃ centrifuge at 20000rpm for 5 min. Carefully pipette the supernatant into a pre-cooled EP tube, mark and store in a freezer at-80 ℃.

(2) Protein concentration measurement and protein denaturation treatment

Each reagent was added as indicated in Table 1 below, followed by the addition of 200. mu.l of the prepared BCA working solution to each enzyme-labeled well.

TABLE 1 amount of working solution added to each enzyme-labeled well

Number of holes	0	1	2	3	4	5	6	7
									Protein Standard solution (ul)	0	1	2	4	8	12	16	20
Deionized water (ul)	20	19	18	16	12	8	4	0
									Corresponding protein content (μ g)	0.0	0.5	1.0	2.0	4.0	6.0	8.0	10.0

① the conditions of the microplate reader are set to 37 ℃ for incubation for 30min in advance, oscillation is carried out for 1 time at an interval of 10sec, the absorbance is 562nm, and after preheating, the microplate is placed into the microplate reader to detect the OD value.

② after measuring the absorbance data of each well, a standard curve was drawn with the protein content (μ g) as ordinate and the absorbance value as abscissa.

(3) Sample protein content determination

And adding 19 mu l of lysine Buffer into 1 mu l of extracted protein stock solution to dilute the sample by 20 times, uniformly mixing, adding 20 mu l of sample diluent into each hole, adding 200 mu l of BCA working solution, and fully and uniformly mixing to ensure that no bubbles exist.

And (3) setting the conditions of the microplate reader to incubate at 37 ℃ for 30min in advance, shaking for 1 time at an interval of 10sec, and detecting the OD value, wherein the absorbance is 562nm, and after preheating, placing the microplate into the microplate reader.

The concentration of the sample (. mu.g/. mu.l) was determined by dividing the amount of protein on the abscissa by the total volume of 20. mu.l, according to the OD value on the ordinate of the previously prepared standard curve, and multiplying the result by the dilution factor.

Adjusting the protein concentration of each sample: adding precooled Lysis Buffer according to the measured protein concentration of the sample, mixing with 5 × Loading Buffer according to the ratio of 4:1, adjusting the protein concentration of the sample to 5ug/μ l, then shaking, mixing uniformly, sealing, boiling in boiling water for 10min, cooling, packaging at-80 ℃, packaging and storing, and avoiding repeated freeze thawing.

(4) Preparing concentrated glue and separation glue

And (3) cleaning the glass plate, washing with flowing water, washing with double distilled water, drying in the air, and then fully wiping with methanol.

The glass plate is placed into the clamp to be aligned, clamped and fixed, and then the preparation of the separation glue is started.

An 8%/12% separation gel was formulated according to the molecular weight of the protein as follows:

TABLE 2 separating glue formulation table

Components	8% gel	12% gel
			Deionized water	4.8ml	3.3ml
30% acrylamide	2.7ml	4.0ml
			4 XTTris/SDS gel buffer (pH 8.8)	2.5ml	2.5ml
10％APS	0.1ml	0.1ml
			TEMED	4μl	4μl

When the gel is prepared in sequence, APS and TEMED are added, vortex and mix evenly immediately and follow-up operation is started:

the 8%/12% separation gel was added slowly from one side of the gap between the two glass plates using a 1000 μ l pipette, stopped at 3/4 height, left the remaining space, and then covered with methanol on top of the separation gel in order to flatten the horizontal line of the gel, drain the air, stand at room temperature until a clear line of reflection between the gel and methanol appeared, pour off the methanol, and suck off the remaining moisture as much as possible with filter paper.

The concentrate was prepared immediately as in table 3 below:

TABLE 3 concentrated gum formulation table

Components	5% gel
		Deionized water	3.4ml
30% acrylamide	0.85ml
		4 XTTris/SDS gel buffer (pH 8.8)	0.65ml
10％APS	50μl
		TEMED	5μl

the concentrated gel was added between the two glass plates using a 1000. mu.l pipette until overflowing, quickly inserted into a special comb and allowed to stand overnight in a refrigerator at 4 ℃.

After the gel had fully coagulated, the comb was removed.

(5) Electrophoresis

Connecting an electrophoresis device, fixing the glass gel plate in an electrophoresis tank, adding a freshly prepared 5 xTris-glycine-SDS buffer solution from an inner tank to a position with 2 scales to ensure that the buffer solution overflows a gel sample adding hole, and adding a recycled electrophoresis solution into an outer tank to a position with 4 scales of the electrophoresis tank.

Mu.l of the sample was added to the wells of the gel using a 10. mu.l pipette and 5. mu.l of protein Marker was added to the wells on both sides.

And after sample adding, connecting a power supply to start electrophoresis, carrying out electrophoresis at a constant voltage of 70V until the color of the protein Marker dye is separated, adjusting the voltage to 120V to continue the electrophoresis, and finishing the electrophoresis after the front end of the protein Marker dye reaches the bottom of the gel.

(5) Rotary film

And (3) placing a nitrocellulose membrane (PVDF membrane) and a membrane transfer filter paper in advance in the membrane transfer solution for soaking 30min before the electrophoresis is finished for standby.

After electrophoresis is finished, the black surface of the membrane rotating clamp faces downwards, a layer of spongy cushion is arranged on the upper surface of the membrane rotating clamp, a layer of filter paper is arranged on the upper surface of the membrane rotating clamp, the required strips and the internal references are paved on the filter paper, the PVDF membrane is arranged on the PVDF membrane, a layer of filter paper and a spongy cushion are arranged on the PVDF membrane, a sandwich form is formed, bubbles are driven away, and the PVDF membrane is clamped tightly. Placing the film in a film rotating groove, wherein the black surface of the clamp corresponds to the black surface of the film rotating groove, the transparent surface corresponds to the red surface of the film rotating groove, adding film rotating liquid until the film rotating liquid overflows, and placing the film rotating groove in an ice box for cooling.

The film transfer current is set to be 200m A, and the film transfer time is set to be 2 h.

Incubation of primary and secondary antibodies: and after the film is transferred, taking out the PVDF film, trimming and discarding the gel.

And (3) sealing: dissolving milk powder in PBST to prepare 5% sealing liquid, and placing the PVDF membrane in the sealing liquid for 2 hours at room temperature. Incubating the primary antibody: incubating the primary antibody: after the sealing is completed, taking out the PVDF membrane, and discarding the sealing liquid. The PVDF membrane was then incubated with the appropriate concentration of primary antibody on a shaker (murine anti-BMPR 2 antibody diluted 1: 1000 with 5% skim milk powder solution, rabbit anti-p-smad 1/5 antibody diluted 1: 1000 with 5% skim milk powder solution, rabbit anti-IL-6 antibody diluted 1: 1000 with 5% skim milk powder solution), overnight at 4 ℃ with care taken to exclude air bubbles. The temperature is restored for 2 hours, the PVDF membrane is carefully taken out, the primary antibody is discarded, and PBST is washed for 3 times for 10min each time.

Incubation of secondary antibody: the goat anti-rabbit IgG secondary antibody is mixed with 5% skimmed milk powder solution in a ratio of 1:2000, mixed uniformly by a vortex oscillator, and incubated on the PVDF membrane for 2h at room temperature.

After the secondary antibody incubation was completed, the PVDF membrane was removed and the secondary antibody was discarded, and washed 3 times with PBST for 15min each.

Exposure and development:

preparing a color developing solution: mixing the reagent A and the reagent B in the ECL chemiluminescence substrate reaction solution in a ratio of 1:1, and shaking uniformly for later use.

Putting the PVDF mould on a tray of a multifunctional molecular imaging system, dropwise adding 100 mu l of prepared ECL reaction liquid, incubating for 2min, setting the exposure time to Auto, and then photographing and recording the real result.

And (4) analyzing results: and (3) measuring and analyzing the accumulated gray value of the target protein band in the experimental result by using a Quantity One image analysis system, and simultaneously correcting errors by comparing with the gray value of the internal reference beta-actin.

2.2 results of the experiment

Effect of isorhamnetin on the expression of p-smad1/5, BMPR2 protein in TNF- α induced HPASMCs: according to the Western bolt result, the expression of the BMPR2 protein is obviously reduced compared with the normal group by p-smad1/5 in the HPASMCs of the TNF-alpha model group (figure 2, 3, p < 0.01); compared with the TNF-alpha model group, the isorhamnetin (100 mu M) group can obviously improve the TNF-alpha induced p-smad1/5 in HPASMCs, and the expression of BMPR2 protein is increased (figure 2, 3, p is less than 0.01, and p is less than 0.05). (III) detecting the expression of Id1 and Id3 mRNA in the HPASMCs induced by the isorhamnetin by a Quantitative real-time PCR (qRT-PCR) method in real time:

experimental methods

3.1 preparation at the early stage: all pipettors, pipette tips, EP tubes and eight-row tubes required for the experiment need high-pressure sterilization and DEPC enzyme removal to reduce RNA enzyme interference experiment results.

3.2 primer design:

according to the human Id1, Id3 and beta-actin gene sequences provided by NCBI Genbank Gene Bank, synthetic primers designed by Shanghai Biotech engineering Co., Ltd were as follows:

3.3 RNA extraction of HPASMCs:

total mRNA was extracted from HPASMCs using an Axygen Total RNA Mini kit. HPASMCs were cultured in 60mm dishes and following molding administration the following were performed:

removing the culture medium, washing with precooled PBS for 3 times, adding 400 mu LBuffer R-I into the culture dish, blowing the bottom of the dish by using a pipette gun until all cells fall off, repeatedly sucking the cells by using an injector with the specification of 1ml for more than 8-10 times, and transferring the cell suspension to a 1.5ml centrifuge tube;

adding 150 mul Buffer R-II into each tube, carrying out vortex oscillation for 15-30 seconds, and then centrifuging at 12000rpm for 5 minutes at 4 ℃;

the supernatant was taken, placed in a new 1.5ml centrifuge tube, 250. mu.l of isopropanol was added, shaken up and down, and mixed well.

Placing the preparation tube in a 2ml centrifuge tube, transferring the mixed solution into the tube, and centrifuging at 6000rpm for 1 minute;

the filtrate was discarded, the preparation tube was put back into a 2ml centrifuge tube, 500. mu.l of Buffer W1A (mixed with the specified volume of absolute ethanol) was added, and centrifugation was carried out at 12000rpm for 1 minute;

the filtrate was discarded, the preparation tube was returned to a 2ml centrifuge tube, and 700. mu.l of buffer W2 (mixed with a specified volume of absolute ethanol) was added and centrifuged at 12000rpm for 1 minute;

washing once again with 700. mu.l buffer W2 in the same manner as in 6);

discarding the filtrate, putting the preparation tube back into a 2ml centrifuge tube, and centrifuging at 12000rpm for 1 minute;

putting the prepared tube into a new 1.5ml centrifuge tube EP tube, and adding 70-100 μ l buffer TE in the center of the prepared tube membrane; the mixture was left standing at room temperature for 1 minute, centrifuged at 12000rpm for 1 minute, and the mRNA was eluted.

3.4 determination of RNA concentration:

the RNA concentration was measured using a ultramicro nucleic acid concentration measuring instrument, and first, the nucleic acid concentration measuring instrument was set to zero, and then 1. mu.l of the sample was aspirated and placed in the measurement well under the conditions OD260/OD 280. If the OD260/OD280 value of the sample is in the range of 1.8-2.0, the purity of the sample is proved to be better, and the sample can be used for subsequent experiments.

3.5 RNA reverse transcription reaction System (20. mu.l System):

after the system is mixed evenly, incubation is carried out in a reverse transcriptase meter, and the incubation conditions are as follows: heating at 25 deg.C for 10min, 42 deg.C for 15min, and 85 deg.C for 5min to terminate the reaction, cooling, packaging, and storing in-80 deg.C refrigerator.

3.6 PCR amplification reaction System (20. mu.l System)

Amplification conditions: at 94 ℃ for 1 min 30s, at 94 ℃ for 30s, at 60 ℃ for 30s, at 72 ℃ for 60 s, at 72 ℃ for 5min, for 35 cycles.

2% agarose gel was prepared, and 10. mu.l of the amplification product was added to the gel well under 120V electrophoresis conditions.

After electrophoresis, the DNA bands were observed and an optimal annealing temperature was selected for each primer.

3.7 fluorescent quantitative reaction system:

qPCR amplification conditions:

3.8 fluorescent quantitative PCR result analysis:

ct value definition: the Ct value is expressed as the number of cycles that the reaction occurs when the intensity of the fluorescence signal of the sample reaches the set detection threshold intensity during the reaction (C represents Cycle and t represents threshold);

when the Ct value is less than 15, the amplification is considered to be still in the range of the baseline period, and the fluorescence threshold value is not reached, and when the Ct value is more than 35, the template initial copy number is less than 1 theoretically, and the amplification is considered to be meaningless.

The fluorescence quantification is calculated by adopting a relative comparative quantification method, wherein the expression multiple is 2-delta Ct, △△ Ct (Ct experimental group target gene-Ct to-be-measured group internal reference gene) — (Ct control group target gene-Ct control group internal reference gene), and beta-actin is a correction initial template.

3.9 analysis of results

The results in FIGS. 4 and 5 show that the expression levels of Id1 and Id3 mRNA in the TNF-. alpha.model group were significantly decreased (P < 0.01) compared to the normal control group, and that the expression levels of Id1 and Id3 in the isorhamnetin (100. mu.M) group were significantly increased (P < 0.01) compared to the TNF-. alpha.model group.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. The application of isorhamnetin in preparing a medicament for inhibiting the proliferation of lung smooth muscle cells is disclosed, wherein the structural formula of the isorhamnetin is shown as a formula (1):

2. the use according to claim 1, wherein the single application dose of isorhamnetin is a dose that does not cause central inhibition.

3. Use according to claim 2, wherein isorhamnetin is applied in a concentration of 25-100 μ M in human pulmonary smooth muscle cells.

4. Use according to claim 3, wherein isorhamnetin is applied at a concentration of 25 μ M.

5. Use according to claim 3, wherein isorhamnetin is applied at a concentration of 50 μ M.

6. Use according to claim 3, wherein isorhamnetin is applied at a concentration of 100. mu.M.

7. Use according to any one of claims 1 to 6, wherein inhibition of lung smooth muscle cell proliferation inhibits pulmonary vascular remodeling.

8. Use according to claim 7, wherein inhibition of pulmonary vascular remodeling inhibits pulmonary hypertension, cardiac malformations, or COPD.

9. A medicament for inhibiting lung smooth muscle cell proliferation comprises isorhamnetin as the only active ingredient and pharmaceutically acceptable auxiliary materials.

10. The medicament according to claim 9, which is capable of inhibiting pulmonary vascular remodeling and further inhibiting pulmonary hypertension, cardiovascularisation or COPD.