CN109846881A - Purposes of the aloperine in preparation treatment pulmonary hypertension drug - Google Patents

Abstract

Purposes the invention discloses aloperine (Aloperine) as treatment pulmonary hypertension drug.It is of the invention the experimental results showed that when being used in safe-dosaging limits, the aloperine that dosage is 50 μM can significantly inhibit the proliferation and DNA synthesis of human pulmonary artery smooth muscle cells, blocks cellular enters the S phase by the G0/G1 phase, improve early apoptosis rate, late apoptic rate and total apoptosis rate, significantly reduce the expression of NF- κ B signal pathway associated protein, it was demonstrated that aloperine has therapeutic effect to pulmonary hypertension.

Description

Purposes of the aloperine in preparation treatment pulmonary hypertension drug

Technical field

The present invention relates to the applications of aloperine, the in particular to purposes in aloperine preparation treatment pulmonary hypertension drug.

Background technique

Pulmonary hypertension (pulmonary arterial hypertension, PAH) be one group by heterologous disease and not Continued to increase with caused by pathogenesis with pulmonary vascular resistance, and leads to right ventricle failure and the dead pulmonary artery remodeling being characterized Property disease, have high incidence, high disability rate, high mortality, it is considered to be " false malignant tumour ".Lung blood caused by inflammatory reaction Vascular smooth muscle cell proliferation caused by endothelial tube damages and pulmonary artery remodeling, lung artery occlusion are considered as pulmonary hypertension hair The main feature of interpretation of the cause, onset and process of an illness system and pathological change.Due to the increase of pulmonary vascular resistance and increasing for right ventricular afterload, cause the right heart Room reconstruct, eventually leads to right ventricle failure.Along with vessel retraction, inflammation, tube wall thickening and microvascular corrosion cast, pulmonary vascular resistance Right heart failure and sudden occurs for gradual raising, gas exchanges function decline, and then anoxic and right cardiac load is caused to increase, serious person Extremely.Pulmonary artery remodeling mainly includes blood vessel surrounding substrate deposition, inflammatory infiltration, vascular smooth muscle cell proliferation and impaired vascular endothelium, And vascular smooth muscle cell proliferation and the long-term prognosis of patient are closely related.The paraplasm hypertrophy of vascular smooth muscle has related generally to blood Platelet source property growth factor (platelet-derived growth factor, PDGF), angiotensinⅡ, fibroblast Growth factor and vascular endothelial growth factor etc..In vitro study shows that PDGF is the effective stimulus factor of vascular smooth muscle, and Pdgf receptor signal path and pulmonary artery remodeling are closely related, lure in idiopathic pulmonary hypertension patient and hypoxemia and monocrotaline In the pulmonary artery remodeling animal model led, the expression of PDGF and its receptor is raised.Pdgf receptor antagonist can be prevented and be reversed The right ventricular pressure that hypoxemia and monocrotaline induce increases and pulmonary artery remodeling.With to Pulmonary Artery Hypertension and pathology Physiologic Studies go deep into and the clinical application of newtype drug, oneself is taken on a new look for the prognosis of patients with pulmonary hypertension, but long-term Validity is limited, and 5 annual survival rates are only 49%.Therefore, it finds to have and inhibits even reversing pulmonary vascular reconstruct, cell is inhibited to increase It grows, for right ventricular function and cheap drug, it will help clinical early intervention and treatment has far-reaching research Meaning.

Sophora alopecuroide (sophora alopecuroidles L) has a large amount of wild and artificial growth resource in Ningxia, is Emphasis supports large area standardized planting and preferential deep development simultaneously in Ningxia Hui Autonomous Region's " 13 science and technology development planning " One of Special plant of industrialization, have the effects that it is clearing heat and detoxicating, dispel pathogenic wind and remove dampness, relieve pain desinsection.Aloperine (Aloperine) is One of main alkaloid extracted from Sophora alopecuroide is easier with source, and it is excellent to extract that yield is relatively high, and toxicity is relatively low etc. Gesture.Domestic and foreign scholars have anti-arrhythmia, anti-cardiac muscle studies have shown that aloperine is especially prominent to the effect of cardiovascular system The effects of ischemic.It should be pointed out that Alkaloids From Sophora Alopecuroides L to the pharmacological action of cardiovascular system be this seminar for a long time The problem in science paid close attention to always, the research discovery of our early periods: matrine can fight phyenlephrinium and draw in Alkaloids From Sophora Alopecuroides L The guinea pig aorta ring risen is shunk, and there is protection to make high cholesterol and/or isoprel myocardial injury and ischemic With；Oxymatrine regulates and controls nitric oxide/ADMA signal path to chronic heart failure caused by isoprel with inverse Transfer to use.Whether aloperine is inhibited to the proliferation of arteria pulmonalis smooth muscle cells, and how is mechanism of action, has no at present Report.Being developed has high potential value and social effect for treatment pulmonary hypertension drug.

Summary of the invention

The purpose of the present invention is it is high in preparation treatment pulmonary artery to provide aloperine by the pharmacological research to aloperine Purposes in pressing object.

The present invention is achieved through the following technical solutions goal of the invention:

The present invention provides purposes of the aloperine in preparation treatment pulmonary hypertension drug, and the structural formula of the aloperine is such as Shown in formula (1):

Specifically, the pulmonary hypertension be 20ng/ml PDGF-BB stimulate for 24 hours human pulmonary artery smooth muscle cells for 24 hours after The pulmonary hypertension of induced synthesis.

Specifically, the aloperine single application dosage is the dosage for not causing CNS inhibition.

Preferably, the aloperine single application dosage is 0.125-1mM.

Preferably, the aloperine single application dosage is 0.25-1mM.

Preferably, the aloperine single application dosage is 0.0.5-1mM.

Specifically, the aloperine single application dosage is human pulmonary artery smooth muscle cells 0.125-1mM, standard weight people 70kg single use amount is 16mg/kg-64mg/kg.

Specifically, the drug is configured to the dosage form given by gastrointestinal tract or parenteral.

Preferably, the dosage form is peroral dosage form, parenteral solution formulation or the powder-injection allowed in pharmacy.

Specifically, the aloperine is preparing the purposes in pulmonary hypertension drug as sole active agent.

Aloperine provided by the invention has the advantages that as the purposes for the treatment of pulmonary hypertension drug

(1) aloperine can significantly inhibit human pulmonary artery smooth muscle cells proliferation and DNA synthesis, promote people's arteria pulmonalis smooth muscle Apoptosis；

(2) aloperine can inhibit inflammatory effect, significantly inhibit the proliferation of human pulmonary artery smooth muscle cells caused by inflammation, and And the expression of NF- κ B p65 signal path GAP-associated protein GAP can be inhibited.

The experimental results showed that aloperine has treatment pulmonary hypertension effect, it can be used for preparing the medicine of pulmonary hypertension Object.

Detailed description of the invention

Fig. 1: survival rate figure of the aloperine to human pulmonary artery smooth muscle cells.

Fig. 2: proliferation rate influence diagram of the aloperine to human pulmonary artery smooth muscle cells.

Fig. 3: aloperine synthesizes influence diagram to the DNA of human pulmonary artery smooth muscle cells.

Fig. 4: Cell cycle influences figure of the aloperine to human pulmonary artery smooth muscle cells.

Fig. 5: influence of the aloperine to IKK α protein expression level in Rats of Pulmonary Hypertension lung tissue is (with normal group ratio Compared with: p > 0.05, compared with model group: p > 0.05 (x ± s, n=6)；Relative optical density: light is close relatively Degree).

Fig. 6: influence of the aloperine to p IKK α protein expression level in Rats of Pulmonary Hypertension lung tissue is (with normal group ratio Compared with: ##p < 0.01, compared with model group: * p < 0.05, * * p < 0.01 (x ± s, n=6)).

Fig. 7: influence of the aloperine to I κ B α protein expression level in Rats of Pulmonary Hypertension lung tissue is (with normal group ratio Compared with: p > 0.05, compared with model group: p > 0.05 (x ± s, n=6)).

Fig. 8: influence of the aloperine to p I κ B α protein expression level in Rats of Pulmonary Hypertension lung tissue is (with normal group ratio Compared with: ##p < 0.01, compared with model group: * p < 0.05, * * p < 0.01 (x ± s, n=6)).

Fig. 9: aloperine to NF- κ B p65 protein expression level in Rats of Pulmonary Hypertension lung tissue influence (with it is normal Group compares: ##p < 0.01, compared with model group: * p < 0.05, * * p < 0.01 (x ± s, n=6)).

Figure 10: influence of the aloperine to TNF-α protein expression level in Rats of Pulmonary Hypertension lung tissue is (with normal group ratio Compared with: ##p < 0.01, compared with model group: * p < 0.05, * * p < 0.01 (x ± s, n=6)).

Figure 11: aloperine to Cyclin E1 protein expression level in Rats of Pulmonary Hypertension lung tissue influence (with it is normal Group compares: ##p < 0.01, compared with model group: * p < 0.05, * * p < 0.01 (x ± s, n=6)).

Figure 12: influence of the aloperine to p27kip1 protein expression level in Rats of Pulmonary Hypertension lung tissue is (with normal group Compare: ##p < 0.01, compared with model group: * p < 0.05, * * p < 0.01 (x ± s, n=6)).

Specific embodiment

Below with reference to embodiment, the present invention is described in further detail, and aloperine used in embodiment is aforementioned Formula (1) compound represented.

Embodiment 1

Purposes of the aloperine as treatment pulmonary hypertension drug, shown in the structural formula of the aloperine such as formula (1):

Wherein, aloperine single application dosage is human pulmonary artery smooth muscle cells 0.5mM, and the dosage form of drug is solution Type.

Embodiment 2

Purposes of the aloperine as treatment pulmonary hypertension drug, shown in the structural formula of the aloperine such as formula (1):

Wherein, aloperine single application dosage is human pulmonary artery smooth muscle cells 0.5mM, and the dosage form of drug is powder-injection Type.

Embodiment 3

Purposes of the aloperine as treatment pulmonary hypertension drug, shown in the structural formula of the aloperine such as formula (1):

Wherein, aloperine single application dosage is human pulmonary artery smooth muscle cells 1mM, and the dosage form of drug is powder-injection type.

The effect of embodiment 1 to 3 can be confirmed by following zoopery and its result:

One, experimental material

1.1 cell

Human pulmonary artery smooth muscle cells (is purchased from upper marine Qiao Xinzhou Biotechnology Co., Ltd)

1.2 primary drugs and reagent

Aloperine (is purchased from Shanghai source leaf biology Co., Ltd), silaenafil (it is purchased from pfizer inc, S42045), DMEM (be purchased from U.S. Gibco company, 11965092), fetal calf serum (it is purchased from U.S. Gibco company, C2027010), trypsase (be purchased from U.S. Gibco company, 15050065), CCK-8 (Beijing Suo Laibao Science and Technology Ltd, M8180-250), dimethyl sulfoxide (DMSO) (being purchased from Sigma Co., USA D8418), BrdU (is purchased from Roche Switzerland), Apoptosis and cycle detection kit (being purchased from green skies biotechnology research institute), holoprotein extracts examination Agent box, protein assay kit (being purchased from Nanjing Keygen Biotech's technical concern Co., Ltd, KGP2100, KGPBCA), TEMED (is purchased from Sigma Co., USA, T8090), and ammonium persulfate (AP) (is purchased from Sigma Co., USA, A8090), 10%SDS (Shanghai Tongshan company, P61014), 30% acrylamide (are purchased from Shanghai D ouble-Helix, P31031), albumen loading Buffer 5 × (being purchased from Beijing TransGen Biotech, P62114), chemiluminescent substrate (it is purchased from U.S. Advansta company, K-12045-D10), NF- κ B p65 monoclonal antibody, IKK alpha monoclonal antibodies, I κ B alpha monoclonal antibodies, p-IKK alpha monoclonal are anti- Body, p-I κ B alpha monoclonal antibodies (being purchased from U.S. Abcam company, #8242, ab32041, ab7217, #2697, #9246), β- Actin monoclonal antibody, α-tubulin monoclonal antibody, rabbit-anti IgG monoclonal antibody are (public purchased from U.S. Proteintech Department, 17168-1-AP, 11224-1-AP, ZB-2301).

1.3 key instrument

Super-clean bench (SW-CJ-2FD, safe and sound Technology Co., Ltd., Suzhou province), carbon dioxide incubator (U.S. Thermo Scinentific), vertical pressure steam sterilizer (Shenan Medical Appliances Factory, Shanghai), centrifuge (Hypothermia, Z323K, Siemens Company), water bath (constant temperature, DK-420S, Shanghai Jinghong experimental facilities company), enzyme It marks instrument (Model 550, U.S. Thermo scientific), BH-N I C-B type inverted microscope (Japanese Olympus Optical company), laser confocal microscope (TCS-SP, German LEICA), electronic analytical balance (AL104 Switzerland, Mettler-Toleclo Group company), low temperature refrigerator (BCD-215KCM, China's Haier), refrigerator (994-80.C, Germany Thermo), snowflake ice machine (SIM-F124, SANYO GS company), ultrapure water system (Milli-Q Academic A10, beauty Millipore company, state), drying box (9140, Shanghai Jinghong experimental facilities company), whirlpool instrument (8K- II, Shanghai operation Instrument factory).

1.4 main agents are prepared

50mM aloperine stores liquid: the aloperine for weighing 116mg is dissolved in after 2ml DMSO plus 8ml ultrapure water formation concentration is The aloperine mother liquor of 50mM, 0.22 μm of filtering with microporous membrane degerming, 4 DEG C are kept in dark place (whole sterile working).

2800ng/ml PDGF-BB stores liquid: the PDGF-BB of 10mg specification being taken to add 3.75ml 100mM's to specifications Acetic acid forms the PDGF-BB mother liquor that concentration is 2800ng/ml, and 0.22 μm of filtering with microporous membrane degerming, 4 DEG C are kept in dark place (whole process Sterile working).

4000 μM of silaenafils store liquid: the active principle that 1 silaenafil contains is 100mg, and 1 silaenafil is molten In 5ml DMSO, take wherein 1ml add 9ml ultrapure water, forming concentration is 4000 μM of mother liquor, and 0.22 μm of filtering with microporous membrane removes Bacterium, 4 DEG C are kept in dark place (whole sterile working).

1.5 recovery step

It should be put into rapidly in the water-bath for filling 37 DEG C with safety goggles and gloves from cryopreservation tube note is taken out in liquid nitrogen container, and It shakes frequently, thaws cut off gauze pocket as early as possible, take out cryopreservation tube, after being sterilized with alcohol wipe, lid is opened on clean bench, is moved to In centrifuge tube, culture solution is added, and blows and beats cell and is sucked out with suction pipe in the culture bottle that cell suspension is added to, is placed in 37 DEG C of cultures Tranquillization culture in case.With complete culture solution culture, the culture solution complete culture solution culture of replacement in second day, backward every one Its culture solution of replacement.

1.6 cell freezing method

Culture solution to be replaced before freezing, and suitable pancreatin is added, microscopically observation removes digestive juice after seeing cellular contraction, Appropriate culture solution is added, the cell digested is collected in centrifuge tube, centrifugation removes supernatant, and addition freezes culture solution, blows It beats cell to be allowed to uniformly, be distributed into cryopreservation tube, closed good cryopreservation tube is successively put in 4 DEG C of 30min, -20 DEG C of 2h, liquid nitrogen table Liquid nitrogen cryopreservation is immersed in face.

1.7 secondary culture

After cell fusion, old culture solution is removed, suitable pancreatin observation is added, adds 1ml culture medium whole after cellular contraction Only digestion removal digestive juice moves into 10ml centrifuge tube 1000rpm and is centrifuged 10min.New culture bottle is taken, the cell that centrifugation is completed The supernatant containing pancreatin is abandoned, is transferred to after being suspended with culture medium piping and druming, culture solution is supplied, microscopically observation record is put into training incubator Culture.Complete culture solution can be changed to the complete culture solution culture containing calf serum by passage cell since the third generation, every other day more Change a culture solution.It is more than generation can at least to pass six for human pulmonary artery smooth muscle cells, and cell can freeze since the second generation It deposits.Well-grown generation cell is taken to carry out follow-up test.

1.8 cell observation

Observe the growing state of cell after cell inoculation under inverted microscope daily, form, size including cell are melted Conjunction situation, have it is pollution-free etc., and take a picture be kept for reference.

1.9 the culture of experimental cell

It takes the cell in logarithmic growth phase to be counted with trypsin digestion with blood counting instrument, is inoculated with by required density In tissue culture plate/bottle.Time culture solution is replaced every other day, until reaching fusion.It changes serum-free medium starvation into, keeps cell same Stepization.Then it collects cell and carries out coherent detection.

Two, experimentation

(1) cytotoxicity of CCK-8 method detection aloperine:

1.1 experimental procedure

(1) digestion and bed board: pancreatin digests logarithmic phase arteria pulmonalis smooth muscle cells, is collected by centrifugation after termination, adjusts cell Suspension concentration, cell count adjust its concentration to 5-10 × 104/ml.It is 100ul that volume, which is added, in every hole, and when bed board makes to be measured thin Born of the same parents' tune density to the hole 5000-10000/, edge hole is filled with sterile PBS.

(2) it is administered: 5%CO₂, 37 DEG C of baking ovens are incubated for, and next day cell monolayer is paved with bottom hole (96 hole flat underside), be added 0mM, The aloperine of 0.125mM, 0.25mM, 0.5mM, 1mM concentration gradient, every hole 100ul.

(3) 5%CO₂, 37 DEG C of incubation 12h observe the function and effect of drug under inverted microscope.

(4) 10 μ L CCK solution are added to every hole (to be careful not to generate bubble in hole again, they will affect the reading of OD value Number).

(5) culture plate is incubated for 4h in incubator.

(6) absorbance at 450nm is measured with microplate reader.

(7) zeroing hole (culture medium, CCK-8) and control wells (cell, culture solution, CCK-8) are set simultaneously.

Vigor calculates:

Cell viability * (%)=[A (dosing)-A (blank)]/[A (0 dosing)-A (blank)] × 100

Wherein: A (dosing): the absorbance in the hole with cell, CCK solution and drug solution；

A (blank): have culture medium and CCK solution without the absorbance in the hole of cell；

A (0 dosing): have cell, CCK solution without the absorbance in the hole of drug solution.

* cell viability: cell Proliferation vigor or cytotoxicity vigor.

1.2 experimental results: various concentration aloperine is on the survival rate of human pulmonary artery smooth muscle cells (HPASMCs) without influence

CCK-8 method detects toxicity of the aloperine (0.125-1mM) to HPASMCs of various concentration.Concentration is 0.125-1mM Aloperine have no significant effect (p > 0.05) in survival rate of 12, the 24 and 48h to HPASMCs, cell viability can maintain about 93%.This result shows that, the survival rate of the HPASMCs of the aloperine pair of experimental concentration is without overt toxicity (Fig. 1).

(2) influence of the CCK-8 method detection aloperine to the proliferation of the PDGF-BB HPASMCs induced

2.1 experimental procedures:

(1) digestion and bed board: pancreatin digests logarithmic phase arteria pulmonalis smooth muscle cells, is collected by centrifugation after termination, adjusts cell Suspension concentration, cell count adjust its concentration to 5-104/ml.100ul is added in every hole, and bed board makes cell tune density to be measured extremely Hole, edge hole are filled with sterile PBS.

(2) modeling and administration: 5%CO₂, 37 DEG C of baking ovens are incubated for, until cell monolayer is paved with bottom hole (96 hole flat underside), normally Control group replaces 100 μ l fresh cultures, remaining each group replaces the fresh culture for the PDGF-BB that 100 μ l contain 20ng/ml, Continue to cultivate 12h.Normal group and model group replace 100 μ l fresh cultures after 12h, and positive drug silaenafil group is added The 100 μ l fresh cultures that concentration is 40 μM, it is bitter that administration group sequentially adds 0.125mM, 0.25mM, 0.5mM, 1mM concentration gradient The fresh culture of beans alkali, every hole 100ul, 5 multiple holes of every group of setting.

(3) 5%CO₂, 37 DEG C of incubation 12h observe the function and effect of drug under inverted microscope.

(4) 10 μ L CCK solution are added to every hole (to be careful not to generate bubble in hole again, they will affect the reading of OD value Number).

(5) culture plate is incubated for 4h in incubator.

(6) absorbance at 450nm is measured with microplate reader.

(7) zeroing hole (culture medium, CCK-8) and control wells (cell, culture solution, CCK-8) are set simultaneously.

2.2 experimental results:

The HPASMCs proliferation that CCK-8 method finds that aloperine induces PDGF-BB is inhibited: the exception of HPASMCs Proliferation can cause the formation of pulmonary artery remodeling.Aloperine is a kind of native compound that can inhibit tumor cell proliferation, still, bitter Whether beans alkali is able to suppress the proliferation of HPASMCs and has no relevant report.In order to determine influence of the aloperine to HPASMCs, I By CCK-8 method and detect the HPASMCs proliferation sound that aloperine induce PDGF-BB.CCK-8 method cell Proliferation testing result Show: PDGF-BB stimulates 24 hour cells proliferation after HPASMCs significant compared with the control group；Aloperine (0.125mM, 0.25mM, 0.5mM, 1mM) intervene for 24 hours after CCK-8 the results show that the PDGF-BB group (model group) of aloperine is not added, The group proliferative amount that aloperine is added declines, and embodies the inhibiting effect to the HPASMCs proliferation of PDGF-BB induction and adds The concentration for entering aloperine is higher (0.125mM, 0.25mM, 0.5mM, 1mM), and proliferation quantity is smaller, it is meant that this inhibiting effect Show concentration dependent (p < 0.01, p < 0.05).The aloperine of 0.5mM concentration makees the inhibition that HPASMCs is proliferated simultaneously With the most significant (p < 0.01) (Fig. 2).

(3) influence of the BrdU method detection aloperine to the DNA synthesis of the PDGF-BB HPASMCs induced

3.1 experimental procedures:

(1) preparation of reagents:

BrdU marking fluid: with aseptic culture medium 1:100 dilution (bottle 1) (final concentration of 100 be μM BrdU).96 holes Plate needs 1ml BrdU marking fluid if cell is inoculated with by 100 holes μ l/, if be inoculated with by 200 holes μ l/, needs 2ml BrdU Marking fluid.Undissolved BrdU marking fluid (1000 ×): 2-8 DEG C is protected from light the storable several months.The BrdU marking fluid of dissolution: 2-8 DEG C It is protected from light storable several weeks.The BrdU marking fluid of long-term preservation, need to dispense and be stored in -20 DEG C.

Anti-BrdU-POD stores liquid: dissolving Anti-BrdU-POD (bottle 3) 10min with 1.1ml distilled water and mixes It is even, it is placed in 2-8 DEG C and is protected from light the storable several months, long-term preservation should dispense preservation to -20 DEG C.

Anti-BrdU-POD working solution: with antibody diluent (bottle 4) 1:100 dilution Anti-BrdU-POD storage Liquid.One 96 orifice plate dilutes 100 μ l, Anti-BrdU-POD working solutions with 10ml antibody diluent (bottle 4) and matches before use System, can not store.

Washing lotion: diluting Washing buffer concentrate (bottle 5) with distilled water 1:10,96 orifice plates, 10ml Washing buffer concentrate (bottle 5) is diluted with 90ml distilled water, can be reserved for several weeks in 2-8 DEG C.

(2) logarithmic phase cell is collected, concentration of cell suspension is adjusted, is inoculated in 96 orifice plates, 5 × 103/ holes, final volume 100 The hole μ l/, 37 DEG C of incubations.

(3) the cell culture time depends on specific experimental method and the cell category for measurement.For most of Experimental program, incubation time is generally in 24-120h.

(4) cell is cultivated by 100 holes μ l/, and BrdU marking fluid is added by 10 holes μ l/, and 37 DEG C are continued to solve hatching cell 2-24h, Cell is cultivated by 200 holes μ l/, and BrdU marking fluid is added by 20 holes μ l/.The general 2h label time, oneself was enough.Extend incubation time The total amount of BrdU infiltration cell DNA will be will increase, absorbance value and sensitivity is caused to increase (this experiment is 2h).

(5) marking fluid is removed, light button or absorption discard marking fluid.Continuous mode can be interrupted after label, if It does not continue to measure, removes marking fluid, cell can be placed in 2-8 DEG C one week.

FixDenat solution (bottle 2) 15-25 DEG C of incubation 30min is added in (6) 200 holes μ l/.

(7) fall or gently buckle by bullet and sufficiently remove FixDenat solution.

(8) Anti-BrdU-POD working solution is added according to 100 holes μ l/, in 15-25 DEG C of incubation about 90min.

(9) antibody conjugates are removed and clean every hole with 200 hole μ l-300 μ l/ washing lotions, are cleaned 3 times.

(10) washing lotion is removed.

Substrate solution is added in (11) 100 holes μ l/.

(12) 15-25 DEG C of incubation 5-30min is until color enough photometric detections.Terminate liquid is not added: being measured with microplate reader Sample absorbance, wavelength 370nm (referring to wavelength 492nm), allows replication (such as 5,10,20min) in different time points.Enzyme The optimum reacting time of substrate specificity depends on different cell categories.Add terminate liquid: the 1mM sulfuric acid that 25 μ l are added in every hole terminates Reaction, 300rpm vibrate about 1min, detect OD value (690nm that reference wavelength is) at microplate reader 450nm wavelength, i.e. BrdU inspection Measured value.Detection need to carry out in 5min after terminate liquid is added.

3.2 experimental result

The synthesis of DNA and cell cycle and cell Proliferation are closely related, so we study hardship by BrdU infiltration method Influence of the beans alkali to the PDGF-BB arteria pulmonalis smooth muscle cells DNA synthesis induced.BrdU detection DNA composite result show: with it is right According to group compared to 24 hours after PDGF-BB stimulation HPASMCs, cell Proliferation is significant (p < 0.01)；Aloperine 0.125mM, 0.25mM, 0.5mM, 1mM) intervene the BrdU after 2h the results show that the PDGF-BB group of aloperine is not added, aloperine is added Group proliferative amount decline, embody the inhibiting effect to the HPASMCs proliferation of PDGF-BB induction, and aloperine be added Concentration it is higher, proliferation quantity it is smaller, the effect of Inhibit proliferaton is more significant, it is meant that this inhibiting effect shows concentration dependant Property (p < 0.01, p < 0.05).And the aloperine of 0.5mM concentration inhibits HPASMCs DNA synthetic effect best (p < 0.01) (Fig. 3).

(4) influence of the Flow cytometry aloperine to the PDGF-BB HPASMCs cell cycle induced

4.1 experimental procedures:

(1) cell culture observes cell fusion up to vitellophag after 80% under six orifice plates, inverted microscope, is centrifuged and receives Collecting cell, 800rpm/min, 5min abandon supernatant, and cell is resuspended 1 time with the PBS of pre-cooling, is centrifuged again, 1000rpm/min, 5min ,-as cell needed for a sample can use 6 orifice plates culture.

(2) 70% ethyl alcohol (dehydrated alcohol+PBS preparation) that 1ml is prepared and is pre-chilled in advance is added, is fixed overnight in 4 DEG C. It has to gently be blown and beaten with liquid-transfering gun and be vortexed to rock to blow cell open when cell is fixed, blow out unicellular.

(3) next day is centrifuged again, 1500rpm/min, 5min, abandons supernatant.

(4) primary, centrifugation of with the PBS of 1ml washing cell again, 1500rpm/min, 5min abandon supernatant, collect cell.

(5) it is needed according to each sample: 0.5mlPI/RNase dye solution is added, slowly and be sufficiently resuspended cell, 4 DEG C it is protected from light incubation 30 minutes.The prepared ethidium bromide staining liquid short time can be stored in 4 DEG C, this experiment is ready-to-use.Dye Each period phase cell percentage of machine testing is gone up after color immediately.

Flow cytometer showed: with standardization program flow cytomery, ten thousand cells are generally counted, as a result use Modfit cell Period fits software analysis, with the sieve of 200 mesh is to filter coherent cell mass before upper machine, avoid clogging machine And there is polyploid interference effect experimental result.

4.2 experimental result

The Flow cytometry cell cycle finds that aloperine has the HPASMCs proliferation that PDGF-BB is induced and inhibits to make With: the cell cycle refers to that cell terminates overall process experienced, cell cycle to division next time since being completed primary division Retardance can influence cell Proliferation.We are using the shadow for passing through flow cytomery aloperine cell cycle after PI dyeing It rings.HPASMCs is in G0/G1 percentage reduction (Fig. 4, p < 0.05) after PDGF-BB stimulation for 24 hours, and S phase cell percentages increase (Fig. 4, p < 0.01).Can make after the intervention of 0.5mM aloperine 24 hours compared with the control group cell G0/G1 percentage increase (Fig. 4, P < 0.05), S phase cell percentages increase (Fig. 4, p < 0.01), and G2/M has no significant effect (Fig. 4, p > to phase cell percentages 0.05).The result shows that aloperine can block G0/G1 phase cell to S phase transition.

(5) Western blot detects IKK α, I κ B α, p-I κ B α, p- in the HPASMCs that aloperine induces PDGF-BB The influence of IKK α, NF- κ B p65, TNF-α, p27kip1, Cyclin E1 protein expression

5.1 experimental procedure

(1) protein extraction (whole low-temperature operation)

Cell prepares: HPASMCs being inoculated in the culture dish of 60mm, when cell fusion is up to 80%, hungry 12h keeps it same Stepization.The fresh culture that 5ml contains 20ng/ml PDGF-BB is added in each culture dish, fresh training is added in Normal group Support base, normal group and model group replace fresh culture after being put into incubator culture for 24 hours, administration group be added 0.5mM aloperine after Continuous culture is for 24 hours.Group of cells is taken out from incubator, is washed 3 times with the PBS of pre-cooling, digests lower cell using pancreatin, is transferred to In the EP pipe of 1.5ml, centrifugation.(1000rpm be centrifuged 10min, 4 DEG C) abandons supernatant, and the PBS that 1mL pre-cooling is added in precipitating is suspended, again Centrifugation, condition are same as above.Aforesaid operations are repeated 2 times (precipitating i.e. cell).

By specification prepares cell pyrolysis liquid: preparing on demand: in every cold Lysis Buffer of 1ml, 10 μ l phosphatases are added Inhibitor, 1 μ l protease inhibitors and 5 μ l 100mM PMSF are saved stand-by on ice.Pay attention to matching while using.

Lytic cell: cell abandons supernatant after 2 centrifugations, and 60 μ l lysates are added into pure cell, and mixing, which is placed on, shakes On bed, rocked under 4 DEG C of environment 30 minutes (shaking speed is adjusted to maximum).

Centrifuge separation: each group EP pipe is centrifuged 5min with centrifugation in 4 DEG C of centrifuges is flat on for 20000rpm.It is careful to draw Supernatant is transferred in the EP pipe of pre-cooling, is marked and is placed at -80 DEG C of refrigerators preservations.

(2) determination of protein concentration and albuminous degeneration processing

Each reagent is added according to mark in the following table 1, is then separately added into 200 μ l preparation into each enzyme mark hole again BCA working solution.

The amount of working solution is added in 1. each enzyme mark hole of table

Kong Hao	0	1	2	3	4	5	6	7
									Protein standard solution (μ l)	0	1	2	4	8	12	16	20
Deionized water (μ l)	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. being in advance 37 DEG C of incubation 30min by microplate reader condition setting, being at interval of 10sec concussion 1 time, absorbance ELISA Plate is put into microplate reader by 562nm after the completion of preheating, detects OD value.

2. being ordinate, light absorption value as abscissa using protein content (μ g) after measuring every hole absorbance data, standard is drawn Curve.

(3) sample determining the protein quantity

19 μ l Lysis Buffer are added by 20 times of Sample Dilution, after mixing, every hole in the albumen stoste that every 1 μ l is extracted 20 μ l of Sample dilution is added, adds 200 μ l of BCA working solution, mixes well and guarantee bubble-free.

It is in advance 37 DEG C of incubation 30min by microplate reader condition setting, shakes 1 time, absorbance 562nm at interval of 10sec, ELISA Plate is put into microplate reader after the completion of preheating, detects OD value.

OD value on the standard curve ordinate drawn according to before, corresponds to protein content on abscissa, divided by 20 μ l's Total volume just obtains sample concentration (μ g/ μ l) multiplied by extension rate.

It adjusts each sample protein concentration: according to the sample protein concentration measured, the Lysis of pre-cooling is proportionally added Buffer is mixed with 5 × Loading Buffer with the ratio of 4:1, and adjustment sample protein concentration is 5ug/ μ l, and then concussion is mixed 10min is boiled in even, sealing in boiling water, and -80 DEG C of packing save after cooling packing, avoid multigelation.

(4) concentration glue and separation gel are prepared

Glass plate is cleaned, flowing washing, distilled water flushing is dried, later with the abundant wiped clean of methanol.

Glass plate is put into alignment clamping in clip and is fixed, then starts to prepare separation gel.

8%/12% separation gel is prepared according to the following table 2 according to the molecular weight of albumen:

2. separation gel formula table of table

Component	8% gel	12% gel
			Deionized water	4.8ml	3.3ml
30% acrylamide	2.7ml	4.0ml
			4 × Tris/SDS separation gel buffer (PH 8.8)	2.5ml	2.5ml
10%APS	0.1ml	0.1ml
			TEMED	4μl	4μl

It is added to be vortexed immediately after APS and TEMED when preparing gel in order and mixes and start subsequent operation:

8%/12% separation gel is slowly added to from the side in the gap between two pieces of glass plates with the liquid-transfering gun of 1000 μ l, It adds to glass plate 3/4 highly to locate to stop, leaving remaining space, then again with methanol is covered at the top of separation gel, it is therefore an objective to be flattened The horizontal line of gel drains air, is stored at room temperature to methanol is removed when occurring clearly Reflect light line between gel and methanol, with filter Paper sucks residual moisture as far as possible.

Immediately concentration glue is prepared according to the following table 3:

Glue formula table is concentrated in table 3.

Component	5% gel
		Deionized water	3.4ml
30% acrylamide	0.85ml
		4 × Tris/SDS separation gel buffer (PH 8.8)	0.65ml
10%APS	50μl
		TEMED	5μl

It is added to be vortexed immediately after APS and TEMED when preparing gel in order and mixes and start subsequent operation:

Glue will be concentrated with the liquid-transfering gun of 1000 μ l to be added between two pieces of glass plates to overflowing, be rapidly inserted into dedicated comb, Resting on 4 DEG C of refrigerators can stay overnight.

Glue to be concentrated all after cohesion, removes comb.

(5) electrophoresis

Electrophoretic apparatus is connected, glass offset plate is fixed in electrophoresis tank, 5 × Tris- that fresh configuration is added from inside groove is sweet At propylhomoserin-SDS buffer to 2 scales, it is ensured that buffer did not had gel well, the electrophoresis liquid that outer groove addition recycles to electricity At 4 scales of swimming slot.

10 μ l samples are added in the well of gel with the liquid-transfering gun of 10 μ l, then the egg of 5 μ l is added in the hole on both sides White Marker.

Power supply is connected after sample-adding starts electrophoresis, it will to after running away between albumen Marker dye colour with constant pressure 70V electrophoresis Voltage, which is adjusted to 120V, continues electrophoresis, terminates electrophoresis after albumen Marker dye front reaches gel bottom.

(5) transferring film

Nitrocellulose filter (NC film) and transferring film filter paper are placed in transferring film liquid by the 30min before electrophoresis terminates in advance It impregnates stand-by.

After electrophoresis, transferring film clip black flour downward, is padding one layer of foam-rubber cushion, then pad one layer of filter paper, by required item above Band and internal reference are layered on filter paper, NC film are placed on, then put one layer of filter paper and a foam-rubber cushion, are formed sandwich formats, are caught up with It walks bubble and clamps.It is placed in transferring film slot, the black flour of clip corresponds to the black flour of transferring film slot, and transparent side corresponds to the red face of transferring film slot, Transferring film liquid is added to overflowing, is put into ice chest cooling.

Transferring film electric current is set as 200m A, the transferring film time is 2h.

It is incubated for primary antibody, secondary antibody: taking out NC film after transferring film and trimmed, discard gel.

Closing: milk powder is dissolved in PBST and is configured to 5% confining liquid, NC film is placed in confining liquid and is placed at room temperature for 2 hours.

It is incubated for primary antibody: taking out NC film after the completion of closing, discard confining liquid.Then NC film is incubated for primary antibody on shaking table.Rabbit Anti- IKK Alpha antibodies press 1 by 1:10000 dilution, 5% skimmed milk power solution of rabbit-anti I κ B Alpha antibodies with 5% skimmed milk power solution: 1000 dilutions, rabbit-anti p-I κ B Alpha antibodies are with 5% skimmed milk power solution by 1:1000 dilution, 5% degreasing of rabbit-anti p-IKK Alpha antibodies Milk power solution is by 1:1000 dilution, rabbit-anti NF- κ B p65 antibody with 5% skimmed milk power solution by 1:50000 dilution, rabbit-anti TNF- Alpha antibodies are dilute by 1:1000 by 1:500 dilution, 5% skimmed milk power solution of rabbit-anti p27kip1 antibody with 5% skimmed milk power solution Release, rabbit-anti Cyclin E1 antibody is diluted with 5% skimmed milk power solution by 1:1000,4 DEG C overnight.

Rewarming 2 hours, the careful NC film that takes out discarded primary antibody, and PBST is washed 3 times, each 10min.

It is incubated for secondary antibody: goat anti-rabbit igg secondary antibody being mixed with 5% skimmed milk power solution with the ratio of 1:2000, with vortex Oscillator is incubated for NC film 2h after mixing at room temperature.

After secondary antibody is incubated for, takes out NC film and discard secondary antibody, washed 3 times with PBST, each 15min.

Exposure development:

Prepare developing solution: by ECL chemiluminescent substrate reaction solution reagent A and reagent B with 1:1 ratio mix, shake up For use.

NC mould is put on the pallet of polyfunctional molecule imaging system, prepared 100 μ l of ECL reaction solution is added dropwise, is incubated for 2min, setting time for exposure are Auto, then photograph to record real result.

Interpretation of result: the accumulation using Quantity One image analysis system to the target protein band in experimental result Gray value measures analysis, while by comparing with internal reference β-actin gray value with correction error.

5.2 experimental result

IKK in the HPASMCs that aloperine induces PDGF-BB, I κ B, p-I κ B, p-IKK, NF- κ B, TNF-α, The influence of p27kip1, Cyclin E1 protein expression: according to Western bolt's the results show that compared with normal group, model Group HPASMCs NF κ B-p65, p-IKK α, p-I κ B α, TNF-α and Cyclin E1 protein expression obviously increase (Fig. 6,8,9, 10,11, p < 0.01, p < 0.05), I κ B α, IKK α protein expression are unchanged (Fig. 5,7, p > 0.05), p27kip1 protein expression It significantly reduces (Figure 12, p < 0.01)；Compared with model group, aloperine group be can obviously improve in the HPASMCs of PDGF-BB induction NF κ B-p65, p-IKK α, p-I κ B α, TNF-α and Cyclin E1 protein expression increase phenomenon (Fig. 6,8,9,10,11, p < 0.01, p < 0.05), it improves p27kip1 protein expression (Figure 12, p < 0.05)；And for I κ B α, IKK α albumen, the equal nothing of each group Significant difference (Fig. 5,7, p > 0.05).

Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not Under the premise of being detached from the invention design, various modifications and improvements can be made, these belong to protection model of the invention It encloses.

Claims (9)

1. purposes of the aloperine in the drug of preparation treatment pulmonary hypertension, it is characterised in that: the pulmonary hypertension is PDGF-BB stimulation for 24 hours afterwards induction human pulmonary artery smooth muscle cells increment and formed；The structural formula of the aloperine such as formula (1) It is shown:

。

2. purposes according to claim 1, it is characterised in that: aloperine single application dosage is not cause CNS inhibition Dosage.

3. purposes according to claim 2, it is characterised in that: aloperine single application dosage is 0.125-1mM.

4. purposes according to claim 3, it is characterised in that: aloperine single application dosage is 0.125mM.

5. purposes according to claim 3, it is characterised in that: aloperine single application dosage is 0.25mM.

6. purposes according to claim 3, it is characterised in that: aloperine single application dosage is 0.5M.

7. purposes according to claim 1, it is characterised in that: aloperine single application dosage is that people's arteria pulmonalis smooth muscle is thin Born of the same parents 0.125-1mM, standard weight people's 70kg single use amount are 16mg/kg-64mg/kg.

8. purposes according to claim 1-7, it is characterised in that: the drug be configured to through gastrointestinal tract or The dosage form that parenteral is given.

9. purposes according to claim 8, it is characterised in that: the dosage form is the peroral dosage form allowed in pharmacy, note Penetrate liquor type or powder-injection.