CN102160866A - Application of tanshinone IIA or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing pulmonary hypertension disease - Google Patents
Application of tanshinone IIA or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing pulmonary hypertension disease Download PDFInfo
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Abstract
The invention discloses application of tanshinone IIA or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing pulmonary hypertension disease. The tanshinone IIA or the pharmaceutically acceptable salts thereof have good treatment effect on the pulmonary hypertension, have high safety, and have good application prospect. Compared with the clinical medicines for treating the pulmonary hypertension at present, the tanshinone IIA or the pharmaceutically acceptable salts thereof are low in price and have good market prospect.
Description
Technical field
The present invention relates to the new application of tanshinone, especially relate to the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone or its materia medica.
Background technology
The pulmonary hypertension disease has cause of disease complexity, sickness rate height, misdiagnosis rate height, hazardness is strong, mortality rate is high characteristics, and its main clinical manifestation is that average pulmonary artery is more than or equal to 25mmHg.The early stage normal no obvious subjective symptoms of pulmonary hypertension, though pulmonary hypertension has caused the plump and chronic high pressure pulmonary heart disease of right ventricle sometimes, but symptom might not be remarkable, and how just to engender out of breath, weak, dyspnea or symptoms such as spitting of blood, cardiopalmus, hoarseness are arranged between 20～40 years old.Because cardiac output reduces, can have that extremity cold, pulse are tiny, property cyanosis, angina pectoris on every side, faint etc., cyanosis is often not serious in early days, but significant cyanosis can occur having under the situation of right-to-left shunt.The method of traditional treatment comprises anticoagulant, digoxin and oxygen uptake, but effect is all undesirable.
Radix Salviae Miltiorrhizae is the dry root and rhizome of labiate Radix Salviae Miltiorrhizae Radix Salvia Miltiorrhiza bge., and the beginning is stated from Shennong's Herbal, bitter in the mouth, cold nature, have broken disease and remove abdominal mass, end stuffy sensation with restlessness, effects such as QI invigorating, classify as top grade, GUIXIN, liver two warps, the successive dynasties book on Chinese herbal medicine all records.Tanshinone is the effective monomer that extracts from salviamiltiorrhizabung, and its molecular formula is C
19H
18O
3, the at present clinical treatment cardiovascular disease aspect that is mainly used in has atherosclerosis, suppresses left ventricular hypertrophy, anti-myocardial ischemia, improves effects such as vascular smooth muscle state, arrhythmia.
Summary of the invention
The object of the present invention is to provide the new purposes of acceptable salt on tanshinone or its materia medica, i.e. tanshinone and derivant thereof the new application in pharmacy.
Particularly, the object of the invention is to provide acceptable salt on tanshinone or its materia medica as the application in the medicine of preparation treatment or prevention pulmonary hypertension disease.
And the pulmonary hypertension disease is divided into following several different type according to the different causes of disease: 1: hypertensive pulmonary arterial disease; 2: left heart disease association hypertensive pulmonary arterial disease; 3: the pulmonary hypertension disease relevant with respiratory system disease and/or anoxia; 4: chronic thromboembolia type pulmonary hypertension disease; 5: pulmonary hypertension disease due to the not clear and definite multiple factor.Although the pathogeny difference of the pulmonary hypertension disease of above-mentioned several types, but discover through the inventor, they all can cause pulmonary hypertension patient's last airway obstruction, thereby cause the chronic hypoxia of lung tissue, this mechanism is considered to the main inducing that the pulmonary vascular contraction strengthens and blood vessel is reinvented.Discover that arteriolar paraplasm of lung and persistence vasoconstriction are the key factors that causes pulmonary hypertension, and Ca in the pulmonary vascular smooth muscle cell
2+Concentration is unbalance to be the key factor of pulmonary hypertension morbidity, causes cellular contraction and smooth muscle cell proliferation just because of the increase of endocellular liberation calcium concentration, thereby causes that pulmonary vascular pressure persistence raises, and causes blood vessel to reinvent, and finally causes pulmonary hypertension.And Ca in the cell
2+The increase of concentration mainly is by following several approach: sarcoplasmic reticulum discharges Ca in (1) cell
2+(2) Ca
2+Rely on Ca from extracellular fluid by L type voltage
2+Passage (VDCC), receptor-operated Ca
2+Passage (ROCC) and calcium pond maneuverability Ca
2+Passage (SOCC) enters in the cell.SOCC raises and is only the main cause that causes calcium ion disbalance in the smooth muscle cell when the inventor discovers hypoxia.
The inventor found through experiments, acceptable salt can suppress the intracellular calcium concentration rising that hypoxia causes on tanshinone or its materia medica, act on the SOCC passage and reduce intracellular calcium concentration, can influence intracellular calcium concentration by the expression that influences TRPC6mRNA, can reduce the proteic expression of pulmonary hypertension model induced lung arterial smooth muscle tissue TRPC1, effectively reduce the proteic expression of pulmonary hypertension model induced lung arterial smooth muscle tissue TRPC6, effectively the pathological change that reduces pulmonary hypertension model rat right ventricular pressure and effectively suppress the right heart compensatory hypertrophy of pulmonary hypertension rat model and can reduce the little vascular smooth muscle layer of lung of Hypoxic Pulmonary Hypertension in Rats rat model illustrates that acceptable salt can be used in the above-mentioned all types of pulmonary hypertension disease of treatment on tanshinone or its materia medica.
Therefore in fact, the present invention relates on tanshinone or its materia medica acceptable salt as the application in the medicine of preparation treatment or prophylaxis of pulmonary hypertension disease.
Relate to acceptable salt on tanshinone or its materia medica as the preparation treatment or prevent application in the medicine of left heart disease association hypertensive pulmonary arterial disease.
Relate to acceptable salt on tanshinone or its materia medica as the application in the medicine of preparation treatment or the prevention pulmonary hypertension disease relevant with respiratory system disease and/or anoxia.
Relate to acceptable salt on tanshinone or its materia medica as the preparation treatment or prevent application in the medicine of chronic thromboembolia type pulmonary hypertension disease.
Relate to acceptable salt on tanshinone or its materia medica as the application in the medicine of pulmonary hypertension disease due to the not clear and definite multiple factor of preparation treatment or prevention.
Acceptable salt can use separately or use with the form of pharmaceutical composition on tanshinone of the present invention or its materia medica, and described pharmaceutical composition comprises acceptable salt and pharmaceutical carrier on tanshinone or its materia medica.Can also comprise in the described pharmaceutical composition that acceptable salt on tanshinone or its materia medica and pharmaceutical carrier and prostacyclin and analog (as epoprostenol) thereof, endothelin-1 receptor pick up the drug regimen of the treatment pulmonary hypertension disease of one or more compositions in anti-agent (as bosentan) and the 5 type phosphodiesterase inhibitors (as sldenafil).
The treatment effective dose of acceptable salt is the dosage that reduces intracellular calcium concentration and reduce the effect of pulmonary artery pressure isoreactivity for producing after the individual administration on described tanshinone or its materia medica.And when giving individual single agent or multiple dose, dosage comprises pharmacokinetic property, route of administration, what characteristic (sex, age, body weight, health condition, build) of patient's situation, symptom degree and the treatment of depositing, therapeutic frequency and the desired effects of acceptable salt on tanshinone or its materia medica according to multiple factor and difference.
Generally, the independent medication of acceptable salt can reach the effect of treatment on described tanshinone or its materia medica.It is 40～80mg/ time that described tanshinone or derivatives thereof acts on individual each dosage as active component, one day twice, effective therapeutic effect of the expectation of right heart compensatory hypertrophy that just can reach remarkable reduction intracellular calcium concentration, reduce pulmonary artery pressure, the inhibition pulmonary hypertension causes and the pathological change that reduces the little vascular smooth muscle layer of lung.
Acceptable salt can carry out individual administration by number of ways on described tanshinone and the materia medica thereof, and that route of administration comprises is oral, intravenous injection etc.Correspondingly, described tanshinone or derivatives thereof can be prepared into oral formulations and injection with pharmaceutically suitable carrier.Wherein, oral formulations comprises tablet, capsule, microcapsule, soft capsule, granule, drop pill, oral liquid and powder.Injection comprises intravenous injection and injectable powder.Can also different pharmaceutically suitable carrier can be equipped with according to the dosage form needs, antioxidant, emulsifying agent, stabilizing agent and antifungus agent can be added simultaneously.
Beneficial effect of the present invention is as follows:
1. the present invention has excavated new medical application to acceptable salt on known compound tanshinone or its materia medica, has opened up a new application.
2. acceptable salt pair pulmonary hypertension has the good curing effect on tanshinone or its materia medica, and safety is good, has good application prospects.And acceptable salt is compared with the present medicine of the treatment pulmonary hypertension of usefulness clinically on tanshinone and the materia medica thereof, and low price has market prospect.
Description of drawings
Fig. 1 detects the influence of tanshinone to basic calcium in the former foster pulmonary artery smooth muscle of being commissioned to train with calcium ion real-time visualization technical method, and there is statistical significance P＜0.05.
Fig. 2 detects the influence of tanshinone to SOCE peak value in the former foster pulmonary artery smooth muscle of being commissioned to train with calcium ion real-time visualization technical method, and there is statistical significance P＜0.01.
Fig. 3 detects the influence of tanshinone to TRPC1mRNA expression in the former foster pulmonary artery smooth muscle of being commissioned to train with the RT-PCR method, and there is statistical significance P＜0.05.
Fig. 4 detects the influence of tanshinone to TRPC6mRNA expression in the former foster pulmonary artery smooth muscle of being commissioned to train with the RT-PCR method, and there is statistical significance P＜0.05.
Fig. 5 detects the influence of tanshinone to pulmonary hypertension rat model arteria pulmonalis smooth muscle tissue TRPC1 expressing quantity with the western-blot method, and there is statistical significance P＜0.05.
Fig. 6 detects the influence of tanshinone to pulmonary hypertension rat model arteria pulmonalis smooth muscle tissue TRPC6 expressing quantity with the western-blot method, and there is statistical significance P＜0.05.
Fig. 7 detects the protein expression electrophoretogram of tanshinone to pulmonary hypertension rat model arteria pulmonalis smooth muscle tissue TRPC1, TRPC6 with the western-blot method.
Fig. 8 carries out the influence of pharmaceutical intervention to the mean pressure of pulmonary hypertension rat model right ventricle with tanshinone, and there is statistical significance P＜0.05.
Fig. 9 carries out the influence of pharmaceutical intervention to pulmonary hypertension rat model right ventricle hypertrophy index with tanshinone, and there is statistical significance P＜0.01.
Figure 10 carries out pharmaceutical intervention to pulmonary hypertension rat model lung tissue HE coloration result with tanshinone
A: the lung tissue of rats section HE coloration result of normal oxygen matched group, arrow shows the lung small artery, and tube wall is not seen hypertrophy, and tube chamber is not seen narrow;
B: the lung tissue of rats section HE coloration result of normal oxygen medicine-feeding group, arrow shows the lung small artery, does not see difference with normal oxygen matched group;
C: hypoxia (10%O
2) the lung tissue of rats section HE coloration result of matched group, arrow shows the lung small artery, visible lung small artery tube wall and smooth muscle layer obviously thicken luminal stenosis;
D: hypoxia (10%O
2) the lung tissue of rats section HE coloration result of medicine-feeding group, lung morphological change and the hypoxia matched group is apparent in view alleviates.
The specific embodiment
Describe the new purposes of acceptable salt in field of medicaments on tanshinone or its materia medica by the following examples, but following examples only are used to illustrate purpose of the present invention, do not limit protection scope of the present invention.
Embodiment one: acceptable salt can suppress the intracellular calcium concentration rising that hypoxia causes on tanshinone or its materia medica
Material and method:
One, main experiment material
1, SPF level SD rat: body weight 200g-250g, Guangdong Province's Experimental Animal Center
2, low sugar DMEM culture medium, hyclone (fetal bovine serum, FBS), the type i collagen enzyme: U.S. Gibico company
3, papain, rat α-actin monoclonal antibody: Sigma company
4, pentobarbital sodium: Guangzhou chemistry two factories
5, Cy3 labelling goat-anti rat lgG:Jackson company
6, Fura-2 fluorescent dye: Invitrogen company
Two, key instrument
1, calcium ion fluoroscopic image system: InCyt Im1
TMCompany
2, fluorescence inverted microscope: Nikon company
3, micropipettor: Gilson company
Three, experimental technique
1, arteria pulmonalis smooth muscle cells former is commissioned to train foster:
SD rat cardiopulmonary tissue is got by the sterile working, place 4 ℃ of balanced salt solutions (Hank ' s Balanced Salt Solution, HBSS).The cleaning stain of dehematizing is isolated the small tremulous pulse of lung with microinstrument under the Stereo microscope.Carefully divest tunica adventitia of artery (fiber fat deposit), after vascular strip is vertically cut off, in face up and strike off inner membrance from top to bottom with the elbow pincet.Middle membrane tissue is placed Digestive system, and (the Digestive system composition is a 1750U/ml type i collagen enzyme, 9.5U/ml papain, the 2mg/ml bovine serum albumin, the 1mM dithiothreitol, DTT, be dissolved in the HBSS liquid of no calcium ion) in 35～37 ℃ digestion 15-20min, soft to piece of tissue, the edge is scared, is broken shape to a certain degree; Add in the low sugar DMEM culture medium of 10% hyclone and Digestive system, centrifugal, absorb supernatant, add that PBS cleans 1 time and centrifugal, absorb supernatant, add new mixed culture medium again, make cell suspension in culture medium, adjusting cell density is 1～2 * 10
5Cell/ml also plants in culture plate, places 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, change culture fluid after 3-4 days,, promptly finish primitive cell culture, obtain the former foster pulmonary artery smooth muscle of being commissioned to train when cell grows to exponential phase.
2, the making of hypoxic cell model:
Be divided into four groups with cultivating the pulmonary artery smooth muscle that obtains: normal oxygen cellular control unit, normal oxygen medicine-feeding group, hypoxia matched group and hypoxia medicine-feeding group, wherein hypoxia matched group and hypoxia medicine-feeding group place hypoxic cell incubator (37 ℃, 5%CO
2, 4%O
2) cultivated 60 hours.Normal oxygen medicine-feeding group and hypoxia medicine-feeding group are all carried out pharmaceutical intervention with the 10mg/L sodium tanshinone IIA sulfate, and normal oxygen matched group and hypoxia matched group add normal saline.
3, survey intracellular calcium concentration:
Survey the former generation arteria pulmonalis smooth muscle cells that each group is obtained before the calcium and after 24 hours, add 5 μ M Fura-2,37 ℃, 5%CO with the cultivation of 0.3% blood serum medium
2Hatched 1 hour, and measured with calcium concentration in the calcium ion fluoroscopic image system pair cell.
Result of the test: as shown in Figure 1, normal oxygen cellular control unit basis calcium is 130.63 ± 16.06nM, and normal oxygen medicine-feeding group cell base calcium is 98.05 ± 10.91nM; Hypoxia cellular control unit basis calcium (4%O
2, 60h) be 237.12 ± 40.13nM, hypoxia medicine-feeding group cell base calcium (4%O
2, 60h, 10mg/L sodium tanshinone IIA sulfate) and be 103.05 ± 20.20nM.
Conclusion: tanshinone can suppress the rising of basic calcium concentration in the cell that hypoxia causes.
Embodiment two: acceptable salt can act on SOCC passage reduction intracellular calcium concentration on tanshinone or its materia medica
Material and method:
One, main experiment material
1, SPF level SD rat: body weight 200g-250g, Guangdong Province's Experimental Animal Center
2, low sugar DMEM culture medium, hyclone (fetal bovine serum, FBS), the type i collagen enzyme: U.S. Gibico company
3, pentobarbital sodium: Guangzhou chemistry two factories
4, papain, CPA, nifedipine: Sigma company
5, Fura-2 fluorescent dye: Invitrogen company
Two, key instrument
1, calcium ion fluoroscopic image system: InCyt Im1
TMCompany
2, fluorescence inverted microscope: Nikon company
3, micropipettor: Gilson company
Three, experimental technique
1, arteria pulmonalis smooth muscle cells former is commissioned to train foster:
With embodiment one.
2, the making of hypoxic cell model:
With embodiment one.
3, survey the variation of SOCE in the cell:
Survey the former generation arteria pulmonalis smooth muscle cells that each group is obtained before the calcium and after 24 hours, add 5 μ M Fura-2,37 ℃, 5%CO with the cultivation of 0.3% blood serum medium
2Hatched 1 hour, successively with the HPSS liquid perfusion cell 5 minutes that calcium is arranged, the HPSS liquid perfusion cell 10min that contains 5uM nifedipine and 10uM CPA of no calcium, the HPSS liquid perfusion cell 10min that contains 5uM nifedipine and 10uM CPA that calcium is arranged, the HPSS liquid perfusion cell 10 minutes that calcium is arranged, change with calcium concentration in the calcium ion fluoroscopic image system pair cell then and measure in real time, analyze the influence that tanshinone pair cell SOCE changes.
Result of the test: as shown in Figure 2, normal oxygen cellular control unit SOCE peak value is 196.64 ± 24.44nM, and normal oxygen medicine-feeding group cell SOCE peak value is 143.82 ± 13.20nM, hypoxia cellular control unit (4%O
2, 60h, normal saline) and the SOCE peak value is 307.12 ± 5.47nM, hypoxia medicine-feeding group cell (4%O
2, 60h, 10mg/L sodium tanshinone IIA sulfate) and the SOCE peak value is 119.01 ± 19.36nM.
Conclusion: the rise of SOCE when acceptable salt can suppress hypoxia on tanshinone or its materia medica illustrates that TANSHINONES can act on the SOCC passage and reduce intracellular calcium concentration.
Embodiment three: acceptable salt influences intracellular calcium concentration by the expression that influences TRPC1mRNA on tanshinone or its materia medica
Material and method:
One, main experiment material
1, SPF level SD rat: body weight 200g-250g, Guangdong Province's Experimental Animal Center
2, pentobarbital sodium: Guangzhou chemistry two factories
3, low sugar DMEM culture medium, hyclone (fetal bovine serum, FBS), the type i collagen enzyme: U.S. Gibico company
5, papain: Sigma company
6, Trizol:Invitrogen company
7, PrimeScript RT reagent Kit:TaKaRa company
8, SsoFast EvaGreen Supermix:Biorad company
Two, key instrument
1, micropipettor: Gilson company
2, regular-PCR instrument, CEX96 quantitative real time PCR Instrument: Biorad company
Three, experimental technique
1, arteria pulmonalis smooth muscle cells former is commissioned to train foster:
With embodiment one.
2, the making of hypoxic cell model:
Method is identical with embodiment one, and normal oxygen medicine-feeding group and hypoxia medicine-feeding group are all carried out pharmaceutical intervention with the 5mg/L sodium tanshinone IIA sulfate, and normal oxygen matched group and hypoxia matched group add normal saline.
3, detect TRPC1mRNA expression difference in the cell:
Extract the total RNA that respectively organizes cell that cultivates according to the Trizol workbook, carry out reverse transcription then, obtain cDNA, the reverse transcription process is as follows:
Reaction system (20ul):
Reaction system is blown and beaten mixing,
Reaction condition:
37 ℃ of reaction 15min react 5sec down at 85 ℃ again.
Primer with TRPC1 carries out the RT-PCR detection
Rat TRPC1 primer: positive-sense strand sequence: 5 '-AGCCTCTTGACAAACGAGGA-3 '
Antisense strand sequence: 5 '-ACCTGACATCTGTCCGAACC-3 '
Reaction system: (15ul)
With reaction system piping and druming evenly, 95 ℃ of following degeneration 3min.
Reaction condition:
95 ℃ of degeneration 5sec; 60 ℃ of degeneration 15sec, totally 40 circulations.
Result of the test: as shown in Figure 3, normal oxygen medicine-feeding group is 97.92 ± 9.03 with respect to the expression of normal oxygen matched group TRPC1mRNA, and the hypoxia matched group is 178.86 ± 9.79 with respect to the expression of normal oxygen matched group, hypoxia medicine-feeding group (4%O
2, 60h, 5mg/L sodium tanshinone IIA sulfate) and be 87.16 ± 6.40 with respect to the expression of normal oxygen matched group.
Conclusion: acceptable salt can reduce the expression of pulmonary hypertension cell model TRPC1mRNA on tanshinone or its materia medica, TRPC1mRNA is by translating into TRPC1 albumen, and TRPC1 albumen is the constitutive protein of SOCC calcium channel, and then acceptable salt can influence intracellular calcium concentration by the expression that influences TRPC1mRNA on tanshinone or its materia medica.
Embodiment four: acceptable salt can influence intracellular calcium concentration by the expression that influences TRPC6mRNA on tanshinone or its materia medica
Material and method:
One, main experiment material
With embodiment three.
Two, key instrument
With embodiment three.
Three, experimental technique
1, arteria pulmonalis smooth muscle cells former is commissioned to train foster:
With embodiment one.
2, the making of hypoxic cell model:
With embodiment three.
3, detect TRPC1mRNA expression difference in the cell:
Response system and condition are with embodiment three.
Rat TRPC6 primer: positive-sense strand sequence: 5 '-TACTGGTGTGCTCCTTGCAG-3 '
The antisense strand sequence: 5 '-GAGCTTGGTGCCTTCAAATC-3 '.
Result of the test: as shown in Figure 4, normal oxygen medicine-feeding group is 126.10 ± 26.31 with respect to the expression of normal oxygen matched group TRPC6mRNA, hypoxia matched group (4%O
2, 60h) expression with respect to normal oxygen matched group is 176.78 ± 19.96, hypoxia medicine-feeding group (4%O
2, 60h, 5mg/L sodium tanshinone IIA sulfate) and be 101.37 ± 7.32 with respect to the expression of normal oxygen matched group.
Conclusion: acceptable salt can reduce the expression of pulmonary hypertension cell model TRPC6mRNA on tanshinone or its materia medica, TRPC6mRNA is by translating into TRPC6 albumen, and TRPC6 albumen is the constitutive protein of SOCC calcium channel, and then acceptable salt can influence intracellular calcium concentration by the expression that influences TRPC6mRNA on tanshinone or its materia medica.
Embodiment five: acceptable salt can effectively reduce the proteic expression of pulmonary hypertension model induced lung arterial smooth muscle tissue TRPC1 on tanshinone or its materia medica
One, main experiment material
1, the Mus TRPC1 of the rabbit Chinese People's Anti-Japanese Military and Political College one is anti-: U.S. Sanda company
2, the goat-anti rabbit Ig-G two of horseradish peroxidase-labeled is anti-: Dalian Bo Ruide company
3, dye molecular weight of albumen Marker in advance: U.S. BIO-RAD company
4, RIPA cell pyrolysis liquid: Chinese green skies biotechnology research institute
5, DC determination of protein concentration test kit: U.S. BIO-RAD company
6, the Mus actin of rabbit Chinese People's Anti-Japanese Military and Political College antibody: U.S. Sigma company
Two, key instrument
1, electrophresis apparatus: U.S. BIO-RAD company
2, change the film instrument: U.S. BIO-RAD company
Three, experimental technique
1, the making of Hypoxic Pulmonary Hypertension in Rats rat model:
Rat is divided into normal oxygen matched group, normal oxygen medicine-feeding group, hypoxia matched group and hypoxia medicine-feeding group.Normal oxygen matched group and normal oxygen medicine-feeding group are raised at normal feeding environment, and hypoxia matched group and hypoxia medicine-feeding treated animal place in the hypoxia animal feeding case, at 10%O
2Raised 21 days under the concentration.Normal oxygen medicine-feeding group and hypoxia medicine-feeding group lumbar injection every day 30mg/kg body weight sodium tanshinone IIA sulfate, normal oxygen matched group and hypoxia matched group give the normal saline of same dose.
2, pulmonary vascular smooth muscle is organized the extraction of total protein
Put to death experimental rat, micro-separating experiment animal pulmonary vascular smooth muscle layer respectively according to grouping.Smooth muscle tissue is added in the EP pipe of protein lysate (pre-cooling), 4 ℃ of centrifugal 20min of 12000rpm get supernatant after the Ultrasonic Pulverization.Aforesaid operations all carries out on ice, to avoid protein degradation.Protein concentration is determined with the DC analytic process.
3, the mensuration of sample protein concentration: adopt the DC development process, measure according to the workbook of DC determination of protein concentration test kit.
4、Western?blotting
(1) the SDS-polyacrylamide separation gel of preparation 10%
The mixed liquor of following reagent is added in the middle of two blocks of glass offset plates of the about 2mm of edge strip, make liquid level reach the about 1cm of application of sample comb lower edge place, slowly add one deck dehydrated alcohol, room temperature leaves standstill treats the natural polymerization coagulation of its gel.
The dehydrated alcohol on careful tipping upper strata after the separation gel polymerization, deionized water rinsing back for several times carefully blot with filter paper, and concentrated glue reinjects.
(2) preparation 4% concentrates glue
After above-mentioned mixing material injects between the layer glass plate, carefully insert the application of sample comb, room temperature leaves standstill, treat carefully to take out the application of sample comb after its polymerization, with electrophoretic buffer flushing well for several times in order to avoid residual in the hole gel arranged.
(3) sample treatment
Get contain equal protein respectively organize sample, add the 2 * albumen sample-loading buffer mixing that contains beta-mercaptoethanol respectively, in boiling water, boil and put into ice rapidly after 5 minutes and make albuminous degeneration.
(4) the SDS-polyacrylamide gel electrophoresis (SDS-polyacrylamidedel gelelectrophoresis, SDS-PAGE)
Sample protein separates with SDS-PAGE.Electrophoresis use earlier constant voltage 80V, when bromophenol blue walks to when concentrating glue with the separation gel intersection, voltage is transferred to 100V continue electrophoresis, at bromjophenol blue end electrophoresis near bottom the separation gel time.
(5) change film
To be gone on the nitrocellulose filter by the albumen electricity of electrophoretic separation on the gel.Unload offset plate, cut the separation gel part that contains separated sample protein, be cut into glue size consistent with nitrocellulose filter 6 of filter paper, be dipped in the transfering buffering liquid, about 10min places 3 of 3 of filter paper, SDS-PAGE gel, nitrocellulose filter, filter paper successively from top to bottom, drives bubble between each layer away with Glass rod, put battery lead plate well, carry out the wet type electrotransfer.
(6) sealing
Film after transfer finishes spends the night with the sealing of 5% Ox blood serum.
(7) hybridize and wash film
1h is hatched in 4 ℃ of hybridization of the anti-mice TRPC6 of film after the sealing and monoclonal rabbit (I is anti-), hybridizes 1h with goat anti-rabbit igg antibody (two the is anti-) incubated at room of horseradish peroxidase-labeled again.Antibody all suitably dilutes with the antibody buffer, and jog antibody makes it even with film, fully contacts during hybridization, and each hybridization finishes and all uses not binding antibody of TTBS flush away remnants.
(8) chemiluminescence and exposure
Nitrocellulose filter is dipped in the DAB colour developing liquid, observes in the dark place, observing has when specific band occurs clearly, reacts with color development stopping with a large amount of water flushing nitrocellulose filters.
5, strip analysis as a result
With gel imaging system sepharose electrophoresis figure and Western blotting exposed plate are carried out image scanning and band optical density semi-quantitative analysis.For eliminating the error in scanning and the analysis, this process of triplicate adopts SPASS13.0 software analysis result, relatively adopts one factor analysis of variance between group, and P＜0.05 is thought statistical significance.
Result of the test: as Fig. 5, shown in Figure 7, normal oxygen medicine-feeding group is 1.02 ± 0.11 with respect to the proteic expression of normal oxygen matched group TRPC1, hypoxia matched group (10%O
2, 21 days) and be 1.91 ± 0.11 with respect to the expression of normal oxygen matched group, hypoxia medicine-feeding group (10%O
2, 21 days, 30mg/kg sodium tanshinone IIA sulfate) and be 1.43 ± 0.12 with respect to the expression of normal oxygen matched group.
Conclusion: acceptable salt can reduce the proteic expression of pulmonary hypertension model induced lung arterial smooth muscle tissue TRPC1 on tanshinone or its materia medica, TRPC1 albumen is the important composition albumen of SOCC calcium channel, and then acceptable salt can influence the contraction of pulmonary artery and reinvents by influencing the proteic expression of TRPC1 on tanshinone or its materia medica.
Embodiment six: acceptable salt can effectively reduce the proteic expression of pulmonary hypertension model induced lung arterial smooth muscle tissue TRPC6 on tanshinone or its materia medica
One, main experiment material
1, the Mus TRPC6 of the rabbit Chinese People's Anti-Japanese Military and Political College one is anti-: U.S. Sanda company
2, the goat-anti rabbit Ig-G two of horseradish peroxidase-labeled is anti-: Dalian Bo Ruide company
3, dye molecular weight of albumen Marker in advance: U.S. BIO-RAD company
4, RIPA cell pyrolysis liquid: Chinese green skies biotechnology research institute
5, DC determination of protein concentration test kit: U.S. BIO-RAD company
6, the Mus actin of rabbit Chinese People's Anti-Japanese Military and Political College antibody: U.S. Sigma company
Two, key instrument
1, electrophresis apparatus: U.S. BIO-RAD company
2, change the film instrument: U.S. BIO-RAD company
Three, experimental technique
With embodiment five.
Result of the test: as Fig. 6, shown in Figure 7, normal oxygen medicine-feeding group is 0.97 ± 0.14 with respect to the proteic expression of normal oxygen matched group TRPC6, hypoxia matched group (10%O
2, 21 days) and be 2.48 ± 0.14 with respect to the expression of normal oxygen matched group, hypoxia medicine-feeding group (10%O
2, 21 days, 30mg/kg sodium tanshinone IIA sulfate) and be 1.72 ± 0.04 with respect to the expression of normal oxygen matched group.
Conclusion: acceptable salt can reduce the proteic expression of pulmonary hypertension model induced lung arterial smooth muscle tissue TRPC6 on tanshinone or its materia medica, TRPC6 albumen is the important composition albumen of SOCC calcium channel, and then acceptable salt can influence the contraction of pulmonary artery and reinvents by influencing the proteic expression of TRPC6 on tanshinone or its materia medica.
Embodiment seven: acceptable salt can effectively reduce pulmonary hypertension model rat right ventricular pressure on tanshinone or its materia medica
Material and method:
One, main experiment material
1, SPF level SD rat: body weight 200g-250g, Guangdong Province's Experimental Animal Center
2, pentobarbital sodium: Guangzhou chemistry two factories
3, heparin sodium
Two, key instrument
1,16 passage toy physiographs: BIOPAC company
2, toy operating theater instruments: Shanghai medical apparatus and instruments company limited operating theater instruments factory
Three, experimental technique
1, the making of Hypoxic Pulmonary Hypertension in Rats rat model:
With embodiment five.
2, rat right ventricular pressure force measurement:
Experimental rat is anaesthetized, be fixed on the toy operating-table, cut off its skin of neck, passivity is separated the rat jugular vein, and the ligation distal end is cut off blood vessel, silica gel tube with the 2mm bore inserts blood vessel, enter right ventricle, be communicated with pressure transducer, right ventricular pressure, heart rate are measured with the toy physiograph.
Result of the test: as shown in Figure 8, normal oxygen control rats right ventricle mean blood pressure is 12.04 ± 0.579mmHg, normal oxygen medicine-feeding group rat right ventricle mean blood pressure is 12.25 ± 0.364mmHg, hypoxia control rats right ventricle mean blood pressure is 26.58 ± 0.812mmHg, and hypoxia medicine-feeding group right ventricle mean blood pressure is 18.43 ± 0.369mmHg.
Conclusion: acceptable salt can effectively reduce pulmonary hypertension model rat right ventricular pressure on tanshinone or its materia medica, can reduce the pulmonary hypertension pulmonary arterial pressure in rats.
Embodiment eight: acceptable salt can effectively suppress the right heart compensatory hypertrophy of pulmonary hypertension rat model on tanshinone or its materia medica
Material and method:
One, main experiment material
1, SPF level SD rat: body weight 200g-250g, Guangdong Province's Experimental Animal Center
2, pentobarbital sodium: Guangzhou chemistry two factories
Two, key instrument
1, analytical balance: Mettler Toledo Inc.
2, toy operating theater instruments: Shanghai medical apparatus and instruments company limited operating theater instruments factory
Three, experimental technique
1, the making of Hypoxic Pulmonary Hypertension in Rats rat model:
With embodiment five.
2, the mensuration of rat right ventricle hypertrophy index
Get each experimental group animal hearts, separate its right ventricle wall and left ventricular wall, the flush away blood stains, suck dry moisture carries out accurate weighing, and right ventricle wall weight/left ventricular wall weight is right ventricle hypertrophy index.
Result of the test: as shown in Figure 9, normal oxygen control rats right ventricle hypertrophy index is 0.26 ± 0.013, normal oxygen medicine-feeding group rat right ventricle hypertrophy index is 0.26 ± 0.012, and hypoxia control rats right ventricle hypertrophy index is 0.55 ± 0.016, and hypoxia medicine-feeding group right ventricle hypertrophy index is 0.39 ± 0.021.
Conclusion: acceptable salt can effectively suppress the right heart compensatory hypertrophy of pulmonary hypertension rat model on tanshinone or its materia medica, prevents that the mistake of the right heart is compensatory.
Embodiment nine: acceptable salt can reduce the pathological change of the little vascular smooth muscle layer of lung of Hypoxic Pulmonary Hypertension in Rats rat model on tanshinone or its materia medica
Material and method:
One, main experiment material
1, SPF level SD rat: body weight 200g-250g, Guangdong Province's Experimental Animal Center
2, pentobarbital sodium: Guangzhou chemistry two factories
3, neutral formalin: Sigma company
4, haematoxylin: Guangzhou chemical reagent two factories
5, Yihong: Guangzhou chemical reagent two factories
Two, key instrument
1, Citadel 1000 tissue processors: Britain Shandon company
2, BM-II water-bath type biological tissue embedding machine: the firm Electronics Factory that reaches is newly pacified in Shenzhen
3, optical microscope: Japanese Olympus company
4, digital photography integral system: Japanese Nikon company
5, fluorescence inverted microscope: German Leica company
Three, experimental technique
1, the making of Hypoxic Pulmonary Hypertension in Rats rat model:
With embodiment five.
2, lung tissue of rats pathological research
Anesthesia is put to death and is respectively organized experimental rat, separates trachea, pours into full lung with 4% paraformaldehyde and fixes, and get lung tissue and carry out routine pathology slicing treatment, HE dyeing, microscopically is observed.
Being prepared as follows of Pathologic specimen:
Getting lung tissue of rats places 4% paraformaldehyde to fix
1) dehydration: 70% ethanol 2h → 80% ethanol 1h → 95% ethanol 1h → 95% ethanol 1h → dehydrated alcohol 2h → dehydrated alcohol 1.5h → dehydrated alcohol 1.5h;
2) transparent: dimethylbenzene 0.5h → dimethylbenzene 1h → dimethylbenzene 0.5h
3) waxdip: low temperature (50 ℃) waxdip 1h → waxdip 1h
4) embedding: the tissue behind the waxdip is put into 60 ℃ of paraffin embeddings
5) section: 4um serial section
6) stand sheet: spread out sheet in 49 ℃ of warm water
7) baking sheet: 55 ℃ of baking 20min
8) transparent and aquation:
Dimethylbenzene 10min → dimethylbenzene 10min → dehydrated alcohol 5min → dehydrated alcohol 5min → 95% ethanol 5min → 80% ethanol 5min → 70% ethanol 5min → flowing water flushing 10min
9) HE dyeing:
Haematoxylin dyeing 1min → flowing water flushing 1min → 1% hydrochloride alcohol decolouring 10sec → flowing water flushing 10min → Yihong dyeing 10sec → flowing water flushing 10min → 55 ℃ baking 20min → neutral gum mounting.
Result of the test: shown in the A and B of Figure 10, normal oxygen matched group and normal oxygen medicine-feeding group lung tissue of rats structure do not have obviously unusual, lung small artery inner membrance complete and smooth, and the flesh layer does not have and thickens; Hypoxia matched group visible lung small artery tube wall and smooth muscle layer obviously thicken luminal stenosis shown in the C of Figure 10 and D; The lung morphological change of hypoxia medicine-feeding group and the hypoxia matched group is apparent in view alleviates.
Conclusion: acceptable salt can reduce the pathological change of the little vascular smooth muscle layer of lung of Hypoxic Pulmonary Hypertension in Rats rat model, pulmonary vascular reinventing when alleviating pulmonary hypertension on tanshinone or its materia medica.
Claims (10)
1. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone or its materia medica.
2. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone according to claim 1 or its materia medica, it is characterized in that described treatment or prevention pulmonary hypertension disease medicament are treatment or prophylaxis of pulmonary hypertension medicine.
3. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone according to claim 1 or its materia medica, it is characterized in that described treatment or prevention pulmonary hypertension disease medicament are for treatment or prevent left heart disease association pulmonary hypertension medicine.
4. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone according to claim 1 or its materia medica, it is characterized in that described treatment or prevention pulmonary hypertension disease medicament are treatment or the prevention pulmonary hypertension medicine relevant with respiratory system disease and/or anoxia.
5. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone according to claim 1 or its materia medica, it is characterized in that described treatment or prevention pulmonary hypertension disease medicament are for treatment or prevent chronic thromboembolia type pulmonary hypertension medicine.
6. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone according to claim 1 or its materia medica, it is characterized in that described treatment or prevention pulmonary hypertension disease medicament are pulmonary hypertension medicine due to the not clear and definite multiple factor of treatment or prevention.
7. according to the application of acceptable salt on the described tanshinone of the arbitrary claim of claim 1～6 or its materia medica in preparation treatment or prevention pulmonary hypertension disease medicament, it is characterized in that acceptable salt uses separately on described tanshinone or its materia medica.
8. according to the application of acceptable salt on the described tanshinone of the arbitrary claim of claim 1～6 or its materia medica in preparation treatment or prevention pulmonary hypertension disease medicament, it is characterized in that, acceptable salt uses with the form of pharmaceutical composition on described tanshinone or its materia medica, and described pharmaceutical composition comprises acceptable salt and pharmaceutical carrier on tanshinone or its materia medica.
9. the application of acceptable salt in preparation treatment or prevention pulmonary hypertension disease medicament on tanshinone according to claim 8 or its materia medica, it is characterized in that, comprise also in the described pharmaceutical composition that prostacyclin, endothelin-1 receptor pick up one or more the compositions in anti-agent and the 5 type phosphodiesterase inhibitors.
10. according to the application of acceptable salt on the described tanshinone of the arbitrary claim of claim 1～6 or its materia medica in preparation treatment or prevention pulmonary hypertension disease medicament, it is characterized in that acceptable salt and pharmaceutically suitable carrier are prepared into oral formulations or injection on described tanshinone or its materia medica.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100922180A CN102160866A (en) | 2011-04-13 | 2011-04-13 | Application of tanshinone IIA or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing pulmonary hypertension disease |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011100922180A CN102160866A (en) | 2011-04-13 | 2011-04-13 | Application of tanshinone IIA or pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing pulmonary hypertension disease |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961383A (en) * | 2012-11-16 | 2013-03-13 | 广州医学院第一附属医院 | Application of tanshinone IIA or pharmaceutically acceptable salt thereof in improving exercise tolerance of pulmonary vascular disease patients |
| CN109498675A (en) * | 2018-12-29 | 2019-03-22 | 王亚峰 | Application of the Salvia przewalskii Maxim in preparation prevention or treatment pulmonary hypertension drug |
| CN114469960A (en) * | 2022-03-01 | 2022-05-13 | 天津欣满和生物科技有限公司 | Application of tanshinone IIA sodium sulfonate in preparation of medicine for treating pulmonary artery remodeling diseases |
-
2011
- 2011-04-13 CN CN2011100922180A patent/CN102160866A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 《Cardiovascular Pharmacology》 20100509 Jing Wang等 Tanshinone IIA modulates pulmonary vascular response to agonist and hypoxia primarily via inhibiting Ca2+ influx and release in normal and hypoxic pulmonary hypertension rats 129-138 1-9 , 第640期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961383A (en) * | 2012-11-16 | 2013-03-13 | 广州医学院第一附属医院 | Application of tanshinone IIA or pharmaceutically acceptable salt thereof in improving exercise tolerance of pulmonary vascular disease patients |
| CN109498675A (en) * | 2018-12-29 | 2019-03-22 | 王亚峰 | Application of the Salvia przewalskii Maxim in preparation prevention or treatment pulmonary hypertension drug |
| CN114469960A (en) * | 2022-03-01 | 2022-05-13 | 天津欣满和生物科技有限公司 | Application of tanshinone IIA sodium sulfonate in preparation of medicine for treating pulmonary artery remodeling diseases |
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Application publication date: 20110824 |