CN109331000A - Mitochondrial complex I reversible inhibitor combines the purposes for preparing ischemical reperfusion injury protection drug with active oxygen scavenger - Google Patents
Mitochondrial complex I reversible inhibitor combines the purposes for preparing ischemical reperfusion injury protection drug with active oxygen scavenger Download PDFInfo
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- CN109331000A CN109331000A CN201811239481.6A CN201811239481A CN109331000A CN 109331000 A CN109331000 A CN 109331000A CN 201811239481 A CN201811239481 A CN 201811239481A CN 109331000 A CN109331000 A CN 109331000A
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Abstract
The invention discloses mitochondrial complex I reversible inhibitors to combine the purposes for preparing ischemical reperfusion injury protection drug with active oxygen scavenger.It is a discovery of the invention that mitochondrial complex I reversible inhibitor can play the protective effect similar with ischemic preconditioning phenomenon, ischemic preconditioning drug may be used as；It is combined with active oxygen scavenger, mitochondrial complex I reversible inhibitor is given before ischemic, active oxygen scavenger is given before Reperfu- sion, the therapeutic effect of ischemia-reperfusion can be significantly improved.Binary drug release double-layer tablets provided by the invention, it is taken before ischemic, the release layer that outer layer contains mitochondrial complex I reversible inhibitor, which discharges rapidly, plays ischemic preconditioning effect, slow release layer slow release that internal layer contains active oxygen scavenger simultaneously reaches maximum plasma concentration before Reperfu- sion, it is effective, convenient to improve the treatment curative effect of ischemia-reperfusion.
Description
This application be submit on 07 01st, 2018 application No. is " 2018107040563 ", entitled " mitochondrias
Composite I reversible inhibitor combine with active oxygen scavenger prepares ischemical reperfusion injury protection drug purposes " patent
The divisional application of application.
Technical field
The invention belongs to field of medicaments, and in particular to mitochondrial complex I reversible inhibitor and active oxygen scavenger join
Conjunction prepares the purposes of ischemical reperfusion injury protection drug.
Background technique
Currently, myocardial ischemia has become the popular word in the common people mouthful, the main reason is that due to myocardial ischemia in recent years
Incidence constantly rises, and the death rate also obviously rises.In recent years, with the raising of clinical diagnosis and treatment level, so that after myocardial ischemia
The Proportion of patients for obtaining blood reperfusion treatment rises.But myocardial ischemia-reperfusion frequently can lead to a series of heart conditions sometimes
Event occurs, thus beneficial effect of the partial offset to heart, referred to as reperfusion injury.It shows as cardiac muscle cell apoptosis, oxygen
Free radical increases, norepinephrine secretion increases, and leads to the generation of malignant cardiac adverse events.Myocardial ischemia-reperfusion damage
Hurting phenomenon, oneself becomes a great problem for hindering reperfusion therapy, thus, mitigating MIRI becomes the effective for the treatment of myocardial ischemia
Approach (bibliography: the Review Study of myocardial ischemic preconditioning, Long Dong institute journal, the 3rd phase of volume 28 in May, 2017).
The curative effect that the Reperfu- sion stage gives antioxidant (such as N acetylcysteine, the drugs such as vitamin E) treatment again is unknown
It is aobvious.In the Ischemia-reperfusions damage process such as cardiac muscle, many factors participate in Reperfu- sion damage, but the outburst of active oxygen ROS is
Most obvious phenomenon.Theoretically antioxidant can be to anti reperfusion injury, but clinical effectiveness is undesirable, it may be possible to because of Reperfu- sion
There is ROS outburst in very short time (10-20 minutes), and the reducing equivalent that antioxidant provides is not enough to inactivate ROS；In addition,
In reducing/oxidizing reaction, antioxidant itself is used as reducing agent, and when restoring ROS, itself is oxidized, it is possible at
For new active oxygen species.
Ischemic preconditioning (IPC) can effectively improve the treatment curative effect in Reperfu- sion stage.The study found that primary in advance or anti-
Ischemic can make cardiac muscle cell and be protected in the ischemic of long period behind in the multiple multiple short time, so that cell be delayed to wither
It dies, this phenomenon is known as cardiac muscle IPC in clinic.With going deep into for research, scholars are had found, chemical damage, drug etc. can also
Cause the universal pre-adaptation phenomenon of patient's body, to play the protective effect (bibliography: myocardial ischemia similar with IPC phenomenon
The Review Study of pre-adaptation, Long Dong institute journal, the 3rd phase of volume 28 in May, 2017).
The drug that protective effect similar with IPC phenomenon can be played is known as ischemic preconditioning drug by us.
The report that mitochondrial complex I reversible inhibitor is used as ischemic preconditioning drug is had not yet to see, more has no it
Combine the report for ischemical reperfusion injury protection with active oxygen scavenger.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide mitochondrial complex I reversible inhibitor and work
Property oxygen scavenger combine prepare ischemical reperfusion injury protection drug purposes.
Above-mentioned purpose of the invention is achieved by following technical solution:
Mitochondrial complex I reversible inhibitor is combined with active oxygen scavenger prepares ischemical reperfusion injury protection drug
Purposes.
Preferably, the mitochondrial complex I reversible inhibitor is melbine or dihydrotanshinone I.
Preferably, the active oxygen scavenger is vitamin E, protocatechualdehyde or N-acetylcystein.
Mitochondrial complex I reversible inhibitor is used as the purposes of ischemic preconditioning drug.
Preferably, the mitochondrial complex I reversible inhibitor is melbine or dihydrotanshinone I.
Melbine or dihydrotanshinone I are used as the purposes of mitochondrial complex I reversible inhibitor.
A kind of binary sustained-release preparation, including immediate release component and release sustaining component, immediate release component includes mitochondrial complex I can
Inverse property inhibitor；Release sustaining component includes active oxygen scavenger.
Preferably, said preparation is a kind of binary drug release double-layer tablets, including outer layer and internal layer；Outer layer is release layer, wherein containing
Active ingredient mitochondrial complex I reversible inhibitor；Internal layer is slow release layer, wherein containing active constituent oxygen scavenging activity
Agent.
Preferably, the mitochondrial complex I reversible inhibitor is melbine or dihydrotanshinone I.
Preferably, the active oxygen scavenger is vitamin E, protocatechualdehyde or N-acetylcystein.
The utility model has the advantages that
1, it is a discovery of the invention that mitochondrial complex I reversible inhibitor can play it is similar with ischemic preconditioning phenomenon
Protective effect may be used as ischemic preconditioning drug；It is combined with active oxygen scavenger, mitochondrial complex is given before ischemic
I reversible inhibitor gives active oxygen scavenger before Reperfu- sion, can significantly improve the therapeutic effect of ischemia-reperfusion；
2, binary provided by the invention releases the drug double-layer tablets, takes before ischemic, outer layer contains the suppression of mitochondrial complex I invertibity
The release layer of preparation, which discharges rapidly, plays ischemic preconditioning effect, and internal layer contains the slow release layer slow release of active oxygen scavenger simultaneously
Reach maximum plasma concentration before Reperfu- sion, so that the treatment curative effect of ischemia-reperfusion is improved, it is effective, convenient.
Detailed description of the invention
Fig. 1 is reversible inhibition of the dihydrotanshinone I to mitochondrial complex I；
Fig. 2 is each group myocyte survival rate (P: protocatechualdehyde；D: dihydrotanshinone I；X: it is not administered)；
Fig. 3 is the shadow of dihydrotanshinone I and/or protocatechualdehyde to rat myocardial infarction model area in ami model
It rings；
Fig. 4 is the influence of melbine and/or vitamin E to rat myocardial infarction model area in ami model；
Fig. 5 is each group Neuronal Survival rate (P: protocatechualdehyde；D: dihydrotanshinone I；X: it is not administered)；
Fig. 6 is each group renal epithelial cell survival rate (P: protocatechualdehyde；D: dihydrotanshinone I；X: it is not administered)；
Fig. 7 is each group myocyte survival rate (NAC:N- acetylcysteine；D: dihydrotanshinone I；X: it is not administered).
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
Embodiment 1: to the therapeutic effect of myocardial ischemia-reperfusion
One, experimental material
HK-2 cell: H9c2 cell, N2a cell are purchased from ATCC company, are saved by this laboratory.DMEM high glucose medium,
Fetal calf serum, 0.25% pancreatin, 100 × penicillin and streptomysin, CalceinAM (Thermo Fisher company of the U.S.), PBS
(Beijing Bo Aosen Bioisystech Co., Ltd), CoCl2, hydrogen peroxide (Sigma Co., USA), PhosSTOP, complete
ULTRATablets, Mini EASYpack (Roche company), CCK8, Fluo-4AM, Rhod-2AM (Shanghai east Renhua subject skill
Co., Ltd), RIPA lysate, LY294002 (the green skies Bioisystech Co., Ltd in Shanghai), AKT, p-AKT, p-GSK3, β-
Actin (U.S. Cell Signaling Technology).Super-clean bench (Thermo company of the U.S., Thermo Scientific
1300A2 type), multi-function microplate reader FLUOstar Omega (German BMG LABTECH company), laser co-focusing (German karr
Zeiss company), electric-heated thermostatic water bath (Guo Hua Electrical Appliances Co., Ltd, HH-4 digital display thermostat water bath), CO2Incubator, centrifugation
Machine (Thermo Fisher company of the U.S.), balance (Beijing Sai Duolisi scientific instrument Co., Ltd, BT224S), cell culture
Consumptive material, micropipettor (German Eppendorff company), vertical electrophoresis apparatus (Bio-RAD company of the U.S.), Tanon are full-automatic
It learns luminescence imaging system (Shanghai Tian Neng Science and Technology Ltd.).
Standard items: protocatechualdehyde, dihydrotanshinone I, melbine, vitamin E, N-acetylcystein purity are not less than
98%.Animal origin: Sprage-Dawley rat is purchased in Shanghai western Poole-Bi Kai experimental animal Co., Ltd.
Two, experimental method
1, myocardial cells culture and passage
(1) cardiac muscle cell recovers
Taking-up freezes the rat myocardial cell (H9c2 cell) in liquid nitrogen container, is put into fast in the water-bath for be preheated to 37 DEG C
Speed is thawed, and cell is aseptically moved in the centrifuge tube of DMEM culture medium of the 9ml containing 10% serum, 1000rmp centrifugation
4min.Liquid is discarded supernatant, cell is resuspended in the above-mentioned culture medium of 1mL, transfers in the culture dish containing new culture medium, with shifting
Liquid rifle, which gently dispels cell, makes it be uniformly distributed in culture dish, is placed in 37 DEG C, 5%CO2CMC model changes liquid afterwards for 24 hours.
(2) cell dissociation
The culture medium in culture dish is discarded, washed once with PBS solution, after 1ml pancreatin is added, culture dish is placed in cell
2min in incubator is added 2ml culture medium and terminates digestion, with liquid-transfering gun by the cell of adherent growth when cellular contraction is rounded
It dispels to completely falling off, it is made to be suspended from culture medium, used to follow-up test.
(3) cell passes on
Culture dish is placed in microscopically observation, when cell grows to fusion 80%~90%, after cell dissociation, and transfer
Into centrifuge tube, 1000rmp is centrifuged 4min, discards supernatant liquid, cell is resuspended in new culture medium, by 1:3 pro rate
Extremely in the culture dish containing new culture medium, it is placed in 37 DEG C, containing 5%CO2Cell incubator in cultivate.
2, the method for building up and pharmaceutical intervention of Myocytes Anoxia reoxygenation model
Cardiac muscle cell's cell it is adherent after with contain 2% serum culture medium starvation 12h, after change anoxic liquid, be placed in anoxic case,
Control anoxic case O2Concentration 1%, CO2Concentration 5% after anoxic 8h, changes the normal culture solution of 2% serum into, continues to cultivate 8h.
Experimental group is as follows:
Control group (control group, Ctrl): the normal culture solution of whole 2% serum is normally trained after cardiac muscle cell is adherent
It supports；
Hypoxia-reoxygenation group (model group, H/R): modeling culture is carried out by the method for building up of above-mentioned Hypoxia-reoxygenation model；
Tanshinone-protocatechualdehyde group (D-P): rear to lack with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I
Oxygen culture 8h changes the normal culture solution containing 2% serum, 40 μM of protocatechualdehydes into and continues to cultivate 8h；
Protocatechualdehyde-tanshinone group (P-D): with the culture medium starvation 12h containing 2% serum, 40 μM of protocatechualdehydes, rear anoxic
8h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
Tanshinone group (D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic culture 8h is changed
Continue to cultivate 8h at the normal culture solution containing 2% serum；
Tanshinone-tanshinone group (D-D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic
8h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
Protocatechualdehyde group (P): with contain 2% serum culture medium starvation 12h, rear anoxic culture 8h, change into containing 2% serum,
The normal culture solution of 40 μM of protocatechualdehydes continues to cultivate 8h；
Protocatechualdehyde-protocatechualdehyde group (P-P): rear to lack with the culture medium starvation 12h containing 2% serum, 40 μM of protocatechualdehydes
Oxygen culture 8h changes the normal culture solution containing 2% serum, 40 μM of protocatechualdehydes into and continues to cultivate 8h.
3, the method for building up and pharmaceutical intervention of acute myocardial infarction of rat model
This experiment fills model [A novel and using coronary artery left anterior descending branch desmurgia production acute myocardial infarction of rat again
efficient model of coronary artery ligation and myocardial infarction in the
mouse,Circ Res.2010；107(12):1445-53].Method are as follows: take healthy SD rat, weight 250-280g, measurement is greatly
Mouse limb lead II leads electrocardiogram, and the normal rat of coring electrograph is performed the operation.Set ventilator parameter are as follows: respiratory rate 80
Secondary/min, tidal volume are 10ml/kg weight, respiratory quotient 2:1.Rat is using 3% yellow Jackets intraperitoneal injection (45mg/kg)
After anesthesia, tracheotomy connects ventilator row artificial respiration, opens ramus descendens anterior arteriae coronariae sinistrae (Left on chest and exposure heart
Anterior descending, LAD) ligation point, at left auricle of heart lower edge 2mm with 3/8 arc 3 × 6 without cingula line ophthalmology suture
Needle (6-0 silk thread) passes through myocardium shallow-layer, ligatures LAD, is obviously above lifted with ECG ST section and myocardium color below ligature is dimmed
For markers of cardiac ischemia, after ligation, chest, layer-by-layer suture rib cage, muscle and skin are closed；After pseudo- operation group opens chest, in modeling equity
Position only threads and does not ligature.
Reperfu- sion: it after ischemic 30min, opens chest and unlocks ligation recovery blood supply, close chest, layer-by-layer suture rib cage, muscle and skin.
Experimental group is as follows, every group of 6 rats:
Pseudo- operation group (control group, Sham): chest is opened according to the above method, is not ligatured；
Ischemia-reperfusion group (model group, H/R): ischemia-reperfusion is carried out by above-mentioned method for establishing model；
Tanshinone-protocatechualdehyde group (D-P): opening 48h stomach-filling before breast knot pricks ischemic and give 0.1mg/kg dihydrotanshinone I,
(or tail vein, similarly hereinafter) is injected intraperitoneally before Reperfu- sion and gives 4mg/kg protocatechualdehyde；
Protocatechualdehyde-tanshinone group (P-D): it opens 48h stomach-filling before breast knot pricks ischemic and gives 4mg/kg protocatechualdehyde, Reperfu- sion
0.1mg/kg dihydrotanshinone I is given in preceding intraperitoneal injection；
Tanshinone-tanshinone group (D-D): opening 48h stomach-filling before breast knot pricks ischemic and give 0.1mg/kg dihydrotanshinone I, then
0.1mg/kg dihydrotanshinone I is given in intraperitoneal injection before perfusion；
Protocatechualdehyde-protocatechualdehyde group (P-P): it opens 48h stomach-filling before breast knot pricks ischemic and gives 4mg/kg protocatechualdehyde, then fill
4mg/kg protocatechualdehyde is given in intraperitoneal injection before note；
Melbine-vitamin E group (M-V): opening 48h stomach-filling before breast knot pricks ischemic and give 6.5mg/kg melbine, then
20mg/kg vitamin E is given in intraperitoneal injection (or tail vein, similarly hereinafter) before perfusion；
Vitamin E-melbine group (V-M): it opens 48h stomach-filling before breast knot pricks ischemic and gives 20mg/kg vitamin E, then fill
6.5mg/kg melbine is given in intraperitoneal injection before note；
Melbine-melbine group (M-M): opening 48h stomach-filling before breast knot pricks ischemic and give 6.5mg/kg melbine, then
6.5mg/kg melbine is given in intraperitoneal injection before perfusion；
Vitamin E-vitamin E group (V-V): it opens 48h stomach-filling before breast knot pricks ischemic and gives 20mg/kg vitamin E, Reperfu- sion
20mg/kg vitamin E is given in preceding intraperitoneal injection.
4, CCK8 method measures the survival rate of each group cardiac muscle cell in Myocytes Anoxia reoxygenation model
Cell inoculation is into 96 orifice plates, 5000, every hole cell, and after the processing of culture, administration and anoxia _ reoxygenation, every hole adds
Enter 10 μ L (the 1/10 of nutrient solution volume) CCK-8 solution, blank group is done with not celliferous culture solution, every group sets 6 multiple holes, sets
37 DEG C, 5%CO2After being incubated for 2~4h in incubator, the absorbance (OD value) in each hole is detected at 450nm with microplate reader.According to institute
It surveys OD value and calculates cell survival rate, cell survival rate=(experimental group OD value-blank group OD value) (control/group OD value-blank group OD
Value) × 100%.With control group survival rate for 100%.
5, in acute myocardial infarction of rat model each group rat myocardial infarction model area measurement
Rat weight, 20% urethane (1g/kg) anesthesia, opens rapidly chest and wins heart, cut off surrounding connective tissue, use ice
Physiological saline cleans, and quick after filter paper suck dry moisture to cross liquid nitrogen flash freezer, heart tissue is uniformly cut to the thin slice of 1~2mm, is placed in
10min is dyed in 37 DEG C of water-baths in 1%TTC dyeing liquor.After TTC is dyed, normal myocardium tissue is in rose, and infarction tissue is in
White.Computer is inputted after shooting photo with digital camera, calculates infarct size hundred with Image-ProPlus6 image analysis software
Divide ratio, calculation formula are as follows: infarct area/left ventricular area × 100%.
6, reversible inhibition of the dihydrotanshinone I to mitochondrial complex I
Seahorse XF experiment flow
Experiment the previous day
(1) it inoculating cell: seeds cells into Seahorse XF tissue culture plate, is trained overnight in growth medium
It supports.Note: cell inoculation time and incubation time can be selected according to cell processing requirement.
(2) preheater apparatus: opening Seahorse instrument and computer, opens software, instrument is made to be warming up to 37 DEG C, pre- overnight
Heat.
(3) aquation probe: Seahorse XF calibration solution is added in Utility Plate, test board is put back to
On Utility Plate, 37 DEG C are placed in without CO2Aquation probe is stayed overnight in incubator.
Experimental day
(1) prepare detection liquid: preparing detection liquid with Seahorse XF Base Medium, substrate required for being added will
Solution is heated to 37 DEG C, and with NaOH tune pH value to 7.4, filtering is set spare in 37 DEG C of water-baths.Note: required addition in detection liquid
Substrate and concentration depend on cell type and experimental design, or be consistent with growth medium.
(2) cell is observed: by cell plates from CO2It is taken out in incubator, observes cell state under the microscope.
(3) cell changes liquid: growth medium being changed to detection liquid, cell is then placed on 37 DEG C without CO2In incubator
1h。
(4) it makes up a prescription: according to kit specification, preparing and dilute drug to required concentration.
(5) dosing: the drug diluted is separately added into tetra- medicine feeding holes of A, B, C, D on test board.Note: according to reality
Test dosing hole number used in design alternative.
(6) software records experiment condition and contrived experiment program contrived experiment template: are used.
(7) machine on: bring into operation experimental arrangement, by the test board of medicine was added and the Utility Plate equipped with calibration solution
It is placed on instrument supporting plate together.
(8) instrument calibrates probe automatically.
(9) cell plates are changed: when prompt information occurs in software, Utility Plate being changed to cell plates.
(10) test board and cell plates are exited according to software prompt information when experiment is completed, and observe cell under the microscope
State.
(11) data are analyzed: being analyzed using wave software and report generator experimental data.
7, statistical method
Experimental result is indicated with mean ± standard deviation (X ± SD).Sample average compares two-by-two, using GraphPad
6 software of Prism carries out Two-wayANOVA inspection, and P < 0.05 thinks significant difference.
Three, experimental result
1, reversible inhibition of the dihydrotanshinone I to mitochondrial complex I
Dihydrotanshinone I is washed away with the method for wash can reverse it to mitochondrial complex I inhibiting effect, root
According to time shaft, 0-16min, in the case where not adding substrate, all group oxygen consumptions are minimum；Mitochondria is being added in 16-24min
After composite I substrate Mal/Pyr or Complex II substrate Suc/Rot, all groups start to generate oxygen consumption, and rise to highest
Point (24min or so), then all groups, which gradually have, slowly reduces (24-40min), starts this moment in 40min, contains composite I
The other dihydrotanshinone I DT group (black box) of substrate group decreased significantly relative to control group (white box), illustrate that DT can press down
Composite I activity processed, but change containing the other DT group of Complex II group (black circle) relative between control group (white circle)
It is unobvious, thus it is smaller to the activity influence of Complex II.But the inhibitor positive drug rotenone wash of mitochondrial complex I
Method wash away and cannot reverse, illustrate that dihydrotanshinone I is a kind of mitochondrial complex I reversible inhibition different from rotenone
Agent.It can be seen that dihydrotanshinone I with it has been reported that mitochondrial complex I reversible inhibitor melbine have the same line grain
Nanocrystal composition rejection characteristic (bibliography: Metformin inhibits mitochondrial complex I ofcancer
cells to reduce tumorigenesis,Elife.2014)。
2, in Myocytes Anoxia reoxygenation model each group cardiac muscle cell survival rate
Each group myocyte survival rate is as shown in Fig. 2, tanshinone-protocatechualdehyde group (D-P) organizes myocyte survival rate most
Height gives the protective effect for awarding protocatechualdehyde to Ischemia-reperfusion Cardiomyocytes before dihydrotanshinone I, reoxygenation before illustrating anoxic
It is optimal.
3, each group rat myocardial infarction model area in acute myocardial infarction of rat model
As shown in figure 3, tanshinone-protocatechualdehyde group rat myocardial infarction model area is minimum compared with ischemia-reperfusion group, and
It is significantly better than protocatechualdehyde-tanshinone group, tanshinone-tanshinone group, protocatechualdehyde-protocatechualdehyde group (P < 0.05).
As shown in figure 4, melbine-vitamin E group rat myocardial infarction model area is minimum compared with ischemia-reperfusion group,
And it is significantly better than vitamin E-melbine group, melbine-melbine group, vitamin E-vitamin E group (P < 0.05).
Protocatechualdehyde and vitamin E are known active oxygen scavenger.
The above results show to award before giving mitochondrial complex I dihydrotanshinone I or melbine, reoxygenation before anoxic
Active oxygen scavenger protocatechualdehyde or vitamin E are optimal to the protective effect of Ischemia-reperfusion Cardiomyocytes.
Embodiment 2: to the therapeutic effect of cerebral ischemia re-pouring
One, experimental method
1, neuronal cell cultures and passage
(1) nerve cell is recovered
Taking-up freezes the nerve cell (N2a cell) in liquid nitrogen container, is put into the water-bath for be preheated to 37 DEG C and solves rapidly
Freeze, cell is aseptically moved in the centrifuge tube of DMEM culture medium of the 9ml containing 10% serum, 1000rmp is centrifuged 4min.
Liquid is discarded supernatant, cell is resuspended in the above-mentioned culture medium of 1mL, transfers in the culture dish containing new culture medium, use liquid-transfering gun
Gently dispelling cell makes it be uniformly distributed in culture dish, is placed in 37 DEG C, 5%CO2CMC model changes liquid afterwards for 24 hours.
(2) cell dissociation
The culture medium in culture dish is discarded, washed once with PBS solution, after 1ml pancreatin is added, culture dish is placed in cell
1min in incubator is added 2ml culture medium and terminates digestion, with liquid-transfering gun by the cell of adherent growth when cellular contraction is rounded
It dispels to completely falling off, it is made to be suspended from culture medium, used to follow-up test.
(3) cell passes on
Culture dish is placed in microscopically observation, when cell grows to fusion 80%~90%, after cell dissociation, and transfer
Into centrifuge tube, 1000rmp is centrifuged 4min, discards supernatant liquid, cell is resuspended in new culture medium, by 1:3 pro rate
Extremely in the culture dish containing new culture medium, it is placed in 37 DEG C, containing 5%CO2Cell incubator in cultivate.
2, the method for building up and pharmaceutical intervention of nerve cell Hypoxia-reoxygenation model
Nerve cell it is adherent after with contain 2% serum culture medium starvation 12h, after change anoxic liquid, be placed in anoxic case,
Control anoxic case O2Concentration 1%, CO2Concentration 5% after anoxic 4h, changes the normal culture solution of 2% serum into, continues to cultivate 8h.
Control group (control group, Ctrl): the normal culture solution of whole 2% serum is normally trained after nerve cell is adherent
It supports；
Hypoxia-reoxygenation group (model group, H/R): modeling culture is carried out by the method for building up of above-mentioned Hypoxia-reoxygenation model；
Tanshinone-protocatechualdehyde group (D-P): rear to lack with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I
Oxygen culture 4h changes the normal culture solution containing 2% serum, 40 μM of protocatechualdehydes into and continues to cultivate 8h；
Protocatechualdehyde-tanshinone group (P-D): with the culture medium starvation 12h containing 2% serum, 40 μM of protocatechualdehydes, rear anoxic
4h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
Tanshinone group (D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic culture 4h is changed
Continue to cultivate 8h at the normal culture solution containing 2% serum；
Tanshinone-tanshinone group (D-D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic
4h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
Protocatechualdehyde group (P): with contain 2% serum culture medium starvation 12h, rear anoxic culture 4h, change into containing 2% serum,
The normal culture solution of 40 μM of protocatechualdehydes continues to cultivate 8h；
Protocatechualdehyde-protocatechualdehyde group (P-P): rear to lack with the culture medium starvation 12h containing 2% serum, 40 μM of protocatechualdehydes
Oxygen culture 4h changes the normal culture solution containing 2% serum, 40 μM of protocatechualdehydes into and continues to cultivate 8h.
3, CCK8 method measures the survival rate of each group nerve cell in nerve cell Hypoxia-reoxygenation model
Cell inoculation is into 96 orifice plates, 5000, every hole cell, and after the processing of culture, administration and anoxia _ reoxygenation, every hole adds
Enter 10 μ L (the 1/10 of nutrient solution volume) CCK-8 solution, blank group is done with not celliferous culture solution, every group sets 6 multiple holes, sets
37 DEG C, 5%CO2After being incubated for 2~4h in incubator, the absorbance (OD value) in each hole is detected at 450nm with microplate reader.According to institute
It surveys OD value and calculates cell survival rate, cell survival rate=(experimental group OD value-blank group OD value) (control/group OD value-blank group OD
Value) × 100%.With control group survival rate for 100%.
Two, experimental result
Neuronal Survival rate such as Fig. 5, tanshinone-protocatechualdehyde group (D-P) organize myocyte survival rate highest, illustrate to lack
It is given before oxygen and awards protocatechualdehyde before dihydrotanshinone I, reoxygenation to the protective effect of cerebral ischemia re-pouring injured nerve cell most
It is excellent.
Embodiment 3: to the therapeutic effect of Ischemia-reperfusion Injury
One, experimental method
1, renal epithelial cell culture and passage
(1) renal epithelial cell is recovered
Taking-up freezes the renal epithelial cell (HK2 cell) in liquid nitrogen container, is put into the water-bath for be preheated to 37 DEG C rapidly
It thaws, cell is aseptically moved in the centrifuge tube of DMEM culture medium of the 9ml containing 10% serum, 1000rmp centrifugation
4min.Liquid is discarded supernatant, cell is resuspended in the above-mentioned culture medium of 1mL, transfers in the culture dish containing new culture medium, with shifting
Liquid rifle, which gently dispels cell, makes it be uniformly distributed in culture dish, is placed in 37 DEG C, 5%CO2CMC model changes liquid afterwards for 24 hours.
(2) cell dissociation
The culture medium in culture dish is discarded, washed once with PBS solution, after 1ml pancreatin is added, culture dish is placed in cell
1min in incubator is added 2ml culture medium and terminates digestion, with liquid-transfering gun by the cell of adherent growth when cellular contraction is rounded
It dispels to completely falling off, it is made to be suspended from culture medium, used to follow-up test.
(3) cell passes on
Culture dish is placed in microscopically observation, when cell grows to fusion 80%~90%, after cell dissociation, and transfer
Into centrifuge tube, 1000rmp is centrifuged 4min, discards supernatant liquid, cell is resuspended in new culture medium, by 1:3 pro rate
Extremely in the culture dish containing new culture medium, it is placed in 37 DEG C, containing 5%CO2Cell incubator in cultivate.
2, the method for building up and pharmaceutical intervention of renal epithelial cell Hypoxia-reoxygenation model
Renal epithelial cell cell it is adherent after with contain 2% serum culture medium starvation 12h, after change anoxic liquid, be placed in anoxic case
In, control anoxic case O2Concentration 1%, CO2Concentration 5% after anoxic 12h, changes the normal culture solution of 2% serum into, continues to cultivate
8h。
Control group (control group, Ctrl): the normal culture solution of whole 2% serum is normal after renal epithelial cell is adherent
Culture；
Hypoxia-reoxygenation group (model group, H/R): modeling culture is carried out by the method for building up of above-mentioned Hypoxia-reoxygenation model；
Tanshinone-protocatechualdehyde group (D-P): rear to lack with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I
Oxygen culture 12h changes the normal culture solution containing 2% serum, 40 μM of protocatechualdehydes into and continues to cultivate 8h；
Protocatechualdehyde-tanshinone group (P-D): with the culture medium starvation 12h containing 2% serum, 40 μM of protocatechualdehydes, rear anoxic
12h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
Tanshinone group (D): with culture medium starvation 12h, rear anoxic culture 12h containing 2% serum, 1 μM of dihydrotanshinone I,
The normal culture solution containing 2% serum is changed into continue to cultivate 8h；
Tanshinone-tanshinone group (D-D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic
12h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
Protocatechualdehyde group (P): with contain 2% serum culture medium starvation 12h, rear anoxic culture 12h, change into containing 2% serum,
The normal culture solution of 40 μM of protocatechualdehydes continues to cultivate 8h；
Protocatechualdehyde-protocatechualdehyde group (P-P): rear to lack with the culture medium starvation 12h containing 2% serum, 40 μM of protocatechualdehydes
Oxygen culture 12h changes the normal culture solution containing 2% serum, 40 μM of protocatechualdehydes into and continues to cultivate 8h.
3, CCK8 method measures the survival rate of each group nerve cell in nerve cell Hypoxia-reoxygenation model
Cell inoculation is into 96 orifice plates, 5000, every hole cell, and after the processing of culture, administration and anoxia _ reoxygenation, every hole adds
Enter 10 μ L (the 1/10 of nutrient solution volume) CCK-8 solution, blank group is done with not celliferous culture solution, every group sets 6 multiple holes, sets
37 DEG C, 5%CO2After being incubated for 2~4h in incubator, the absorbance (OD value) in each hole is detected at 450nm with microplate reader.According to institute
It surveys OD value and calculates cell survival rate, cell survival rate=(experimental group OD value-blank group OD value) (control/group OD value-blank group OD
Value) × 100%.With control group survival rate for 100%.
Two, experimental result
Renal epithelial cell survival rate such as Fig. 6, tanshinone-protocatechualdehyde group (D-P) organize myocyte survival rate highest, explanation
Protective effect of the protocatechualdehyde to renal epithelial cell ischemical reperfusion injury is awarded before giving dihydrotanshinone I, reoxygenation before anoxic
It is optimal.
Embodiment 4: the therapeutic effect of dihydrotanshinone I and N-acetylcystein to myocardial ischemia-reperfusion
One, experimental method
1, myocardial cells culture and passage
(1) cardiac muscle cell recovers
Taking-up freezes the rat myocardial cell (H9c2 cell) in liquid nitrogen container, is put into fast in the water-bath for be preheated to 37 DEG C
Speed is thawed, and cell is aseptically moved in the centrifuge tube of DMEM culture medium of the 9ml containing 10% serum, 1000rmp centrifugation
4min.Liquid is discarded supernatant, cell is resuspended in the above-mentioned culture medium of 1mL, transfers in the culture dish containing new culture medium, with shifting
Liquid rifle, which gently dispels cell, makes it be uniformly distributed in culture dish, is placed in 37 DEG C, 5%CO2CMC model changes liquid afterwards for 24 hours.
(2) cell dissociation
The culture medium in culture dish is discarded, washed once with PBS solution, after 1ml pancreatin is added, culture dish is placed in cell
2min in incubator is added 2ml culture medium and terminates digestion, with liquid-transfering gun by the cell of adherent growth when cellular contraction is rounded
It dispels to completely falling off, it is made to be suspended from culture medium, used to follow-up test.
(3) cell passes on
Culture dish is placed in microscopically observation, when cell grows to fusion 80%~90%, after cell dissociation, and transfer
Into centrifuge tube, 1000rmp is centrifuged 4min, discards supernatant liquid, cell is resuspended in new culture medium, by 1:3 pro rate
Extremely in the culture dish containing new culture medium, it is placed in 37 DEG C, containing 5%CO2Cell incubator in cultivate.
2, the method for building up and pharmaceutical intervention of Myocytes Anoxia reoxygenation model
Cardiac muscle cell's cell it is adherent after with contain 2% serum culture medium starvation 12h, after change anoxic liquid, be placed in anoxic case,
Control anoxic case O2Concentration 1%, CO2Concentration 5% after anoxic 8h, changes the normal culture solution of 2% serum into, continues to cultivate 8h.
Experimental group is as follows:
Control group (control group, Ctrl): the normal culture solution of whole 2% serum is normally trained after cardiac muscle cell is adherent
It supports；
Hypoxia-reoxygenation group (model group, H/R): modeling culture is carried out by the method for building up of above-mentioned Hypoxia-reoxygenation model；
Tanshinone-N-acetylcystein group (D-NAC): hungry with the culture medium containing 2% serum, 1 μM of dihydrotanshinone I
12h, rear anoxic culture 8h change the normal culture solution containing 2% serum, 500 μM of N-acetylcysteins into and continue to cultivate 8h；
N-acetylcystein-tanshinone group (NAC-D): with the culture containing 2% serum, 500 μM of N-acetylcysteins
Base starvation 12h, rear anoxic culture 8h change the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I into and continue to cultivate 8h；
Tanshinone group (D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic culture 8h is changed
Continue to cultivate 8h at the normal culture solution containing 2% serum；
Tanshinone-tanshinone group (D-D): with the culture medium starvation 12h containing 2% serum, 1 μM of dihydrotanshinone I, rear anoxic
8h is cultivated, the normal culture solution containing 2% serum, 1 μM of dihydrotanshinone I is changed into and continues to cultivate 8h；
N-acetylcystein group (NAC): with the culture medium starvation 12h containing 2% serum, rear anoxic culture 8h is changed into and is contained
2% serum, 500 μM of N-acetylcysteins normal culture solution continue cultivate 8h；
N-acetylcystein-N-acetylcystein group (NAC-NAC): with containing 2% serum, 500 μM of half Guangs of N- acetyl
Culture medium starvation 12h, the rear anoxic culture 8h of propylhomoserin change the normal culture containing 2% serum, 500 μM of N-acetylcysteins into
Liquid continues to cultivate 8h.
3, CCK8 method measures the survival rate of each group cardiac muscle cell in Myocytes Anoxia reoxygenation model
Cell inoculation is into 96 orifice plates, 5000, every hole cell, and after the processing of culture, administration and anoxia _ reoxygenation, every hole adds
Enter 10 μ L (the 1/10 of nutrient solution volume) CCK-8 solution, blank group is done with not celliferous culture solution, every group sets 6 multiple holes, sets
37 DEG C, 5%CO2After being incubated for 2~4h in incubator, the absorbance (OD value) in each hole is detected at 450nm with microplate reader.According to institute
It surveys OD value and calculates cell survival rate, cell survival rate=(experimental group OD value-blank group OD value) (control/group OD value-blank group OD
Value) × 100%.With control group survival rate for 100%.
Two, experimental result
Each group Neuronal Survival rate is as shown in fig. 7, tanshinone-N-acetylcystein group (D-NAC) organizes cardiac muscle cell
Survival rate highest, illustrates to give before anoxic and awards N-acetylcystein before dihydrotanshinone I, reoxygenation and damage to cerebral ischemia re-pouring
The protective effect for hurting nerve cell is optimal.N-acetylcystein is known active oxygen scavenger.
Embodiment 5: binary drug release double-layer tablets
One, laboratory apparatus and material
DP30A single-punch tablet press；YD-20 intelligence hardness tester；ZB-1C intelligent disintegration tester；FT-2000 friability somascope；
ZRS-8G intelligence dissolving-out tester；Agilent-1200 high performance liquid chromatograph.
Protocatechualdehyde, dihydrotanshinone I, melbine, vitamin E purity are not less than 98%.
Auxiliary material is the customary adjuvant that market is commercially available.
Two, experimental method
1, the preparation of double-layer tablets
(1) dihydrotanshinone I-protocatechualdehyde binary drug release double-layer tablets
A release layer prescription:
PVP-K30 is configured to binder with 50% ethyl alcohol.Weigh the dihydrotanshinone I of recipe quantity, lauryl sodium sulfate,
PVPP and appropriate pregelatinized starch are uniformly mixed, with binder softwood.14 mesh nylon mesh are crossed, wet granular is in 50 DEG C of dryings.
Dry particl crosses 14 mesh nylon mesh, after whole grain, appropriate magnesium stearate is added and is uniformly mixed.Contain 50mg dihydro in 200mg supplementary material
The weight ratio of Tanshinone I, PVP-K30, PVPP and lauryl sodium sulfate is 20:1:1.5.
B slow release layer prescription:
Protocatechualdehyde, HPMC (hydroxypropyl methyl cellulose), MCC (microcrystalline cellulose) are taken, is sieved with 100 mesh sieve, is mixed, is used
The wetting of 95% appropriate amount of ethanol, is made softwood, crosses the granulation of 20 meshes, and 18 mesh sieves are crossed in 50 DEG C of dryings, adds appropriate magnesium stearate mixed
It is even.Contain 100mg protocatechualdehyde in 200mg supplementary material, the weight ratio of HPMC and MCC are 1:1.
C tabletting
Above-mentioned release layer and slow release layer are subjected to tabletting, slow release layer is first pressed, presses release layer afterwards.Every slow release layer Central Plains catechu
The weight of aldehyde is 40 times of dihydrotanshinone I weight in release layer.Every slice weight 840mg.
(2) melbine-vitamin E binary drug release double-layer tablets
A release layer prescription:
PVP-K30 is configured to binder with 50% ethyl alcohol.Weigh the melbine of recipe quantity, lauryl sodium sulfate,
PVPP and appropriate pregelatinized starch are uniformly mixed, with binder softwood.14 mesh nylon mesh are crossed, wet granular is in 50 DEG C of dryings.
Dry particl crosses 14 mesh nylon mesh, after whole grain, appropriate magnesium stearate is added and is uniformly mixed.Contain 50mg diformazan in 200mg supplementary material
The weight ratio of biguanides, PVP-K30, PVPP and lauryl sodium sulfate is 20:1:1.5.
B slow release layer prescription:
Vitamin E, HPMC (hydroxypropyl methyl cellulose), MCC (microcrystalline cellulose) are taken, is sieved with 100 mesh sieve, is mixed, is used
The wetting of 95% appropriate amount of ethanol, is made softwood, crosses the granulation of 20 meshes, and 18 mesh sieves are crossed in 50 DEG C of dryings, adds appropriate magnesium stearate mixed
It is even.Contain 100mg vitamin E in 200mg supplementary material, the weight ratio of HPMC and MCC are 1:1.
C tabletting
Above-mentioned release layer and slow release layer are subjected to tabletting, slow release layer is first pressed, presses release layer afterwards.Vitamin in every slow release layer
The weight of E is 3 times of melbine weight in release layer.Every slice weight 500mg.
2, vitro release measures
Above-mentioned double-layer tablets are taken respectively, and the release in vitro of release layer and slow release layer index components is investigated by drug release determination method
Degree.
Drug release determination method: it with reference to 2015 editions second annex dissolution rates of Chinese Pharmacopoeia and drug release determination method, uses
Basket method, revolving speed 100r/min, using 900mL de aerated water as dissolution medium, temperature is (37 ± 0.5) DEG C, is operated according to methods.In difference
Time point respectively samples 5mL, is filtered with 0.45 μm of filter membrane, subsequent filtrate is as test solution；Dihydrotanshinone I, original are separately taken respectively
Catechu aldehyde, melbine, vitamin E reference substance solution are measured by the chromatographic condition drafted respectively, calculate each sample time spot speed
Release the Accumulation dissolution of layer, slow release layer index components.
Three, experimental result
Drug release determination is the results show that in above-mentioned binary drug release double-layer tablets, and release layer 15min accumulative releasing degree is 99%
More than；Slow release layer 1h accumulative releasing degree is no more than 15%, 8h accumulative releasing degree and is no more than 55%, 12h accumulative releasing degree about 70%.
By optimization formulation, the release time of release layer, slow release layer can be further controlled.1~3 effect experiment can in conjunction with the embodiments
Know, binary drug release double-layer tablets can be taken before ischemic, contain mitochondrial complex I reversible inhibitor dihydrotanshinone I
Or the release layer of melbine discharges rapidly and plays ischemic preconditioning effect, the slow release layer slow release containing active oxygen scavenger
And reach maximum plasma concentration before Reperfu- sion, so that the treatment curative effect of ischemia-reperfusion is improved, it is effective, convenient.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (3)
1. the purposes that mitochondrial complex I reversible inhibitor is used as ischemic preconditioning drug.
2. purposes according to claim 1, it is characterised in that: the mitochondrial complex I reversible inhibitor is diformazan
Biguanides or dihydrotanshinone I.
3. the purposes that melbine or dihydrotanshinone I are used as mitochondrial complex I reversible inhibitor.
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