CN109966288A - A kind for the treatment of or prevention atherosclerosis drug and application - Google Patents
A kind for the treatment of or prevention atherosclerosis drug and application Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology fields, disclose a kind for the treatment of or prevention atherosclerosis drug and application, the treatment or prevention atherosclerosis drug is butylphenyl phthaleine, and there is antioxidant activity Keap1 inhibitor to be formed by butylphenyl phthaleine and pharmaceutically acceptable auxiliaries.Butylphenyl phthaleine of the present invention generates the mechanism of action for reducing oxidative stress by targeted inhibition Kelch sample epoxychloropropane associated protein 1 (Keap1), achievees the effect that treat atherosclerosis；The present invention solves new application of the butylphenyl phthaleine in terms for the treatment of or preventing atherosclerosis and provides its new application for alleviating oxidative stress by targeted inhibition Keap1 albumen.
Description
Technical field
The invention belongs to pharmaceutical technology field more particularly to a kind for the treatment of or prevention atherosclerosis drug and applications.
Background technique
Currently, the immediate prior art:
With the development of economy, the prevalence of aging of population and various risk factors, the number of cardiovascular and cerebrovascular patient is not
It is disconnected to increase.Atherosclerosis (Atherosclerosis, AS) is arterial occlusive cardiovascular and cerebrovascular diseases such as Acute Coronary Syndrome
The important pathogenic factor of sign, cerebral apoplexy etc..Under the main Digital arteries of atherosclerosis and its blood vessel wall inner membrance and inner membrance of branch
Lipid it is calm, while being migrated with the inner membrance of medial smooth muscle cells, to make vascellum tunica interna incrassation and form athero- lesion
And a kind of disease of fiber Lipid Plaque lesion.The many factors such as hyperlipidemia, obesity, hypertension, hyperglycemia, smoking and heredity are equal
It can lead to atherosclerosis, wherein hyperlipidemia is one of most important reason.
Currently, the prior art commonly used in the trade is such that
Drug therapy is that control atherosclerotic plaque forms most effective measure.Clinical treatment AS is mainly gone back at present
It is based on the drugs such as reducing blood lipid, platelet aggregation-against.But because there is respective specific contraindication or patient not in said medicine
It is resistant to treated situation and seriously limits their application.Therefore, it is badly in need of excavating other mechanism for the treatment of atherosclerosis
And new therapeutic agent is found to improve the prognosis of atherosclerotic.The pathogenic process of atherosclerosis is more multiple
It is miscellaneous, relate generally to the mechanism such as lipid precipitation, endothelial cell damage, monocytes/macrophages infiltration, smooth muscle cell migration proliferation.
Current academic viewpoint thinks, vascular tissue's oxidative stress be important pathology in atherosclerotic plaque forming process because
Element, oxidative stress theory obtain the occurrence and development process interpretations of atherosclerosis more comprehensive.From hyperlipidemia (mainly blood
OxLDL ELISA (oxLDL) concentration increases in circulation) stimulate vascular endothelial cell and monocyte to start, oxidative stress
Increase with active oxygen (ROS) level just through the whole process of atherosclerosis occurrence and development, and with advancing of disease
Gradually cascade is amplified with positive feedback.The oxidative stress reduced in vascular tissue can effectively prevent atherosclerosis
Patch is formed.It can be seen that excavating the oxidative stress during progression of atherosclerosis, treatment Atherosclerosis is found
The novel drugs and novel targets of change have important research significance.
Kelch sample epoxychloropropane associated protein 1 (Keap1) is Keap1-Nrf2- Antioxidant responsive element (ARE) signal
The master selector of access can turn on or off Keap1-Nrf2-ARE access according to intracellular redox state.?
Under physiological condition, Keap1 is incorporated into endochylema in the form of ubiquitination Binding Capacity albumen with Nrf2, therefore Keap1 dominates Nrf2
Ubiquitination degradation, is allowed to maintain normal level, to inhibit the expression of downstream gene.After cell experiences stimulation, Keap1
Conformation change, Nrf2 and Keap1, which occur to dissociate, to be simultaneously transferred into nucleus, identify the binding site on ARE and with
Combination, start downstream antioxidant genes transcription, improve cell resistance external source sexual stimulus ability.As it can be seen that Keap1-Nrf2-
ARE signal shaft is the important channel of intracellular regulation response to oxidative stress.
Butylphenyl phthaleine (n-Butylphthalide, NBP), also known as Butylphthalide, belong to simple phthalide-type, are China's thirds
Possess the chemicals of independent intellectual property right, and in the world dedicated for the chemical new drug for the treatment of cerebrovascular disease.2004, fourth
Phthalide is approved for treatment cerebral ischemia in China.Butylphenyl phthaleine has outstanding, safe monolithic entity structure, while having a variety of
Pharmacological action, such as: energetic supersession is improved, it is anti-oxidant, promote microcirculation, reduces nerve cell apoptosis and inhibition inflammation etc. and make
With.Butylphenyl phthaleine can complete treatment cerebral arterial thrombosis, significantly reduce infraction after neurological deficit and improve minimal invasive treatment's energy
Power state, and the side effect in preclinical and clinical research is slight.In terms of cardiovascular protection, it has been reported that butylphenyl phthaleine inhibits
Oxidative stress simultaneously alleviates acute myocardial infarction of rat.It is big that the discovery butylphenyl phthaleine such as Wang, Y.G. can protect ischemia/reperfusion to induce
Rat cardiomyocyte apoptosis.To sum up, drug of the butylphenyl phthaleine as clinical treatment cerebral arterial thrombosis, while also having to ischemic
Cardiopathic protective effect.Atherosclerosis is the common etiology base of cerebral arterial thrombosis and acute ischemic heart disease
Plinth, but due to there is no the Related Experimental Study carried out using atherosclerosis animal and cell model to butylphenyl phthaleine at present, because
This, effect of the butylphenyl phthaleine in prevention and treatment atherosclerosis is unclear.
Butylphenyl phthaleine has preferable antioxidant activity.Wang, S. etc. have found that butylphenyl phthaleine passes through activation PI3K/Akt and Mash1
Access come protect H2O2 induce neural stem cells injury.In Alzheimer sample lesion, butylphenyl phthaleine can be by raising Nrf2
To inhibit the oxidative stress in neuronal cell.In addition, butylphenyl phthaleine can also by lower tissue in ROS content come alleviate Ah
Mycin and the performance of lipopolysaccharide-induced depressed sample.
But current research only has detected butylphenyl phthaleine to the regulating and controlling effect of anti-oxidant signal path, does not sieve further
Butylphenyl phthaleine is selected to play the targeting proteins of antioxidation, therefore, the anti-oxidant target spot of butylphenyl phthaleine is unclear.
In conclusion problem of the existing technology is:
Currently used treatment atherosclerosis drug is not resistant to the feelings such as treatment because of contraindication existing for it or patient
Condition seriously limits their application.
Effect and mechanism of the butylphenyl phthaleine in atherosclerosis are still not clear, and solve the problems, such as that this can be acute with primary prevention
Cerebral infarction and acute coronary syndrome have preferable clinical value.The direct anti-oxidant target spot of butylphenyl phthaleine is unclear, solves
The problem will be applied to clinical treatment cardiovascular and cerebrovascular disease for butylphenyl phthaleine and provide theoretical foundation.
Solve the difficulty of above-mentioned technical problem:
Currently, shortage butylphenyl phthaleine acts in atherosclerosis animal and cell model and the research of mechanism；In addition, fourth
The anti-oxidant target spot and Study on Molecular Mechanism of phthalide are unclear.
Solve the meaning of above-mentioned technical problem:
One aspect of the present invention provides new drug and new mechanism of action and target for treatment atherosclerosis disease
Point；On the other hand, the anti-oxidant target spot of butylphenyl phthaleine is disclosed, is applied to clinical treatment cardiovascular and cerebrovascular disease for butylphenyl phthaleine and theory is provided
Foundation.
Summary of the invention
In view of the problems of the existing technology, it the present invention provides a kind for the treatment of or prevention atherosclerosis drug and answers
With.It is related to purposes of the butylphenyl phthaleine in preparation treatment atherosclerosis drug, further relates to butylphenyl phthaleine in preparation with anti-oxidant
Purposes in active Keap1 inhibitor.
The invention is realized in this way a kind for the treatment of or prevention atherosclerosis drug, the treatment or prevention artery
Anti-atherosclerotic agent is butylphenyl phthaleine.Injection and oral capsule of the butylphenyl phthaleine for injection.
Another object of the present invention is to provide a kind of using made of the treatment or prevention atherosclerosis drug
Injection (10-20mg/kg/ days).
Another object of the present invention is to provide a kind of using made of the treatment or prevention atherosclerosis drug
Oral capsule (10-20mg/kg/ days).
Another object of the present invention is to provide a kind of using made of the treatment or prevention atherosclerosis drug
It is described to have antioxidant activity Keap1 inhibitor by butylbenzene with antioxidant activity Keap1 inhibitor (10-20mg/kg/ days)
Phthalein and pharmaceutically acceptable auxiliaries into (" auxiliary material " refers to the pharmaceutical carrier of pharmaceutical field routine, such as: diluent, tax
Shape agent such as water etc., filler such as starch, sucrose etc.；Adhesive such as cellulose derivative, alginates, gelatin and polyvinylpyrrolidine
Ketone；Wetting agent such as glycerol；Disintegrating agent such as agar, calcium carbonate and sodium bicarbonate；Sorbefacient such as quaternary ammonium compound；Surface-active
Agent such as hexadecanol；Absorption carrier such as kaolin and soap clay；Lubricant such as talcum powder, calcium stearate/magnesium, polyethylene glycol etc..
Configuration proportion is butylphenyl phthaleine by taking 0.9% sodium chloride as an example: 0.9% sodium chloride=1:20).
Further, acceptable auxiliary material is acceptable carrier or filler or adhesive or wetting agent or disintegrating agent or suction
Receive promotor or surfactant or absorption carrier or lubricant or 0.9% sodium chloride；
In mass ratio, butylphenyl phthaleine and pharmaceutically acceptable auxiliary material proportion are 1:20.
There is piece made of antioxidant activity Keap1 inhibitor using described another object of the present invention is to provide a kind of
Agent.
There is ball made of antioxidant activity Keap1 inhibitor using described another object of the present invention is to provide a kind of
Agent.
Using using described there is antioxidant activity Keap1 inhibitor to be made another object of the present invention is to provide a kind of
Suspension.
Using using described there is antioxidant activity Keap1 inhibitor to be made another object of the present invention is to provide a kind of
Granule.
There is cream made of antioxidant activity Keap1 inhibitor using described another object of the present invention is to provide a kind of
Liquor.
In conclusion advantages of the present invention and good effect are as follows:
Present invention discover that butylphenyl phthaleine generates the mechanism of action for reducing oxidative stress by targeted inhibition Keap1, reach treatment
The effect of atherosclerosis；The present invention solve new application of the butylphenyl phthaleine in terms for the treatment of or preventing atherosclerosis and/
Or provide its new application that oxidative stress is reduced by targeted inhibition Keap1 albumen.
In the present invention, the therapeutic effect that first discovery butylphenyl phthaleine forms atherosclerotic plaque.Using high in fat
Diet (HFD) feeds ApoE and knocks out (ApoE-/-) cause Atherosclerosis Model within mouse 12 weeks, and give mouse peritoneal injection
Butylphenyl phthaleine (12 weeks 20mg/kg/2 days).As a result prompt butylphenyl phthaleine significantly alleviates the ApoE of HFD induction-/-Mouse aorta bow, master
Atherosclerotic plaque accumulation increases (embodiment 1) in endarterium and aorta petal.Meanwhile the present invention has detected each group mouse
As a result blood lipid level prompts butylphenyl phthaleine to have no effect on the ApoE of HFD induction-/-Lipid of mice changes (embodiment 1).
Butylphenyl phthaleine is to play treatment or prevention of arterial congee and inhibiting or reducing oxidative stress in vascular tissue
The effect of sample hardening.In the present invention, it was discovered that butylphenyl phthaleine restrained effectively the ApoE of HFD induction-/-Rat aorta tissue oxidizing
Stress level increases and (embodiment 2).In addition, it has also been found that butylphenyl phthaleine can weaken OxLDL ELISA (oxLDL
50ug/ml) induce vascular endothelial cell (HUVEC) in products of oxidative stress level change (embodiment 2).
Butylphenyl phthaleine is the Mechanisms and therapy atherosclerosis by inhibiting Keap1 to generate reduction oxidative stress
Effect.When cell is in normal condition, Nrf2 is anchored in cytoplasm, and is combined closely and interacted with Keap1.
Keap1 is maintained at a lower level by making Nrf2 that ubiquitination degradation occur.The present invention utilizes immunofluorescence skill
Art discovery butylphenyl phthaleine can inhibit HFD to induce ApoE-/-Keap1 expression quantity increases in rat aorta tissue, and butylphenyl phthaleine can also increase
The content (embodiment 3) of Nrf2 in artery.In addition, the present invention can be inhibited using co-immunoprecipitation test discovery butylphenyl phthaleine
Keap1 and Nrf2 albumen is compound in HUVEC, and further, using plasmid, high expressing K eap1 can then weaken fourth in HUVEC
Activation (embodiment 3) of the phthalide to Nrf2.
Butylphenyl phthaleine may be present with Keap1 albumen and bind directly, for antioxidant activity Keap1 inhibitor.Benefit of the invention
It may be present with Keap1 albumen with molecular docking technology and molecular dynamics (MD) simulation and forecast discovery butylphenyl phthaleine and bound directly and moved
Mechanics is stablized, wherein being successively Val-465, Gly-417, Val- by the key amino acid site of both hydrophobic effect connections
418 equal (embodiments 4).
When concrete application, the drug is butylphenyl phthaleine and pharmaceutically acceptable auxiliary material and the various preparations being configured to.
Wherein it is preferable over the injection and oral capsule for injection.Preparation of the invention can as unit of dosage form, as injection,
Capsule (including releasing pattern is released in sustained release or delay), tablet, pill, suspension, granule, tincture, syrup, lotion
The dosage forms such as agent, suspension and various sustained-release dosage types to be suitble to various administration modes, such as take orally, parenteral injection, glue
The form of medication of film, muscle, intravenous, subcutaneous, intraocular, intradermal or process skin etc. (is wherein preferable over the needle for injection
Agent and oral capsule).
Detailed description of the invention
Fig. 1 is the ApoE that butylphenyl phthaleine provided in an embodiment of the present invention induces HFD-/-Atherosclerotic plaque in mice heap
The increased influence diagram of product.
Fig. 2 is the ApoE that butylphenyl phthaleine provided in an embodiment of the present invention induces HFD-/-The influence that lipid of mice level changes
Figure.
Fig. 3 is the ApoE that butylphenyl phthaleine provided in an embodiment of the present invention induces HFD-/-Rat aorta tissue oxidizing stress water
The influence diagram of the change of products of oxidative stress level in the HUVEC that flat raising and butylphenyl phthaleine induce oxLDL.
Fig. 4 is regulation of the butylphenyl phthaleine provided in an embodiment of the present invention to Keap1/Nrf2 signal in mouse blood vessel and HUVEC
Figure.
Fig. 5 is structure and dynamic analysis figure of the butylphenyl phthaleine provided in an embodiment of the present invention in conjunction with Keap1.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Currently used treatment atherosclerosis drug is not resistant to the feelings such as treatment because of contraindication existing for it or patient
Condition seriously limits their application.Effect and mechanism of the butylphenyl phthaleine in atherosclerosis are still not clear.Butylphenyl phthaleine it is straight
It is unclear to connect anti-oxidant target spot.
To solve the above problems, the present invention is described in detail with reference to the accompanying drawing.
The embodiment of the present invention provides a kind for the treatment of or prevention atherosclerosis drug, and the treatment or prevention artery is athero-
Hardening drug is butylphenyl phthaleine (10-20mg/kg/ days).Injection and oral capsule of the butylphenyl phthaleine for injection.
In the present invention, atherosclerosis includes that hyperlipidemia, obesity, hypertension, hyperglycemia, smoking and heredity etc. are various
One or more of atherosclerosis caused by factor.Atherosclerosis turns to Atherosclerosis caused by hyperlipidemia
Change.
In the present invention, the atherosclerotic plaque that butylphenyl phthaleine is used to reduce hyperlipidemia induction is formed.
Butylphenyl phthaleine is to play treatment or prevention and inhibiting or reducing the oxidative stress that hyperlipidemia induces in the artery
The effect of atherosclerosis.
Butylphenyl phthaleine is by targeted inhibition oxidative stress modulin Kelch sample epoxychloropropane associated protein 1 (Keap1)
Play the effect for reducing the oxidative stress that hyperlipidemia induces in the artery.
The embodiment of the present invention provides a kind of butylphenyl phthaleine and pharmaceutically acceptable auxiliary material, and (wherein, term is " pharmaceutically acceptable
Auxiliary material " be well-known to those skilled in the art, including pharmaceutically acceptable carrier, excipient, diluent etc., they with
Active constituent is compatible.Specific configuration proportion is depending on each adjunct ingredient depending on) be formulated with antioxidant activity Keap1 inhibitor
(10-20mg/kg/ days).
It is described to have antioxidant activity Keap1 inhibitor by butylphenyl phthaleine and pharmaceutically acceptable auxiliaries into (" auxiliary
Material " refers to the pharmaceutical carrier of pharmaceutical field routine, such as: diluent, excipient such as water etc., filler such as starch, sucrose etc.；Bonding
Agent such as cellulose derivative, alginates, gelatin and polyvinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, calcium carbonate
And sodium bicarbonate；Sorbefacient such as quaternary ammonium compound；Surfactant such as hexadecanol；Absorption carrier such as kaolin and soap are viscous
Soil；Lubricant such as talcum powder, calcium stearate/magnesium, polyethylene glycol etc..Configuration proportion is butylphenyl phthaleine by taking 0.9% sodium chloride as an example:
0.9% sodium chloride=1:20).
The invention will be further described combined with specific embodiments below.
Embodiment 1
The ApoE that butylphenyl phthaleine induces HFD-/-Atherosclerotic plaque in mice accumulates increased influence:
The ApoE of male C57BL/6 background-/-Mouse purchase is raised from Beijing HFK Bio-Technology Co., Ltd.
In Institutes Of Jiaxing's animal experimental center.Mouse standard rodent animal food and water raising are in the constant temperature 12-12h rhythm and pace of moving things round the clock
In animal house.Animal at least spends week age to carry out environmental suitability growth before experiment starts.It is related to the association that animal uses
View all obtains the approval (approval documents: 2018JXSA27) of Jiaxing City No.2 Hospital animal policy and the welfare committee.It is testing
Used in butylphenyl phthaleine be the manufactured dosage form for dissolving in 9% sodium chloride.The pH value of solution is 7.36 and filters through 0.22 micropore
Film filtering.8-10 week old male ApoE-/-Mouse is randomly divided into 4 groups: normally nursing group (ND), normal nursing+butylphenyl phthaleine group (ND+
Butylphenyl phthaleine), high fat diet group (HFD), high fat diet+butylphenyl phthaleine group (HFD+ butylphenyl phthaleine), every group 8.Normal diet includes
10kcal.% fat, 20kcal.% protein and 70kcal.% carbohydrate, high fat diet include 40kcal.% fat,
20kcal.% protein and 20kcal.% carbohydrate；Butylphenyl phthaleine is subcutaneous administrations (12 weeks 10mg/kg/2 days).Knot
Fruit prompt butylphenyl phthaleine significantly alleviates the ApoE of HFD induction-/-Atherosclerotic plaque accumulation increases (Figure 1A) at mouse aorta bow.
The result prompt butylphenyl phthaleine of oil red O stain significantly alleviates the ApoE of HFD induction-/-Mouse aorta inner membrance (Figure 1B-C) and active
The accumulation of arteries and veins valve (Fig. 1 D-F) atherosclerotic plaque increases.Meanwhile the present invention has detected the blood lipid level of each group mouse, as a result prompts
Butylphenyl phthaleine has no effect on the ApoE of HFD induction-/-Lipid of mice changes, as cholesterol (TCH) increases (Fig. 2A), triglycerides
(TG) increase (Fig. 2 B), low-density lipoprotein (LDL) increases (Fig. 2 C) and high-density lipoprotein (HDL) reduction (Fig. 2 D).More than
As a result prompt butylphenyl phthaleine has therapeutic effect to what atherosclerotic plaque was formed, and this effect is not by regulation blood lipid
It realizes.
Wherein, what Figure 1A was indicated is that butylphenyl phthaleine alleviates HFD induction ApoE under light microscopic-/-Mouse aorta bends atherosis spot
Block is formed；Figure 1B indicates that oil red O stain dye aorta total preparation shows that butylphenyl phthaleine alleviates HFD and induces ApoE-/-Mouse aorta
Inner membrance atherosclerotic plaque is formed；Fig. 1 C indicates that each group is with respect to plaque area statistical chart in Figure 1B；Fig. 1 D indicates oil red O stain dye
Aortic root sample shows that butylphenyl phthaleine alleviates HFD and induces ApoE-/-Mouse aorta valve atherosclerotic plaque is formed；Fig. 1 E
And F indicates aortic root plaque area statistics.These results suggest that butylphenyl phthaleine alleviates the ApoE of high fat diet induction-/-Mouse
Aorta atheromatous plague accumulation increases.
Wherein, what Fig. 2A was indicated is that butylphenyl phthaleine does not influence HFD induction ApoE-/-Mice serum cholesterol (TCH) content liter
It is high；What Fig. 2 B was indicated is that butylphenyl phthaleine does not influence HFD induction ApoE-/-Mice serum triglycerides (TG) content increases；Fig. 2 C is indicated
Be butylphenyl phthaleine do not influence HFD induction ApoE-/-Mice serum low-density lipoprotein (LDL) content increases；What Fig. 2 D was indicated is fourth
Phthalide does not influence HFD induction ApoE-/-Mice serum high-density lipoprotein (HDL) content reduces.
Embodiment 2
Butylphenyl phthaleine is to the raised effect of atherosclerotic blood vessel tissue oxygen stress level:
Oxidative stress with atherosclerosis occurrence and development.Early period, the present invention had detected in each group rat aorta
ROS content and oxidative stress marker 3-NT are horizontal, as a result, it has been found that butylphenyl phthaleine intraperitoneal injection restrained effectively HFD induction
ApoE-/-Rat aorta tissue ROS content increases (Fig. 3 A) and 3-NT expression quantity increases (Fig. 3 B).
Using butylphenyl phthaleine, to oxLDL stimulation HUVEC oxidative stress, (malonaldehyde (MDA) increases and superoxide dismutase
(SOD) reducing) method that influences tests the antioxidation activity in vitro of butylphenyl phthaleine, and the specific method is as follows: 1.2 × 10^6It is a
HUVEC cell is incubated at 37 DEG C with DMEM culture solution, and culture solution is updated after 24 hours, and it is (final concentration of that tested butylphenyl phthaleine is added
10 μM) pretreatment 1 hour, then continued with 12 hours with oxLDL (50 μ g/ml), cell detection MDA and SOD content is collected, is received
Collect cell detection total protein concentration, MDA and SOD result is divided by calibrated with corresponding total protein concentration, and each compound repeats to survey
Examination 3 times calculates average value and error amount.The endothelial cell MDA level that butylphenyl phthaleine mitigates oxLDL induction as the result is shown increases (figure
3D) and SOD level reduces (Fig. 3 E).The invention shows butylphenyl phthaleines, and atherosclerotic blood vessel tissue oxygen can be effectively relieved
Stress level increases.
Wherein, that Fig. 3 A is indicated is the ApoE that DHE dyeing prompt butylphenyl phthaleine inhibits HFD induction-/-Rat aorta ROS content
It increases；That Fig. 3 B is indicated is the ApoE that immunohistochemical staining prompt butylphenyl phthaleine inhibits HFD induction-/-Rat aorta 3-NT level increases
It is high；What Fig. 3 C was indicated is influence of the butylphenyl phthaleine to HUVEC cell activity；What Fig. 3 D was indicated is that butylphenyl phthaleine mitigation oxLDL is induced
MDA level increases in HUVEC；Fig. 3 E indicates that butylphenyl phthaleine mitigates the reduction of SOD level in the HUVEC of oxLDL induction.
Embodiment 3
Regulation of the butylphenyl phthaleine to Keap1/Nrf2 signal in mouse blood vessel and HUVEC:
Keap1-Nrf2-ARE signal shaft is the important anti-oxidant signaling mechanism of body.Wherein Keap1 is the main regulation of Nrf2
Device, it can turn on or off the access according to intracellular redox state.The present invention has investigated NBP in previous experiments
Influence to Keap1/Nrf2 signal, the Keap1 expression quantity for as a result prompting NBP that atherosclerosis can be inhibited to induce increase
(Fig. 4 A), NBP can also increase the content (Fig. 4 B) of Nrf2 in artery.
Meanwhile the present invention can inhibit Keap1 and Nrf2 albumen in HUVEC using co-immunoprecipitation test discovery butylphenyl phthaleine
It is compound.The present invention has detected butylphenyl phthaleine using co-immunoprecipitation experiment and lives to the external inhibition of this Keap1 and Nrf2 complex
Property.Experimental data is shown in Fig. 4 C.Experiment shows that butylphenyl phthaleine significantly inhibits anchoring of the Keap1 to Nrf2 in HUVEC, promotes releasing for Nrf2
It puts.Further, Keap1 plasmid (Sino Biological lnc., D236H mutations, Beijing, China) is utilized
And Lipofectamine 2000 (Invitrogen, Carlsbad, California) transfects HUVEC, high expressing K eap1 albumen
(Fig. 4 D).It is continued with 6 hours with oxLDL (50 μ g/ml) again, collects culture solution with RT-qPCR method and detect Nrf2 mRNA expression
The case where, high expressing K eap1 can weaken the expression of Nrf2mRNA caused by butylphenyl phthaleine and increase (Fig. 4 E) as the result is shown.The invention shows
Butylphenyl phthaleine can inhibit Keap1 to activate Nrf2.
Embodiment 4
Butylphenyl phthaleine and Keap1 albumen have direct combination and dynamic stabilization:
It is closed using molecular docking test and the combination of molecule power (MD) analogue technique analysis butylphenyl phthaleine and Keap1 albumen
System: molecular dynamics simulation is completed using the PMEMD module of 11 software of AMBER.Before molecular dynamics simulation, Suo Youti
System passes through the optimization of six steps in our current research.System local energy is excessively high in order to prevent, and it is excellent to carry out energy to system first
Change.The 7th step carries out molecular dynamics simulation system ancestor's simulation of 50ns later.The truncation distance of nonbonding is set as 9A, uses Particle
Mesh Ewald (PME) method calculates the electrostatic interaction of long-range, and hydrogen atom bond distance is limited using SHAKE method, temperature control
System uses Langevin algorithm, and wherein collision frequency γ is set as 2ps-1.System pressure is 1atm, integration step 2fs, is total to
25000000 steps save track every 10ps (500 step) and are used for subsequent analysis.Root-mean-square-deviation (RMSD) the results show that
Keap1 albumen and NBP have reached dynamic equilibrium (Fig. 5 A), can be used for subsequent Conjugated free energy and structural analysis.DCC analysis shows that
Keap1/NBP compound has reached relatively stable (Fig. 5 B) in stimulating course.Then, MM/GBSA amino acid residue point is utilized
Solution, the present invention predict that the critical amino acid residues of Keap1 albumen and NBP during being mutually distinguishable are successively Val-465,
Gly-417, Val-418, Gly-464, Ile-559, Val-512, Ala-366, Val-606, Cys-513, Ala-556 (figure
5C).It is analyzed in conjunction with critical amino acid residues Energy Decomposition and composite structure, main phase interaction of the Keap1 albumen in conjunction with NBP
With for hydrophobic effect (Fig. 5 D).
Below with reference to the preparation method of drug, the invention will be further described.
Treatment or prevention atherosclerosis process for preparing medicine provided in an embodiment of the present invention includes: medicine group of the present invention
The various dosage forms for closing object can be prepared according to the conventional production process of pharmaceutical field.Such as make active constituent butylphenyl phthaleine and it is a kind of or
The mixing of variety carrier auxiliary material, is then made into required dosage form, such as injection, capsule and tablet.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind for the treatment of or prevention atherosclerosis drug, which is characterized in that the treatment or prevention Atherosclerosis chemical drug
Object is butylphenyl phthaleine.
2. a kind of utilize injection made for the treatment of or prevention atherosclerosis drug described in claim 1.
3. a kind of utilize oral capsule made for the treatment of or prevention atherosclerosis drug described in claim 1.
4. a kind of have antioxidant activity using made for the treatment of or prevention atherosclerosis drug described in claim 1
Keap1 inhibitor, which is characterized in that described to have antioxidant activity Keap1 inhibitor by butylphenyl phthaleine and pharmaceutically acceptable
Auxiliaries form.
5. being treated or prevented made of atherosclerosis drug as claimed in claim 4, there is antioxidant activity Keap1 to inhibit
Agent, which is characterized in that acceptable auxiliary material is acceptable carrier or filler or adhesive or wetting agent or disintegrating agent or suction
Receive promotor or surfactant or absorption carrier or lubricant or 0.9% sodium chloride；
In mass ratio, butylphenyl phthaleine and pharmaceutically acceptable auxiliary material proportion are 1:20.
6. a kind of utilize has tablet made of antioxidant activity Keap1 inhibitor described in claim 4.
7. a kind of utilize has pill made of antioxidant activity Keap1 inhibitor described in claim 4.
8. a kind of utilize has suspension made of antioxidant activity Keap1 inhibitor described in claim 4.
9. a kind of utilize has granule made of antioxidant activity Keap1 inhibitor described in claim 4.
10. a kind of utilize has emulsion agent made of antioxidant activity Keap1 inhibitor described in claim 4.
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