CN110200976A - Purposes of the Cryptotanshinone in the drug that preparation promotes diabetic's wound healing - Google Patents
Purposes of the Cryptotanshinone in the drug that preparation promotes diabetic's wound healing Download PDFInfo
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- CN110200976A CN110200976A CN201910593197.7A CN201910593197A CN110200976A CN 110200976 A CN110200976 A CN 110200976A CN 201910593197 A CN201910593197 A CN 201910593197A CN 110200976 A CN110200976 A CN 110200976A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The embodiment of the invention provides purposes of the Cryptotanshinone in the drug that preparation promotes diabetic's wound healing, Cryptotanshinone provided in an embodiment of the present invention can promote surface of a wound edge tissues angiogenesis, promote venous endothelial cell at pipe, mitigate the inflammatory reaction of surface of a wound edge tissues, inhibit surface of a wound edge tissues ECM degradation, thus has the function of promoting diabetic's wound healing.
Description
Technical field
The present invention relates to Cryptotanshinone new application technical fields, promote patient of diabetes in preparation more particularly to Cryptotanshinone
Purposes in the drug of person's wound healing.
Background technique
One serious complication of diabetes is exactly that the surface of a wound of patient's formation is difficult to heal, including due to organizing to lack
Surface of a wound caused by the surface of a wound caused by blood, ulcer and wound etc..Normal wound healing is a complexity and orderly pathology mistake
Journey relates generally to all too many levels, the different reasons such as inflammatory cell, repair cell, extracellular matrix (ECM) and cell factor and causes
The surface of a wound, agglutination all includes the processes such as cell Proliferation, migration, angiogenesis, epithelium regeneration.But in diabetes environment
Under, wound is postponed, and leads to impaired wound healing conjunction or disunion.
Cryptotanshinone (Cryptotanshinone, CT) belongs to diterpene quinone, is the master of Lamiaceae plant Radix Salviae Miltiorrhizae
Want fat-soluble extract；Its molecular mass is 296.360, molecular formula C19H20O3, structural formula is as shown in Equation 1.
Existing research shows that Cryptotanshinone has anti-inflammatory, anti-oxidant, antibacterial, antitumor, anti-angiogenic rebirth, anti-obesity
Equal pharmacological activity.Currently, main pharmacological known to Cryptotanshinone has: (1) anti-inflammatory, antioxidant activity: 1. CT can inhibit
Inflammatory mediator generates in RAW264.7, reduces CD14 and TLR4 expression, inhibits the TAK1 phosphorylation of LPS induction.2. passing through IL-15
Promote transcription factor, such as Id2, GATA3, T-bet, Ets-1 and p38MAPK Expression of phosphorylated, so that NK be induced to break up.
3. enhancing intracellular ROS level, CD95 (APO-1/CD95), Bcl-2 and PPAR- γ expression are lowered, Bax, AMPK and p- are raised
AMPK expression, and cause caspase-3 and the PARP cracking of activation, make JNK and ERK activation and phosphorylation, and then play and inhibit
Effect.(2) antibacterial activity: to staphylococcus aureus, methicillin-resistant staphylococcus aureus and beta-lactam enzyme positive gold
Staphylococcus aureus all has obvious inhibiting effect.It can destroy bacteria cell wall and membrane structure, cause cell membrane penetration
Property increase, cause cellular content to leak.Moreover, it also has certain influence to bacterio protein synthesis, bacterium vivo protein can be made
Matter reduces and hinders its expression, eventually leads to the forfeiture of protein normal physiological function.(3) antitumor, anti-angiogenic rebirth is active: 1.
Inhibit the proliferation of human hepatocarcinoma BEL-7402, reduces the mRNA expression of MAP2K1 albumen.2. inhibiting HepG-2 cell Bcl-2
Expression promotes Bax expression, regulates and controls Bcl-2/Bax ratio, activate downstream effect factor caspase-3, induces HepG-2 apoptosis.
3. activating AMPK protein kinase signal access, induction HepG-2 blocks in G1 cell cycle phase, promotes partial-length apoptosis,
And it is dead to induce it that autophagy occurs by AMPK.4. PI3K/Akt expression in Human umbilical vein endothelial cells is reduced,
Inducing endothelial cell apoptosis inhibits vascular endothelial cell proliferation, to play its antitumor action indirectly.5. inhibiting DU145 thin
JNK and p38MAPK activity in born of the same parents inhibits Bcl-2 expression, adjusts tumor necrosis factor Fas (APO1/CD95) and increases its sensitivity
Property, to induce the Apoptosis.(4) anti-obesity activity: activation MAPK access promotes glycometabolism, takes Cryptotanshinone energy for a long time
Reduce the weight and blood glucose of diabetic mice.
Summary of the invention
The present inventor passes through the further investigation to Cryptotanshinone, it has unexpectedly been found that Cryptotanshinone can promote glycosuria
The healing of disease model wounds in mice, and find that Cryptotanshinone can promote people by up-regulation vascular endothelial growth factor (VEGFA) expression
Venous endothelial cell increases surface of a wound edge tissues capillary density, capillary density increase can be new at approach such as pipes
Raw tissue provides oxygen and nutriment, promotes tissue newborn, and then promote surface of a wound wound healing, and complete based on above-mentioned discovery
The present invention.Specific technical solution is as follows:
First aspect present invention provides use of the Cryptotanshinone in the drug that preparation promotes diabetic's wound healing
On the way.
In the present invention, Cryptotanshinone promotes diabetic's surface of a wound by increasing surface of a wound edge tissues capillary density
Healing.
In the present invention, Cryptotanshinone increases surface of a wound edge tissues capillary by promoting people's venous endothelial cell at pipe
Density.
In the present invention, the Cryptotanshinone increases surface of a wound edge tissues capillary density by promoting vegf expression.
In the present invention, the Cryptotanshinone also passes through the inflammatory reaction for inhibiting excessive, promotes diabetic's wound healing.
In wound, after inflammatory reaction occurs mainly in surface of a wound generation, such as 1-3 days, postoperative 1 day after operation
It peaks, after 1 day, inflammatory reaction is gradually subsided, but in the surface of a wound of diabetic, after surface of a wound generation after at least 3 days, example
A large amount of inflammatory factor and chemotactic factor (CF) there are a large amount of inflammatory cell and are secreted in 16 days surface of a wound edges after such as performing the operation, with normal phase
Than showing as excessive inflammatory reaction.
Second aspect of the present invention additionally provides a kind of composition containing Cryptotanshinone and promotes diabetic's wound in preparation
Purposes in the drug of face healing.
Cryptotanshinone in some embodiments of second aspect of the present invention, in the composition containing Cryptotanshinone
It is to be provided with monomeric form, or provided in the form of the plant extracts comprising Cryptotanshinone, the mainly liposoluble of Radix Salviae Miltiorrhizae
Property form of extract provide.Cryptotanshinone can be bought by commercial sources；The Radix Salviae Miltiorrhizae of Cryptotanshinone described in herein
Fat-soluble extract can be prepared using known preparation method, and the present invention is herein without limiting.
In some embodiments of second aspect of the present invention, the composition also includes any pharmaceutically acceptable load
Body and/or excipient.
In some embodiments of second aspect of the present invention, the pharmaceutically acceptable carrier or excipient are selected from molten
Agent, diluent, dispersing agent, suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, adhesive, lubricant,
Stabilizer, hydrating agents, emulsification accelerator, buffer, absorbent, colorant, flavouring agent, sweetener, ion-exchanger, demoulding
One of agent, smears, corrigent or antioxidant are a variety of.
In some embodiments of second aspect of the present invention, the composition is formulated as powder, tablet, capsule, ball
Any one dosage form in agent, pill, injection, emulsion, suspension or tincture.
The routine techniques in formulation art can be used, obtain this by extraction separation and purification means common in pharmaceutical production
The effective component of the raw material of invention pharmaceutical composition, mixes with one or more of pharmaceutically acceptable carriers, then forms institute
The dosage form needed, to prepare pharmaceutical composition of the invention.
As used herein, it is " pharmaceutically acceptable " indicate when with usual dosage using when do not have tangible toxicity
Effect, so as to ratify or be approved for animal, more specifically to people by government or with its comparable international organization,
Or it is registered in pharmacopeia.
Available " pharmaceutically acceptable carrier " can be any in field of pharmaceutical preparations in pharmaceutical composition of the present invention
The selection of conventional carrier, specific support will depend on the administration mode for being used to treat particular patient or disease type and state.
The preparation method of said synthetic processes for specific administration mode is completely in the knowledge of drug field technical staff.
For example, can be used as solvent, diluent that pharmaceutically acceptable carrier includes pharmaceutical field routine, dispersing agent, suspending agent, surface
Activating agent, isotonic agent, thickener, emulsifier, adhesive, lubricant, stabilizer, hydrating agents, emulsification accelerator, buffer, suction
Receive agent, colorant, ion-exchanger, release agent, smears, corrigent and antioxidant etc..It when necessary, can also be in drug
Flavouring agent, preservative and sweetener etc. are added in composition.
As used herein, term " pharmaceutical composition " has its general sense.In addition, " medicine group of the present invention
Close object " can also exist or provide in the form of health care product, functional food, food, food additives etc..Pharmaceutical field can be used
Routine techniques especially in formulation art obtains medicine of the invention by extraction separation and purification means common in pharmaceutical production
The effective component of the raw material of compositions, optionally mixes with one or more of pharmaceutically acceptable carriers, then forms institute
The dosage form needed, to prepare pharmaceutical composition of the invention.Pharmaceutical composition according to the present invention, for can be adapted for taking orally to
The pharmaceutical preparation of medicine, parenteral or local administration, topical administration.Tablet, powder can be made in pharmaceutical composition of the invention
The diversified forms such as agent, granule, capsule, oral solution.The drug of above-mentioned various dosage forms can be according to the routine side of pharmaceutical field
Method preparation.Specifically, pharmaceutical composition according to the present invention, the pharmaceutical dosage form includes but is not limited to: tablet, capsule
Agent, pulvis, injection, injectable powder, transdermal patch, ointment, gelling agent, suppository, oral administration solution, takes orally and mixes granule
Suspension, emulsion for injection, Orally taken emulsion etc., sustained-release tablet, Dospan.The drug of above-mentioned various dosage forms can be according to pharmacy
It is prepared by the conventional method in field.
Dosage forms for oral administration may include such as tablet, pill, hard or soft capsule agent, solution, suspension, cream
Agent, syrup, powder, pulvis, granula subtilis, granule, pilule, elixir etc., however it is not limited to this.Other than active constituent, this
A little preparations also may include diluent (such as lactose, dextrose, sucrose, mannitol, D-sorbite, cellulose and glycine),
Lubricant (such as silica, talcum, stearic acid or its magnesium salts, calcium salt and polyethylene glycol).Tablet also may include adhesive, example
Such as aluminium-magnesium silicate, gelatinized corn starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine.When necessary,
It also may include medical additive, such as disintegrating agent (such as starch, agar, alginic acid or its sodium salt), absorbent, colorant, perfume (or spice)
Taste agent, sweetener etc..Tablet can be prepared according to common mixing, the method for being granulated or being coated.
Dosage form for parenterally application may include such as injection, medicinal drops, ointment, lotion, gelling agent, cream
Cream, spray, suspension, emulsion, suppository, patch etc., however it is not limited to this.
According to the pharmaceutical composition of the disclosure is orally available or non-bowel such as per rectum, through part, through skin, through vein
It is interior, through it is intramuscular, intraperitoneally or subcutaneously apply.
The pharmaceutically acceptable dosage of active constituent Cryptotanshinone, i.e. administration dosage, can be according to the year of object to be treated
Age, gender and weight, disease specific to be treated or pathological state, the severity of disease or pathological state, administration method and
The judgement of diagnosis person and change.Consider that these factors determine administration dosage in the horizontal extent of those skilled in the art.Generally
Dosage can be 0.01-1000mg/kg/ days, specifically 1-100mg/kg/ days.However, the scope of the present disclosure is not with any side
Formula is limited to the administration dosage.
Cryptotanshinone provided in an embodiment of the present invention can promote surface of a wound edge tissues angiogenesis, promote venous endothelial thin
Born of the same parents mitigate the inflammatory reaction of surface of a wound edge tissues at pipe, inhibit surface of a wound edge tissues ECM degradation, thus have and promote diabetes
The effect of wounds healing.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 shows that Cryptotanshinone promotes type II diabetes model mice wound healing.
The inflammation that Fig. 2 shows that Cryptotanshinone inhibits type II diabetes model mice surface of a wound edge skin histology excessive is anti-
It answers.
Fig. 3 shows that Cryptotanshinone can promote type II diabetes model mice surface of a wound edge skin histology angiogenesis.
Fig. 4 shows that Cryptotanshinone can promote HUVECs cell at pipe.
Fig. 5 shows that Cryptotanshinone can inhibit the degradation of ECM in type II diabetes model mice surface of a wound edge skin histology.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The experimental material that may be used in following each embodiments is illustrated first, it should without detailed experimental material
It is interpreted as material or reagent customary in the art.
8-12 weeks SPF grades of (no-special pathogen, Specific pathogen Free) male C57BL/6JNju (db/+)
Mouse and diabetic mice BKS.Cg-Dock7m+ /+Lepr db/JNju (db/db) mouse (are purchased from Nanjing University Nanjing
Biomedical research institute, experimental program is ratified by Ethics Committee, Tianjin University Of Traditional Chinese Medicine in Mice Body, and mouse is in Chinese medicine
It is raised under the conditions of academy of sciences Radiation Medicine Institute of Botany (Tianjin) Yu Wendu 22-25 DEG C and relative humidity 50%-60%)；It is hidden
Tanshinone (article No.: DY0011 is purchased from Chengdu De Site biology Co., Ltd, purity >=98%)；RIPA lysate (R0010) purchase
From Beijing Suo Laibao Science and Technology Ltd.Protease inhibitors (0469132001), inhibitors of phosphatases (0490837001), rouge
Enzyme inhibitor (P0100) is purchased from German Roche.TRIzol (15596-026) is purchased from U.S. Life Technology.
PrimeScript RT Master Mix and SYBR Premix Ex Taq (11636103001) is purchased from German Roche.Note
It penetrates with agglutinin Lectin I (Vector laboratories), sheep anti mouse Lectin I primary antibody (Vector
Laboratories, USA), Lectin I secondary antibody (594ANTI-GOAT IgG (H+L), Vector
Laboratories, USA)；DAPI (Suo Laibao, 10 μ g/ml instants), β-actin (4970S) rabbit polyclonal antibody is purchased from beauty
State Cell Signaling Technology.MMP2 (WL01579a) rabbit polyclonal antibody is purchased from Shenyang Wanleibio.VEGF
Rabbit polyclonal antibody (ab46154), CD45 rabbit polyclonal antibody (ab10558), Ang-1 Mouse Polyclonal Antibody (ab8451),
MMP9 rabbit polyclonal antibody (ab38898) is purchased from Britain Abcam..Blood glucose meter and blood sugar test paper (Roche group, Basel, Sweden)；
Other common agents such as methanol are that domestic analysis is pure, and experimental water is deionized water.
Embodiment 1: wounds in mice model foundation
Mouse peritoneal injects Avertin (A Wei tincture) solution, and (solute ethobrom, solvent are tert-pentyl alcohol, concentration 15g/
L) 16.5ml/kg after going hair removal from back surfaces under anaesthesia, creates the full-thickness excisional skin of a 6mm on mouse middle line
The skin surface of a wound.Silica gel plate is fixed on around the surface of a wound with adhesive, and is sutured with 5-0 suture.Drip gentamicin on the surface of a wound
Afterwards, with sterile transparent dressing flap coverage, replacement in every two days is once.It is postoperative to be randomly divided into db/+ and db/db mouse by weight
Three groups: db/+ model group (db/+Model), db/db model group (db/db Model), db/db Cryptotanshinone group (db/db
CT).Every group 9, daily gastric infusion is primary, wherein db/+Model group and db/db Model group give CMC-Na's respectively
The concentration of normal saline solution, dosage 10ml/kg, CMC-Na is 2mg/ml, and db/db CT group gives Cryptotanshinone
CMC-Na solution, dosage 10ml/kg, the concentration of Cryptotanshinone are 3mg/ml；Postoperative 0 day to postoperative 16 days, successive administration
17 days.
Embodiment 2, CT promote the healing of type II diabetes wounds in mice
It is postoperative to be tracked with healing state of the camera to three groups of wounds in mice models that embodiment 1 is established, every two days
Tracking is primary.Wound healing rate is quantified with Image J software, Wound healing rate is with X day wound healing area and the 0th
Its surface of a wound area percentage indicates, calculates are as follows: 1- [(the day X surface of a wound area/surface of a wound area of day 0 day)] × 100%.Postoperative 16
It takes the skin histology at surface of a wound edge to be fixed with 4% paraformaldehyde, and 5 μm of slice is made after paraffin embedding.60 DEG C were incubated for
Then night passes through dimethylbenzene and a series of ethyl alcohol (dipped dehydrated alcohol (I)/15min-dehydrated alcohol (II)/10min-respectively
95% ethyl alcohol (I)/95% ethyl alcohol of 8min-(II)/80% ethyl alcohol of 5min -/75% ethyl alcohol of 5min -/5min) dewaxing, carry out HE
Dyeing.HE dyeing is technological means commonly used in the art, and this will not be repeated here by the present invention.
Shown in the A-E figure of experimental result as shown in figure 1, wherein the A figure of Fig. 1 show db/+Model, db/db Model with
And db/db CT group wounds in mice healing state, the B figure of Fig. 1 show the quantitative result of Wound healing rate；From A figure and B figure
As can be seen that db/db Model group mouse was substantially reduced in 4-16 days Wound healing rates, with db/ compared with db/+Model group
Db Model is compared, and db/db CT group wounds in mice increased in 8-16 days wound healing rates, and 8-14 days obviously compare db/db
The Wound healing rate of Model group mouse increases；Statistical result is mean+SD, n=9,**P < 0.05,***P < 0.01, with db/+
Model group is compared；#P < 0.05, compared with db/db Model group.
The result of HE dyeing is as shown in the C figure of Fig. 1, the quantitative result of granulation tissue thickness and epithelial space such as D figure and E figure
It is shown；From C figure-E figure as can be seen that compared with db/db Model group, db/db CT group mouse granulation tissue thickness and epithelium
Change obviously increases.Statistical result is mean+SD, n=9,*P < 0.05,**P < 0.01, compared with db/db Model group.This reality
Result explanation is tested, compared with normal mouse (db/+ mouse), type II diabetes model mouse (db/db mouse) wound healing is slow；
And Cryptotanshinone can promote the wound healing rate of db/db mouse.
Embodiment 3, CT inhibit excessive inflammatory reaction
The wounds in mice model established using embodiment 1 takes surface of a wound edge skin histology to carry out for postoperative 16 days as research object
CD45 immunohistochemical staining；Flesh tissue is separately taken to be used to extract RNA, RT-PCR detects inflammatory factor in surface of a wound edge tissues and becomes
Change the expression of the factor.
CD45 immunohistochemical staining: the skin histology at surface of a wound edge is fixed with 4% paraformaldehyde, is made after paraffin embedding
5 μm of slice, 60 DEG C of overnight incubations, is then dewaxed by dimethylbenzene and a series of ethyl alcohol, and PBS is washed 3 times, 5min/ times, uses lemon
Sour antigen retrieval buffers carry out (92-95 DEG C) of high temperature reparation, and 3% H is added dropwise2O2, 37 DEG C of incubation 15min, inactivation, PBS washes 3 times,
5min/ times, lowlenthal serum closes 1h, and 4 DEG C of CD45 primary antibody (CD45 rabbit polyclonal antibody, anti-mouse) (PBS 1:100 dilution) is incubated
It educates overnight, PBS is washed 3 times, 5min/ times, is added dropwise secondary antibody (goat antirabbit lgG biotin) (PBS 1:200 dilution), 37 DEG C of incubations
30min, PBS are washed 3 times, and 5min/ times, strepto- avidin (PBS 1:200 dilution) 37 DEG C of incubation 20min are added dropwise, and DAB dye is added dropwise
Liquid, while observing under an optical microscope, after having apparent positive signal, with tap water repeated flushing, reaction is terminated, will be improved
The haematoxylin dye liquor (Beijing Suo Laibao Science and Technology Ltd) of type is added drop-wise to tissue, and tap water repeated flushing distinguishes slice
Dipped 75% ethyl alcohol (I)/80% dehydrated alcohol of 5min-(II)/95% ethyl alcohol of 5min-(I)/95% ethyl alcohol of 2min-(II)/
2min-dehydrated alcohol/1min-dehydrated alcohol/1min-dimethylbenzene/1min-dimethylbenzene/1min dehydration, resinene envelope
Piece.Observation is taken pictures under inverted microscope, and Image J calculates the number of positive cell in unit area under 3 random fields.
RT-PCR detection: surface of a wound edge fresh skin tissue (400mg) is taken to shred into the fritter of 3mm × 3mm or so, as far as possible
The non-purpose tissue such as adipose tissue and connective tissue is removed, is washed twice with PBS, 1ml TRIzol is added after centrifuging and taking precipitating
(Life Technologies), is transferred in the sterilizing glass homogenizer being pre-chilled in advance, on ice homogenate manually, until homogenizer bottom
Wall does not have apparent tissue block, and homogenate is transferred in 1.5ml centrifuge tube, and the chloroform of 0.08ml is added in every pipe, stands on ice
15min, every 5min shake 1 time, each 30s, after concussion 3 times, with 12000 × g centrifugation 15 minutes at 4 DEG C.Upper strata aqueous phase is taken,
- 20 DEG C are placed in overnight after isometric isopropanol concussion uniformly is added.4 DEG C, 12000 × g is centrifuged 20min, abandons supernatant, is added
After 75% ethyl alcohol of DEPC water process gently shakes, 4 DEG C, 8000 × g is centrifuged 10min, abandons supernatant, dry, and 20 μ are added
Precipitating is resuspended in LRNase, after 55 DEG C of water-bath 15min with e-spect instrument (Japanese Malcom company) read RNA concentration and
260/280 numerical value is inverted using Transcriptor First Strand Cdna Synthesis Kit (Roche) and is recorded
Standby cDNA.With FastStart Universal SYBR Green Master (Roche) and7500Real-time
PCR system (Applied Biosystems) detects expression of each target gene in different samples.25 μ L of total system；Amplification condition
Are as follows: 95 DEG C of 15min；94 DEG C of 15s, 55 DEG C of 30s, 70 DEG C of 30s are a circulation, coamplification 40 circulations.Primer information such as 1 institute of table
Show.
Table 1
Experimental result is as shown in A-C figure in Fig. 2, and wherein A figure is CD45 immunohistochemical staining results, it can be seen that and db/
Db Model is compared, and the quantity of db/db CT group wounds in mice edge C D45 positive cell significantly reduces；RT-PCR result such as Fig. 2
Shown in middle B-C, it is seen that the expression of the mRNA of Chemokines CC xcl1, Cxcl2 significantly reduce, statistical result mean+SD, * P <
0.05, * * P < 0.01, * * * P < 0.001, compared with db/db Model group, n >=6.It is thin to illustrate that Cryptotanshinone can be reduced inflammatory
The infiltration of born of the same parents and the expression of chemotactic factor (CF), inhibit excessive inflammatory reaction.
Embodiment 4, CT promote angiogenesis
The wounds in mice model established using embodiment 1 postoperative 16 days, takes db/db Model group and db/db as research object
CT group mouse each 8, with Lectin I (HPS 5mg/ml dilution) tail vein injection, injection volume 2.5mL/kg.It takes pictures observation
Surface of a wound marginal vessel, Immunofluorescence test surface of a wound edge skin histology capillary density, fresh skin tissue are used to extract egg
White, western blotting detects the expression of VEGF and Ang-1.
TRIzol immunofluorescence dyeing: after tail vein injection Lectin I hours, putting to death, take surface of a wound edge skin histology,
It is fixed with paraformaldehyde, 5 μm of slice is made after paraffin embedding.60 DEG C of overnight incubations, then pass through dimethylbenzene and a series of second
Alcohol (referring to embodiment 2) dewaxing, PBS are washed 3 times, 5min/ times, are repaired liquid with citric acid antigen and carry out high temperature reparation, and PBS is washed 3 times,
It washes 3 times, 5min/ times, is added dropwise Lectin I primary antibody (PBS 1:100 dilution), 4 DEG C of mistakes for 5min/ times, 37 DEG C of BSA closing 1h, PBS
Night.It being added dropwise Lectin I secondary antibody (PBS 1:200 dilution), DAPI redyes nucleus, and it observes and takes pictures under inverted fluorescence microscope,
Imaje J calculates the number of unit area inner capillary tube under 3 random fields.
Western blotting: surface of a wound edge fresh skin tissue is taken, shreds into the fritter of 3mm × 3mm or so, as far as possible
The non-purpose tissue such as adipose tissue and connective tissue is removed, is washed twice with PBS, tissue is put into the lysate of pre-cooling
(10ml lysate :+50 μ l phenylmethylsulfonyl fluoride of 10ml RIPA+0.1g protease inhibitors+0.1g inhibitors of phosphatases
(PMSF)), grinding is broken, and 4 DEG C, 12000rpm is centrifuged 15min, Aspirate supernatant, and BCA method measures protein concentration.10%
Sample is separated on SDS-PAGE, and is transferred on pvdf membrane.It (is dissolved in and being contained with 5% bovine serum albumin(BSA) or skim milk
Have the Tris-HCl buffer of 0.1%Tween-20, TBST) after closing 3 hours, it is ((molten with an anti-vegf and Ang-1 at 4 DEG C
In 5% skimmed milk power, dilution ratio 1:1000)) be incubated overnight, after thoroughly washed with TBST.Then, by film be connected with it is peppery
(article No. is that ZB-2301 is purchased from Beijing Zhong Shan Golden Bridge biology skill to the secondary antibody goat antirabbit lg/G horseradish enzyme label of root peroxidase
Art Co., Ltd) and goat anti-mouse lg/G horseradish enzyme label (article No. ZB-2305, purchased from Beijing Zhong Shan Golden Bridge biotechnology
Co., Ltd) (dilution ratio 1:10000) be incubated at room temperature 1h.With the chemiluminescence detection reagent of enhancing, (kit is
Chemiluminescent HRP Substrate is purchased from U.S. MILLIPORE company) carry out trace detection.With Image J amount
Change the gray-scale intensity of protein band, and is normalized with the β-actin in each sample.
Experimental result as shown in figure 3, wherein A figure be skin heart photo, B figure is Lectin I immunofluorescence dyeing knot
Fruit and Lectin I immunofluorescence dyeing quantitative result；From A figure as can be seen that compared with db/db Model group, db/db CT
Group wounds in mice edgewick vessel density obviously increases, and B figure also shows db/db CT group wounds in mice edge tissues
Lectin positive cell dramatically increases；C figure in Fig. 3 shows that the expression of phosphorylation eNOS, D figure show the egg of VEGF
White expression, E figure show the expression of Ang-1 albumen, it can be seen that b/db CT group wounds in mice edge tissues phosphorus
Acidification eNOS, VEGF and Ang-1 protein expression obviously increase, statistical result be mean+SD.*P < 0.05, * * P < 0.01, with
Db/db Model group is compared, n >=6；VEGF (vascular endothelial growth factor, vascular endothelial growth factor
Son) be a kind of high degree of specificity rush vascular endothelial growth factor, have promote vasopermeability increase, promote extracellular base
Qualitative change promotees the effects of migration of vascular endothelial cells, proliferation and vascularization.(it is raw to promote blood vessel to Ang-1 by angiopoietin-1
It 1) works during new vessels segment dislocation at element, stablizes blood vessel structure.It is activated after eNOS phosphorylation, generates one
Nitrogen oxide (NO) promotes the growth of endothelial cell and promotes angiogenesis.Thus embodiment can illustrate that Cryptotanshinone can promote
Surface of a wound edge tissues angiogenesis, and this process may be with the increase of angiogenesis albumen eNOS phosphorylation, VEGF and Ang-1
Expression quantity increases related.
Embodiment 5, CT promote the expression at pipe and VEGF of HUVECs cell
RT-PCR detection: HUVECs cell (being purchased from ATCC) uses DMSO, 2.5 μM of Cryptotanshinone (0.2% CMC-Na water
Solution) after processing 24 hours, RT-PCR detects the expression of VEGF mRNA, and primer sequence is as shown in table 2, and method is referring to embodiment
3。
Table 2
ELISA detection: after HUVECs cell DMSO, 2.5 μM of Cryptotanshinone are handled 24 hours, cell conditioned medium is collected
Liquid, with the expression of VEGF ELISA kit (VEGF ELISA kit (DVE00) is purchased from U.S. R&D company) detection VEGF.
HUVECs cell is at pipe: matrigel matrigel (Corning Incorporated) being laid in 96 orifice plates, bubble is avoided to produce
Raw, this process operates on ice.37 DEG C are put into, 5%CO2It is incubated for 1 hour in incubator.HUVECs cell is containing 5%FBS's
EGM-2 complete medium (Lonza company, the U.S.) routine culture, until with the digestion of 0.25% pancreatin when 80% fusion, respectively with containing
(2.5 μM of CT of complete medium of the complete medium (DMSO group) of 0.1%DMSO, 2.5 μM of Cryptotanshinones and 0.1%DMSO
Group) processing HUVECs cell 18 hours, with 2 × 104A cell per well is inoculated in 96 orifice plates for completing matrigel.It is placed in 37
DEG C, 5%CO2It cultivates in incubator, takes pictures after cell culture 18h, calculate the number of vessel branch point.
Experimental result is as shown in A-D figure in Fig. 4, and wherein A figure is HUVECs cell into pipe experimental result, and B figure is HUVECs
Cell is at the quantitative result of pipe, and as can be seen from the results, Cryptotanshinone can promote HUVECs at pipe, experimental result mean+
SD, * * P < 0.01, compared with DMSO group, n >=3；C figure is VEGF mRNA expression, D figure vegf protein expression, from
As a result it can be seen that compared with the HUVECs cell of DMSO processing, 2.5 μM of Cryptotanshinone can increase in HUVECs cell
The expression of the mRNA and albumen of VEGF, experimental result mean+SD, * P < 0.05, * * P < 0.01, P < 0.001 * * *, with DMSO
The cell of processing is compared, n >=3.By Cryptotanshinone to HUVECs at the effect of pipe and vegf expression, illustrate that Cryptotanshinone can
The expression and release for promoting VEGF in vitro, promote angiogenesis.
Embodiment 6, CT inhibit the degradation of ECM (extracellular matrix)
Collagen is the chief component of extracellular matrix, and the degradation of ECM is reduced, and collagen deposits, and leads to ECM weight
Modeling, can promote wound healing.
Using embodiment 1 establish wounds in mice model as research object, postoperative 16 days, take db/db Model group mouse with
Db/db CT group mouse each 8, surface of a wound edge skin histology is taken, immunohistochemistry detects surface of a wound edge skin histology collagen deposition,
Fresh skin tissue is used to extract albumen, and western blotting detects the expression of Col-1, Col-3, MMP2 and MMP9.
Masson immunohistochemistry: the skin histology at surface of a wound edge is fixed with 4% paraformaldehyde, and 5 μ are made after paraffin embedding
The slice of m.60 DEG C of overnight incubations carry out Masson dye then by dimethylbenzene and a series of ethyl alcohol (referring to embodiment 2) dewaxing
Color.
Western blotting: WB operation is carried out using conventional method, (is dissolved at 4 DEG C with primary antibody MMP2 and MMP9
5% skimmed milk power, dilution ratio 1:1000) be incubated overnight, after washed with TBST；The secondary antibody of horseradish peroxidase-labeled
Goat antirabbit lg/G horseradish enzyme label (ratio of releasing is 1:10000) is incubated at room temperature 1 hour.With the chemiluminescence detection of enhancing
Reagent carries out trace detection.Quantify the gray-scale intensity of protein band with Image J, and is carried out with the β-actin in each sample
Normalization.
Experimental result is as shown in A-C figure in Fig. 5, and wherein A figure is Masson coloration result, as can be seen from the results, with
Db/db Model group is compared, and db/db CT group wounds in mice edge collagen deposition obviously increases, fibroblast marshalling,
Illustrate that Cryptotanshinone can promote ECM to remold；B figure-C figure shows the protein expression WB and quantitative result of MMP2 and MMP9, can be with
Find out that the protein expression of MMP2 and MMP9 in db/db Model group wounds in mice edge tissues is decreased obviously, experimental result is
Mean+SD, * P < 0.05, * * p < 0.01, compared with db/db Model group, n >=6, MMP2 and MMP9 are matrix metalloproteases
Enzyme can promote the degradation of collagen, and MMP2 and both protein decreaseds of MMP9 illustrate that collagen degradation is reduced, and collagen deposition increases in turn
Add, promotes ECM remodeling, promote wound healing.It is possible thereby to illustrate, Cryptotanshinone is able to suppress the degradation of collagen, and then inhibits
ECM degradation, promotes the deposition of ECM.
The experimental result of above embodiments is shown: 1, Cryptotanshinone can promote type II diabetes wounds in mice to heal；2,
Cryptotanshinone can promote type II diabetes wounds in mice edge tissues angiogenesis；3, Cryptotanshinone can promote people intravenous
Chrotoplast is at pipe；4, Cryptotanshinone can promote surface of a wound edge tissues or people's venous endothelial cell VEGF expression；5, Cryptotanshinone
It can reduce type II diabetes wounds in mice edge tissues inflammatory reaction；6, Cryptotanshinone is able to suppress type II diabetes mouse wound
Face edge tissues ECM degradation.As can be seen from the above results, Cryptotanshinone can be by promoting surface of a wound edge tissues angiogenesis
With venous endothelial cell at pipe, increase surface of a wound edge tissues vessel density, and vessel density increase can be by promoting tissue new
Wound healing from birth, during wound healing, blood vessel provides oxygen and nutriment, promotion group for newborn tissue
Knit new life.And then promote；On the other hand, Cryptotanshinone promotes the surface of a wound by the excessive inflammatory reaction of mitigation surface of a wound edge tissues
Healing；In addition, Cryptotanshinone can also inhibit the degradation of ECM, wound healing；The work that Cryptotanshinone passes through this three aspect
With promotion diabetic's wound healing.
Each embodiment in this specification is all made of relevant mode and describes, same and similar portion between each embodiment
Dividing may refer to each other, and each embodiment focuses on the differences from other embodiments.Especially for system reality
For applying example, since it is substantially similar to the method embodiment, so being described relatively simple, related place is referring to embodiment of the method
Part explanation.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
SEQUENCE LISTING
<110>Tianjin University Of Traditional Chinese Medicine
<120>purposes of the Cryptotanshinone in the drug that preparation promotes diabetic's wound healing
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<170> PatentIn version 3.5
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Claims (10)
1. purposes of the Cryptotanshinone in the drug that preparation promotes diabetic's wound healing.
2. purposes according to claim 1, which is characterized in that the Cryptotanshinone is by increasing surface of a wound edge tissues capillary
Vessel density promotes diabetic's wound healing.
3. purposes according to claim 2, which is characterized in that the Cryptotanshinone by promote people's venous endothelial cell at
Pipe increases surface of a wound edge tissues capillary density.
4. purposes according to claim 1, which is characterized in that the Cryptotanshinone is excessive by inhibiting surface of a wound edge tissues
Inflammatory reaction, promote diabetic's wound healing.
5. purposes according to claim 1, which is characterized in that the Cryptotanshinone is by inhibiting surface of a wound edge tissues ECM
Degradation promotes diabetic's wound healing.
6. a kind of purposes of composition containing Cryptotanshinone in the drug that preparation promotes diabetic's wound healing.
7. purposes according to claim 6, which is characterized in that the Cryptotanshinone be provided with monomeric form, or
It is provided in the form of the plant extracts comprising Cryptotanshinone.
8. purposes according to claim 6, which is characterized in that the composition also includes any pharmaceutically acceptable load
Body and/or excipient.
9. purposes according to claim 8, which is characterized in that the pharmaceutically acceptable carrier or excipient are selected from molten
Agent, diluent, dispersing agent, suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, adhesive, lubricant,
Stabilizer, hydrating agents, emulsification accelerator, buffer, absorbent, colorant, flavouring agent, sweetener, ion-exchanger, demoulding
One of agent, smears or antioxidant are a variety of.
10. purposes according to claim 6, which is characterized in that the composition is formulated as powder, tablet, capsule, ball
Any one dosage form in agent, pill, injection, emulsion, suspension or tincture.
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Cited By (1)
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| CN114796596A (en) * | 2022-04-22 | 2022-07-29 | 青岛大学 | Nanofiber hydrogel dressing loaded with salvia miltiorrhiza and radix puerariae and preparation process thereof |
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| CN101254165A (en) * | 2008-03-27 | 2008-09-03 | 泰山医学院 | Skin osmosis promoting absorbing agent of cryptotanshinone |
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| CN101254165A (en) * | 2008-03-27 | 2008-09-03 | 泰山医学院 | Skin osmosis promoting absorbing agent of cryptotanshinone |
| CN108619158A (en) * | 2017-03-17 | 2018-10-09 | 南京柯菲平盛辉制药有限公司 | Application of the sodium tanshinon Ⅱa silate in preparing the drug for promoting angiogenesis |
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Application publication date: 20190906 |