CN104127403A - Application of flavonoid quercetin in inhibition of skin scar formation and skin fibration - Google Patents

Abstract

The invention relates to an application of flavonoid quercetin in inhibition of skin scar formation and skin fibration. The application verifies that quercetin has no toxicity to fibroblast, human dermal fibroblast I/III-type collagen mRNA expression and protein synthesis can be inhibited by inhibiting a TGF beta1 signal pathway in cells, fibroblast can be inhibited to myofibroblast differentiation, so that skin scar formation or dermal collagen deposition (fibration) can be inhibited, Animal in-vitro experiment confirms that quercetin is effected on mice skin, bleomycin-induced mice skin fibration and tension force-induced mice skin scar formation can be inhibited. Because that quercetin has the characteristics of small molecular weight, high lipid solubility and good transdermal absorption capability, the molecules can be used for preparing the medicine for resisting skin scar formation and skin fibration, and have wide application prospect in medical, shaping and cosmetic fields.

Description

Flavone compound Quercetin is in the application suppressing in cicatrix of skin formation and fibrosis of skin

technical field

The present invention relates to a kind of new medical usage of flavone compound Quercetin, specifically, relate to flavone compound Quercetin in the application suppressing in cicatrix of skin formation and fibrosis of skin.

background technology

Cicatrization is one of modal complication after wound healing.After cicatrization, affect patient's appearance, and with the pain of itching, and the contracture producing can cause dysfunction in various degree, as tendon contracture, joint dislocation, dyskinesia, job psychograph obstacle etc., reduce patient's quality of life, for anaphase brings heavy economy and psychological burden.According to statistics, the incidence rate of compatriots' hypertrophic scar is 91.4%, if the burn wound healing time exceedes 2 weeks, hypertrophic cicatrix incidence rate is up to 74.67%, far above 38.00% of external report.The study on prevention of cicatrix is a not only ancient but also novel problem.Research find scar hyperplasia be human body to a kind of excessive healing reaction of wound cause taking fibroblast abnormality proliferation, collagen in a large number synthetic, collagen over-deposit as the skin fiber proliferative disease of histological characteristic, but its pathogenesis is illustrated not yet completely, therefore its treatment is at present still a rather thorny clinically difficult problem.The generation of Chinese scholars to cicatrix for many years, develop and the mechanism that disappears has been carried out multi-angle, multifaceted further investigation, but so far the research of its mechanism be there is no to clear and definite conclusion, prevent and treat aspect and also there is no good recipe, more consistent view has: 1. the main effector lymphocyte of scar hyperplasia is fibroblast, and taking cell hyperproliferation and extracellular matrix over-deposit as feature; 2. collagenic supersession disorder is its main aspect performance biology; 3. TGF-β 1/Smad signal path and fibroblastic propagation, break up, migrate, the multiple physiology such as apoptosis and collagenic supersession, pathological process is closely related, according to different typings, to Collagen of Fibroblasts, metabolism has double regulation control effect to Smads.

In clinical treatment, treatment cicatrix method has pressure therapy, corticosteroid hormone associating 5-FU injection for curing, pellosil and aerogel dressing treatment, laser therapy, roentgenotherapia, cold therapy etc. at present.But, can resist the small-molecule drug of cicatrix still very limited.Mostly current anti-cicatrix medicine is anti-inflammatory agent (as: steroid hormone) or cell toxicity medicament (as: 5-FU), almost seldom have and can directly suppress the synthetic medicine of Collagen of Fibroblasts, therefore the effect of its anti-cicatrix is faint and unstable, and has toxic and side effects widely.Therefore finding the fat-soluble molecule that a kind of molecular weight is little, can directly penetrate epidermal barrier and act on skin corium fibroblast, is very promising treatment means thereby suppress the expression of collagen synthetic.

Quercetin is a kind of flavone compound, English name Quercetin, chemical formula C ₁₅h ₁₀o ₇, molecular weight 302.24, structural formula is as shown in Figure 1A.It is in glacial acetic acid, and it is yellow that alkaline aqueous solution is, water-soluble, alcoholic solution hardly; Taste is very bitter.Can be used as medicine, have preferably eliminate the phlegm, antitussive action, and have certain antiasthmatic effect.In addition reduce blood pressure in addition, strengthen capillary resistance, reduce capillary fragility, blood fat reducing, coronary artery dilator, increase the effects such as coronary flow, can be used for treating chronic bronchitis, coronary heart disease and hyperpietic are also had to auxiliary therapeutic action, and research at present also finds to possess certain antitumor action.

But have not been reported in the effect aspect the formation of inhibition cicatrix of skin and fibrosis of skin about Quercetin at present.

summary of the invention

The object of the invention is for deficiency of the prior art, the new purposes of Quercetin is provided.

For achieving the above object, the technical scheme that the present invention takes is:

First aspect, provides the purposes of Quercetin, and for the preparation of suppressing, cicatrix of skin forms and/or the reagent of fibrosis of skin.Described reagent is medicine or cosmetics.

Second aspect, provides the purposes of Quercetin, and for the preparation of reagent, described reagent is used for:

A) suppress skin flbroblast Collagen I/III mrna expression; Or

B) suppressing skin flbroblast α-SMA expresses; Or

C) suppress skin flbroblast to myofibroblast differentiation; Or

D) suppress skin flbroblast smad2/3 signal protein phosphorylation; Or

E) suppress skin flbroblast smad4 signal protein and enter nucleus.

Described skin flbroblast is normal skin fibroblast.

Described skin flbroblast is dermal fibroblast.

The third aspect, provides the purposes of Quercetin, for the preparation of the reagent that suppresses mice corium fabric model collagen deposition.For reducing mice dermis thickness.

Fourth aspect, provides the purposes of Quercetin, for the preparation of the synulotic reagent of mice that suppresses tension force induction.For reducing mice cicatrix volume.

The invention has the advantages that:

The present invention confirms that flavone compound Quercetin has no significant effect fibroblastic normal function and physiologically active, also can not cause the apoptosis of cell, to fibroblast avirulence.Expression and albumen that Quercetin can suppress human skin fibroblast I/III collagen mRNA synthesize, thereby and can be suppressed to fibrocyte suppresses cicatrix of skin formation or generation from fibrosis of skin to myofibroblast differentiation.The present invention has further probed into Quercetin and has suppressed I/III Collagen Type VI synthetic biological mechanism, finds that Quercetin is effectively to suppress the synthetic of collagen in human fibroblasts by suppressing TGF β 1/Smad signal path in cell.Animal has confirmed the mouse skin cicatrization of fibrosis of skin and the tension force induction that can suppress bleomycin induction after Quercetin acts on mouse skin in body experiment.Because quercetin molecule amount is little and fat-soluble height, Transdermal absorption ability is good, and therefore this molecule can be used for preparing the medicine of anti-cicatrix of skin formation and anti-fibrosis of skin, has broad application prospects at medical treatment, shaping and cosmetic field.

brief description of the drawings

Figure 1A is quercetin molecule structural formula.

Figure 1B is control media (DMSO), Quercetin (10 μ M) and TGF β 1(10ng/ml) effect 3 days after, the result of cell density and cell Toluidine blue staining.

Fig. 1 C is that the Quercetin (100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M, 50nM) of variable concentrations acts on respectively l cell (NIH3T3) and the primary fibroblast of people Collagen I/III mrna expression horizontal detection result after 3 days.

Fig. 1 D is that variable concentrations Quercetin (100 μ M, 25 μ M, 5 μ M, 1 μ M, 0.1 μ M) acts on l cell (NIH3T3) and extracellular matrix dyeing (toluidine blue) and cell dyeing (crystal violet) result of the primary fibroblast of people after 3 days.

Fig. 1 E is that variable concentrations Quercetin (100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M, 50nM) acts on the primary fibroblast of people Collagen I/III protein expression level testing result after 5 days.

Fig. 2 A is high concentration (50 μ M) and low concentration (1 μ M) Quercetin effect other types collagen (Collagen II/X) and Mmp2/Mmp9/Timp1 mrna expression horizontal detection result after 3 days.

Fig. 2 B is that 50 μ M, 10 μ M Quercetins act on apoptosis testing result and the statistical analysis of the primary fibroblast of people after 3 days.

Fig. 2 C is that 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M Quercetin are made the primary fibroblast of employment MTT testing result after 48 hours.

Fig. 3 A is that normal saline+DMSO, normal saline+Quercetin, bleomycin+DMSO, bleomycin+Quercetin are injected into respectively injected in mice position skin HE dyeing and Masson trichrome stain result after mice subcutaneous (once a day) January and add up with dermis thickness.

Fig. 3 B be after three groups of mouse skin holostromes cut, smear respectively DMSO, stretching wound+smear DMSO, stretching wound+smear Quercetin after 2 weeks mice cicatrix position skin substantially see, HE dyeing, Masson trichrome stain result and cicatrix cardinal principle area statistics and the statistical result of cicatrix cross section area.

Fig. 4 A is that 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M Quercetin are made the primary fibroblast of employment α-SMA mrna expression horizontal detection result after 3 days.

Fig. 4 B is control media (DMSO), Quercetin (10 μ M), TGF β 1(5ng/ml) and Quercetin+TGF β 1 act on 3 days after actin (F-actin) and α-smooth muscle actin (α-SMA) detection of expression result.

Fig. 4 C is that 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M Quercetin are made the primary fibroblast of employment α-SMA protein expression testing result after 5 days.

Fig. 4 D is that normal saline+DMSO, normal saline+Quercetin, bleomycin+DMSO, bleomycin+Quercetin are injected into respectively injected in mice position skin α-SMA immunohistochemical staining result and the statistical result of α-SMA expression after mice subcutaneous (once a day) January.

Fig. 4 E smears respectively DMSO, stretching wound+smear DMSO, stretching wound+smear Quercetin mice cicatrix position skin α-SMA immunohistochemical staining result and the statistical result of α-SMA expression afterwards in 2 weeks after three groups of mouse skin holostromes cut.

Fig. 5 A be 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M Quercetin do the primary fibroblast of employment after 3 days TGF β 1 mrna expression horizontal detection result and Quercetin make the primary fibroblast of employment TGF β 1 protein level testing result after 5 days.

Fig. 5 B is that 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M Quercetin are made the primary fibroblast of employment TGF β RI, TGF β RII mrna expression horizontal detection result after 3 days.

Fig. 5 C is control media (DMSO), Quercetin (10 μ M), TGF β 1(5ng/ml) and Quercetin+TGF β 1 act on after 1 hour, Smad4 enters core situation testing result.

Fig. 5 D is TGF β 1 signal path (Smad2/P-Smad2/ Smad3/P-Smad3/Smad4/Smad6/Smad7), BMP-2 signal path (Smad1/5/8, P-Smad1/5/8) protein expression testing result.

Fig. 6 is the Biological mechanism of action schematic diagram of Quercetin.

Detailed description of the invention

Below in conjunction with accompanying drawing, detailed description of the invention provided by the invention is elaborated.

embodiment 1

one, experiment material and method

1, quercetin molecule physicochemical property

Test Quercetin used (CAS 117-39-5) molecule purchased from Selleck company (cat. no. S2391).

Medicine sees through horny layer and epidermis enters the main path that corium is medicine absorbing transmission from skin.Medicine will reach the effect of Transdermal absorption, need to reach following requirement: dosage is little and effect is strong; The more difficult horny layer that passes through of material that molecular weight is greater than 600; Organic monoacid or organic weak base medicine exist with molecular forms, have larger percutaneous to see through ability; Fat-soluble medicine Transdermal absorption is satisfactory for result, and ionic drug is generally difficult for seeing through horny layer.Quercetin molecule amount is 302.24, structural formula is as Figure 1A, belong to organic monoacid medicine, and have well fat-solublely, can reach well the effect of Transdermal absorption, and Transdermal absorption reaches after skin corium, because it is good fat-soluble, can be diffused into blood capillary by hydrophilic tissue absorption and enter body circulation, therefore can reach the effect of local action, and can not act on other histoorgans except skin histology.

2, Cell isolation and culture

Fibroblast: skin-derived is in the child's of the 9th the People's Hospital Urology Surgery excision prepuce tissues (agreeing to through patient).After prepuce tissues sterilization is clean, subcutaneus adipose tissue in prepuce tissues is removed and (ensured to remove clean, do not damage skin corium) simultaneously, dermatological specimens is cut into little strip, be soaked in the medium-sized protease (dispase I (Roche of 8 U/ml, USA)), in 4 DEG C of refrigerators, digest and spend the night.Digestion 12-16 hour, separates epidermal area to abandon from skin corium with microforceps, and remaining dermal tissue, shakes 37 DEG C of digestion half an hour frequently with the type i collagen enzyme (Sigma-Aldrich) of 375 U/ml.Digestive system is with after strainer filtering, by the centrifugal filtrate cell precipitation that obtains, by the DMEM-high glucose medium re-suspended cell precipitation containing 10% hyclone (Gibco), 100U/ml penicillin, 100 μ g/ml streptomycins, be inoculated in 10mm culture dish and carry out cell culture, not adherent dead cell is sucked away changed liquid after 24 hours time.The cell in the 2nd to 5 generations is for follow-up experimental analysis.

3, extracellular matrix dyeing

Extracellular matrix mainly contains the composition such as collagen and acid mucopolysaccharide (hyaluronic acid).Can make extracellular collagen painted with toluidine blue, present bluish violet.By the depth of Toluidine blue staining, can reflect intuitively the number of the collagenogenic amount in extracellular.

Concrete grammar is as follows:

1. after dosing finishes action time, suck culture medium, rinse cell 2 times with aseptic PBS, remove residual culture medium;

2. add 4% paraformaldehyde room temperature fixed cell 15 minutes;

3. suck paraformaldehyde, rinse cell 3 times with aseptic PBS, remove residual paraformaldehyde;

4. add the Toluidine blue staining liquid that is preheated to 37 DEG C, Tissue Culture Plate is put into 37 DEG C of dyeing 5min;

5. suck aniline blue dyeing liquor, rinse cell 3 times with aseptic PBS, remove residual dyeing liquor;

6. scanner scanning or inverted microscope are taken dyeing situation.

4, cytoactive analysis

MTT(tetrazolium bromide) cell viability is measured is that succinate dehydrogenase based in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value by microplate reader at 490nm wavelength place, and within the scope of certain cell number, the amount that MTT crystallization forms is directly proportional to cell number.According to the absorbance recording (OD value), judge living cells quantity.Respectively at before adding the Quercetin of 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M concentration (solvent is DMSO) and dosing after 48 hours, be detected as fibrocellular proliferation activity with MTT reagent.Matched group is processed cell with DMSO.

Concrete grammar is as follows:

1. after dosing finishes action time, suck culture medium, rinse cell 2 times with aseptic PBS, remove residual culture medium;

2. under lucifuge condition, change the fresh MTT working solution that is dissolved in serum-free medium to cell, the 96 every holes of orifice plate add 100 μ l, 37 ° of C, 5%CO ₂under saturated humidity condition, hatch 4 hours;

3. under lucifuge condition, suck MTT working solution, add the every hole 100 μ l of DMSO, shaking table shakes 15min.Optical density (OD value) by microplate reader in the each hole of 490nm wavelength measurement, contrast adds the cell proliferation difference of Quercetin group and DMSO group.

5, Apoptosis by Flow Cytometry

Fibroblast uses respectively Quercetin (50 μ M, 10 μ M) and DMSO to process after 72 hours, remove culture fluid, wash and once use afterwards 0.25% trypsinization with PBS, collecting cell, centrifugal rear by the abundant re-suspended cell precipitation of PBS, add 10 μ l iodate pyridines (propidium iodide) and 10 μ l film mucin V(annexin V) working solution, under room temperature, lucifuge is hatched 10 minutes, centrifugal rear PBS washed cell 2 times of using, by flow cytometer detection (FACScalibur system (Becton Dickinson Biosciences)) apoptosis situation.The two positive marks' of iodate pyridine and film mucin V cell is considered to apoptotic cell.

6, reverse transcription-polymerase chain reaction real-time quantitative PCR-gene expression detects

After fibroblast uses respectively Quercetin (100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M, 50nM) and DMSO to process, according to test kit explanation, the cell of collecting extracts total RNA in cell with TRIzol reagent (Invitrogen), after reverse transcription, carry out real-time quantitative PCR detection at ABI 7900HT instrument with SYBR fluorescent protein systems.PCR reaction condition is as follows: each reaction system comprises the each 0.1 μ l of upstream and downstream primer, SYBR 5 μ l, cDNA 0.3 μ l, ddH ₂o 4.5 μ l.PCR response procedures is as follows: 10 seconds → 95 ° C of 95 ° of C degeneration 10 seconds, 60 ° of C 30 seconds, continue 40 solubility curves that circulate → enter.Computational methods: GAPDH is as internal reference.Remainder data is analyzed according to Ct value (2 ^{-Δ Δ Ct}) method.In result figure, be shown as the multiple value changing with respect to the expression of internal reference.The primer sequence is as follows:

7, Western blot detects protein expression

After fibroblast uses respectively Quercetin (100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M, 50nM) and DMSO to process, collecting cell, with cell pyrolysis liquid cell lysis on ice 30 minutes.Cell pyrolysis liquid composition is: 50 mM Tris – HCl, pH 7.4,150 mM NaCl, 1% Nonidet P-40,0.1% SDS, protease inhibitor cocktail (10 mg/ml leupeptin, 10 mg/ml pepstatin A and 10 mg/ml aprotinin).Repeatedly blow and beat to lysis, hatch 30min on ice.4 ° of C, the centrifugal 5min of 12000rpm, shifts supernatant for subsequent use.Determination of protein concentration adopts BCA determination of protein concentration test kit.Protein sample applied sample amount is 15 μ g, runs glue transferring film to PVDF albuminous coat at 8% SDS – PAGE.Pvdf membrane is dipped in to primary antibodie Incubating Solution, and primary antibodie anti-smads dilution ratio used is 1:1000(Cell Signaling Technology), anti-collagen I(1:1000, Abcam), anti-collagen III(1:1000, Abcam).GAPDH is as internal reference antibody, dilution ratio 1:10000.4 ° of C spend the night, and wash film with TBST.Pvdf membrane is immersed containing in two anti-Incubating Solutions of HRP labelling, at room temperature rock and hatch 90-120min, then wash film with TBST, method is the same.Adopt Tanon imager scanning protein band.

8, immunofluorescence dyeing

1. α-SMA and phalloidin dyeing

Fibroblast is divided into 4 groups, is respectively: 1 group of matched group, 1 group of TGF-β, Quercetin group, Quercetin+TGF β.Use respectively DMSO, TGF-β 1(5ng/ml), Quercetin (10 μ M), Quercetin+TGF β 1 process 3 days, removes culture fluid, with after PBS washing once, with 4% paraformaldehyde fixed cell 15 minutes under room temperature, remove fixative, with PBS washing 3 times, each 10 minutes.Add permeable membrane liquid 0.25% TritonX100, permeable membrane 10 minutes.Add 3% tire bovine albumin to carry out non-specific protein binding site sealing.After sealing, add primary antibodie anti-α-SMA(1:200, Abcam) and the phalloidin Phalloidin(1:200 of Alexa Fluor 488 labellings, Cytoskeleton, Inc, Denver, CO, USA) under room temperature, dye 2 hours.With PBST concussion washing 3 times, each 10 minutes.Add two anti-(Invitrogen) with fluorescently-labeled goat-anti rabbit or anti-Mus accordingly by 1:500 dilution proportion, under room temperature, hatch 1 hour, after dyeing finishes, add 100 μ l DAPI(2mg/ml, Invitrogen) carry out nucleus dyeing 10 minutes.After dyeing finishes, under laser confocal microscope, observe.

2. Smad4 dyeing

Fibroblast is divided into 4 groups, is respectively: 1 group of matched group, 1 group of TGF-β, Quercetin group, Quercetin+TGF β.Use respectively DMSO, TGF-β 1(5ng/ml), Quercetin (10 μ M), Quercetin+TGF β 1 process 1 hour, removes culture fluid, with after PBS washing once, with 4% paraformaldehyde fixed cell 10 minutes under room temperature, remove fixative, with PBS washing 3 times, each 5 minutes.Add permeable membrane liquid 0.25% TritonX100, permeable membrane 10 minutes.Add 3% tire bovine albumin to carry out non-specific protein binding site sealing.After sealing, add primary antibodie anti-Smad4(1:200, Bioworld), under room temperature, hatch 2 hours, dyeing liquor is washed away, with PBST concussion washing 3 times, each 10 minutes.Add two anti-(Invitrogen) with fluorescently-labeled goat-anti rabbit or anti-Mus accordingly by 1:500 dilution proportion, under room temperature, hatch 1 hour, after dyeing finishes, add 100 μ l DAPI (2mg/ml, Invitrogen) transfect cell cores, dye core after 10 minutes, dyeing liquor is washed away, with PBST concussion washing 3 times, each 10 minutes, as previously mentioned.Dyeing finishes with anti-fluorescent quenching mountant mounting.Under laser confocal microscope, observe albumen at thin inner cellular localization.

9, collagen in zoopery, immunohistochemical staining and Masson trichrome stain-display organization

9.1 zooperies:

1. the fibrosis of skin model of bleomycin injection induction:

Get 18 close ages of body weight and be the male C57/BL6 mice of 8 weeks, back hair is rejected clean, be divided at random Quercetin group, bleomycin group and bleomycin+Quercetin group, 6 every group.Every mouse back skin right side injection Experimental agents, left side injecting normal saline (0.9% NaCl) is as own control.The Quercetin (100 μ l in 0.9% NaCl) of Quercetin group mouse subcutaneous injection 10 μ M concentration once a day, bleomycin group subcutaneous injection bleomycin 100 μ l(1 mg/ml in 0.9% NaCl) once a day, bleomycin+Quercetin group subcutaneous injection bleomycin and Quercetin mixed solution 100 μ l(bleomycin 1 mg/ml, Quercetin 10 μ M in 0.9% NaCl).After January, mice is put to death, get mouse back skin, under room temperature, fixedly spend the night with 4% paraformaldehyde.Flowing water is cleaned, and according to standard paraffin organization embedding method, skin histology is dewatered, transparent, waxdip, and embedding, the processing such as section, (5 μ are m) to obtain skin histology paraffin section.

2. the scar model of tension force induction:

Get 12 close ages of body weight and be the male mouse of kunming of 8 weeks, back hair is rejected clean, be divided at random tension force group, tension force+Quercetin group, 6 every group.Every mouse back skin center line up and down vertical two places' skin holostromes cuts, 1.5 centimetres, every otch, and two otch distances are greater than 2 centimetres, and otch does holostrome interrupted suture.After 4 days, take out stitches, use suture self-retaining retractor, below otch does not apply tension force, and top otch applies tension force.Tension force group up otch is smeared DMSO mono-day three times, and tension force+Quercetin group up otch is smeared Quercetin (concentration 10 μ M) one day three times.Mice was put to death in the 14th day, get mice incision skin, under room temperature, fixedly spend the night with 4% paraformaldehyde.Flowing water is cleaned, and according to standard paraffin organization embedding method, skin histology is dewatered, transparent, waxdip, and embedding, the processing such as section, (5 μ are m) to obtain skin histology paraffin section.

9.2 immunohistochemical stainings:

Section is after fully dewaxing, clean with PBS.Carry out antigen retrieval the endogenous peroxydase of putting out a fire with EDTA repair liquid.Add 5% tire bovine albumin to carry out non-specific protein binding site sealing.Add primary antibodie anti-α-SMA(Abcam), overnight incubation at 4 DEG C.TBS washing specimen 3 times.Add with two of HRP labelling and resist.Under room temperature, hatch 15 minutes.TBS washes specimen 3 times, and (wash 5 minutes, concussion, wash clean is for well) gets rid of unnecessary liquid at every turn.DAB dyeing 10 minutes.Haematoxylin is redyed 1 minute, 1% hydrochloride alcohol differentiation.Redye rear tap water and rinse well, with 100%, 95%, 85%, 75% dehydration of alcohol, dimethylbenzene is transparent twice, each 5 minutes.Resinene mounting, in optical microphotograph Microscopic observation immunohistochemical staining result.

9.3 Masson trichrome stains:

Masson dyeing is one of main method of fibrin dyeing in display organization, is the authoritative and classical technical method of collagen fiber protein staining.This method dyeing theory is relevant with the infiltration of anionic dye bulk of molecule and tissue.Bulk of molecule is embodied by molecular weight, the easy penetrant structure densification of micromolecule amount, the tissue that permeability is low, and macromolecule can only enter tissue short texture, that permeability is high.Pale green molecular weight maximum.Therefore Masson dyeing muscle fiber takes on a red color, and it is blue that collagen fiber albumen is, and is mainly used in distinguishing collagen fiber albumen and parapeptone.With Masson complex staining liquid dyeing 5min, washing, phosphomolybdic acid dyeing 5min, dries, aniline blue dyeing 5min, washing, can break up if desired.Flowing water rinses 10min, breaks up fast with 95% ethanol, and anhydrous alcohol dewaters, and dimethylbenzene is transparent, sealing.

two, experimental result

1, Quercetin suppresses the synthetic I/III Collagen Type VI of human fibroblasts

The Quercetin effect that we give 100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M, 50nM concentration to fibroblast is after 3 days, be extracted into fibrocellular mRNA and carry out fluorescent quantitation PCR in real time detection discovery, at collagen mrna expression, Quercetin can significantly be suppressed to the synthetic of fibrocyte I/III Collagen Type VI, and is dose dependent.When activity reaches after 25 μ M, type i collagen mRNA level is reduced to 0.4 times of left and right of matched group, and its inhibitory action to III Collagen Type VI is stronger, and expression is less than 0.2 times of matched group.Quercetin is respectively 1 μ M and 0.5 μ M(Fig. 1 C to the minimum effective drug concentration of I type and III Collagen Type VI).On the other hand, by Western Blot, the detection of collagen protein level is found, when giving 100 μ M, 50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M, 0.5 μ M, 0.1 μ M, 50nM Quercetin acted on fibroblast after 5 days, compared with processing with matched group DMSO, I/III collagen type level all significantly reduces (Fig. 1 E).

2, Quercetin has no significant effect fibroblastic proliferation activity and apoptosis

The Quercetin stimulation that we give variable concentrations (50 μ M, 25 μ M, 10 μ M, 5 μ M, 1 μ M) to fibroblast detected cytoactive with mtt assay after 48 hours.Found that, under the mass action of 25 μ M, 10 μ M, 5 μ M and 1 μ M, cytoactive uninfluenced (Fig. 2 C), after processing with matched group DMSO, cytoactive approaches.Further apoptotic testing result is found, DMSO compares with matched group, gives 50 μ M, 10 μ M Quercetin apoptotic cell ratio after treatment does not increase (Fig. 2 B).Therefore, Quercetin has no significant effect fibroblastic propagation and apoptosis.

3, Quercetin does not affect the expression of other types collagen and matrix metalloproteinase and TIMP

The impact of other types collagen protein being expressed in order to inquire into Quercetin, and whether affect the expression of matrix metalloproteinase (MMP) and mortifier (TIMP) thereof, we are detected and are found that the Quercetin of variable concentrations does not affect the mrna expression level of II type and X-type collagen by fluorescent quantitation PCR in real time, and do not affect the expression (Fig. 2 A) of MMP2/9 and TIMP1.Illustrate that Quercetin is not that degraded by affecting collagen plays a role.

4, Quercetin suppresses the mouse skin fibrosis of bleomycin induction

For the effect that confirms that quercetin molecule suppresses collagen, we utilize bleomycin subcutaneous injection to manufacture mice corium fabric model, and Quercetin is acted on to mouse skin, survey the situation of change (Fig. 3 A) at intradermal collagen content in health check-up.Giving after Quercetin effect, there is not ulceration, any untoward reaction such as rubescent in mouse skin.Thereby HE dyeing can reflect dermis thickness observe dermal collagen deposition number, Masson Coloration experiment can be dyed whole collagen protein blueness, the blue depth also reflected collagen content number.Result shows, the fibrosis of skin model of contrast bleomycin induction, when giving after quercetin molecule effect, by mouse skin tissue slice HE and Masson dyeing, mice dermis thickness and painted all significantly lower than bleomycin group, similar to matched group (DMSO).Dermis thickness statistics also demonstrates statistical significant difference.Can show by above experiment, Quercetin acts on after mouse skin, can suppress mice dermal collagen deposition.

5, Quercetin suppresses the mice cicatrization of tension force induction

In addition, we have set up the mice scar model of tension force induction, thereby by tissue retractor, skin incision are applied tension force and caused the formation of skin hyperplasia cicatrix.Compared with applying separately tension force group, smear Quercetin every day for three times and can significantly reduce sight substantially and the cross section size of cicatrix.Statistics matched group, tension force group and tension force+Quercetin group mice cicatrix are seen and cross section size substantially, ask its meansigma methods, are respectively large bulk area: 3.79mm ²; 38.84mm ²; 12.59mm ², cross-sectional area: 0.07mm ²; 1.56mm ²; 0.17mm ², large bulk area and cross-sectional area, smearing Quercetin and applying tension force separately all has significant difference (P<0.05) (Fig. 3 B).Illustrate that Quercetin has significantly suppressed the mice hypertrophic cicatrix formation of tension force induction, this is synthetic relevant with its inhibition collagen.

6, Quercetin is suppressed to fibrocellular activation

Fibroblast in corium under normal circumstances major part remains static, under wound or inflammation etc. stimulate, fibroblast activation, expressing smooth muscle actin (α-SMA) becomes myofibroblast, and it can cause cicatrix to shrink and secrete a large amount of collagen protein.Therefore, myofibroblast is the important target cell of causing skin collagen deposition and cicatrix to shrink.We find, are giving after Quercetin effect, and the mRNA of fibroblast α-SMA and expressing quantity be significantly decline (Fig. 4 A, Fig. 4 C) all.Immunofluorescence dyeing shows, do not have in irritant situation and α-SMA expression that TGF-β 1 induces under, give after Quercetin effect, the expression of α-SMA significantly declines.Quercetin does not affect cytoskeleton (F-actin) (Fig. 4 B) simultaneously.In the mouse skin fibrosis model of bleomycin induction and the mice scar model of tension force induction, immunohistochemical staining shows that α-SMA expresses all significantly decline (Fig. 4 D, Fig. 4 E).Therefore Quercetin can significantly be suppressed to fibrocyte suppresses fibrosis of skin or generation from cicatrix to myofibroblast differentiation.

7, Quercetin suppresses the conduction of TGF-β 1/Smad2/3 signal path

Next we have probed into the synthetic biological mechanism of Quercetin inhibition collagen.Find by the detection to smad signal protein family, quercetin molecule acted on fibroblast after 48 hours, the smad albumen of the smad2/3(phosphorylation of phosphorylation shows that this signal protein is activated) level obviously raises, and total smad2/3 protein level remains unchanged.And smad6, smad7 signal protein does not all change, and total smad4 protein level also remains unchanged.Illustrate that Quercetin has suppressed smad2/3 signal protein phosphorylation.

Meanwhile, we reflect that by the method for immunofluorescence dyeing quercetin molecule can suppress TGF-β 1/Smad2/3 signal path intuitively.We are by green fluorescence on smad4 protein labeling, nucleus labelling blue fluorescence, found that, give after TGF-β 1 stimulation, blue-fluorescence overlaps with part green fluorescence, show that smad4 signal protein has entered in nucleus, and give TGF-β 1 and Quercetin group Green fluorescence all separates with blue-fluorescence simultaneously, show that smad4 major part is all positioned at cytoplasm.Therefore prove by the experiment of immunofluorescence that intuitively smad4 signal protein suppressed entering in nucleus under quercetin molecule effect plays a role, thereby suppress I/III Collagen Type VI gene expression mRNA, and then suppressed the expression of collagen protein.

three, conclusion

Micromolecule flavone compound Quercetin is can be by suppressing TGF-β 1/Smad2/3 signal path in cell in without overt toxicity situation to cell, effectively suppress the synthetic of collagen in human fibroblasts.Its concrete Biological mechanism of action can be referring to Fig. 6.Quercetin is the little and fat-soluble high molecule of a kind of molecular weight, has good Transdermal absorption ability, and therefore this molecule can be applied to that anti-cicatrix of skin forms and the treatment of anti-fibrosis of skin.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and the supplementary protection scope of the present invention that also should be considered as.

SEQUENCE LISTING

<110> Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch

<120> flavone compound Quercetin is in the application suppressing in cicatrix of skin formation and fibrosis of skin

<130> /

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<170> PatentIn version 3.3

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Claims (9)

1. the purposes of Quercetin, is characterized in that, for the preparation of suppressing, cicatrix of skin forms and/or the reagent of fibrosis of skin.

2. purposes according to claim 1, is characterized in that, described reagent is medicine or cosmetics.

3. the purposes of Quercetin, is characterized in that, for the preparation of reagent, described reagent is used for:

A) suppress skin flbroblast Collagen I/III mrna expression; Or

B) suppressing skin flbroblast α-SMA expresses; Or

C) suppress skin flbroblast to myofibroblast differentiation; Or

D) suppress skin flbroblast smad2/3 signal protein phosphorylation; Or

E) suppress skin flbroblast smad4 signal protein and enter nucleus.

4. purposes according to claim 3, is characterized in that, described skin flbroblast is normal skin fibroblast.

5. purposes according to claim 4, is characterized in that, described skin flbroblast is dermal fibroblast.

6. the purposes of Quercetin, is characterized in that, for the preparation of the reagent that suppresses mice corium fabric model collagen deposition.

7. purposes according to claim 6, is characterized in that, for reducing mice dermis thickness.

8. the purposes of Quercetin, is characterized in that, for the preparation of the synulotic reagent of mice that suppresses tension force induction.

9. purposes according to claim 8, is characterized in that, for reducing mice cicatrix volume.