CN106176675A - The application in treatment hepatic fibrosis of the honokiol nano-particle of targeting sinusoidal endothelial cell - Google Patents

The application in treatment hepatic fibrosis of the honokiol nano-particle of targeting sinusoidal endothelial cell Download PDF

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CN106176675A
CN106176675A CN201610555432.8A CN201610555432A CN106176675A CN 106176675 A CN106176675 A CN 106176675A CN 201610555432 A CN201610555432 A CN 201610555432A CN 106176675 A CN106176675 A CN 106176675A
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endothelial cell
sinusoidal endothelial
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particle
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CN106176675B (en
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姜颖
贺福初
劳远翔
李严严
魏汉东
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Academy of military medicine, PLA Academy of Military Sciences
BEIJING PROTEOME RESEARCH CENTER
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Abstract

The invention discloses the application in treatment hepatic fibrosis of the honokiol nano-particle of targeting sinusoidal endothelial cell.The invention provides the following purposes of the Honokiol nano-particle of targeting sinusoidal endothelial cell: 1) preparation treatment hepatic fibrosis disease product；2) preparation suppression hepatic fibrosis product；3) preparation promotes or maintains or recover sinusoidal endothelial cell to be in differentiation state product；4) preparation improves sinusoidal endothelial cell and promotes liver repair product；5) HGF protein expression and/or Wnt2 protein expression product during preparation improves sinusoidal endothelial cell；6) preparation suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates product.Utilize and packed Honokiol suppression hepatic fibrosis by the PEG nano-particle of cRGD labelling.

Description

The honokiol nano-particle of targeting sinusoidal endothelial cell is in treatment hepatic fibrosis Application
Technical field
The present invention relates to biological technical field, particularly relate to the honokiol nano-particle of a kind of targeting sinusoidal endothelial cell Application in treatment hepatic fibrosis.
Background technology
Honokiol is a class Magnoliaceae deciduous tree Cortex Magnoliae Officinalis extract.There is obvious, lasting central muscle pine Relax, Central nervous depressant, antiinflammatory, antibacterial, resisting pathogenic microbes, antiulcer, antioxidation, defying age, antitumor, reduces gallbladder The pharmacological actions such as sterin.It can suppress Akt phosphorylation, and also can promote ERK1/2 phosphorylation, in research report before this, Honokiol can by the protective effect of vascular endothelial cell being suppressed the formation of arterial thrombus, further investigations have shown that and Magnolol can also be by promoting the apoptosis generating suppression vascular endothelial cell of prostacyclin, it addition, honokiol is people The expression of controllable iNOS/eNOS in huve cell, and then maintain vascular endothelial cell by the synthesis of regulation NO Stable state.
In terms of chronic hepatic injury research, the research of honokiol is few, but its dependency with chronic hepatopathy also has report Road.Under condition of culture, honokiol can produce impact to activated hepatic stellate cells HSC in vitro, and it passes through mitochondrial cytochrome The release of C and the activation of Caspase 3 promote the HSC apoptosis of activation, and parenchyma does not cause great damage.Additionally Honokiol also adjustable liver damage repair process, it can suppress the disorganization that chronic injury is caused significantly, show Go out the response to treatment stronger to liver tissue injury.
The PEG nanoparticle of rgd peptide labelling is the drug delivery system of endothelial-cell specific, can firmly be incorporated into by targeting The inflammation part of Surface of Vascular Endothelial Cells is it is considered to be target vascular therapy endotheliocyte treats preferable drug-supplying system.PEG is coated The research of honokiol also early has been reported that.
Summary of the invention
It is an object of the present invention to provide the new application of the Honokiol nano-particle of targeting sinusoidal endothelial cell.
The Honokiol nano-particle of the targeting sinusoidal endothelial cell that the present invention provides is following 1)-10) at least one Application:
1) preparation treatment hepatic fibrosis disease product；
2) preparation suppression hepatic fibrosis product；
3) preparation promotes or maintains or recover sinusoidal endothelial cell to be in differentiation state product；
4) preparation improves sinusoidal endothelial cell and promotes liver repair product；
5) HGF protein expression and/or Wnt2 protein expression product during preparation improves sinusoidal endothelial cell；
6) preparation suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates product；
7) preparation reduces serum alt content and/or AST content product；
8) Akt phosphorylation Horizontal production in preparation suppression sinusoidal endothelial cell；
9) Erk1/2 phosphorylation level product during preparation promotes sinusoidal endothelial cell；
10) eNOS expression and Nox2 expression ratio product during preparation improves sinusoidal endothelial cell.
The application in preparation treatment hepatic fibrosis disease product of the Honokiol nano-particle of targeting sinusoidal endothelial cell Also it is the scope of protection of the invention.
The application in preparation suppression hepatic fibrosis product of the Honokiol nano-particle of targeting sinusoidal endothelial cell is also The scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell promotes in preparation or maintains or recover sinusoidal endothelial cell Being in the application in differentiation state product is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell improves sinusoidal endothelial cell in preparation and promotes liver reparation Application in effect product is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell is HGF protein expression in preparation improves sinusoidal endothelial cell And/or the application in Wnt2 protein expression product is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell is formed at preparation suppression hepatic fibrosis tuberosity or promotes liver The application that fibrosis tuberosity eliminates in product is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell reduces serum alt content and/or AST content in preparation Application in product is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell is Akt phosphorylation water in preparation suppression sinusoidal endothelial cell Application in flat products is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell is Erk1/2 phosphoric acid in preparation promotes sinusoidal endothelial cell The application changed in Horizontal production is also the scope of protection of the invention.
The Honokiol nano-particle of targeting sinusoidal endothelial cell is eNOS expression in preparation improves sinusoidal endothelial cell Also it is the scope of protection of the invention with the application in Nox2 expression ratio product.
Promote or maintain or recover sinusoidal endothelial cell to be in the material of differentiation state following 1)-10) at least one Application be also the model that the present invention protects:
1) preparation treatment hepatic fibrosis disease product；
2) preparation suppression hepatic fibrosis product；
4) preparation improves sinusoidal endothelial cell and promotes liver repair product；
5) HGF protein expression and/or Wnt2 protein expression product during preparation improves sinusoidal endothelial cell；
6) preparation suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates product；
7) preparation reduces serum alt content and/or AST content product；
8) Akt phosphorylation Horizontal production in preparation suppression sinusoidal endothelial cell；
9) Erk1/2 phosphorylation level product during preparation promotes sinusoidal endothelial cell；
10) eNOS expression and Nox2 expression ratio product during preparation improves sinusoidal endothelial cell.
It is targeting sinusoidal endothelial cell that above-mentioned promotion or maintenance or recovery sinusoidal endothelial cell are in the material of differentiation state Honokiol nano-particle.
In above-mentioned application, described product is medicine.
Another object of the present invention is to provide one and has following 1)-10) in the product of at least one function.
The product that the present invention provides, including the Honokiol nano-particle of targeting sinusoidal endothelial cell；
1) treatment hepatic fibrosis disease；
2) suppression hepatic fibrosis；
3) promote or maintain or recover sinusoidal endothelial cell and be in differentiation state；
4) improve sinusoidal endothelial cell and promote liver repair；
5) HGF protein expression and Wnt2 protein expression in sinusoidal endothelial cell is improved；
6) suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates；
7) serum alt content and/or AST content are reduced；
8) Akt phosphorylation level in suppression sinusoidal endothelial cell；
9) Erk1/2 phosphorylation level in sinusoidal endothelial cell is promoted；
10) eNOS expression and Nox2 expression ratio in sinusoidal endothelial cell are improved.
In the said goods, the Honokiol nano-particle of described targeting sinusoidal endothelial cell is for use targeting sinusoidal endothelial cell Albumen or fusion protein containing this albumen or be coated, containing the complex of this albumen, the granule that honokiol obtains.
In the said goods, described product is medicine.
In above-mentioned, the Honokiol nano-particle of described targeting sinusoidal endothelial cell is with the egg of targeting sinusoidal endothelial cell Fusion protein in vain or containing this albumen or the complex containing this albumen are coated the granule that honokiol obtains, the present invention's In embodiment, the polypeptide of targeting sinusoidal endothelial cell is cRGD, and the Honokiol nano-particle of targeting sinusoidal endothelial cell is for using CRGD-PEG-PLGA is coated the honokiol nano-particle that honokiol (Honokiol) obtains, and makes the most as follows Standby:
1) by PEG-PLGA and cRGD in the medium mixed in molar ratio of DMSO solution, PEG-cRGD is obtained；
2) it is that 10:1 mixes in DMSO solution by PEG-cRGD and PLGA according to mol ratio, reaction, obtain cRGD-PEG- PLGA；
3) cRGD-PEG-PLGA solution (50mg/ml) is dripped in the Honokiol solution (10mg/ml) stirred according to body Long-pending ratio mixes for 3:4, and reaction, obtaining cRGD-PEG-PLGA is coated the nano-particle of honokiol, is Honokiol nanometer Grain.
The 3rd purpose of the present invention is to provide a kind of method suppressing hepatic fibrosis.
The method that the present invention provides, for using LSEC as target cell, maintains LSEC to be in differentiation state, thus reaches suppression The purpose of hepatic fibrosis.
The experiment proves that, utilize and packed Honokiol by the PEG nano-particle of cRGD labelling, make endotheliocyte Special drug delivery system, enters in Mice Body via lumbar injection, can effectively maintain phenotypic differentiation and the rush of LSEC Regeneration function, thus in the most effective process suppressing hepatic fibrosis of chronic hepatic injury, induction LSEC regulates and controls hepatic tissue simultaneously Injury repairing.And reversing the phase in hepatic fibrosis, Honokiol can accelerate the elimination efficiency of intrahepatic deposition fiber, and recovers rapidly The phenotypic differentiation of LSEC and rush regeneration function so that it is can react rapidly to exercise the directive function that hepatic injury is repaired.Therefore, with LSEC, as target cell, suppresses hepatic fibrosis to develop, for antifibrosis therapy by regulating and controlling its phenotypic differentiation Research has certain directive significance.
Accompanying drawing explanation
Fig. 1 is p-Erk1/2 in each group of LSEC, the expression of p-Akt, Erk1/2, Akt, eNOS, Nox2.
Fig. 2 be (A) scanning electron microscope image display fibrosis 4 weeks with in 6 weeks chronic hepatic injury groups and Honokiol suppression group The change of LSEC surface fenestration；(B) scanning electron microscope image display fibrosis reverse 2 weeks phases and 4 weeks chronic hepatic injury groups and Honokiol promotees the change of LSEC surface fenestration in reverse group.
Fig. 3 is that (A) Western blot result display fibrosis suppressed with Honokiol for 4 weeks with 6 weeks chronic hepatic injury groups The change that in group LSEC, HGF and Wnt2 expresses；(B) Western blot result display chronic hepatic injury group is controlled with Honokiol The change that in treatment group LSEC, HGF and Wnt2 expresses.
Fig. 4 pressed down with Honokiol for (A) Masson trichrome stain image display fibrosis for 4 weeks with 6 weeks chronic hepatic injury groups The change of tissue fibers deposition in processed group；(B) Masson trichrome stain image display fibrosis reverses 2 weeks and damages with 4 weeks Chronic Livers Hinder reverse group and promote the change of tissue fibers deposition in reverse group with Honokiol.
Fig. 5 is that ALT and AST serum-concentration monitoring result display Honokiol can significantly reduce this two class transaminase at liver fibre The content of (B) in dimensionization process (A) and reversal procedures.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The mice used in following embodiment is C57BL/6J mice, limited purchased from Beijing dimension tonneau China laboratory animal technology Company；
The honokiol nano-particle (hereinafter referred to as Honokiol nano-particle) of targeting LSEC in following embodiment: for CRGD-PEG-PLGA is coated the honokiol nano-particle that honokiol (Honokiol) obtains；
Honokiol nanometer grain preparation method is with reference to such as Publication about Document: Qing Xu, Yuexian Liu, Shishuai Su, Wei Li,Chunying Chen*,Yan Wu*.Anti-tumor activity of paclitaxel through dual- targeting carrier of cyclic RGD and transferrin conjugated hyperbranched Copolymer nanoparticles, Biomaterials, 2012,33,1627-1639, specifically comprise the following steps that
Precision weighs PEG-PLGA (14.7 μm ol, bank paddy nanometer) 50mg to the round-bottomed flask containing 500 μ L DMSO solution In, under room temperature after stirring and dissolving, add equimolar polypeptide cRGD (Suzhou shine by force biology) stirring 2h, use thin layer chromatography (TLC) tracking reaction is analyzed, until cRGD is wholly absent.Boil off solvent under vacuum condition, then again dissolve with distilled water, then Removing small-molecule substance with deionized water dialysis, bag filter (retains relative molecular weight 3500), concentrates stand-by, after being concentrated PEG-cRGD, wherein, the addition mol ratio of PEG-PLGA and cRGD is 1:1.
Take the PEG-cRGD after concentration (14.7 μm ol) to be dissolved completely in DMSO solution 500 μ L, be subsequently adding 1.47 μ The PLGA solution (solvent is DMSO 500 μ L) of mol, stirs 18h, TLC tracking and is reacted to terminate under room temperature.Steam under vacuum condition Remove solvent, then again dissolve with distilled water, then dialyse with deionized water, bag filter (retaining relative molecular weight is 14000), warp Lyophilization, obtains cRGD-PEG-PLGA solid product, and wherein, the addition mol ratio of PEG-cRGD and PLGA is 10:1.
CRGD-PEG-PLGA and Honokiol is made into respective concentration (solvent is DMSO) the most respectively, by cRGD- PEG-PLGA solution (50mg/ml) drips in the Honokiol solution (10mg/ml) of stirring, and incubated at room 30min makes body The long-pending complex than 3:4 ratio, after complex solution aseptic filtration 4 DEG C standby, granular size～200nm, obtain Honokiol Nano-particle.
The application in treatment hepatic fibrosis of embodiment 1, Honokiol nano-particle
One, Honokiol nano-particle the chronic hepatic injury stage to Hepar Mus sinusoidal endothelial cells in the work of correlative protein expression With
Chronic hepatic injury can cause hepatic fibrosis, and this part Experiment is to prove that Honokiol is by raising p-Erk1/ The ratio regulation and control normal physiological function of sinusoidal endothelial cell of 2/p-Akt.
1, Honokiol nano-particle acts on the hepatic fibrosis Mouse Method that chronic hepatic injury causes
Experiment packet:
1) negative control group: normal drinking water, normal mouse chow are fed, free choice feeding, change weekly 3 drinking waters with And feedstuff, only inject aseptic olive oil and normal saline, frequency injection corresponding experimental group corresponding with dosage；
2) chronic hepatic injury pathologic group (CCl4Cause chronic liver damage model): normal drinking water, normal mouse chow are fed Support, free choice feeding, change weekly 3 drinking waters and feedstuff, 2 CCl of lumbar injection weekly4With mixed with olive oil thing (volume Ratio 1:7, dosage 1mg/kg) and normal saline, injection 6 weeks continuously.Put to death mice 2 weeks, 4 weeks and 6 weeks respectively to detect.
3) reverse group: normal drinking water, normal mouse chow are fed, free choice feeding, changes weekly 3 drinking waters and raises Material, 6 weeks CCl of continuous lumbar injection4With mixed with olive oil thing (volume ratio 1:7) and normal saline, stop injection CCl4, normally drink Food, allows it recover voluntarily, and intraperitoneal injection of saline reverses simultaneously, within 2 weeks and 4 weeks, puts to death mice in reverse respectively and detects.
4) Honokiol nano-particle suppression group: normal drinking water, normal mouse chow are fed, free choice feeding, the most more Change 3 drinking waters and feedstuff, 6 weeks CCl of continuous lumbar injection4(volume ratio 1:7, dosage 1mg/kg need with mixed with olive oil thing Normal saline) and normal saline, Honokiol nano-particle is entered Mice Body according to 0.2mg/kg ratio together lumbar injection In, injection 6 weeks continuously.Put to death mice 2 weeks, 4 weeks and 6 weeks respectively to detect.
5) Honokiol nano-particle promotees reverse group: normal drinking water, normal mouse chow are fed, free choice feeding, weekly Change 3 drinking waters and feedstuff, 6 weeks CCl of continuous lumbar injection4With mixed with olive oil thing (volume ratio 1:7, dosage 1mg/kg) and Normal saline, stops injection CCl4, normal diet, allow it recover voluntarily, simultaneously by Honokiol nano-particle according to 0.2mg/ Kg ratio together lumbar injection enters in Mice Body, continuously injection 4 weeks.Within 4 weeks, put to death mice in reverse 2 weeks and reverse respectively to carry out Detection.
Above-mentioned feeding environment condition: temperature range 20-22 DEG C, humidity range 50-55%, each 12 hours of light and shade.Experiment is dynamic Thing ad lib and drinking-water, 4 cages are raised.
2, detection
1) Western blotting detection
Extract the Mouse Liver sinusoidal endothelial cells whole protein that in above-mentioned 1, in each group, different time points is put to death, pass through Western Blotting detect in each group Erk1/2 in the Mouse Liver sinusoidal endothelial cells of different time points, Akt, p-Erk1/2, p-Akt, ENOS and Nox2 protein expression level, concrete detection method is as follows:
First albumen is carried out Native-PAGE (SDS-PAGE), after electricity forwards on nitrocellulose filter Put into and confining liquid (5% defatted milk powder in TBST) is closed 1h, at the Erk1/2 antibody of 1:200 dilution, Akt antibody, p-Erk1/ 2 antibody, p-Akt antibody and eNOS antibody (being all from CST), Nox2 antibody (Proteintech company), one anti-in 4 DEG C incubate Educating overnight, TBST washes film 5 times, adds anti-(Kang Wei company) the incubated at room temperature 1h of 1:5000 dilution Mus two, and film 5 washed by TBST buffer Secondary, chemical luminous substrate ECL test kit (Thermo, 32106) develops the color.
Result puts to death mice as it is shown in figure 1,2w, 4w, 6w are chronic hepatic injury pathologic group 2 weeks, 4 weeks and 6 weeks；2w+H、4w+ H, 6w+H are Honokiol nano-particle suppression group 2 weeks, 4 weeks and 6 weeks and put to death mice；R2w, R4w are reverse group 2 weeks, 4 weeks and put to death Mice；R2w+H, R4w+H are that Honokiol nano-particle rush reverse group puts to death mice in 2 weeks, 4 weeks；It can be seen that Honokiol receives Rice grain has more significantly inhibitory action to the phosphorylation of Akt in chronic hepatic injury 4-6 week and 4 weeks reverse phases LSEC, Meanwhile can promote the phosphorylation of Erk1/2, and be obviously improved the ratio of downstream eNOS/Nox2.Honokiol sends out in fibrosis Be able to maintain that the ratios of p-Erk1/2/p-Akt in LSEC when life, and be obviously improved downstream eNOS/Nox2 ratio and then Maintain the phenotypic differentiation of LSEC and normal physiological function.
Two, Honokiol nano-particle is at the phenotypic differentiation of chronic hepatic injury stage regulation and control LSEC
Honokiol is able to maintain that the ratios of p-Erk1/2/p-Akt in LSEC when fibrosis occurs, and then keeps The phenotypic differentiation of LSEC and normal physiological function.
Known Honokiol can suppress the phosphorylation of Akt albumen, and promotes the phosphorylation of Erk1/2, and FDA ratifies Honokiol is clinically used as the cardiovascular disease such as blood vessel protective agent, treatment myocardial hypertrophy.Honokiol nanometer shown in Fig. 1 Granule has more significantly inhibitory action to the phosphorylation of Akt in chronic hepatic injury 4-6 week and 4 weeks reverse phases LSEC, with This can promote the phosphorylation of Erk1/2 simultaneously, and is obviously improved the ratio of downstream eNOS/Nox2.Therefore scanning electricity is used further The method of mirror, confirms whether Honokiol nano-particle has regulation and control in the chronic hepatic injury stage to the phenotypic differentiation of LSEC and make With.
1, whether Honokiol nano-particle has regulating and controlling effect in the chronic hepatic injury stage to the phenotypic differentiation of LSEC
Concrete grammar is as follows:
1) draw materials: piece of tissue is fixed on filter paper or ivory board, fully to expose tissue surface to be seen, with operation Cutter is cut into area and reaches 8mm × 8mm, and thickness reaches the fritter of 5mm；
2) cleaning of sample: clean with isotonic PBS；
3) fixing: 3% glutaraldehyde is fixed.4 DEG C overnight；
4) secondary daily PBS cell, successively with 50%, 70%, 80%, 90% and 100% ethanol dehydration, wherein 100% ethanol dehydration 3 times, each 10min；
5) ethanol is used: the mixed solution dehydration of the tert-butyl alcohol=1:1；
6) pure tert-butyl alcohol dehydration is used 2 times, each 10min；
7) the fresh tert-butyl alcohol is added not have tissue sample to be advisable；
8) lyophilization；
9) sample metal spraying；
10) scanning electron microscope observation.
Honokiol nano-particle is to each group of different time points tissue sinus hepaticus surface endothelial cell differentiation mark-fenestra knot The maintenance situation of structure puts to death mice as in figure 2 it is shown, 2w, 4w, 6w are chronic hepatic injury pathologic group 2 weeks, 4 weeks and 6 weeks；2w+H、4w+ H, 6w+H are Honokiol nano-particle suppression group 2 weeks, 4 weeks and 6 weeks and put to death mice；R2w, R4w are reverse group 2 weeks, 4 weeks and put to death Mice；R2w+H, R4w+H are that Honokiol nano-particle rush reverse group puts to death mice in 2 weeks, 4 weeks；It can be seen that Honokiol receives Rice grain can well maintain the phenotypic differentiation of LSEC (surface fenestration) for 4 weeks and 6 weeks to chronic hepatic injury, and in the phase of reverse In 2 weeks and 4 weeks, Honokiol nano-particle can substantially speed up the recovery of LSEC phenotypic differentiation.
2, Honokiol nano-particle improves LSEC in the chronic hepatic injury stage and promotes liver repair ability
It addition, LSEC has preferable repair to hepatic injury, HGF and Wnt2 of its secretion promotes damage location liver The propagation of parenchyma.Therefore the method using WB detection, confirms that Honokiol nano-particle is in the chronic hepatic injury stage pair Whether the rush repair ability of LSEC has regulating and controlling effect.Concrete grammar is as follows:
Extract the Mouse Liver sinusoidal endothelial cells whole protein that in above-mentioned 1, in each group, different time points is put to death, pass through Western In each group of blotting detection, HGF and Wnt2 of different time points (promotes hepatic parenchymal cells reparation, promotees to repair phenotype mark for LSEC Will thing) expression:
First sample is carried out Native-PAGE (SDS-PAGE), after electricity forwards on nitrocellulose filter Put into and confining liquid (5% defatted milk powder in TBST) is closed 1h, at HGF antibody and the Wnt2 antibody of 1:200 dilution 4 DEG C of overnight incubation during (Proteintech company) is anti-, TBST washes film 5 times, adds 1:5000 dilution Mus two anti-(Kang Wei company) Incubated at room temperature 1h, TBST buffer washes film 5 times, and chemical luminous substrate ECL test kit (Thermo, 32106) develops the color.
Honokiol nano-particle each group of different time points LSEC is promoted repair ability maintenance situation as it is shown on figure 3,2w, 4w, 6w are chronic hepatic injury pathologic group 2 weeks, 4 weeks and 6 weeks and put to death mice；2w+H, 4w+H, 6w+H are Honokiol nano-particle Suppression group puts to death mice in 2 weeks, 4 weeks and 6 weeks；R2w, R4w are reverse group 2 weeks, 4 weeks and put to death mice；R2w+H, R4w+H are Honokiol nano-particle promotees reverse group 2 weeks, 4 weeks and puts to death mice；It can be seen that Honokiol promotes that HGF and Wnt2 expresses, table Bright, Honokiol can well maintain the plerosis function of LSEC for 4 weeks and 6 weeks to chronic hepatic injury, and is reversing 2 weeks phases and 4 Zhou Zhong, Honokiol nano-particle can substantially speed up the recovery of LSEC phenotypic differentiation.
Three, Honokiol nano-particle application in treatment chronic hepatic injury stage hepatic fibrosis disease
Honokiol nano-particle shown in known Fig. 1 is to chronic hepatic injury 4-6 week and reverses Akt in 4 weeks phases LSEC Phosphorylation has more significantly inhibitory action, meanwhile can promote the phosphorylation of Erk1/2, and be obviously improved downstream eNOS/ The ratio of Nox2.Honokiol nano-particle shown in Fig. 2 can well maintain the differentiation of LSEC for 4 weeks and 6 weeks to chronic hepatic injury Phenotype, and in reversing 2 weeks phases and 4 weeks, Honokiol nano-particle can substantially speed up the recovery of LSEC phenotypic differentiation.Fig. 3 institute Show that Honokiol nano-particle can well maintain the plerosis function of LSEC for 4 weeks and 6 weeks to chronic hepatic injury, and in the phase of reverse In 2 weeks and 4 weeks, Honokiol nano-particle can substantially speed up the recovery of LSEC phenotypic differentiation.Therefore further hepatic tissue section Masson trichrome stain and Serum ALT, (hepatic injury Testing index assists Masson staining evaluation hepatic fibrosis disease to AST content Seriousness) method that detects, confirm that Honokiol nano-particle targeting intervenes LSEC in the hepatic fibrosis of chronic hepatic injury stage Whether disease has therapeutical effect, and concrete grammar is as follows
1, tissue slice Masson trichrome stain
A, the configuration of reagent
1) haematoxylin dye liquor
2) Masson complex staining liquid: Ponceaux (ponceau 2R) 0.7g, acid fuchsin (acid fuchsin) 0.3g, Distilled water 99ml, glacial acetic acid (glacial acetic acid) 1ml.
3) viride nitens dyeing liquor: viride nitens (light green) 1g, glacial acetic acid (glacial acetic acid) 1ml, distillation Water 99ml.
4) 1% phosphomolybdic acid aqueous solution: phosphomolybdic acid (phosphomolybdic acid) 1g, distilled water adds to 100ml.
5) 2% aniline blue liquid: aniline blue (aniline blue) 2g, glacial acetic acid (glacial acetic acid) 2ml, Distilled water adds to 100ml.
B, staining procedure:
The murine liver tissue that different time points in above-mentioned 1 each group is put to death is carried out 10% formalin fix, paraffin section, Conventional dewaxing is to water.
1) haematoxylin dye liquor 5～10min.
2) flowing water is slightly washed, 1% hydrochloric acid differentiation.
3) flowing water rinses several minutes.
4) Masson complex staining liquid 5～10min.
5) distilled water slightly rinses.
6) 1% phosphotungstic acid liquid processes about 5min.
7) do not wash with water, directly redye 5min with viride nitens dyeing liquor (or aniline blue liquid).
8) 1% glacial acetic acid water processes 1min.
9) 95% dehydration of alcohol is repeatedly.
10) dehydrated alcohol dehydration, dimethylbenzene is transparent, neutral gum sealing.
The suppression (A) to hepatic fibrosis process of the Honokiol nano-particle and the acceleration to hepatic fibrosis reverse (B) are made Masson dyeing checking as shown in Figure 4,2w, 4w, 6w be chronic hepatic injury pathologic group 2 weeks, 4 weeks and 6 weeks execution mice；2w + H, 4w+H, 6w+H are Honokiol nano-particle suppression group 2 weeks, 4 weeks and 6 weeks and put to death mice；R2w, R4w be reverse group 2 weeks, 4 Week puts to death mice；R2w+H, R4w+H are that Honokiol nano-particle rush reverse group puts to death mice in 2 weeks, 4 weeks；It can be seen that Honokiol nano-particle targeting LSEC suppression chronic hepatic injury 4 weeks and the formation of 6 weeks hepatic fibrosis tuberositys, and reversing the phase 2 Week, Honokiol nano-particle can substantially speed up the elimination of hepatic fibrosis tuberosity with in 4 weeks.
2, Serum ALT, AST content detection
The mice that in above-mentioned 1, in each group, different time points is put to death plucks eyeball blood sampling, and blood room temperature stands 1-2 hour, 3000r/min be centrifuged, collect supernatant liquid, transfer to the Chinese People's Liberation Army 307 hospital laboratory carry out glutamate pyruvate transaminase and Glutamic oxaloacetic transaminase, GOT expression detects, and concrete grammar is as follows:
1) preparation of reagents
(1) 0.1mol/L phosphate buffer (pH7.4)
Weigh NaH2PO4.2H2O 2.96495g, Na2HPO4.12H2O 29.0142g, is dissolved in distilled water, and constant volume arrives 1000mL。
(2) 20 μm ol/L Sodium Pyruvate standard solution
Weigh 22mg Sodium Pyruvate, be dissolved in 0.1mol/L phosphate buffer (pH7.4) 100mL.Now with the current.
(3) ALT matrix liquid
Weighing α-ketoglutaric acid 29.2mg, DL-Alanine 1.78mg (ALANINE 0.85mg) is dissolved in 30mL0.1mol/ In L phosphate buffer (pH7.4), dissolve post-equalization pH to 7.4, then be settled to 100mL with the phosphate buffer of pH7.4, put ice Case saves backup (can preserve one week).Chloroform few drops anticorrosion can be added.
(4) 0.02%2,4 dinitro benzene hydrazine solutions
Weighing 20mg2,4 dinitrophenylhydrazines are first dissolved in 10mL pure hydrochloric acid, and heating by electric cooker hydrotropy treats 2,4 dinitros After base phenylhydrazine all dissolves, with distilled water diluting to 100mL, it is contained in after filtration in brown, puts in refrigerator and save backup.
(5) 1mol/L NaOH solution:
Weigh after 4g NaOH is dissolved in distilled water, constant volume to 100mL.
(6) 0.4mol/L NaOH solution
Weigh after 16g NaOH is dissolved in distilled water, constant volume to 1000mL.
2) experimental technique
(1) process of sample is carried out by 3.5 methods.
(2) making of ALT standard curve
1. all will be placed in water bath outside mensuration agents useful for same dehydrogenation sodium hydroxide solution, pre-temperature 37 DEG C is to then using. Take 6 test tubes, prepare by table 1
The preparation of table 1 ALT standard curve
2. measure light absorption value OD with 0 for comparison spectrophotometric colo after cooling520nm。
3. with acetone acid content as vertical coordinate, light absorption value OD520nmStandard curve is drawn for abscissa
The suppression (A) to hepatic fibrosis process of the Honokiol nano-particle and the acceleration to hepatic fibrosis reverse (B) are made Serum ALT, the checking of AST content detection is as shown in Figure 5, it can be seen that Honokiol nano-particle targeting LSEC reduce Chronic hepatic injury 4 weeks and 6 weeks serum alt and the content of AST, and in reversing 2 weeks phases and 4 weeks, Honokiol nano-particle The reduction of the content of serum alt and AST can be substantially speeded up.

Claims (10)

1. the Honokiol nano-particle of targeting sinusoidal endothelial cell is following 1)-10) at least one application:
1) preparation treatment hepatic fibrosis disease product；
2) preparation suppression hepatic fibrosis product；
3) preparation promotes or maintains or recover sinusoidal endothelial cell to be in differentiation state product；
4) preparation improves sinusoidal endothelial cell and promotes liver repair product；
5) HGF protein expression and/or Wnt2 protein expression product during preparation improves sinusoidal endothelial cell；
6) preparation suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates product；
7) preparation reduces serum alt content and/or AST content product；
8) Akt phosphorylation Horizontal production in preparation suppression sinusoidal endothelial cell；
9) Erk1/2 phosphorylation level product during preparation promotes sinusoidal endothelial cell；
10) eNOS expression and Nox2 expression ratio product during preparation improves sinusoidal endothelial cell.
2. the Honokiol nano-particle of targeting sinusoidal endothelial cell application in preparation treatment hepatic fibrosis disease product；
Or, the application in preparation suppression hepatic fibrosis product of the Honokiol nano-particle of targeting sinusoidal endothelial cell；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell promotes in preparation or maintains or recover at sinusoidal endothelial cell Application in differentiation state product；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell improves sinusoidal endothelial cell in preparation and promotes that liver reparation is made With the application in product；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell is HGF protein expression in preparation improves sinusoidal endothelial cell And/or the application in Wnt2 protein expression product；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell is formed at preparation suppression hepatic fibrosis tuberosity or promotes that liver is fine Dimensionization tuberosity eliminates the application in product；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell reduces serum alt content and/or AST content in preparation Application in product；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell is Akt phosphorylation water in preparation suppression sinusoidal endothelial cell Application in flat products；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell is Erk1/2 phosphorylation in preparation promotes sinusoidal endothelial cell Application in Horizontal production；
Or, the Honokiol nano-particle of targeting sinusoidal endothelial cell preparation improve in sinusoidal endothelial cell eNOS expression with Application in Nox2 expression ratio product.
Application the most according to claim 1 and 2, it is characterised in that:
The Honokiol nano-particle of described targeting sinusoidal endothelial cell is to use the albumen of targeting sinusoidal endothelial cell or containing being somebody's turn to do The fusion protein of albumen or the complex containing this albumen are coated the granule that honokiol obtains.
4. promote or maintain or recover sinusoidal endothelial cell to be in the material of differentiation state following 1)-10) at least one Application:
1) preparation treatment hepatic fibrosis disease product；
2) preparation suppression hepatic fibrosis product；
4) preparation improves sinusoidal endothelial cell and promotes liver repair product；
5) HGF protein expression and/or Wnt2 protein expression product during preparation improves sinusoidal endothelial cell；
6) preparation suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates product；
7) preparation reduces serum alt content and/or AST content product；
8) Akt phosphorylation Horizontal production in preparation suppression sinusoidal endothelial cell；
9) Erk1/2 phosphorylation level product during preparation promotes sinusoidal endothelial cell；
10) eNOS expression and Nox2 expression ratio product during preparation improves sinusoidal endothelial cell.
Application the most according to claim 4, it is characterised in that: described promotion or maintenance or recovery sinusoidal endothelial cell are in The material of differentiation state is the Honokiol nano-particle of targeting sinusoidal endothelial cell.
6. according to described application arbitrary in claim 1-5, it is characterised in that: described product is medicine.
7. have following 1)-10) in the product of at least one function, the Honokiol including targeting sinusoidal endothelial cell receives Rice grain；
1) treatment hepatic fibrosis disease；
2) suppression hepatic fibrosis；
3) promote or maintain or recover sinusoidal endothelial cell and be in differentiation state；
4) improve sinusoidal endothelial cell and promote liver repair；
5) HGF protein expression and Wnt2 protein expression in sinusoidal endothelial cell is improved；
6) suppression hepatic fibrosis tuberosity is formed or promotes that hepatic fibrosis tuberosity eliminates；
7) serum alt content and/or AST content are reduced；
8) Akt phosphorylation level in suppression sinusoidal endothelial cell；
9) Erk1/2 phosphorylation level in sinusoidal endothelial cell is promoted；
10) eNOS expression and Nox2 expression ratio in sinusoidal endothelial cell are improved.
Product the most according to claim 7, it is characterised in that:
The Honokiol nano-particle of described targeting sinusoidal endothelial cell is to use the albumen of targeting sinusoidal endothelial cell or containing being somebody's turn to do The fusion protein of albumen or the complex containing this albumen are coated the granule that honokiol obtains.
9. according to the application described in claim 7 or 8, it is characterised in that: described product is medicine.
10. the method suppressing hepatic fibrosis, for using LSEC as target cell, maintains LSEC to be in differentiation state, thus reaches Purpose to suppression hepatic fibrosis.
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