CN110402894A - A kind of drug diathesis liver injury model and its construction method and application - Google Patents
A kind of drug diathesis liver injury model and its construction method and application Download PDFInfo
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- CN110402894A CN110402894A CN201910698098.5A CN201910698098A CN110402894A CN 110402894 A CN110402894 A CN 110402894A CN 201910698098 A CN201910698098 A CN 201910698098A CN 110402894 A CN110402894 A CN 110402894A
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Abstract
The application discloses a kind of evaluation model of immune diathesis hepatic injury, and the evaluation model includes NLRP3 inflammation corpusculum and the ingredient for promoting the activation of NLRP3 inflammation corpusculum, wherein the ingredient for promoting the activation of NLRP3 inflammation corpusculum is icariside I.The construction method of the evaluation model is also disclosed in the application, and the construction method includes: the preparation of icariside I solution；Mouse primary bone marrow macrophage is prepared, and LPS is added to be stimulated；Mouse primary bone marrow macrophage, which is added, in the solution of preparation stimulates；ATP, which is added, to be stimulated；Western blot detects the expression of the small body associated protein of NLRP3 inflammation, and ELISA detects IL-1 β, the content of TNF-α and Caspase-1 determination of activity in cell conditioned medium, and carries out statistical analysis.
Description
Technical field
This application involves drug screening field, especially but not limited to being related to a kind of drug diathesis liver injury model and its structure
Construction method and application.
Background technique
Drug induced hepatic injury refers to the hepar damnification as caused by drug or its metabolite, is medicament research and development failure or removes city
One of the major reasons.Drug hepatic injury can be divided into own type and diathesis type, and own type hepatic injury research is more universal, be by
Caused by the direct toxicity of drug or its metabolite, with predictability and drug dose dependence.And diathesis type drug
Property hepatic injury (IDILI) incidence it is extremely low and there is unpredictability, it is explicitly corresponding with the shortages such as drug dose, time
Relationship, but it is often closely related with the bodies disease state such as gene pleiomorphism, immune inflammation.Just because of diathesis type Drug
Hepatic injury has sporadic and does not have effectively evaluating standard still, is difficult to find risk of liver injury before causing some drugs to list,
Great risk is brought to clinical application.Generation and body disease state due to IDILI is closely related, with very big
Body otherness, leads at this stage that there are no particularly suitable IDILI evaluation models.Currently, the mechanism research based on IDILI
Incremental advances are had been achieved for, and have formd the Mechanism Hypothesis of a variety of IDILI, including injury of mitochondria hypothesis, inflammation are answered
Swash damage hypothesis etc., has been set up Syndrome model for part hypothesis and evaluated for IDILI, though these animal models are certain
Disease basis and the mechanism of some drugs diathesis hepatic injury are illustrated in degree, but there is also evaluate the problems such as unstable.
Summary of the invention
It is the general introduction to the theme being described in detail herein below.This general introduction is not the protection model in order to limit claim
It encloses.
NLRP3 inflammation corpusculum is related by the adaptor protein apoptosis in intracellular inherent immunity receptor protein NLRP3 and downstream
Speck-like protein (Apoptosis associated speck like protein, ASC) and caspases are special
More eggs that protease 1 (Cysteinyl aspartate specific proteinase, Caspase-1) is formed as core
White compound, activation need the collaboration of two signal paths to complete: TLRs receptor (Toll-like receptor) and lipopolysaccharides
Ligand bindings such as (Lipopolysaccharides, LPS) induce NF- κ B signal signal pathway activated, to mediate NLRP3, pro-
The NLRP3 inflammation corpusculum such as IL-1 β expression, in addition, being acted in ATP equivalent damage associated molecular pattern and pathogen-associated molecular pattern
Under, NLRP3 albumen and ASC form polymeric complex, so that pro-Caspase-1 self splicing is induced to activate, activation
On the one hand Caspase-1 can be died with inducing cell coke, on the other hand then can induce pro-IL-1 β and pro-IL-18 shearing activation
IL-1 β and IL-18 are formed, immunocyte is recruited, to participate in the occurrence and development of the numerous diseases of the mankind.At this stage, had big
Quantity research discovery, NLRP3 inflammation corpusculum play a significant role in panimmunity related disease, especially there is document report
NLRP3 inflammation corpusculum has played important function in the drug-induced diathesis course of liver damage such as amodiaquine.However, Chinese medicine
Whether small molecule compound can use NLRP3 inflammation corpusculum activation model, and to carry out toxicity assessment, there is not been reported.In recent years
Research finds that drug induced hepatic injury and immunity of organism are closely related especially very close with NLRP3 activation relationship, in Drug
Hepatic injury is especially immunized in diathesis hepatic injury and plays an important role.Regulate and control NLRP3 activation, mitigate liver inflammation reaction and
Pathology damage degree, it is considered to be the new way of the drug diathesis treating liver injury of great prospect.And to the relevant medicine of NLRP3
The discussion of object diathesis hepatic injury pathogenesis and the screening and evaluation of potential treatment drug, are largely dependent upon disease
The foundation and application of relevant internal external model.But it is available still to lack stable, reliable animal model so far, gives drug diathesis
The expansion for the treatment of liver injury strategy and new drug development have caused great difficulties.
Existing diathesis hepatic injury research is mostly based on traditional toxicological evaluation mode, not yet using fuselage state as core
Considerations.This research method is damaged based on diathesis hepatic injury immune response feature and NLRP3 inflammation corpusculum in diathesis liver
The important function played in wound establishes a kind of drug diathesis liver injury model, and inquires into its application.The invention solves skill
Art problem is to provide a kind of hepatotoxicant formula induction, is suitable for routine experiment animal, simple and easy, activates NLRP3 dynamic
Object model, and labor intensive financial resources are few, experimental period is short, and it is high at mould rate, it is suitable for drug diathesis hepatic injury pathogenesis
Research and diathesis liver injury medicament evaluation, provided for the expansion and new drug development of drug diathesis treating liver injury strategy
A kind of stabilization, reliable animal model.
Specifically, the application provides a kind of evaluation model of immune diathesis hepatic injury, and the evaluation model includes NLRP3
Inflammation corpusculum and the ingredient for promoting the activation of NLRP3 inflammation corpusculum, wherein it is excessive for promoting the ingredient of NLRP3 inflammation corpusculum activation
The sheep leaves of pulse plants time glycosides I.
In this application, the evaluation model using NLRP3 inflammation corpusculum and promote NLRP3 inflammation corpusculum activation at
Divide and is constructed.In this application, the evaluation model can also use NLRP3 inflammation corpusculum and promote NLRP3 inflammation small
The ingredient of body activation is constructed as marker.
The application also provides the construction method of the evaluation model, and the construction method includes:
S100. cellular level is evaluated；
S101. the preparation of icariside I solution；
S102. mouse primary bone marrow macrophage is prepared, and LPS is added to be stimulated；
S103. the solution prepared in step S101 addition mouse primary bone marrow macrophage is stimulated；
S104. ATP, which is added, to be stimulated；
S105.Western blot detects the expression of the small body associated protein of NLRP3 inflammation, and ELISA is detected in cell conditioned medium
IL-1 β, the content of TNF-α and Caspase-1 determination of activity, and carry out statistical analysis.
In this application, the method can also include CCK-8 cytotoxicity experiment, and CCK-8 cytotoxicity experiment includes:
IBMDM and L02 cell is taken, is inoculated with the cell suspending liquid of iBMDM and L02, in the incubator preculture, CCK- is added
8 solution measure the absorbance at 450nm, and calculate maximal non-toxic dosage and IC50 value in being incubated in incubator.
In this application, the maximal non-toxic dosage of icariside can be 10 μm of ol-20 μm of ol, and IC50 is 120 μ
mol-200μmol。
In this application, the construction method includes:
S100. cellular level is evaluated；
S101. the preparation of icariside I solution；
S102. mouse primary bone marrow macrophage is prepared, and LPS is added to be stimulated；
S103. the solution prepared in step S101 addition mouse primary bone marrow macrophage is stimulated；
S104. ATP, which is added, to be stimulated；
S105.Western blot detects the expression of the small body associated protein of NLRP3 inflammation, and ELISA is detected in cell conditioned medium
IL-1 β, the content of TNF-α and Caspase-1 determination of activity, and carry out statistical analysis；
S200. animal assessment of levels；
S201. mouse tail vein injection LPS；
S202. intraperitoneal injection promotes the solution of the ingredient of NLRP3 inflammation corpusculum activation；
S203. the content of serum alt, ASTI, L-1 β, TNF-α is measured, and carries out tunnel dyeing.
In this application, the dosage of the icariside I can be 25mg/kg-100mg/kg, preferably
50mg/kg。
Mg/kg refers to the dosage in terms of mg of every kg the weight of animals in this application.
In this application, the administration time of the icariside I can be 1 hour -3 small after lipopolysaccharides injection
When, preferably 2 hours.
In this application, the dosage of lipopolysaccharides can be 0.5mg/kg-3mg/kg, preferably 2mg/kg.
In this application, in step s105, the small body associated protein of NLRP3 inflammation may include IL-1 β p17,
Any one of caspase-1 p20, NLRP3, ASC, pro-Caspase-1 and pro-IL-1 β or more.
The application also provides purposes of the icariside I in evaluation drug diathesis hepatic injury.
In this application, the dosage of icariside I can be 25mg/kg-100mg/kg, preferably 50mg/kg.
The application also provides the combination of MCC950, lipopolysaccharides and icariside I in evaluation drug diathesis hepatic injury
Purposes.
In this application, the dosage of MCC950 can be 50mg/kg-80mg/kg, and the dosage of lipopolysaccharides can be with
For 0.5mg/kg-3mg/kg, the dosage of icariside I can be 25 mg/kg-100mg/kg.
Mould is evaluated in lipopolysaccharides/icariside I (LPS/icarisid I) diathesis hepatic injury that the present patent application provides
Type, can from different perspectives, and accurately delicately whether evaluation Traditional Chinese Herb compound has diathesis hepatic injury attribute.
MCC950 can specifically inhibit NLRP3 inflammation corpusculum to activate.MCC950/LPS/icarisid I is directed to drug diathesis liver
Whether the evaluation model of damage screening hepatic accurately delicately can evaluate Traditional Chinese Herb compound from different perspectives
With the attribute for inhibiting the hepatic injury of drug diathesis, be Chinese medicine or Traditional Chinese Herb compound hepatotoxicity wind agitation evaluation model building,
The clinical safety reasonable development application of the research of science mechanism, Chinese medicine or tcm components provides important evidence.
Other features and advantage will illustrate in the following description, also, partly become from specification
It obtains it is clear that being understood and implementing the application.The purpose of the application and other advantages can be by specifications, right
Specifically noted structure is achieved and obtained in claim and attached drawing.
Detailed description of the invention
Attached drawing is used to provide to further understand technical scheme, and constitutes part of specification, with this
The embodiment of application is used to explain the technical solution of the application together, does not constitute the limitation to technical scheme.
Fig. 1 is the CCK-8 toxicity test of icariside I.
Fig. 2 shows icariside Is to activate NLRP3 inflammation at ATP and nigericin (Nigericin) stimulation
Corpusculum.
Fig. 3 shows LPS/ icariside I building diathesis liver injury model, and wherein Con is control group, and Mod is model
Group, the icariside I that I ca refers to.
Fig. 4, which shows LPS/ icariside I, leads to hepar damnification, and wherein Con is control group, and Mod is model group.
Fig. 5 is the diathesis liver injury model based on LPS/ icariside I, can screen targeting NLRP3 inflammation corpusculum
Liver protection model drug (MCC950), wherein Con is control group, and Mod is model group, the icariside I that I ca refers to, what MCC referred to
It is MCC950.
Fig. 6 shows MCC950 and alleviates hepar damnification caused by LPS/ icariside I, and wherein Con is control group, and Mod is
Model group.
Specific embodiment
For the purposes, technical schemes and advantages of the application are more clearly understood, below in conjunction with attached drawing to the application
Embodiment be described in detail.It should be noted that in the absence of conflict, in the embodiment and embodiment in the application
Feature can mutual any combination.
Drug:
Herba Epimedii monomer component (icariside I) is purchased from TargetMol company, and the storage of 10 mmol/L is made
Concentration is dissolved in DMSO, is stored in -20 DEG C, spare.Final administration concentration: 10 μm of ol/L dilute institute by Opti-MEM culture medium
.
Icariside I needed for zoopery is purchased from Chengdu Pu Feide Bioisystech Co., Ltd, before use with containing 10%
The PBS buffer solution of Tween 80 dissolves, and is made into 25,50,100mg/kg respectively.
LPS needed for zoopery: it is purchased from U.S. Sigma Aldrich, is configured to 2mg/ with PBS buffer solution before use
kg。
Reagent, material:
DMEM culture medium, fetal calf serum (HyClone company, the U.S.)；(U.S. Gibco is public for Opti-MEM serum free medium
Department)；Trypsase, dual anti-(Pen .- Strep solution) (stepping morning science and technology Beijing Co., Ltd)；Atriphos (ATP), Buddhist nun
Day Leah rhzomorph (Nigericin), lipopolysaccharides (LPS), dimethyl sulfoxide (DMSO) (U.S.'s Sigma Aldrich)；
MCC950 is purchased from TargetMol company；NLRP3, ASC, Caspase-1, pro-Caspase-1, pro-IL1 β, GAPDH are anti-
Body (Cell Signaling Technology company, the U.S.)；CCK-8 kit (MCE)；Caspase-1 activit assay kits
Box (Promega company, the U.S.)；TNF-α, IL-1 β ELISA detection kit (it is Bioisystech Co., Ltd that Beijing, which reaches section)；5
(Beijing Ding Guochang wins the limited public affairs of biotechnology by × SDS protein electrophoresis sample-loading buffer, tri- color pre-dyed albumen Marker of PM2510
Department)；Electrophoresis liquid, transfer liquid, 10 × TBST buffer, Coomassie brilliant blue (Beijing Bo Maide gene technology Co., Ltd)；Methanol,
Acetic acid (Beijing Chemical Plant)；RIPA lysate (the green skies Bioisystech Co., Ltd in Shanghai)；A, B developer solution, pvdf membrane (beauty
Millipore company, state).
Instrument:
Carbon dioxide incubator (Thermo Scientific company, the U.S.)；Microplate reader (Promega company, the U.S.)；It is low
Warm supercentrifuge (Sigma Co., USA)；Electrophoresis tank, transfer groove, gel electrophoresis analysis system (Bio-Rad company, the U.S.)；
Tissue culture plate shaker (German IKA company).
Product prepares embodiment:
1. icariside I activates NLRP3 inflammation corpusculum situation
1.1 icariside Is are dissolved into 10mM with DMSO.- 20 DEG C of storages, it is spare；
The preparation of 1.2 mouse primary bone marrow macrophages (BMDMs): taking 10 week old C57BL/6 female mices, separates humerus
And femur, with DMEM culture medium (10% fetal calf serum) repeatedly bone marrow extraction cell and by 1: 4000 be added mouse colony stimulation
After the factor (MCSF) is broken up 5 days, vitellophag simultaneously presses 9 × 105/ml kind plate (24 orifice plates, 0.5ml/well), after adherent overnight
Carry out next step experiment.
1.3 LPS stimulation: the mode for changing liquid is taken, BMDMs is added to simultaneously with the concentration (0.4ml/well) of 50ng/ml
Stimulate 4h.
1.4 administrations: icariside I is diluted to 10 μM with Opti-MEM culture medium, LPS is siphoned away, in a manner of changing liquid
It is added to BMDMs (0.4ml/well), stimulates 1h.
The stimulation of 1.5 NLRP3 stimulating factors: the mode of fluid infusion is taken, final concentration of 5mM ATP (0.02ml/ is separately added into
Well 1h, 10 μM of nigericin (0.02ml/well)) is stimulated to stimulate 0.5h.
1.6 receive samples: cell receives sample, is directly added into 1 × RIPA Loading cracking (120 hole μ L/), shakes rapidly even, makes
Loading covers all cells, and again in this way, 3 times repeatedly after 5mins, Loading is sucked in EP pipe.At cell conditioned medium sample
Reason: room temperature 4000rpm is centrifuged 5mins, receives supernatant, and 1/4 volume trichloroacetic acid (TCA) is added, after 4 DEG C of standing 1h or more, 4 DEG C,
12000rpm is centrifuged 15mins；Supernatant is abandoned, ice acetone (500 hole μ L/) mixing of turning upside down is added, 4 DEG C, 12000rmp is centrifuged
15mins abandons supernatant, and 95 DEG C of metal baths volatilize acetone, and after acetone volatilization plus 1 × RIPA Loading, 35 μ L oscillation mixes,
Water-bath is boiled, and is used as Supernatant samples after cooling.
1.7 Western blot: IL-1 β p17, caspase-1 p20 protein expression level in detection supernatant, detection are thin
NLRP3, ASC, pro-Caspase-1 in cellular lysate liquid, pro-IL-1 β protein level.Cell conditioned medium protein sample uses 12%
The SDS- polyacrylamide gel electrophoresis of concentration, cell pyrolysis liquid sample are electric using the SDS- polyacrylamide gel of 10% concentration
Swimming, powers on, and sets constant voltage 80V, and voltage is changed to 135V after 45mins, when bromophenol blue reaches separation gel bottom, terminates
Electrophoresis installs transfer device.After cell conditioned medium terminates electrophoresis, glue is cut out along the position maker 60kD, 60kD or more uses coomassie
Brilliant blue dye 30mins, destainer (water: methanol: acetic acid=5: 4: 1), until background blue take off it is transparent；The cathode paving 4 of transfer groove
The remaining gel of 60kD or less is laid on filter paper, and in glue upper berth PVDF film, 5 layers of filter paper is layered on the upper of film by layer filter paper
Side.Power on, set with constant current, 450mA, 3h.After transferring film, 5% defatted milk room temperature closes 1h, different primary antibodies (1: 1000)
4 DEG C of solution overnight incubations, PVDF film washes film 3 times through 1%TBS-T solution interval 5mins, then with the secondary antibodies of respective markers (1:
5000) solution carries out reaction 1h, abandons secondary antibody, is washed film 3 times with 1%TBS-T solution interval 5mins, by two kinds of developer solution A and B
It after reagent mixes in equal volume, is added on film immediately, darkroom is developed using X-ray film tabletting Exposure mode.
1.8 Caspase-1 determinations of activity: it is operated according to Caspase-1 Activity Assay Kit specification.First room
Temperature balance1 buffer, Z-WEHD fluorescein substrate and MG-132 inhibitor, then in 10mL Z-WEHD-
60 μ L MG-132 (120 μM) inhibitor sufficient vortexes are added in Aminofluorescein substrate, to being thoroughly mixed, to add1 buffer, sufficient vortex form final concentration of 60 μM of fluorescence detection mixture, for use.In 96 orifice plates
The cell conditioned medium sample of 50 μ L is added in every hole, then every hole is added 30 μ L and prepares stand-by fluorescence detection mixture, tissue culture plate
Oscillator vibrates 1min, and room temperature, which is protected from light balance, combines sample sufficiently with fluorogenic substrate, tests and analyzes after 1h through microplate reader
Caspase-1 fluorescent value.
1.9 ELISA detection: measuring the content of IL-1 β in cell supernatant, TNF-α, and operating procedure by specification carries out.
OD value is read in 450nm wavelength, sample concentration is calculated according to standard curve.
1.10 statistical methods: data are counted using GraphPad Prism 6.0 (GraphPad, San Diego, CA)
Software is handled, and group difference analysis selects One-way ANOVA method to carry out.It is poor that P < 0.05 is considered that there are statistics
It is different.
2. icariside I CCK-8 cytotoxicity experiment
Take iBMDM the and L02 cell of 80~90% abundance of culture extremely, inoculating cell suspension (the 100 μ L/ in 96 orifice plates
Hole), 2.5 × 105cells/mL of density.IBMDM and L02 cell is divided into the administration of normal group, 0.1%DMSO group, various concentration
Group, every group sets 3 multiple holes, and culture plate is put preculture in the incubator and for 24 hours, the CCK-8 solution of 10 μ L, culture are added to every hole
Plate is placed in incubator after incubation 15min, measures the absorbance at 450nm with microplate reader, calculates monomer according to absorbance value
The maximal non-toxic dosage and IC50 value of ingredient.
3. being based on LPS model, specifying LPS/icarisid I can lead to serious diathesis hepar damnification
3.1 determine LPS administration time using C57BL/6 female mice
3.1.1 6-8 weeks C57BL/6 female mice tail vein injection LPS (2mg/kg)
3.1.2 icariside I (50mg/kg) is injected intraperitoneally after 1h, 2h, 3h
3.1.3 mouse orbit takes blood after 6h, and serum alt, the content of AST are surveyed in 3500rpm/min centrifugation.
3.2 LPS joint icariside I causes the LPS dosage of hepatic injury to determine
3.2.1 6-8 weeks C57BL/6 female mice tail vein injection LPS (0.5mg/kg, 1mg/kg, 2mg/kg, 3mg/
kg)
3.2.2 icariside I (50mg/kg) is injected intraperitoneally after 2h
3.2.3 mouse orbit takes blood after 6h, and serum alt, the content of AST are surveyed in 3500rpm/min centrifugation.
3.3 cause the objective attribute of diathesis hepatic injury based on LPS model evaluation icariside I
3.3.1 6-8 weeks C57BL/6 female mice tail vein injection LPS (2mg/kg)
3.3.2 icariside I (25,50,100mg/kg) are injected intraperitoneally after 2h
3.3.3 after 6h, mouse orbit takes blood, and IL-1 β, TNF-α, ALT, AST in serum are surveyed in 3500rpm/min centrifugation
Content；Mouse is dissected, hepatomegaly leaf is put into 5% formalin, and HE dyeing detection hepatocellular injury, it is thin that tunnel dyeing detects liver
Born of the same parents' apoptosis.
3.4 MCC950 can be used as a kind of liver protection model drug for targeting NLRP3 inflammation corpusculum.
3.4.1 C57BL/6 female mice intraperitoneal injection MCC950 (50mg/kg) in 6-8 weeks；
3.4.2 tail vein injection LPS (2mg/kg) after 2h；
3.4.3 icariside I (50mg/kg) is injected intraperitoneally after 2h；
3.4.5 after 6h, mouse orbit takes blood, and IL-1 β, TNF-α, ALT, AST in serum are surveyed in 3500rpm/min centrifugation
Content；Mouse is dissected, hepatomegaly leaf is put into 5% formalin, and HE dyeing detection hepatocellular injury, it is thin that tunnel dyeing detects liver
Born of the same parents' apoptosis.
As a result
1. icariside I CCK-8 toxicity test
It can be seen that in L02 cell, iBMDMs, BMDM cell from CCK-8 experimental result, IC50 value is both greater than
120μmol/L.Therefore, when next step inquires into icariside I to NLRP3 inflammation corpusculum activation, under non-toxic,
It is tested when i.e. concentration is 40 μm of ol/L or less.2. be based on BMDMs cell, icariside I LPS and ATP, LPS and
Under nigericin Co stituation, its activation situation to NLRP3 inflammation corpusculum is studied
Select its activation situation small to NLRP3 inflammation of the following concentration studies of icariside I: 5,10,20,40 μm of ol/
L.Cell 4h first is handled with LPS (50ng/ml), then changes culture medium into various concentration that serum-free opti-MEM is formulated
Icariside I, handle 1h, and then collect cell conditioned medium after adding ATP (5mM) or nigericin (10 μM), 1h and split
It solves liquid and carries out index of correlation detection.As can be seen from Figure 2 at LPS and the effect of ATP (nigericin) Co stituation, western
Blot is as the result is shown in BMDMs cell conditioned medium, the expressing quantity of caspase-1p20 and IL-1 β p17 is with icariside
The increase of I concentration and gradient is incremented by；Caspase-1 determination of activity the result shows that in BMDMs cell conditioned medium caspase-1 amount
Increase as icariside I concentration increases in dose dependent；ELISA the result shows that in cell conditioned medium IL-1 β content with
Icariside I concentration increase in dose dependent increase, and to non-NLRP3 inflammation corpusculum rely on TNF-α content without
It influences.The above result of study shows icariside I under LPS and ATP (nigericin) stimulation, and caspase-1 is further
Shearing activation, and then promote the activation of NLRP3 inflammation corpusculum, cause inflammatory factor IL-1 β to increase significantly, so as to cause more serious
Immunization inflammatory reaction, aggravate diathesis hepatic injury.
3. causing the objective attribute of diathesis hepatic injury based on LPS model evaluation icariside I
6-8 week old female C57BL/6 mouse gives the excessive sheep of various concentration after LPS (2mg/kg) tail vein injection 2h
The leaves of pulse plants time glycosides I (25,50,100mg/kg) handles 6h, and eye socket takes blood, and TNF-α, ALT, AST in serum are surveyed in 3500rpm/min centrifugation
Content；Mouse hepatomegaly leaf is put into 10% neutral formalin fixer, HE dyeing detection hepatocellular injury, tunnel dyeing inspection
Survey hepatocellular apoptosis.Experimental result shows that compared with the control group, LPS group Serum TNF-α significantly increases.And it is administered alone group
(25,50,100mg/kg) compared with the control group TNF-α, there was no significant difference for the content of ALT, AST, prompt icariside I
Hepar damnification can not be directly resulted in.And compared with LPS group, LPS+ administration group (25,50,100mg/kg) Serum ALT, AST,
TNF-α significantly increases, and prompts in the diathesis liver injury model that LPS is induced, and icariside I will lead to hepar damnification, and
Degree of injury and inflammatory factor TNF- α are closely related, show LPS/icarisid I really and can lead to diathesis hepatic injury.
4.MCC950 can be used as a kind of liver protection model drug for targeting NLRP3 inflammation corpusculum.
6-8 week old female C57BL/6 mouse peritoneal injects MCC950 (50mg/kg), quiet through LPS (2mg/kg) tail after 2h
Arteries and veins is injected, and after 2h, gives icariside I (I) (50mg/kg) processing 6h, eye socket takes blood, and serum is surveyed in 3500rpm/min centrifugation
Middle IL1 β, TNF-α, the content of ALT, AST；Mouse hepatomegaly leaf is put into 10% neutral formalin fixer, HE dyeing detection liver
Cellular damage, tunnel dyeing detection hepatocellular apoptosis.Experimental result shows, compared with the control group, 1 β of LPS group serum IL,
TNF-α significantly increases.And be administered alone group (50mg/kg) there was no significant difference compared with the control group, prompt icariside I,
MCC950 can not directly result in hepar damnification.Compared with LPS group, LPS+MCC950 (LM), LPS+MCC950+ Herba Epimedii
Glycosides I (LIM), IL1 β, TNF-α, the not significant change of the content of ALT, AST in serum prompt MCC950 to simulate well
NLRP3-/- knocks out state, hepar damnification can not be led to by giving icariside I on this basis.And compared with LPS group,
LPS+ icariside I (LI), IL1 β, TNF-α, the content of ALT, AST dramatically increase in serum, this experiment shows LPS/
Icarisid I can lead to immune diathesis hepatic injury, and the diathesis liver injury medicament of NLRP3 is activated for relying on, MCC950
There is effective hepatoprotective effect again.
Although embodiment disclosed by the application is as above, the content only for ease of understanding the application and use
Embodiment is not limited to the application.Technical staff in any the application fields, is taken off not departing from the application
Under the premise of the spirit and scope of dew, any modification and variation, but the application can be carried out in the form and details of implementation
Scope of patent protection, still should be subject to the scope of the claims as defined in the appended claims.
Claims (11)
1. a kind of evaluation model of immune diathesis hepatic injury, the evaluation model includes NLRP3 inflammation corpusculum and promotion
The ingredient of NLRP3 inflammation corpusculum activation, wherein the ingredient for promoting the activation of NLRP3 inflammation corpusculum is icariside I.
2. the construction method of evaluation model described in claim 1, the construction method include:
S100. cellular level is evaluated；
S101. the preparation of icariside I solution；
S102. mouse primary bone marrow macrophage is prepared, and LPS is added to be stimulated；
S103. the solution prepared in step S101 addition mouse primary bone marrow macrophage is stimulated；
S104. ATP, which is added, to be stimulated；
S105.Western blot detects the expression of the small body associated protein of NLRP3 inflammation, and ELISA detects IL-1 in cell conditioned medium
β, the content of TNF-α and Caspase-1 determination of activity, and carry out statistical analysis.
3. construction method according to claim 2, wherein the construction method further includes CCK-8 cytotoxicity experiment,
CCK-8 cytotoxicity experiment includes:
IBMDM and L02 cell is taken, the cell suspending liquid of iBMDM and L02 is inoculated with, carries out preculture, CCK-8 solution is added and is incubated
It educates, measures the absorbance at 450nm, and calculate maximal non-toxic dosage and IC50 value.
4. construction method according to claim 3, wherein the maximal non-toxic dosage of icariside I is 10 μm of ol-20 μ
Mol, IC50 are 120 μm of ol-200 μm of ol.
5. construction method according to claim 2, wherein the construction method includes:
S100. cellular level is evaluated；
S101. the preparation of icariside I solution；
S102. mouse primary bone marrow macrophage is prepared, and LPS is added to be stimulated；
S103. the solution prepared in step S101 addition mouse primary bone marrow macrophage is stimulated；
S104. ATP, which is added, to be stimulated；
S105.Western blot detects the expression of the small body associated protein of NLRP3 inflammation, and ELISA detects IL-1 in cell conditioned medium
β, the content of TNF-α and Caspase-1 determination of activity, and carry out statistical analysis；
S200. animal assessment of levels；
S201. mouse tail vein injection LPS；
S202. intraperitoneal injection promotes the solution of the ingredient of NLRP3 inflammation corpusculum activation；
S203. the content of serum alt, ASTI, L-1 β, TNF-α is measured, and carries out tunnel dyeing.
6. construction method according to claim 5, wherein the dosage of the icariside I is 25mg/kg-
100mg/kg。
7. construction method according to claim 5 or 6, wherein the dosage of lipopolysaccharides is 0.5mg/kg-3mg/kg.
8. purposes of the icariside I in evaluation drug diathesis hepatic injury.
9. purposes according to claim 8, wherein the dosage of icariside I is 25mg/kg-100mg/kg.
Purposes of the combination of 10.MCC950, lipopolysaccharides and icariside I in evaluation drug diathesis hepatic injury.
11. purposes according to claim 10, wherein the dosage of MCC950 is 50mg/kg-80mg/kg, lipopolysaccharides
Dosage be 0.5mg/kg-3mg/kg, the dosage of icariside I is 25mg/kg-100mg/kg.
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Application publication date: 20191105 |