CN110368495A - Application of the NLRP3/ASC inflammation corpusculum as treatment recurrent ischemia headstroke target spot - Google Patents
Application of the NLRP3/ASC inflammation corpusculum as treatment recurrent ischemia headstroke target spot Download PDFInfo
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Abstract
Application the invention discloses NLRP3/ASC inflammation corpusculum as treatment recurrent ischemia headstroke target spot.Target spot of the NLRP3/ASC inflammation corpusculum as prevention and treatment recurrent headstroke in the present invention, compared with IL molecule, target spot of the NLRP3/ASC as prophylactic treatment recidivity apoplexy, more precisely, it is more special, there is critically important meaning for the prevention and treatment of recurrent headstroke, is worth further investigation, can significantly improve the control efficiency of clinical patients recurrent headstroke.
Description
Technical field
The present invention relates to the prophylactic treatment technical fields of ischemia apoplexy, more particularly, to NLRP3/ASC inflammation
Application of the corpusculum as prophylactic treatment recurrent ischemia headstroke target spot.
Background technique
Headstroke is one group using cerebral ischemic and heamorrhagic lesions symptom as the disease of main clinical manifestation, also known as cerebral apoplexy
Or cerebrovascular accident, there is high case fatality rate and disability rate, be broadly divided into hemorrhagic cerebral apoplexy (cerebral hemorrhage or cavum subarachnoidale
Bleeding) and ischemia apoplexy (cerebral infarction, cerebral thrombosis) two major classes, it is most commonly seen with cerebral infarction.Headstroke morbidity is anxious, disease
Dead rate is high, is one of most important fatal disease in the world.The death rate of apoplexy also has the tendency that increasing and rising with the age,
Due to lacking effective remedy measures always, it is now recognized that prevention is best measure.
There are about 1/3 paralytics may recurrence in 5 years.From recurrence time, recidivist accounts for about 2 8% in 1 year
Recurrence accounts for 25% in 30%, 2 year, and 3 years to 5 years recidivists account for 16%, and person accounts for about 29% within 5 years or more.It is first in terms of recurrent number
It is secondary to account for 74%, 22% is accounted for for the second time.WHO points out that the probability for occurring apoplexy again in headstroke latter week is very high, and and apoplexy
Dull-witted generation is closely related afterwards.
C reactive protein, the inflammatory factors such as IL-6 and apoplexy again have an obvious correlation, and can be at some extent as
Predict the recurrent biological indicator of apoplexy, human monoclonal antibodies canakinumab is significantly dropped by targeted therapy IL-1 β at present
Low plasma IL -6, high-sensitivity C-reactive protein, the intervention means as prevention recidivity angiocarpy infraction enter clinical test,
But the missing of IL function can reduce blood vessel and nerve regeneratl, aggravate the chronic cerebral lesion of bring after apoplexy.
Lack a kind of marker of effective prevention recidivity headstroke, and the target for the treatment of recidivity headstroke at present
Point.
Summary of the invention
The purpose of the invention is to overcome the deficiencies in the prior art, prepare single-shot using photochemical method and recurrent lacks
Hemorrhagic apoplexy model provides application of the NLRP3/ASC inflammation corpusculum as prophylactic treatment recurrent ischemia headstroke target spot.
To achieve the goals above, the present invention is achieved by the following technical programs:
This patent prepares single-shot and recurrent ischemia apoplexy model using photochemical method, and clear apoplexy for the first time is as the
One stimulus signal modifies NLRP3 albumen, and recurrent apoplexy activates NLRP3 albumen as second stimulus signal, and causes
The aggregation of ASC albumen, so that inflammation corpusculum access is activated, it is final to activate IL1 β, aggravate cerebral lesion.
According to previous document report, ASC is present in intracerebral spongiocyte, can be from inflammation after the sexual stimulus that comes to harm
Disease cell discharges and keeps activity, and then is swallowed and activated by periphery inflammatory cell Caspase-1 and IL1- β, has PrPC
Whether characteristic, ASC albumen activate inflammation in the state for depending on NLRP3.
ASC is discharged from damaged cell after inventor has found apoplexy for the first time, and can keep activity, but under physiological conditions, is not led
Cause obvious inflammatory reaction；When being stimulated by recurrent apoplexy, ASC can be dependent on NLRP3 amplification inflammatory reaction, aggravate apoplexy knot
Office, therefore NLRP3 gene knockout can eliminate the effect of ASC in recurrent apoplexy.
Therefore claimed the following contents:
NLRP3 gene and/or albumen are as the application for preventing and/or treating headstroke and complication medicine target spot.
The inhibitor of NLRP3 gene and/or albumen is as the application for preventing and/or treating headstroke drug.
ASC gene and/or albumen are as the application for preventing and/or treating headstroke and complication medicine target spot.
The inhibitor of ASC gene and/or albumen is as the application for preventing and/or treating headstroke drug.
NLRP3/ASC inflammation corpusculum is as the application for preventing and/or treating headstroke and complication medicine target spot.
The inhibitor of NLRP3/ASC inflammation corpusculum is as the application for preventing and/or treating headstroke drug.
Preferably, the headstroke is recurrent headstroke.
Preferably, the complication is inflammatory reaction.
Preferably, the complication is cerebral injury caused by inflammatory reaction.
Compared with prior art, the invention has the following beneficial effects:
Target spot of the NLRP3/ASC inflammation corpusculum as prevention and treatment recurrent headstroke in the present invention, compared with IL molecule,
Target spot of the NLRP3/ASC as prophylactic treatment recidivity apoplexy, more precisely, more specifically, for recurrent headstroke prevention and
Treatment has critically important meaning, is worth further investigation, can significantly improve the control efficiency of clinical patients recurrent headstroke.
Detailed description of the invention
Nissl's staining and immunofluorescence contaminate after parietal cortex infarct on the left of Fig. 1 wild type WT and NLRP3 knock out mice
Colour analysis；A: on the left of single after parietal cortex apoplexy, WT mouse and NLRP gene knockout (NLRP3Knock out) mouse cortex
Nissl's staining, infarct location at red-label；B: top on the left of independent sample t check analysis WT and NLRP3 knock out mice
The injury scores of infarct；C: 1 immunofluorescence label periinfarct microglia of Iba is used；D: independent samples t test analysis
WT mouse and NLRP3 knock out mice left cortical periinfarct microglia quantity；E:WT and NLRP3 gene knockout
The immunofluorescence dyeing of mouse periinfarct M1 type (CD-16 is positive) microglia；F: independent sample t check analysis WT is small
The M1 type microglia quantity of mouse and NLRP3 knock out mice periinfarct；G:WT and NLRP3 knock out mice obstruct
The immunofluorescence dyeing of dead surrounding M2 type (CD-206 is positive) microglia: H: independent samples t test analyze WT mouse with
The M2 type microglia quantity of NLRP3 knock out mice periinfarct；* P < 0.05, * * P < 0.01, * * * P < 0.001, n=
6。
Fig. 2 is the expression of inflammation corpusculum after unilateral left side parietal cortex infarct；On the left of A:WT and NLRP3KO mouse (L) and
Right side (R) NFKB and β-actin imaging；B: on the left of two-way analysis of variance after (L) parietal cortex infarct, WT is small with NLRP3KO
(L) and right side (R) NLRP3 and β-actin imaging on the left of mouse；(L) and right side (R) NLRC4 on the left of F.WT and NLRP3KO mouse,
AIM2 albumen and β-actin imaging；G. on the left of two-way analysis of variance after (L) parietal cortex infarct, WT mouse and NLRP3 KO
The ratio Analysis of the NLRC4 and AIM2 protein expression of (L) and right side (R) and β-actin albumen on the left of mouse；H. left cortical
(L) after apoplexy, (L) and the imaging of right side (R) ASC albumen and β-actin on the left of WT and NLRP3KO mouse；I. dual factors variance point
After (L) the parietal cortex infarct of analysis left side, the ASC protein expression and β-of WT and NLRP3KO mouse left side (L) and right side (R)
The ratio Analysis of actin albumen；* P < 0.05, * * P < 0.01, P < 0.001.n=6.
Fig. 3 is the expression of infarct size and periinfarct microglia after recidivity apoplexy；A:WT mouse and NLRP3KO
After mouse bilateral apoplexy, Nissl's staining shows infarct location (red-label)；B: dual factors method analysis it is more secondary (right side,
R) and for the first time (left side, L) post-stroke injury score；It is secondary (right side, R) after C:WT mouse and NLRP3KO mouse bilateral apoplexy
The immunofluorescence dyeing of (left side, L) periinfarct microglia for the first time；D:WT mouse and NLRP3KO mouse bilateral apoplexy
Afterwards, secondary (right side, R) and for the first time comparison of (left side, L) periinfarct microglia quantity；E:WT mouse and NLRP3KO
After mouse bilateral apoplexy, secondary (right side, R) and for the first time (left side, L) periinfarct M2 type (CD-206 is positive) microglia
Immunofluorescence dyeing；After F:WT mouse and NLRP3 KO mouse bilateral apoplexy, secondary (right side, R) and for the first time (left side, L) infarct
The comparison of surrounding M2 type (CD-206 is positive) microglia quantity；After G:WT mouse and NLRP3KO mouse bilateral apoplexy, two
The immunofluorescence dyeing of secondary (right side, R) and for the first time (left side, L) periinfarct M1 type (CD-16 is positive) microglia；H:WT
After mouse and NLRP3KO mouse bilateral apoplexy, second (right side, R) and first time (left side, L) periinfarct M1 type (CD-16
It is positive) comparison of microglia quantity；* P < 0.05, * * P < 0.01, P < 0.001.n=6.
Fig. 4 is the expression and activation of inflammation corpusculum after bilateral parietal cortex infarct；A: after bilateral apoplexy, WT mouse and
NLRP3 knock out mice for the first time (L) and second stroke (R) periinfarct NLRP3, NLRC4, AIM2 band and β-actin at
Picture；B: NLRP3 inflammation corpusculum and two-way analysis of variance WT are small after independent samples t test WT mouse bilateral parietal cortex infarct
Mouse and NLRP3 the knock out mice NLRC4 after (L) and secondary (R) apoplexy for the first time, AIM2 protein expression and β-actin albumen
Ratio Analysis；C: after bilateral apoplexy, WT mouse and NLRP3 knock out mice infarct week after (L) and secondary (R) apoplexy for the first time
Enclose ASC albumen and β-actin imaging；D: WT mouse and NLRP3 clpp gene after two-way analysis of variance bilateral parietal cortex infarct
Except periinfarct ASC monomer, the expression of dimer and multimeric protein and β-actin after mouse for the first time (L) and secondary (R) apoplexy
The ratio Analysis of albumen；E: after bilateral apoplexy, WT mouse and NLRP3 knock out mice obstruct after (L) and secondary (R) apoplexy for the first time
Dead surrounding IL1- β albumen and β-actin imaging；F: WT mouse and NLRP3 after two-way analysis of variance bilateral parietal cortex infarct
The knock out mice expression of the Caspase-1 albumen of periinfarct and β-actin albumen after (L) and secondary (R) apoplexy for the first time
Ratio Analysis；G: after bilateral apoplexy, WT and NLRP3KO mouse periinfarct IL1- β egg after (L) and secondary (R) apoplexy for the first time
Bletilla β-actin imaging；H: WT and NLRP3KO mouse be for the first time (L) and two after two-way analysis of variance bilateral parietal cortex infarct
The ratio Analysis of the expression of the IL1- β albumen of periinfarct and β-actin albumen after secondary (R) apoplexy.*P<0.05,**P<0.01,
P < 0.001.n=6.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
One, experimental animal
This research is approved and agreed through the animal welfare committee, No.1 Hospital Affiliated to Zhongshan Univ., all animal treatment measures
It all reduces animal injury to the greatest extent and reduces the size of animal for research.Using 6-8 weeks WT mouse and NLRP3 gene knockout
(NLRP3knockout) mouse, WT mouse are bought from Zhongshan University's Experimental Animal Center, NLRP3-/- mouse (B6/N-
Nlrp3tm1/Nju (article No.: T000352)) is bought from model animal research institute, Nanjing University, animal monitoring is raised in Guangdong Province
It supports and breeds.
NLRP3 knock out mice: model animal research institute, Nanjing University buys NLRP3KO mouse (B6/N-
Nlrp3tm1/Nju), bred, which is built upon on C57BL/6J genetic background, therefore selects C57BL/6J mouse
For wild type control.
Two, experimental method
1, materials slice
Ischemic stroke model constructs after a week, using 4% paraformaldehyde heart perfusion, then uses 4% paraformaldehyde
12-16 hours are fixed after progress, is embedded using OCT, and freezing microtome carries out frozen section, coronal section, every 200 μm
It cuts, with a thickness of 10 μm, is used for histological stain afterwards or deposits in -80 DEG C of refrigerators.
2, Injury score after infarct
Infarct hemisphere tissue is divided into 4 areas, calculates the cumulative score in 4 areas, 0 point is normal；1 point < 10% nerve
Member damage, 2 points of neure damages for 20%-50%；3 points be > 50% neure damage, 4 points be bulk portion neuron
Necrosis, 5 points are involved hippocampus for damage.
3, Nissl's staining:
Slides are taken out from -80 DEG C of refrigerators, rewarming 30 minutes；It is rinsed 3 times using PBS, each 5min；Change a ring with group,
It covers Nissl's staining liquid and is fixed on around tissue, Nissl's staining liquid in drop, room temperature (or 37 degree of baking ovens) dyes 40min；It adopts
Dyestuff is cleaned with distilled water；It is respectively placed in 70%, 80%, 95% ethyl alcohol 2min, 100% ethyl alcohol I, 100% ethyl alcohol II mono- minute
It is dehydrated and uses dimethylbenzene transparent；Finally, using neutral gum mounting.
4, immunofluorescence dyeing
Slides are taken out from -80 DEG C of refrigerators, rewarming 30 minutes；It is rinsed 3 times using 1 × PBS, each 5min；It is placed in citric acid
In salt buffer, antigen retrieval, microwave ingle 5min, room temperature or the cooling 30min of cold bath are carried out；Glass is dried with blotting paper
Piece changes a ring with group, in addition 100 rupture of membranes 20min of 0.3%Triton；PBS is washed three times, each 5min；Confining liquid is added,
Reaction 1 hour；After drying slide, it is added primary antibody (1:100 or 1:300), slide is placed in wet box, 4 DEG C of refrigerators reacted
Night；Room temperature rewarming 30min；1 × PBS is washed 3 times, each 5min；Slide is dried, secondary antibody, normal-temperature reaction 1h is added；1 × PBS washes 3
It is secondary, each 5min；Mounting is carried out using the anti-fluorescence quenching containing DAPI.
5, Western blot Protein Detection
(1) preparation method of buffer
(A) configuration of histone lysate: by RIPA protein lysate and PMSF protease inhibitors according to 100:1 ratio
Example configuration, carries out on ice, ready-to-use.
(B) configuration of APS: configuration 10%APS solution weighs 0.1g APS pulvis and is dissolved in 1ml ultrapure water, and packing is simultaneously
It is saved in -20 DEG C of refrigerators.
(C) 10 × electrophoretic buffer: glycine 144g Tris base 30.3g SDS 10g deionized water is settled to
1000ml, room temperature preservation.
(D) 1 × electrophoretic buffer: take 10 × electrophoretic buffer of 100ml, 900ml deionized water be added and is made into 1000ml
1 × electrophoretic buffer, it is ready-to-use.
(E) 10 × transferring film buffer: glycine 144g Tris base+30.3g deionized water is settled to 1000ml,
Room temperature preservation.
(F) 1 × transferring film buffer: 10 × transferring film liquid 100ml+ methanol 200ml+ deionized water 700ml, be settled to
1000ml is ready-to-use
(G) 10 × TBS buffer: Tris base 24.2g+NaCL 80g+ deionized water is settled to 1000ml, room temperature
It saves
(H) 1 × TBST buffer: 10 × TBS 100ml+Tween-20 1ml+ deionized water is settled to 1000ml, room temperature
Configuration saves
(I) 5%BSA confining liquid: 1 × TBST of BSA 2.5g is settled to 50ml
(J) glue formula: ddH is concentrated2O 4.1ml+30% polyacrylamide gel 1.0ml+1M Tris-HCL (pH 6.8)
60 μ l+10%APS 60ul+TEMED of 0.75ml+10%SDS, 6 μ l
(K) 10% separation glue formula: ddH2O 4ml+30% polyacrylamide gel 3.3ml+1.5M Tris-HCL (pH
8.8) 100 μ l+10%APS 100ul+TEMED of+2.5ml 10%SDS, 4 μ l.
(L) 12% separation glue formula: ddH2O 3.3m+30% polyacrylamide gel 4.0m+1.5M Tris-HCL (pH
8.8) 100 μ l+10%APS 100ul+TEMED of 2.5ml+10%SDS, 4 μ l.
(M) 15% separation glue formula: ddH2O 2.3ml+30% polyacrylamide gel 5.0ml+1.5M Tris-HCL
100 μ l+10%APS 100ul+TEMED of (PH 8.8)+2.5ml+10%SDS, 4 μ l.
(2) protein extraction
(A) after carrying out mouse heart perfusion using ice business salt water, Fresh Frozen brain tissue is taken, according to 100ul/10mg
Weight is added RIPA protein lysate and is ground, and carries out ultrasonic cracking with Ultrasound Instrument, stands 30min；
(B) 4 DEG C of centrifuge 12000rpm are centrifuged 20 minutes, and supernatant is albumen, and supernatant is drawn in another EP pipe -80 DEG C
It saves.
(C) protein concentration is surveyed using BCA method；Referring to BCA protein concentration detection kit specification configuration standard product, concentration
Respectively 0,25,125,250,500,750,1000,1500,2000 μ g/ml；Solvent A and solvent B are mixed in the ratio of 50:1
It closes and configures suitable BCA working solution；C. in 96 orifice plates, standard items and sample are all provided with 2 secondary orifices, and 10ul standard is added in every hole
200ul BCA working solution is added in product or 1ul sample to be tested+9ul RIPA protein lysate, every hole, and 37 DEG C are incubated for 30 points
Clock；D. using microplate reader measurement sample in the absorption photometric value of 562nm and according to the concentration of standard items drafting, and ensure R2>
0.98；The concentration of sample to be tested albumen is calculated according to standard curve；Using concentration of specimens × 10/1000 of calculating and final dense
Degree unit is ug/ul.
(3)Western blot
(A) albuminous degeneration: according to concentration, 20 μ g protein contents are calculated, and are configured to same volume, 5 × SDS is added
Loading buffer make its final concentration of 1 ×, boiling water bath 5min；The SDS- of glue and 10%, 12% or 15% is concentrated in configuration
PAGE glue is stored at room temperature 30min and waits being gelled admittedly；Albumen loading and electrophoresis loading: prepared gel is put into electrophoresis tank, is added
Electrophoresis liquid takes out stripping fork, and albumen Marker and protein sample is added；
(B) electrophoresis: with connect constant pressure electrophoresis apparatus, constant pressure 60V when gel electrophoresis is concentrated, after sample electrophoresis enters separation gel,
Voltage is changed to 120V constant pressure, continues electrophoresis and stops electrophoresis when bromophenol blue is close to glue bottom surface；
(C) transferring film: removing gel, after pvdf membrane is put into methanol in 10s, puts as in 1 × transferring film liquid, by from filter paper,
Glue, film, filter paper sequence are put into slot after clipping, and pour into transferring film liquid, slot is placed in ice water foam box, and transferring film condition is 150V,
90min；
(D) it closes: after transferring film, taking out pvdf membrane, close 1h on 5%BSA confining liquid room temperature shaker；
(E) it antibody incubation: after antibody is configured well with 5%BSA confining liquid by proper proportion (usual 1:1000), is incubated in antibody
Educate 4 DEG C of overnight incubations in box.Film is taken out, is washed 3 times, every time 10 minutes with TBST；Secondary antibody is configured with BSA confining liquid, room temperature is shaken
It is incubated on bed 1 hour, is then washed 3 times, every time 10 minutes with TBST；
(F) develop: A liquid in ECL luminescent solution and B liquid are pressed into 1:1 mixed configuration development work liquid；It takes and drips to pvdf membrane in right amount
It goes up and impregnates the several seconds, image is acquired with chemiluminescence analysis system.
(4) statistical analysis
Cell number number and western blot strip analysis are carried out using IMageJ software；Using 5.0graphpad
Prism maps, and total data is for statistical analysis on 21.0 software of SPSS, and all data use mean ± standard
Poor form is shown.To single apoplexy model, wild-type mice and NLRP3 knock out mice are detected using independent samples t test
Between the biological indicators such as infarct size, microglia；In all statistical methods, P < 0.05 indicates that difference has statistics
Learn meaning.
Embodiment 1NLRP3 gene delection not shadow single cortical ischemia apoplexy ring infarct after injury scores and small colloid it is thin
The expression of born of the same parents
One, experimental method
(1) damage after apoplexy, specific apoplexy damage model are carried out to WT mouse and NLRP3 knock out mice respectively
Building it is as follows:
1, chloraldurate (4.2%, 0.1ml/10g) is injected intraperitoneally, partly sterilised is carried out using alcohol, cuts off scalp, is stood
Body position indicator positions parietal cortex on the left of mouse (2mm after bregma, middle line on the outside of 1.7mm)；
2, the cranium window that wears down skull, prepare diameter be 3mm soft with cranial drill, using the fixed skull of special iron plate and sudden and violent
Reveal cranium window, covers non-irradiated region skull with impermeable ray film；
3, tail vein injection 0.2ml (10 μ l/g) rose-red dye liquor, mouse cranium window is placed under Two Photon Fluorescence, is focused
Keep cortex blood vessel high-visible, apoplexy model is prepared using laser irradiation, irradiation time is 15 minutes, laser intensity 150W.
4, iron plate, partly sterilised, and skin suture are removed.Mouse is placed in insulation blanket, until putting back to small mouse cage after revival, is made
For the damage model of single apoplexy.
(2) ischemic stroke model constructs after a week, using paraformaldehyde heart perfusion, coronal section, every 200 μm
It cuts, with a thickness of 10 μm；It samples and Nissl's staining is used to determine infarct lesion, immunofluorescence dyeing and Wstern blot detection point
The activation of Inflammatory Pathway after analysis apoplexy.
Two, experimental result
By tail vein injection rose-red dye liquor and pass through laser illumination, irradiation time 15min, laser irradiation public affairs
In be 150W, perfusion materials after a week, using Nissl's staining pathological staining as it can be seen that left side parietal cortex cause it is typical and stable
Infarction Model (Fig. 1 .A).Using independent samples t test, find WT mouse and NLRP3 knock out mice after single apoplexy
Injury scores no significant difference (P > 0.05, Fig. 1 .B)；For immunofluorescence dyeing the study found that after the parietal cortex infarct of left side, WT is small
Mouse and NLRP3 knock out mice infarct week microglia quantity no significant difference (P > 0.05, Fig. 1 .C&D), and M1 type
(CD-16 positive) microglia quantity and M2 type (CD-206 is positive) equal no significant difference of microglia quantity (P >
0.05, P > 0.05) (Fig. 1 .E-H).
Influence of the embodiment 2NLRP3 gene knockout to single cortical ischemia apoplexy
Using the damage model sample of the WT mouse of the acquisition of embodiment 1 and NLRP3 knock out mice single apoplexy as inspection
Object is surveyed, influence of the NLRP3 gene knockout to ishemic stroke is probed into.
One, mechanism of action of the NLRP3 inflammation corpusculum in single apoplexy
1, experimental method
The activation of NLRP3 inflammation corpusculum needs two signal stimulus, and first stimulus signal (Signal 1) makes NLRP3
Inflammation corpusculum, which carries out modification, increases the mRNA expression of NLRP3, and NFKB plays emphatically the modification of NLRP3 inflammation corpusculum
It acts on, can be activated when the NLRP3 of modified encounters second inflammatory stimulus signal, and then cause a series of waterfall types scorching
Disease reaction.The present invention has detected the expression of NFkB albumen, the mRNA of NLRP3 and albumen respectively, small with clear NLRP3 inflammation
Mechanism of action of the body in single apoplexy.
2, experimental result
(Fig. 2) shown in two-way analysis of variance result, compared with undamaged right side cortex (R), left side (L) cortex apoplexy
Afterwards, the NFKB protein expression of WT mouse and NLRP3 knock out mice increases (P < 0.001, P < 0.01), but WT with
No significant difference (P > 0.05) between NLRP3KO mouse.NLRP3 albumen table compared with not damaging survey with right side, on the left of WT mouse
It is obviously increased (P>0.05) up to nothing, but the mRNA level in-site of NLRP3 dramatically increases (P<0.001).
Two, after apoplexy periinfarct NLRC4 and AIM2 expression
1, experimental method
Research shows that pathology damage of the NLRC4 and AIM2 inflammation corpusculum after ishemic stroke occupies important function, this hair
The bright expression that periinfarct NLRC4 and AIM2 after apoplexy are detected using western blot.
2, experimental result
(Fig. 2 .F&G) as the result is shown is had found compared with not damaging side with right side, WT mouse using dual factors variance statistic analysis
After NLRP3KO left cortical apoplexy, NLRC4 the and AIM2 inflammation corpusculum expression of periinfarct dramatically increases (P < 0.01；P<
0.001), but between two groups compare without obvious statistical difference (P > 0.05)
Three, the expression of ASC monomer, dimer and polymer
1, experimental method
It is constructed as the important composition between connection inflammation corpusculum and Caspase-1, ASC regulatory protein inflammation after a stroke
Starting and the loss of neuron play an important role, therefore further have detected the table of ASC monomer, dimer and polymer
It reaches.
2, experimental result
The results show that compared with not damaging opposite side, after the apoplexy of WT and NLRP3 knock out mice unilateral side, ASC monomer
It expresses no significant difference (P>0.05), and ASC dimer obviously increases (P<0.001), but without bright between WT and NLRP3KO mouse
Significant difference is different (P > 0.05), and expresses (Fig. 2 .H&I) without obvious ASC polymer.
Embodiment 3NLRP3 gene delection mitigates recidivity apoplexy cerebral injury and microglia reaction
Research finds that the probability that apoplexy again occurs after apoplexy is very big, and damage it is serious compared with apoplexy for the first time, but to its shadow
Sound Mechanism Study report is different, and mechanism is not yet clear.Influence of the inflammatory reaction generated after apoplexy for the first time to recidivity apoplexy is made
With being gradually taken seriously, research shows that there are ASC inflammation corpusculums in cerebrospinal fluid after cerebral injury, the final result of apoplexy can be predicted.Cause
This, further studies mechanism of action of the NLRP3 in recidivity apoplexy.
It is right using the preparation of same method after a week first using parietal cortex apoplexy on the left of photochemical model preparation mouse
Side parietal cortex apoplexy completes the preparation of recidivity apoplexy model.After a week, perfusion materials.
One, experimental method
The damage model of single apoplexy is constructed according to the method for embodiment 1.After a week, it is prepared using above-mentioned same method right
Side (opposite side) parietal cortex ischemic stroke model, as recidivity apoplexy model.
Ischemic stroke model constructs after a week, and using paraformaldehyde heart perfusion, coronal section is cut every 200 μm,
With a thickness of 10 μm；It samples and Nissl's staining is used to determine infarct lesion, during immunofluorescence dyeing and Wstern blot are tested and analyzed
The activation of Inflammatory Pathway after wind.
Two, experimental result
Using two-way analysis of variance the study found that in WT mouse, second stroke (right side, R) injury scores are (left for the first time
Side, L) increase (P < 0.001) after apoplexy, and in the second stroke of NLRP3KO mouse (right side, R) and apoplexy (left side, L) for the first time
Injury scores no significant difference (P > 0.05) (Fig. 1 .A& B).It is found using immunofluorescence dyeing, in WT mouse, second stroke
Quantity significantly increases (P < 0.01) apoplexy (left side, L) the microglia quantity of (right side, R) afterwards more for the first time afterwards, and
NLRP3KO mouse, twice the microglia quantity no significant difference (P > 0.05) after apoplexy.Further use immunofluorescence
Study on dyeing discovery, WT mouse second stroke (right side, R) M2 (CD-206 positive) microglia quantity apoplexy more for the first time afterwards
(left side, L) quantity reduces (P < 0.001) afterwards, and M1 (CD-16 positive) microglia quantity apoplexy (left side, L) more for the first time
Significantly increase afterwards (P < 0.001), and second of apoplexy (right side, R) of NLRP3 KO mouse M1/M2 type (CD-16/CD-206 afterwards
It is positive) microglia quantity more for the first time after (left side, L) apoplexy quantity without significant difference (P > 0.05) (Fig. 3 .E-H).
Embodiment 4NLRP3 gene delection mitigates the activation of Caspase-1 and IL-1 β after recidivity apoplexy
One, experimental method
For activation situation of the clear NLRP3 inflammation corpusculum after recidivity apoplexy, we are using western blot to WT
The inflammation corpusculum and inflammatory factor of peri-infarct tissue after mouse and NLRP3 knock out mice (NLRP3KO) bilateral apoplexy
Expression detected.
Further having detected WT mouse and NLRP3 knock out mice respectively, Caspase-1 albumen is split after apoplexy twice
The activation of solution and IL1- β albumen.
Two, experimental result
Compare with apoplexy for the first time (left side, L), the NLRP3 inflammation corpusculum expression of (right side, R) is bright after WT mouse second stroke
It is aobvious to increase (P < 0.001), and expression of the NLRP3KO mouse without obvious NLRP3 albumen.WT mouse and NLRP3KO mouse it is secondary
The expression of NLRC4 and AIM2 inflammation corpusculum between apoplexy and for the first time apoplexy without significant difference (P > 0.05) (Fig. 4 .A&B) and
No significant difference (P > 0.05) between WT and NLRP3 KO mouse.Compare afterwards with apoplexy for the first time (left side, L), WT mouse it is secondary
The expression of apoplexy (right side, R) ASC protein monomer and dimer afterwards is without obviously increasing (P > 0.05), but the expression of ASC polymer is aobvious
It writes and increases (P < 0.001), and the ASC monomer, dimer and polymer after the second stroke of NLRP3KO mouse and for the first time apoplexy
Express equal no significant difference (P > 0.05) (Fig. 4 .C &D)
Two-way analysis of variance cracks the Caspase-1 of state the results show that after WT mouse second stroke (right side, R)
Expression increases (P < 0.001, P < 0.001) after apoplexy more for the first time for expression with the IL1- β of the state of activation, and NLRP3 clpp gene
Except the Caspase-1 and state of activation of the cracking of mouse IL1- β expression apoplexy twice express no significant difference (P >
0.05).In addition, Caspase-1 that WT mouse and NLRP3KO mouse crack after (left side, L) apoplexy for the first time and the state of activation
The expression no significant difference (P > 0.05) of IL1- β, but the Caspase-1 that (right side, R) is cracked after NLRP3KO mouse second stroke
Expression with the IL1- β of the state of activation, which is expressed compared with WT mouse, significantly reduces (P < 0.001).
Claims (9)
1.NLRP3 gene and/or albumen are as the application for preventing and/or treating headstroke and complication medicine target spot.
The inhibitor of 2.NLRP3 gene and/or albumen is as the application for preventing and/or treating headstroke drug.
3.ASC gene and/or albumen are as the application for preventing and/or treating headstroke and complication medicine target spot.
The inhibitor of 4.ASC gene and/or albumen is as the application for preventing and/or treating headstroke drug.
5.NLRP3/ASC inflammation corpusculum is as the application for preventing and/or treating headstroke and complication medicine target spot.
The inhibitor of 6.NLRP3/ASC inflammation corpusculum is as the application for preventing and/or treating headstroke drug.
7. any application according to claim 1~6, which is characterized in that the headstroke is recurrent headstroke.
8. any application according to claim 1~6, which is characterized in that the complication is inflammatory reaction.
9. any application according to claim 1~6, which is characterized in that the complication is brain caused by inflammatory reaction
Damage.
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| US20100233168A1 (en) * | 2009-03-11 | 2010-09-16 | Alan Wanderer | Rationale for IL-1 Beta targeted therapy in sickle cell disease for ischemia-reperfusion induced complications |
| CN108601762A (en) * | 2016-02-08 | 2018-09-28 | 客乐谐控股株式会社 | Inflammatory body activates inhibitor |
| CN107648248A (en) * | 2017-09-18 | 2018-02-02 | 暨南大学 | Lamp-dish flower acetic is preparing the application in treating NLRP3 relevant disease medicines |
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