CN104258398B - DRD1 and its activator are preparing the purposes in treating NLRP3 inflammation corpusculum related inflammation disease medicaments - Google Patents
DRD1 and its activator are preparing the purposes in treating NLRP3 inflammation corpusculum related inflammation disease medicaments Download PDFInfo
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Abstract
The invention discloses DRD1 albumen and its activator to prepare the purposes in treating NLRP3 inflammation corpusculum related inflammation disease medicaments.The albumen induces the degraded of inflammation corpusculum important component NLRP3 albumen in the presence of its endogenous agonist dopamine, the effect for suppressing that the inflammatory factors such as the β of IL 1 are ripe and secrete is played, so as to suppress the activation of NLRP3 inflammation corpusculums.The invention provides application of the DRD1 albumen as the target spot of NLRP3 inflammation corpusculum related inflammations disease (peripheral-system inflammation and cental system inflammation), can be used for the medicine of screening research and development NLRP3 inflammation corpusculum related inflammation diseases.Present invention also offers the artificial synthesized activator A 68930 of DRD1 albumen, the hydrochloride of (±) SKF 38393, the Chloro PB hydrobromide of SKF 89626 and 6 antiphlogistic effects, for these activators or its active component prepares anti-inflammatory drug and treatment diseases associated with inflammation provides original thinking and theories integration.
Description
Technical field
The present invention relates to DRD1 albumen and its activator to prepare treatment NLRP3 inflammation corpusculum related inflammation disease medicaments
In purposes.
Background technology
NLRP3 inflammation corpusculums are a kind of polyprotein compounds of intracellular, and it is mainly by NLRP3 (NACHT, LRR and
PYD domains-containing protein 3), ASC (apoptosis-associated speck-like protein
Containing a CARD) and tri- kinds of protein of caspase-1 formed.NLRP3 inflammation corpusculum and its signal path are starting
There is vital effect (Davis et al., 2011, Annu Rev in inflammation and its related immune answering
Immunol；Marltinon et al.,2009,Immunol).So activation of NLRP3 inflammation corpusculums, it is necessary to by accurate
And strict regulation and control.If the imbalance of inflammatory reaction, such as excessively amplification or persistently presence, can all trigger body injury, very
To the generation for causing diseases associated with inflammation.The abnormal activation of NLRP3 inflammation corpusculums can promote a variety of mankind's major diseases to send out
Exhibition, such as type-II diabetes (Zhou et al., 2010, Nat Immunol；Yan et al., 2013, Immunity) it is, dynamic
Pulse atherosclerosis (Duewell et al., 2010, Nature), gout (Martinon et al., 2006, Nature), enteritis
(Siegmund et al., 2001, Proc Natl Acad Sci USA.), hepatitis (Negash et al., 2013, PLoS
Pathog), skin sunburn (the Watanabe et that silicosis (Dostert et al., 2008, Science), ultraviolet are induced
al.,2007,J Investig D ermatol.；Feldmeyer et al., 2007, Curr Biol) and contact it is super quick anti-
Answer (Watanabe et al., 2007, J Investig Dermatol；Sutterwala et al.,2006,Immunity)、
Septicopyemia (Mao et al., 2013, Cell Res), even tumour (Ghiringhelli et al., 2009, Nat
Med；Allen et al., 2010, J Exp Med) and nerve degenerative diseases (Halle et al., 2008, Nat
Immunol) etc..
Since the inflammatory reaction of NLRP3 inflammation corpusculum and its induction participates in the generating process of a variety of mankind's major diseases,
NLRP3 inflammation corpusculum is naturally with regard to as the potential target spot prevented and treated to these diseases.At present the field basis and
Clinical research is concentrated mainly on using IL-1 acceptors neutrality antibody or antagonist to block IL-1 to be combined with its acceptor, so as to
Suppress inflammatory reaction (Dinarello et al., 2012, Annu Rev Immunol).But although IL-1 is NLRP3 activation
The effector molecule of a caused very important transmitting inflammation afterwards, but be not unique molecule.NLRP3 inflammation corpusculum is lived
Change can also produce the inflammatory mediator such as IL-18, IL-33, HMGB1 and the lipid material of proinflammatory disease, thus it is possible to suppress IL-1 activity
Good antiphlogistic effects (von Moltke et al., 2012, Nature) can't be produced.With for IL-1 and its acceptor
Intervention means are compared, and the intervention means of targeting NLRP3 inflammation corpusculum activation then can fundamentally suppress IL-1, IL-18, IL-33
With the inflammatory reaction of the mediation such as HMGB1, it is possible to obtain more preferable antiphlogistic effects.But so far, also without effective pin
The anti-inflammatory drug of NLRP3 inflammation corpusculums is come out.
Dopamine (DA) is a kind of important neurotransmitter.It can not only adjust the behavior of body, motion, endocrine,
Angiocarpy, kidney and digestive system function, and it is also used as the molecule bridge of nervous system and immune system.In almost institute
In some immunocytes, dopamine receptor has expression.Dopamine receptor has found five hypotypes altogether, is Drd1, Drd2 respectively,
Drd3, Drd4 and Drd5, wherein, Drd1 and Drd5 are referred to as D1 sample dopamine receptors, and Drd2, Drd3 and Drd4 are referred to as D2
Sample dopamine receptor.Multinomial report points out, dopamine can effectively adjust the activation of immunocyte by its each acceptor,
Secretion (Beck the et al., 2004, Crit Care of propagation and cell factor；Shao et al., 2013, Nature).Separately
Outside, dopamine receptor Drd2 defects mouse suffers from neuroinflamation, and this has prompted dopamine and its downstream signaling pathway to have anti-inflammatory
Function (Shao et al., 2013, Nature).Moreover, it is reported that, dopamine continuous low-level is sent out with Parkinson's disease
Immune system disorder during disease and inflammation of the central nervous system it is closely related (Wullner＆Klockgether, 2003, J
Neurol；Meredith et al., 2005, Immunology).These work prompt us, and dopamine is likely to anti-inflammatory
Function, either in periphery, or in cental system.But the basis in the field and clinical research are concentrated mainly on use at present
The activator of dopamine and dopamine receptor is treated and alleviates Parkinson's disease, also without being used to treat NLRP3 inflammation corpusculums
The successful example of related diseases associated with inflammation.
We study the target receptor of dopamine by determining inhibitory action of the dopamine to NLRP3 inflammation corpusculums.DOPA
The determination of amine anti-inflammatory target spot, can be provided for the related diseases associated with inflammation of a variety of NLRP3 inflammation corpusculums treatment thoughts and theory according to
According to, more can for screen and prepare treatment diseases associated with inflammation medicine provide molecular target and preparation thinking.
The content of the invention
It is an object of the invention to provide DRD1 albumen as the diseases associated with inflammation for preparing treatment NLRP3 inflammation corpusculum correlation
Medicinal application, there is provided the activator dopamine of DRD1 albumen, A-68930, (±)-SKF-38393hydrochloride, SKF
The medicine of 89626 diseases associated with inflammation related as treatment NLRP3 inflammation corpusculums to 6-Chloro-PB hydrobromide
The application of active component.
Specifically, the first aspect of the invention provides DRD1 albumen and is used to prepare treatment diseases associated with inflammation medicine or examination
Agent box, as treatment NLRP3 inflammation corpusculum related inflammation diseases medicine target spot and for screen treat NLRP3 inflammation
The purposes of corpusculum related inflammation disease medicament.
In a preferred embodiment, the amino acid sequence of the DRD1 albumen such as SEQ ID No：Shown in 1.
In a preferred embodiment, the diseases associated with inflammation is the related diseases associated with inflammation of NLRP3 inflammation corpusculum.
In a preferred embodiment, the diseases associated with inflammation be type-II diabetes, atherosclerosis, gout,
Skin sunburn, contact hypersensitivity, septicopyemia tumour or the nervus retrogression that enteritis, hepatitis, silicosis, ultraviolet are induced
Disease.
In a preferred embodiment, the inflammation includes cental system inflammation and peripheral-system inflammation, is preferably
Periphery Abdominal inflammation.
The second aspect of the invention provide DRD1 albumen activator be used for prepare treatment diseases associated with inflammation medicine or
The purposes of kit.
In a preferred embodiment, the activator passes through DRD1 protein exhibits therapeutic actions.
In a preferred embodiment, the diseases associated with inflammation is the related diseases associated with inflammation of NLRP3 inflammation corpusculum.
In a preferred embodiment, the diseases associated with inflammation be type-II diabetes, atherosclerosis, gout,
Skin sunburn, contact hypersensitivity, septicopyemia tumour or the nervus retrogression that enteritis, hepatitis, silicosis, ultraviolet are induced
Disease.
In a preferred embodiment, the inflammation includes cental system inflammation and peripheral-system inflammation, is preferably
Periphery Abdominal inflammation.
In a preferred embodiment, the activator of the DRD1 albumen be selected from dopamine, A-68930, (±)-
SKF-38393hydrochloride, SKF 89626 and 6-Chloro-PB hydrobromide or its combination.
In a preferred embodiment, the activator of the DRD1 albumen can suppress IL-1 β secretion, pass through it
Acceptor DRD1 suppresses the activation of NLRP3 inflammation corpusculums, induces NLRP3 degraded on film, so as to realize treatment diseases associated with inflammation, especially
It is the related diseases associated with inflammation of NLRP3 inflammation corpusculum.
Brief description of the drawings
Fig. 1 DA suppress IL-1 β secretion
Fig. 2 DA suppress the activation of NLRP3 inflammation corpusculums
Fig. 3 DA suppress the activation of NLRP3 inflammation corpusculums by acceptor DRD1 on its film
Fig. 4 DA induce NLRP3 degraded
Fig. 5 DA induce NLRP3 degraded by acceptor DRD1 on its film
Fig. 6 DRD1 activators suppress the activation of NLRP3 inflammation corpusculums
Fig. 7 DRD1 activators A-68930 induces NLRP3 degraded
Fig. 8 DA can suppress the activation of microglia inflammation corpusculum by DRD1
Fig. 9 DA can suppress the activation of astroglia inflammation corpusculum by DRD1
Figure 10 DRD1 albumen can suppress cental system inflammation
Figure 11 DRD1 activators A-68930 can suppress cental system inflammation
Figure 12 DA can suppress the peripheral-system inflammation of LPS inductions
Figure 13 DRD1 albumen can suppress the peripheral-system inflammation of LPS inductions
Figure 14 DA can suppress the periphery Abdominal inflammation of MSU inductions
Figure 15 DRD1 albumen can suppress the periphery Abdominal inflammation of MSU inductions
Embodiment
Following example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment side in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is from often unless otherwise specified
Rule biochemical reagents shop is commercially available.
BMDM (macrophage of derived from bone marrow)：Preparation method is shown in document (Yan et al., 2013, Immunity).
Microglia：Preparation method is shown in document (Shao et al., 2012, Nature).
Astroglia：Preparation method is shown in document (Shao et al., 2012, Nature).
Drd1 defect mouse:Source see document (Martinon et al., 2006, Nature；Kelly et al., 1997,
Neuron；Drago et al., 1994, Proc Natl Acad Sci USA).
Anti- mouse IL-1 β antibody:R&D,AF-401-NA.
Anti- mouse caspase-1 antibody:Adipogen,AG-20B-0042.
Anti- NLRP3 antibody:Adipogen,AG-20B-0014.
Anti- β-actin antibody:Abmart,P30002.
Anti- ASC antibody:Santa Cruz,sc-22514.
DA (dopamine)：Sigma, H8502.
Ultrapure LPS：Invivogen, tlrl-3pelps.
Nigericin (nigericin)：Sigma, N7143.
MSU (uric acid crystal)：Sigma, U0881.
ATP：Sigma, A1852.
Imject-Alum (aluminium)：Pierce Biochemicals, 77161.
MPTP：Sigma, M0896.
LPS：Sigma, L2630.
A-68930：Sigma, A8852.
(±)-SKF-38393hydrochloride：Sigma, D047.
SKF 89626：Sigma, S3066.
6-Chloro-PB hydrobromide：Sigma, S143.
Embodiment 1, DA and its activator suppress the activation of macrophage NLRP3 inflammation corpusculums in vitro
First, DA suppresses IL-1 β secretion
1, first day, BMDM is assigned on 12 orifice plates, each hole 5*105Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.It is divided into five groups, every group of three multiple holes.
First group, negative control group.
Second group, final concentration of 10 μM of Nigericin half an hour is added per hole.
3rd group, final concentration of 0.15mM tri- hours of DA are added per hole, add final concentration of 10 μM
Nigericin half an hour.
4th group, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration of 10 μM
Nigericin half an hour.
5th group, final concentration of 0.25mM tri- hours of DA are added per hole, add final concentration of 10 μM
Nigericin half an hour.
3, the cell after step 2 processing, collects cell conditioned medium (SN) and cell pyrolysis liquid (Input) is resisted with anti-mouse IL-1 β
Body and anti-mouse caspase-1 antibody carry out western blotting analyses.As a result see that (swimming lane 1 is first group to Fig. 1, and swimming lane 2 is
Second group, swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group, and swimming lane 5 is the 5th group).
Under the first signal LPS and secondary signal Nigericin collective effect, the activation of inflammation corpusculum, p20 and IL-1 β
Maturation is simultaneously secreted among supernatant (swimming lane 2), DA addition, restrained effectively p20 and IL-1 β maturations and secretion, also, this
Kind effect is in dose dependent (swimming lane 3, swimming lane 4 and swimming lane 5).
2nd, DA suppresses the activation of NLRP3 inflammation corpusculums
1, first day, BMDM is assigned on 12 orifice plates, each hole 5*105Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.It is divided into ten groups, every group of three multiple holes.
First group, negative control group.
Second group, final concentration of 150 μ g/ml MSU is added per hole six hours.
3rd group, final concentration of 10 μM of Nigericin half an hour is added per hole.
4th group, final concentration of 2.5mM ATP half an hour is added per hole.
5th group, final concentration of 300 μ g/ml Alum is added per hole six hours.
6th group, final concentration of 0.2mM tri- hours of DA are added per hole.
7th group, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration of 150 μ g/ml MSU
Six hours.
8th group, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration of 10M Nigericin
Half an hour.
9th group, final concentration of 0.2mM tri- hours of DA are added per hole, the ATP half for adding final concentration of 2.5mM is small
When.
Tenth group, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration of 300 μ g/ml's
Alum six hours.
3, the cell after step 2 processing, collects cell conditioned medium (SN) and cell pyrolysis liquid (Input) is resisted with anti-mouse IL-1 β
Body and anti-mouse caspase-1 antibody carry out western blotting analyses.As a result see that (swimming lane 1 is first group to Fig. 2, and swimming lane 2 is
Second group, swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group, and swimming lane 5 is the 5th group, and swimming lane 6 is the 6th group, and swimming lane 7 is the 7th group,
Swimming lane 8 is the 8th group, and swimming lane 9 is the 9th group, and swimming lane 10 is the tenth group).
Nigericin, MSU, ATP and Alum be almost it is known it is all can activate NLRP3 inflammation corpusculums second letter
Number.Under the collective effect of the first signal LPS and these secondary signals, the activation of inflammation corpusculum, p20 and IL-1 β are ripe and secrete
To among supernatant (swimming lane 2, swimming lane 3, swimming lane 4 and swimming lane 5), DA addition, it restrained effectively what these stimulants were induced
P20 and IL-1 β are ripe and secrete (swimming lane 7, swimming lane 8, swimming lane 9 and swimming lane 10).
3rd, DA suppresses the activation of NLRP3 inflammation corpusculums by acceptor DRD1 on its film
1, first day, the BMDM of wild mouse (WT) and Drd1 defect mouse is assigned on 12 orifice plates, each hole 5*105It is individual thin
Born of the same parents.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.Two kinds of BMDM are respectively divided into three groups, totally six groups, every group of three multiple holes.
First group, WT mouse BMDM, negative control group.
Second group, WT mouse BMDM, final concentration of 10 μM of Nigericin half an hour is added per hole.
3rd group, WT mouse BMDM, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration of 10 μM
Nigericin half an hour.
4th group, Drd1 defect mouse BMDM, negative control group.
5th group, Drd1 defect mouse BMDM, final concentration of 10 μM of Nigericin half an hour is added per hole.
6th group, Drd1 defect mouse BMDM, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration
For 10M Nigericin half an hour.
3, the cell after step 2 processing, collects cell conditioned medium (SN) and cell pyrolysis liquid (Input) is resisted with anti-mouse IL-1 β
Body and anti-mouse caspase-1 antibody carry out western blotting analyses.As a result see that (swimming lane 1 is first group to Fig. 3, and swimming lane 2 is
Second group, swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group, and swimming lane 5 is the 5th group, and swimming lane 6 is the 6th group).
For the macrophage of wild-type mice, DA addition, the p20 for effectively inhibiting Nigericin to be induced
With IL-1 β maturations and secretion (swimming lane 3), that is to say, that the effective activation for inhibiting NLRP3 inflammation corpusculums.And for Drd1
The macrophage of defect mouse, DA addition, it can not effectively suppress the activation (swimming lane 6) of NLRP3 inflammation corpusculums.Therefore, DA
It is to play a part of suppression NLRP3 inflammation corpusculum by acceptor DRD1 on its film to activate.
4th, DA induces NLRP3 degraded
1, first day, BMDM is assigned on 12 orifice plates, each hole 2*105Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.It is divided into six groups, every group of three multiple holes.
First group, negative control group.
Second group, final concentration of 0.15mM tri- hours of DA are added per hole.
3rd group, final concentration of 0.2mM tri- hours of DA are added per hole.
4th group, final concentration of 0.25mM tri- hours of DA are added per hole.
5th group, final concentration of 0.2mM two hours of DA are added per hole.
6th group, final concentration of 0.2mM tetra- hours of DA are added per hole.
3, the cell after step 2 processing, collect the anti-NLRP3 antibody of cell pyrolysis liquid, anti-ASC antibody, anti-mouse
Caspase-1 antibody and anti-β-actin antibody carry out western blotting analyses.As a result seeing Fig. 4 A and Fig. 4 B, (Fig. 4 A swim
Road 1 is first group, and swimming lane 2 is second group, and swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group.Fig. 4 B swimming lanes 1 are first group, swimming lane 2
For the 5th group, swimming lane 3 is the 3rd group, and swimming lane 4 is the 6th group).
DA can cause the NLRP3 in BMDM to degrade, also, this degraded is that dosage and time dependence is presented.This
It can prove, DA can induce NLRP3 to degrade, and this may is that the reason for DA suppresses the activation of NLRP3 inflammation corpusculum.
Five DA induce NLRP3 degraded by acceptor DRD1 on its film
1, first day, the BMDM of wild mouse (WT) and Drd1 defect mouse is assigned on 12 orifice plates, each hole 2*105It is individual thin
Born of the same parents.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.Two kinds of BMDM are respectively divided into two groups, totally four groups, every group of three multiple holes.
First group, WT mouse BMDM, negative control group.
Second group, WT mouse BMDM, final concentration of 0.2mM tri- hours of DA are added per hole.
3rd group, Drd1 defect mouse BMDM, negative control group.
4th group, Drd1 defect mouse BMDM, final concentration of 0.2mM tri- hours of DA are added per hole.
3, the cell after step 2 processing, collect the anti-NLRP3 antibody of cell pyrolysis liquid, anti-ASC antibody, anti-mouse
Caspase-1 antibody and anti-β-actin antibody carry out western blotting analyses.As a result see Fig. 5 (swimming lane 1 be first group,
Swimming lane 2 is second group, and swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group).
DA has five acceptors, DRD1, DRD2, DRD3, DRD4 and DRD5 on film, next, we see whether DA is logical
Cross DRD1 and exercise its function.DA can induce the degraded of NLRP3 in wild mouse BMDM, still, in Drd1 defect mouse, DA's
This effect is just without therefore, DA is worked by acceptor Drd1 on its film.
6th, DRD1 activators suppress the activation of NLRP3 inflammation corpusculums
1, first day, BMDM is assigned on 12 orifice plates, each hole 5*105Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.It is divided into 15 groups, every group of three multiple holes.
First group, negative control group.
Second group, final concentration of 10 μM of Nigericin half an hour is added per hole.
3rd group, 10 μM final concentration of of tri- hours of A-68930 are added per hole, add final concentration of 10 μM
Nigericin half an hour.
4th group, final concentration of 20 μM of tri- hours of A-68930 are added per hole, add final concentration of 10 μM
Nigericin half an hour.
5th group, final concentration of 30 μM of tri- hours of A-68930 are added per hole, add final concentration of 10 μM
Nigericin half an hour.
6th group, positive controls.Final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration of 10 μ
M Nigericin half an hour.
7th group, final concentration of 20 μM of three hours of (±)-SKF-38393hydrochloride are added per hole, then are added
Enter final concentration of 10 μM of Nigericin half an hour.
8th group, final concentration of 40 μM of three hours of (±)-SKF-38393hydrochloride are added per hole, then are added
Enter final concentration of 10 μM of Nigericin half an hour.
9th group, final concentration of 100 μM of three hours of (±)-SKF-38393hydrochloride are added per hole, then
Add final concentration of 10 μM of Nigericin half an hour.
Tenth group, final concentration of 20 μM of 89,626 3 hours of SKF are added per hole, add final concentration of 10 μM
Nigericin half an hour.
11st group, final concentration of 40 μM of 89,626 3 hours of SKF are added per hole, add final concentration of 10 μM
Nigericin half an hour.
12nd group, final concentration of 100 μM of 89,626 3 hours of SKF are added per hole, add final concentration of 10 μM
Nigericin half an hour.
13rd group, final concentration of 20 μM of tri- hours of 6-Chloro-PB hydrobromide are added per hole, then are added
Enter final concentration of 10 μM of Nigericin half an hour.
14th group, final concentration of 40 μM of tri- hours of 6-Chloro-PB hydrobromide are added per hole, then are added
Enter final concentration of 10 μM of Nigericin half an hour.
15th group, final concentration of 100 μM of tri- hours of 6-Chloro-PB hydrobromide are added per hole, then are added
Enter final concentration of 10 μM of Nigericin half an hour.
3, the cell after step 2 processing, collects cell conditioned medium (SN) and cell pyrolysis liquid (Input) is resisted with anti-mouse IL-1 β
Body and anti-mouse caspase-1 antibody carry out western blotting analyses.As a result see that (Fig. 6 A swimming lanes 1 are the to Fig. 6 A and Fig. 6 B
One group, swimming lane 2 is second group, and swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group, and swimming lane 5 is the 5th group, and swimming lane 6 is the 6th group；)
Fig. 6 B swimming lanes 1 are first group, and swimming lane 2 is second group, and swimming lane 3 is the 6th group, and swimming lane 4 is the 7th group, and swimming lane 5 is the 8th group, swimming
Road 6 is the 9th group, and swimming lane 7 is the tenth group, and swimming lane 8 is the 11st group, and swimming lane 9 is the 12nd group, and swimming lane 10 is the 13rd group, swimming
Road 11 is the 14th group, and swimming lane 12 is the 15th group).
As shown in Figure 6A, under the first signal LPS and secondary signal Nigericin collective effect, the activation of inflammation corpusculum,
P20 and IL-1 β are ripe to be simultaneously secreted among supernatant (swimming lane 2), A-68930 addition, restrained effectively p20 with IL-1 β into
Ripe and secretion, also, this effect is in dose dependent (swimming lane 3, swimming lane 4 and swimming lane 5).Fig. 6 B have studied other several
DRD1 activator (±)-SKF-38393hydrochloride, SKF 89626 and 6-Chloro-PB hydrobromide.
The addition of these activators, can restrained effectively that p20 and IL-1 β are ripe and secretion, also, this effect be in dosage according to
Rely property (swimming lane 4, swimming lane 5 and swimming lane 6 show (±)-SKF-38393hydrochloride inhibition；Swimming lane 7, swimming
Road 8 and swimming lane 9 show SKF 89626 inhibition；Swimming lane 10, swimming lane 11 and swimming lane 12 show 6-Chloro-PB
Hydrobromide inhibition).
Seven DRD1 activators A-68930 induce NLRP3 degraded
1, first day, BMDM is assigned on 12 orifice plates, each hole 2*105Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.It is divided into six groups, every group of three multiple holes.
First group, negative control group.
Second group, final concentration of 20 μM of tri- hours of A-68930 are added per hole.
3rd group, final concentration of 30 μM of tri- hours of A-68930 are added per hole.
4th group, final concentration of 40 μM of tri- hours of A-68930 are added per hole.
5th group, final concentration of 30 μM of two hours of A-68930 are added per hole.
6th group, final concentration of 30 μM of tetra- hours of A-68930 are added per hole.
3, the cell after step 2 processing, collect the anti-NLRP3 antibody of cell pyrolysis liquid, anti-ASC antibody, anti-mouse
Caspase-1 antibody and anti-β-actin antibody carry out western blotting analyses.As a result seeing Fig. 7 A and Fig. 7 B, (A of figure seven swims
Road 1 is first group, and swimming lane 2 is second group, and swimming lane 3 is the 3rd group, and swimming lane 4 is the 4th group.Fig. 7 B swimming lanes 1 are first group of swimming lane 2
For the 5th group, swimming lane 3 is the 3rd group, and swimming lane 4 is the 6th group).
DRD1 activators A-68930 can cause the NLRP3 in BMDM to degrade, also, identical with DA, and this degraded is to be in
Existing dosage and time dependence.
Embodiment 2, DRD1 albumen can suppress cental system inflammation
First, DA can suppress the activation of microglia inflammation corpusculum by DRD1
1, first day, the microglia of wild mouse (WT) and Drd1 defect mouse is assigned on 12 orifice plates, each hole 1*
106Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.Two kinds of microglias are respectively divided into three groups, totally six groups, every group of three multiple holes.
First group, WT mouse microglias, negative control group.
Second group, WT mouse microglias, the Nigericin half an hour of final concentration of 10 μM of addition per hole.
3rd group, WT mouse microglias, final concentration of 0.2mM tri- hours of DA are added per hole, add final concentration
For 10 μM of Nigericin half an hour.
4th group, Drd1 defect mouse microglias, negative control group.
5th group, Drd1 defect mouse microglias, the Nigericin half an hour of final concentration of 10 μM of addition per hole.
6th group, Drd1 defect mouse microglias, final concentration of 0.2mM tri- hours of DA are added per hole, are added
Final concentration of 10 μM of Nigericin half an hour.
3, the cell after step 2 processing, collect the survey that cell conditioned medium carries out the cell factors such as IL-1 β with ELISA method
It is fixed.As a result Fig. 8 is seen.
For the microglia of wild-type mice, DA addition, effectively inhibit what Nigericin was induced
IL-1 β secretion, that is to say, that the effective activation for inhibiting NLRP3 inflammation corpusculums.And for the small colloid of Drd1 defect mouse
Cell, DA addition, it can not effectively suppress the activation of NLRP3 inflammation corpusculums.Therefore, it is similar with macrophage, in small glue
Among cell plastid, DA is also to play a part of suppression NLRP3 inflammation corpusculum by acceptor DRD1 on its film to activate.
2nd, DA can suppress the activation of astroglia inflammation corpusculum by DRD1
1, first day, the astroglia of wild mouse (WT) and Drd1 defect mouse is assigned on 12 orifice plates, Mei Gekong
1*106Individual cell.
2, second day, supernatant is first sucked, the opti-MEM that 500 μ l contain ultra-LPS (500ng/ml), processing are added per hole
Three hours.Two kinds of astroglias are respectively divided into three groups, totally six groups, every group of three multiple holes.
First group, WT mouse astroglias, negative control group.
Second group, WT mouse astroglias, the Nigericin half an hour of final concentration of 10 μM of addition per hole.
3rd group, WT mouse astroglias, final concentration of 0.2mM tri- hours of DA are added per hole, are added dense eventually
Spend the Nigericin half an hour for 10 μM.
4th group, Drd1 defect mouse astroglias, negative control group.
5th group, Drd1 defect mouse astroglias, the Nigericin half an hour of final concentration of 10 μM of addition per hole.
6th group, Drd1 defect mouse astroglias add final concentration of 0.2mM tri- hours of DA per hole, then add
Enter final concentration of 10 μM of Nigericin half an hour.
3, the cell after step 2 processing, collect the survey that cell conditioned medium carries out the cell factors such as IL-1 β with ELISA method
It is fixed.As a result Fig. 9 is seen.
For the astroglia of wild-type mice, DA addition, effectively inhibit what Nigericin was induced
IL-1 β secretion, that is to say, that the effective activation for inhibiting NLRP3 inflammation corpusculums.And for the star glue of Drd1 defect mouse
Cell plastid, DA addition, it can not effectively suppress the activation of NLRP3 inflammation corpusculums.Therefore, it is thin with macrophage and small colloid
Born of the same parents are similar, and among astroglia, DA is also to play suppression NLRP3 inflammation corpusculum by acceptor DRD1 on its film to activate
Effect.
Three DRD1 albumen can suppress cental system inflammation
1, first day, the wild mouse (WT) and Drd1 defect mouse of selection 8~12 weeks.Two kinds of mouse are respectively divided into two groups, every group three
Only, totally four groups.
First group, wild-type mice, PBS is as negative control for intraperitoneal injection, every two hours injects once, co-injection five times.
Second group, wild-type mice, MPTP is injected intraperitoneally, injection dosage 20mg/kg, every two hours injects once, altogether note
Penetrate five times.
3rd group, Drd1 defect mouse, PBS is as negative control for intraperitoneal injection, every two hours injects once, co-injection five
It is secondary.
4th group, Drd1 defect mouse, MPTP is injected intraperitoneally, injection dosage 20mg/kg, every two hours injects once, altogether
Injection five times.
2, second day, blood is collected with the method for pulling out eyeball, room temperature is placed half an hour, then low-speed centrifugal half an hour, is received
Collect serum.
3, the serum that step 2 obtains carries out the measure of the cell factors such as IL-1 β and IL-18 with ELISA method.As a result see
Figure 10.
The background cytokine levels of Drd1 defect mouse are similar with wild-type mice, still, under MPTP effects, Drd1 defects
The IL-1 β of mouse compare wild-type mice with proinflammatory cytokines such as IL-18 obvious rise, and this expression, DRD1 is in body
Suppress to serve important function in cental system inflammation, so, its missing, it can cause body Secretion of Inflammatory Factors is horizontal to rise
It is high.
Four DRD1 activators A-68930 can suppress cental system inflammation
1, first day, the wild mouse (WT) of selection 8~12 weeks.Mouse is divided into three groups, every group three.
First group, wild-type mice, PBS is as negative control for intraperitoneal injection, every two hours injects once, co-injection five times.
Second group, wild-type mice, MPTP is injected intraperitoneally, injection dosage 20mg/kg, every two hours injects once, altogether note
Penetrate five times.
3rd group, wild-type mice, A-68930 is injected intraperitoneally, injection dosage 5mg/kg, injects once within every eight hours, altogether
Injection is three times；Also, after first time injection A-68930 eight hours, it is injected intraperitoneally MPTP, injection dosage 20mg/kg, every two
Hour injects once, co-injection five times.
2, second day, blood is collected with the method for pulling out eyeball, room temperature is placed half an hour, then low-speed centrifugal half an hour, is received
Collect serum.
3, the serum that step 2 obtains carries out the measure of the cell factors such as IL-1 β and IL-18 with ELISA method.As a result see
Figure 11.
Under MPTP effects, the proinflammatory cytokines such as IL-1 β and IL-18 of mouse have obvious rise, and note simultaneously
Penetrate the proinflammatory cytokines such as DRD1 activators A-68930 mouse, IL-1 β and IL-18 to be remarkably decreased, this expression, DRD1
Activator A-68930 can effectively suppress the high expression for the inflammatory cytokine that MPTP is induced, that is to say, that DRD1 excitements
Agent A-68930 can suppress cental system inflammation.
Embodiment 3, DRD1 albumen can suppress peripheral-system inflammation
First, DA can suppress the peripheral-system inflammation of LPS inductions
1, the wild mouse (WT) of 8~12 weeks is chosen, mouse is divided into three groups, every group three.
2, first group, wild-type mice, PBS is as negative control for intraperitoneal injection
Second group, wild-type mice, LPS, injection dosage 20mg/kg is injected intraperitoneally
3rd group, wild-type mice, while LPS and DA is injected intraperitoneally, wherein, LPS injection dosage is 20mg/kg, DA's
Injection dosage is 50mg/kg
After 3, four hours, blood is collected with the method for pulling out eyeball, room temperature is placed half an hour, then low-speed centrifugal half an hour,
Collect serum.
4, the serum that step 3 obtains carries out the measure of the cell factors such as IL-1 β and IL-18 with ELISA method.As a result see
Figure 12.
Under LPS effects, the proinflammatory cytokines such as IL-1 β and IL-18 have an obvious rise, and DA addition can be with
Effectively suppress the level of these inflammatory factors, this expression, DA can effectively suppress the peripheral inflammation of LPS inductions.
2nd, DRD1 albumen can suppress the peripheral-system inflammation of LPS inductions
1, choose the wild mouse (WT) and Drd1 defect mouse of 8~12 weeks, two kinds of mouse are respectively divided into two groups, every group three, and totally four
Group.
2, first group, wild-type mice, PBS is as negative control for intraperitoneal injection
Second group, wild-type mice, LPS, injection dosage 20mg/kg is injected intraperitoneally
3rd group, Drd1 defect mouse, PBS is as negative control for intraperitoneal injection
4th group, Drd1 defect mouse, LPS, injection dosage 20mg/kg is injected intraperitoneally
After 3, four hours, blood is collected with the method for pulling out eyeball, room temperature is placed half an hour, then low-speed centrifugal half an hour,
Collect serum.
4, the serum that step 3 obtains carries out the measure of the cell factors such as IL-1 β and IL-18 with ELISA method.As a result see
Figure 13.
The background cytokine levels of Drd1 defect mouse are similar with wild-type mice, still, under LPS effects, Drd1 defects
The IL-1 β of mouse compare wild-type mice with proinflammatory cytokines such as IL-18 obvious rise, and this expression, DRD1 is in body
Suppress to serve important function in peripheral inflammation, so, its missing, the horizontal rise of body Secretion of Inflammatory Factors can be caused.
3rd, DA can suppress the periphery Abdominal inflammation of MSU inductions
1, the wild mouse (WT) of 8~12 weeks is chosen, mouse is divided into three groups, every group three.
2, first group, wild-type mice, PBS is as negative control for intraperitoneal injection
Second group, wild-type mice, MSU is injected intraperitoneally, injection dosage is every mouse 0.5mg
3rd group, wild-type mice, while MSU and DA is injected intraperitoneally, wherein, MSU injection dosage is every mouse 0.5mg,
DA injection dosage is 50mg/kg
After 3, six hours, 10mlPBS is squeezed into the abdominal cavity of mouse, and take peritoneal lavage fluid.
4, the peritoneal lavage fluid that step 3 obtains carries out the measure of the cell factors such as IL-1 β with ELISA method.As a result see
Figure 14.
In the presence of MSU, the proinflammatory cytokines such as IL-1 β have obvious rise, and DA addition can be effective
Suppression these inflammatory factors level, this expression, DA can effectively suppress MSU induction periphery Abdominal inflammation.
4th, DRD1 albumen can suppress the periphery Abdominal inflammation of MSU inductions
1, choose the wild mouse (WT) and Drd1 defect mouse of 8~12 weeks, two kinds of mouse are respectively divided into two groups, every group three, and totally four
Group.
2, first group, wild-type mice, PBS is as negative control for intraperitoneal injection
Second group, wild-type mice, MSU is injected intraperitoneally, injection dosage is every mouse 0.5mg
3rd group, Drd1 defect mouse, PBS is as negative control for intraperitoneal injection
4th group, Drd1 defect mouse, MSU is injected intraperitoneally, injection dosage is every mouse 0.5mg
After 3, six hours, 10mlPBS is squeezed into the abdominal cavity of mouse, and take peritoneal lavage fluid.
4, the peritoneal lavage fluid that step 3 obtains carries out the measure of the cell factors such as IL-1 β with ELISA method.As a result see
Figure 15.
The background cytokine levels of Drd1 defect mouse are similar with wild-type mice, still, under MSU effects, Drd1 defects
The proinflammatory cytokines such as the IL-1 β of mouse have obvious rise compared to wild-type mice, and this expression, DRD1 suppresses periphery in body
Important function is served in Abdominal inflammation, so, its missing, the horizontal rise of body Secretion of Inflammatory Factors can be caused.
Claims (4)
1.DRD1 albumen is used for the purposes for screening treatment NLRP3 inflammation corpusculum related inflammation disease medicaments, wherein the NLRP3
The related diseases associated with inflammation of inflammation corpusculum is type-II diabetes, atherosclerosis, gout, enteritis, hepatitis, silicosis, ultraviolet
Skin sunburn, contact hypersensitivity or the septicopyemia tumour induced, the amino acid sequence such as SEQ of the DRD1 albumen
ID No：Shown in 1.
The activator of 2.DRD1 albumen is used for the medicine or kit for preparing the related diseases associated with inflammation for the treatment of NLRP3 inflammation corpusculum
Purposes, wherein the related diseases associated with inflammation of the NLRP3 inflammation corpusculum is type-II diabetes, atherosclerosis, gout, intestines
Skin sunburn, contact hypersensitivity or the septicopyemia tumour that inflammation, hepatitis, silicosis, ultraviolet are induced.
3. purposes according to claim 2, the activator passes through DRD1 protein exhibits therapeutic actions.
4. purposes according to claim 2, the activator of the DRD1 albumen be selected from dopamine, A-68930, (±)-
SKF-38393hydrochloride, SKF 89626 and 6-Chloro-PB hydrobromide or its combination.
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