CN106511367A

CN106511367A - Application of C41 molecule in preparation of medicine for treating various different pathogen-associated molecular patterns mediated inflammatory reactions

Info

Publication number: CN106511367A
Application number: CN201610894022.6A
Authority: CN
Inventors: 李彦; 刘万成; 杨雪姣; 郑江
Original assignee: First Affiliated Hospital of TMMU
Current assignee: First Affiliated Hospital of TMMU
Priority date: 2016-10-13
Filing date: 2016-10-13
Publication date: 2017-03-22

Abstract

The invention relates to application of C41 molecule in preparation of a medicine for treating various different pathogen-associated molecular patterns mediated inflammatory reactions. According to the application, based on the C41 molecule targeting UNC93B protein, the C41 molecule has multiple immunosuppressive effects on membrane Toll-like receptor and NOD-like receptor mediated inflammatory reactions, so as to interfere with various different pathogen-associated molecular patterns mediated inflammatory reactions. Therefore, the C41 molecule has an adjuvant therapeutic effect on allergic diseases and autoimmune diseases and the like.

Description

C41 molecules are preparing the various Different Kinds of Pathogens model molecule inducing inflammatory reaction medicines for the treatment of Application in thing

Technical field

The present invention relates to field of pharmaceutical biology, more particularly to a kind of C41 molecules are in the various Different Kinds of Pathogens patterns of preparation treatment Application in numerator mediated inflammatory reaction medicine.

Background technology

After pathogenic microorganism infection body, by specific pattern recognition receptors (PRRs) and pathogen-associated molecular pattern (PAMPs) combine, quick activation inherent immunity system.Important PRRs includes Toll-like receptor (TLRs) and NOD sample receptors (NLRs)。

TLRs is mainly expressed in inherent immunity cell, such as dendritic cell, macrophage.According to TLRs cell positioning It is divided into two big class：One class includes TLR1,2,4,5,6,11, and they are mainly expressed in film surface, identification such as lipid, lipoprotein and egg The film constituent of the microorganisms such as white matter；It is another kind of including TLR3,7,8,9, they are predominantly located at intracellular vesicle, such as endoplasm Net, endosome etc., the nucleic acid of main identification pathogenic microorganism.TLR not only should in the inherent immunity for resisting pathogenic microorganism invasion Play a significant role in answering, and it connects inherent immunity and adaptive immunity reaction.

NLRP3 inflammation corpusculums are formed by NLRs family member's NLRP3 albumen, adaptor protein ASC and caspase-1 Individual multiprotein complex, can promote various inflammatory cytokines such as IL-1 β, IL-18 ripe and secretion, send out in inflammation after its activation Play a crucial role during life.

C41 is a kind of particular sequence nucleic acid fragment that applicant's early stage is filtered out by bioinformatics, and its sequence is shown in CN105541947A.At present, by state natural sciences fund (No.81373133) and ChongQing, Chongqing City city forward position and application foundation The subsidy of project key project (cstc2013jjB0128), further furthers investigate the effect of TLR9 blocker c41 molecules Mechanism.

The content of the invention

It is an object of the invention to provide a kind of C41 molecules are anti-in the various Different Kinds of Pathogens model molecule transmitting inflammations of preparation treatment Answer the application in medicine, the application is to act on UNC93B albumen based on C41 is molecular targeted, internal membranous type Toll-like receptor and The receptor-mediated inflammatory reaction of NOD samples has Multiple immunizations depression effect, so as to intervene various Different Kinds of Pathogens model molecule guiding Inflammatory reaction, thus, to anaphylactic disease and autoimmune disease etc. have auxiliary therapeutic action.

The technical scheme is that：

The anaphylactic disease and autoimmunity of C41 molecules inner membrance receptor mediation in treatment inhibition of innate immune system Application in disease medicament.

Further, C41 is molecular targeted acts on UNC93B albumen, so as to internal membranous type Toll-like receptor and NOD sample receptors The inflammatory reaction of mediation has Multiple immunizations depression effect.

Further, C41 molecules are received in the release of anticusp inflammation factor and NOD samples for preparing membranous type Toll-like receptor induction in alleviation Application in the medicine that the inflammation corpusculum of body mediation is formed.

Further, C41 molecules are being prepared by various Different Kinds of Pathogens model molecules by the struvite disease of inner membrance receptor induction Application in the medicine of disease.

Applicant observes the internalization process of CpG-c41 by immunofluorescence technique, flutters and grasps C41 with a kind of crucial transhipment , there is common location in kytoplasm in albumen-UNC93B1.UNC93B is a kind of multispan transmembrane domain protein, by with identification nucleic acid Between TLRs, physical property interacts, and controls the transhipment of TLRs, so as to affect downstream signal transmission.Simultaneously bacteria RNA must be according to Rely Caspase-1 is activated in UNC93B.The experiment of applicant shows, after interior membranous type TLRs and NLRs is activated, UNC93B albumen Internalization transhipment is participated in, but, in the process, C41 blocks the signal of various intracellular receptor mediations by targeting UNC93B albumen Transmission, so that reach Multiple immunizations depression effect.

The present invention does not only disclose Multiple immunizations depression effects of the C41 in congenital immunologic process, has pointed out targeting yet UNC93B, can intervene the inflammatory reaction of various Different Kinds of Pathogens model molecule mediations.This for membranous type TLRs in future therapeutic and The related anaphylactic disease of NLRs overactivities and autoimmune disease, there is provided new therapy target and strategy.

The following examples can explain the present invention, but not limit the present invention with this.

Description of the drawings

Fig. 1 is that the C41 specificitys suppress bone marrow macrophage TLR3,7/8,9 activation；

Fig. 2 is the formation and activation of the inflammation corpusculum that the nucleic acid fragment suppresses Jing RAW264.7 cells NLR mediations；

Fig. 3 is the nucleic acid fragment and UNC93B albumen common locations.

Specific embodiment

C41 sequences of the present invention and preparation method are shown in the patent application of applicant's Application No. 201610015280.2.

1 C41 specificitys of embodiment suppress bone marrow macrophage TLR3,7/8,9 activation

1.1 main materials, reagent and equipment：

1) laboratory animal：Balb/c mices (magnificent Fukang biotech inc, China).

2)M-CSF from mouse：Sigma

3) cell culture medium：DMEM (Gibco, Inc., USA).

4) Elisa test kits：Anti- Mus TNF-a, IL-6 (eBioscience, Inc., USA).

5) cell culture incubator：FORMA 3111.

6) microplate reader：All-wave length multi-function microplate reader (Thermo Scientific, USA).

1.2 experimental technique：Balb/c mouse femur medullary cells are taken from, 96 orifice plates (200 μ L/ holes) are added, M-CSF is dense eventually Degree 50ng/ml, puts 37 DEG C, 5%CO₂Incubator culture washed non-attached cell off using PBS after 2 days, added containing 50ng/ml The DMEM culture medium culturings of M-CSF were divided into the following group after changing the DMEM culture medium without M-CSF to the 4th day：DMEM groups, TLR3 agonist Poly (I：C) (5 μ l, 100 μ g/ml of final concentration) group, TLR4 agonist LPS (5 μ l, final concentration 100ng/ml) Group, TLR7/8 agonist R848 (5 μ l, 0.2 μ g/ml of final concentration) group, TLR9 agonist CpG-ODN1826 (5 μ l, 1 μ of final concentration M) group, above each group are all provided with 8 multiple holes.Then, each group separates 4 multiple holes, plus 5 μ l of culture fluid, used as placebo；Each group remaining 4 Multiple holes, add 5 μ l of c41 molecules (2 μM of final concentration) of the present invention as treatment group.After continuing culture 24h, supernatant is collected, is entered Row Elisa is detected, with each hole absorbance (OD determined at 450nm₄₅₀) value, obtain TNF-a, IL-6 concentration.

1.3 experimental result：Placebo group, TLR3,4,7,9 are normally activated, and stimulate the release of bone marrow macrophage cell a large amount of TNF-a and IL-6,；Treatment group, under medicine c41 molecular actions of the present invention, TNF-a, IL-6 level of TLR3,7,9 is received To significantly inhibiting (P ＜ 0.01), but TLR4 there are no depression effect.So, the medicine energy specificity effectively suppresses interior model TLRs is activated.As shown in Figure 1.

2 C41 of embodiment suppresses the formation and activation of the inflammation corpusculum of RAW264.7 cell Jing NLR mediations

2.1 main materials, equipment：

1) cell strain：RAW264.7 (ATCC, USA).

2) cell culture medium：DMEM (Gibco, Inc., USA).

3) cell culture incubator：FORMA 3111.

4) chemiluminescence imaging system：ChemioDoc XRS+(USA)

2.2 experimental technique：

2.2.1 main agents and activity

Reagent

Reagent	Final concentration	Dosage/hole	Source
				C41	10μM	20μl	Raw work
Poly(I：C)	100μg/ml	20μl	Ivivogen
				LPS	100ng/ml	20μl	Sigma
R848	1μg/ml	20μl	Ivivogen
				CpG-1826	3μM	20μl	Raw work
ATP	5mM	20μl	Sigma

Antibody

2.2.2 packet and stimulation

1) separation and culture of RAW264.7 cells

2) plating cells, 6 orifice plates, 4X10⁶/ ml, 2ml/ are per hole, and according to the form below institute grouping Processing makees liquid, add c41 after 12h With each TLR agonist, add ATP after 4h, sample after 0.5h.

Placebo group	DMEM	DMEM+Poly(I：C)	DMEM+LPS	DMEM+R848	DMEM+1826
						Treatment group	C41	C41+Poly(I：C)	C41+LPS	C41+R848	C41+1826

2.2.3 detection：

1) sample：Culture medium is absorbed, adds PBS to wash 2 times, finally plus 1ml PBS are put in 20min on ice, the big portion of cell Divide and come off.Scraped using cell, cell scraped down, 1000 turns/min 5min absorb PBS and add 120 μ l cell pyrolysis liquids, Ice bath 30min.12000 turns/min, 4 DEG C of 15min, stay supernatant.

2) detect protein concentration：Protein concentration is detected using BCA methods, applied sample amount is determined (loading total protein is 25 μ g)；

3) boiled egg is white：105 DEG C, 5min；

4) match somebody with somebody glue：With 13.5% separation gel；

5) loading：Applied sample amount according to determining is added to upper strata glue duct, and plus Marker.

Upper strata glue 70V 30min, lower floor glue 120V 90min；

6) transferring film：250mA 90min；

7) close：5%BSA closes 1h；

8) incubate one to resist：4 DEG C of incubator overnights.

9) wash film：5min, 3 times

10) incubate two to resist：1.5h

11) wash film：15min, 4 times；

12) expose：

2.3 experimental result：

Relative to placebo group, LPS, R848, CpG-1826 can effectively raise the expression of NLRP3, and can activate Inflammation corpusculum produces Caspase10, and causes IL-1beta to secrete；Treatment group, in medicine c41 molecular actions of the present invention Under, NLRP3, Caspase10 (Fig. 2A) of R848, CpG-1826 effect group and IL-1beta (Fig. 2 B) level are significantly pressed down System (P ＜ 0.01). as different from R848, CpG-1826 acts only on interior membrane receptor, and LPS may also act to receptor on film, So c41 there are no depression effect to LPS effect groups.So, c41 medicine energy specificity suppresses membranous type in cause of disease model molecule Jing The inflammation corpusculum activation of NLR mediations.

3 C41 of embodiment and UNC93B common locations.

3.1 main materials, reagent and equipment：

1) cell strain：RAW264.7 (ATCC, USA).

2) cell culture medium：DMEM (Gibco, Inc., USA)..

3) cell culture incubator：FORMA 3111.

4) laser co-focusing culture dish：20mm (NEST, USA)

5) laser confocal microscope：SLM 780 (Germany)

6)C41-cy3：Raw work is biological

7) rabbit anti-mouse UNC93B antibody：Abcam

3.2 experimental technique：

1) separation and culture of RAW264.7 cells；

2) plating cells, laser co-focusing culture dish, 1 × 10⁵/ ml, 0.5ml/ add CpG-1826 afterwards per hole, overnight, eventually 1 μM of concentration；Add c41-FAM, 2 μM of final concentration simultaneously.

1.5h absorbs culture medium, and PBS washes twice；

3) paraformaldehyde fixes 10min, and PBS washes 2 times；

4) 0.1%Triton perforation 5min, PBS wash 3 times, 5min/ time；

5) room temperature 5%BSA closing 1h；

6) concentration is added to be that 20 μ g/ml UNC93B and Tubulin mono- resist 50 μ l, 37 DEG C of 2h, PBS wash 3 times, 5min/ time；

7) goat antirabbit two is added to resist, 37 DEG C, 50min, PBS wash 3 times, 15min/ time；

8) take pictures under laser co-focusing.

3.3 experimental result：

As c41 can suppress the activation of various PRRs mediations, and the signal path in these different PRRs downstreams is not Together, point out the scorching link of c41 suppressions unrelated with downstream signaling pathway, and during should be at internalization transhipment.Then, we pass through Laser confocal microscope, observes and internalization transfer related protein, including clathrin, actin, UNC93B1 albumen Deng whether related to these albumen with the suppression function of verifying c41.

The result shows, after nonirritant c41 individually enters born of the same parents, we do not observe any common location (Fig. 3 A), have The meaning, after being stimulated using interior membranous type TLRs agonist, distributions of the UNC93b in endochylema has been spread, and and C41 The significant common location of generation (Fig. 3 B), as shown in Figure 3.Comparatively speaking, c41 (is schemed without common location with matched group tubulin albumen 3C).UNC93B1 is the new albumen for finding in recent years, still comprehensive to the understanding of its function, and previously reported confirmation UNC93B1 is one Kind chaperone can adjust all intracellulars TLRs and transport into lysosome, then depend on the approach of MyD88/TRIF to transmit letter Number.Thus illustrate, the motion of UNC93b after c41 individually enters born of the same parents, will not be excited, but, after interior membranous type PRRs is stimulated, UNC93B occurrence dynamics are called, and the specific binding with c41 just occur, so as to interference stimulation signal is downstream transmitted.

Claims

The anaphylactic disease and autoimmunity disease of 1.C41 molecules inner membrance receptor mediation in treatment inhibition of innate immune system Application in medicine.
2. application according to claim 1, it is characterised in that：C41 is molecular targeted to act on UNC93B albumen, so as to internal Membranous type Toll-like receptor and the receptor-mediated inflammatory reaction of NOD samples have Multiple immunizations depression effect.
3. application according to claim 1, it is characterised in that：C41 molecules membranous type Toll-like receptor in preparation is alleviated is lured Application in the medicine that the release of anticusp inflammation factor and the receptor-mediated inflammation corpusculum of NOD samples led is formed.
4. application according to claim 1, it is characterised in that：C41 molecules are being prepared by various Different Kinds of Pathogens model molecules Application in the medicine of diseases associated with inflammation is induced by inner membrance receptor.