CN107723304A - Applications of the PRKAR2A in inflammatory resolution - Google Patents

Applications of the PRKAR2A in inflammatory resolution Download PDF

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CN107723304A
CN107723304A CN201610651255.3A CN201610651255A CN107723304A CN 107723304 A CN107723304 A CN 107723304A CN 201610651255 A CN201610651255 A CN 201610651255A CN 107723304 A CN107723304 A CN 107723304A
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macrophage
protein active
prkar2a
gene
reagent
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余鹰
孔德平
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00

Abstract

The present invention relates to applications of the PRKAR2A in inflammatory resolution.Specifically, the present invention relates to a kind of method that external regulation and control macrophage polarizes to M1 types macrophage or M2 types macrophage, methods described to include：(1) type of macrophage D prostaglandin receptors 1 is regulated and controled, i.e. DP1, the step of gene expression or protein active；And/or the step of (2) regulation and control PRKAR2A gene expressions or protein active；And/or the step of (3) regulation and control IFN γ acceptor IFNGR2 gene expressions or protein active；And/or the expression of gene or the step of protein active in (4) regulation and control JAK2/STAT1 paths.The present invention also relates to corresponding inhibitor or the pharmaceutical applications of activator.

Description

Applications of the PRKAR2A in inflammatory resolution
Technical field
The present invention relates to applications of the PRKAR2A in inflammatory resolution.
Background technology
Prostaglandin (PG) D2It is that arachidonic acid (AA) passes through cyclo-oxygenase (COX) and PGD2Synzyme it is a series of anti- The bioactivity lipid amboceptor answered and generated.PGD2Its effect is played by activating D prostaglandin receptors (DP1 and DP2).Most Closely, PGD is proved on evidence2It is related to the regression of inflammation.For example, observed in the latter half (48h) of the inflammation of carrageenan induction COX-2 is expressed and PGD2Production increases again；Being handled with cox 2 inhibitor deteriorates inflammation digestion, adds external source PGD2It is reversible The deterioration.PGD2And PGE2Both can adjust lipoxin (LX) and cooperate with lipoxin to promote inflammatory resolution.Hematopoiesis PGD2Synzyme (H-PGDS) defect causes mouse inflammatory resolution to be damaged.But this area does not know PGD still2The molecular mechanism of the regression of mediation.
Macrophage plays an important role in the former innate immunity and acquired immunity are caused a disease in response, their transmitting inflammation mistakes Journey, disappeared by secreting anti-inflammatory cytokines and eliminating dead cell and matrix chip to participate in the activity of inflammation.Macrophage With plasticity and heterogeneity, this causes these cells that character mutation occurs when responding varying environment signal.Miocardial infarction (MI, a kind of sterile inflammatory reaction) is related to two kinds of different subgroups of macrophage in recovery process afterwards.In the process, MI Occurs within 1-3 days the macrophage (Ly6C of early stage response afterwardshighCD11b+CD206-), they express high-caliber M1 features base Cause, such as cachectin (TNF α), interleukin (IL) -6, Chemokines CC-C motifs part (CCL) 2, and nitric oxide Synzyme (NOS) 2, and the macrophage (Ly6C of later stage responselowCD11b+CD206+) then occur within 4-7 days after MI, it passes through The increase of M2 samples revision points (including IL-10, ARG1 and TGF β 1) is expressed and stimulates inflammatory resolution.
DP1 altimeters in monocyte/macrophage reach, but PGD2/ DP1 paths disappear in macrophage polarization and inflammation Effect in moving back is still unknown.
The content of the invention
First aspect present invention provides a kind of external regulation and control macrophage to M1 types macrophage or M2 type macrophages pole The method of change, methods described include：
(1) type of macrophage D prostaglandin receptors 1 is regulated and controled, i.e. DP1, the step of gene expression or protein active； And/or
(2) the step of regulating and controlling PRKAR2A gene expressions or protein active；And/or
(3) the step of regulating and controlling IFN γ acceptor IFNGR2 gene expressions or protein active；And/or
(4) expression of gene is regulated and controled in JAK2/STAT1 paths or the step of protein active.
In one or more specific embodiments, what methods described polarized to promote macrophage to M1 types macrophage Method, including：
(1) DP1 gene expressions or protein active are suppressed；And/or
(2) PRKAR2A gene expressions or protein active are suppressed；And/or
(3) IFN γ acceptor IFNGR2 gene expressions or protein active are promoted；And/or
(4) expression of gene or protein active in JAK2/STAT1 paths are promoted.
In one or more specific embodiments, what methods described polarized to promote macrophage to M2 types macrophage Method, including：
(1) DP1 gene expressions or protein active are promoted；And/or
(2) PRKAR2A gene expressions or protein active are promoted；And/or
(3) IFN γ acceptor IFNGR2 gene expressions or protein active are suppressed；And/or
(4) expression of gene or protein active in JAK2/STAT1 paths are suppressed.
In one or more specific embodiments, implement methods described by the following means：
(a) gene knockout；
(b) it is overexpressed gene；And/or
(c) activator or inhibitor are given.
Second aspect of the present invention provides following reagents and is preparing regulation and control macrophage to M1 types macrophage or M2 type macrophages Purposes in the medicine of polarization：
(1) reagent of the regulation and control type of macrophage D prostaglandin receptors 1, i.e. DP1, gene expression or protein active； And/or
(2) reagent of PRKAR2A gene expressions or protein active is regulated and controled；And/or
(3) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is regulated and controled；And/or
(4) expression of gene or the reagent of protein active in JAK2/STAT1 paths are regulated and controled.
In one or more specific embodiments, the medicine is used to promote macrophage to M1 type macrophages pole Change, the reagent includes：
(1) reagent of DP1 gene expressions or protein active is suppressed；And/or
(2) reagent of PRKAR2A gene expressions or protein active is suppressed；And/or
(3) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is promoted；And/or
(4) expression of gene or the reagent of protein active in JAK2/STAT1 paths are promoted；Or
In one or more specific embodiments, the medicine is used to promote macrophage to M2 type macrophages pole Change, the reagent includes：
(a) reagent of DP1 gene expressions or protein active is promoted；And/or
(b) reagent of PRKAR2A gene expressions or protein active is promoted；And/or
(c) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is suppressed；And/or
(d) expression of gene or the reagent of protein active in JAK2/STAT1 paths are suppressed.
In one or more specific embodiments, the medicine is used to treat inflammation, preferably benefits from macrophage The inflammation to be polarized to M2 types macrophage.
In one or more specific embodiments, the inflammation be selected from asthma, inflammatory bowel disease (including Crohn disease and burst Ulcer colitis), appendicitis, blepharitis, capillary bronchitis, bronchitis, bursal synovitis, cervicitis, cholangitis, cholecystitis, knot Enteritis, conjunctivitis, cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, small intestine colon Inflammation, epicondylitis, epididymitis, fascitis, fibrositis, gastritis, gastroenteritis, hepatitis, suppurative hidradenitis, laryngitis, mastitis, Meningitis, myelitis, myocarditis, myositis, ephritis, oaritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, Peritonitis, pharyngitis, pleurisy, phlebitis, pneumonia, rectitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, mouth Inflammation, synovitis, tendonitis, tonsillitis, uveitis, vaginitis, vasculitis or vulvitis.
In one or more specific embodiments, the reagent is selected from：DP1 activators, JAK2 inhibitor, STAT1 suppressions Preparation, PRKAR2A activators and IFNGR2 inhibitor.
In one or more specific embodiments, the reagent is nucleic acid, protein or micromolecular compound.
In one or more specific embodiments, the DP1 activators are BW245C.
In one or more specific embodiments, the JAK2 inhibitor is selected from：AT9283、AZD-1480、 Baricitinib or its phosphate, BMS-911543, CEP-33779, CYT387 or its mesylate or sulfate, GLP0634 And LY2784544.
In one or more specific embodiments, the STAT1 inhibitor is selected from：Fludarabine, Stattic, chlorine nitre Willow amine and nifuroxazide.
In one or more specific embodiments, the PRKAR2A activators are Foskolin.
In one or more specific embodiments, the IFNGR2 inhibitor is PRKAR2A or its activator.
Third aspect present invention provides PRKAR2A or IFNGR2 and regulates and controls macrophage to M1 type macrophages in screening as target spot The material or the application in the medicine for preparing treatment inflammation of cell or the polarization of M2 types macrophage.
Brief description of the drawings
Fig. 1：DP1 knocks out the M1 types polarization for promoting macrophage.(A) in macrophage after LPS/IFN γ and IL-4 processing Cyclo-oxygenase (COX), prostaglandin D2 synzyme (PGDS) and prostaglandin D2(PGD2) acceptor (DP).With solvent (Vehicle) compare, * p<0.05, * * p<0.01；N=6.(B) DP1 knocks out M1 mark response LPS/IFN proinflammatory to macrophage The influence of the expression of γ processing.Compared with wild type, * p<0.05, * * p<0.01；N=6.(C) DP1 knocks out promotees to macrophage The influence of the expression of scorching M1 marks response IL-4 processing.Compared with wild type, * p<0.05, * * p<0.01；N=6.(D) DP2 strikes Except the influence for the expression that response LPS/IFN γ processing is marked to the proinflammatory M1 of macrophage.Compared with the solvent of response, * p< 0.05, * * p<0.01；N=6.(E) DP2 knocks out the influence of the expression of M1 marks response IL-4 processing proinflammatory to macrophage.With The solvent of response is compared, * p<0.05, * * p<0.01；N=6.All figures are represented with average value ± SEM.Data are at least two only The result of vertical experiment.Measure significance,statistical is examined using unpaired T.
Fig. 2：DP1 verifies to the streaming and immunofluorescence of M2 types polarization facilitation.(A-C) flow cytometry DP1 is knocked out to M1 (F4/80+CD206-) and M2 (F4/80+CD206+) macrophage distribution influence.(A) representational streaming Cytometry results, (B, C) F4/80+CD206-And F4/80 (M1)+CD206+(M2) macrophage quantifies.With wild type phase Than * p<0.05；Compared with solvent, #p<0.05, ##p<0.01；N=4.(D-F) knocked out using the DP1 of immunofluorescence assays To M1 (CD68+NOS2+) and M2 (CD68+NOS2-) macrophage distribution influence.(D) representational immunofluorescence figure, unit Length is 5 μm；(E、F)M1(CD68+NOS2+) and M2 (CD68+NOS2-) macrophage quantifies.Compared with wild type, * p< 0.05；Compared with solvent, #p<0.05, ##p<0.01；N=4.
Fig. 3：DP1 is overexpressed the influence to macrophage polarization.Detected in the DP1- that DP1 is expressed again/- macrophage DP1mRNA levels (A), NOS2 and YM1mRNA (B, C) and protein level (D, E).Compared with carrier, * * p<0.01；With solvent phase Than #p<0.05, ##p<0.01；N=4.All figures are represented with average value ± SEM.Data are the knots of at least two independent experiments Fruit.Measure significance,statistical is examined using unpaired T.
Fig. 4：DP1 whole bodies, which knock out, passes through PGD2/ DP1 paths influence inflammatory regression of the macrophage in peritonitis.(A) COX and PGDS mRNA level in-sites in peritoneal macrophages；(B) PGD in peritoneal lavage2Production (B)；In inflammation most serious (12h) gives PGD2(100ug/kg) or BW (1mg/kg), the depletion exponential being calculated, including Tmax(PMN reaches peak Time point), T50(PMN was reduced to about 50% time point) and Ri(interval of disappearing, TmaxAnd T50Between interval).For ferment Inflammation caused by female glycan, Tmax,~12h；T50,~24h；Ri,~12h.Add PGD2Afterwards：Tmax,~12h；T50,~21h； Ri,~9h.After adding BW, Tmax,~12h；T50,~22h；Ri,~10h.N=4-6.
Fig. 5：The structure of DP1 macrophage specific knockdown mouse and checking.(A) complete DP1WT、DP1flox-neoEquipotential base Cause, DP1floxThe schematic diagram of allele, DP1KO allele.Filled box presentation code extron, dotted line represent to be used for together The region of source restructuring；Gray triangles represent the primer for genotyping；Neo refers to neo expression cassette.(B) it is derived from DP1flox/floxLysCreThe PCR analyses of the genomic DNA of mouse (Mac-DP1KO) each organ.(C)DP1flox/floxLysCre And DP1 (Mac-DP1KO)flox/flox(WT) DP1mRNA is horizontal in the macrophage of mouse.Compared with wild type, * * p<0.01, n =6.Data are represented with average value ± SEM.Measure significance,statistical is examined using unpaired T.
Fig. 6：DP1 macrophages specific knockdown influences the polarization of M2 type macrophages in mouse peritoneum inflammation and inflammatory disappeared Move back.Total lymphocyte infiltration (A), total macrophage since mouse zymosan Induced Peritonitis to its regression period analysis is thin Born of the same parents (B), F4/80+CD206+And F4/80+CD206-Cell ratio (C) and the mRNA of pro-inflammation genes (D-F) and anti-inflammation gene (G-I) Expression.Compared with wild type, * p<0.05, * * p<0.01；N=4-6.All data are represented with average value ± SEM.Use Unpaired T examines measure significance,statistical.
Fig. 7：DP1 inhibitor influences the polarization of M2 type macrophages and inflammatory regression in mouse peritoneum inflammation.Gathered with yeast Sugar is stimulated after mouse 24 hours, gives DP1 antagonists MK0524 (4mg/kg/ days), and then at the appointed time point collection peritonaeum is huge Phagocyte.Total macrophage (A), F4/80 are calculated in different time points+CD206+And F4/80+CD206-Cell (B).Yeast gathers Collect macrophage within 72 hours after sugar stimulates, detect its pro-inflammation genes expression (C) and anti-inflammation gene expression (D).With solvent phase Than * p<0.05, * * p<0.01；N=4-6.Data are represented with average value ± SEM.Measure statistics is examined to show using unpaired T Work property.
Fig. 8：DP1 knocks out promotes M1 types to polarize by activating JAK2/STAT1 signal paths, suppresses STAT6 M2 types pole Change.(A, B) analysis DP1 activators (BW245C；BW) the JAK2/STAT1 phosphoric acid of the LPS/IFN γ inductions of the macrophage of processing Change (A) and NOS2 expression (B).The JAK2/STAT1 phosphorylations (C) that LPS/IFN γ are induced in (C, D) detection DP1KO macrophages With the STAT6 phosphorylations (D) of IL-4 inductions.
Fig. 9：JAK2/STAT1 pathway inhibitors can reverse DP1 to knock out the M1 macrophages polarization promoted.(A-D) use JAK2 inhibitor Cs EP33791 (CEP) pretreatments DP1 knocks out macrophage and wild type (WT) macrophage, and detects JAK2/ STAT1 phosphorylations (A), pro-inflammation genes expression (C), STAT6 phosphorylations (B) and anti-inflammation gene expression (D).Compared with wild type, * p<0.05, * * p<0.01；Compared with unused CEP,#p<0.05,##p<0.01；N=4.(E-H) STAT1 inhibitor Fludara is used (FLU) pre-process DP1 and knock out macrophage and wild type macrophage, and assess JAK2/STAT1 phosphorylations (E), pro-inflammation genes Express (G), STAT6 phosphorylations (F) and anti-inflammation gene expression (H).Compared with wild type, * p<0.05, * * p<0.01；With it is unused FLU is compared,#p<0.05,##p<0.01；N=4.Data are represented with average value ± SEM.Measure statistics is examined using unpaired T Conspicuousness.In triplicate, other be also distributed about in two independent experiments carries out inflammation to all proteins trace.
Figure 10：The suppression of JAK2/STAT1 signal paths can reverse DP1 to knock out the inflammatory resolution suppressed in peritonitis. Zymosan gives FLU (40mg/kg/12h) for 24 hours after stimulating wild type hour and Mac-DP1KO mouse.Then specifying Time point collecting peritoneum cell.A-E represent respectively total infiltrating cells (A) in regression stage Mac-DP1KO mouse macrophages, Total CD11b+F4/80+Cell (B), M2 ratios (C), M1 gene expressions (D) and M2 gene expressions (E).As shown in FIG., * p< 0.05, * * p<0.01；N=4.Data are represented with average value ± SEM.Using two analysis of variance (two-wayANOVA) and with Bonferroni post-hoc afterwards examine (A-C) or unpaired S to examine (D, E) to carry out statistical analysis.
Figure 11：DP1 activation is played a role by PKA regulation and control subunit.(A) wild type is handled respectively with DP1 activators Macrophage and DP1 knock out macrophage, change horizontal monitoring intracellular cAMP.Compared with wild type, * p<0.05；##p< 0.01；N=6.(B) suppression for the JAK2/STAT1 phosphorylations that Rp-cAMPS (RP) and H89 mediates to forskolin in macrophage Influence.(C-E) the JAK2/STAT1 phosphorylations that Rp-cAMPS (10 μM, 50 μM and 100 μM) mediates to BW- in macrophage (C), the influence of M1 gene expressions (D) and M2 gene expressions (E).As shown in FIG., * p<0.05, * * p<0.01；N=4.(F、G) H89 is to the M1 gene expressions (F) mediated of BW in macrophage and the influence of M2 gene expressions (G).As shown in FIG., * p< 0.05, * * p<0.01；N=4.Data are represented with average value ± SEM, and are the results of at least two independent experiments.Using not matching somebody with somebody Measure significance,statistical is examined to T.
Figure 12：DP1 macrophages are overexpressed the structure of (Mac-DP1-Tg) mouse.(A) Mac-DP1-Tg constructions；Its In, SP146 refers to the promoter original paper of the synthesis of clone 146；P47 refers to 138bp p47phox promoters, is that promoter synthesizes Minimal promoter, and as the comparison other of promoter selection.(B) genotyping of Mac-DP1-Tg mouse.(C)Mac- DP1 expression in DP1-Tg mouse macrophages.Compared with wild type, * * p<0.01；N=4-6.Data with average value ± SEM is represented.Measure significance,statistical is examined using unpaired T.PCR is analyzed in triplicate.
Figure 13：The suppression of PKA regulation and control subunits can suppress the inflammatory regression as caused by being overexpressed DP1.(A-E) yeast gathers Sugar gives Rp-cAMPS in 24 hours after stimulating Mac-DP1 transgenosis (Tg) mouse.Collect peritonaeum to compare, monitor CD45+Cell (A), total CD11b+F4/80+Cell (B), M2 ratios (C), M1 gene expressions (D) and M2 gene expressions (E).As illustrated, * p< 0.05, * * p<0.01；N=4-6.Data are represented with average value ± SEM, and are the data of at least two independent experiments.Use two Analysis of variance (two-way ANOVA) and subsequent Bonferroni post-hoc examine (A-C) or unpaired S to examine (D, E) carries out statistical analysis.
Figure 14：Interaction between IFNGR2 and PRKAR2A be present.(A) the representativeness silver of anti-IFNGR2 immunoprecipitates Dyeing.The albumen of mark is produced from LC-MS/MS.(B-E) using anti-FLAG (B, D) and anti-HA immunoprecipitations (IP) (C, E) analysis IFNGR2-PRKAR2A interacts.
Figure 15：DP1 activation can promote the combination of PRKAR2A and IFNGR2 in macrophage.(A, B) BW processing is to huge The influence of PRKAR2A and IFNGR2 common locations in phagocyte.Compared with wild type, * p<0.05；Compared with unused BW,##p< 0.01；Unit length is 5 μm；N=4.(C) IFNGR2- in the wild type macrophage and Mac-DP1KO macrophages of BW processing The analysis of PRKAR2A interactions.(D) Rp-cAMPS (10 μM, 50 μM and 100 μM) is induced BW in macrophage The influence of IFNGR2-PRKAR2A interactions.
Figure 16：The identification of IFNGR2 and PRKAR2A interaction sites.(A, B) is by FLAG-IFNGR2 and HA-PRKAR2A Truncated mutant (A) cotransfection enter in 293T cells.IP analyses are carried out using FLAG antibody, protein is carried out using HA antibody Engram analysis (B).(C, D) HA-PRKAR2A and FLAG-IFNGR2 truncated mutant (C) cotransfection enters 293T cells.Use HA antibody carries out IP analyses, and western blot analysis (D) are carried out using FALG antibody.
Figure 17：Direct interaction be present in IFNGR2 and PRKAR2A.(A-D) using GST and His drop-down detections Direct interaction between the IFNGR2 and PRKAR2A of (pulldown assay) detection purifying, include IFNGR2 total length egg (A, B) and TMR fragments and PRKAR2A PBC pieces intersegmental (C, D) interact in vain.
Figure 18：R, which is overexpressed, can reactivate M2 polarization caused by DP1 activation.A to D is respectively IFNGR2TMR domains It is overexpressed suppression (A), STAT6 phosphorylations (B), the pro-inflammation genes expression of the JAK2/STAT1 activity induced BW in macrophage (C) and anti-inflammation gene expresses (D).As illustrated, * p<0.05, * * p<0.01；N=4.Data are represented with average value ± SEM.Number According to the result for being at least two independent experiments.Measure significance,statistical is examined using unpaired T.
Figure 19：Prkar2a rises in M2 polarization.(A) the PBC knots of 4 kinds of PKA R subunits (RI α, RI β, RII α and RII β) Structure domain.(B) LPS/IFN γ processing and the expression of Prkar1a and Prkar2a after IL-4 processing are responded in macrophage.**p< 0.01；N=6.Data are represented with average value ± SEM.Measure significance,statistical is examined using unpaired T.
Figure 20：PRKAR2A knocks out the M2 macrophages polarization for slackening DP1 activation inductions.(A)Prkar2a-/-Macrophage Middle Prkar1a and Prkar2a expression.Prkar2a missings are to STAT6's in (B, C) BW245C (BW) pretreatment macrophages Influence (B) and the influence (C) to JAK2/STAT1 activity.(D-G) BW245C (BW) pre-processes Prkar2a in macrophage and lacked Influence (D-F) to M1 gene expressions and the influence (G-I) to M2 gene expressions.Data are represented with average value ± SEM.Using not Paired-samples T-test determines significance,statistical.All proteins trace is in triplicate, other to be also distributed about in two independent experiments Carry out inflammation.
Figure 21：PRKAR2A knocks out the inflammatory resolution for slackening DP1 activation inductions.(A-E) zymosan stimulates PRKAR2A-/- Give BW within 24 hours after mouse.Peritoneum inflammation cell is collected, monitors CD45+Cell (A), total CD11b+F4/80+Cell (B), M2 Ratio (C), M1 gene expressions (D) and M2 gene expressions (E).As illustrated, * p<0.05, * * p<0.01；Compared with solvent,#p< 0.05；N=4-6.Data represent with average value ± SEM, use two analysis of variance (two-way ANOVA) and subsequent Bonferroni post-hoc examine (A-C) or unpaired S to examine (D, E) to carry out statistical analysis.
Embodiment
This experiment mainly using construction of recombinant plasmid technology, laser scanning confocal microscopy, Real-time round pcrs, The advanced biology techniques means such as flow cytometry, immunoblot assay and proteomic image, with wild type, DP1 macrophages The mouse that cell-specific knocks out, it is object that specificity, which is overexpressed mouse and PRKAR2A knock-out mices, utilizes peritonitis model Study effect and its molecular mechanism of the DP1 signal paths in inflammatory resolution.Result of study shows：First, In vitro cell experiment It was found that DP1 missing can promote macrophage to be polarized to M1 types, suppress the M2 types polarization of macrophage；Secondly in vivo Find that missings of the DP1 in macrophage can suppress inflammatory resolution in peritonitis model experiment, especially inhibit inflammatory resolution During conversion of the macrophage to M2 types, be embodied in proinflammatory factor rise and most of anti-inflammatory factors reduction.Grind Study carefully the key signal path for finding DP1 missing high level activation JAK2/STAT1 this M1 polarization regulation and control.Further study show that DP1 activation can activate the separation of PKA (protein kinase A) regulation and control subunits and catalytic subunit, identified, a kind of tune discharged Controlling subunit PRKAR2A can interact with IFN γ acceptor IFNGR2 transmembrane region, stablize its conformation and then prevent downstream The activation of JAK2/STAT1 signal paths.It can be seen that free PRKAR2A subunits have certain inhibitory action for M1 types polarization path, This is further verified in PRKAR2A knock-out mices：The inventors discovered that DP1 activation can promote the polarization of M2 types and inflammation The regression of disease, but after PRKAR2A is lacked, regression reaction caused by DP1 activation can be totally constrained.In summary number is tested According to the present inventor draws a conclusion：PGD2/DP1 signal paths can be by promoting suppression of the PRKAR2A to JAK2/STAT1 paths To promote the regression of the polarization of M2 macrophages and inflammation.So in clinical treatment inflammatory resolution relevant disease, macrophage Each molecule in upper PGD2/DP1 signal paths, including DP1 and PRKAR2A, can serve as potential target spot.
Therefore, the present invention provides the method that regulation and control macrophage polarizes to M1 types macrophage or M2 types macrophage, with And the application of corresponding inhibitor or activator in the medicine for preparing treatment inflammation.Methods described can be vivo approaches, It can be in-vitro method.
Herein, " regulation and control " include suppressing and promoted.Using the method for the present invention, macrophage can be promoted to be polarized to M1 types Or suppress macrophage and polarized to M2 types, or promote macrophage to be polarized to the polarization of M2 types or suppression macrophage to M1 types.Should Understand, when promoting macrophage to be polarized to M1 types, it is then suppressed to the polarization of M2 types；Similarly, when promote macrophage to When M2 types polarize, it is suppressed to the polarization of M1 types.
It can realize that macrophage polarizes to M1 or M2 types using following means：(1) the macrophage D prostaglandins are regulated and controled Receptor type 1 (DP1) gene expression or protein active；And/or (2) regulation and control PRKAR2A gene expressions or protein active；And/or (3) Regulate and control IFN γ acceptor IFNGR2 gene expressions or protein active；And/or (4) regulate and control the expression of gene in JAK2/STAT1 paths Or protein active., can (1) suppression DP1 gene expressions or albumen when needing to promote macrophage to polarize to M1 types macrophage Activity；And/or (2) suppress PRKAR2A gene expressions or protein active；And/or (3) promote IFN γ acceptor IFNGR2 gene tables Reach or protein active；And/or (4) promote the expression of gene or protein active in JAK2/STAT1 paths.When need promote macrophage The method that cell polarizes to M2 types macrophage, can (1) promotion DP1 gene expressions or protein active；And/or (2) promote PRKAR2A gene expressions or protein active；And/or (3) suppress IFN γ acceptor IFNGR2 gene expressions or protein active；And/or (4) expression of gene or protein active in JAK2/STAT1 paths are suppressed.
Herein, technology well known in the art can be used to suppress or promote target gene such as PGD2、PD1、PRKAR2A、 The expression of IFNGR2, JAK2 and STAT1 gene.For example, for suppressing, the expression of RNAi technology interference target gene can be used, Or target gene is knocked out using homologous recombination technique., can be by giving corresponding reagent to realize in these embodiments State suppression.For example, the expression of the target gene can be lowered using the siRNA of target gene.Or will using targeting vector Target gene full genome knocks out.Therefore, this kind of reagent includes but is not limited to siRNA and targeting vector.For promoting, mesh can be given The expression vector of gene realize, or give the activators of the upstream regulating genes of target gene and realize.For example, for DP1 genes, to promote its expression, PGD can be given2Activator.
Technology well known in the art can be used to suppress or promote (up-regulation) destination protein such as PGD2、PD1、PRKAR2A、 The activity of IFNGR2, JAK2 and STAT1 albumen.For example, for suppress, can by it is previously described suppression its gene expression, strike Except its gene or the mutation for making its gene cause protein active to inactivate, make its protein content reduction expressed or expressed egg White activity is relative to wild type decline or inactive；Or give inhibitor well known in the art.For promoting, on the one hand can give The expression vector of its gene is given, improves expression quantity of the albumen in macrophage, so as to realize its activity up-regulation；On the other hand Corresponding activator can be given, directly acts on destination protein, improves its activity.Herein, " activity " refers to destination protein and is used for Realize the activity of the object of the invention.For example, the activity of DP1 albumen adjusts point of subunit PRKAR2A and catalytic subunit for regulation PKA From PRKAR2A activity is is combined with IFNGR2, and IFNGR2 activity is activation JAK2/STAT1, and JAK2 and STAT1 work Property then for regulation macrophage M1M2 types polarization.
Therefore, the present invention can be implemented by the following means：(a) gene knockout；(b) it is overexpressed gene；And/or (c) gives Corresponding activator or inhibitor.
Reagent for the present invention includes：DP1 inhibitor and activator, JAK2 inhibitor and activator, STAT1 suppressions Preparation and activator, PRKAR2A inhibitor and activator and IFNGR2 inhibitor and activator.This kind of reagent can be this area Known reagent, can be nucleic acid molecules, albumen such as antibody or micromolecular compound.For example, DP1 activators can be with It is BW245C；DP1 inhibitor can be AH 6809 (Cat.No.HY-10418), Laropiprant (Cat.No.HY-50175) Deng.JAK2 inhibitor can be AT9283, AZD-1480, Baricitinib or its phosphate, BMS-911543, CEP- 33779th, CYT387 or its mesylate or sulfate, GLP0634 and LY2784544.STAT1 inhibitor can be that fluorine reaches drawing Shore (Fludarabine), Stattic, niclosamidum (Niclosamide) and nifuroxazide etc..JAK2 and STAT1 excitement Agent can be IFNGR2 or its expression vector, or IFNGR2 activator.PRKAR2A inhibitor can be such as H89 or Its dihydrochloride；PRKAR2A activator can promote the PKA regulation and control subunits reagent that also catalytic subunit separates, such as Foskolin (it promotes cAMP rises, so as to promote the separation of PKA regulation and control subunits and catalytic subunit).IFNGR2 inhibitor can be with It is such as PRKAR2A and its activator；IFNGR2 activator can for example suppress PRKAR2A activity or the examination of its expression Agent, or its TMR over-express vector.
In certain embodiments, the present invention is more particularly directed to regulate and control macrophage to M1 types or M2 by target spot of PRKAR2A The method of type polarization, or PRKAR2A is as application of the target for modulation in macrophage into M1 types or the polarization of M2 types.Protein kinase A (PKA) is also known as the protein kinase A dependent on cAMP, is made up of four subunits, and two of which is regulation subunit, and another two is to urge Change subunit.In no cAMP to be passivated complex form presence, cAMP is combined with regulation subunit, changes regulation subunit conformation, Regulation subunit is set to be separated with catalytic subunit.It is PRKAR2A to adjust subunit present document relates to the one of PKA.
As it was noted above, PRKAR2A activator or inhibitor can be directly given, so as to regulate and control its biological activity.Or The activity of the albumen of person, the expression of controllable PRKAR2A upstream genes or its coding, to realize the regulation and control of PRKAR2A activity.Example Such as, in certain embodiments, DP1 activator is given, up-regulation DP1 activity, promotes the separation of PRKAR2A and catalytic subunit, So as to realize the up-regulation of PRKAR2A activity.In certain embodiments, expression DP1 expression vector is given, improves DP1 table Up to amount, it can also promote the separation of PRKAR2A and catalytic subunit, so as to realize the up-regulation of PRKAR2A activity.In some embodiments In, the separation of PKA regulation and control subunits and catalytic subunit can be promoted by raising intracellular cAMP level.Therefore, any other rise is thin Means or reagent horizontal intracellular cAMP are used equally for the present invention.In certain embodiments, it can also give expression PRKAR2A's Expression vector, the expression quantity of PRKAR2A in cell is directly improved, so as to improve its biological activity.It should be understood that above-mentioned raising The various means of PRKAR2A activity individually or can be used in any combination.
When needing to suppress PRKAR2A, such as PRKAR2A inhibitor can be given, or knocks out PKA expression, or it is broken PRKAR2A sequence (sequence for especially encoding PRKAR2A PBC regions) is encoded in bad PKA coded sequences so that it is expressed PRKAR2A is inactive or reduced activity, or give DP1 inhibitor or knock out DP1 genes or reduce DP1 expression, or DP1 genes is undergone mutation and cause the DP1 loss of activity or decrease of expression, and any combination of above-mentioned means.
Promotion macrophage of the present invention is used for resolution to the method that M2 types macrophage polarizes.
Herein, inflammation preferably benefits from the inflammation that macrophage polarizes to M2 types macrophage, and this kind of inflammation includes But it is not limited to asthma, inflammatory bowel disease (including Crohn disease and ulcerative colitis), appendicitis, blepharitis, capillary bronchitis, branch Tracheitis, bursal synovitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, cystitis, dacryoadenitis, dermatitis, dermatomyositis, brain Inflammation, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fascitis, fibrositis, gastritis, Gastroenteritis, hepatitis, suppurative hidradenitis, laryngitis, mastitis, meningitis, myelitis, myocarditis, myositis, ephritis, oaritis, testis It is ball inflammation, osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, pneumonia, rectitis, preceding Row adenositis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillitis, uveitis, vaginitis, Vasculitis or vulvitis etc..
Therefore, in certain embodiments, the present invention provides a kind of inflammation treatment or regression method, and methods described includes giving The suitable reagent of object of needs is given, to promote the macrophage in the subject to be polarized to M2 types macrophage.This kind of reagent Can be previously described various reagents.The amount given is therapeutically effective amount, can be according to the age of individual, body weight, sex, disease Species and the order of severity are determined.Object can be mammal, especially people, mouse, rabbit, pig, sheep and dog etc..To prescription Method is the conventional method in this area, for example, orally, injection etc., and can be adjusted for different reagents.
The present invention also provides purposes of following reagents in the medicine for preparing treatment inflammation：
(1) reagent of DP1 gene expressions or protein active is promoted；And/or
(2) reagent of PRKAR2A gene expressions or protein active is promoted；And/or
(3) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is suppressed；And/or
(4) expression of gene or the reagent of protein active in JAK2/STAT1 paths are suppressed.
In certain embodiments, the reagent is as described above.
In certain embodiments, the present invention is more particularly directed to using PRKAR2A as target treatment inflammation, or prepare with PRKAR2A is the medicine of the treatment inflammation of target spot.Method using PRKAR2A as target treatment inflammation can be as mentioned before.With PRKAR2A is that the medicine of the treatment inflammation of target spot especially includes those reagents that PRKAR2A can be promoted to express or raise its activity, Including nucleic acid, albumen (such as antibody) and micromolecular compound.For example, these reagents can be PRKAR2A activator, such as appoint What can promote the elevated reagents of intracellular cAMP, such as Foskolin；The either expression of controllable PRKAR2A upstream genes or its volume The activity of the albumen of code, to realize the regulation and control of PRKAR2A activity, including DP1 activator and expression DP1 expression vector. It can be the expression vector for expressing PRKAR2A.
The medicine of the present invention can contain active agent as described herein and pharmaceutically acceptable carrier.It is pharmaceutically acceptable Carrier be typically safe and nontoxic, and broadly may include to be used to prepare any known of pharmaceutical composition in pharmaceutical industries Material, such as filler, diluent, coagulating agent, binder, lubricant, glidant, stabilizer, colouring agent, wetting agent, disintegrant Deng.When selection is applied to deliver the excipient of synthetic peptide, the administering mode of this pharmaceutical composition, this area skill need to be mainly considered The known technique of art personnel.The content of active agent described in medicine of the present invention can be determined according to different therapeutical uses.Can Aforementioned pharmaceutical compositions are prepared according to known pharmaceutical procedures, for example《Remington's Pharmaceutical Science》(Remington’s Pharmaceutical Sciences) (the 17th edition, Alfonoso R.Gennaro are compiled, Mike publishing company (Mack Publishing Company), Easton, Pennsylvania (1985)) be documented in detail in a book.The medicine of the present invention can To be various suitable formulations, including but not limited to tablet, capsule, injection etc..
The present invention will be described in a manner of specific embodiment below.It should be understood that these embodiments are only illustrative, and Unrestricted protection scope of the present invention.In embodiment, unless otherwise stated, being implemented using this area conventional technical means each Plant method, step and use various reagents.
First, method and material
1.1 experiment material
1.1.1 laboratory apparatus
Electrophoresis apparatus (Tanon), sterile super-clean bench (Thermo), Biohazard Safety Equipment (Thermo), electric homogenizer (IKA), Pipettor (Eppendorf), PCR instrument (Eppendorf and Bio-Rad), ultra low temperature freezer (Nuair), toy anesthesiaing system, Flow cytometer (BD Biosciences), 5415D types table model high speed centrifuge (Eppendorf), Ultrospec2100pro Ultraviolet specrophotometer (Amersham), Dounce homogenizers (Kontes), constant incubator (Medcenter), gel imaging System (Tanon), cell culture incubator (Heraeus), liquid nitrogen container (Thermolyne), inverted fluorescence microscope (Olympus), essence Close balance (Mettler Toledo), ABI-7900 real-time fluorescence quantitative PCR systems, CFX real-time fluorescence PCR systems (Bio- Rad)。
In addition to above-mentioned instrument, conventional instrument：Sample beveller, water-bath, dry type metal bath, Horizontal electrophoresis tank and match somebody with somebody Cover power supply, Ultrasonic Pulverization instrument, pH meter, magnetic stirring apparatus, micro-wave oven, hybrid heater, vertical laminar flow clean bench, biochemical culture Case, refrigerator, electric heating constant temperature tank, shaking table, JT502N assay balances, eddy mixer etc..
1.1.2 experimental animal
Used mouse is C57BL/6 backgrounds in this experiment, raise in SPF level Animal Houses, give constant temperature (22 ± 1 DEG C) constant humidity, and the condition of circulation in 12 hours round the clock.Mouse freely activity can obtain drinking-water and mouse grain.DP1flox/floxIt is small Mouse and LysMCre+Mouse is hybridized, and obtains DP1flox/floxLysMCre+Mouse, i.e. DP1 genes specifically knock out in macrophage Mouse, Mac-DP1KO mouse are simply named as herein.Corresponding control group mice is DP1flox/floxLysMCre-It is small Mouse, WT mouse are simply named as herein.In addition it is special in macrophage in Sai Ye companies to construct DP1 genes by the present inventor Property be overexpressed mouse, be named as Mac-DP1-Tg mouse, obtain two overexpression strains：C systems and E systems.All zoopery behaviour The animal protection of Shanghai Inst. of Life Science, CAS nutrition science research institute is followed strictly with using rules Correlative detail.
1.1.3 experimental cell
It is a large amount of macrophage derived as follows in peritoneal macrophage, specific acquisition and cultural method used in this experiment：
1) every TGA of mouse peritoneal injection 1ml 3%；
2) after 3 days, mouse is put to death with dislocation method, four limbs are fixed, with alcohol spray disinfectant mouse web portion；
3) every sterile PBS of mouse peritoneal injection 10ml, with the soft belly of cotton balls；
4) suction out intraperitoneal liquid with syringe and centrifuge 5min in 800g；
5) cell precipitation is resuspended with 1ml culture mediums (RPMI1640+10%FBS+1%PS), is counted with tally.
6) cell density is finally diluted to 5 × 105/ ml, it is taped against 6 orifice plates or 12 orifice plates.
7) after 6 hours, culture medium is sucked, is washed one time with PBS, fresh culture is added and is cultivated or handled.
The induction of M1 type macrophages：LPS(1μg/mL)+IFNγ(20ng/mL)
The induction of M2 type macrophages：IL-4(20ng/mL)
1.1.4 experiment reagent
IFN γ and IL-4 are purchased from Peprotech (Rocky Hill, NJ, USA).LPS, niacin (niacin), yeast gather Sugar, thioglycolate salt, IBMX and ProteoSilver Plus Stain kits purchased from Sigma-Aldrich (St.Louis, MO,USA).H89 and BW245C is purchased from Cayman Chemical (Ann Arbor, MI, USA).Forskolin is purchased from Enzo (Farmingdale,NY,USA).Fludara and CEP33791 is purchased from Selleck Chemical (Houston, TX, USA). MK0524 is purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Trizol is purchased from Invitrogen.Luciferin is purchased from Xenogen Biotechnology.M-MLV reverse transcriptions try Agent box and Dual luciferase reporter system are purchased from Promega.Taq enzyme, restriction enzyme and T4DNA connect Connect enzyme and be purchased from TaKaRa.Small amount plasmid extraction agent box is purchased from Beijing ancient cooking vessel state purchased from Shen energy lottery industry, glue reclaim kit.Bacterium Culture is purchased from tryptone, agar powder, dusty yeast, protease inhibitors (Protease inhibitor cocktail) Sigma.Serine/threonine inhibitor of phospholipase enzymes (Phosphatase inhibitor cocktail C) is purchased from Santa Cruz. Pvdf membrane and 0.22 μM and 0.45 μM of filter are purchased from Millipore purchased from Millipore.Western blot chemical illuminating reagentWest Pico Chemiluminescent Substrate are purchased from Pierce, and X-ray film is purchased from Kodak. Tissue Culture Dish, culture plate, cryopreservation tube and centrifuge tube are purchased from Corning.
Antibody sources and dilution ratio are as follows：COX-1(1:1000；Cayman Chemical), COX-2 (1:1000； Cayman Chemical), DP1 (1:200；Cayman Chemical), HA-tag (1:1000；Cell Signaling), phospho-JAK1(1:1000；Cell Signaling), JAK1 (1:1000；Cell Signaling), phospho-JAK2 (1:1000；Cell Signaling), JAK2 (1:1000；ABclonal), phospho-JAK3 (1:1000；Cell Signaling), JAK3 (1:1000；Cell Signaling), phospho-STAT1 (58D6) (1:1000；Cell Signaling), STAT1 (1:1000；ABclonal), phospho-STAT6 (pY641) (1:1000；BD Biosciences), STAT6 (1:1000；ABclonal), Ym1 (1:1000；R＆D), NOS2 (1:500, Abcam), FLAG M2 (1:2000, ABclonal), His-tag (1:2000；Cell Signaling), GST-tag (1:2000；ABclonal), IFNGR2(1:500；Santa Cruz Biotechnology), PRKAR2A (1:500；Santa Cruz Biotechnology), Actin (1:2000；Sigma-Aldrich), GAPDH (1:2000；Cell Signaling).
1.2 experimental method
1.2.1 peritonitis model
1) every mouse peritoneal injection 500ul is dissolved with the PBS solution of 1mg A type zymosans, thus Induced Peritonitis mould Type；
2) specific time point puts to death mouse with dislocation method in an experiment, and four limbs are fixed, with alcohol spray disinfectant mouse abdomen Portion；
3) every sterile PBS of mouse peritoneal injection 10ml, with the soft belly of cotton balls；
4) suction out intraperitoneal liquid with syringe and 800g centrifuges 5min；
5) cell conditioned medium freezes after collecting is used for the detection of follow-up prostaglandin level in -80 DEG C；
6) cell precipitation is used for streaming dyeing or cAMP detections or RNA, Protein Detection.
1.2.2 streaming dyeing and analysis
1) cell sample for needing to carry out streaming staining analysis is diluted to 106/ml；
2) corresponding antibodies press 1：200 are diluted in PBS solution；
3) each sample is resuspended with 50ul antibody-solutions, and 4 DEG C stand dyeing 40min；
4) dyeing, 5000rpm centrifugations 5min are terminated with 1ml PBS solutions；
5) cell precipitation is resuspended with 1% paraformaldehyde, subsequently upper machine testing.
1.2.3 the detection of prostaglandin extraction
1) 500 μ l abdominal cavities leachates are taken, add 2ul PGE2 and PGF respectively in same pipe2a, PGD2, PGI2, TXA2It is interior Mark (1ng/ul)；
2) 40ul 1M citric acids and 5ul 10%BHT are added to avoid free radical catalysed oxidn；
3) abdominal cavity leachate extracts, and adds 1ml n-hexane/ethyl acetate (1:1) vortex concussion mixing 1min；
4) after 3000g, 4 DEG C of centrifugation 10min, suction out upper organic phase and preserve；
5) aqueous phase n-hexane/ethyl acetate (1:1) reextraction；
6) it organic twice will mix, blown with nitrogen evaporator nitrogen；
7) dissolved with the acetonitriles of 100ul 10%, with 0.2um filter 12000g centrifugation 2min filterings；
8) filtrate is collected, you can upper machine testing, final each internal standard concentration is 20ng/ml.
1.2.4 the immunofluorescence dyeing of cell climbing sheet
1) culture medium is suctioned out, 1 × PBS of 1ml is added and washes once, to remove residual media, edge culture when paying attention to adding PBS Plate hole wall is slowly added into, in order to avoid cell is washed away；
2) PBS is suctioned out, 4% paraformaldehyde for being slowly added into about 400ul fixes cell, is incubated at room temperature 10min；
3) paraformaldehyde is suctioned out, is washed 2 times with PBS；
4) PBS is suctioned out, the PBS containing 0.2%TritonX-100 is added, to improve the permeability of cell, is stored at room temperature 5min；
5) liquid, the liquid especially around slide in exhaustion hole, and not make slide contact hole wall；
6) gently add primary antibody about 40ul on slide, it is spilt over outside slide, be incubated at room temperature 1h；Primary antibody dilution should make With the PBS solution containing 1%BSA；
7) 1ml PBS are washed 1 time, remove uncombined primary antibody；Exhaust slide surrounding liquid, and slide is touched wall；
8) gently add secondary antibody about 40ul on slide, it is spilt over outside slide, lucifuge room temperature 30min；
9) PBS is washed 2 times, and last time liquid not suction out；
10) (about 20ul) mounting liquid (Mow Oil) is dripped on a slide that is clean and performing mark, with a tip Tweezers take out slide, and dry liquids are dipped on a clean paper handkerchief.Slide is overturn, makes cell face-down, and mounting liquid end thereof contacts, Slide is slowly put down, avoids producing bubble；
11) fluorescence microscopy Microscopic observation, take pictures.
1.2.5 co-immunoprecipitation
1) the ice-cold PBS of cell, IP lysis buffers (50mM Tris, 150mM in pH 7.4 are cracked NaCl, 1%NP40,1mM EDTA, 1mM Na3VO4, 10mM NaF, 2.5mg/ml Aprotinins and leupeptin, 1mM β-phosphoglycerol And AEBSF, 10mM acetoiodide) in；
2) cell pyrolysis liquid is incubated 15 minutes on ice, 4 DEG C, 5000g is centrifuged off cell fragment；
3) by 4 DEG C of overnight incubations of cell pyrolysis liquid and corresponding antibody；
4) albumin A-Sepharose (Amersham Biosciences) pearl is added (with 1:1 volume ratio and 50mM Tris buffer solutions (pH7.0) mix), it is incubated again at 4 DEG C 4 hours；
5) immunoprecipitation is washed 3 times with Tris buffer solutions；
6) 2X sample loading buffers of the 40 μ l containing 0.02% bromophenol blue and 2% beta -mercaptoethanol is added, is boiled 10 minutes, then carry out Western Blot are analyzed.
1.2.6 quantitative fluorescent PCR
1.2.6.1 the preparation of cell total rna
Completed with reference to Invitrogen company's T rizol reagents specification.
1) attached cell is cultivated：It is not necessary to digest, can be directly digested, cracked with Trizol, Trizol volumes are pressed 10cm2/ ml ratios add；Suspension cell can directly be collected, cracked, per 1mlTrizol cleavables 5 × 106Animal, plant or ferment Mother cell, or 107Bacterial cell；
2) after cell adds Trizol, room temperature places 5min, it is fully cracked；
3) chloroform is added by 200 μ l chloroforms/ml Trizol, vibration mixes 15s, and room temperature places 10min；
4) by sample, 12000g centrifuges 15min under the conditions of 4 DEG C；
5) upper strata aqueous phase is carefully drawn to be transferred in new 1.5ml centrifuge tubes (no RNase)；
6) isopropanol is added by 0.5ml isopropanols/ml Trizol, fully mixed, 5-10min is placed in room temperature；
7) sample is centrifuged into 10min in 4 DEG C of 12000g, abandons supernatant, RNA is sunken to ttom of pipe；
8) 75% ethanol (deionized water after DEPC processing is prepared) is added by 75% ethanol of 1ml/ml Trizol, gently Centrifuge tube is vibrated, suspend washing precipitation；
9) sample is centrifuged into 5min in 4 DEG C of 8000g, abandons supernatant as far as possible；
10) 5-10min is dried or be dried in vacuo to room temperature；
11) 20-50 μ lDEPC H2O dissolving RNA samples, 55-60 DEG C of 5-10min can be used；
12) the quantitative RNA concentration of O.D. values is surveyed.
1.2.6.2 the reverse transcription of cell total rna
Completed with reference to Takara companies reverse transcription reagent box specification.
1) genomic DNA removes dereaction；
2) reverse transcription reaction.
1.2.6.3 quantitative PCR reacts
Completed with reference to ABI company SYBR reagents specification.The primer that quantitative PCR uses see the table below 1：
Table 1
1.2.7 protein immunoblotting
The cell of culture is cleaned 2 times with PBS solution, adds RIPA lysates afterwards, with thin after cracking 15 minutes on ice Born of the same parents, which scrape, scrapes cell, and 12,000rpm rotating speeds centrifuge 15min at 4 DEG C, draws supernatant solution, is surveyed afterwards with Brandford methods Determine protein concentration.After quantitative, protein concentration leveling will be loaded, adds 6 × sample loading buffer, in concentration is 10% after denatured by boiling Or 12% SDS-PAGE on carry out electrophoresis.Treat that albumen is transferred into PVDF by wet turn after the completion of electrophoresis On (polyvinylidene difluoride membranes) film (Millipore), with the TBST (25mM containing 5%BSA Tris, 150mM NaCl, 0.05%Tween20, pH 7.5) room temperature closes 1 hour, added after TBST rinsings once corresponding Primary antibody, 4 DEG C of overnight incubations.Next day removes primary antibody, and the secondary antibody for adding horseradish peroxidase afterwards three times, room are washed with TBST Temperature is incubated 1 hour, is washed twice with TBST, TBS is washed once, is finally entered with Enhanced Chemiluminescene (Pierce) Row colour developing.
1.2.8 gene overexpression
Mouse DP1 genes and TMR fragments are cloned into carrier pcDNA3.1, then by corresponding plasmid PT-103-01N respectively The special transfection reagent of jetPEI macrophages carries out plasmid transfection, comprises the following steps that：
1st, cell changes serum free medium into before transfecting；
2nd, DNA-PEI compounds are prepared, it is as follows：
A, with 50ul NaCl 150mM reagent dilutions DNA, gently mix；
B, gently mix before PEI uses, with 50ul NaCl 150mM reagent dilutions PEI, gently mix；
C, b is added in a immediately, gently mixed, is incubated at room temperature 20 minutes；
3rd, 100ul DNA-PEI compounds are added in culture medium, it is gently front and rear to shake up；
4th, it is put into after cultivating 4-6 in 37 degree of incubators, changes complete medium into；
5th, transfection 48 as a child carries out subsequent treatment or experiment.
1.2.9 statistical analysis
Every group of mouse number included or sample number illustrate in the example shown in each experiment, the reality of the present inventor Numerical value is tested to represent with means standard deviation.During analyze data, whether difference has statistics between control group and experimental group Learn meaning use non-matching group between double tail t detected, the difference of multigroup with using again after two-way ANOVA detections Bonferroni post-test (GraphPad Prism 5software) are detected, and work as p<When 0.05, it is believed that have between group Notable significant difference.
2nd, experimental result
1st, DP1 knocks out the M1 types polarization for promoting macrophage
First, the present inventor studies DP1 to M1M2 types by the macrophage of in vitro culture with a variety of detection methods The influence of macrophage polarization.Genetic test finds COX2/H-PGDS expression rise (Figure 1A) during the polarization of M1 types macrophage, DP1 expression is expressed higher (Figure 1A) in M2 type macrophages.After DP1 is knocked out, macrophage to M1 polarize when, pro-inflammation genes Expression substantially increase (Figure 1B)；When being polarized to M2, the expression of M2 type genes is relatively low (Fig. 1 C).This shows that DP1 strikes Polarized except macrophage is advantageous to M1 types.Meanwhile the present inventor is to PGD2The shadow that another acceptor DP2 polarizes to macrophage Sound is also detected, the results showed that polarization of the DP2 to macrophage M1M2 does not have a significant impact (Fig. 1 D, 1E).
DP1 knocks out the promotion to the polarization of M1 types and tested by flow cytometer showed (Fig. 2A -2C) and immunofluorescence (Fig. 2 D-2E) Verified to many-side.Found when the present inventor is knocked out after macrophage carries out DP1 covering expression (Fig. 3 A) to DP1, pro-inflammation genes Expression be inhibited (Fig. 3 B, 3D), the expression of M2 type genes rise (Fig. 3 C, 3E).Result above demonstrates DP1 and struck in vitro Except macrophage can be promoted to be polarized to M1 types, suppress the M2 types polarization of macrophage.
2nd, DP1 knocks out the polarization of macrophage during influence inflammatory resolution
In mouse peritonitis model, the inventors discovered that the upstream COX/PGDS signal path related to DP1 is in inflammation The regression stage (24h), which has, substantially raises (Fig. 4 A), PGD in corresponding abdominal cavity leachate2Content also risen (Fig. 4 B).This PGD in the rat pleural inflammation model reported before2Dynamic change it is consistent.This prompting PGD2It may be sent out in the inflammatory resolution stage Wave certain effect.Following the present inventor removes quantitative testing PGD with the regression index of classics2And DP1 activators BW245C (BW) Whether there is pro-inflammatory effect.PGD is injected in inflammatory reaction peak 12h2Or BW can reduce Ri(cell quantity is down to half Time used) about 2-3 hour (Fig. 4 C), this shows PGD2/ DP1 paths have the function that to promote inflammatory resolution.
In order to further verify effects of the DP1 in Macrophage Inflamatory regression, it is thin that the present inventor constructs DP1 macrophages Born of the same parents' specific knockdown mouse (Fig. 5).
As a result after finding that DP1 macrophages specifically knock out, inflammatory resolution substantially slows down (Fig. 6 A), especially macrophage Inflammatory resolution (Fig. 6 B).Following the present inventor carries out M1M2 type analysis to macrophage, and M1 types are huge after as a result showing DP1 knockouts Phagocyte ratio is significantly raised (Fig. 6 C), and genetic test finds that the expression of pro-inflammation genes is also of a relatively high (Fig. 6 D-6F), M2 type bases Because expression is relatively low (Fig. 6 G-6I).Result above shows that DP1 is knocked out and inhibits during inflammatory resolution macrophage to M2 types Polarization.
DP1 knock out to Macrophage Inflamatory disappear inhibitory action DP1 inhibitor MK0524 using when tested Card：The regression of macrophage is suppressed (Fig. 7 A) after DP1 suppresses, and analysis shows the significantly raised (figure of M1 type macrophage quantity 7B), genetic test finds that the expression of pro-inflammation genes is also of a relatively high (Fig. 7 C), the relatively low (figure of M2 type anti-inflammation gene expressions 7D)。
3rd, DP1 knocks out promotes M1 types to polarize by activating JAK2/STAT1 signal paths
Known STAT1/STAT6 balance plays a significant role in macrophage M1/M2 polarization, and the present inventor is just to phase The JAK/STAT signal paths of pass are detected.When as a result showing M1 polarization, DP1 activation inhibits JAK2/STAT1 signals to lead to Road (Fig. 8 A), the expression of M1 gene NOSs 2 reduce (Fig. 8 B)；Accordingly when M2 polarizes, DP1 excitement can improve STAT6's Phosphorylation (Fig. 8 A), M2 genes YM1 expression also rise (Fig. 8 B).On the other hand, after DP1 is knocked, JAK2/STAT1 leads to Road suppresses (Fig. 8 D) by advanced activation (Fig. 8 C), STAT6 signal paths by relative.This shows that DP1 is knocked out by activating JAK2/ STAT1 signal paths promote the polarization of M1 types, suppress the M2 types polarization of STAT6 mediations.
The present inventor handles M1M2 type macrophages with JAK2 inhibitor Cs EP, after as a result finding that JAK2 is suppressed, DP1 The difference of STAT1/STAT6 caused by knockout is eliminated, and M1/M2 gene expressions also reach similar level (Fig. 9 A-9D).Further Ground, STAT1 inhibitor FLU also it is similar eliminate DP1 knock out caused by difference (Fig. 9 E-9H).This further demonstrates DP1 and struck Except being to promote M1 types to polarize by activating JAK2/STAT1 signal paths.
4th, the suppression of JAK2/STAT1 signal paths can reverse DP1 to knock out the inflammatory resolution suppressed
Following the present inventor verifies that the covering the DP1 whether suppression of STAT1 signal paths can be similar is knocked out in peritonaeum Effect in inflammation.As a result show, in WT and Mac-DP1KO mouse, after STAT1 is suppressed, macrophage disappears in inflammation Move back (Figure 10 A, 10B), the ratio (Figure 10 C) of M2 type macrophages, and the expression of M1/M2 type genes all reaches similar horizontal (Figure 10 D, 10E).
5th, DP1 activation suppresses JAK2/STAT1 signal paths by promoting PRKAR2A and IFNGR2 combination
DP1 is as a kind of GSCoupling protein, it activates the level that can increase cAMP.The inventors discovered that DP1 knocks out it Afterwards, cAMP level is suppressed significantly (Figure 11 A) in macrophage.CAMP can combine with PKA catalytic subunits, and then promote PKA regulates and controls the separation of subunit and catalytic subunit.The present inventor is pressed down with RP and H89 to regulation and control subunit and catalytic subunit respectively System.As a result finding, the inhibitor RP that JAK2/STAT1 suppression caused by DP1 activation can be adjusted subunit is reactivated, and And RP has concentration gradient effect (Figure 11 B-11E)；The inhibitor H89 of catalytic subunit to JAK2/STAT1 signal paths and M1/M2 gene expressions all have no significant effect (Figure 11 B, 11F, 11G).So DP1 activation is played by PKA regulation and control subunit Effect.
In order to further verify, the present inventor constructs DP1 macrophages and is overexpressed mouse (Figure 12).As a result find, In peritonitis in model, DP1 can promote the inflammatory regression (Figure 13 A, 13B) of macrophage in the overexpression of macrophage, The ratio (Figure 13 C) of increase M2 type macrophages is mainly manifested in, reduces the expression (Figure 13 D) of pro-inflammation genes, improves M2 type bases The expression (Figure 13 E) of cause.But the suppression of PKA regulation and control subunits can suppress inflammatory regression (Figure 13 A- as caused by being overexpressed DP1 13E)。
How present inventors studied PKA regulation and control subunits suppresses JAK2/STAT1 signal paths.Known JAK2 and IFNGR2 It is combined, ligand i FNr combination can promote JAK2 autophosphorylation and then recruit STAT1, promote its phosphorylation.Therefore, The present inventor carries out IP to IFNGR2, and proteomic image analysis is carried out to IP products, as a result finds to be primarily present one in IP products Kind albumen PRKAR2A, analysis find that it is a kind of hypotype (Figure 14 A) in PKA regulation and control subunits.By being overexpressed IP experiments in vitro It was found that interaction (Figure 14 B-14E) between PRKAR2A and IFNGR2 be present.
Immunofluorescence experiment finds that DP1 activation can promote the combination (figure of PRKAR2A and IFNGR2 in macrophage 15A, 15B).IP, which is tested, also demonstrates the interaction of PRKAR2A and IFNGR2 in primary macrophage, and this makees apparatus There is RP concentration gradients effect (Figure 15 C, 15D).
The present inventor further study action site between the two.Found by being segmented IP experiments, PRKAR2A's PBC regions and IFNGR2 trans-membrane region (TMR) are main function site (Figure 16).
Whether the effect for being research between both is directly to act on, and the present inventor purifies two hatching eggs with GST and His respectively White total length and effect fragment.As a result show direct interaction (Figure 17) between the two be present.
When having served as Expression of TM R fragments, JAK2/STAT1 suppression caused by DP1 activation is reactivated (Figure 18 A), proinflammatory The expression of gene rises (Figure 18 C) again；Equally, when having served as Expression of TM R fragments, STAT6 activation caused by DP1 activation is weighed New to suppress (Figure 18 B), the expression of M2 genes is suppressed (Figure 18 D).
6th, PRKAR2A knockout can suppress the polarization of M2 type macrophages and inflammatory resolution caused by DP1 activation
PKA, which regulates and controls subunit, in mouse four kinds, PRKAR1A, PRKAR1B, PRKAR2A, PRKAR2B.It is main in macrophage Express Prkar1a and Prkar2a.Simultaneously homology (Figure 19 A) is not present in Prkar1a and Prkar2a PBC regions.External M1/ M2 type macrophages Induction experiments find that Prkar2a has conspicuousness to rise (Figure 19 B) in M2 type macrophages.
The present inventor introduces PRKAR2A knock-out mices (Figure 20 A).As a result after showing that PRKAR2A is knocked out, DP1 activation The suppression of JAK2/STAT1 signal paths and M1 pro-inflammation genes (Figure 20 C, 20D-20F) can not be caused；STAT6 in M2 polarization The expression of phosphorylation and M2 genes can not be activated (Figure 20 B, 20G-20I) by DP1 activation.
Equally, in peritonitis model, the inventors discovered that PRKAR2A, which is knocked out, can eliminate the inflammation that DP1 activation promotes Disappear (Figure 21).

Claims (10)

1. a kind of method that external regulation and control macrophage polarizes to M1 types macrophage or M2 types macrophage, methods described bag Include：
(1) type of macrophage D prostaglandin receptors 1 is regulated and controled, i.e. DP1, the step of gene expression or protein active；And/or
(2) the step of regulating and controlling PRKAR2A gene expressions or protein active；And/or
(3) the step of regulating and controlling IFN γ acceptor IFNGR2 gene expressions or protein active；And/or
(4) expression of gene is regulated and controled in JAK2/STAT1 paths or the step of protein active.
2. the method as described in claim 1, it is characterised in that
The method that methods described polarizes for promotion macrophage to M1 types macrophage, including：
(1) DP1 gene expressions or protein active are suppressed；And/or
(2) PRKAR2A gene expressions or protein active are suppressed；And/or
(3) IFN γ acceptor IFNGR2 gene expressions or protein active are promoted；And/or
(4) expression of gene or protein active in JAK2/STAT1 paths are promoted；Or
The method that methods described polarizes for promotion macrophage to M2 types macrophage, including：
(a) DP1 gene expressions or protein active are promoted；And/or
(b) PRKAR2A gene expressions or protein active are promoted；And/or
(c) IFN γ acceptor IFNGR2 gene expressions or protein active are suppressed；And/or
(d) expression of gene or protein active in JAK2/STAT1 paths are suppressed.
3. such as the method any one of claim 1-2, it is characterised in that implement methods described by the following means：
(a) gene knockout；
(b) it is overexpressed gene；And/or
(c) activator or inhibitor are given.
4. following reagents are preparing regulation and control macrophage into M1 types macrophage or the medicine of M2 type macrophage polarization Purposes：
(1) reagent of the regulation and control type of macrophage D prostaglandin receptors 1, i.e. DP1, gene expression or protein active；And/or
(2) reagent of PRKAR2A gene expressions or protein active is regulated and controled；And/or
(3) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is regulated and controled；And/or
(4) expression of gene or the reagent of protein active in JAK2/STAT1 paths are regulated and controled.
5. purposes as claimed in claim 4, it is characterised in that the medicine is used to promote macrophage to M1 type macrophages Polarization, the reagent include：
(1) reagent of DP1 gene expressions or protein active is suppressed；And/or
(2) reagent of PRKAR2A gene expressions or protein active is suppressed；And/or
(3) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is promoted；And/or
(4) expression of gene or the reagent of protein active in JAK2/STAT1 paths are promoted.
6. purposes as claimed in claim 4, it is characterised in that the medicine is used to promote macrophage to M2 type macrophages Polarization, the reagent include：
(a) reagent of DP1 gene expressions or protein active is promoted；And/or
(b) reagent of PRKAR2A gene expressions or protein active is promoted；And/or
(c) reagent of IFN γ acceptor IFNGR2 gene expressions or protein active is suppressed；And/or
(d) expression of gene or the reagent of protein active in JAK2/STAT1 paths are suppressed.
7. purposes as claimed in claim 6, it is characterised in that the medicine is used to treat inflammation, preferably benefits from macrophage The inflammation that cell polarizes to M2 types macrophage；
It is highly preferred that the inflammation is selected from asthma, inflammatory bowel disease (including Crohn disease and ulcerative colitis), appendicitis, eye Blepharitis, capillary bronchitis, bronchitis, bursal synovitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, cystitis, tear Adenositis, dermatitis, dermatomyositis, encephalitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, manadesma Inflammation, fibrositis, gastritis, gastroenteritis, hepatitis, suppurative hidradenitis, laryngitis, mastitis, meningitis, myelitis, myocarditis, It is myositis, ephritis, oaritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, quiet Arteries and veins inflammation, pneumonia, rectitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis, tonsillotome Inflammation, uveitis, vaginitis, vasculitis or vulvitis.
8. purposes as claimed in claim 7, it is characterised in that
The reagent is selected from：DP1 activators, JAK2 inhibitor, STAT1 inhibitor, PRKAR2A activators and IFNGR2 suppress Agent；And/or
The reagent is nucleic acid, protein or micromolecular compound.
9. method as claimed in claim 8, it is characterised in that
The DP1 activators are BW245C；
The JAK2 inhibitor is selected from：AT9283, AZD-1480, Baricitinib or its phosphate, BMS-911543, CEP- 33779th, CYT387 or its mesylate or sulfate, GLP0634 and LY2784544；
The STAT1 inhibitor is selected from：Fludarabine, Stattic, niclosamidum and nifuroxazide；
The PRKAR2A activators are Foskolin；With
The IFNGR2 inhibitor is PRKAR2A or its activator.
10.PRKAR2A or IFNGR2 regulates and controls macrophage to M1 types macrophage or M2 type macrophages as target spot in screening The material of polarization or the application in the medicine for preparing treatment inflammation.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111549000A (en) * 2020-06-18 2020-08-18 中国医学科学院整形外科医院 Recombinant adipose-derived stem cell for over-expression of Hpgds, preparation method and application thereof
CN115006386A (en) * 2022-03-28 2022-09-06 中国人民解放军海军军医大学 Application of nifuratel in preparing medicine for resisting infection of tick-borne encephalitis virus West Nile virus yellow fever virus and chikungunya virus
CN116036285A (en) * 2023-02-07 2023-05-02 河北大学附属医院 Application of substance for inhibiting STAT6 site ubiquitination in regulating macrophage polarization

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103038219A (en) * 2010-07-01 2013-04-10 阿勒根公司 Compounds acting at multiple prostaglandin receptors to give a general anti-inflammatory response
CN103119039A (en) * 2010-08-20 2013-05-22 阿勒根公司 Compounds act at multiple prostaglandin receptors giving a general anti-inflammatory response

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103038219A (en) * 2010-07-01 2013-04-10 阿勒根公司 Compounds acting at multiple prostaglandin receptors to give a general anti-inflammatory response
CN103119039A (en) * 2010-08-20 2013-05-22 阿勒根公司 Compounds act at multiple prostaglandin receptors giving a general anti-inflammatory response

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ADAM C. LABONTE ET AL.,: ""The Role of Macrophage Polarization in Infectious and Inflammatory Diseases"", 《MOL. CELLS》 *
CHRISTOPHERD.BUCKLEY ET AL.,: ""Proresolving Lipid Mediators and Mechanisms in the Resolution of Acute Inflammation"", 《IMMUNITY》 *
DEREK ET AL.,: ""Inducible cyclooxygenase may have anti-inflammatory properties"", 《NATURE MEDICINE》 *
MIKAYASUI ET AL.,: ""The Prostaglandin E2 Receptor EP4R egulates Obesity-Related Inflammation and Insulin Sensitivity"", 《PLOS ONE》 *
WS KANG ET AL.,: ""A macrophage-specific synthetic promoter for therapeutic application of adiponectin"", 《GENETHERAPY》 *
应延风等: ""***素及其受体对相关炎症细胞调控功能研究进展"", 《金华职业技术学院学报》 *
曹春水等: ""***素生物代谢与炎症性疾病"", 《中华医学会急诊医学分会第17次全国急诊医学会学术年会》 *
阮静瑶等: ""巨噬细胞M1/M2极化的信号通路研究进展"", 《免疫学杂志》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111549000A (en) * 2020-06-18 2020-08-18 中国医学科学院整形外科医院 Recombinant adipose-derived stem cell for over-expression of Hpgds, preparation method and application thereof
CN115006386A (en) * 2022-03-28 2022-09-06 中国人民解放军海军军医大学 Application of nifuratel in preparing medicine for resisting infection of tick-borne encephalitis virus West Nile virus yellow fever virus and chikungunya virus
CN115006386B (en) * 2022-03-28 2024-03-26 中国人民解放军海军军医大学 Application of nifurazite in preparation of medicines for resisting tick-borne encephalitis virus west nile virus yellow fever virus and chikungunya fever virus infection
CN116036285A (en) * 2023-02-07 2023-05-02 河北大学附属医院 Application of substance for inhibiting STAT6 site ubiquitination in regulating macrophage polarization
CN116036285B (en) * 2023-02-07 2024-02-02 河北大学附属医院 Application of substance for inhibiting STAT6 site ubiquitination in regulating macrophage polarization

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