CN104548060B - The method and the compound of compound regulation TAK1 activity are used for the application for treating hand-foot-and-mouth disease - Google Patents
The method and the compound of compound regulation TAK1 activity are used for the application for treating hand-foot-and-mouth disease Download PDFInfo
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Abstract
The invention provides a kind of method whether authenticating compound adjusts mammal TAK1 activity, and compound 2,4,6 trimethyl N (trifluoromethyls of meta 3) benzsulfamide (m 3M3FBS) and/or its physiologically tolerable salt and pharmaceutically acceptable carrier be used for the purposes for preparing pharmaceutical composition, the pharmaceutical composition is used to treat the hand-foot-and-mouth disease as caused by enterovirns type 71 or coxsackie virus A 16.Experimental study shows, can significantly inhibit TAK1 phosphorylation and the map kinase in the macrophage that dsRNA is stimulated and the activity of NF к В paths with m 3M3FBS processing, also be substantially reducing at poly (I:C) macrophage stimulated includes the expression of TNF, IL 6 and IL 12 proinflammatory cytokine.Moreover, reducing the clinical score of the mouse of CVA16 infection with m 3M3FBS processing, the survival of the mouse of CVA16 infection is also extended, and reduces their histopathology development in lung and musculature.
Description
Technical field
It is used to prepare treatment by enteron aisle disease the present invention relates to a kind of method of compound regulation TAK1 activity and the compound
The pharmaceutical composition of hand-foot-and-mouth disease and its application caused by malicious 71 types or coxsackie virus A 16.
Background technology
Hand-foot-and-mouth disease (Hand, foot and mouth disease, HFMD) is [with COxsackie A groups 16 by enterovirus
Type (CVA16), enterovirns type 71 (EV71) are common] caused by acute infectious disease, be usually expressed as with herpangina, pharynx
Gentle and self-limited disease that laryngalgia and heating are characterized (Huang, C.C., C.C.Liu, Y.C.Chang, C.Y.Chen,
S.T.Wang,and T.F.Yeh.1999.Neurologic complications in children with
enterovirus 71infection,N Engl J Med.341:936-942).A small number of HFMD cases may occur in which aseptic brain
Film inflammation, BBE and acute hemorrhagic necrotizing pancrease (a kind of infantile paralysis is like syndrome).
The HFMD as caused by such as CVA16 with EV71 enterovirus brings into millions of the infected and hundreds of dead
Die, and be presently considered to be important infection disease (Solomon, T., P.Lewthwaite, D.Perera,
M.J.Cardosa,P.McMinn,and M.H.Ooi.2010.Virlogy,epidemiology,pathogenesis,and
control of enterovirus 71.The Lancet Infectious Diseases.10:778-790).Currently without
Effective vaccine or specific antiviral therapy, therefore the prevention or treatment means developed effectively for hand-foot-and-mouth disease are very
Urgent.
It is HFMD morbidity original that high-caliber IL-6, which is reported,.Although what is induced after being stimulated through pathogen is further amounts of proinflammatory
Cell factor should activate acute phase reaction and for pathogen remove T cell and B cell stimulation (Kanda, T.,
J.E.McManus,R.Nagai,S.Imai,T.Suzuki,D.Yang,B.M.McManus,and
I.Kobayashi.1996.Modification of viral myocarditis in mice by interleukin-
6.Circ.Res.78:848-856；Kopf,M.,H.Baumann,G.Freer,M.Freudenberg,M.Lamers,
T.Kishimoto,R.Zinkernagel,H.Bluethmann,and G.1994.Impaired immune and
acute-phase responses in interleukin-6-deficient mice.Nature.368:339-342), receive
The evidence of collection but shows that the proinflammatory cytokine of excessive level can overwhelm Immune defense, and cause serious immune-inflammatory disease.
Some clinical researches show that the induction of a large amount of proinflammatory cytokines is that EV71 virus infection causes serious morbidity former
Cause.In fact, being infected from EV71 in the serum and celiolymph (cerebral spinal fluid, CSF) gathered in patient
Containing high-caliber proinflammatory cytokine, such as IL-6 is persistently reported, for example：
1.Lin,T.Y.,L.Y.Chang,Y.C.Huang,K.H.Hsu,C.H.Chiu,and
K.D.Yang.2002.Different proinflammatory reactions in fatal and non-fatal
enterovirus 71infections:implications for early recognition and therapy.Acta
Paediatr.91:632-635；
2.Lin,T.Y.,S.H.Hsia,Y.C.Huang,C.T.Wu,and
L.Y.Chang.2003.Proinflammatory cytokine reactions in enterovirus 71infections
of the central nervous system.Clin Infect Dis.36:269-274；
3.Wang,S.M.,H.Y.Lei,M.C.Huang,L.Y.Su,H.C.Lin,C.K.Yu,J.L.Wang,and
C.C.Liu.2006.Modulation of cytokine production by intravenous immunoglobulin
in patients with enterovirus 71-associated brainstem
encephalitis.J.Clin.Virol.37:47-52；
4.Wang,S.M.,H.Y.Lei,L.Y.Su,J.M.Wu,C.K.Yu,J.R.Wang,and
C.C.Liu.2007.Cerebrospinal fluid and cytokines in enterovirus 71brain stem
encephalitis and echovirus meningitis infections of varying
severity.Clin.Microbiol.Infect.13:677-682；
5.Weng,K.F.,L.L.Chen,P.N.Huang,and S.R.Shih.2010.Neural pathogenesis
of enterovirus 71infection.Microbes Infect.12:a505-510；
6.Wang,S.M.,H.Y.Lei,and Liu,C.C.2012.Cytokine immunopathogenesis of
enterovirus 71brain stem encephalitis.Clin Dev Immunol.2012:876241。
In mouse, it is the pathogenic factor of the EV71 in baby's mouse model that the IL-6 of continual high levels, which is reported,
(Khong,W.X.,D.G.Foo,S.L.Trasti,E.L.Tan,and S.Alonso.2011.Sustained high
levels of interleukin-6contribute to the pathogenesis of enterovirus 71in a
neonate mouse model.J Virol.85:3067-3076)。
However, IL-6 generation is how to influence HFMD progress still unknown.
The content of the invention
What the present invention proposed to solve above mentioned problem of the prior art.
The invention provides a kind of method whether authenticating compound adjusts mammal TAK1 activity, wherein TAK1 activity
It is labeled with the proinflammatory cytokines induced the pathogen for producing the hand-foot-and-mouth disease as caused by enterovirns type 71 or coxsackie virus A 16
The factor plays a crucial role.
It is used to treat the hand-foot-and-mouth disease as caused by enterovirns type 71 or coxsackie virus A 16 present invention also offers one kind
Pharmaceutical composition, the composition includes albumen, polypeptide or their activating compounds with the activity of phospholipase Cβ 2, and/
Or its physiologically tolerable salt and pharmaceutically acceptable carrier.
To achieve the above object, the present invention uses following technical scheme：
The first aspect of the invention is to provide a kind of method whether authenticating compound adjusts mammal TAK1 activity,
Comprise the following steps：
Step 1, by mammalian cell compound incubation；
Step 2, the cell of step 1 virus and viral antigen composition derivant stimulated；
Step 3, the TAK1 activity for determining the cell from step 2；
Wherein, the TAK1 activity marks to producing the brothers as caused by enterovirns type 71 or coxsackie virus A 16
The proinflammatory cytokine that the pathogen of stomatosis induces plays a crucial role.
Preferably, the mammalian cell of the step 1 is the cell of rodent, and the rodent is more preferably
Mouse, more preferably mouse.
Preferably, the mammalian cell of the step 1 is the cell of the mankind.
Wherein, the cell is selected from pancreatic cell, myocyte, liver cell, nephrocyte, brain cell, fat cell, macrophage
Any one or more, more preferably macrophage.
Cell culture of the cell of mammal from mouse.
Preferably, the virus and viral antigen composition of the step 2 are selected from poly I:poly C poly (I:C)、H5N1、
Any one or more in NDV, CAV16, more preferably poly I:poly C poly (I:C).
Wherein, the phospholipase Cβ 2 in the compound activating cell.
The second aspect of the invention is to provide one kind and caused for treating by enterovirns type 71 or coxsackie virus A 16
Hand-foot-and-mouth disease pharmaceutical composition, the composition includes albumen, polypeptide or their activation with the activity of phospholipase Cβ 2
Compound, and/or its physiologically tolerable salt, and pharmaceutically acceptable carrier or excipient.
The compound is preferably 2,4,6- trimethyls-N- (meta-3- trifluoromethyl-phenyls)-benzsulfamide (2,4,6-
trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide,m-3M3FBS)。
The third aspect of the invention be to provide compound m-3M3FBS and/or its physiologically tolerable salt be used for make
The purposes of standby pharmaceutical composition, the pharmaceutical composition is used to treat the hand as caused by enterovirns type 71 or coxsackie virus A 16
Sufficient stomatosis.
The present invention uses above-mentioned technical proposal, compared with prior art, have the following technical effect that：
Experimental study shows, in phosphorylation and macrophage that TAK1 can be significantly inhibited with m-3M3FBS processing
The activity of map kinase and NF- к В paths, also significantly reduce macrophage include TNF, IL-6 and IL-12 proinflammatory cytokines because
The expression of son.Moreover, reducing the clinical score of the mouse of CVA16- infection with m-3M3FBS processing, CVA16- senses are also extended
The survival of the mouse of dye, and reduce their histopathology development in lung and musculature.
Brief description of the drawings
Fig. 1：In HFMD sufferers, the PLC β 2 with higher expression combine appearance with lower p-TAK1 and blood serum IL-6；
PLC β's 2 in the PMBC (PBMC) of (A, B) from blank control group and HFMD sufferer groups is immune
Trace (IB) , ﹡ p ＜ 0.05；
IL-6 EUSAs (ELISA) , ﹡ p ＜ 0.05 in the serum of (C, D) from HFMD sufferers；
The Western blotting (IB) of PLC β 2 and p-TAK1 in the PBMC of (E, F) from HFMD sufferers；Intensity based on every band
PLC β 2 and p-TAK1 levels are divided into basic, normal, high, and are sorted in the percentage of the sufferer of each species and are described in (F)
In block diagram；
(G) Western blotting of the PLC β 2 in the Turnover of Mouse Peritoneal Macrophages of infection CVA16 viral one specified time
(IB)；
(H) come the poly (I that use by oneself:C) (10mg/ml) stimulates the dissolving production of the Turnover of Mouse Peritoneal Macrophages of one specified time
The Western blotting (IB) of PLC β 2 in thing；
(I) come the poly (I that use by oneself:C) (10mg/ml) stimulates the wild type (WT) and TLR3 of one specified time-/-Mouse abdomen
The Western blotting (IB) of PLC β 2 in the lysate of chamber macrophage；
(J) with poly (I:C) (10mg/ml) is located in advance before stimulating one specified time with p38 inhibitor SB203580
The Western blotting (IB) of PLC β 2 in the reason Turnover of Mouse Peritoneal Macrophages of 1 hour；
G, H, I, J represent at least three kinds independent experiments.
Fig. 2：PLCβ2-/-(PLC β 2- are not enough) mouse is easier to be infected by CVA16；
(A, B) uses poly (I:C) (10mg/ml) stimulates WT the or PLC β 2 of one specified time-/-It is relative in macrophage
Tnf (A) and IL-6 (B) mRNA level in-site Q-PCR analysis；14 -day-old of WT or PLC β 2-/-10 μ l of infection in mouse peritoneal
CVA16 is (per mouse 4 × 105PUF) 0-5 days；
(C, D, E) comes from WT or PLC β 2-/-Relative Tnf (C), IL-6 (D) and IL-12 (E) in the musculature of mouse
The Q-PCR analyses (n=3) of mRNA level in-site；
(F) 0 day (being uninfected by) or WT the or PLC β 2 of 5 days are infected according to the method described above-/-The tissue disease of Muscle Tissue
Reason figure；Original magnification：100 × (top) or 200 × (bottom)；
(G) CVA16 expression (n=in the replicated rna sample obtained from the musculature of the mouse handled through the above method
3)；
WT the or PLC β 2 of (H, I) designated method processing-/-The clinical score (H) or survival rate (I) of mouse；These data
Represent at least three kinds independent experiments (average value and the , ﹡ ﹡ p ＜ 0.01 of s.e.m) , ﹡ p ＜ 0.05.
Fig. 3：PLC β 2 suppress MAPKs and NF- к В activity by targetting TAK1 ubiquitinations；
(A, B) expression in HEK293T cells carries hemagglutinin (HA) or Flag TAK1 or PLC β 2 cell
The immunoprecipitation (IP) and Western blotting (IB) of pyrolysis product；
(C) from expression Flag-PLC β 1-3 and HA-TAK1 HEK293T cells lysate immunoprecipitation (IP) and
Western blotting (IB)；
(D) the external GST precipitation examinations combined using the PLC β 2 with histidine (HIS) label of purifying with GST-TAK1
Test；
(E) with the GST-TAK1 of the purifications combined of endogenous PLC β 2 immunoprecipitation experiment；
(F) through poly (I:C) in the main macrophage stimulated PLC β 2 and TAK1 endogenous interaction relationship；
(G) come the Diagnosis of Sghistosomiasis of the pyrolysis product of the HEK293T cells for the phosphoric acid-TAK1 antibody expression PLC β 2 and TAK1 that uses by oneself
Mark is analyzed；
(H, I) uses phosphoric acid-TAK1 antibody (H) or phosphorylation-antibody (I), come the poly (I that use by oneself:C) (10mg/ml) is pierced
Swash WT the or PLC β 2 of one specified time-/-The immunoblotting assay of the lysate of macrophage；
(J) there is or lacking the pyrolysis product that PLC β express K63-Ub, TAK1 and TAB1 HEK293T cells for 2 time
Immunoprecipitation (IP) and Western blotting (IB) analysis；
(K) with poly (I:C WT or PLC β 2) are stimulated-/-The one specified time of mouse, and by product of cell lysis with anti-
TAK1 immunoprecipitations；With anti-Ub (K63) antibody analysis immunoprecipitate；
(L) in presence or the immunoprecipitation of the lysate for the HEK293T cells for lacking 2 times expression TAK1 and TAB1 of PLC β
(IP) analyzed with Western blotting (IB)；These data represent at least three kinds independent experiments.
Fig. 4：M-3M3FBS mitigates hand-foot-and-mouth disease caused by CVA16 virus infection；
The peritoneal macrophage and then use phosphorylation that (A, B) originates come DMSO or m-3M3FBS pretreatment of mice of using by oneself-
Antibody is to poly (I:C the pyrolysis product of the macrophage of one specified time) is stimulated to carry out Western blotting (IB) analysis；
(C, D, E) 14 -day-old of WT mouse are with DMSO or m-3M3FBS pretreatments and then infected 0-5 days with CVA16；Come from
The Q-PCR analyses (n=3) of relative Tnf (C), IL-6 (D) and IL-12 (E) mRNA level in-site in two groups of musculatures；
(F) from the histopathology figure of two groups of musculature obtained；Original magnification：100 × (top) or 200 ×
(bottom)；
The clinical score (G) or survival rate (H) for the mouse that (G, H) is handled through the above method；These data represent at least three
Plant independent experiment (average value and s.e.m)；
(I) chart is described by viral-induced PLC β 2, by targetting TAK1 ubiquitinations, negative regulation congenital immunity
Reaction.
Embodiment
The present invention is described in more detail below by specific embodiment, for a better understanding of the present invention,
But following embodiments are not intended to limit the scope of the invention.
Test material and method：
Cell culture and reagent：
By HEK293T cell culture in DMEM culture mediums (Hyclone), through 10% (v/v) heat-inactivated fetal bovine serum
(FBS, Gibco) and 100U/ml penicillin and streptomysin.HeLa cells and peritoneal macrophage are in RPMI-1640 culture mediums
Middle culture, through 10% (v/v) FBS and 100U/ml penicillin and streptomysin.Vero cells (African green monkey kidney cell) are in VP-
Cultivated in SFM culture mediums (Gibco), 4mML- glutamates (Gibco).These cell culture are with 5%CO237 DEG C of balance
In incubator.The micro-carriers cell culture bioremediation reported before use, the CVA16 bacterial strains clinically separated are put
Bred in Vero cells.The virus stocks are stored in -80 DEG C.Dripped using the plaque-forming assay method Test Virus of standard
Degree.
Use following antibody：
The specific poly- ubiquitin (5621) of rabbit-anti K63- connections, rabbit-anti phosphoric acid TAK1 (Thr187) (4536), rabbit-anti phosphoric acid TAK1
(Ser412) (9339), rabbit-anti phosphoric acid p65 (3033), rabbit-anti phosphoric acid Erk1/2 (9101), rabbit-anti phosphoric acid p38 (9215) and rabbit-anti
Phosphoric acid Jnk (9251), these antibody come from Cell Signaling Technology；
Rabbit-anti PLC β 2 (sc-206), rabbit-anti phosphoric acid TAK1 (Ser192) (sc-130219) and rabbit-anti TAK1 (sc-7162),
These antibody come from Santa Cruz Biotechnology；
Rabbit-anti TAB1 (ab27983, from Abcam)；The anti-Flag M2 affinity gels (A2220) of mouse of monoclonal, monoclonal
The anti-HA of mouse (H9658), rabbit-anti HA (H6908), rabbit-anti GAPDH (SAB2701826) and rabbit-anti Flag (F7425), these antibody
From Sigma；
And Protein G SepharoseTM 4Fast Flow(GE Healthcare)。
Use following compounds：
M-3M3FBS (T5699) and SB203580 (P38 inhibitor, S8307), both is from Sigma.
Plasmid and plasmid construction：
The TAK1 and its deletion mutant of the cDNAs PLC β 2 encoded and its form truncated and cDNAs codings are inserted into
In Flag-pcDNA3 carriers.PLC β 2 and TAK1cDNAs are inserted into HA-pcDNA3 expression vectors.CDNAs is encoded
TAB1 and TAB2 are inserted into pcDNA3 carriers, and the cDNAs PLC β 2 encoded and TAK1 are inserted respectively into pET28a and p-
In GEX-4T-1 carriers.
Mouse species：
Homozygous PLC β 2 and TLR3 gene knockout mices are in Shanghai Laboratory Animal Center without specific pathogen bar
Raised under part.The 14 -day-old or 6 weeks big knock out mice of PLC β 2 and wild type blank control are used in the experiment of the present invention
Mouse.All zooscopies are looked after with being ratified using group by the Institutional Animal of Tongji University.
The separation of macrophage：
Peritoneal macrophage is small after being injected 4 days by Brewer improvement thioglycollate broths (BD, Sparks, MD)
Obtained in mouse.These cells poly (I:C) (10mg/ml), LPS (100ng/ml), PGN (25 μ g/ml), H5N1 (MOI=1)
Or NDV (MOI=1) stimulates one time specified.
RT-PCR is analyzed：
First, these cells are hatched 12 hours in the case of serum-free.With DMSO or m-3M3FBS (10mM) preincubation
After 30 minutes, these cells poly (I:C) (10mg/ml) stimulates one time specified.According to manufacturers instruction
(Invitrogen) instruction, total serum IgE is extracted with 1ml TRIzol reagents.Then, using ReverTraqPCR RT
Kit (Toyobo, FSQ-101), is indicated according to manufacturers instruction, by 1 μ g total serum IgE reverse transcriptions.LightCycler(Roche,
) and SYBR RT-PCR kit (Toyobo, QPK-212) are used for quantitative Real time RT-PCR analysis LC480.Expression value is by standard
Turn to those expression values obtained with respective gene Gapdh (coding GAPDH).
Luciferase reporter gene is tested：
Mammal 293T cell lines are used for luciferase reporter gene experiment.Cell is in DMEM (high glucoses
Content；Bibco, Life Technologies, Grand Island, NY, USA) in cultivated with 10%FBS., will before transfection
Cell is accessed in 96 porocyte culture plates.After 24 hours, Lipofectamine is usedTM2000 transfection reagents (Invitrogen,
Life Technologies, Grand Island, NY, USA) by reporter plasmid (per hole 10ng altogether) and show plasmid (every hole
100ng) common transfection.After transfection after about 48 hours, according to the instruction of manufacturers instruction, in ModulusTMSingle tube multifunctional enzyme
Mark and Luciferase Assay Reagent II (LAR are used on instrument (Turner Biosystems, Sunnyvale, CA, USA)
II) (Promega, Madison, WI, USA), quantitative Renilla and firefly luciferase activity.Transfection is triplicate.Match somebody with somebody
T is examined and is used for determining significant difference.
Western blot test and co-immunoprecipitation reaction：
To occur immune precipitation, method is infected altogether using calcium phosphate-DNA and is transiently transfected in=293T cells.48
After hour, cell is washed with PBS, and then in lysis buffer (20mM Tris (pH7.5), 150mM NaCl, 1%Triton
X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin) in cell crack, add 1% albumen enzyme level
Agent composition (Sigma, P8340), 1mM NaF and 1mM Na3VO4 49.Be positioned on ice after 30 minutes, by lysate with
13523 × g speed, centrifugation 15 minutes at 4 DEG C, to remove fragment.Product of cell lysis is affine with anti-Flag M2
Gel, the incubated overnight at 4 DEG C.For raw immune precipitation, peritoneal macrophage poly (I in occurring:C one section of finger) is stimulated
The fixed time.Cell is then cleaved, and by the lysate antibody of PLC β 2 and Protein G SepharoseTM 4Fast
Flow, the incubated overnight at 4 DEG C.Agarose sample is centrifuged, is washed with cell lysis buffer solution three times, and use SDS loadings
Buffer solution boils 8 minutes.
Gst fusion protein and precipitation test：
The albumen of TAK1 and PLC β 2 is expanded by PCR, and is subcloned respectively to pGEX-4T1 (Amersham) and pET28a
(Novagen) in carrier.Indicated according to manufacturers instruction, by His and gst fusion protein expression in BL-21 (DE3) bacterium
(Invitrogen) in.According to the method described above in dissolving RAW264.7 cells.By lysate or the albumen of His-PLC β 2 of purification
Hatch 4 hours with the appropriate fusion protein of equivalent, at 4 DEG C, be attached on glutathione pearl.Peptide pearl is centrifuged, washed
Wash, boiled with SDS sample-loading buffers 8 minutes, and then pass through immunoblotting assay.
Cell dyeing and confocal microscope：
Indicated according to manufacturers instruction, use LipofectamineTM2000 (11668-019, Invitrogen) are transfected
HeLa cells.After transfection 48 hours, cell is fixed 30 minutes with 4% formaldehyde at 25 DEG C.Cell is colored, and using being equipped with
The Leica confocal microscopes of analysis software are detected.
Zoopery：
With syringe by 10 μ l CVA16 (per mouse 4 × 105PUF) intraperitoneal injection no-special pathogen 14 -day-old of WT or
The knock out mice of PLC β 2.The clinical symptoms and death toll of 1 mouse of observation, are observed 14 days daily.By DMSO or m-3M3FBS
(5mg/kg) be expelled to CVA16 infection mouse in, injection 3 times (infection 1 day before, infection 2 days after and infection 5 days after).
Clinical disease presses following scoring：
0, health；
1, flanging fur and bow-backed outward appearance；
2, consumption；
3, limbs fatigue；
4, quadriplegia；
5, it is dying and dead.
After infection 5 days, lung, muscle, spleen tissue sample are gathered from the gene knockout mices of WT or PLC β 2, and be fixed on
In 10% neutral buffered formalin (Sigma-Aldriich).These tissue by with FFPE, incision, use Yihong stain
(H＆E) dye.Obtain × 100 or × 200 enlargement ratio bright-field microscope photo.All zooscopies pass through Tongji University
Institutional Animal manage with being ratified using group.
Statistical analysis：
Statistical significance between two groups is examined by double tail student t and two-way ANOVA (ANOVA) is determined.As p ＜
When 0.05, it is considered to be have marked difference.
Ethics is stated：
Present invention work receives batch of the clinical ethics committee from the attached Xinhua Hospital of pharmaceutical college of Shanghai Communications University
Standard, meets the guilding principle and institutional policy of national government.Obtain from the minor/youngster participated in present invention research
The Written informed consent of virgin participant signature under the accompanying of relative, guardian or ward.Ethics Committee clearly ratifies
The program of this part of letter of consent.
Human sample：
Whole blood sample is obtained from the children less than 10 years old.According to ministry of Health of China HFMD diagnostic criteria (2008)
(http://www.moh.gov.cn/mohbgt/s9503/200812/38494.shtml) clinical diagnosis goes out 6 children and suffers from
HFMD.In addition, the healthy children without HFMD histories of matching in 5 years old is included as blank control group.To carry out Diagnosis of Sghistosomiasis
Mark is tested, and according to the instruction of manufacturers instruction, whole blood sample is handled with red blood cell lysis buffer (eBioscience)；So
Afterwards, remaining cell is dissolved in lysis buffer.
Test result analysis：
Blood sample is gathered from 5 sufferers of healthy control group and 6 clinical diagnosises with HFMD without HFMD histories, and
These samples are then analyzed with western blot test.It was found that phospholipase C (phospholipase C, PLC) β 2 protein level
(a kind of to be used for the crucial enzyme that effective signal conversion and white blood cell are activated) is much higher than control group (figure in HFMD patient
1A and Figure 1B).Then, the collection of blood sample is extended into 30 HFMD sufferers, and analyzes transforming growth factor β activation kinases
1 (transforming growth factor- β-activated kinase 1, TAK1), regulation nuclear factor-κB
(nuclear factor, NF- к В) and MAPK (mitogen-activated protein kinase,
MAPK the activity of intracellular central element), they are labeled with to producing the path that proinflammatory cytokine plays a crucial role.
According to the level of TAK1 phosphorylations, preceding 12 samples are divided into two groups.Second group of sufferer has higher than first group
Level TAK1 activity however the PLC β 2 of more low expression (Fig. 1 E).Moreover, being found in one group of 30 HFMD clinical sample
There is inverse correlation (Fig. 1 F) in TAK1 activity and the protein levels of PLC β 2.Moreover, second group of HFMD sufferer produces significantly lower IL-6
(Fig. 1 C and Fig. 1 D).These results show that PLC β 2 can suppress TAK1 activity, and which reduces IL-6 generation.
Then, it is determined that whether PLC β 2 are to be induced by the infection of CVA16 viruses.In fact, infection CVA16 can induce
PLC β 2 expression (Fig. 1 G).Would generally be produced in virus infection dsRNA (Weber, F., V.Wagner,
S.B.Rasmussen,R.Hartmann,and S.R.Paludan.2006.Double-stranded RNA is produced
by positive-strand RNA viruses and DNA viruses but not in detectable amounts
by negative-strand RNA viruses.J Virol.80:5059-5064).With poly I:poly C poly (I:
C the generation (Fig. 1 H) for also improving PLC β 2) is stimulated.However, in TLR3-/-Macrophage or wild type (wild-type, WT) are huge
In phagocyte, by poly (I:C) PLC β 2 up-regulation is stimulated to be weakened, with SB203580 processing (Fig. 1 I and Fig. 1 J).These knots
Fruit shows that PLC β 2 can be induced by dsRNA by TLR3-p38 paths.
To check whether PLC β 2 work to the CVA16 generations of proinflammatory cytokine induced, with poly (I:C) stimulate
From PLC β 2-/-(PLC β 2- are not enough) mouse or the macrophage of WT mouse, and it is proinflammatory thin with quantitative RT-PCR assay analysis
The generation of intracellular cytokine.When with poly (I:C after) stimulating, PLC β 2-/-Macrophage show than WT macrophage produce it is higher
Proinflammatory cytokine TNF and IL-6 (Fig. 2A and Fig. 2 B).To investigate the functions of PLC β 2 in vivo, by two weeks big WT
Mouse or PLC β 2-/-Mouse CVA16 viruses infection 5 days, the expression quantitative RT-PCR assay of proinflammatory cytokine
Analysis.PLCβ2-/-The muscle of mouse has the TNF (Fig. 2 C) more significantly higher than WT mouse, IL-6 (Fig. 2 D), IL-12 (Fig. 2 E)
MRNA level in-site, illustrate the viral-induced expression of proinflammatory factor in the negative regulation bodies of PLC β 2.By WT mouse two weeks big or
PLCβ2-/-Mouse CVA16 viruses infection 5 days, further investigates functional meanings of the PLC β 2 in host anti-virus reaction.
The PLC β 2 of CVA16 infection-/-Mouse facing with the virus titer and Geng Gao higher than WT mouse in their musculature
Bed scoring (Fig. 2 G and Fig. 2 H).The PLC β 2 of CVA16 infection-/-Mouse is faster more dead (Fig. 2 I) than WT mouse.It is small from infection CVA16
The lung (not providing) or the histological indications of musculature that mouse is isolated show, PLC β 2-/-Mouse has more than WT mouse
High histopathology development (Fig. 2 F).PLCβ2-/-Mouse be easier by CVA16 virus infection the fact that illustrate, PLC
β 2 plays a part of the negative regulation agent of In vivo infection CVA16 morbidity original.
The mechanism of the viral-induced function for producing proinflammatory cytokine, PLC β 2 are being adjusted for further investigation PLC β 2
It is whether interrelated detected with TAK1.In the instantaneous Human embryonic kidney (human for infecting expression TAK1 and PLC β 2
Embryonic kidney, HEK293T) in cell, immune precipitation (Fig. 3 A and figure are occurred into together with PLC β 2 for TAK1
3B).However, the not no immunoprecipitations (Fig. 3 C) together with PLC β other isotype PLC β 1 or PLC β 3 of TAK1.Paddy Guang in vivo
Purified recombinant protein is used in sweet peptide sulfurtransferase (glutathione S-transferase, the GST) precipitation method, is found
TAK1 albumen and the purified restructuring His-PLC β 2 or interior raw PLC β 2 from RAW264.7 cells be united (Fig. 3 D and
Fig. 3 E).Finally, with poly (I:C the phase for improving interior raw TAK1 and interior raw PLC β 2 in Turnover of Mouse Peritoneal Macrophages) is stimulated
Interaction (Fig. 3 F), it is to stimulate to rely on to illustrate the interaction between TAK1 and PLC β 2.PLC β 2 include four domains：PH_
PLC, Plcc, C2_2 and PLC- β C.To determine the region being connected in PLC β 2 with TAK1, PLC β 2 deletion mutant is built, and
Analyze their interactions with TAK1.Plcc and C2_2 and TAK1 interacts, and PH_PLC and PLC- β C do not have (to carry
For).As a result ground, confocal microscope analysis display, in HeLa cells overexpression TAK1 and in the mutant of PLC β 2 are consistent
In, only Plcc cooperates together with TAK1 and (not provided).
Because PLC β 2 and TAK1 interact, 2 couples of activation TAK1 of PLC β effect is examined.PLC β 2 overexpression is obvious
Ground shows phosphorylations (Fig. 3 G) of the TAK1 at Thr187, Ser192 and Ser417.After being activated, TAK1 phosphorylation IKK β
Or MKKs, cause NF- к В, JNK and p38 kinases of path label activation (Adhikari, A., M.Xu, and Z.J.Chen,
2007.Ubiquitin-mediated activation of TAK1and IKK.Oncogene.26:3214-3226).Enter one
Walk the effect that 2 couples of activation NF- к В and AP-1 of PLC β are assessed by reporter gene assay.TAK1 and TAB1 is in HEK293T cells
Expression significantly improve NF- к В and AP-1 reporter gene activity, and PLC β 2 expression is inhibited in a dose-dependent manner
This growth (not providing).To further determine that whether PLC β 2 influence TAK1 activity, analyze and be isolated from PLC β 2-/-Mouse
Macrophage in dsRNA induce TAK1 activity.With poly (I:C) stimulate, in PLC β 2-/-Compare in the macrophage of mouse
In the macrophage of wild-type mice, phosphorylations (Fig. 3 H) of more induction TAK1 at Thr187, Ser192 and Ser417.
The negative regulation TAK1 of these as shown by data PLC β 2 phosphorylation.As a result ground is consistent, when with poly (I:C) stimulate, PLC β
2-/-Macrophage show the higher activity of map kinase and NF- к В paths (Fig. 3 I) than the macrophage of wild type.These
As shown by data, PLC β 2 also inhibits the activity that the map kinase in downstream and the TAK1- in NF- к В paths are mediated.
TAK1 activation needs the poly ubiquitination of TAK1 K63- connections, and TAK1 ubiquitination by with TAK1-
The protein TAB1's and TAB2 of connection combines to adjust.PLC β 2 significantly reduce TAB1 and TAK1 in dose-dependent mode
Joint (Fig. 3 L).TAB1 improves the poly ubiquitination of TAK1 K63- connections, but PLC β 2 overexpression is significantly reduced
The ubiquitination (Fig. 3 J) of TAK1 K63- connections.On the contrary, poly (I:C) the poly ubiquitination of the TAK1 of induction K63- connections exists
PLCβ2-/-Macrophage in ratio be significantly enhanced in WT macrophage (Fig. 3 K).These results jointly show, PLC β
2 can target TAK1-TAB1 complexs, and by the poly ubiquitination for the K63- connections for suppressing TAK1, negative regulation TAK1's
Activity.
Reactive compound 2,4,6- trimethyls-N- (meta-3- trifluoromethyl-phenyls)-benzsulfamide (2,4,6-
Trimethyl-N- (meta-3-trifluoromethyl-phenyl)-benzenesulfonamide, m-3M3FBS) can be straight
Connecing stimulates the activity of phospholipase C.Due to having identified that PLC β 2, as the negative regulation albumen of virus infection, are next assessed
Effects of the m-3M3FBS in antiviral immunity reaction.With m-3M3FBS processing can significantly inhibit TAK1 phosphorylation and
The activity (Fig. 4 A and Fig. 4 B) of map kinase and NF- к В paths in the macrophage that dsRNA- is stimulated.As a result ground is consistent,
M-3M3FBS is substantially reducing at poly (I:C the macrophage of)-stimulation includes TNF (Fig. 4 C), IL-6 (Fig. 4 D) and IL-12
The expression of the proinflammatory cytokine of (Fig. 4 E).Moreover, the clinic that the mouse of CVA16- infection is reduced with m-3M3FBS processing is commented
Divide (Fig. 4 G).M-3M3FBS also extends the survival (Fig. 4 H) of the mouse of CVA16- infection, and reduces them and (do not carried in lung
For) and musculature (Fig. 4 F) in histopathology development.These results indicate that m-3M3FBS can be as controlling
The potential medicine of CVA16 virus infection.
In the present invention, clinical samples of the as shown by data PLC β 2 in the macrophage from HFMD sufferers or CVA16- infection
Highly induced in this.Mouse that the PLC β 2- infected by CVA16 lack has the proinflammatory cytokine and Geng Gao of higher amount
Virus titer, and be easier to be infected by the virus.These results indicate that the inhibitor that PLC β 2 can feed back as negative sense, to subtract
The proinflammatory cytokine of the weak viral-induced generations of internal CVA16.Moreover, can be reduced with PLC activity agent m-3M3FBS processing
Histopathology development of the CVA16- infecting mouses in lung and musculature, and extend the survival of CVA- infecting mouses.Therefore, mark
It is beneficial to remember that 2 couples for the treatment of such as HFMD of PLC β infect disease.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, and the present invention is not limited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (6)
1. whether authenticating compound can mitigate the method for hand-foot-and-mouth disease caused by enterovirns type 71 or coxsackie virus A 16,
It is characterised in that it includes following steps：
Step 1, by mammalian cell addition compound be incubated；
Step 2, the cell of step 1 virus or viral antigen composition derivant stimulated；
Step 3, the TAK1 activity for determining the cell from step 2；
Step 4, according to TAK1 activity height judge whether the compound can mitigate enterovirns type 71 or Coxsackie virus
Hand-foot-and-mouth disease caused by A16；
Wherein, the compound is the phospholipase Cβ 2 in activating cell；
Wherein, the TAK1 activity marks to producing the hand-foot-and-mouth disease as caused by enterovirns type 71 or coxsackie virus A 16
Pathogen induce proinflammatory cytokine play a crucial role；
Wherein, the virus or viral antigen composition of the step 2 are selected from poly I:poly C poly (I:C), in H5N1, NDV
Any one or it is any several；
Wherein, the step 3 is the TAK1 activity that the cell from step 2 is determined by western blot test.
2. according to the method described in claim 1, it is characterised in that the mammalian cell of the step 1 is rodent
Cell.
3. method according to claim 2, it is characterised in that the rodent is mouse.
4. according to the method described in claim 1, it is characterised in that the mammalian cell of the step 1 is the cell of the mankind.
5. the method according to claim 1-4 any one, it is characterised in that the cell is selected from macrophage.
6. according to the method described in claim 1, it is characterised in that the cell of the mammal comes from cell culture.
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