CN110038020A - Saliva acid mimic inhibits the application under infection of macrophages or state of activation in the drug of caused inflammation in preparation - Google Patents
Saliva acid mimic inhibits the application under infection of macrophages or state of activation in the drug of caused inflammation in preparation Download PDFInfo
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Abstract
The invention discloses saliva acid mimics to inhibit the application under infection of macrophages or state of activation in the drug of caused inflammation in preparation, and saliva acid mimic is the mixture of 3 '-saliva lactose, 6 '-saliva lactose sodium salts or 3 '-saliva lactose, 6 '-saliva lactose sodium salts.The present invention is commercialized saliva acid mimic using two kinds and handles the inflammation of the Macrophage Model as caused by mode picornavirus infection or representative pro-inflammatory cytokine；By Western blot, real-time quantitative RT-PCR experimental identification, both saliva acid mimics all have effective anti-inflammatory properties, can be applied to anti-inflammatory drug research and development.Result of study provided by the invention enriches autarcesiology, provides new thinking and strategy for follow-up study body inflammatory and anti-inflammatory mechanisms.
Description
Technical field
The present invention relates to immunologys and cell biology, inhibit macrophage in preparation more particularly to saliva acid mimic
Application under cell infection or state of activation in the drug of caused inflammation.
Background technique
Inflammation (inflammation) is that one kind that body generates destructive stimulus (such as pathogen and pro-inflammatory cytokine) is natural
Be immunoreacted, panimmunity cell (such as Monocytes/Macrophages) is participated in body: immunocyte inflammatory signals access is crucial
The factors activated B cell κ light chain enhancer of molecular core (nuclear factor kappa-light-chain-enhancer of
Activated B cells, NF- κ B), mitogen-activated protein kinase (mitogen-activated protein
Kinases, MAPKs) family member p38MAPK, extracellular signal-regulated kinase (extracellular signal-
Regulated kinases, ERKs), c-Jun N terminal kinase (c-Jun N-terminal kinase, JNK) etc. occurs
It is phosphorylated-activated and generate tumor necrosis factor (tumor necrosis factor α, TNF-α), interleukins
The pro-inflammatory cytokines such as (interleukin, IL) -6, IL-1 β.Under normal circumstances, inflammation is to removing pathogenic infection, reparation
Body injury etc. has beneficial effect；However excessive inflammatory reaction can cause such as allergy (allergy), atherosclerosis
(atherosclerosis), acquired immunodeficiency syndrome (acquired immune deficiency syndrome,
AIDS), the diseases associated with inflammation such as cancer, septicemia (sepsis) seriously endanger human health.Although there are many for controlling at present
The anti-inflammatory drug for treating diseases associated with inflammation comes out, but anti-inflammatory drug research and development still need to constantly carry out.
Summary of the invention
Demand is researched and developed for current anti-inflammatory drug, the object of the present invention is to provide saliva acid mimics to inhibit macrophage in preparation
Application under cell infection or state of activation in the drug of caused inflammation.
To achieve the goals above, the technical scheme adopted by the invention is that:
Saliva acid mimic inhibits answering in the drug of caused inflammation under infection of macrophages or state of activation in preparation
With.
Saliva acid mimic is 3 '-saliva lactose, 6 '-saliva lactose sodium salts or 3 '-saliva lactose, 6 '-saliva lactose
The mixture of sodium salt.
Saliva acid mimic further include using the saliva acid mimic as core, it is any that the saliva acid mimic is changed
It makes, optimize.
Saliva acid mimic is inhibiting the pass of inflammatory signals access caused by virus infection or pro-inflammatory cytokine activating macrophage
Application in key molecule activation.
Saliva acid mimic is inhibiting pro-inflammatory cytokine caused by virus infection or pro-inflammatory cytokine activating macrophage
Application in generation.
Macrophage is mouse monokaryon macrophage Raw264.7.
The present invention utilizes two kinds of commercialization salivas acid mimic (3 '-saliva lactose, English names 3 '-
Sialyllactose, referred to as 3 ' sia and 6 '-saliva lactose sodium salts, 6 '-Sialyllactose sodium of English name
Salt, referred to as 6 ' sia) to by mode RNA virus (such as vesicular stomatitis virus, English name vesicular stomatitis
Virus, abbreviation VSV) and infection or representative pro-inflammatory cytokine (such as lipopolysaccharides, English name lipopolysaccharide, referred to as
LPS the inflammation of Macrophage Model caused by) (such as mouse monokaryon macrophage Raw264.7) is handled；Pass through immunoblotting
Method (immunoblotting, IB), real-time quantitative RT-PCR (real-time PCR) experimental identification, the simulation of both sialic acids
Object all has effective anti-inflammatory properties, can be applied to anti-inflammatory drug research and development.
The invention has the advantages that:
1, the present invention comprehensive utilization technologies such as immunology and cell biology, find two kinds of saliva acid mimics to generation
The macrophage of inflammation has anti-inflammatory effect；
2, saliva acid mimic provided by the invention can be applied to anti-inflammatory drug research and development；
3, result of study provided by the invention enriches autarcesiology, mentions for follow-up study body inflammatory and anti-inflammatory mechanisms
For new thinking and strategy.
Detailed description of the invention
Fig. 1 is that two kinds of saliva acid mimics inhibit the inflammatory signals access as caused by VSV infection Raw264.7 cell to close
The activation of key molecule.
In figure, " Mock " is that VSV is uninfected by and is not added with saliva acid mimic group, and " PBS " is phosphate buffer
(phosphate buffered saline) represents VSV infection and is not added with saliva acid mimic group, infects with VSV and adds
Saliva acid mimic (" 3 ' sia " or " 6 ' sia " or " 3 ' sia+6 ' sia ") group compares；Inflammatory signals access key molecule phosphorus
For the gentle protein level of acidifying water respectively with " P- " and " Total- " mark, " p65 " is NF- κ B ingredient, and " I κ B α " is NF- κ B
Inhibit albumen, " ERK1/2 " is ERKs, and " p38 " is p38MAPK, and " β-actin " is beta-actin, as IB cell internal reference.
Fig. 2 is that two kinds of saliva acid mimics inhibit the pro-inflammatory cytokine as caused by VSV infection Raw264.7 cell
Generation.
In figure, " Mock-infection " is that VSV is uninfected by and is not added with saliva acid mimic group, and " VSV " is VSV infection
And it is not added with saliva acid mimic group, " VSV+3 ' sia " is that VSV infects and adds saliva acid mimic (3 ' sia) group, " VSV+
6 ' sia " are that VSV infects and add saliva acid mimic (6 ' sia) group, and " VSV+3 ' sia+6 ' sia " is that VSV infects and adds saliva
Liquid acid mimic (3 ' sia+6 ' sia) group；Pro-inflammatory cytokine generation is indicated with mRNA abundance；RT-PCR result is counted
Credit analysis is infected using VSV and is not added with saliva acid mimic group as standard, and " * " indicates p < 0.05, i.e. statistically significant, " * * "
Indicate p < 0.01, i.e. statistics is extremely significant.
Fig. 3 is that two kinds of saliva acid mimics inhibit the inflammatory signals access as caused by LPS stimulation Raw264.7 cell to close
The activation of key molecule.
In figure, " Mock " is that LPS does not stimulate and be not added with saliva acid mimic group, and " PBS " is phosphate buffer
(phosphate buffered saline) represents LPS stimulation and is not added with saliva acid mimic group, stimulates with LPS and adds
Saliva acid mimic (" 3 ' sia " or " 6 ' sia " or " 3 ' sia+6 ' sia ") group compares；" WCL " is full cell pyrolysis liquid
(whole cell lysate)；Inflammatory signals access key molecule phosphorylation level and protein level respectively with " P- " and
" Total- " mark, " p65 " are NF- κ B ingredient, and " I κ B α " is that NF- κ B inhibits albumen, and " β-actin " is that β-flesh moves egg
It is white, as IB cell internal reference.
Fig. 4 is that two kinds of saliva acid mimics inhibit the pro-inflammatory cytokine as caused by LPS stimulation Raw264.7 cell
Generation.
In figure, " Mock " is that LPS does not stimulate and be not added with saliva acid mimic group, and " LPS " is that LPS is stimulated and is not added with saliva
Liquid acid mimic group, " LPS+3 ' sia " are that LPS stimulates and add saliva acid mimic (3 ' sia) group, and " LPS+6 ' sia " is LPS
Stimulation and addition saliva acid mimic (6 ' sia) group, " LPS+3 ' sia+6 ' sia " are that LPS is stimulated and added saliva acid mimic
(3 ' sia+6 ' sia) group；Pro-inflammatory cytokine generation is indicated with mRNA abundance；RT-PCR result carries out statistical analysis, with
It is standard that LPS, which is stimulated and is not added with saliva acid mimic group, and " * " indicates p < 0.05, i.e., statistically significant, " * * " expression p <
0.01, i.e. statistics is extremely significant.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
3 '-saliva lactose are purchased from Sigma-Aldrich company.
6 '-saliva lactose sodium salts are purchased from Sigma-Aldrich company.
1. two kinds of saliva acid mimics of embodiment are crucial to the inflammatory signals access as caused by VSV infection Raw264.7 cell
The inhibition of molecule activation
The Raw264.7 cell of 1.1 two kinds of saliva acid mimic processing VSV infection
Raw264.7 cell is pressed 1 × 105A/mL is laid with 24 porocyte culture plates (500 μ L), removes original cell culture
Liquid, addition contain 500 μ of DMEM culture medium of 10% (v/v) heat-inactivated fetal bovine serum, 100U/mL penicillin, 100 μ g/mL streptomysins
L (Gibco company, the U.S.), in 37 DEG C of CO212h is cultivated in incubator；Saliva is added into Raw264.7 cell culture fluid respectively
Acid mimic 3 '-saliva lactose PBS solution (10 μM of final concentration), 6 '-saliva lactose sodium salt PBS solutions (10 μM of final concentration) or two
Person's mixture PBS solution (final concentration is 10 μM), in 37 DEG C of incubation 1h；The Indiana VSV blood is diluted using DMEM culture medium
Clear type (Indiana serotype) strain to virus infection plural (MOI) is 20, in 37 DEG C of infection Raw264.7 cell 6h (into
Row culture solution is replaced to maintain 500 μ L total volumes)；Metainfective cell is harvested, RIPA lysate (the green skies biology in Shanghai is used
Technology Co., Ltd.) cracking, centrifugation, harvest WCL progress next step detection (VSV infection and addition saliva acid mimic group).Together
When, parallel laboratory test is carried out with the PBS solution substitution PBS solution of acid mimic containing saliva according to the above method, infects as VSV and does not add
Add saliva acid mimic group, infects and add saliva acid mimic group compareing with VSV；It is replaced with DMEM culture medium containing 20MOI's
VSV DMEM culture medium, the PBS solution substitution PBS solution of acid mimic containing saliva progress parallel laboratory test, are uninfected by and not as VSV
Saliva acid mimic (Mock) group is added, infects with VSV and be not added with saliva acid mimic group compareing.
1.2IB detects two kinds of saliva acid mimics and infects Raw264.7 cellular inflammation signal path key molecule to by VSV
The influence of activation
Using same amount β-actin as standard adjustment WCL applied sample amount；Utilize the dodecyl of 8-15% mass concentration gradient
Sodium sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) carries out Protein Separation to WCL；It will be separated by electric robin (15V, 1h)
Protein delivery afterwards is to vinylidene fluoride film (PVDF, Merck Millipore company, the U.S.)；Using containing 5% (v/v) defatted milk
PBST in 37 DEG C of closing 30min；Respectively with mouse β-actin (8H10D10) monoclonal antibody (goods purchased from U.S. CST company
Number #3700), rabbit phosphorylation NF- κ B p65 (Ser536) (93H1) monoclonal antibody (article No. #3033), rabbit NF- κ B p65
(D14E12)Monoclonal antibody (article No. #8242), mouse phosphorylation I κ B α (Ser32/36) (5A5) monoclonal antibody (article No. #
9246), mouse I κ B α (L35A5) monoclonal antibody (article No. #4814), rabbit phosphorylation p44/42MAPK Erk1/2 (Thr202/
Tyr204)(D13.14.4E)Monoclonal antibody (article No. #4370), rabbit p44/42MAPK Erk1/2 (137F5) monoclonal
Antibody (article No. #4695), phospho p38 MAPK antibody (article No. #9211), p38MAPK antibody (article No. #9212) are used as primary antibody,
37 DEG C of incubation 1h；PBST is washed 3 times, the sheep anti mouse or rabbit antibody (U.S. Abbkine marked with horseradish peroxidase (HRP)
Company) it is used as secondary antibody, 37 DEG C of incubation 30min；PBST is washed 3 times, uses enhanced chemical luminescence reagent (Beijing Suo Laibao science and technology
Co., Ltd) respectively to inflammatory signals access key molecule NF- κ B p65, I κ B α, p38MAPK, ERKs phosphorylation and albumen water
It is flat to develop the color.
As a result as shown in Figure 1, compared with Mock group, VSV, which infects and is not added with saliva acid mimic group, passes through phosphorylation NF-
κ B inhibits protein I κ B α (band thickening) that I κ B α is caused to degrade (band decrease), so that NF- κ B ingredient p65 phosphorylation be made to swash
Living (band thickening), in addition, phosphorylation activation (band thickening) equally occurs for p38MAPK and ERKs does not have significant changes, shows
VSV infection can cause Raw264.7 cellular inflammation signal path key molecule activation, that is, cause inflammatory reaction；However, being added two
It (infects with VSV and not plus compared with saliva acid mimic group) after kind saliva acid mimic, presses down although being activated to p38MAPK, ERKs
Effect processed is unobvious, but cause I κ B α phosphorylation level decline (band decrease) and it is non-degradable (band thickening), to inhibit
NF- κ B ingredient p65 phosphorylation activation (band decrease), table particularly evident particularly with the addition of the two mixture inhibiting effect
Bright two kinds of saliva acid mimics can inhibit living by the caused Raw264.7 cellular inflammation signal path key molecule of VSV infection
Change, that is, there is anti-inflammatory effect.
2. two kinds of saliva acid mimics of embodiment are raw to the pro-inflammatory cytokine as caused by VSV infection Raw264.7 cell
At inhibition
The Raw264.7 cell of 2.1 two kinds of saliva acid mimic processing VSV infection
Raw264.7 cell is pressed 1 × 105A/mL is laid with 24 porocyte culture plates (500 μ L), removes original cell culture
Liquid, addition contain 500 μ of DMEM culture medium of 10% (v/v) heat-inactivated fetal bovine serum, 100U/mL penicillin, 100 μ g/mL streptomysins
L (Gibco company, the U.S.), in 37 DEG C of CO212h is cultivated in incubator；Saliva is added into Raw264.7 cell culture fluid respectively
Acid mimic 3 '-saliva lactose PBS solution (10 μM of final concentration), 6 '-saliva lactose sodium salt PBS solutions (10 μM of final concentration) or two
Person's mixture PBS solution (final concentration is 10 μM), in 37 DEG C of incubation 1h；The Indiana VSV blood is diluted using DMEM culture medium
Clear type strain to virus infection plural (MOI) is 20, (carries out culture solution displacement in 37 DEG C of infection Raw264.7 cell 6h to maintain
500 μ L total volumes), it harvests metainfective cell and carries out next step detection (VSV infection and addition saliva acid mimic group).Together
When, parallel laboratory test is carried out with the PBS solution substitution PBS solution of acid mimic containing saliva according to the above method, infects as VSV and does not add
Add saliva acid mimic group, infects and add saliva acid mimic group compareing with VSV；It is replaced with DMEM culture medium containing 20MOI's
VSV DMEM culture medium, the PBS solution substitution PBS solution of acid mimic containing saliva progress parallel laboratory test, are uninfected by and not as VSV
Saliva acid mimic (Mock) group is added, infects with VSV and be not added with saliva acid mimic group compareing.
2.2RT-PCR detects two kinds of saliva acid mimics to raw by VSV infection Raw264.7 cell pro-inflammatory cytokine
At influence
The total serum IgE that reagent TRIzol (Invitrogen company, the U.S.) extracts cell is extracted using RNA, is used
PrimeScriptTMReverse transcription reagent box (TaKaRa company) generates complementary DNA (cDNA) and is used as quantitative fluorescent PCR template.Using
Relative fluorescence quantitative PCR surveys pro-inflammatory cytokine (TNF-α, IL-6, IL-1 β) mRNA abundance in infection cell
It is fixed, with glyceraldehyde 3-phosphate dehydro-genase (GAPDH) for internal reference, using 2-ΔΔCtMethod analyzes data.Primer and reaction system
See Tables 1 and 2, response procedures carry Fast sequencing (U.S. Applied Biosystems public affairs using ABI fluorescence quantitative PCR instrument
Department).The experiment independently carries out three times, and three repetitions every time, experimental data is with cell mean and standard error of mean (SEM) table
Show, statistical analysis is examined using the double tail Student t of Origin software non-matching and carried out；" * " indicates p < 0.05, i.e. statistics
Significantly, " * * " indicates p < 0.01, i.e. statistics is extremely significant.
1 relative fluorescence quantification PCR primer sequence of table
2 relative fluorescence quantitative PCR reaction system of table
As a result as shown in Fig. 2, compared with Mock group, VSV infection (being not added with saliva acid mimic) can cause Raw264.7
Cell representativeness pro-inflammatory cytokine (TNF-α, IL-6, IL-1 β) mRNA abundance increases, and shows that VSV infection can cause
Raw264.7 cell pro-inflammatory cytokine generates, that is, causes inflammatory reaction；However, after two kinds of saliva acid mimics are added (with
VSV infects and saliva acid mimic group is not added to compare) but cause representative pro-inflammatory cytokine mRNA abundance to decline, especially
It is particularly evident that the two mixture effect is added, shows that two kinds of saliva acid mimics can inhibit caused by VSV infection
Raw264.7 cell pro-inflammatory cytokine generates, that is, has anti-inflammatory effect.
3. two kinds of saliva acid mimics of embodiment are crucial to the inflammatory signals access as caused by LPS stimulation Raw264.7 cell
The inhibition of molecule activation
The Raw264.7 cell of 3.1 two kinds of saliva acid mimic processing LPS stimulations
Raw264.7 cell is pressed 1 × 105A/mL is laid with 24 porocyte culture plates (500 μ L), removes original cell culture
Liquid, addition contain 500 μ of DMEM culture medium of 10% (v/v) heat-inactivated fetal bovine serum, 100U/mL penicillin, 100 μ g/mL streptomysins
L (Gibco company, the U.S.), in 37 DEG C of CO212h is cultivated in incubator；Saliva is added into Raw264.7 cell culture fluid respectively
Acid mimic 3 '-saliva lactose PBS solution (10 μM of final concentration), 6 '-saliva lactose sodium salt PBS solutions (10 μM of final concentration) or two
Person's mixture PBS solution (final concentration is 10 μM), in 37 DEG C of incubation 1h；Add (the U.S. LPS of 10 μ g/mL of final concentration
Sigma-Aldrich company, article No. #L3024), in 37 DEG C of stimulation Raw264.7 cell 1h；Post-stimulatory cell is harvested, is used
RIPA lysate (the green skies Bioisystech Co., Ltd in Shanghai) cracking is harvested by centrifugation, and WCL carries out next step detection (LPS stimulation
And addition saliva acid mimic group).Meanwhile according to the above method with PBS solution substitution the PBS solution of acid mimic containing saliva carry out it is flat
Row experiment, stimulates as LPS and is not added with saliva acid mimic group, stimulates with LPS and addition saliva acid mimic group compares；With
LPS is not stimulated, the PBS solution substitution PBS solution of acid mimic containing saliva carries out parallel laboratory test, is not stimulated and is not added with as LPS
Saliva acid mimic (Mock) group stimulates with LPS and is not added with saliva acid mimic group and compares.
3.2IB, which detects two kinds of saliva acid mimics, stimulates Raw264.7 cellular inflammation signal path key molecule to by LPS
The influence of activation
Using same amount β-actin as standard adjustment WCL applied sample amount；Utilize the dodecyl of 8-15% mass concentration gradient
Sodium sulphate polyacrylamide gel electrophoresis (SDS-PAGE) carries out Protein Separation to WCL；It will be separated by electric robin (15V, 1h)
Protein delivery afterwards is to vinylidene fluoride film (PVDF, Merck Millipore company, the U.S.)；Using containing 5% (v/v) defatted milk
PBST in 37 DEG C of closing 30min；Respectively with mouse β-actin (8H10D10) monoclonal antibody (goods purchased from U.S. CST company
Number #3700), rabbit phosphorylation NF- κ B p65 (Ser536) (93H1) monoclonal antibody (article No. #3033), rabbit NF- κ B p65
(D14E12)Monoclonal antibody (article No. #8242), mouse phosphorylation I κ B α (Ser32/36) (5A5) monoclonal antibody (article No. #
9246), mouse I κ B α (L35A5) monoclonal antibody (article No. #4814) is used as primary antibody, 37 DEG C of incubation 1h；PBST is washed 3 times, with peppery
The sheep anti mouse or rabbit antibody (Abbkine company, the U.S.) of root peroxidase (HRP) label are used as secondary antibody, 37 DEG C of incubation 30min；
PBST is washed 3 times, using enhanced chemical luminescence reagent (Beijing Suo Laibao Science and Technology Ltd) respectively to inflammatory signals access
Key molecule NF- κ B p65, I κ B α phosphorylation and protein level develop the color.
As a result as shown in figure 3, compared with Mock group, LPS, which is stimulated and is not added with saliva acid mimic group, passes through phosphorylation NF-
κ B inhibits protein I κ B α (band thickening) that I κ B α is caused to degrade (band decrease), so that NF- κ B ingredient p65 phosphorylation be made to swash
(band thickening) living shows that LPS stimulation can cause Raw264.7 cellular inflammation signal path key molecule activation, that is, causes inflammation
Reaction；However, (stimulating with LPS and not plus compared with saliva acid mimic group) after two kinds of saliva acid mimics are added, but cause I κ B
α phosphorylation level decline (band decrease) and it is non-degradable (band thickening), to inhibit NF- κ B ingredient p65 phosphorylation
It activates (band decrease), it is particularly evident particularly with the addition of the two mixture inhibiting effect, show two kinds of saliva acid mimics
Inhibit the Raw264.7 cellular inflammation signal path key molecule activation caused by LPS stimulation, that is, there is anti-inflammatory effect.
4. two kinds of saliva acid mimics of embodiment are raw to the pro-inflammatory cytokine as caused by LPS stimulation Raw264.7 cell
At inhibition
The Raw264.7 cell of 4.1 two kinds of saliva acid mimic processing LPS stimulations
Raw264.7 cell is pressed 1 × 105A/mL is laid with 24 porocyte culture plates (500 μ L), removes original cell culture
Liquid, addition contain 500 μ of DMEM culture medium of 10% (v/v) heat-inactivated fetal bovine serum, 100U/mL penicillin, 100 μ g/mL streptomysins
L (Gibco company, the U.S.), in 37 DEG C of CO212h is cultivated in incubator；Saliva is added into Raw264.7 cell culture fluid respectively
Acid mimic 3 '-saliva lactose PBS solution (10 μM of final concentration), 6 '-saliva lactose sodium salt PBS solutions (10 μM of final concentration) or two
Person's mixture PBS solution (final concentration is 10 μM), in 37 DEG C of incubation 1h；Add (the U.S. LPS of 10 μ g/mL of final concentration
Sigma-Aldrich company, article No. #L3024), in 37 DEG C of stimulation Raw264.7 cell 1h；Post-stimulatory cell is harvested to carry out down
One step detection (LPS stimulation and addition saliva acid mimic group).Meanwhile garden sorrel containing saliva is substituted with PBS solution according to the above method
Quasi- object PBS solution carries out parallel laboratory test, and saliva acid mimic group is stimulated and be not added with as LPS, stimulates with LPS and adds saliva
The control of acid mimic group；It is not stimulated with LPS, the PBS solution substitution PBS solution of acid mimic containing saliva progress parallel laboratory test, as
LPS does not stimulate and is not added with saliva acid mimic (Mock) group, stimulates with LPS and is not added with saliva acid mimic group and compares.
4.2RT-PCR detects two kinds of saliva acid mimics to raw by LPS stimulation Raw264.7 cell pro-inflammatory cytokine
At influence
The total serum IgE that reagent TRIzol (Invitrogen company, the U.S.) extracts cell is extracted using RNA, is used
PrimeScriptTMReverse transcription reagent box (TaKaRa company) generates complementary DNA (cDNA) and is used as quantitative fluorescent PCR template.Using
Relative fluorescence quantitative PCR surveys pro-inflammatory cytokine (TNF-α, IL-6, IL-1 β) mRNA abundance in infection cell
It is fixed, with glyceraldehyde 3-phosphate dehydro-genase (GAPDH) for internal reference, using 2-ΔΔCtMethod analyzes data.Primer and reaction system
See Tables 1 and 2, response procedures carry Fast sequencing (U.S. Applied Biosystems public affairs using ABI fluorescence quantitative PCR instrument
Department).The experiment independently carries out three times, and three repetitions every time, experimental data is with cell mean and standard error of mean (SEM) table
Show, statistical analysis is examined using the double tail Student t of Origin software non-matching and carried out；" * " indicates p < 0.05, i.e. statistics
Significantly, " * * " indicates p < 0.01, i.e. statistics is extremely significant.
As a result as shown in figure 4, compared with Mock group, LPS stimulation (being not added with saliva acid mimic) can cause Raw264.7
Cell representativeness pro-inflammatory cytokine (TNF-α, IL-6, IL-1 β) mRNA abundance increases, and shows that LPS stimulation can cause
Raw264.7 cell pro-inflammatory cytokine generates, that is, causes inflammatory reaction；However, after two kinds of saliva acid mimics are added (with
LPS is stimulated and saliva acid mimic group is not added to compare) but cause representative pro-inflammatory cytokine mRNA abundance to decline, especially
It is particularly evident that the two mixture effect is added, shows that two kinds of saliva acid mimics can inhibit caused by LPS stimulation
Raw264.7 cell pro-inflammatory cytokine generates, that is, has anti-inflammatory effect.
Sequence table
<110>Henan Academy of Agricultural Sciences
<120>saliva acid mimic inhibits under infection of macrophages or state of activation in the drug of caused inflammation in preparation
Using
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Claims (6)
1. saliva acid mimic inhibits the application under infection of macrophages or state of activation in the drug of caused inflammation in preparation.
2. application according to claim 1, which is characterized in that saliva acid mimic is 3 '-saliva lactose, 6 '-salivas cream
The mixture of sugared sodium salt or 3 '-saliva lactose, 6 '-saliva lactose sodium salts.
3. application according to claim 2, which is characterized in that saliva acid mimic further includes with the saliva acid mimic
For core, any transformation to the saliva acid mimic, optimization.
4. application according to claim 1, which is characterized in that saliva acid mimic is inhibiting virus infection or pro-inflammatory cytokine
Application in the activation of inflammatory signals access key molecule caused by activating macrophage.
5. application according to claim 1, which is characterized in that saliva acid mimic is inhibiting virus infection or pro-inflammatory cytokine
Application in the generation of pro-inflammatory cytokine caused by activating macrophage.
6. application according to claim 1-5, which is characterized in that the macrophage is that mouse monokaryon macrophage is thin
Born of the same parents Raw264.7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910328620.0A CN110038020A (en) | 2019-04-23 | 2019-04-23 | Saliva acid mimic inhibits the application under infection of macrophages or state of activation in the drug of caused inflammation in preparation |
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